A preliminary survey of the diversity of soil algaeand cyanoprokaryotes on mafic and ultramaficsubstrates in South Africa

Arthurita VenterAC Anatoliy LevanetsA Stefan SiebertA and Nishanta RajakarunaAB

AUnit for Environmental Sciences and Management North-West UniversityPrivate Bag X6001 Potchefstroom 2520 South Africa

BCollege of the Atlantic 105 Eden Street Bar Harbor ME 04609 USACCorresponding author Email 10066551nwuacza

Abstract Despite a large body of work on the serpentine-substrate effect on vascular plants little work has beenundertaken to describe algal communities found on serpentine soils derived from peridotite and other ultramafic rocks Wereport a preliminary study describing the occurrence of algae and cyanoprokaryotes on mafic and ultramafic substratesfrom South Africa Results suggest that slope and aspect play a key role in species diversity and community compositionand although low pH nutrients and metal content do not reduce species richness these edaphic features also influencespecies composition Further typical soil genera such as Leptolyngbya Microcoleus Phormidium ChlamydomonasChlorococcum and Hantzschia were found at most sites Chroococcus sp Scytonema ocellatum Nostoc linckiaChlorotetraedron sp Hormotilopsis gelatinosa Klebsormidium flaccidium Pleurococcus sp and Tetracystis ellipticawere unique to one serpentine site The preliminary survey provides directions for future research on the serpentine-substrate effect on algal and cyanoprokaryote diversity in South Africa

Additional keywords algae cryptogamic ecology serpentine geoecology species diversity

Received 25 August 2014 accepted 2 February 2015 published online 21 April 2015

Introduction

A range of soils can develop from ultramafic rocks depending onclimate time relief chemical composition of the parentmaterialsas well as biotic factors especially plants and microbes (ProctorandWoodell 1975Cardace et al 2014)Serpentine soil is derivedfrom serpentinite a rock formed primarily by the hydration andmetamorphic transformation of the ultramafic rock peridotiteUltramafic rocks can vary greatly in chemical and mineralcomposition and can be composed of various combinations ofthe minerals olivine orthorhombic and monoclinic pyroxeneshornblende as well as the secondary products of these mineralssuch as serpentine group minerals including fibrous amphibolesand talc (Alexander et al 2007) Serpentine soils have elevatedlevels of heavy metals such as nickel (Ni) and chromium (Cr)near-neutral to alkaline pH values and calcium magnesium(Ca Mg) ratios lt1 (Rajakaruna et al 2009) Serpentine soilsare also generally characterised by nutrient deficienciesespecially nitrogen (N) phosphorus (P) and potassium (K)(Daghino et al 2012) The distinctive chemistry of ultramaficrocks and resulting serpentine soils restricts the growth of manyplants making such sites refuges for those plants that can thriveunder the serpentine influence (Alexander et al 2007) Serpentinehabitats are known to harbour high numbers of endemic plant

species (Siebert et al 2002 OrsquoDell and Rajakaruna 2011) andare model settings for the study of plant ecology and evolution(Harrison and Rajakaruna 2011) However cryptogamic species(ie such as lichens bryophytes algae cyanoprokaryotes) showlow levels of edaphic endemism (Bramwell andCaujape-Castells2011 Rajakaruna et al 2012) and appear to be broadly tolerant ofsubstrate resulting in wide geographic distributions and rangedisjunctions that frequently span more than one continent(Schuster 1983) Species with wide distributions sometimesundergo environmental modification and exhibit habitat-associated vagrant forms (ie morphotypes) For exampleRosentreter and McCune (1992) documented how soil biotaandclimate can interact toproducepartiallyor completelyvagrantlife forms in several lichen genera

According to Belnap and Lange (2001) algae andcyanoprokaryotes can colonise almost all soil types Terlizziand Karlander (1979) found members of the CyanophyceaeChlorophyceae and Bacillariophyceae in serpentine soilsamples collected at Soldiers Delight Maryland USA butconcluded that the composition of the soil flora at the divisionlevel is similar to that of more favourable soil types Serpentinesoil environments are comparable tometal-enrichedmine tailingsthat include stressors such as nutrient deficiencies unfavourable

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Australian Journal of Botanyhttpdxdoiorg101071BT14207

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soil structure water stress and toxic concentrations of metals(Reddy et al 2001 OrsquoDell and Rajakaruna 2011) Orlekowskyet al (2013) found that algae and cyanoprokaryotes are ableto colonise mine tailings despite the harsh conditions andspeculated that the presence of higher plants might haveprovided a microenvironment for the establishment of theseorganisms Rosentreter and McCune (1992) found that vascularplants create windbreaks and shade influencing moisture contentand light intensity at the soil surface and creating suitable habitatsfor microbes Cabala et al (2011) concluded that crust formationin soils with a low pH and heavy-metal contamination ispossible but an increase in moisture and pH promotes algaldevelopment According to Lukesova (2001) BacillariophyceaeandCyanophyceae aremore characteristic of alkaline environmentswhereas species of Chlorophyceae can colonise more acidic soils

Moisture is a key factor in the establishment of algae andcyanoprokaryotes The low plant cover often associated withexposed and rocky serpentine outcrops contributes to hot and drysoil surfaces (Kruckeberg 2002)making it uninhabitable for soil-dwelling algae and cyanoprokaryotes Surface temperature is alsoimportant because it regulates many ecosystem functions such asrates of N and carbon fixation soil water evaporation andmicrobial activity (Belnap 2003) Kruckeberg (2002) proposedthat the microbial biota of serpentine soil would be species poorparalleling the scanty vegetation but admitted that very little isknown about the microbes of serpentine soils

SinceKruckeberg (2002) numerous studies havedocumentedthe diversity of bacteria (Oline 2006 Rajkumar et al 2008) fungi(DeGrood et al 2005 Daghino et al 2012 Southworth et al2014) and lichens (Favero-Longo et al 2004 Rajakaruna et al2012) in serpentine soil yet investigations of algae andcyanoprokaryotes in ultramafic soil (Terlizzi and Karlander1979 Couteacute et al 1999 Hauer 2008) are still scarce The aimof the present study was to conduct a preliminary survey of thealgal and cyanoprokaryote composition in mafic- and ultramafic-derived soil to improve our understanding of the microbialdiversity of serpentine and related soils

Materials and methodsSampling sitesSoil was sampled in February 2012 at different samplinglocalities in Mpumalanga and Limpopo provinces of SouthAfrica (Fig 1 Table 1) Ohrigstad sites (Sites 1 and 2 whichwere siliciclastic rocks and Silverton Formation)were situated onthemetasediments of the Paleoproterozoic Transvaal Supergroupthat forms the floor to the maficndashultramafic Rustenburg LayeredSuite of the Bushveld Igneous Complex (Clarke et al 2009) TheBushveld Complex was formed 2000million years ago whenenormous volumes of magma intruded the upper levels of theearth crust (Clarke et al 2009) Steelpoort sites (Sites 3 and 4)were situated on the ultramafic pyroxenite hills of theVlakfonteinSubsuite of the Rustenburg Layered Suite and the Burgersfortsites (Sites 5 and 6) on mafic hills of Kolobeng norite Thevegetation of Sites 1ndash6 (Table 1) had an open to dense woodylayer including woody and herbaceous shrubs and an open toclosed grass layer (Mucina andRutherford 2006) It was found onmoderate to steep mountainsides

Sites 7 and 8 were located on amphibolites and serpentiniteof the lower-most greenstone formations of the BarbertonSupergroup (Norman and Whitfield 2006) These lowerformations (Sites 7 and 8) are metamorphic and made up ofthe volcano-sedimentary Onverwacht Group that consists ofultramafic rocks that formed 3500million years ago and minorfelsic volcanic and sedimentary rocks that were deposited inan ancient marine environment (Hofmann et al 2012) Thevegetation of Sites 7 and 8 is considered herbaceous savannawith an open woody layer and dense forb and grass layer(Mucina and Rutherford 2006) It is generally associated withhilly terrain with steep to moderate slopes

Mean annual temperatures of the study area vary between167C and 18C and amean annual summer rainfall ranges from518 to 1194mm (Mucina and Rutherford 2006 see Table 1)

At each site a disposable plastic teaspoon was used to scrapesoil up to a depth of 3mm at three localities not more than 50mapart namely underneath low-growing vegetation (mostlygrasses) in bare soil and between rocks A composite samplecomprising nine subsamples (teaspoons) was made for each ofthe eight sites and mixed thoroughly

IdentificationA combination of direct-determination and enrichment-culturetechniques were used to detect cyanoprokaryotes and eukaryoticalgae (Langhans et al 2009) A direct investigation wasconducted according to the method described in Lund (1945)Subsamples of 10 g of soil were wetted with distilled water inPetri dishes and incubated at 20C and a light intensity of35mmolmndash2 sndash1 Three sterile coverslips were placed on thesoil in each Petri dish after 24 h Algal communities thatestablished underneath the coverslips were examined andenumerated using the semiquantitative scale of Starmach(1963) which classifies algae and cyanoprokaryotes assubdominant if 30ndash50 specimens are present on the coverslipand dominant if more than 50 specimens are present

The enrichment-culture techniques included the use ofagar plates and liquid cultures For the agar plates 10-g soilsubsamples were incubated on 15 agar plates enriched witheither Boldrsquos basal growth medium (BBM Brown et al 1964) asdescribed in Stein (1973) or GBG11 growth medium (Kruumlger1978) and incubated at 20C and a light intensity of35mmolmndash2 sndash1 For the liquid cultures subsamples of 10 gof soil each were enriched with either 100mL BBM (Stein1973) or 100mL GBG11 growth medium (Kruumlger 1978) andincubated at a temperature of 20C and a continuous lightintensity of 15mmolmndash2 sndash1 to stimulate the growth of algaeand cyanoprokaryotes present in low concentrations Twodifferent growth media were used because green algal growthis enhanced by BBM (Stein 1973) and cyanoprokaryotes byGBG11 growth medium (Kruumlger 1978) Cyanoprokaryotesalso prefer lower light intensities and this was implementedduring the incubation of the cultures

The species were identified microscopically using a Nikon80i microscope with differential interference contrast lsquon 60XPlan Apochromatic 14 numerical aperture (NA) oil violet-corrected with a 14 NA oil condenser Literature used foridentification included Ettl et al (1999) Hindak (2008)

B Australian Journal of Botany A Venter et al

Huumlber-Pestalozzi (1961) John et al (2002) Komaacuterek andAnagnostidis (2005) Taylor et al (2007) and Wehr andSheath (2003)

Soil analysesAt each site the soil was randomly sampled from five of thenine subsamples per locality (minimum one and maximum twoper site) andpooled tomake a composite sampleThe soil analysiswas performed in accordance with the standards set out by thecontrol schemes of the Agricultural Laboratory Association ofSouthern Africa and the International Soil Analytical Exchange(ISE) Wageningen The Netherlands Exchangeable Ca Mg Kand sodium (Na) were estimated by 1M ammonium acetate(pH=7) P was estimated by P-Bray 1 extraction pH wasestimated via 1 25 extraction and electrical conductivity (EC)was determined with a saturated extraction These methodsfollowed NSSSA (1990) All heavy metals including aluminium(Al) arsenic (As) cadmium (Cd) cobalt (Co) copper (Cu) Criron (Fe) mercury (Hg) manganese (Mn) molybdenum (Mo) Nilead (Pb) and vanadium (V) as well asNandSwere estimatedby EPA Method 3050b (US EPA 1996)

Data analysisStatistica version 12 software (StatSoft Inc Tulda OK USA)was used to perform Studentrsquos t-tests to determine whether thedata from one site differed significantly (P lt 05) from those fromanother site The algal composition of a site was compared withthe algal composition of another site and repeated until each sitewas compared with all the other sites The approach was alsorepeated for the edaphic features of each site Similarities amongthe species compositions of the different sites were analysed byusing the BrayndashCurtis dissimilarity index (Hahs and McDonnell2006) This was performed with the software program Primer 5(Clarke and Gorley 2001) CANOCO version 45 software(Cambridge University Press Cambridge UK) was used toperform principal component analysis (PCA) on the chemicalvariables of the different sites Canonical correspondenceanalysis (CCA) was performed on the species data as well asthe chemical variables (Table 2) which included theMonte Carlopermutation test for significance (Ter Braak and Smilauer 1998)The species matrix for the analysis was compiled by allocatingvalues to the species thatwere absent (0) present (1) subdominant(2) and dominant (3)

N

S

Towns Onverwacht Group Barberton Supergroup

Silverton Formation Pretoria Group

Vlakfontein Subsuite amp Kolobeng Norite Rustenburg Layered Suite

0 50

MALELANEKAAPSEHOOP

OHRIGSTADBURGERSFORT

STEELPOORT

Mpumalanga

Limpopo

100 200 300 400

Kilometers

Africa

Mpumalanga

Limpopo

EW

Fig 1 Geology and locations of towns nearest to the sampling localities Sites 1 2 are near Ohrigstad Sites 3 4 are near Steelpoort Sites 5 6 are nearBurgersfort Site 7 is near Malelane and Site 8 is near Kaapschehoop Sites 7 and 8 are serpentinite-derived

Soil algae and cyanoprokaryotes Australian Journal of Botany C

Tab

le1

Listof

algaean

dsoilsamplinglocalitiesin

Mpu

malan

gaan

dLim

popo

Sou

thAfrica

includ

ingselected

environm

entalv

ariables

anddescriptions

Site

number

Nearesttown

Strataname

Lith

icclass

Aspect

Vegetationun

itMeanannu

alrainfall(m

m)

Meanannual

temperature

(C)

Coordinates

1Ohrigstad

Silv

ertonFormation

PretoriaGroup

Siliciclastic

rocks

Quartzite

Foo

tslopen

orthern

aspect

Ohrigstad

Mountain

Bushv

eld

645

180

24 4558

60S3

023

307

0 E

2Ohrigstad

Silv

ertonFormation

PretoriaGroup

Siliciclastic

rocks

Quartzite

Upp

erslop

eno

rthern

aspect

Ohrigstad

Mountain

Bushv

eld

645

180

24 4553

50S3

023

304

0 E

3Steelpo

ort

Vlakfon

tein

Subsuite

RustenburgLayered

Suite

Ultram

aficrocks

Pyrox

enite

Foo

tslopew

estern

aspect

Sekhu

khun

eMountain

Bushv

eld

609

175

24 2967

30S3

007

993

0 E

4Steelpo

ort

Vlakfon

tein

Subsuite

RustenburgLayered

Suite

Ultram

aficrocks

Pyrox

enite

Upp

erslop

ewestern

aspect

Sekhu

khun

eMountain

Bushv

eld

609

175

24 2961

80S3

008

020

0 E

5Burgersfort

Vlakfontein

Subsuite

RustenburgLayered

Suite

Maficrocks

Norite

Upp

erslop

enorth-western

aspect

Sekhu

khun

ePlains

Bushv

eld

518

190

24 4036

40S3

016

743

0 E

6Burgersfort

Vlakfontein

Subsuite

RustenburgLayered

Suite

Maficrocks

Norite

Foo

tslope

north-western

aspect

Sekhu

khun

ePlains

Bushv

eld

518

190

24 4036

90S3

016

731

0 E

7Malelane

Onverwacht

Group

BabertonSup

ergrou

pMetam

orphicrocks

Amph

ibolite

serpentin

eFoo

tslopen

orthern

aspect

Barberton

Serpentine

Sou

rveld

884

184

25 3199

70S3

131

050

0 E

8Kaapschehoop

Onverwacht

Group

BabertonSup

ergrou

pMetam

orphicrocks

Amph

ibolite

serpentin

eRocky

ridge

north-western

aspect

Barberton

Serpentine

Sou

rveld

1194

167

25 3289

20S3

048

962

0 E

D Australian Journal of Botany A Venter et al

Results

Soil analyses

All soils were slightly acidic except for serpentine Site 7 wherea pH (H2O) of 761 was reported (Table 2) The highestconcentrations of K and Al were measured at the upper slopeof the siliciclastic soils at Ohrigstad (Site 2) and the highestconcentrations of Cd and Hgwere measured at the foot-slope sitenear Steelpoort (Site 3) Burgersfort mafic soils had the highestconcentrations of Ca (Site 5) and P (Site 6)

The first two axes of the PCA of the soil characteristics andthe sampling sites explained 716 of the total variance (Fig 2)It showed a clear association of heavy metals such as As CoCu Cr Fe Mn Mo Ni Pd and V with the serpentine site atKaapschehoop (Site 8) that also features other characteristicsof serpentine soils such as low concentrations of essentialnutrients including a Ca Mg ratio of lt1 This wassubstantially lower than for other ultramafic and mafic rocks(Sites 3 4 6) with ratios lt1 All mafic and ultramafic sites hadheavy-metal (mostly Ni) concentrations exceeding those of thesedimentary rocks and comparable to concentrations found atthe two serpentine sites

The cation exchange capacity (CEC) of the serpentine soil atSite 8 was the lowest among the sites (376 cmol(+) kgndash1) This isbelow the minimum standard of 8 cmol(+) kgndash1 proposed foragricultural top soil in South Africa by the Soil ClassificationWorking Group (1991) The EC of Site 8 soil was low compared

to the other sites likely resulting from low K concentrationspresent at this site

A Studentrsquos t-test indicated that the edaphic features (seeTable 2 for soil features tested) of the siliciclastic Site 2 andthe serpentine Site 8 differ significantly (P= 0001) Sites 1 2 and8 had the highest concentrations of Al a known soil toxin inSouth Africa but the pH of Site 2 and Site 8 was lower (554and 591 respectively) than the pH of Site 1 (627) which couldplay a role in the bioavailability of heavy metals For instance atan acidic pH Al exists only in one oxidation state (+3) and canreact with other matter in the environment to form variouscomplexes (ATSDR 2008)

Algal diversity

Site 1 had the highest number of species (24) followed byserpentine Sites 7 (22) and 8 (20) (Table 3) The lowestnumber of species (6) was found on the pyroxenite-derivedsoil at Site 3 Cyanophyceae diversity was highest at Sites 14 7 and 8 and dominated at all sites except at Site 3 whereHantzschia amphioxys (Bacillariophyceae) was the dominantalgae Leptolyngbya sp was dominant at Sites 1 4 5 7 and 8and subdominant at Site 6 Phormidiumwas dominant at Sites 24 5 and7and subdominant atSites 3 6and8Microcoleus spwassubdominant in the siliciclastic sites near Burgersfort (Sites 1 2)as well as the serpentine sites (Sites 7 and 8) and dominant at themafic site near Burgersfort (Site 6) Leptolyngbya foveolarum

Table 2 Chemical characteristics for composite soil samples collected from the eight sitesEC electrical conductivity CEC cation exchange capacity

Soil characteristic Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Site 7 Site 8

pH (H2O) 627 554 646 636 686 631 761 591pH (KCl) 531 481 521 549 615 538 68 534EC (mS mndash1) 23 31 28 31 58 39 55 10CEC (cmol(+) kgndash1) 1144 1349 2468 2520 2595 2518 1839 376Base saturation 6625 4801 5436 6784 9317 7214 1094 469MacronutrientsCalcium (Ca) (cmol(+) kgndash1) 507 349 582 617 1461 84 822 036Magnesium (Mg) (cmol(+) kgndash1) 198 223 1125 1065 906 918 1151 136Ca Mg ratio 256 157 052 058 161 092 071 026Potassium (K) (mg kgndash1) 1955 281 1029 95 1885 218 1365 15Sodium (Na) (mg kgndash1) 6 8 7 7 6 7 9 9Phosphorus (P) (mg kgndash1) 36 43 212 108 39 377 42 17Nitrogen 046 044 052 049 058 063 047 04Sulfur 021 0 001 001 0 004 0 001

Micronutrients and heavy metals (mg kgndash1)Aluminium 2746 3514 826 921 1959 1514 1555 3021Arsenic 008 01 01 008 007 015 043 406Cadmium 0001 0001 001 0001 00008 0003 00008 00009Cobalt 138 179 348 392 22 479 378 1514Copper 117 153 085 06 174 126 435 831Chromium 626 794 8529 9397 3245 1244 5542 1342Iron 3098 3733 3832 3377 2271 4701 4274 8814Mercury 00004 00007 00046 00005 00002 00004 00006 00004Manganese 4733 6021 6035 7873 5168 1088 936 167Molybdenum 002 003 003 002 002 004 002 004Nickel 402 503 3625 2988 1748 3127 3386 4833Palladium 001 002 001 001 001 002 002 003Vanadium 851 1011 424 339 463 521 796 2062

Soil algae and cyanoprokaryotes Australian Journal of Botany E

Phormidium ambiguum and Hantzschia amphioxys were foundat all sites Chlorophyceae was the most diverse group with thehighest diversity at Site 1 whereas Bacillariophyceae diversitywas the highest at Sites 1 4 and 5 The CyanophyceaeChlorophyceae and Bacillariophyceae were represented atevery site However Eustigmatophyceae did not occur at Sites3 and 4 and the Xantophyceae occurred only at Sites 2 6 and 7

Preliminary correlations between edaphic featuresand algal diversity

Multivariate analysis confirms the strong association of heavymetals with the serpentine soils at Site 8 (Figs 2 3) The first twoaxes of the CCA (Fig 3) explained only 362 of the varianceVariables with high inflation factors (Ca Mg P and N) wereremoved to improve the analysis but eigenvalues or theexplanation of the total variance did not change The forwardselection of factors lowered the P-value however it was stillnot significant so we kept all the variables Even with the lowvalues the CCA helps to visualise the species distribution withrespect to the different sites and presents preliminary informationon how different edaphic features are correlated with algaldiversity Hormotilopsis gelatinosa Klebsormidium flaccidumPleurococcus species Chlorotetraedron species and Tetracystiselliptica belonging to the Chlorophyceae and Chroococcus

species Scytonema ocellatum Nostoc linckia as well as anunknown Nostoc species from the Cyanophyceae were closelyassociated with Site 8 (Fig 3) High concentrations of K wereassociatedwith theupper slopeof thesedimentary soils inOhrigstad(Site 2 Figs 2 3) as well as the green algae Characiopsis minimaChlamydomonas macrostellata Bumilleriopsis filiformis from theXanthophyceae and the cyanoprokaryote Oscillatoria raoiCalcium Mg S and N appear to play a role in the similarity inspecies composition of Sites 1 3 4 and 5

BrayndashCurtis dissimilarity index (Hahs and McDonnell2006) grouped samples from lower slopes (Sites 1 6 and7 indicated with black square in Fig 3) whereas the othersites were scattered (stress value 005) Cyanoprokaryotessuch as Leptolyngbya foveolarum Microcoleus vaginatus andPhormidium ambiguum Chlorophyceae such asChlamydomonasand Chlorococcum species Eustigmatophyceae such asEustigmatos magnus and Hantzschia amphioxys fromBacillariophyceae occurred on all the lower-slope sites (Sites 16 and 7) but were not unique to these sites The cyanoprokaryotePhormidium ambiguum was dominant in all the upper-slope sites(Sites 2 4 and 5) It was interesting that the serpentine sites(Sites 7 and 8) did not form a group Nor did the mafic andultramafic sites (Sites 3 4 5 and 6) or siliciclastic sites (Sites 1and 2) This random pattern with a lack of grouping for sitesshowed that slope position (upper vs lower) had a larger effect on

Legend sites rarr environmental variables

Axis 1 2 3 4

Eigen values 048 0235 0093 0091

Cumulative percentage variance

Of species 48 716 808 90

ndash10 15ndash10

10

Ca

Mg

K

Na

P

N

S

Al

As

Cd

Co

Cu

Cr

Fe

Hg

MnMo

Ni

Pd

V

12

3

4

5

6

7

8

Fig 2 Principal component analysis (PCA) to show the relationship between soil characteristics and the sampling sites

F Australian Journal of Botany A Venter et al

Table 3 Algae and cyanoprokaryotes identified from the different soil samples collected from the eight sitesdom dominant sdom subdominant + present

Species Abbreviations Siliciclastic rocks Mafic and ultramafic rocks Serpentineused in Fig 3 Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Site 7 Site 8

CyanophyceaeChroococcus sp Chro +Komvophoron cf schmidlei (Jaag)Anagnostids et Komaacuterek

Kom +

Leptolyngbya foveolarum (Rabenhorstex Gomont) Anagnostidiset Komaacuterek

Lepto dom + + dom dom sdom dom dom

Lyngbya major Meneghini Lyn1 + + +Lyngbya cf truncicola Ghose Lyn2 +Microcoleus vaginatus (Vauch) Gom Micro sdom sdom dom sdom sdomNostoc commune Vauch sensu Elenk Nos1 +Nostoc linckia (Roth) Born et Flah

in sensu ElenkNos2 sdom

Nostoc punctiforme (Kuumltz) Hariot Nos3 +Nostoc sp Nos4 +Oscillatoria limosa Ag Osc1 + +Oscillatoria raoi De Toni Osc2 +Phormidium ambiguum Gomont Pho1 + dom sdom dom dom sdom dom sdomPhormidium animale (Agardh ex

Gomont) Anagnostidis et KomaacuterekPho2 +

Phormidium cf corium (Ag) Kuumltzex Gomont

Pho3 +

Phormidium cf jenkelianum Schmid Pho4 +Phormidium jadinianum Gomont Pho5 +Phormidium mucicola Naum et Hub-

PestalozziPho6 +

Pseudanabaena cf minima (GSAn)Anagnostidis

Pseu1 +

Pseudanabaena frigida (Fritsch)Anagnostidis

Pseu2 +

Pseudanabaena sp Pseu3 + + +Scytonema myochrous (Dillw) Ag

ex Born et FlahScyt1 + dom

Scytonema ocellatum Born et Flah Scyt2 domScytonema sp Scyt3 +Synechocystis crassa Woronich Syn +Total Cyanophyceae 7 4 4 9 4 5 8 8

ChlorophyceaeBracteacoccus cf grandis Bischoff

et BoldBrac1 +

Bracteacoccus minor (Chod) Petrovaacute Brac2 +Bracteacoccus sp Brac3 +Characiopsis minima Pascher Chara +Chlamydomonas macrostellata Lund Chlamy sdomChlamydomonas sp1 Chlam1 + + + + + +Chlamydomonas sp2 Chlam2 +ChlorellamunitissimaFott etNovaacutekovaacute Chlo1 +Chlorella vulgaris Beijerinck Chlo2 + sdom +Chlorella sp Chlo3 + +Chlorococcum echinozygotum Starr Chlc1 sdomChlorococcum infusionum (Schrank)

MeneghiniChlc2 + + + + +

Chlorococcum oviforme Archibaldet Bold

Chlc3 +

Chlorococcum vacuolatum Starr Chlc4 +Chlorococcum sp1 Chlc5 + +Chlorococcum sp2 Chlc6 + + + + +

(continued next page )

Soil algae and cyanoprokaryotes Australian Journal of Botany G

diversity Cyanoprokaryote species were less associated withthe foot slope of pyroxenitendashmagnesium rich soil in Steelpoort(Site 3) with a Studentrsquos t-test supporting a significant differencefrom those at Sites 1 (P=003) 4 (P=003) 7 (P=0007) and 8(P=001) The pattern also showed that the lithic class may beless important than the slope position in determining the algaland cyanoprokayote species present at the sites

Discussion

It is widely reported that cyanoprokaryotes are well adapted toa wide range of environmental conditions and have an ability

to growon avariety of substrates such asmine tailings (Lukesova2001 Orlekowsky et al 2013) barren artic soils (Michaud et al2012) arid regions (Rehaacutekovaacute et al 2011 Muumlhlsteinovaacute et al2014) and alkaline and serpentinising springs (Blank et al 2009Suzuki et al 2013) Our preliminary study documented thatcyanoprokaryotes and algae are found on mafic and ultramaficsoils in SouthAfrica and that slope position plays a role in speciescomposition Leptolyngbya foveolarumMicrocoleus vaginatusPhormidium ambiguum Chlamydomonas ChlorococcumEustigmatos magnus as well as Hantzschia amphioxys werepresent on all three foot-slope sites at Ohrigstad BurgersfortandMalelane (Sites 1 6 and7) Environmental conditions such as

Table 3 (continued )

Species Abbreviations Siliciclastic rocks Mafic and ultramafic rocks Serpentineused in Fig 3 Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Site 7 Site 8

Chlorosarcinopsis aggregata Arceet Bold

Chls1 +

Chlorosarcinopsis minor (Gerneck)Herndon

Chls2 sdom + +

Chlorotetraedron sp Chlt +Hormotilopsis gelatinosa Trainoret Bold

Hormo +

Interfilum sp Inter + +Klebsormidium flaccidum (Kuumltz)PCSilva Mattox et WHBlackwell

Kleb +

Myrmecia biatorellae (Tschermak-Woess) JBPetersen

Myrm +

Pleurococcus Pleur +Scenedesmus sp Scene +Scotiellopsis rubescens Vinatzer Scot1 +Scotiellopsis terrestris (Reisigl)Puncochaacuterovaacute et Kalina

Scot2 + + +

Spongiochloris sp1 Spong1 + +Spongiochloris sp2 Spong2 + + +Tetracystis aggregata Brown et Bold Tetra1 + +Tetracystis elliptica Nakano Tetra2 +Tetracystis sp Tetra3 + +Total Chlorophyceae 12 7 1 7 5 8 10 9

EustigmatophyceaeEustigmatos magnus (JBPetersen)Hibberd

Eust + + + sdom +

Monodopsis subterranea (JBPetersen)Hibberd

Mono + +

Total Eustigmatophyceae 1 1 0 0 1 1 1 2

XantophyceaeBotrydiopsis arhiza Borzi Botry1 sdomBotrydiopsis sp Botry2 +Bumilleriopsis filiformis Vischer Bum1 sdomTotal Xantophyceae 0 1 0 0 0 1 1 0

BacillariophyceaeAmphora veneta Kuumltzing Amph1 +Pinnularia borealis Ehrenberg Pin + + sdomNavicula mutica Kuumltzing Nav1 + +Navicula pelliculosa (Kuumltzing) Hilse Nav2 + +Navicula veneta Kuumltzing Nav3 +Hantzschia amphioxys (Ehrenberg)Grunow

Hant + sdom dom sdom sdom + + +

Total Bacillariophyceae 4 1 1 3 4 1 2 1

Total species richness 24 14 6 19 14 16 22 20

H Australian Journal of Botany A Venter et al

an increase in soil moisture as a result of runoff and theaccumulation of organic matter (Chen et al 2007) could haveplayed a role in the assembly of certain species on foot slopes Thecyanoprokaryotes Leptolyngbya foveolarum and Phormidiumambiguum were also present at the foot slope of the Mg-richpyroxenite soil (Site 3) whereas Microcoleus vaginatusChlamydomonas Chlorococcum and Eustigmatos magnus didnot occur at this site The absence of these algae as well as the lowdiversity observed at Site 3 could be the result of the highconcentrations of Cd (001mg kgndash1) and Hg (00046mg kgndash1)measured at this site According to Fernandez-Pintildeas et al (1995)Cd induces progressive disorganisation and degradation ofthylakoid membranes as well as a decrease in the mobilisationof the polyphosphate granules leading to phosphorus starvationPinto et al (2003) showed that Cd andHg inhibit photosynthesisHowever further experimentation is clearly needed to determinewhether Cd and Hg were responsible for the reduced diversityobserved at this site

Most of the cyanoprokaryotes were represented by filamentousspecies such as Leptolyngbya foveolarumMicrocoleus vaginatusand Phormidium ambiquum and were found at all eight samplingsites These filamentous cyanoprokaryotes can wind throughoutthe uppermost soil layers forming a net-like structure that bindstogether soil particles (Rosentreter et al 2007) This formssoil aggregates that create pathways for water infiltration andsurfaces for nutrient transformations while also increasing thesoil resistance to wind and water erosion Once the filamentouscyanoprokaryotes stabilise thesoil single-celledcyanoprokaryotessuch as Synechocystis sp are able to colonise the substrate(Rosentreter et al 2007) The Chlorophyceae representedby mainly coccoid species such as Chlamydomonas andChlorococcum were found on all the sites except at Site 3Chlorococcum echinozygotum and Chlamydomonas macrostellawere subdominant on the siliciclastic-derived soils of Sites 1 and 2respectively Bacillariophyceae such as Hantzschia amphioxyswas found on all the sites and was subdominant at Sites 2 4 and 5

Legend Δ species and sites rarr environmental variables

Axis

ndash06

ndash10 15

10

1 2 3 4

Eigen values 052 0411 0388 0368

Cumulative percentage variance

Of species 202 362 513 656

Of environmental relations 202 362 513 656

Test of significance of first canonical axes p = 10

Test of significance of all canonical axes p = 10

Fig 3 Canonical correspondence analysis (CCA) of metal concentrations and algal and cyanoprokaryote species of the different sampling sitesAbbreviations of species are given in Table 3

Soil algae and cyanoprokaryotes Australian Journal of Botany I

and dominant at Site 3 According to Metting (1981)Leptolyngbya Microcoleus Phormidium ChlamydomonasChlorococcum and Hantzschia species are all typicalcosmopolitan soil algae

Shields and Durell (1964) and Starks and Shubert (1982)suggested that the species composition of soil algal populationsis affected less by the chemical nature of the substrate than bycertain physical properties that influence soil moisture levelsDuring February 2012 when sampling took place the study areahad received between 50 and 200mm rain which accounts formore than 75 of the expected average summer rainfall for thearea (South African Weather Service 2012) Moisture wastherefore not a limiting factor and the chemical nature of thesoils along with other abiotic habitat features appear to haveinfluenced species richness and composition In addition tothe abiotic conditions such as edaphic features biotic factorsespecially the type of vegetation can influence diversity andcommunity composition of cryptogamic biota For exampleRosentreter et al (2007) documented that vagrant populationsofDermatocarponoccur on poorly drained basaltflats dominatedby Artemisia rigida A papposa Antennaria flagellaris andPoa sandbergii The ecological conditions found at such sitescan support completely or partially vagrant life forms inDermatocarpon and other lichen genera The differences invegetation cover of the plant communities at the sampling sites(Sites 1ndash8) could have created microenvironments that favourspecific algal and cyanoprokaryote species assemblages Sites1ndash7 had moderate cover whereas Site 8 was an open savannacharacterised by numerous bare soil patches (serpentine effect)

Twenty different species were identified at Site 8 (serpentine)despite this site showing elevated concentrations of heavymetalsincluding Co Cr Fe Mn and Ni (Table 2) Chroococcus spScytonema ocellatum Nostoc linckia Chlorotetraedron spHormotilopsis gelatinosa Klebsormidium flaccidiumPleurococcus sp and Tetracystis elliptica were unique to thissite Metals can be divided into those that are required byorganisms in small quantities such as Fe Cu and Zn whichare essential in some biochemical reactions (Raven et al 1999)andnon-essentialmetals such asAs Pb andHgwhichmaycausesevere harm to organisms even at very low concentrationsbecause they do not provide any known biochemical function(Monteiro et al 2012) However the toxic effects of metallicelements on microalgae are complex and differ markedly amongspecies depending on the element itself and the prevailingenvironmental conditions (Monteiro et al 2012) Stark andShubert (1979) reported a positive correlation between Mn NP silica (Si) Al zinc (Zn) and Pb concentrations and algalabundance and a negative correlation with Na Cd Culithium (Li) Mo and strontium (Sr) Cyanoprokaryotes havedeveloped efficient strategies for metal uptake andaccumulation (Shcolnick and Keren 2006) According toKeren et al (2002) cyanoprokaryotes are able to accumulatehigh concentrations of Mn in the envelope layers of their cellsWhether the highMn supports the growth of cyanoprokaryotes orreduces competition by other groups that are less tolerant of Mnwas unclear Soil pH plays a critical role in the bioavailability ofheavy metals (Rajakaruna and Boyd 2008) and althoughserpentine Site 7 had high metal concentrations the relativelyhigher pH at this site may have made those metals less

bioavailable (Neilson and Rajakaruna 2012) Hence it wasunclear whether the higher algal diversity at this site resultedfrom metal tolerance or reduced metal toxicity owing to lowermetal availabilityHowever algae are able togrow in thepresenceof heavy metals as a result of a variety of tolerance mechanismsfor example binding to cell wall precipitation in vacuole andsynthesis of heavy metal-binding compounds such as proteinsorganic acids and phenolic compounds (Metha and Gaur 2005)Serpentine soils are renowned for low Ca Mg ratio howeverDeGrood et al (2005) found that low Ca Mg ratio does notsignificantly explain variation in microbial community patternsalso our study did not show a strong correlation between algaldiversity and Ca Mg Microbes may therefore be respondingto increased soil organicmatter concentrations probably becauseof the associated increases in nutrient availability and water-holding capacity

Algae as a group are physiologically heterogeneous makingany generalisation about their soil relations difficult (OrsquoKelly1974) However favourable soil pH moisture conditions andnutrient content contribute to species diversity and communitycomposition (Shields andDurell 1964)Themultivariate analysesdid not suggest a distinct species association for serpentine sites(Sites 7 and8) Thiswas also true for themafic andultramafic sites(Sites 3 4 5 and 6) as well as the siliciclastic sites (Sites 1 and 2)The lack of a strong correlation with lithic type and a strongercorrelation with slope position suggests that the type of slopemay bemore important in influencing diversity Site pairs 1 and 23 and 4 as well as 5 and 6 were sampled at different slopepositions and at times shared similar species associationsbased on slope position (Fig 3)

Conclusions

It seems unlikely that soil chemistry alone was responsiblefor determining species diversity and no unique algal florafor serpentine soils was confirmed However the serpentinesoil at Kaapschehoop (Site 8) did have a unique speciesassemblage comprising Chroococcus sp Chlorotetraedronsp Hormotilopsis gelatinosa Klebsormidium flaccidiumPleurococcus sp and Tetracystis elliptica However thisrequires further investigation to determine whether it was alithic or climatic effect

Soil features alongwith other biotic (vegetation composition)and abiotic (slope exposure) habitat characteristics mayinfluence the presence and dominance of some algal andcyanoprokaryote species in harsh edaphic settings Our resultssuggested that topography (ie slope position) rather than thechemistry of the lithic class (ie rock type) wasmost important ininfluencing species diversity However high concentrations ofheavy metals also influenced species richness as well ascommunity composition

The characterisation of microbial communities using anisolation approach can be biased because this approach tendsto favour some groups of microbes over others Therefore in thefuture we plan to incorporate molecular approaches (Bjellandet al 2011 Daae et al 2013) to characterise the diversity ofcyanoprokaryotes and soil algae at our sites Additionally theecological heterogeneity within each site should be taken intoconsideration because our sampling strategy (ie pooling of soil

J Australian Journal of Botany A Venter et al

samples) did not allow for detecting species that may berestricted to distinct microhabitats found within our sites Acomposite sample does not allow for a correlation with bioticand abiotic factors but gives a general and preliminary view onthe diversity at each site Although our findings are preliminarythe study sets the stage for detailed investigations on the relativeimportance of edaphic versus other habitat features on algal andcyanoprokaryote diversity and community assembly

Acknowledgements

We thank the staff at Eko-Analitika North-West University Potchefstroomfor the soil analyses Christel Pretorius for drawing the map of the study areaand two anonymous reviewers for their useful suggestions to improve thequality of our manuscript

References

Alexander EB Coleman RG Keeler-Wolf T Harrison SP (2007)lsquoSerpentine geoecology of western North America geology soilsand vegetationrsquo (Oxford University Press New York)

ATSDR (Agency for Toxic Substances and Disease Registry) (2008)lsquoToxicological profile for aluminiumrsquo (US Department of Health andHuman Services Public Health Service Atlanta GA) Available at httpwwwatsdrcdcgovToxProfilestp22pdf [Verified 19 August 2014]

Belnap J (2003) The world at your feet desert biological soil crustsFrontiers in Ecology and the Environment 1 181ndash189doi1018901540-9295(2003)001[0181TWAYFD]20CO2

Belnap J Lange OL (2001) lsquoBiological soil crusts structure function andmanagementrsquo (Springer Berlin)

Bjelland T Grube M Hoem S Jorgensen SL Daae FL Thorseth IH OvrearingsL (2011) Microbial metacommunities in the lichen-rock habitatEnvironmental Microbiology Reports 3 434ndash442doi101111j1758-2229201000206x

Blank JG Green SJ Blake D Valley JW Kita NT Treiman A Dobson PF(2009) An alkaline spring system within the Del Puerto Ophiolite(California USA) a Mars analog site Planetary and Space Science57 533ndash540 doi101016jpss200811018

Bramwell D Caujape-Castells J (2011) lsquoThe biology of island florarsquo(Cambridge Press Cambridge UK)

Brown RM Larson DA Bold HC (1964) Airborne algae their abundanceand heterogeneity Science 143 583ndash585doi101126science1433606583

Cabala J Rahmonov O Jablonska M Teper E (2011) Soil algal colonizationand its ecological role in an environment polluted by past ZnndashPb miningand smelting activity Water Air and Soil Pollution 215 339ndash348doi101007s11270-010-0482-1

Cardace D Meyer-Dombard DR Olsen A Parenteau MN (2014) Bedrockand geochemical controls on extremophile habitats In lsquoPlant ecologyand evolution in harsh environmentsrsquo (Eds N Rajakaruna RS BoydTB Harris) pp 1ndash32 (Nova Science Publishers New York)

Chen YN Wang Q Li WH Ruan X (2007) Microbiotic crusts andthere interrelations with environmental factors in the Gurbantonggutdesert western China Environmental Geology 52 691ndash700doi101007s00254-006-0505-9

Clarke KR Gorley RN (2001) lsquoPRIMER v5 user manualtutorialrsquo(PRIMER-E Plymouth UK)

Clarke B Uken R Reinhardt J (2009) The geometry and emplacementmechanics of a Bushveld complex peridotite body South Africa SouthAfrican Journal of Geology 112 141ndash162 doi102113gssajg1122141

Couteacute A Tell G Therezien Y (1999) Aerophytic Cyanophyceae fromNew Caledonia Cryptogamie Algologie 20 301ndash344doi101016S0181-1568(00)88147-7

Daae FLOslashkland I DahleH Joslashrgensen SL Thorseth IH Pedersen RB (2013)Microbial life associated with low-temperature alteration of ultramafic

rocks in the Leka ophiolite complex Geobiology 11 318ndash339doi101111gbi12035

Daghino S Murat C Sizzano E Girlanda M Perotto S (2012) Fungaldiversity is not determined by mineral and chemical differences inserpentine substrates PLoS ONE 7(9) e44233doi101371journalpone0044233

DeGrood SH Claassen VP Scow KM (2005) Microbial communitycomposition on native and drastically disturbed serpentine soils SoilBiology amp Biochemistry 37 1427ndash1435doi101016jsoilbio200412013

Ettl H Gaumlrtner G Heynig H Mollenhauer D Komaacuterek J Anagnostidis K(1999) lsquoSuumlsswasser von Mittleeuropa Cyanoprokaryota Teil 1Chroococcalesrsquo (Gustav Fisher Verlag Stuttgart Germany)

Favero-Longo SE Isocrono D Piervittori R (2004) Lichens and ultramaficrocks a review Lichenologist (London England) 36 391ndash404doi101017S0024282904014215

Fernandez-Pintildeas F Mateo P Bonilla I (1995) Ultrastructural changesinduced by selected cadmium concentrations in the cyanobacteriumNostoc UAM208 Journal of Plant Physiology 147 452ndash456doi101016S0176-1617(11)82182-6

Hahs AK McDonnell MJ (2006) Selecting independent measures toquantify Melbournersquos urbanndashrural gradient Landscape and UrbanPlanning 78 435ndash448 doi101016jlandurbplan200512005

Harrison SP Rajakaruna N (2011) lsquoSerpentine evolution and ecology in amodel systemrsquo (University of California Press Berkeley CA)

Hauer T (2008) Epilithic cyanobacterial flora of Mohelenskaacute Hadcovaacutesteppe nature reseve (western Moravia Czech Republic) 70 years agoand now Fottea 8 129ndash132

Hindak F (2008) lsquoAtlas of cyanophytesrsquo (VEDA Bratislava Slovakia)Hofmann A Wilson A Kroumlner A Guy B Beukes N (2012) lsquoWorkshop

on craton formation and destruction post-conference excursionguidebook early craton formation stabilisation plume-relatedmagmatism and meteorite impact (Vredefort Witwatersrand Bushveldand Barberton)rsquo (Burlington House Piccadilly London) Availableat httpwwwgssaorgzaDocumentsCraton20Formation20and20Destruction20Excursion20Guidepdf [Verified 27November 2013]

Huumlber-Pestalozzi G (1961) lsquoDas Phytoplankton des Suumlsswassers SystematikundBiologie Tiel 5 Chlorophyceae (Gruumlnalgen) OrdnungVolvocalesrsquo(Verlagbuchhandlung Stuttgart Germany)

John DM Whitton BA Brook AJ (2002) lsquoFreshwater algal flora of theBritish Isles a guide to freshwater and terrestrial algaersquo (CambridgeUniversity Press Cambridge UK)

Keren N Kidd MJ Penner-Hahn JE Pakrasi HB (2002) A light-dependentmechanism formassive accumulation ofmanganese in the photosyntheticbacterium Synechocystis sp PCC 6803Biochemistry 41 15 085ndash15 092doi101021bi026892s

Komaacuterek J Anagnostidis K (2005) lsquoCyanoprokaryota Oscillatorialesrsquo(Spektrum Akademischer Verlag Heidelberg Germany)

Kruckeberg AR (2002) lsquoGeology and plant life the effects oflandforms and rock type on plantsrsquo (University Washington PressSeattle WA)

Kruumlger GHJ (1978) lsquoThe effect of physico-chemical factors on growthrelevant to the mass culture of Microcystis under sterile conditionsrsquo(University of the Orange Free State Bloemfontein South Africa)

Langhans TM Storm C Schwabe A (2009) Community assembly ofbiological soil crusts of different successional stages in a temperatesand ecosystem as assessed by direct determination and enrichmenttechniques Microbial Ecology 58 394ndash407doi101007s00248-009-9532-x

Lukesova A (2001) Soil algae in brown coal lignite post-mining areas incentral Europe (Czech Republic and Germany) Restoration Ecology 9341ndash350 doi101046j1526-100X200194002x

Lund JWG (1945) Observations on soil algae I The ecology size andtaxonomy of British soil diatoms Part 1 New Phytologist 44 196ndash219doi101111j1469-81371945tb05033x

Soil algae and cyanoprokaryotes Australian Journal of Botany K

Huumlber-Pestalozzi (1961) John et al (2002) Komaacuterek andAnagnostidis (2005) Taylor et al (2007) and Wehr andSheath (2003)

Soil analysesAt each site the soil was randomly sampled from five of thenine subsamples per locality (minimum one and maximum twoper site) andpooled tomake a composite sampleThe soil analysiswas performed in accordance with the standards set out by thecontrol schemes of the Agricultural Laboratory Association ofSouthern Africa and the International Soil Analytical Exchange(ISE) Wageningen The Netherlands Exchangeable Ca Mg Kand sodium (Na) were estimated by 1M ammonium acetate(pH=7) P was estimated by P-Bray 1 extraction pH wasestimated via 1 25 extraction and electrical conductivity (EC)was determined with a saturated extraction These methodsfollowed NSSSA (1990) All heavy metals including aluminium(Al) arsenic (As) cadmium (Cd) cobalt (Co) copper (Cu) Criron (Fe) mercury (Hg) manganese (Mn) molybdenum (Mo) Nilead (Pb) and vanadium (V) as well asNandSwere estimatedby EPA Method 3050b (US EPA 1996)

Data analysisStatistica version 12 software (StatSoft Inc Tulda OK USA)was used to perform Studentrsquos t-tests to determine whether thedata from one site differed significantly (P lt 05) from those fromanother site The algal composition of a site was compared withthe algal composition of another site and repeated until each sitewas compared with all the other sites The approach was alsorepeated for the edaphic features of each site Similarities amongthe species compositions of the different sites were analysed byusing the BrayndashCurtis dissimilarity index (Hahs and McDonnell2006) This was performed with the software program Primer 5(Clarke and Gorley 2001) CANOCO version 45 software(Cambridge University Press Cambridge UK) was used toperform principal component analysis (PCA) on the chemicalvariables of the different sites Canonical correspondenceanalysis (CCA) was performed on the species data as well asthe chemical variables (Table 2) which included theMonte Carlopermutation test for significance (Ter Braak and Smilauer 1998)The species matrix for the analysis was compiled by allocatingvalues to the species thatwere absent (0) present (1) subdominant(2) and dominant (3)

N

S

Towns Onverwacht Group Barberton Supergroup

Silverton Formation Pretoria Group

Vlakfontein Subsuite amp Kolobeng Norite Rustenburg Layered Suite

0 50

MALELANEKAAPSEHOOP

OHRIGSTADBURGERSFORT

STEELPOORT

Mpumalanga

Limpopo

100 200 300 400

Kilometers

Africa

Mpumalanga

Limpopo

EW

Fig 1 Geology and locations of towns nearest to the sampling localities Sites 1 2 are near Ohrigstad Sites 3 4 are near Steelpoort Sites 5 6 are nearBurgersfort Site 7 is near Malelane and Site 8 is near Kaapschehoop Sites 7 and 8 are serpentinite-derived

Soil algae and cyanoprokaryotes Australian Journal of Botany C

Tab

le1

Listof

algaean

dsoilsamplinglocalitiesin

Mpu

malan

gaan

dLim

popo

Sou

thAfrica

includ

ingselected

environm

entalv

ariables

anddescriptions

Site

number

Nearesttown

Strataname

Lith

icclass

Aspect

Vegetationun

itMeanannu

alrainfall(m

m)

Meanannual

temperature

(C)

Coordinates

1Ohrigstad

Silv

ertonFormation

PretoriaGroup

Siliciclastic

rocks

Quartzite

Foo

tslopen

orthern

aspect

Ohrigstad

Mountain

Bushv

eld

645

180

24 4558

60S3

023

307

0 E

2Ohrigstad

Silv

ertonFormation

PretoriaGroup

Siliciclastic

rocks

Quartzite

Upp

erslop

eno

rthern

aspect

Ohrigstad

Mountain

Bushv

eld

645

180

24 4553

50S3

023

304

0 E

3Steelpo

ort

Vlakfon

tein

Subsuite

RustenburgLayered

Suite

Ultram

aficrocks

Pyrox

enite

Foo

tslopew

estern

aspect

Sekhu

khun

eMountain

Bushv

eld

609

175

24 2967

30S3

007

993

0 E

4Steelpo

ort

Vlakfon

tein

Subsuite

RustenburgLayered

Suite

Ultram

aficrocks

Pyrox

enite

Upp

erslop

ewestern

aspect

Sekhu

khun

eMountain

Bushv

eld

609

175

24 2961

80S3

008

020

0 E

5Burgersfort

Vlakfontein

Subsuite

RustenburgLayered

Suite

Maficrocks

Norite

Upp

erslop

enorth-western

aspect

Sekhu

khun

ePlains

Bushv

eld

518

190

24 4036

40S3

016

743

0 E

6Burgersfort

Vlakfontein

Subsuite

RustenburgLayered

Suite

Maficrocks

Norite

Foo

tslope

north-western

aspect

Sekhu

khun

ePlains

Bushv

eld

518

190

24 4036

90S3

016

731

0 E

7Malelane

Onverwacht

Group

BabertonSup

ergrou

pMetam

orphicrocks

Amph

ibolite

serpentin

eFoo

tslopen

orthern

aspect

Barberton

Serpentine

Sou

rveld

884

184

25 3199

70S3

131

050

0 E

8Kaapschehoop

Onverwacht

Group

BabertonSup

ergrou

pMetam

orphicrocks

Amph

ibolite

serpentin

eRocky

ridge

north-western

aspect

Barberton

Serpentine

Sou

rveld

1194

167

25 3289

20S3

048

962

0 E

D Australian Journal of Botany A Venter et al

Results

Soil analyses

All soils were slightly acidic except for serpentine Site 7 wherea pH (H2O) of 761 was reported (Table 2) The highestconcentrations of K and Al were measured at the upper slopeof the siliciclastic soils at Ohrigstad (Site 2) and the highestconcentrations of Cd and Hgwere measured at the foot-slope sitenear Steelpoort (Site 3) Burgersfort mafic soils had the highestconcentrations of Ca (Site 5) and P (Site 6)

The first two axes of the PCA of the soil characteristics andthe sampling sites explained 716 of the total variance (Fig 2)It showed a clear association of heavy metals such as As CoCu Cr Fe Mn Mo Ni Pd and V with the serpentine site atKaapschehoop (Site 8) that also features other characteristicsof serpentine soils such as low concentrations of essentialnutrients including a Ca Mg ratio of lt1 This wassubstantially lower than for other ultramafic and mafic rocks(Sites 3 4 6) with ratios lt1 All mafic and ultramafic sites hadheavy-metal (mostly Ni) concentrations exceeding those of thesedimentary rocks and comparable to concentrations found atthe two serpentine sites

The cation exchange capacity (CEC) of the serpentine soil atSite 8 was the lowest among the sites (376 cmol(+) kgndash1) This isbelow the minimum standard of 8 cmol(+) kgndash1 proposed foragricultural top soil in South Africa by the Soil ClassificationWorking Group (1991) The EC of Site 8 soil was low compared

to the other sites likely resulting from low K concentrationspresent at this site

A Studentrsquos t-test indicated that the edaphic features (seeTable 2 for soil features tested) of the siliciclastic Site 2 andthe serpentine Site 8 differ significantly (P= 0001) Sites 1 2 and8 had the highest concentrations of Al a known soil toxin inSouth Africa but the pH of Site 2 and Site 8 was lower (554and 591 respectively) than the pH of Site 1 (627) which couldplay a role in the bioavailability of heavy metals For instance atan acidic pH Al exists only in one oxidation state (+3) and canreact with other matter in the environment to form variouscomplexes (ATSDR 2008)

Algal diversity

Site 1 had the highest number of species (24) followed byserpentine Sites 7 (22) and 8 (20) (Table 3) The lowestnumber of species (6) was found on the pyroxenite-derivedsoil at Site 3 Cyanophyceae diversity was highest at Sites 14 7 and 8 and dominated at all sites except at Site 3 whereHantzschia amphioxys (Bacillariophyceae) was the dominantalgae Leptolyngbya sp was dominant at Sites 1 4 5 7 and 8and subdominant at Site 6 Phormidiumwas dominant at Sites 24 5 and7and subdominant atSites 3 6and8Microcoleus spwassubdominant in the siliciclastic sites near Burgersfort (Sites 1 2)as well as the serpentine sites (Sites 7 and 8) and dominant at themafic site near Burgersfort (Site 6) Leptolyngbya foveolarum

Table 2 Chemical characteristics for composite soil samples collected from the eight sitesEC electrical conductivity CEC cation exchange capacity

Soil characteristic Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Site 7 Site 8

pH (H2O) 627 554 646 636 686 631 761 591pH (KCl) 531 481 521 549 615 538 68 534EC (mS mndash1) 23 31 28 31 58 39 55 10CEC (cmol(+) kgndash1) 1144 1349 2468 2520 2595 2518 1839 376Base saturation 6625 4801 5436 6784 9317 7214 1094 469MacronutrientsCalcium (Ca) (cmol(+) kgndash1) 507 349 582 617 1461 84 822 036Magnesium (Mg) (cmol(+) kgndash1) 198 223 1125 1065 906 918 1151 136Ca Mg ratio 256 157 052 058 161 092 071 026Potassium (K) (mg kgndash1) 1955 281 1029 95 1885 218 1365 15Sodium (Na) (mg kgndash1) 6 8 7 7 6 7 9 9Phosphorus (P) (mg kgndash1) 36 43 212 108 39 377 42 17Nitrogen 046 044 052 049 058 063 047 04Sulfur 021 0 001 001 0 004 0 001

Micronutrients and heavy metals (mg kgndash1)Aluminium 2746 3514 826 921 1959 1514 1555 3021Arsenic 008 01 01 008 007 015 043 406Cadmium 0001 0001 001 0001 00008 0003 00008 00009Cobalt 138 179 348 392 22 479 378 1514Copper 117 153 085 06 174 126 435 831Chromium 626 794 8529 9397 3245 1244 5542 1342Iron 3098 3733 3832 3377 2271 4701 4274 8814Mercury 00004 00007 00046 00005 00002 00004 00006 00004Manganese 4733 6021 6035 7873 5168 1088 936 167Molybdenum 002 003 003 002 002 004 002 004Nickel 402 503 3625 2988 1748 3127 3386 4833Palladium 001 002 001 001 001 002 002 003Vanadium 851 1011 424 339 463 521 796 2062

Soil algae and cyanoprokaryotes Australian Journal of Botany E

Phormidium ambiguum and Hantzschia amphioxys were foundat all sites Chlorophyceae was the most diverse group with thehighest diversity at Site 1 whereas Bacillariophyceae diversitywas the highest at Sites 1 4 and 5 The CyanophyceaeChlorophyceae and Bacillariophyceae were represented atevery site However Eustigmatophyceae did not occur at Sites3 and 4 and the Xantophyceae occurred only at Sites 2 6 and 7

Preliminary correlations between edaphic featuresand algal diversity

Multivariate analysis confirms the strong association of heavymetals with the serpentine soils at Site 8 (Figs 2 3) The first twoaxes of the CCA (Fig 3) explained only 362 of the varianceVariables with high inflation factors (Ca Mg P and N) wereremoved to improve the analysis but eigenvalues or theexplanation of the total variance did not change The forwardselection of factors lowered the P-value however it was stillnot significant so we kept all the variables Even with the lowvalues the CCA helps to visualise the species distribution withrespect to the different sites and presents preliminary informationon how different edaphic features are correlated with algaldiversity Hormotilopsis gelatinosa Klebsormidium flaccidumPleurococcus species Chlorotetraedron species and Tetracystiselliptica belonging to the Chlorophyceae and Chroococcus

species Scytonema ocellatum Nostoc linckia as well as anunknown Nostoc species from the Cyanophyceae were closelyassociated with Site 8 (Fig 3) High concentrations of K wereassociatedwith theupper slopeof thesedimentary soils inOhrigstad(Site 2 Figs 2 3) as well as the green algae Characiopsis minimaChlamydomonas macrostellata Bumilleriopsis filiformis from theXanthophyceae and the cyanoprokaryote Oscillatoria raoiCalcium Mg S and N appear to play a role in the similarity inspecies composition of Sites 1 3 4 and 5

BrayndashCurtis dissimilarity index (Hahs and McDonnell2006) grouped samples from lower slopes (Sites 1 6 and7 indicated with black square in Fig 3) whereas the othersites were scattered (stress value 005) Cyanoprokaryotessuch as Leptolyngbya foveolarum Microcoleus vaginatus andPhormidium ambiguum Chlorophyceae such asChlamydomonasand Chlorococcum species Eustigmatophyceae such asEustigmatos magnus and Hantzschia amphioxys fromBacillariophyceae occurred on all the lower-slope sites (Sites 16 and 7) but were not unique to these sites The cyanoprokaryotePhormidium ambiguum was dominant in all the upper-slope sites(Sites 2 4 and 5) It was interesting that the serpentine sites(Sites 7 and 8) did not form a group Nor did the mafic andultramafic sites (Sites 3 4 5 and 6) or siliciclastic sites (Sites 1and 2) This random pattern with a lack of grouping for sitesshowed that slope position (upper vs lower) had a larger effect on

Legend sites rarr environmental variables

Axis 1 2 3 4

Eigen values 048 0235 0093 0091

Cumulative percentage variance

Of species 48 716 808 90

ndash10 15ndash10

10

Ca

Mg

K

Na

P

N

S

Al

As

Cd

Co

Cu

Cr

Fe

Hg

MnMo

Ni

Pd

V

12

3

4

5

6

7

8

Fig 2 Principal component analysis (PCA) to show the relationship between soil characteristics and the sampling sites

F Australian Journal of Botany A Venter et al

Table 3 Algae and cyanoprokaryotes identified from the different soil samples collected from the eight sitesdom dominant sdom subdominant + present

Species Abbreviations Siliciclastic rocks Mafic and ultramafic rocks Serpentineused in Fig 3 Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Site 7 Site 8

CyanophyceaeChroococcus sp Chro +Komvophoron cf schmidlei (Jaag)Anagnostids et Komaacuterek

Kom +

Leptolyngbya foveolarum (Rabenhorstex Gomont) Anagnostidiset Komaacuterek

Lepto dom + + dom dom sdom dom dom

Lyngbya major Meneghini Lyn1 + + +Lyngbya cf truncicola Ghose Lyn2 +Microcoleus vaginatus (Vauch) Gom Micro sdom sdom dom sdom sdomNostoc commune Vauch sensu Elenk Nos1 +Nostoc linckia (Roth) Born et Flah

in sensu ElenkNos2 sdom

Nostoc punctiforme (Kuumltz) Hariot Nos3 +Nostoc sp Nos4 +Oscillatoria limosa Ag Osc1 + +Oscillatoria raoi De Toni Osc2 +Phormidium ambiguum Gomont Pho1 + dom sdom dom dom sdom dom sdomPhormidium animale (Agardh ex

Gomont) Anagnostidis et KomaacuterekPho2 +

Phormidium cf corium (Ag) Kuumltzex Gomont

Pho3 +

Phormidium cf jenkelianum Schmid Pho4 +Phormidium jadinianum Gomont Pho5 +Phormidium mucicola Naum et Hub-

PestalozziPho6 +

Pseudanabaena cf minima (GSAn)Anagnostidis

Pseu1 +

Pseudanabaena frigida (Fritsch)Anagnostidis

Pseu2 +

Pseudanabaena sp Pseu3 + + +Scytonema myochrous (Dillw) Ag

ex Born et FlahScyt1 + dom

Scytonema ocellatum Born et Flah Scyt2 domScytonema sp Scyt3 +Synechocystis crassa Woronich Syn +Total Cyanophyceae 7 4 4 9 4 5 8 8

ChlorophyceaeBracteacoccus cf grandis Bischoff

et BoldBrac1 +

Bracteacoccus minor (Chod) Petrovaacute Brac2 +Bracteacoccus sp Brac3 +Characiopsis minima Pascher Chara +Chlamydomonas macrostellata Lund Chlamy sdomChlamydomonas sp1 Chlam1 + + + + + +Chlamydomonas sp2 Chlam2 +ChlorellamunitissimaFott etNovaacutekovaacute Chlo1 +Chlorella vulgaris Beijerinck Chlo2 + sdom +Chlorella sp Chlo3 + +Chlorococcum echinozygotum Starr Chlc1 sdomChlorococcum infusionum (Schrank)

MeneghiniChlc2 + + + + +

Chlorococcum oviforme Archibaldet Bold

Chlc3 +

Chlorococcum vacuolatum Starr Chlc4 +Chlorococcum sp1 Chlc5 + +Chlorococcum sp2 Chlc6 + + + + +

(continued next page )

Soil algae and cyanoprokaryotes Australian Journal of Botany G

diversity Cyanoprokaryote species were less associated withthe foot slope of pyroxenitendashmagnesium rich soil in Steelpoort(Site 3) with a Studentrsquos t-test supporting a significant differencefrom those at Sites 1 (P=003) 4 (P=003) 7 (P=0007) and 8(P=001) The pattern also showed that the lithic class may beless important than the slope position in determining the algaland cyanoprokayote species present at the sites

Discussion

It is widely reported that cyanoprokaryotes are well adapted toa wide range of environmental conditions and have an ability

to growon avariety of substrates such asmine tailings (Lukesova2001 Orlekowsky et al 2013) barren artic soils (Michaud et al2012) arid regions (Rehaacutekovaacute et al 2011 Muumlhlsteinovaacute et al2014) and alkaline and serpentinising springs (Blank et al 2009Suzuki et al 2013) Our preliminary study documented thatcyanoprokaryotes and algae are found on mafic and ultramaficsoils in SouthAfrica and that slope position plays a role in speciescomposition Leptolyngbya foveolarumMicrocoleus vaginatusPhormidium ambiguum Chlamydomonas ChlorococcumEustigmatos magnus as well as Hantzschia amphioxys werepresent on all three foot-slope sites at Ohrigstad BurgersfortandMalelane (Sites 1 6 and7) Environmental conditions such as

Table 3 (continued )

Species Abbreviations Siliciclastic rocks Mafic and ultramafic rocks Serpentineused in Fig 3 Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Site 7 Site 8

Chlorosarcinopsis aggregata Arceet Bold

Chls1 +

Chlorosarcinopsis minor (Gerneck)Herndon

Chls2 sdom + +

Chlorotetraedron sp Chlt +Hormotilopsis gelatinosa Trainoret Bold

Hormo +

Interfilum sp Inter + +Klebsormidium flaccidum (Kuumltz)PCSilva Mattox et WHBlackwell

Kleb +

Myrmecia biatorellae (Tschermak-Woess) JBPetersen

Myrm +

Pleurococcus Pleur +Scenedesmus sp Scene +Scotiellopsis rubescens Vinatzer Scot1 +Scotiellopsis terrestris (Reisigl)Puncochaacuterovaacute et Kalina

Scot2 + + +

Spongiochloris sp1 Spong1 + +Spongiochloris sp2 Spong2 + + +Tetracystis aggregata Brown et Bold Tetra1 + +Tetracystis elliptica Nakano Tetra2 +Tetracystis sp Tetra3 + +Total Chlorophyceae 12 7 1 7 5 8 10 9

EustigmatophyceaeEustigmatos magnus (JBPetersen)Hibberd

Eust + + + sdom +

Monodopsis subterranea (JBPetersen)Hibberd

Mono + +

Total Eustigmatophyceae 1 1 0 0 1 1 1 2

XantophyceaeBotrydiopsis arhiza Borzi Botry1 sdomBotrydiopsis sp Botry2 +Bumilleriopsis filiformis Vischer Bum1 sdomTotal Xantophyceae 0 1 0 0 0 1 1 0

BacillariophyceaeAmphora veneta Kuumltzing Amph1 +Pinnularia borealis Ehrenberg Pin + + sdomNavicula mutica Kuumltzing Nav1 + +Navicula pelliculosa (Kuumltzing) Hilse Nav2 + +Navicula veneta Kuumltzing Nav3 +Hantzschia amphioxys (Ehrenberg)Grunow

Hant + sdom dom sdom sdom + + +

Total Bacillariophyceae 4 1 1 3 4 1 2 1

Total species richness 24 14 6 19 14 16 22 20

H Australian Journal of Botany A Venter et al

an increase in soil moisture as a result of runoff and theaccumulation of organic matter (Chen et al 2007) could haveplayed a role in the assembly of certain species on foot slopes Thecyanoprokaryotes Leptolyngbya foveolarum and Phormidiumambiguum were also present at the foot slope of the Mg-richpyroxenite soil (Site 3) whereas Microcoleus vaginatusChlamydomonas Chlorococcum and Eustigmatos magnus didnot occur at this site The absence of these algae as well as the lowdiversity observed at Site 3 could be the result of the highconcentrations of Cd (001mg kgndash1) and Hg (00046mg kgndash1)measured at this site According to Fernandez-Pintildeas et al (1995)Cd induces progressive disorganisation and degradation ofthylakoid membranes as well as a decrease in the mobilisationof the polyphosphate granules leading to phosphorus starvationPinto et al (2003) showed that Cd andHg inhibit photosynthesisHowever further experimentation is clearly needed to determinewhether Cd and Hg were responsible for the reduced diversityobserved at this site

Most of the cyanoprokaryotes were represented by filamentousspecies such as Leptolyngbya foveolarumMicrocoleus vaginatusand Phormidium ambiquum and were found at all eight samplingsites These filamentous cyanoprokaryotes can wind throughoutthe uppermost soil layers forming a net-like structure that bindstogether soil particles (Rosentreter et al 2007) This formssoil aggregates that create pathways for water infiltration andsurfaces for nutrient transformations while also increasing thesoil resistance to wind and water erosion Once the filamentouscyanoprokaryotes stabilise thesoil single-celledcyanoprokaryotessuch as Synechocystis sp are able to colonise the substrate(Rosentreter et al 2007) The Chlorophyceae representedby mainly coccoid species such as Chlamydomonas andChlorococcum were found on all the sites except at Site 3Chlorococcum echinozygotum and Chlamydomonas macrostellawere subdominant on the siliciclastic-derived soils of Sites 1 and 2respectively Bacillariophyceae such as Hantzschia amphioxyswas found on all the sites and was subdominant at Sites 2 4 and 5

Legend Δ species and sites rarr environmental variables

Axis

ndash06

ndash10 15

10

1 2 3 4

Eigen values 052 0411 0388 0368

Cumulative percentage variance

Of species 202 362 513 656

Of environmental relations 202 362 513 656

Test of significance of first canonical axes p = 10

Test of significance of all canonical axes p = 10

Fig 3 Canonical correspondence analysis (CCA) of metal concentrations and algal and cyanoprokaryote species of the different sampling sitesAbbreviations of species are given in Table 3

Soil algae and cyanoprokaryotes Australian Journal of Botany I

and dominant at Site 3 According to Metting (1981)Leptolyngbya Microcoleus Phormidium ChlamydomonasChlorococcum and Hantzschia species are all typicalcosmopolitan soil algae

Shields and Durell (1964) and Starks and Shubert (1982)suggested that the species composition of soil algal populationsis affected less by the chemical nature of the substrate than bycertain physical properties that influence soil moisture levelsDuring February 2012 when sampling took place the study areahad received between 50 and 200mm rain which accounts formore than 75 of the expected average summer rainfall for thearea (South African Weather Service 2012) Moisture wastherefore not a limiting factor and the chemical nature of thesoils along with other abiotic habitat features appear to haveinfluenced species richness and composition In addition tothe abiotic conditions such as edaphic features biotic factorsespecially the type of vegetation can influence diversity andcommunity composition of cryptogamic biota For exampleRosentreter et al (2007) documented that vagrant populationsofDermatocarponoccur on poorly drained basaltflats dominatedby Artemisia rigida A papposa Antennaria flagellaris andPoa sandbergii The ecological conditions found at such sitescan support completely or partially vagrant life forms inDermatocarpon and other lichen genera The differences invegetation cover of the plant communities at the sampling sites(Sites 1ndash8) could have created microenvironments that favourspecific algal and cyanoprokaryote species assemblages Sites1ndash7 had moderate cover whereas Site 8 was an open savannacharacterised by numerous bare soil patches (serpentine effect)

Twenty different species were identified at Site 8 (serpentine)despite this site showing elevated concentrations of heavymetalsincluding Co Cr Fe Mn and Ni (Table 2) Chroococcus spScytonema ocellatum Nostoc linckia Chlorotetraedron spHormotilopsis gelatinosa Klebsormidium flaccidiumPleurococcus sp and Tetracystis elliptica were unique to thissite Metals can be divided into those that are required byorganisms in small quantities such as Fe Cu and Zn whichare essential in some biochemical reactions (Raven et al 1999)andnon-essentialmetals such asAs Pb andHgwhichmaycausesevere harm to organisms even at very low concentrationsbecause they do not provide any known biochemical function(Monteiro et al 2012) However the toxic effects of metallicelements on microalgae are complex and differ markedly amongspecies depending on the element itself and the prevailingenvironmental conditions (Monteiro et al 2012) Stark andShubert (1979) reported a positive correlation between Mn NP silica (Si) Al zinc (Zn) and Pb concentrations and algalabundance and a negative correlation with Na Cd Culithium (Li) Mo and strontium (Sr) Cyanoprokaryotes havedeveloped efficient strategies for metal uptake andaccumulation (Shcolnick and Keren 2006) According toKeren et al (2002) cyanoprokaryotes are able to accumulatehigh concentrations of Mn in the envelope layers of their cellsWhether the highMn supports the growth of cyanoprokaryotes orreduces competition by other groups that are less tolerant of Mnwas unclear Soil pH plays a critical role in the bioavailability ofheavy metals (Rajakaruna and Boyd 2008) and althoughserpentine Site 7 had high metal concentrations the relativelyhigher pH at this site may have made those metals less

bioavailable (Neilson and Rajakaruna 2012) Hence it wasunclear whether the higher algal diversity at this site resultedfrom metal tolerance or reduced metal toxicity owing to lowermetal availabilityHowever algae are able togrow in thepresenceof heavy metals as a result of a variety of tolerance mechanismsfor example binding to cell wall precipitation in vacuole andsynthesis of heavy metal-binding compounds such as proteinsorganic acids and phenolic compounds (Metha and Gaur 2005)Serpentine soils are renowned for low Ca Mg ratio howeverDeGrood et al (2005) found that low Ca Mg ratio does notsignificantly explain variation in microbial community patternsalso our study did not show a strong correlation between algaldiversity and Ca Mg Microbes may therefore be respondingto increased soil organicmatter concentrations probably becauseof the associated increases in nutrient availability and water-holding capacity

Algae as a group are physiologically heterogeneous makingany generalisation about their soil relations difficult (OrsquoKelly1974) However favourable soil pH moisture conditions andnutrient content contribute to species diversity and communitycomposition (Shields andDurell 1964)Themultivariate analysesdid not suggest a distinct species association for serpentine sites(Sites 7 and8) Thiswas also true for themafic andultramafic sites(Sites 3 4 5 and 6) as well as the siliciclastic sites (Sites 1 and 2)The lack of a strong correlation with lithic type and a strongercorrelation with slope position suggests that the type of slopemay bemore important in influencing diversity Site pairs 1 and 23 and 4 as well as 5 and 6 were sampled at different slopepositions and at times shared similar species associationsbased on slope position (Fig 3)

Conclusions

It seems unlikely that soil chemistry alone was responsiblefor determining species diversity and no unique algal florafor serpentine soils was confirmed However the serpentinesoil at Kaapschehoop (Site 8) did have a unique speciesassemblage comprising Chroococcus sp Chlorotetraedronsp Hormotilopsis gelatinosa Klebsormidium flaccidiumPleurococcus sp and Tetracystis elliptica However thisrequires further investigation to determine whether it was alithic or climatic effect

Soil features alongwith other biotic (vegetation composition)and abiotic (slope exposure) habitat characteristics mayinfluence the presence and dominance of some algal andcyanoprokaryote species in harsh edaphic settings Our resultssuggested that topography (ie slope position) rather than thechemistry of the lithic class (ie rock type) wasmost important ininfluencing species diversity However high concentrations ofheavy metals also influenced species richness as well ascommunity composition

The characterisation of microbial communities using anisolation approach can be biased because this approach tendsto favour some groups of microbes over others Therefore in thefuture we plan to incorporate molecular approaches (Bjellandet al 2011 Daae et al 2013) to characterise the diversity ofcyanoprokaryotes and soil algae at our sites Additionally theecological heterogeneity within each site should be taken intoconsideration because our sampling strategy (ie pooling of soil

J Australian Journal of Botany A Venter et al

samples) did not allow for detecting species that may berestricted to distinct microhabitats found within our sites Acomposite sample does not allow for a correlation with bioticand abiotic factors but gives a general and preliminary view onthe diversity at each site Although our findings are preliminarythe study sets the stage for detailed investigations on the relativeimportance of edaphic versus other habitat features on algal andcyanoprokaryote diversity and community assembly

Acknowledgements

We thank the staff at Eko-Analitika North-West University Potchefstroomfor the soil analyses Christel Pretorius for drawing the map of the study areaand two anonymous reviewers for their useful suggestions to improve thequality of our manuscript

References

Alexander EB Coleman RG Keeler-Wolf T Harrison SP (2007)lsquoSerpentine geoecology of western North America geology soilsand vegetationrsquo (Oxford University Press New York)

ATSDR (Agency for Toxic Substances and Disease Registry) (2008)lsquoToxicological profile for aluminiumrsquo (US Department of Health andHuman Services Public Health Service Atlanta GA) Available at httpwwwatsdrcdcgovToxProfilestp22pdf [Verified 19 August 2014]

Belnap J (2003) The world at your feet desert biological soil crustsFrontiers in Ecology and the Environment 1 181ndash189doi1018901540-9295(2003)001[0181TWAYFD]20CO2

Belnap J Lange OL (2001) lsquoBiological soil crusts structure function andmanagementrsquo (Springer Berlin)

Bjelland T Grube M Hoem S Jorgensen SL Daae FL Thorseth IH OvrearingsL (2011) Microbial metacommunities in the lichen-rock habitatEnvironmental Microbiology Reports 3 434ndash442doi101111j1758-2229201000206x

Blank JG Green SJ Blake D Valley JW Kita NT Treiman A Dobson PF(2009) An alkaline spring system within the Del Puerto Ophiolite(California USA) a Mars analog site Planetary and Space Science57 533ndash540 doi101016jpss200811018

Bramwell D Caujape-Castells J (2011) lsquoThe biology of island florarsquo(Cambridge Press Cambridge UK)

Brown RM Larson DA Bold HC (1964) Airborne algae their abundanceand heterogeneity Science 143 583ndash585doi101126science1433606583

Cabala J Rahmonov O Jablonska M Teper E (2011) Soil algal colonizationand its ecological role in an environment polluted by past ZnndashPb miningand smelting activity Water Air and Soil Pollution 215 339ndash348doi101007s11270-010-0482-1

Cardace D Meyer-Dombard DR Olsen A Parenteau MN (2014) Bedrockand geochemical controls on extremophile habitats In lsquoPlant ecologyand evolution in harsh environmentsrsquo (Eds N Rajakaruna RS BoydTB Harris) pp 1ndash32 (Nova Science Publishers New York)

Chen YN Wang Q Li WH Ruan X (2007) Microbiotic crusts andthere interrelations with environmental factors in the Gurbantonggutdesert western China Environmental Geology 52 691ndash700doi101007s00254-006-0505-9

Clarke KR Gorley RN (2001) lsquoPRIMER v5 user manualtutorialrsquo(PRIMER-E Plymouth UK)

Clarke B Uken R Reinhardt J (2009) The geometry and emplacementmechanics of a Bushveld complex peridotite body South Africa SouthAfrican Journal of Geology 112 141ndash162 doi102113gssajg1122141

Couteacute A Tell G Therezien Y (1999) Aerophytic Cyanophyceae fromNew Caledonia Cryptogamie Algologie 20 301ndash344doi101016S0181-1568(00)88147-7

Daae FLOslashkland I DahleH Joslashrgensen SL Thorseth IH Pedersen RB (2013)Microbial life associated with low-temperature alteration of ultramafic

rocks in the Leka ophiolite complex Geobiology 11 318ndash339doi101111gbi12035

Daghino S Murat C Sizzano E Girlanda M Perotto S (2012) Fungaldiversity is not determined by mineral and chemical differences inserpentine substrates PLoS ONE 7(9) e44233doi101371journalpone0044233

DeGrood SH Claassen VP Scow KM (2005) Microbial communitycomposition on native and drastically disturbed serpentine soils SoilBiology amp Biochemistry 37 1427ndash1435doi101016jsoilbio200412013

Ettl H Gaumlrtner G Heynig H Mollenhauer D Komaacuterek J Anagnostidis K(1999) lsquoSuumlsswasser von Mittleeuropa Cyanoprokaryota Teil 1Chroococcalesrsquo (Gustav Fisher Verlag Stuttgart Germany)

Favero-Longo SE Isocrono D Piervittori R (2004) Lichens and ultramaficrocks a review Lichenologist (London England) 36 391ndash404doi101017S0024282904014215

Fernandez-Pintildeas F Mateo P Bonilla I (1995) Ultrastructural changesinduced by selected cadmium concentrations in the cyanobacteriumNostoc UAM208 Journal of Plant Physiology 147 452ndash456doi101016S0176-1617(11)82182-6

Hahs AK McDonnell MJ (2006) Selecting independent measures toquantify Melbournersquos urbanndashrural gradient Landscape and UrbanPlanning 78 435ndash448 doi101016jlandurbplan200512005

Harrison SP Rajakaruna N (2011) lsquoSerpentine evolution and ecology in amodel systemrsquo (University of California Press Berkeley CA)

Hauer T (2008) Epilithic cyanobacterial flora of Mohelenskaacute Hadcovaacutesteppe nature reseve (western Moravia Czech Republic) 70 years agoand now Fottea 8 129ndash132

Hindak F (2008) lsquoAtlas of cyanophytesrsquo (VEDA Bratislava Slovakia)Hofmann A Wilson A Kroumlner A Guy B Beukes N (2012) lsquoWorkshop

on craton formation and destruction post-conference excursionguidebook early craton formation stabilisation plume-relatedmagmatism and meteorite impact (Vredefort Witwatersrand Bushveldand Barberton)rsquo (Burlington House Piccadilly London) Availableat httpwwwgssaorgzaDocumentsCraton20Formation20and20Destruction20Excursion20Guidepdf [Verified 27November 2013]

Huumlber-Pestalozzi G (1961) lsquoDas Phytoplankton des Suumlsswassers SystematikundBiologie Tiel 5 Chlorophyceae (Gruumlnalgen) OrdnungVolvocalesrsquo(Verlagbuchhandlung Stuttgart Germany)

John DM Whitton BA Brook AJ (2002) lsquoFreshwater algal flora of theBritish Isles a guide to freshwater and terrestrial algaersquo (CambridgeUniversity Press Cambridge UK)

Keren N Kidd MJ Penner-Hahn JE Pakrasi HB (2002) A light-dependentmechanism formassive accumulation ofmanganese in the photosyntheticbacterium Synechocystis sp PCC 6803Biochemistry 41 15 085ndash15 092doi101021bi026892s

Komaacuterek J Anagnostidis K (2005) lsquoCyanoprokaryota Oscillatorialesrsquo(Spektrum Akademischer Verlag Heidelberg Germany)

Kruckeberg AR (2002) lsquoGeology and plant life the effects oflandforms and rock type on plantsrsquo (University Washington PressSeattle WA)

Kruumlger GHJ (1978) lsquoThe effect of physico-chemical factors on growthrelevant to the mass culture of Microcystis under sterile conditionsrsquo(University of the Orange Free State Bloemfontein South Africa)

Langhans TM Storm C Schwabe A (2009) Community assembly ofbiological soil crusts of different successional stages in a temperatesand ecosystem as assessed by direct determination and enrichmenttechniques Microbial Ecology 58 394ndash407doi101007s00248-009-9532-x

Lukesova A (2001) Soil algae in brown coal lignite post-mining areas incentral Europe (Czech Republic and Germany) Restoration Ecology 9341ndash350 doi101046j1526-100X200194002x

Lund JWG (1945) Observations on soil algae I The ecology size andtaxonomy of British soil diatoms Part 1 New Phytologist 44 196ndash219doi101111j1469-81371945tb05033x

Soil algae and cyanoprokaryotes Australian Journal of Botany K

Tab

le1

Listof

algaean

dsoilsamplinglocalitiesin

Mpu

malan

gaan

dLim

popo

Sou

thAfrica

includ

ingselected

environm

entalv

ariables

anddescriptions

Site

number

Nearesttown

Strataname

Lith

icclass

Aspect

Vegetationun

itMeanannu

alrainfall(m

m)

Meanannual

temperature

(C)

Coordinates

1Ohrigstad

Silv

ertonFormation

PretoriaGroup

Siliciclastic

rocks

Quartzite

Foo

tslopen

orthern

aspect

Ohrigstad

Mountain

Bushv

eld

645

180

24 4558

60S3

023

307

0 E

2Ohrigstad

Silv

ertonFormation

PretoriaGroup

Siliciclastic

rocks

Quartzite

Upp

erslop

eno

rthern

aspect

Ohrigstad

Mountain

Bushv

eld

645

180

24 4553

50S3

023

304

0 E

3Steelpo

ort

Vlakfon

tein

Subsuite

RustenburgLayered

Suite

Ultram

aficrocks

Pyrox

enite

Foo

tslopew

estern

aspect

Sekhu

khun

eMountain

Bushv

eld

609

175

24 2967

30S3

007

993

0 E

4Steelpo

ort

Vlakfon

tein

Subsuite

RustenburgLayered

Suite

Ultram

aficrocks

Pyrox

enite

Upp

erslop

ewestern

aspect

Sekhu

khun

eMountain

Bushv

eld

609

175

24 2961

80S3

008

020

0 E

5Burgersfort

Vlakfontein

Subsuite

RustenburgLayered

Suite

Maficrocks

Norite

Upp

erslop

enorth-western

aspect

Sekhu

khun

ePlains

Bushv

eld

518

190

24 4036

40S3

016

743

0 E

6Burgersfort

Vlakfontein

Subsuite

RustenburgLayered

Suite

Maficrocks

Norite

Foo

tslope

north-western

aspect

Sekhu

khun

ePlains

Bushv

eld

518

190

24 4036

90S3

016

731

0 E

7Malelane

Onverwacht

Group

BabertonSup

ergrou

pMetam

orphicrocks

Amph

ibolite

serpentin

eFoo

tslopen

orthern

aspect

Barberton

Serpentine

Sou

rveld

884

184

25 3199

70S3

131

050

0 E

8Kaapschehoop

Onverwacht

Group

BabertonSup

ergrou

pMetam

orphicrocks

Amph

ibolite

serpentin

eRocky

ridge

north-western

aspect

Barberton

Serpentine

Sou

rveld

1194

167

25 3289

20S3

048

962

0 E

D Australian Journal of Botany A Venter et al

Results

Soil analyses

All soils were slightly acidic except for serpentine Site 7 wherea pH (H2O) of 761 was reported (Table 2) The highestconcentrations of K and Al were measured at the upper slopeof the siliciclastic soils at Ohrigstad (Site 2) and the highestconcentrations of Cd and Hgwere measured at the foot-slope sitenear Steelpoort (Site 3) Burgersfort mafic soils had the highestconcentrations of Ca (Site 5) and P (Site 6)

The first two axes of the PCA of the soil characteristics andthe sampling sites explained 716 of the total variance (Fig 2)It showed a clear association of heavy metals such as As CoCu Cr Fe Mn Mo Ni Pd and V with the serpentine site atKaapschehoop (Site 8) that also features other characteristicsof serpentine soils such as low concentrations of essentialnutrients including a Ca Mg ratio of lt1 This wassubstantially lower than for other ultramafic and mafic rocks(Sites 3 4 6) with ratios lt1 All mafic and ultramafic sites hadheavy-metal (mostly Ni) concentrations exceeding those of thesedimentary rocks and comparable to concentrations found atthe two serpentine sites

The cation exchange capacity (CEC) of the serpentine soil atSite 8 was the lowest among the sites (376 cmol(+) kgndash1) This isbelow the minimum standard of 8 cmol(+) kgndash1 proposed foragricultural top soil in South Africa by the Soil ClassificationWorking Group (1991) The EC of Site 8 soil was low compared

to the other sites likely resulting from low K concentrationspresent at this site

A Studentrsquos t-test indicated that the edaphic features (seeTable 2 for soil features tested) of the siliciclastic Site 2 andthe serpentine Site 8 differ significantly (P= 0001) Sites 1 2 and8 had the highest concentrations of Al a known soil toxin inSouth Africa but the pH of Site 2 and Site 8 was lower (554and 591 respectively) than the pH of Site 1 (627) which couldplay a role in the bioavailability of heavy metals For instance atan acidic pH Al exists only in one oxidation state (+3) and canreact with other matter in the environment to form variouscomplexes (ATSDR 2008)

Algal diversity

Site 1 had the highest number of species (24) followed byserpentine Sites 7 (22) and 8 (20) (Table 3) The lowestnumber of species (6) was found on the pyroxenite-derivedsoil at Site 3 Cyanophyceae diversity was highest at Sites 14 7 and 8 and dominated at all sites except at Site 3 whereHantzschia amphioxys (Bacillariophyceae) was the dominantalgae Leptolyngbya sp was dominant at Sites 1 4 5 7 and 8and subdominant at Site 6 Phormidiumwas dominant at Sites 24 5 and7and subdominant atSites 3 6and8Microcoleus spwassubdominant in the siliciclastic sites near Burgersfort (Sites 1 2)as well as the serpentine sites (Sites 7 and 8) and dominant at themafic site near Burgersfort (Site 6) Leptolyngbya foveolarum

Table 2 Chemical characteristics for composite soil samples collected from the eight sitesEC electrical conductivity CEC cation exchange capacity

Soil characteristic Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Site 7 Site 8

pH (H2O) 627 554 646 636 686 631 761 591pH (KCl) 531 481 521 549 615 538 68 534EC (mS mndash1) 23 31 28 31 58 39 55 10CEC (cmol(+) kgndash1) 1144 1349 2468 2520 2595 2518 1839 376Base saturation 6625 4801 5436 6784 9317 7214 1094 469MacronutrientsCalcium (Ca) (cmol(+) kgndash1) 507 349 582 617 1461 84 822 036Magnesium (Mg) (cmol(+) kgndash1) 198 223 1125 1065 906 918 1151 136Ca Mg ratio 256 157 052 058 161 092 071 026Potassium (K) (mg kgndash1) 1955 281 1029 95 1885 218 1365 15Sodium (Na) (mg kgndash1) 6 8 7 7 6 7 9 9Phosphorus (P) (mg kgndash1) 36 43 212 108 39 377 42 17Nitrogen 046 044 052 049 058 063 047 04Sulfur 021 0 001 001 0 004 0 001

Micronutrients and heavy metals (mg kgndash1)Aluminium 2746 3514 826 921 1959 1514 1555 3021Arsenic 008 01 01 008 007 015 043 406Cadmium 0001 0001 001 0001 00008 0003 00008 00009Cobalt 138 179 348 392 22 479 378 1514Copper 117 153 085 06 174 126 435 831Chromium 626 794 8529 9397 3245 1244 5542 1342Iron 3098 3733 3832 3377 2271 4701 4274 8814Mercury 00004 00007 00046 00005 00002 00004 00006 00004Manganese 4733 6021 6035 7873 5168 1088 936 167Molybdenum 002 003 003 002 002 004 002 004Nickel 402 503 3625 2988 1748 3127 3386 4833Palladium 001 002 001 001 001 002 002 003Vanadium 851 1011 424 339 463 521 796 2062

Soil algae and cyanoprokaryotes Australian Journal of Botany E

Phormidium ambiguum and Hantzschia amphioxys were foundat all sites Chlorophyceae was the most diverse group with thehighest diversity at Site 1 whereas Bacillariophyceae diversitywas the highest at Sites 1 4 and 5 The CyanophyceaeChlorophyceae and Bacillariophyceae were represented atevery site However Eustigmatophyceae did not occur at Sites3 and 4 and the Xantophyceae occurred only at Sites 2 6 and 7

Preliminary correlations between edaphic featuresand algal diversity

Multivariate analysis confirms the strong association of heavymetals with the serpentine soils at Site 8 (Figs 2 3) The first twoaxes of the CCA (Fig 3) explained only 362 of the varianceVariables with high inflation factors (Ca Mg P and N) wereremoved to improve the analysis but eigenvalues or theexplanation of the total variance did not change The forwardselection of factors lowered the P-value however it was stillnot significant so we kept all the variables Even with the lowvalues the CCA helps to visualise the species distribution withrespect to the different sites and presents preliminary informationon how different edaphic features are correlated with algaldiversity Hormotilopsis gelatinosa Klebsormidium flaccidumPleurococcus species Chlorotetraedron species and Tetracystiselliptica belonging to the Chlorophyceae and Chroococcus

species Scytonema ocellatum Nostoc linckia as well as anunknown Nostoc species from the Cyanophyceae were closelyassociated with Site 8 (Fig 3) High concentrations of K wereassociatedwith theupper slopeof thesedimentary soils inOhrigstad(Site 2 Figs 2 3) as well as the green algae Characiopsis minimaChlamydomonas macrostellata Bumilleriopsis filiformis from theXanthophyceae and the cyanoprokaryote Oscillatoria raoiCalcium Mg S and N appear to play a role in the similarity inspecies composition of Sites 1 3 4 and 5

BrayndashCurtis dissimilarity index (Hahs and McDonnell2006) grouped samples from lower slopes (Sites 1 6 and7 indicated with black square in Fig 3) whereas the othersites were scattered (stress value 005) Cyanoprokaryotessuch as Leptolyngbya foveolarum Microcoleus vaginatus andPhormidium ambiguum Chlorophyceae such asChlamydomonasand Chlorococcum species Eustigmatophyceae such asEustigmatos magnus and Hantzschia amphioxys fromBacillariophyceae occurred on all the lower-slope sites (Sites 16 and 7) but were not unique to these sites The cyanoprokaryotePhormidium ambiguum was dominant in all the upper-slope sites(Sites 2 4 and 5) It was interesting that the serpentine sites(Sites 7 and 8) did not form a group Nor did the mafic andultramafic sites (Sites 3 4 5 and 6) or siliciclastic sites (Sites 1and 2) This random pattern with a lack of grouping for sitesshowed that slope position (upper vs lower) had a larger effect on

Legend sites rarr environmental variables

Axis 1 2 3 4

Eigen values 048 0235 0093 0091

Cumulative percentage variance

Of species 48 716 808 90

ndash10 15ndash10

10

Ca

Mg

K

Na

P

N

S

Al

As

Cd

Co

Cu

Cr

Fe

Hg

MnMo

Ni

Pd

V

12

3

4

5

6

7

8

Fig 2 Principal component analysis (PCA) to show the relationship between soil characteristics and the sampling sites

F Australian Journal of Botany A Venter et al

Table 3 Algae and cyanoprokaryotes identified from the different soil samples collected from the eight sitesdom dominant sdom subdominant + present

Species Abbreviations Siliciclastic rocks Mafic and ultramafic rocks Serpentineused in Fig 3 Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Site 7 Site 8

CyanophyceaeChroococcus sp Chro +Komvophoron cf schmidlei (Jaag)Anagnostids et Komaacuterek

Kom +

Leptolyngbya foveolarum (Rabenhorstex Gomont) Anagnostidiset Komaacuterek

Lepto dom + + dom dom sdom dom dom

Lyngbya major Meneghini Lyn1 + + +Lyngbya cf truncicola Ghose Lyn2 +Microcoleus vaginatus (Vauch) Gom Micro sdom sdom dom sdom sdomNostoc commune Vauch sensu Elenk Nos1 +Nostoc linckia (Roth) Born et Flah

in sensu ElenkNos2 sdom

Nostoc punctiforme (Kuumltz) Hariot Nos3 +Nostoc sp Nos4 +Oscillatoria limosa Ag Osc1 + +Oscillatoria raoi De Toni Osc2 +Phormidium ambiguum Gomont Pho1 + dom sdom dom dom sdom dom sdomPhormidium animale (Agardh ex

Gomont) Anagnostidis et KomaacuterekPho2 +

Phormidium cf corium (Ag) Kuumltzex Gomont

Pho3 +

Phormidium cf jenkelianum Schmid Pho4 +Phormidium jadinianum Gomont Pho5 +Phormidium mucicola Naum et Hub-

PestalozziPho6 +

Pseudanabaena cf minima (GSAn)Anagnostidis

Pseu1 +

Pseudanabaena frigida (Fritsch)Anagnostidis

Pseu2 +

Pseudanabaena sp Pseu3 + + +Scytonema myochrous (Dillw) Ag

ex Born et FlahScyt1 + dom

Scytonema ocellatum Born et Flah Scyt2 domScytonema sp Scyt3 +Synechocystis crassa Woronich Syn +Total Cyanophyceae 7 4 4 9 4 5 8 8

ChlorophyceaeBracteacoccus cf grandis Bischoff

et BoldBrac1 +

Bracteacoccus minor (Chod) Petrovaacute Brac2 +Bracteacoccus sp Brac3 +Characiopsis minima Pascher Chara +Chlamydomonas macrostellata Lund Chlamy sdomChlamydomonas sp1 Chlam1 + + + + + +Chlamydomonas sp2 Chlam2 +ChlorellamunitissimaFott etNovaacutekovaacute Chlo1 +Chlorella vulgaris Beijerinck Chlo2 + sdom +Chlorella sp Chlo3 + +Chlorococcum echinozygotum Starr Chlc1 sdomChlorococcum infusionum (Schrank)

MeneghiniChlc2 + + + + +

Chlorococcum oviforme Archibaldet Bold

Chlc3 +

Chlorococcum vacuolatum Starr Chlc4 +Chlorococcum sp1 Chlc5 + +Chlorococcum sp2 Chlc6 + + + + +

(continued next page )

Soil algae and cyanoprokaryotes Australian Journal of Botany G

diversity Cyanoprokaryote species were less associated withthe foot slope of pyroxenitendashmagnesium rich soil in Steelpoort(Site 3) with a Studentrsquos t-test supporting a significant differencefrom those at Sites 1 (P=003) 4 (P=003) 7 (P=0007) and 8(P=001) The pattern also showed that the lithic class may beless important than the slope position in determining the algaland cyanoprokayote species present at the sites

Discussion

It is widely reported that cyanoprokaryotes are well adapted toa wide range of environmental conditions and have an ability

to growon avariety of substrates such asmine tailings (Lukesova2001 Orlekowsky et al 2013) barren artic soils (Michaud et al2012) arid regions (Rehaacutekovaacute et al 2011 Muumlhlsteinovaacute et al2014) and alkaline and serpentinising springs (Blank et al 2009Suzuki et al 2013) Our preliminary study documented thatcyanoprokaryotes and algae are found on mafic and ultramaficsoils in SouthAfrica and that slope position plays a role in speciescomposition Leptolyngbya foveolarumMicrocoleus vaginatusPhormidium ambiguum Chlamydomonas ChlorococcumEustigmatos magnus as well as Hantzschia amphioxys werepresent on all three foot-slope sites at Ohrigstad BurgersfortandMalelane (Sites 1 6 and7) Environmental conditions such as

Table 3 (continued )

Species Abbreviations Siliciclastic rocks Mafic and ultramafic rocks Serpentineused in Fig 3 Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Site 7 Site 8

Chlorosarcinopsis aggregata Arceet Bold

Chls1 +

Chlorosarcinopsis minor (Gerneck)Herndon

Chls2 sdom + +

Chlorotetraedron sp Chlt +Hormotilopsis gelatinosa Trainoret Bold

Hormo +

Interfilum sp Inter + +Klebsormidium flaccidum (Kuumltz)PCSilva Mattox et WHBlackwell

Kleb +

Myrmecia biatorellae (Tschermak-Woess) JBPetersen

Myrm +

Pleurococcus Pleur +Scenedesmus sp Scene +Scotiellopsis rubescens Vinatzer Scot1 +Scotiellopsis terrestris (Reisigl)Puncochaacuterovaacute et Kalina

Scot2 + + +

Spongiochloris sp1 Spong1 + +Spongiochloris sp2 Spong2 + + +Tetracystis aggregata Brown et Bold Tetra1 + +Tetracystis elliptica Nakano Tetra2 +Tetracystis sp Tetra3 + +Total Chlorophyceae 12 7 1 7 5 8 10 9

EustigmatophyceaeEustigmatos magnus (JBPetersen)Hibberd

Eust + + + sdom +

Monodopsis subterranea (JBPetersen)Hibberd

Mono + +

Total Eustigmatophyceae 1 1 0 0 1 1 1 2

XantophyceaeBotrydiopsis arhiza Borzi Botry1 sdomBotrydiopsis sp Botry2 +Bumilleriopsis filiformis Vischer Bum1 sdomTotal Xantophyceae 0 1 0 0 0 1 1 0

BacillariophyceaeAmphora veneta Kuumltzing Amph1 +Pinnularia borealis Ehrenberg Pin + + sdomNavicula mutica Kuumltzing Nav1 + +Navicula pelliculosa (Kuumltzing) Hilse Nav2 + +Navicula veneta Kuumltzing Nav3 +Hantzschia amphioxys (Ehrenberg)Grunow

Hant + sdom dom sdom sdom + + +

Total Bacillariophyceae 4 1 1 3 4 1 2 1

Total species richness 24 14 6 19 14 16 22 20

H Australian Journal of Botany A Venter et al

an increase in soil moisture as a result of runoff and theaccumulation of organic matter (Chen et al 2007) could haveplayed a role in the assembly of certain species on foot slopes Thecyanoprokaryotes Leptolyngbya foveolarum and Phormidiumambiguum were also present at the foot slope of the Mg-richpyroxenite soil (Site 3) whereas Microcoleus vaginatusChlamydomonas Chlorococcum and Eustigmatos magnus didnot occur at this site The absence of these algae as well as the lowdiversity observed at Site 3 could be the result of the highconcentrations of Cd (001mg kgndash1) and Hg (00046mg kgndash1)measured at this site According to Fernandez-Pintildeas et al (1995)Cd induces progressive disorganisation and degradation ofthylakoid membranes as well as a decrease in the mobilisationof the polyphosphate granules leading to phosphorus starvationPinto et al (2003) showed that Cd andHg inhibit photosynthesisHowever further experimentation is clearly needed to determinewhether Cd and Hg were responsible for the reduced diversityobserved at this site

Most of the cyanoprokaryotes were represented by filamentousspecies such as Leptolyngbya foveolarumMicrocoleus vaginatusand Phormidium ambiquum and were found at all eight samplingsites These filamentous cyanoprokaryotes can wind throughoutthe uppermost soil layers forming a net-like structure that bindstogether soil particles (Rosentreter et al 2007) This formssoil aggregates that create pathways for water infiltration andsurfaces for nutrient transformations while also increasing thesoil resistance to wind and water erosion Once the filamentouscyanoprokaryotes stabilise thesoil single-celledcyanoprokaryotessuch as Synechocystis sp are able to colonise the substrate(Rosentreter et al 2007) The Chlorophyceae representedby mainly coccoid species such as Chlamydomonas andChlorococcum were found on all the sites except at Site 3Chlorococcum echinozygotum and Chlamydomonas macrostellawere subdominant on the siliciclastic-derived soils of Sites 1 and 2respectively Bacillariophyceae such as Hantzschia amphioxyswas found on all the sites and was subdominant at Sites 2 4 and 5

Legend Δ species and sites rarr environmental variables

Axis

ndash06

ndash10 15

10

1 2 3 4

Eigen values 052 0411 0388 0368

Cumulative percentage variance

Of species 202 362 513 656

Of environmental relations 202 362 513 656

Test of significance of first canonical axes p = 10

Test of significance of all canonical axes p = 10

Fig 3 Canonical correspondence analysis (CCA) of metal concentrations and algal and cyanoprokaryote species of the different sampling sitesAbbreviations of species are given in Table 3

Soil algae and cyanoprokaryotes Australian Journal of Botany I

and dominant at Site 3 According to Metting (1981)Leptolyngbya Microcoleus Phormidium ChlamydomonasChlorococcum and Hantzschia species are all typicalcosmopolitan soil algae

Shields and Durell (1964) and Starks and Shubert (1982)suggested that the species composition of soil algal populationsis affected less by the chemical nature of the substrate than bycertain physical properties that influence soil moisture levelsDuring February 2012 when sampling took place the study areahad received between 50 and 200mm rain which accounts formore than 75 of the expected average summer rainfall for thearea (South African Weather Service 2012) Moisture wastherefore not a limiting factor and the chemical nature of thesoils along with other abiotic habitat features appear to haveinfluenced species richness and composition In addition tothe abiotic conditions such as edaphic features biotic factorsespecially the type of vegetation can influence diversity andcommunity composition of cryptogamic biota For exampleRosentreter et al (2007) documented that vagrant populationsofDermatocarponoccur on poorly drained basaltflats dominatedby Artemisia rigida A papposa Antennaria flagellaris andPoa sandbergii The ecological conditions found at such sitescan support completely or partially vagrant life forms inDermatocarpon and other lichen genera The differences invegetation cover of the plant communities at the sampling sites(Sites 1ndash8) could have created microenvironments that favourspecific algal and cyanoprokaryote species assemblages Sites1ndash7 had moderate cover whereas Site 8 was an open savannacharacterised by numerous bare soil patches (serpentine effect)

Twenty different species were identified at Site 8 (serpentine)despite this site showing elevated concentrations of heavymetalsincluding Co Cr Fe Mn and Ni (Table 2) Chroococcus spScytonema ocellatum Nostoc linckia Chlorotetraedron spHormotilopsis gelatinosa Klebsormidium flaccidiumPleurococcus sp and Tetracystis elliptica were unique to thissite Metals can be divided into those that are required byorganisms in small quantities such as Fe Cu and Zn whichare essential in some biochemical reactions (Raven et al 1999)andnon-essentialmetals such asAs Pb andHgwhichmaycausesevere harm to organisms even at very low concentrationsbecause they do not provide any known biochemical function(Monteiro et al 2012) However the toxic effects of metallicelements on microalgae are complex and differ markedly amongspecies depending on the element itself and the prevailingenvironmental conditions (Monteiro et al 2012) Stark andShubert (1979) reported a positive correlation between Mn NP silica (Si) Al zinc (Zn) and Pb concentrations and algalabundance and a negative correlation with Na Cd Culithium (Li) Mo and strontium (Sr) Cyanoprokaryotes havedeveloped efficient strategies for metal uptake andaccumulation (Shcolnick and Keren 2006) According toKeren et al (2002) cyanoprokaryotes are able to accumulatehigh concentrations of Mn in the envelope layers of their cellsWhether the highMn supports the growth of cyanoprokaryotes orreduces competition by other groups that are less tolerant of Mnwas unclear Soil pH plays a critical role in the bioavailability ofheavy metals (Rajakaruna and Boyd 2008) and althoughserpentine Site 7 had high metal concentrations the relativelyhigher pH at this site may have made those metals less

bioavailable (Neilson and Rajakaruna 2012) Hence it wasunclear whether the higher algal diversity at this site resultedfrom metal tolerance or reduced metal toxicity owing to lowermetal availabilityHowever algae are able togrow in thepresenceof heavy metals as a result of a variety of tolerance mechanismsfor example binding to cell wall precipitation in vacuole andsynthesis of heavy metal-binding compounds such as proteinsorganic acids and phenolic compounds (Metha and Gaur 2005)Serpentine soils are renowned for low Ca Mg ratio howeverDeGrood et al (2005) found that low Ca Mg ratio does notsignificantly explain variation in microbial community patternsalso our study did not show a strong correlation between algaldiversity and Ca Mg Microbes may therefore be respondingto increased soil organicmatter concentrations probably becauseof the associated increases in nutrient availability and water-holding capacity

Algae as a group are physiologically heterogeneous makingany generalisation about their soil relations difficult (OrsquoKelly1974) However favourable soil pH moisture conditions andnutrient content contribute to species diversity and communitycomposition (Shields andDurell 1964)Themultivariate analysesdid not suggest a distinct species association for serpentine sites(Sites 7 and8) Thiswas also true for themafic andultramafic sites(Sites 3 4 5 and 6) as well as the siliciclastic sites (Sites 1 and 2)The lack of a strong correlation with lithic type and a strongercorrelation with slope position suggests that the type of slopemay bemore important in influencing diversity Site pairs 1 and 23 and 4 as well as 5 and 6 were sampled at different slopepositions and at times shared similar species associationsbased on slope position (Fig 3)

Conclusions

It seems unlikely that soil chemistry alone was responsiblefor determining species diversity and no unique algal florafor serpentine soils was confirmed However the serpentinesoil at Kaapschehoop (Site 8) did have a unique speciesassemblage comprising Chroococcus sp Chlorotetraedronsp Hormotilopsis gelatinosa Klebsormidium flaccidiumPleurococcus sp and Tetracystis elliptica However thisrequires further investigation to determine whether it was alithic or climatic effect

Soil features alongwith other biotic (vegetation composition)and abiotic (slope exposure) habitat characteristics mayinfluence the presence and dominance of some algal andcyanoprokaryote species in harsh edaphic settings Our resultssuggested that topography (ie slope position) rather than thechemistry of the lithic class (ie rock type) wasmost important ininfluencing species diversity However high concentrations ofheavy metals also influenced species richness as well ascommunity composition

The characterisation of microbial communities using anisolation approach can be biased because this approach tendsto favour some groups of microbes over others Therefore in thefuture we plan to incorporate molecular approaches (Bjellandet al 2011 Daae et al 2013) to characterise the diversity ofcyanoprokaryotes and soil algae at our sites Additionally theecological heterogeneity within each site should be taken intoconsideration because our sampling strategy (ie pooling of soil

J Australian Journal of Botany A Venter et al

samples) did not allow for detecting species that may berestricted to distinct microhabitats found within our sites Acomposite sample does not allow for a correlation with bioticand abiotic factors but gives a general and preliminary view onthe diversity at each site Although our findings are preliminarythe study sets the stage for detailed investigations on the relativeimportance of edaphic versus other habitat features on algal andcyanoprokaryote diversity and community assembly

Acknowledgements

We thank the staff at Eko-Analitika North-West University Potchefstroomfor the soil analyses Christel Pretorius for drawing the map of the study areaand two anonymous reviewers for their useful suggestions to improve thequality of our manuscript

References

Alexander EB Coleman RG Keeler-Wolf T Harrison SP (2007)lsquoSerpentine geoecology of western North America geology soilsand vegetationrsquo (Oxford University Press New York)

ATSDR (Agency for Toxic Substances and Disease Registry) (2008)lsquoToxicological profile for aluminiumrsquo (US Department of Health andHuman Services Public Health Service Atlanta GA) Available at httpwwwatsdrcdcgovToxProfilestp22pdf [Verified 19 August 2014]

Belnap J (2003) The world at your feet desert biological soil crustsFrontiers in Ecology and the Environment 1 181ndash189doi1018901540-9295(2003)001[0181TWAYFD]20CO2

Belnap J Lange OL (2001) lsquoBiological soil crusts structure function andmanagementrsquo (Springer Berlin)

Bjelland T Grube M Hoem S Jorgensen SL Daae FL Thorseth IH OvrearingsL (2011) Microbial metacommunities in the lichen-rock habitatEnvironmental Microbiology Reports 3 434ndash442doi101111j1758-2229201000206x

Blank JG Green SJ Blake D Valley JW Kita NT Treiman A Dobson PF(2009) An alkaline spring system within the Del Puerto Ophiolite(California USA) a Mars analog site Planetary and Space Science57 533ndash540 doi101016jpss200811018

Bramwell D Caujape-Castells J (2011) lsquoThe biology of island florarsquo(Cambridge Press Cambridge UK)

Brown RM Larson DA Bold HC (1964) Airborne algae their abundanceand heterogeneity Science 143 583ndash585doi101126science1433606583

Cabala J Rahmonov O Jablonska M Teper E (2011) Soil algal colonizationand its ecological role in an environment polluted by past ZnndashPb miningand smelting activity Water Air and Soil Pollution 215 339ndash348doi101007s11270-010-0482-1

Cardace D Meyer-Dombard DR Olsen A Parenteau MN (2014) Bedrockand geochemical controls on extremophile habitats In lsquoPlant ecologyand evolution in harsh environmentsrsquo (Eds N Rajakaruna RS BoydTB Harris) pp 1ndash32 (Nova Science Publishers New York)

Chen YN Wang Q Li WH Ruan X (2007) Microbiotic crusts andthere interrelations with environmental factors in the Gurbantonggutdesert western China Environmental Geology 52 691ndash700doi101007s00254-006-0505-9

Clarke KR Gorley RN (2001) lsquoPRIMER v5 user manualtutorialrsquo(PRIMER-E Plymouth UK)

Clarke B Uken R Reinhardt J (2009) The geometry and emplacementmechanics of a Bushveld complex peridotite body South Africa SouthAfrican Journal of Geology 112 141ndash162 doi102113gssajg1122141

Couteacute A Tell G Therezien Y (1999) Aerophytic Cyanophyceae fromNew Caledonia Cryptogamie Algologie 20 301ndash344doi101016S0181-1568(00)88147-7

Daae FLOslashkland I DahleH Joslashrgensen SL Thorseth IH Pedersen RB (2013)Microbial life associated with low-temperature alteration of ultramafic

rocks in the Leka ophiolite complex Geobiology 11 318ndash339doi101111gbi12035

Daghino S Murat C Sizzano E Girlanda M Perotto S (2012) Fungaldiversity is not determined by mineral and chemical differences inserpentine substrates PLoS ONE 7(9) e44233doi101371journalpone0044233

DeGrood SH Claassen VP Scow KM (2005) Microbial communitycomposition on native and drastically disturbed serpentine soils SoilBiology amp Biochemistry 37 1427ndash1435doi101016jsoilbio200412013

Ettl H Gaumlrtner G Heynig H Mollenhauer D Komaacuterek J Anagnostidis K(1999) lsquoSuumlsswasser von Mittleeuropa Cyanoprokaryota Teil 1Chroococcalesrsquo (Gustav Fisher Verlag Stuttgart Germany)

Favero-Longo SE Isocrono D Piervittori R (2004) Lichens and ultramaficrocks a review Lichenologist (London England) 36 391ndash404doi101017S0024282904014215

Fernandez-Pintildeas F Mateo P Bonilla I (1995) Ultrastructural changesinduced by selected cadmium concentrations in the cyanobacteriumNostoc UAM208 Journal of Plant Physiology 147 452ndash456doi101016S0176-1617(11)82182-6

Hahs AK McDonnell MJ (2006) Selecting independent measures toquantify Melbournersquos urbanndashrural gradient Landscape and UrbanPlanning 78 435ndash448 doi101016jlandurbplan200512005

Harrison SP Rajakaruna N (2011) lsquoSerpentine evolution and ecology in amodel systemrsquo (University of California Press Berkeley CA)

Hauer T (2008) Epilithic cyanobacterial flora of Mohelenskaacute Hadcovaacutesteppe nature reseve (western Moravia Czech Republic) 70 years agoand now Fottea 8 129ndash132

Hindak F (2008) lsquoAtlas of cyanophytesrsquo (VEDA Bratislava Slovakia)Hofmann A Wilson A Kroumlner A Guy B Beukes N (2012) lsquoWorkshop

on craton formation and destruction post-conference excursionguidebook early craton formation stabilisation plume-relatedmagmatism and meteorite impact (Vredefort Witwatersrand Bushveldand Barberton)rsquo (Burlington House Piccadilly London) Availableat httpwwwgssaorgzaDocumentsCraton20Formation20and20Destruction20Excursion20Guidepdf [Verified 27November 2013]

Huumlber-Pestalozzi G (1961) lsquoDas Phytoplankton des Suumlsswassers SystematikundBiologie Tiel 5 Chlorophyceae (Gruumlnalgen) OrdnungVolvocalesrsquo(Verlagbuchhandlung Stuttgart Germany)

John DM Whitton BA Brook AJ (2002) lsquoFreshwater algal flora of theBritish Isles a guide to freshwater and terrestrial algaersquo (CambridgeUniversity Press Cambridge UK)

Keren N Kidd MJ Penner-Hahn JE Pakrasi HB (2002) A light-dependentmechanism formassive accumulation ofmanganese in the photosyntheticbacterium Synechocystis sp PCC 6803Biochemistry 41 15 085ndash15 092doi101021bi026892s

Komaacuterek J Anagnostidis K (2005) lsquoCyanoprokaryota Oscillatorialesrsquo(Spektrum Akademischer Verlag Heidelberg Germany)

Kruckeberg AR (2002) lsquoGeology and plant life the effects oflandforms and rock type on plantsrsquo (University Washington PressSeattle WA)

Kruumlger GHJ (1978) lsquoThe effect of physico-chemical factors on growthrelevant to the mass culture of Microcystis under sterile conditionsrsquo(University of the Orange Free State Bloemfontein South Africa)

Langhans TM Storm C Schwabe A (2009) Community assembly ofbiological soil crusts of different successional stages in a temperatesand ecosystem as assessed by direct determination and enrichmenttechniques Microbial Ecology 58 394ndash407doi101007s00248-009-9532-x

Lukesova A (2001) Soil algae in brown coal lignite post-mining areas incentral Europe (Czech Republic and Germany) Restoration Ecology 9341ndash350 doi101046j1526-100X200194002x

Lund JWG (1945) Observations on soil algae I The ecology size andtaxonomy of British soil diatoms Part 1 New Phytologist 44 196ndash219doi101111j1469-81371945tb05033x

Soil algae and cyanoprokaryotes Australian Journal of Botany K

Results

Soil analyses

All soils were slightly acidic except for serpentine Site 7 wherea pH (H2O) of 761 was reported (Table 2) The highestconcentrations of K and Al were measured at the upper slopeof the siliciclastic soils at Ohrigstad (Site 2) and the highestconcentrations of Cd and Hgwere measured at the foot-slope sitenear Steelpoort (Site 3) Burgersfort mafic soils had the highestconcentrations of Ca (Site 5) and P (Site 6)

The first two axes of the PCA of the soil characteristics andthe sampling sites explained 716 of the total variance (Fig 2)It showed a clear association of heavy metals such as As CoCu Cr Fe Mn Mo Ni Pd and V with the serpentine site atKaapschehoop (Site 8) that also features other characteristicsof serpentine soils such as low concentrations of essentialnutrients including a Ca Mg ratio of lt1 This wassubstantially lower than for other ultramafic and mafic rocks(Sites 3 4 6) with ratios lt1 All mafic and ultramafic sites hadheavy-metal (mostly Ni) concentrations exceeding those of thesedimentary rocks and comparable to concentrations found atthe two serpentine sites

The cation exchange capacity (CEC) of the serpentine soil atSite 8 was the lowest among the sites (376 cmol(+) kgndash1) This isbelow the minimum standard of 8 cmol(+) kgndash1 proposed foragricultural top soil in South Africa by the Soil ClassificationWorking Group (1991) The EC of Site 8 soil was low compared

to the other sites likely resulting from low K concentrationspresent at this site

A Studentrsquos t-test indicated that the edaphic features (seeTable 2 for soil features tested) of the siliciclastic Site 2 andthe serpentine Site 8 differ significantly (P= 0001) Sites 1 2 and8 had the highest concentrations of Al a known soil toxin inSouth Africa but the pH of Site 2 and Site 8 was lower (554and 591 respectively) than the pH of Site 1 (627) which couldplay a role in the bioavailability of heavy metals For instance atan acidic pH Al exists only in one oxidation state (+3) and canreact with other matter in the environment to form variouscomplexes (ATSDR 2008)

Algal diversity

Site 1 had the highest number of species (24) followed byserpentine Sites 7 (22) and 8 (20) (Table 3) The lowestnumber of species (6) was found on the pyroxenite-derivedsoil at Site 3 Cyanophyceae diversity was highest at Sites 14 7 and 8 and dominated at all sites except at Site 3 whereHantzschia amphioxys (Bacillariophyceae) was the dominantalgae Leptolyngbya sp was dominant at Sites 1 4 5 7 and 8and subdominant at Site 6 Phormidiumwas dominant at Sites 24 5 and7and subdominant atSites 3 6and8Microcoleus spwassubdominant in the siliciclastic sites near Burgersfort (Sites 1 2)as well as the serpentine sites (Sites 7 and 8) and dominant at themafic site near Burgersfort (Site 6) Leptolyngbya foveolarum

Table 2 Chemical characteristics for composite soil samples collected from the eight sitesEC electrical conductivity CEC cation exchange capacity

Soil characteristic Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Site 7 Site 8

pH (H2O) 627 554 646 636 686 631 761 591pH (KCl) 531 481 521 549 615 538 68 534EC (mS mndash1) 23 31 28 31 58 39 55 10CEC (cmol(+) kgndash1) 1144 1349 2468 2520 2595 2518 1839 376Base saturation 6625 4801 5436 6784 9317 7214 1094 469MacronutrientsCalcium (Ca) (cmol(+) kgndash1) 507 349 582 617 1461 84 822 036Magnesium (Mg) (cmol(+) kgndash1) 198 223 1125 1065 906 918 1151 136Ca Mg ratio 256 157 052 058 161 092 071 026Potassium (K) (mg kgndash1) 1955 281 1029 95 1885 218 1365 15Sodium (Na) (mg kgndash1) 6 8 7 7 6 7 9 9Phosphorus (P) (mg kgndash1) 36 43 212 108 39 377 42 17Nitrogen 046 044 052 049 058 063 047 04Sulfur 021 0 001 001 0 004 0 001

Micronutrients and heavy metals (mg kgndash1)Aluminium 2746 3514 826 921 1959 1514 1555 3021Arsenic 008 01 01 008 007 015 043 406Cadmium 0001 0001 001 0001 00008 0003 00008 00009Cobalt 138 179 348 392 22 479 378 1514Copper 117 153 085 06 174 126 435 831Chromium 626 794 8529 9397 3245 1244 5542 1342Iron 3098 3733 3832 3377 2271 4701 4274 8814Mercury 00004 00007 00046 00005 00002 00004 00006 00004Manganese 4733 6021 6035 7873 5168 1088 936 167Molybdenum 002 003 003 002 002 004 002 004Nickel 402 503 3625 2988 1748 3127 3386 4833Palladium 001 002 001 001 001 002 002 003Vanadium 851 1011 424 339 463 521 796 2062

Soil algae and cyanoprokaryotes Australian Journal of Botany E

Phormidium ambiguum and Hantzschia amphioxys were foundat all sites Chlorophyceae was the most diverse group with thehighest diversity at Site 1 whereas Bacillariophyceae diversitywas the highest at Sites 1 4 and 5 The CyanophyceaeChlorophyceae and Bacillariophyceae were represented atevery site However Eustigmatophyceae did not occur at Sites3 and 4 and the Xantophyceae occurred only at Sites 2 6 and 7

Preliminary correlations between edaphic featuresand algal diversity

Multivariate analysis confirms the strong association of heavymetals with the serpentine soils at Site 8 (Figs 2 3) The first twoaxes of the CCA (Fig 3) explained only 362 of the varianceVariables with high inflation factors (Ca Mg P and N) wereremoved to improve the analysis but eigenvalues or theexplanation of the total variance did not change The forwardselection of factors lowered the P-value however it was stillnot significant so we kept all the variables Even with the lowvalues the CCA helps to visualise the species distribution withrespect to the different sites and presents preliminary informationon how different edaphic features are correlated with algaldiversity Hormotilopsis gelatinosa Klebsormidium flaccidumPleurococcus species Chlorotetraedron species and Tetracystiselliptica belonging to the Chlorophyceae and Chroococcus

species Scytonema ocellatum Nostoc linckia as well as anunknown Nostoc species from the Cyanophyceae were closelyassociated with Site 8 (Fig 3) High concentrations of K wereassociatedwith theupper slopeof thesedimentary soils inOhrigstad(Site 2 Figs 2 3) as well as the green algae Characiopsis minimaChlamydomonas macrostellata Bumilleriopsis filiformis from theXanthophyceae and the cyanoprokaryote Oscillatoria raoiCalcium Mg S and N appear to play a role in the similarity inspecies composition of Sites 1 3 4 and 5

BrayndashCurtis dissimilarity index (Hahs and McDonnell2006) grouped samples from lower slopes (Sites 1 6 and7 indicated with black square in Fig 3) whereas the othersites were scattered (stress value 005) Cyanoprokaryotessuch as Leptolyngbya foveolarum Microcoleus vaginatus andPhormidium ambiguum Chlorophyceae such asChlamydomonasand Chlorococcum species Eustigmatophyceae such asEustigmatos magnus and Hantzschia amphioxys fromBacillariophyceae occurred on all the lower-slope sites (Sites 16 and 7) but were not unique to these sites The cyanoprokaryotePhormidium ambiguum was dominant in all the upper-slope sites(Sites 2 4 and 5) It was interesting that the serpentine sites(Sites 7 and 8) did not form a group Nor did the mafic andultramafic sites (Sites 3 4 5 and 6) or siliciclastic sites (Sites 1and 2) This random pattern with a lack of grouping for sitesshowed that slope position (upper vs lower) had a larger effect on

Legend sites rarr environmental variables

Axis 1 2 3 4

Eigen values 048 0235 0093 0091

Cumulative percentage variance

Of species 48 716 808 90

ndash10 15ndash10

10

Ca

Mg

K

Na

P

N

S

Al

As

Cd

Co

Cu

Cr

Fe

Hg

MnMo

Ni

Pd

V

12

3

4

5

6

7

8

Fig 2 Principal component analysis (PCA) to show the relationship between soil characteristics and the sampling sites

F Australian Journal of Botany A Venter et al

Table 3 Algae and cyanoprokaryotes identified from the different soil samples collected from the eight sitesdom dominant sdom subdominant + present

Species Abbreviations Siliciclastic rocks Mafic and ultramafic rocks Serpentineused in Fig 3 Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Site 7 Site 8

CyanophyceaeChroococcus sp Chro +Komvophoron cf schmidlei (Jaag)Anagnostids et Komaacuterek

Kom +

Leptolyngbya foveolarum (Rabenhorstex Gomont) Anagnostidiset Komaacuterek

Lepto dom + + dom dom sdom dom dom

Lyngbya major Meneghini Lyn1 + + +Lyngbya cf truncicola Ghose Lyn2 +Microcoleus vaginatus (Vauch) Gom Micro sdom sdom dom sdom sdomNostoc commune Vauch sensu Elenk Nos1 +Nostoc linckia (Roth) Born et Flah

in sensu ElenkNos2 sdom

Nostoc punctiforme (Kuumltz) Hariot Nos3 +Nostoc sp Nos4 +Oscillatoria limosa Ag Osc1 + +Oscillatoria raoi De Toni Osc2 +Phormidium ambiguum Gomont Pho1 + dom sdom dom dom sdom dom sdomPhormidium animale (Agardh ex

Gomont) Anagnostidis et KomaacuterekPho2 +

Phormidium cf corium (Ag) Kuumltzex Gomont

Pho3 +

Phormidium cf jenkelianum Schmid Pho4 +Phormidium jadinianum Gomont Pho5 +Phormidium mucicola Naum et Hub-

PestalozziPho6 +

Pseudanabaena cf minima (GSAn)Anagnostidis

Pseu1 +

Pseudanabaena frigida (Fritsch)Anagnostidis

Pseu2 +

Pseudanabaena sp Pseu3 + + +Scytonema myochrous (Dillw) Ag

ex Born et FlahScyt1 + dom

Scytonema ocellatum Born et Flah Scyt2 domScytonema sp Scyt3 +Synechocystis crassa Woronich Syn +Total Cyanophyceae 7 4 4 9 4 5 8 8

ChlorophyceaeBracteacoccus cf grandis Bischoff

et BoldBrac1 +

Bracteacoccus minor (Chod) Petrovaacute Brac2 +Bracteacoccus sp Brac3 +Characiopsis minima Pascher Chara +Chlamydomonas macrostellata Lund Chlamy sdomChlamydomonas sp1 Chlam1 + + + + + +Chlamydomonas sp2 Chlam2 +ChlorellamunitissimaFott etNovaacutekovaacute Chlo1 +Chlorella vulgaris Beijerinck Chlo2 + sdom +Chlorella sp Chlo3 + +Chlorococcum echinozygotum Starr Chlc1 sdomChlorococcum infusionum (Schrank)

MeneghiniChlc2 + + + + +

Chlorococcum oviforme Archibaldet Bold

Chlc3 +

Chlorococcum vacuolatum Starr Chlc4 +Chlorococcum sp1 Chlc5 + +Chlorococcum sp2 Chlc6 + + + + +

(continued next page )

Soil algae and cyanoprokaryotes Australian Journal of Botany G

diversity Cyanoprokaryote species were less associated withthe foot slope of pyroxenitendashmagnesium rich soil in Steelpoort(Site 3) with a Studentrsquos t-test supporting a significant differencefrom those at Sites 1 (P=003) 4 (P=003) 7 (P=0007) and 8(P=001) The pattern also showed that the lithic class may beless important than the slope position in determining the algaland cyanoprokayote species present at the sites

Discussion

It is widely reported that cyanoprokaryotes are well adapted toa wide range of environmental conditions and have an ability

to growon avariety of substrates such asmine tailings (Lukesova2001 Orlekowsky et al 2013) barren artic soils (Michaud et al2012) arid regions (Rehaacutekovaacute et al 2011 Muumlhlsteinovaacute et al2014) and alkaline and serpentinising springs (Blank et al 2009Suzuki et al 2013) Our preliminary study documented thatcyanoprokaryotes and algae are found on mafic and ultramaficsoils in SouthAfrica and that slope position plays a role in speciescomposition Leptolyngbya foveolarumMicrocoleus vaginatusPhormidium ambiguum Chlamydomonas ChlorococcumEustigmatos magnus as well as Hantzschia amphioxys werepresent on all three foot-slope sites at Ohrigstad BurgersfortandMalelane (Sites 1 6 and7) Environmental conditions such as

Table 3 (continued )

Species Abbreviations Siliciclastic rocks Mafic and ultramafic rocks Serpentineused in Fig 3 Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Site 7 Site 8

Chlorosarcinopsis aggregata Arceet Bold

Chls1 +

Chlorosarcinopsis minor (Gerneck)Herndon

Chls2 sdom + +

Chlorotetraedron sp Chlt +Hormotilopsis gelatinosa Trainoret Bold

Hormo +

Interfilum sp Inter + +Klebsormidium flaccidum (Kuumltz)PCSilva Mattox et WHBlackwell

Kleb +

Myrmecia biatorellae (Tschermak-Woess) JBPetersen

Myrm +

Pleurococcus Pleur +Scenedesmus sp Scene +Scotiellopsis rubescens Vinatzer Scot1 +Scotiellopsis terrestris (Reisigl)Puncochaacuterovaacute et Kalina

Scot2 + + +

Spongiochloris sp1 Spong1 + +Spongiochloris sp2 Spong2 + + +Tetracystis aggregata Brown et Bold Tetra1 + +Tetracystis elliptica Nakano Tetra2 +Tetracystis sp Tetra3 + +Total Chlorophyceae 12 7 1 7 5 8 10 9

EustigmatophyceaeEustigmatos magnus (JBPetersen)Hibberd

Eust + + + sdom +

Monodopsis subterranea (JBPetersen)Hibberd

Mono + +

Total Eustigmatophyceae 1 1 0 0 1 1 1 2

XantophyceaeBotrydiopsis arhiza Borzi Botry1 sdomBotrydiopsis sp Botry2 +Bumilleriopsis filiformis Vischer Bum1 sdomTotal Xantophyceae 0 1 0 0 0 1 1 0

BacillariophyceaeAmphora veneta Kuumltzing Amph1 +Pinnularia borealis Ehrenberg Pin + + sdomNavicula mutica Kuumltzing Nav1 + +Navicula pelliculosa (Kuumltzing) Hilse Nav2 + +Navicula veneta Kuumltzing Nav3 +Hantzschia amphioxys (Ehrenberg)Grunow

Hant + sdom dom sdom sdom + + +

Total Bacillariophyceae 4 1 1 3 4 1 2 1

Total species richness 24 14 6 19 14 16 22 20

H Australian Journal of Botany A Venter et al

an increase in soil moisture as a result of runoff and theaccumulation of organic matter (Chen et al 2007) could haveplayed a role in the assembly of certain species on foot slopes Thecyanoprokaryotes Leptolyngbya foveolarum and Phormidiumambiguum were also present at the foot slope of the Mg-richpyroxenite soil (Site 3) whereas Microcoleus vaginatusChlamydomonas Chlorococcum and Eustigmatos magnus didnot occur at this site The absence of these algae as well as the lowdiversity observed at Site 3 could be the result of the highconcentrations of Cd (001mg kgndash1) and Hg (00046mg kgndash1)measured at this site According to Fernandez-Pintildeas et al (1995)Cd induces progressive disorganisation and degradation ofthylakoid membranes as well as a decrease in the mobilisationof the polyphosphate granules leading to phosphorus starvationPinto et al (2003) showed that Cd andHg inhibit photosynthesisHowever further experimentation is clearly needed to determinewhether Cd and Hg were responsible for the reduced diversityobserved at this site

Most of the cyanoprokaryotes were represented by filamentousspecies such as Leptolyngbya foveolarumMicrocoleus vaginatusand Phormidium ambiquum and were found at all eight samplingsites These filamentous cyanoprokaryotes can wind throughoutthe uppermost soil layers forming a net-like structure that bindstogether soil particles (Rosentreter et al 2007) This formssoil aggregates that create pathways for water infiltration andsurfaces for nutrient transformations while also increasing thesoil resistance to wind and water erosion Once the filamentouscyanoprokaryotes stabilise thesoil single-celledcyanoprokaryotessuch as Synechocystis sp are able to colonise the substrate(Rosentreter et al 2007) The Chlorophyceae representedby mainly coccoid species such as Chlamydomonas andChlorococcum were found on all the sites except at Site 3Chlorococcum echinozygotum and Chlamydomonas macrostellawere subdominant on the siliciclastic-derived soils of Sites 1 and 2respectively Bacillariophyceae such as Hantzschia amphioxyswas found on all the sites and was subdominant at Sites 2 4 and 5

Legend Δ species and sites rarr environmental variables

Axis

ndash06

ndash10 15

10

1 2 3 4

Eigen values 052 0411 0388 0368

Cumulative percentage variance

Of species 202 362 513 656

Of environmental relations 202 362 513 656

Test of significance of first canonical axes p = 10

Test of significance of all canonical axes p = 10

Fig 3 Canonical correspondence analysis (CCA) of metal concentrations and algal and cyanoprokaryote species of the different sampling sitesAbbreviations of species are given in Table 3

Soil algae and cyanoprokaryotes Australian Journal of Botany I

and dominant at Site 3 According to Metting (1981)Leptolyngbya Microcoleus Phormidium ChlamydomonasChlorococcum and Hantzschia species are all typicalcosmopolitan soil algae

Shields and Durell (1964) and Starks and Shubert (1982)suggested that the species composition of soil algal populationsis affected less by the chemical nature of the substrate than bycertain physical properties that influence soil moisture levelsDuring February 2012 when sampling took place the study areahad received between 50 and 200mm rain which accounts formore than 75 of the expected average summer rainfall for thearea (South African Weather Service 2012) Moisture wastherefore not a limiting factor and the chemical nature of thesoils along with other abiotic habitat features appear to haveinfluenced species richness and composition In addition tothe abiotic conditions such as edaphic features biotic factorsespecially the type of vegetation can influence diversity andcommunity composition of cryptogamic biota For exampleRosentreter et al (2007) documented that vagrant populationsofDermatocarponoccur on poorly drained basaltflats dominatedby Artemisia rigida A papposa Antennaria flagellaris andPoa sandbergii The ecological conditions found at such sitescan support completely or partially vagrant life forms inDermatocarpon and other lichen genera The differences invegetation cover of the plant communities at the sampling sites(Sites 1ndash8) could have created microenvironments that favourspecific algal and cyanoprokaryote species assemblages Sites1ndash7 had moderate cover whereas Site 8 was an open savannacharacterised by numerous bare soil patches (serpentine effect)

Twenty different species were identified at Site 8 (serpentine)despite this site showing elevated concentrations of heavymetalsincluding Co Cr Fe Mn and Ni (Table 2) Chroococcus spScytonema ocellatum Nostoc linckia Chlorotetraedron spHormotilopsis gelatinosa Klebsormidium flaccidiumPleurococcus sp and Tetracystis elliptica were unique to thissite Metals can be divided into those that are required byorganisms in small quantities such as Fe Cu and Zn whichare essential in some biochemical reactions (Raven et al 1999)andnon-essentialmetals such asAs Pb andHgwhichmaycausesevere harm to organisms even at very low concentrationsbecause they do not provide any known biochemical function(Monteiro et al 2012) However the toxic effects of metallicelements on microalgae are complex and differ markedly amongspecies depending on the element itself and the prevailingenvironmental conditions (Monteiro et al 2012) Stark andShubert (1979) reported a positive correlation between Mn NP silica (Si) Al zinc (Zn) and Pb concentrations and algalabundance and a negative correlation with Na Cd Culithium (Li) Mo and strontium (Sr) Cyanoprokaryotes havedeveloped efficient strategies for metal uptake andaccumulation (Shcolnick and Keren 2006) According toKeren et al (2002) cyanoprokaryotes are able to accumulatehigh concentrations of Mn in the envelope layers of their cellsWhether the highMn supports the growth of cyanoprokaryotes orreduces competition by other groups that are less tolerant of Mnwas unclear Soil pH plays a critical role in the bioavailability ofheavy metals (Rajakaruna and Boyd 2008) and althoughserpentine Site 7 had high metal concentrations the relativelyhigher pH at this site may have made those metals less

bioavailable (Neilson and Rajakaruna 2012) Hence it wasunclear whether the higher algal diversity at this site resultedfrom metal tolerance or reduced metal toxicity owing to lowermetal availabilityHowever algae are able togrow in thepresenceof heavy metals as a result of a variety of tolerance mechanismsfor example binding to cell wall precipitation in vacuole andsynthesis of heavy metal-binding compounds such as proteinsorganic acids and phenolic compounds (Metha and Gaur 2005)Serpentine soils are renowned for low Ca Mg ratio howeverDeGrood et al (2005) found that low Ca Mg ratio does notsignificantly explain variation in microbial community patternsalso our study did not show a strong correlation between algaldiversity and Ca Mg Microbes may therefore be respondingto increased soil organicmatter concentrations probably becauseof the associated increases in nutrient availability and water-holding capacity

Algae as a group are physiologically heterogeneous makingany generalisation about their soil relations difficult (OrsquoKelly1974) However favourable soil pH moisture conditions andnutrient content contribute to species diversity and communitycomposition (Shields andDurell 1964)Themultivariate analysesdid not suggest a distinct species association for serpentine sites(Sites 7 and8) Thiswas also true for themafic andultramafic sites(Sites 3 4 5 and 6) as well as the siliciclastic sites (Sites 1 and 2)The lack of a strong correlation with lithic type and a strongercorrelation with slope position suggests that the type of slopemay bemore important in influencing diversity Site pairs 1 and 23 and 4 as well as 5 and 6 were sampled at different slopepositions and at times shared similar species associationsbased on slope position (Fig 3)

Conclusions

It seems unlikely that soil chemistry alone was responsiblefor determining species diversity and no unique algal florafor serpentine soils was confirmed However the serpentinesoil at Kaapschehoop (Site 8) did have a unique speciesassemblage comprising Chroococcus sp Chlorotetraedronsp Hormotilopsis gelatinosa Klebsormidium flaccidiumPleurococcus sp and Tetracystis elliptica However thisrequires further investigation to determine whether it was alithic or climatic effect

Soil features alongwith other biotic (vegetation composition)and abiotic (slope exposure) habitat characteristics mayinfluence the presence and dominance of some algal andcyanoprokaryote species in harsh edaphic settings Our resultssuggested that topography (ie slope position) rather than thechemistry of the lithic class (ie rock type) wasmost important ininfluencing species diversity However high concentrations ofheavy metals also influenced species richness as well ascommunity composition

The characterisation of microbial communities using anisolation approach can be biased because this approach tendsto favour some groups of microbes over others Therefore in thefuture we plan to incorporate molecular approaches (Bjellandet al 2011 Daae et al 2013) to characterise the diversity ofcyanoprokaryotes and soil algae at our sites Additionally theecological heterogeneity within each site should be taken intoconsideration because our sampling strategy (ie pooling of soil

J Australian Journal of Botany A Venter et al

samples) did not allow for detecting species that may berestricted to distinct microhabitats found within our sites Acomposite sample does not allow for a correlation with bioticand abiotic factors but gives a general and preliminary view onthe diversity at each site Although our findings are preliminarythe study sets the stage for detailed investigations on the relativeimportance of edaphic versus other habitat features on algal andcyanoprokaryote diversity and community assembly

Acknowledgements

We thank the staff at Eko-Analitika North-West University Potchefstroomfor the soil analyses Christel Pretorius for drawing the map of the study areaand two anonymous reviewers for their useful suggestions to improve thequality of our manuscript

References

Alexander EB Coleman RG Keeler-Wolf T Harrison SP (2007)lsquoSerpentine geoecology of western North America geology soilsand vegetationrsquo (Oxford University Press New York)

ATSDR (Agency for Toxic Substances and Disease Registry) (2008)lsquoToxicological profile for aluminiumrsquo (US Department of Health andHuman Services Public Health Service Atlanta GA) Available at httpwwwatsdrcdcgovToxProfilestp22pdf [Verified 19 August 2014]

Belnap J (2003) The world at your feet desert biological soil crustsFrontiers in Ecology and the Environment 1 181ndash189doi1018901540-9295(2003)001[0181TWAYFD]20CO2

Belnap J Lange OL (2001) lsquoBiological soil crusts structure function andmanagementrsquo (Springer Berlin)

Bjelland T Grube M Hoem S Jorgensen SL Daae FL Thorseth IH OvrearingsL (2011) Microbial metacommunities in the lichen-rock habitatEnvironmental Microbiology Reports 3 434ndash442doi101111j1758-2229201000206x

Blank JG Green SJ Blake D Valley JW Kita NT Treiman A Dobson PF(2009) An alkaline spring system within the Del Puerto Ophiolite(California USA) a Mars analog site Planetary and Space Science57 533ndash540 doi101016jpss200811018

Bramwell D Caujape-Castells J (2011) lsquoThe biology of island florarsquo(Cambridge Press Cambridge UK)

Brown RM Larson DA Bold HC (1964) Airborne algae their abundanceand heterogeneity Science 143 583ndash585doi101126science1433606583

Cabala J Rahmonov O Jablonska M Teper E (2011) Soil algal colonizationand its ecological role in an environment polluted by past ZnndashPb miningand smelting activity Water Air and Soil Pollution 215 339ndash348doi101007s11270-010-0482-1

Cardace D Meyer-Dombard DR Olsen A Parenteau MN (2014) Bedrockand geochemical controls on extremophile habitats In lsquoPlant ecologyand evolution in harsh environmentsrsquo (Eds N Rajakaruna RS BoydTB Harris) pp 1ndash32 (Nova Science Publishers New York)

Chen YN Wang Q Li WH Ruan X (2007) Microbiotic crusts andthere interrelations with environmental factors in the Gurbantonggutdesert western China Environmental Geology 52 691ndash700doi101007s00254-006-0505-9

Clarke KR Gorley RN (2001) lsquoPRIMER v5 user manualtutorialrsquo(PRIMER-E Plymouth UK)

Clarke B Uken R Reinhardt J (2009) The geometry and emplacementmechanics of a Bushveld complex peridotite body South Africa SouthAfrican Journal of Geology 112 141ndash162 doi102113gssajg1122141

Couteacute A Tell G Therezien Y (1999) Aerophytic Cyanophyceae fromNew Caledonia Cryptogamie Algologie 20 301ndash344doi101016S0181-1568(00)88147-7

Daae FLOslashkland I DahleH Joslashrgensen SL Thorseth IH Pedersen RB (2013)Microbial life associated with low-temperature alteration of ultramafic

rocks in the Leka ophiolite complex Geobiology 11 318ndash339doi101111gbi12035

Daghino S Murat C Sizzano E Girlanda M Perotto S (2012) Fungaldiversity is not determined by mineral and chemical differences inserpentine substrates PLoS ONE 7(9) e44233doi101371journalpone0044233

DeGrood SH Claassen VP Scow KM (2005) Microbial communitycomposition on native and drastically disturbed serpentine soils SoilBiology amp Biochemistry 37 1427ndash1435doi101016jsoilbio200412013

Ettl H Gaumlrtner G Heynig H Mollenhauer D Komaacuterek J Anagnostidis K(1999) lsquoSuumlsswasser von Mittleeuropa Cyanoprokaryota Teil 1Chroococcalesrsquo (Gustav Fisher Verlag Stuttgart Germany)

Favero-Longo SE Isocrono D Piervittori R (2004) Lichens and ultramaficrocks a review Lichenologist (London England) 36 391ndash404doi101017S0024282904014215

Fernandez-Pintildeas F Mateo P Bonilla I (1995) Ultrastructural changesinduced by selected cadmium concentrations in the cyanobacteriumNostoc UAM208 Journal of Plant Physiology 147 452ndash456doi101016S0176-1617(11)82182-6

Hahs AK McDonnell MJ (2006) Selecting independent measures toquantify Melbournersquos urbanndashrural gradient Landscape and UrbanPlanning 78 435ndash448 doi101016jlandurbplan200512005

Harrison SP Rajakaruna N (2011) lsquoSerpentine evolution and ecology in amodel systemrsquo (University of California Press Berkeley CA)

Hauer T (2008) Epilithic cyanobacterial flora of Mohelenskaacute Hadcovaacutesteppe nature reseve (western Moravia Czech Republic) 70 years agoand now Fottea 8 129ndash132

Hindak F (2008) lsquoAtlas of cyanophytesrsquo (VEDA Bratislava Slovakia)Hofmann A Wilson A Kroumlner A Guy B Beukes N (2012) lsquoWorkshop

on craton formation and destruction post-conference excursionguidebook early craton formation stabilisation plume-relatedmagmatism and meteorite impact (Vredefort Witwatersrand Bushveldand Barberton)rsquo (Burlington House Piccadilly London) Availableat httpwwwgssaorgzaDocumentsCraton20Formation20and20Destruction20Excursion20Guidepdf [Verified 27November 2013]

Huumlber-Pestalozzi G (1961) lsquoDas Phytoplankton des Suumlsswassers SystematikundBiologie Tiel 5 Chlorophyceae (Gruumlnalgen) OrdnungVolvocalesrsquo(Verlagbuchhandlung Stuttgart Germany)

John DM Whitton BA Brook AJ (2002) lsquoFreshwater algal flora of theBritish Isles a guide to freshwater and terrestrial algaersquo (CambridgeUniversity Press Cambridge UK)

Keren N Kidd MJ Penner-Hahn JE Pakrasi HB (2002) A light-dependentmechanism formassive accumulation ofmanganese in the photosyntheticbacterium Synechocystis sp PCC 6803Biochemistry 41 15 085ndash15 092doi101021bi026892s

Komaacuterek J Anagnostidis K (2005) lsquoCyanoprokaryota Oscillatorialesrsquo(Spektrum Akademischer Verlag Heidelberg Germany)

Kruckeberg AR (2002) lsquoGeology and plant life the effects oflandforms and rock type on plantsrsquo (University Washington PressSeattle WA)

Kruumlger GHJ (1978) lsquoThe effect of physico-chemical factors on growthrelevant to the mass culture of Microcystis under sterile conditionsrsquo(University of the Orange Free State Bloemfontein South Africa)

Langhans TM Storm C Schwabe A (2009) Community assembly ofbiological soil crusts of different successional stages in a temperatesand ecosystem as assessed by direct determination and enrichmenttechniques Microbial Ecology 58 394ndash407doi101007s00248-009-9532-x

Lukesova A (2001) Soil algae in brown coal lignite post-mining areas incentral Europe (Czech Republic and Germany) Restoration Ecology 9341ndash350 doi101046j1526-100X200194002x

Lund JWG (1945) Observations on soil algae I The ecology size andtaxonomy of British soil diatoms Part 1 New Phytologist 44 196ndash219doi101111j1469-81371945tb05033x

Soil algae and cyanoprokaryotes Australian Journal of Botany K

Phormidium ambiguum and Hantzschia amphioxys were foundat all sites Chlorophyceae was the most diverse group with thehighest diversity at Site 1 whereas Bacillariophyceae diversitywas the highest at Sites 1 4 and 5 The CyanophyceaeChlorophyceae and Bacillariophyceae were represented atevery site However Eustigmatophyceae did not occur at Sites3 and 4 and the Xantophyceae occurred only at Sites 2 6 and 7

Preliminary correlations between edaphic featuresand algal diversity

Multivariate analysis confirms the strong association of heavymetals with the serpentine soils at Site 8 (Figs 2 3) The first twoaxes of the CCA (Fig 3) explained only 362 of the varianceVariables with high inflation factors (Ca Mg P and N) wereremoved to improve the analysis but eigenvalues or theexplanation of the total variance did not change The forwardselection of factors lowered the P-value however it was stillnot significant so we kept all the variables Even with the lowvalues the CCA helps to visualise the species distribution withrespect to the different sites and presents preliminary informationon how different edaphic features are correlated with algaldiversity Hormotilopsis gelatinosa Klebsormidium flaccidumPleurococcus species Chlorotetraedron species and Tetracystiselliptica belonging to the Chlorophyceae and Chroococcus

species Scytonema ocellatum Nostoc linckia as well as anunknown Nostoc species from the Cyanophyceae were closelyassociated with Site 8 (Fig 3) High concentrations of K wereassociatedwith theupper slopeof thesedimentary soils inOhrigstad(Site 2 Figs 2 3) as well as the green algae Characiopsis minimaChlamydomonas macrostellata Bumilleriopsis filiformis from theXanthophyceae and the cyanoprokaryote Oscillatoria raoiCalcium Mg S and N appear to play a role in the similarity inspecies composition of Sites 1 3 4 and 5

BrayndashCurtis dissimilarity index (Hahs and McDonnell2006) grouped samples from lower slopes (Sites 1 6 and7 indicated with black square in Fig 3) whereas the othersites were scattered (stress value 005) Cyanoprokaryotessuch as Leptolyngbya foveolarum Microcoleus vaginatus andPhormidium ambiguum Chlorophyceae such asChlamydomonasand Chlorococcum species Eustigmatophyceae such asEustigmatos magnus and Hantzschia amphioxys fromBacillariophyceae occurred on all the lower-slope sites (Sites 16 and 7) but were not unique to these sites The cyanoprokaryotePhormidium ambiguum was dominant in all the upper-slope sites(Sites 2 4 and 5) It was interesting that the serpentine sites(Sites 7 and 8) did not form a group Nor did the mafic andultramafic sites (Sites 3 4 5 and 6) or siliciclastic sites (Sites 1and 2) This random pattern with a lack of grouping for sitesshowed that slope position (upper vs lower) had a larger effect on

Legend sites rarr environmental variables

Axis 1 2 3 4

Eigen values 048 0235 0093 0091

Cumulative percentage variance

Of species 48 716 808 90

ndash10 15ndash10

10

Ca

Mg

K

Na

P

N

S

Al

As

Cd

Co

Cu

Cr

Fe

Hg

MnMo

Ni

Pd

V

12

3

4

5

6

7

8

Fig 2 Principal component analysis (PCA) to show the relationship between soil characteristics and the sampling sites

F Australian Journal of Botany A Venter et al

Table 3 Algae and cyanoprokaryotes identified from the different soil samples collected from the eight sitesdom dominant sdom subdominant + present

Species Abbreviations Siliciclastic rocks Mafic and ultramafic rocks Serpentineused in Fig 3 Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Site 7 Site 8

CyanophyceaeChroococcus sp Chro +Komvophoron cf schmidlei (Jaag)Anagnostids et Komaacuterek

Kom +

Leptolyngbya foveolarum (Rabenhorstex Gomont) Anagnostidiset Komaacuterek

Lepto dom + + dom dom sdom dom dom

Lyngbya major Meneghini Lyn1 + + +Lyngbya cf truncicola Ghose Lyn2 +Microcoleus vaginatus (Vauch) Gom Micro sdom sdom dom sdom sdomNostoc commune Vauch sensu Elenk Nos1 +Nostoc linckia (Roth) Born et Flah

in sensu ElenkNos2 sdom

Nostoc punctiforme (Kuumltz) Hariot Nos3 +Nostoc sp Nos4 +Oscillatoria limosa Ag Osc1 + +Oscillatoria raoi De Toni Osc2 +Phormidium ambiguum Gomont Pho1 + dom sdom dom dom sdom dom sdomPhormidium animale (Agardh ex

Gomont) Anagnostidis et KomaacuterekPho2 +

Phormidium cf corium (Ag) Kuumltzex Gomont

Pho3 +

Phormidium cf jenkelianum Schmid Pho4 +Phormidium jadinianum Gomont Pho5 +Phormidium mucicola Naum et Hub-

PestalozziPho6 +

Pseudanabaena cf minima (GSAn)Anagnostidis

Pseu1 +

Pseudanabaena frigida (Fritsch)Anagnostidis

Pseu2 +

Pseudanabaena sp Pseu3 + + +Scytonema myochrous (Dillw) Ag

ex Born et FlahScyt1 + dom

Scytonema ocellatum Born et Flah Scyt2 domScytonema sp Scyt3 +Synechocystis crassa Woronich Syn +Total Cyanophyceae 7 4 4 9 4 5 8 8

ChlorophyceaeBracteacoccus cf grandis Bischoff

et BoldBrac1 +

Bracteacoccus minor (Chod) Petrovaacute Brac2 +Bracteacoccus sp Brac3 +Characiopsis minima Pascher Chara +Chlamydomonas macrostellata Lund Chlamy sdomChlamydomonas sp1 Chlam1 + + + + + +Chlamydomonas sp2 Chlam2 +ChlorellamunitissimaFott etNovaacutekovaacute Chlo1 +Chlorella vulgaris Beijerinck Chlo2 + sdom +Chlorella sp Chlo3 + +Chlorococcum echinozygotum Starr Chlc1 sdomChlorococcum infusionum (Schrank)

MeneghiniChlc2 + + + + +

Chlorococcum oviforme Archibaldet Bold

Chlc3 +

Chlorococcum vacuolatum Starr Chlc4 +Chlorococcum sp1 Chlc5 + +Chlorococcum sp2 Chlc6 + + + + +

(continued next page )

Soil algae and cyanoprokaryotes Australian Journal of Botany G

diversity Cyanoprokaryote species were less associated withthe foot slope of pyroxenitendashmagnesium rich soil in Steelpoort(Site 3) with a Studentrsquos t-test supporting a significant differencefrom those at Sites 1 (P=003) 4 (P=003) 7 (P=0007) and 8(P=001) The pattern also showed that the lithic class may beless important than the slope position in determining the algaland cyanoprokayote species present at the sites

Discussion

It is widely reported that cyanoprokaryotes are well adapted toa wide range of environmental conditions and have an ability

to growon avariety of substrates such asmine tailings (Lukesova2001 Orlekowsky et al 2013) barren artic soils (Michaud et al2012) arid regions (Rehaacutekovaacute et al 2011 Muumlhlsteinovaacute et al2014) and alkaline and serpentinising springs (Blank et al 2009Suzuki et al 2013) Our preliminary study documented thatcyanoprokaryotes and algae are found on mafic and ultramaficsoils in SouthAfrica and that slope position plays a role in speciescomposition Leptolyngbya foveolarumMicrocoleus vaginatusPhormidium ambiguum Chlamydomonas ChlorococcumEustigmatos magnus as well as Hantzschia amphioxys werepresent on all three foot-slope sites at Ohrigstad BurgersfortandMalelane (Sites 1 6 and7) Environmental conditions such as

Table 3 (continued )

Species Abbreviations Siliciclastic rocks Mafic and ultramafic rocks Serpentineused in Fig 3 Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Site 7 Site 8

Chlorosarcinopsis aggregata Arceet Bold

Chls1 +

Chlorosarcinopsis minor (Gerneck)Herndon

Chls2 sdom + +

Chlorotetraedron sp Chlt +Hormotilopsis gelatinosa Trainoret Bold

Hormo +

Interfilum sp Inter + +Klebsormidium flaccidum (Kuumltz)PCSilva Mattox et WHBlackwell

Kleb +

Myrmecia biatorellae (Tschermak-Woess) JBPetersen

Myrm +

Pleurococcus Pleur +Scenedesmus sp Scene +Scotiellopsis rubescens Vinatzer Scot1 +Scotiellopsis terrestris (Reisigl)Puncochaacuterovaacute et Kalina

Scot2 + + +

Spongiochloris sp1 Spong1 + +Spongiochloris sp2 Spong2 + + +Tetracystis aggregata Brown et Bold Tetra1 + +Tetracystis elliptica Nakano Tetra2 +Tetracystis sp Tetra3 + +Total Chlorophyceae 12 7 1 7 5 8 10 9

EustigmatophyceaeEustigmatos magnus (JBPetersen)Hibberd

Eust + + + sdom +

Monodopsis subterranea (JBPetersen)Hibberd

Mono + +

Total Eustigmatophyceae 1 1 0 0 1 1 1 2

XantophyceaeBotrydiopsis arhiza Borzi Botry1 sdomBotrydiopsis sp Botry2 +Bumilleriopsis filiformis Vischer Bum1 sdomTotal Xantophyceae 0 1 0 0 0 1 1 0

BacillariophyceaeAmphora veneta Kuumltzing Amph1 +Pinnularia borealis Ehrenberg Pin + + sdomNavicula mutica Kuumltzing Nav1 + +Navicula pelliculosa (Kuumltzing) Hilse Nav2 + +Navicula veneta Kuumltzing Nav3 +Hantzschia amphioxys (Ehrenberg)Grunow

Hant + sdom dom sdom sdom + + +

Total Bacillariophyceae 4 1 1 3 4 1 2 1

Total species richness 24 14 6 19 14 16 22 20

H Australian Journal of Botany A Venter et al

an increase in soil moisture as a result of runoff and theaccumulation of organic matter (Chen et al 2007) could haveplayed a role in the assembly of certain species on foot slopes Thecyanoprokaryotes Leptolyngbya foveolarum and Phormidiumambiguum were also present at the foot slope of the Mg-richpyroxenite soil (Site 3) whereas Microcoleus vaginatusChlamydomonas Chlorococcum and Eustigmatos magnus didnot occur at this site The absence of these algae as well as the lowdiversity observed at Site 3 could be the result of the highconcentrations of Cd (001mg kgndash1) and Hg (00046mg kgndash1)measured at this site According to Fernandez-Pintildeas et al (1995)Cd induces progressive disorganisation and degradation ofthylakoid membranes as well as a decrease in the mobilisationof the polyphosphate granules leading to phosphorus starvationPinto et al (2003) showed that Cd andHg inhibit photosynthesisHowever further experimentation is clearly needed to determinewhether Cd and Hg were responsible for the reduced diversityobserved at this site

Most of the cyanoprokaryotes were represented by filamentousspecies such as Leptolyngbya foveolarumMicrocoleus vaginatusand Phormidium ambiquum and were found at all eight samplingsites These filamentous cyanoprokaryotes can wind throughoutthe uppermost soil layers forming a net-like structure that bindstogether soil particles (Rosentreter et al 2007) This formssoil aggregates that create pathways for water infiltration andsurfaces for nutrient transformations while also increasing thesoil resistance to wind and water erosion Once the filamentouscyanoprokaryotes stabilise thesoil single-celledcyanoprokaryotessuch as Synechocystis sp are able to colonise the substrate(Rosentreter et al 2007) The Chlorophyceae representedby mainly coccoid species such as Chlamydomonas andChlorococcum were found on all the sites except at Site 3Chlorococcum echinozygotum and Chlamydomonas macrostellawere subdominant on the siliciclastic-derived soils of Sites 1 and 2respectively Bacillariophyceae such as Hantzschia amphioxyswas found on all the sites and was subdominant at Sites 2 4 and 5

Legend Δ species and sites rarr environmental variables

Axis

ndash06

ndash10 15

10

1 2 3 4

Eigen values 052 0411 0388 0368

Cumulative percentage variance

Of species 202 362 513 656

Of environmental relations 202 362 513 656

Test of significance of first canonical axes p = 10

Test of significance of all canonical axes p = 10

Fig 3 Canonical correspondence analysis (CCA) of metal concentrations and algal and cyanoprokaryote species of the different sampling sitesAbbreviations of species are given in Table 3

Soil algae and cyanoprokaryotes Australian Journal of Botany I

and dominant at Site 3 According to Metting (1981)Leptolyngbya Microcoleus Phormidium ChlamydomonasChlorococcum and Hantzschia species are all typicalcosmopolitan soil algae

Shields and Durell (1964) and Starks and Shubert (1982)suggested that the species composition of soil algal populationsis affected less by the chemical nature of the substrate than bycertain physical properties that influence soil moisture levelsDuring February 2012 when sampling took place the study areahad received between 50 and 200mm rain which accounts formore than 75 of the expected average summer rainfall for thearea (South African Weather Service 2012) Moisture wastherefore not a limiting factor and the chemical nature of thesoils along with other abiotic habitat features appear to haveinfluenced species richness and composition In addition tothe abiotic conditions such as edaphic features biotic factorsespecially the type of vegetation can influence diversity andcommunity composition of cryptogamic biota For exampleRosentreter et al (2007) documented that vagrant populationsofDermatocarponoccur on poorly drained basaltflats dominatedby Artemisia rigida A papposa Antennaria flagellaris andPoa sandbergii The ecological conditions found at such sitescan support completely or partially vagrant life forms inDermatocarpon and other lichen genera The differences invegetation cover of the plant communities at the sampling sites(Sites 1ndash8) could have created microenvironments that favourspecific algal and cyanoprokaryote species assemblages Sites1ndash7 had moderate cover whereas Site 8 was an open savannacharacterised by numerous bare soil patches (serpentine effect)

Twenty different species were identified at Site 8 (serpentine)despite this site showing elevated concentrations of heavymetalsincluding Co Cr Fe Mn and Ni (Table 2) Chroococcus spScytonema ocellatum Nostoc linckia Chlorotetraedron spHormotilopsis gelatinosa Klebsormidium flaccidiumPleurococcus sp and Tetracystis elliptica were unique to thissite Metals can be divided into those that are required byorganisms in small quantities such as Fe Cu and Zn whichare essential in some biochemical reactions (Raven et al 1999)andnon-essentialmetals such asAs Pb andHgwhichmaycausesevere harm to organisms even at very low concentrationsbecause they do not provide any known biochemical function(Monteiro et al 2012) However the toxic effects of metallicelements on microalgae are complex and differ markedly amongspecies depending on the element itself and the prevailingenvironmental conditions (Monteiro et al 2012) Stark andShubert (1979) reported a positive correlation between Mn NP silica (Si) Al zinc (Zn) and Pb concentrations and algalabundance and a negative correlation with Na Cd Culithium (Li) Mo and strontium (Sr) Cyanoprokaryotes havedeveloped efficient strategies for metal uptake andaccumulation (Shcolnick and Keren 2006) According toKeren et al (2002) cyanoprokaryotes are able to accumulatehigh concentrations of Mn in the envelope layers of their cellsWhether the highMn supports the growth of cyanoprokaryotes orreduces competition by other groups that are less tolerant of Mnwas unclear Soil pH plays a critical role in the bioavailability ofheavy metals (Rajakaruna and Boyd 2008) and althoughserpentine Site 7 had high metal concentrations the relativelyhigher pH at this site may have made those metals less

bioavailable (Neilson and Rajakaruna 2012) Hence it wasunclear whether the higher algal diversity at this site resultedfrom metal tolerance or reduced metal toxicity owing to lowermetal availabilityHowever algae are able togrow in thepresenceof heavy metals as a result of a variety of tolerance mechanismsfor example binding to cell wall precipitation in vacuole andsynthesis of heavy metal-binding compounds such as proteinsorganic acids and phenolic compounds (Metha and Gaur 2005)Serpentine soils are renowned for low Ca Mg ratio howeverDeGrood et al (2005) found that low Ca Mg ratio does notsignificantly explain variation in microbial community patternsalso our study did not show a strong correlation between algaldiversity and Ca Mg Microbes may therefore be respondingto increased soil organicmatter concentrations probably becauseof the associated increases in nutrient availability and water-holding capacity

Algae as a group are physiologically heterogeneous makingany generalisation about their soil relations difficult (OrsquoKelly1974) However favourable soil pH moisture conditions andnutrient content contribute to species diversity and communitycomposition (Shields andDurell 1964)Themultivariate analysesdid not suggest a distinct species association for serpentine sites(Sites 7 and8) Thiswas also true for themafic andultramafic sites(Sites 3 4 5 and 6) as well as the siliciclastic sites (Sites 1 and 2)The lack of a strong correlation with lithic type and a strongercorrelation with slope position suggests that the type of slopemay bemore important in influencing diversity Site pairs 1 and 23 and 4 as well as 5 and 6 were sampled at different slopepositions and at times shared similar species associationsbased on slope position (Fig 3)

Conclusions

It seems unlikely that soil chemistry alone was responsiblefor determining species diversity and no unique algal florafor serpentine soils was confirmed However the serpentinesoil at Kaapschehoop (Site 8) did have a unique speciesassemblage comprising Chroococcus sp Chlorotetraedronsp Hormotilopsis gelatinosa Klebsormidium flaccidiumPleurococcus sp and Tetracystis elliptica However thisrequires further investigation to determine whether it was alithic or climatic effect

Soil features alongwith other biotic (vegetation composition)and abiotic (slope exposure) habitat characteristics mayinfluence the presence and dominance of some algal andcyanoprokaryote species in harsh edaphic settings Our resultssuggested that topography (ie slope position) rather than thechemistry of the lithic class (ie rock type) wasmost important ininfluencing species diversity However high concentrations ofheavy metals also influenced species richness as well ascommunity composition

The characterisation of microbial communities using anisolation approach can be biased because this approach tendsto favour some groups of microbes over others Therefore in thefuture we plan to incorporate molecular approaches (Bjellandet al 2011 Daae et al 2013) to characterise the diversity ofcyanoprokaryotes and soil algae at our sites Additionally theecological heterogeneity within each site should be taken intoconsideration because our sampling strategy (ie pooling of soil

J Australian Journal of Botany A Venter et al

samples) did not allow for detecting species that may berestricted to distinct microhabitats found within our sites Acomposite sample does not allow for a correlation with bioticand abiotic factors but gives a general and preliminary view onthe diversity at each site Although our findings are preliminarythe study sets the stage for detailed investigations on the relativeimportance of edaphic versus other habitat features on algal andcyanoprokaryote diversity and community assembly

Acknowledgements

We thank the staff at Eko-Analitika North-West University Potchefstroomfor the soil analyses Christel Pretorius for drawing the map of the study areaand two anonymous reviewers for their useful suggestions to improve thequality of our manuscript

References

Alexander EB Coleman RG Keeler-Wolf T Harrison SP (2007)lsquoSerpentine geoecology of western North America geology soilsand vegetationrsquo (Oxford University Press New York)

ATSDR (Agency for Toxic Substances and Disease Registry) (2008)lsquoToxicological profile for aluminiumrsquo (US Department of Health andHuman Services Public Health Service Atlanta GA) Available at httpwwwatsdrcdcgovToxProfilestp22pdf [Verified 19 August 2014]

Belnap J (2003) The world at your feet desert biological soil crustsFrontiers in Ecology and the Environment 1 181ndash189doi1018901540-9295(2003)001[0181TWAYFD]20CO2

Belnap J Lange OL (2001) lsquoBiological soil crusts structure function andmanagementrsquo (Springer Berlin)

Bjelland T Grube M Hoem S Jorgensen SL Daae FL Thorseth IH OvrearingsL (2011) Microbial metacommunities in the lichen-rock habitatEnvironmental Microbiology Reports 3 434ndash442doi101111j1758-2229201000206x

Blank JG Green SJ Blake D Valley JW Kita NT Treiman A Dobson PF(2009) An alkaline spring system within the Del Puerto Ophiolite(California USA) a Mars analog site Planetary and Space Science57 533ndash540 doi101016jpss200811018

Bramwell D Caujape-Castells J (2011) lsquoThe biology of island florarsquo(Cambridge Press Cambridge UK)

Brown RM Larson DA Bold HC (1964) Airborne algae their abundanceand heterogeneity Science 143 583ndash585doi101126science1433606583

Cabala J Rahmonov O Jablonska M Teper E (2011) Soil algal colonizationand its ecological role in an environment polluted by past ZnndashPb miningand smelting activity Water Air and Soil Pollution 215 339ndash348doi101007s11270-010-0482-1

Cardace D Meyer-Dombard DR Olsen A Parenteau MN (2014) Bedrockand geochemical controls on extremophile habitats In lsquoPlant ecologyand evolution in harsh environmentsrsquo (Eds N Rajakaruna RS BoydTB Harris) pp 1ndash32 (Nova Science Publishers New York)

Chen YN Wang Q Li WH Ruan X (2007) Microbiotic crusts andthere interrelations with environmental factors in the Gurbantonggutdesert western China Environmental Geology 52 691ndash700doi101007s00254-006-0505-9

Clarke KR Gorley RN (2001) lsquoPRIMER v5 user manualtutorialrsquo(PRIMER-E Plymouth UK)

Clarke B Uken R Reinhardt J (2009) The geometry and emplacementmechanics of a Bushveld complex peridotite body South Africa SouthAfrican Journal of Geology 112 141ndash162 doi102113gssajg1122141

Couteacute A Tell G Therezien Y (1999) Aerophytic Cyanophyceae fromNew Caledonia Cryptogamie Algologie 20 301ndash344doi101016S0181-1568(00)88147-7

Daae FLOslashkland I DahleH Joslashrgensen SL Thorseth IH Pedersen RB (2013)Microbial life associated with low-temperature alteration of ultramafic

rocks in the Leka ophiolite complex Geobiology 11 318ndash339doi101111gbi12035

Daghino S Murat C Sizzano E Girlanda M Perotto S (2012) Fungaldiversity is not determined by mineral and chemical differences inserpentine substrates PLoS ONE 7(9) e44233doi101371journalpone0044233

DeGrood SH Claassen VP Scow KM (2005) Microbial communitycomposition on native and drastically disturbed serpentine soils SoilBiology amp Biochemistry 37 1427ndash1435doi101016jsoilbio200412013

Ettl H Gaumlrtner G Heynig H Mollenhauer D Komaacuterek J Anagnostidis K(1999) lsquoSuumlsswasser von Mittleeuropa Cyanoprokaryota Teil 1Chroococcalesrsquo (Gustav Fisher Verlag Stuttgart Germany)

Favero-Longo SE Isocrono D Piervittori R (2004) Lichens and ultramaficrocks a review Lichenologist (London England) 36 391ndash404doi101017S0024282904014215

Fernandez-Pintildeas F Mateo P Bonilla I (1995) Ultrastructural changesinduced by selected cadmium concentrations in the cyanobacteriumNostoc UAM208 Journal of Plant Physiology 147 452ndash456doi101016S0176-1617(11)82182-6

Hahs AK McDonnell MJ (2006) Selecting independent measures toquantify Melbournersquos urbanndashrural gradient Landscape and UrbanPlanning 78 435ndash448 doi101016jlandurbplan200512005

Harrison SP Rajakaruna N (2011) lsquoSerpentine evolution and ecology in amodel systemrsquo (University of California Press Berkeley CA)

Hauer T (2008) Epilithic cyanobacterial flora of Mohelenskaacute Hadcovaacutesteppe nature reseve (western Moravia Czech Republic) 70 years agoand now Fottea 8 129ndash132

Hindak F (2008) lsquoAtlas of cyanophytesrsquo (VEDA Bratislava Slovakia)Hofmann A Wilson A Kroumlner A Guy B Beukes N (2012) lsquoWorkshop

on craton formation and destruction post-conference excursionguidebook early craton formation stabilisation plume-relatedmagmatism and meteorite impact (Vredefort Witwatersrand Bushveldand Barberton)rsquo (Burlington House Piccadilly London) Availableat httpwwwgssaorgzaDocumentsCraton20Formation20and20Destruction20Excursion20Guidepdf [Verified 27November 2013]

Huumlber-Pestalozzi G (1961) lsquoDas Phytoplankton des Suumlsswassers SystematikundBiologie Tiel 5 Chlorophyceae (Gruumlnalgen) OrdnungVolvocalesrsquo(Verlagbuchhandlung Stuttgart Germany)

John DM Whitton BA Brook AJ (2002) lsquoFreshwater algal flora of theBritish Isles a guide to freshwater and terrestrial algaersquo (CambridgeUniversity Press Cambridge UK)

Keren N Kidd MJ Penner-Hahn JE Pakrasi HB (2002) A light-dependentmechanism formassive accumulation ofmanganese in the photosyntheticbacterium Synechocystis sp PCC 6803Biochemistry 41 15 085ndash15 092doi101021bi026892s

Komaacuterek J Anagnostidis K (2005) lsquoCyanoprokaryota Oscillatorialesrsquo(Spektrum Akademischer Verlag Heidelberg Germany)

Kruckeberg AR (2002) lsquoGeology and plant life the effects oflandforms and rock type on plantsrsquo (University Washington PressSeattle WA)

Kruumlger GHJ (1978) lsquoThe effect of physico-chemical factors on growthrelevant to the mass culture of Microcystis under sterile conditionsrsquo(University of the Orange Free State Bloemfontein South Africa)

Langhans TM Storm C Schwabe A (2009) Community assembly ofbiological soil crusts of different successional stages in a temperatesand ecosystem as assessed by direct determination and enrichmenttechniques Microbial Ecology 58 394ndash407doi101007s00248-009-9532-x

Lukesova A (2001) Soil algae in brown coal lignite post-mining areas incentral Europe (Czech Republic and Germany) Restoration Ecology 9341ndash350 doi101046j1526-100X200194002x

Lund JWG (1945) Observations on soil algae I The ecology size andtaxonomy of British soil diatoms Part 1 New Phytologist 44 196ndash219doi101111j1469-81371945tb05033x

Soil algae and cyanoprokaryotes Australian Journal of Botany K

Table 3 Algae and cyanoprokaryotes identified from the different soil samples collected from the eight sitesdom dominant sdom subdominant + present

Species Abbreviations Siliciclastic rocks Mafic and ultramafic rocks Serpentineused in Fig 3 Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Site 7 Site 8

CyanophyceaeChroococcus sp Chro +Komvophoron cf schmidlei (Jaag)Anagnostids et Komaacuterek

Kom +

Leptolyngbya foveolarum (Rabenhorstex Gomont) Anagnostidiset Komaacuterek

Lepto dom + + dom dom sdom dom dom

Lyngbya major Meneghini Lyn1 + + +Lyngbya cf truncicola Ghose Lyn2 +Microcoleus vaginatus (Vauch) Gom Micro sdom sdom dom sdom sdomNostoc commune Vauch sensu Elenk Nos1 +Nostoc linckia (Roth) Born et Flah

in sensu ElenkNos2 sdom

Nostoc punctiforme (Kuumltz) Hariot Nos3 +Nostoc sp Nos4 +Oscillatoria limosa Ag Osc1 + +Oscillatoria raoi De Toni Osc2 +Phormidium ambiguum Gomont Pho1 + dom sdom dom dom sdom dom sdomPhormidium animale (Agardh ex

Gomont) Anagnostidis et KomaacuterekPho2 +

Phormidium cf corium (Ag) Kuumltzex Gomont

Pho3 +

Phormidium cf jenkelianum Schmid Pho4 +Phormidium jadinianum Gomont Pho5 +Phormidium mucicola Naum et Hub-

PestalozziPho6 +

Pseudanabaena cf minima (GSAn)Anagnostidis

Pseu1 +

Pseudanabaena frigida (Fritsch)Anagnostidis

Pseu2 +

Pseudanabaena sp Pseu3 + + +Scytonema myochrous (Dillw) Ag

ex Born et FlahScyt1 + dom

Scytonema ocellatum Born et Flah Scyt2 domScytonema sp Scyt3 +Synechocystis crassa Woronich Syn +Total Cyanophyceae 7 4 4 9 4 5 8 8

ChlorophyceaeBracteacoccus cf grandis Bischoff

et BoldBrac1 +

Bracteacoccus minor (Chod) Petrovaacute Brac2 +Bracteacoccus sp Brac3 +Characiopsis minima Pascher Chara +Chlamydomonas macrostellata Lund Chlamy sdomChlamydomonas sp1 Chlam1 + + + + + +Chlamydomonas sp2 Chlam2 +ChlorellamunitissimaFott etNovaacutekovaacute Chlo1 +Chlorella vulgaris Beijerinck Chlo2 + sdom +Chlorella sp Chlo3 + +Chlorococcum echinozygotum Starr Chlc1 sdomChlorococcum infusionum (Schrank)

MeneghiniChlc2 + + + + +

Chlorococcum oviforme Archibaldet Bold

Chlc3 +

Chlorococcum vacuolatum Starr Chlc4 +Chlorococcum sp1 Chlc5 + +Chlorococcum sp2 Chlc6 + + + + +

(continued next page )

Soil algae and cyanoprokaryotes Australian Journal of Botany G

diversity Cyanoprokaryote species were less associated withthe foot slope of pyroxenitendashmagnesium rich soil in Steelpoort(Site 3) with a Studentrsquos t-test supporting a significant differencefrom those at Sites 1 (P=003) 4 (P=003) 7 (P=0007) and 8(P=001) The pattern also showed that the lithic class may beless important than the slope position in determining the algaland cyanoprokayote species present at the sites

Discussion

It is widely reported that cyanoprokaryotes are well adapted toa wide range of environmental conditions and have an ability

to growon avariety of substrates such asmine tailings (Lukesova2001 Orlekowsky et al 2013) barren artic soils (Michaud et al2012) arid regions (Rehaacutekovaacute et al 2011 Muumlhlsteinovaacute et al2014) and alkaline and serpentinising springs (Blank et al 2009Suzuki et al 2013) Our preliminary study documented thatcyanoprokaryotes and algae are found on mafic and ultramaficsoils in SouthAfrica and that slope position plays a role in speciescomposition Leptolyngbya foveolarumMicrocoleus vaginatusPhormidium ambiguum Chlamydomonas ChlorococcumEustigmatos magnus as well as Hantzschia amphioxys werepresent on all three foot-slope sites at Ohrigstad BurgersfortandMalelane (Sites 1 6 and7) Environmental conditions such as

Table 3 (continued )

Species Abbreviations Siliciclastic rocks Mafic and ultramafic rocks Serpentineused in Fig 3 Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Site 7 Site 8

Chlorosarcinopsis aggregata Arceet Bold

Chls1 +

Chlorosarcinopsis minor (Gerneck)Herndon

Chls2 sdom + +

Chlorotetraedron sp Chlt +Hormotilopsis gelatinosa Trainoret Bold

Hormo +

Interfilum sp Inter + +Klebsormidium flaccidum (Kuumltz)PCSilva Mattox et WHBlackwell

Kleb +

Myrmecia biatorellae (Tschermak-Woess) JBPetersen

Myrm +

Pleurococcus Pleur +Scenedesmus sp Scene +Scotiellopsis rubescens Vinatzer Scot1 +Scotiellopsis terrestris (Reisigl)Puncochaacuterovaacute et Kalina

Scot2 + + +

Spongiochloris sp1 Spong1 + +Spongiochloris sp2 Spong2 + + +Tetracystis aggregata Brown et Bold Tetra1 + +Tetracystis elliptica Nakano Tetra2 +Tetracystis sp Tetra3 + +Total Chlorophyceae 12 7 1 7 5 8 10 9

EustigmatophyceaeEustigmatos magnus (JBPetersen)Hibberd

Eust + + + sdom +

Monodopsis subterranea (JBPetersen)Hibberd

Mono + +

Total Eustigmatophyceae 1 1 0 0 1 1 1 2

XantophyceaeBotrydiopsis arhiza Borzi Botry1 sdomBotrydiopsis sp Botry2 +Bumilleriopsis filiformis Vischer Bum1 sdomTotal Xantophyceae 0 1 0 0 0 1 1 0

BacillariophyceaeAmphora veneta Kuumltzing Amph1 +Pinnularia borealis Ehrenberg Pin + + sdomNavicula mutica Kuumltzing Nav1 + +Navicula pelliculosa (Kuumltzing) Hilse Nav2 + +Navicula veneta Kuumltzing Nav3 +Hantzschia amphioxys (Ehrenberg)Grunow

Hant + sdom dom sdom sdom + + +

Total Bacillariophyceae 4 1 1 3 4 1 2 1

Total species richness 24 14 6 19 14 16 22 20

H Australian Journal of Botany A Venter et al

an increase in soil moisture as a result of runoff and theaccumulation of organic matter (Chen et al 2007) could haveplayed a role in the assembly of certain species on foot slopes Thecyanoprokaryotes Leptolyngbya foveolarum and Phormidiumambiguum were also present at the foot slope of the Mg-richpyroxenite soil (Site 3) whereas Microcoleus vaginatusChlamydomonas Chlorococcum and Eustigmatos magnus didnot occur at this site The absence of these algae as well as the lowdiversity observed at Site 3 could be the result of the highconcentrations of Cd (001mg kgndash1) and Hg (00046mg kgndash1)measured at this site According to Fernandez-Pintildeas et al (1995)Cd induces progressive disorganisation and degradation ofthylakoid membranes as well as a decrease in the mobilisationof the polyphosphate granules leading to phosphorus starvationPinto et al (2003) showed that Cd andHg inhibit photosynthesisHowever further experimentation is clearly needed to determinewhether Cd and Hg were responsible for the reduced diversityobserved at this site

Most of the cyanoprokaryotes were represented by filamentousspecies such as Leptolyngbya foveolarumMicrocoleus vaginatusand Phormidium ambiquum and were found at all eight samplingsites These filamentous cyanoprokaryotes can wind throughoutthe uppermost soil layers forming a net-like structure that bindstogether soil particles (Rosentreter et al 2007) This formssoil aggregates that create pathways for water infiltration andsurfaces for nutrient transformations while also increasing thesoil resistance to wind and water erosion Once the filamentouscyanoprokaryotes stabilise thesoil single-celledcyanoprokaryotessuch as Synechocystis sp are able to colonise the substrate(Rosentreter et al 2007) The Chlorophyceae representedby mainly coccoid species such as Chlamydomonas andChlorococcum were found on all the sites except at Site 3Chlorococcum echinozygotum and Chlamydomonas macrostellawere subdominant on the siliciclastic-derived soils of Sites 1 and 2respectively Bacillariophyceae such as Hantzschia amphioxyswas found on all the sites and was subdominant at Sites 2 4 and 5

Legend Δ species and sites rarr environmental variables

Axis

ndash06

ndash10 15

10

1 2 3 4

Eigen values 052 0411 0388 0368

Cumulative percentage variance

Of species 202 362 513 656

Of environmental relations 202 362 513 656

Test of significance of first canonical axes p = 10

Test of significance of all canonical axes p = 10

Fig 3 Canonical correspondence analysis (CCA) of metal concentrations and algal and cyanoprokaryote species of the different sampling sitesAbbreviations of species are given in Table 3

Soil algae and cyanoprokaryotes Australian Journal of Botany I

and dominant at Site 3 According to Metting (1981)Leptolyngbya Microcoleus Phormidium ChlamydomonasChlorococcum and Hantzschia species are all typicalcosmopolitan soil algae

Shields and Durell (1964) and Starks and Shubert (1982)suggested that the species composition of soil algal populationsis affected less by the chemical nature of the substrate than bycertain physical properties that influence soil moisture levelsDuring February 2012 when sampling took place the study areahad received between 50 and 200mm rain which accounts formore than 75 of the expected average summer rainfall for thearea (South African Weather Service 2012) Moisture wastherefore not a limiting factor and the chemical nature of thesoils along with other abiotic habitat features appear to haveinfluenced species richness and composition In addition tothe abiotic conditions such as edaphic features biotic factorsespecially the type of vegetation can influence diversity andcommunity composition of cryptogamic biota For exampleRosentreter et al (2007) documented that vagrant populationsofDermatocarponoccur on poorly drained basaltflats dominatedby Artemisia rigida A papposa Antennaria flagellaris andPoa sandbergii The ecological conditions found at such sitescan support completely or partially vagrant life forms inDermatocarpon and other lichen genera The differences invegetation cover of the plant communities at the sampling sites(Sites 1ndash8) could have created microenvironments that favourspecific algal and cyanoprokaryote species assemblages Sites1ndash7 had moderate cover whereas Site 8 was an open savannacharacterised by numerous bare soil patches (serpentine effect)

Twenty different species were identified at Site 8 (serpentine)despite this site showing elevated concentrations of heavymetalsincluding Co Cr Fe Mn and Ni (Table 2) Chroococcus spScytonema ocellatum Nostoc linckia Chlorotetraedron spHormotilopsis gelatinosa Klebsormidium flaccidiumPleurococcus sp and Tetracystis elliptica were unique to thissite Metals can be divided into those that are required byorganisms in small quantities such as Fe Cu and Zn whichare essential in some biochemical reactions (Raven et al 1999)andnon-essentialmetals such asAs Pb andHgwhichmaycausesevere harm to organisms even at very low concentrationsbecause they do not provide any known biochemical function(Monteiro et al 2012) However the toxic effects of metallicelements on microalgae are complex and differ markedly amongspecies depending on the element itself and the prevailingenvironmental conditions (Monteiro et al 2012) Stark andShubert (1979) reported a positive correlation between Mn NP silica (Si) Al zinc (Zn) and Pb concentrations and algalabundance and a negative correlation with Na Cd Culithium (Li) Mo and strontium (Sr) Cyanoprokaryotes havedeveloped efficient strategies for metal uptake andaccumulation (Shcolnick and Keren 2006) According toKeren et al (2002) cyanoprokaryotes are able to accumulatehigh concentrations of Mn in the envelope layers of their cellsWhether the highMn supports the growth of cyanoprokaryotes orreduces competition by other groups that are less tolerant of Mnwas unclear Soil pH plays a critical role in the bioavailability ofheavy metals (Rajakaruna and Boyd 2008) and althoughserpentine Site 7 had high metal concentrations the relativelyhigher pH at this site may have made those metals less

bioavailable (Neilson and Rajakaruna 2012) Hence it wasunclear whether the higher algal diversity at this site resultedfrom metal tolerance or reduced metal toxicity owing to lowermetal availabilityHowever algae are able togrow in thepresenceof heavy metals as a result of a variety of tolerance mechanismsfor example binding to cell wall precipitation in vacuole andsynthesis of heavy metal-binding compounds such as proteinsorganic acids and phenolic compounds (Metha and Gaur 2005)Serpentine soils are renowned for low Ca Mg ratio howeverDeGrood et al (2005) found that low Ca Mg ratio does notsignificantly explain variation in microbial community patternsalso our study did not show a strong correlation between algaldiversity and Ca Mg Microbes may therefore be respondingto increased soil organicmatter concentrations probably becauseof the associated increases in nutrient availability and water-holding capacity

Algae as a group are physiologically heterogeneous makingany generalisation about their soil relations difficult (OrsquoKelly1974) However favourable soil pH moisture conditions andnutrient content contribute to species diversity and communitycomposition (Shields andDurell 1964)Themultivariate analysesdid not suggest a distinct species association for serpentine sites(Sites 7 and8) Thiswas also true for themafic andultramafic sites(Sites 3 4 5 and 6) as well as the siliciclastic sites (Sites 1 and 2)The lack of a strong correlation with lithic type and a strongercorrelation with slope position suggests that the type of slopemay bemore important in influencing diversity Site pairs 1 and 23 and 4 as well as 5 and 6 were sampled at different slopepositions and at times shared similar species associationsbased on slope position (Fig 3)

Conclusions

It seems unlikely that soil chemistry alone was responsiblefor determining species diversity and no unique algal florafor serpentine soils was confirmed However the serpentinesoil at Kaapschehoop (Site 8) did have a unique speciesassemblage comprising Chroococcus sp Chlorotetraedronsp Hormotilopsis gelatinosa Klebsormidium flaccidiumPleurococcus sp and Tetracystis elliptica However thisrequires further investigation to determine whether it was alithic or climatic effect

Soil features alongwith other biotic (vegetation composition)and abiotic (slope exposure) habitat characteristics mayinfluence the presence and dominance of some algal andcyanoprokaryote species in harsh edaphic settings Our resultssuggested that topography (ie slope position) rather than thechemistry of the lithic class (ie rock type) wasmost important ininfluencing species diversity However high concentrations ofheavy metals also influenced species richness as well ascommunity composition

The characterisation of microbial communities using anisolation approach can be biased because this approach tendsto favour some groups of microbes over others Therefore in thefuture we plan to incorporate molecular approaches (Bjellandet al 2011 Daae et al 2013) to characterise the diversity ofcyanoprokaryotes and soil algae at our sites Additionally theecological heterogeneity within each site should be taken intoconsideration because our sampling strategy (ie pooling of soil

J Australian Journal of Botany A Venter et al

samples) did not allow for detecting species that may berestricted to distinct microhabitats found within our sites Acomposite sample does not allow for a correlation with bioticand abiotic factors but gives a general and preliminary view onthe diversity at each site Although our findings are preliminarythe study sets the stage for detailed investigations on the relativeimportance of edaphic versus other habitat features on algal andcyanoprokaryote diversity and community assembly

Acknowledgements

We thank the staff at Eko-Analitika North-West University Potchefstroomfor the soil analyses Christel Pretorius for drawing the map of the study areaand two anonymous reviewers for their useful suggestions to improve thequality of our manuscript

References

Alexander EB Coleman RG Keeler-Wolf T Harrison SP (2007)lsquoSerpentine geoecology of western North America geology soilsand vegetationrsquo (Oxford University Press New York)

ATSDR (Agency for Toxic Substances and Disease Registry) (2008)lsquoToxicological profile for aluminiumrsquo (US Department of Health andHuman Services Public Health Service Atlanta GA) Available at httpwwwatsdrcdcgovToxProfilestp22pdf [Verified 19 August 2014]

Belnap J (2003) The world at your feet desert biological soil crustsFrontiers in Ecology and the Environment 1 181ndash189doi1018901540-9295(2003)001[0181TWAYFD]20CO2

Belnap J Lange OL (2001) lsquoBiological soil crusts structure function andmanagementrsquo (Springer Berlin)

Bjelland T Grube M Hoem S Jorgensen SL Daae FL Thorseth IH OvrearingsL (2011) Microbial metacommunities in the lichen-rock habitatEnvironmental Microbiology Reports 3 434ndash442doi101111j1758-2229201000206x

Blank JG Green SJ Blake D Valley JW Kita NT Treiman A Dobson PF(2009) An alkaline spring system within the Del Puerto Ophiolite(California USA) a Mars analog site Planetary and Space Science57 533ndash540 doi101016jpss200811018

Bramwell D Caujape-Castells J (2011) lsquoThe biology of island florarsquo(Cambridge Press Cambridge UK)

Brown RM Larson DA Bold HC (1964) Airborne algae their abundanceand heterogeneity Science 143 583ndash585doi101126science1433606583

Cabala J Rahmonov O Jablonska M Teper E (2011) Soil algal colonizationand its ecological role in an environment polluted by past ZnndashPb miningand smelting activity Water Air and Soil Pollution 215 339ndash348doi101007s11270-010-0482-1

Cardace D Meyer-Dombard DR Olsen A Parenteau MN (2014) Bedrockand geochemical controls on extremophile habitats In lsquoPlant ecologyand evolution in harsh environmentsrsquo (Eds N Rajakaruna RS BoydTB Harris) pp 1ndash32 (Nova Science Publishers New York)

Chen YN Wang Q Li WH Ruan X (2007) Microbiotic crusts andthere interrelations with environmental factors in the Gurbantonggutdesert western China Environmental Geology 52 691ndash700doi101007s00254-006-0505-9

Clarke KR Gorley RN (2001) lsquoPRIMER v5 user manualtutorialrsquo(PRIMER-E Plymouth UK)

Clarke B Uken R Reinhardt J (2009) The geometry and emplacementmechanics of a Bushveld complex peridotite body South Africa SouthAfrican Journal of Geology 112 141ndash162 doi102113gssajg1122141

Couteacute A Tell G Therezien Y (1999) Aerophytic Cyanophyceae fromNew Caledonia Cryptogamie Algologie 20 301ndash344doi101016S0181-1568(00)88147-7

Daae FLOslashkland I DahleH Joslashrgensen SL Thorseth IH Pedersen RB (2013)Microbial life associated with low-temperature alteration of ultramafic

rocks in the Leka ophiolite complex Geobiology 11 318ndash339doi101111gbi12035

Daghino S Murat C Sizzano E Girlanda M Perotto S (2012) Fungaldiversity is not determined by mineral and chemical differences inserpentine substrates PLoS ONE 7(9) e44233doi101371journalpone0044233

DeGrood SH Claassen VP Scow KM (2005) Microbial communitycomposition on native and drastically disturbed serpentine soils SoilBiology amp Biochemistry 37 1427ndash1435doi101016jsoilbio200412013

Ettl H Gaumlrtner G Heynig H Mollenhauer D Komaacuterek J Anagnostidis K(1999) lsquoSuumlsswasser von Mittleeuropa Cyanoprokaryota Teil 1Chroococcalesrsquo (Gustav Fisher Verlag Stuttgart Germany)

Favero-Longo SE Isocrono D Piervittori R (2004) Lichens and ultramaficrocks a review Lichenologist (London England) 36 391ndash404doi101017S0024282904014215

Fernandez-Pintildeas F Mateo P Bonilla I (1995) Ultrastructural changesinduced by selected cadmium concentrations in the cyanobacteriumNostoc UAM208 Journal of Plant Physiology 147 452ndash456doi101016S0176-1617(11)82182-6

Hahs AK McDonnell MJ (2006) Selecting independent measures toquantify Melbournersquos urbanndashrural gradient Landscape and UrbanPlanning 78 435ndash448 doi101016jlandurbplan200512005

Harrison SP Rajakaruna N (2011) lsquoSerpentine evolution and ecology in amodel systemrsquo (University of California Press Berkeley CA)

Hauer T (2008) Epilithic cyanobacterial flora of Mohelenskaacute Hadcovaacutesteppe nature reseve (western Moravia Czech Republic) 70 years agoand now Fottea 8 129ndash132

Hindak F (2008) lsquoAtlas of cyanophytesrsquo (VEDA Bratislava Slovakia)Hofmann A Wilson A Kroumlner A Guy B Beukes N (2012) lsquoWorkshop

on craton formation and destruction post-conference excursionguidebook early craton formation stabilisation plume-relatedmagmatism and meteorite impact (Vredefort Witwatersrand Bushveldand Barberton)rsquo (Burlington House Piccadilly London) Availableat httpwwwgssaorgzaDocumentsCraton20Formation20and20Destruction20Excursion20Guidepdf [Verified 27November 2013]

Huumlber-Pestalozzi G (1961) lsquoDas Phytoplankton des Suumlsswassers SystematikundBiologie Tiel 5 Chlorophyceae (Gruumlnalgen) OrdnungVolvocalesrsquo(Verlagbuchhandlung Stuttgart Germany)

John DM Whitton BA Brook AJ (2002) lsquoFreshwater algal flora of theBritish Isles a guide to freshwater and terrestrial algaersquo (CambridgeUniversity Press Cambridge UK)

Keren N Kidd MJ Penner-Hahn JE Pakrasi HB (2002) A light-dependentmechanism formassive accumulation ofmanganese in the photosyntheticbacterium Synechocystis sp PCC 6803Biochemistry 41 15 085ndash15 092doi101021bi026892s

Komaacuterek J Anagnostidis K (2005) lsquoCyanoprokaryota Oscillatorialesrsquo(Spektrum Akademischer Verlag Heidelberg Germany)

Kruckeberg AR (2002) lsquoGeology and plant life the effects oflandforms and rock type on plantsrsquo (University Washington PressSeattle WA)

Kruumlger GHJ (1978) lsquoThe effect of physico-chemical factors on growthrelevant to the mass culture of Microcystis under sterile conditionsrsquo(University of the Orange Free State Bloemfontein South Africa)

Langhans TM Storm C Schwabe A (2009) Community assembly ofbiological soil crusts of different successional stages in a temperatesand ecosystem as assessed by direct determination and enrichmenttechniques Microbial Ecology 58 394ndash407doi101007s00248-009-9532-x

Lukesova A (2001) Soil algae in brown coal lignite post-mining areas incentral Europe (Czech Republic and Germany) Restoration Ecology 9341ndash350 doi101046j1526-100X200194002x

Lund JWG (1945) Observations on soil algae I The ecology size andtaxonomy of British soil diatoms Part 1 New Phytologist 44 196ndash219doi101111j1469-81371945tb05033x

Soil algae and cyanoprokaryotes Australian Journal of Botany K

diversity Cyanoprokaryote species were less associated withthe foot slope of pyroxenitendashmagnesium rich soil in Steelpoort(Site 3) with a Studentrsquos t-test supporting a significant differencefrom those at Sites 1 (P=003) 4 (P=003) 7 (P=0007) and 8(P=001) The pattern also showed that the lithic class may beless important than the slope position in determining the algaland cyanoprokayote species present at the sites

Discussion

It is widely reported that cyanoprokaryotes are well adapted toa wide range of environmental conditions and have an ability

to growon avariety of substrates such asmine tailings (Lukesova2001 Orlekowsky et al 2013) barren artic soils (Michaud et al2012) arid regions (Rehaacutekovaacute et al 2011 Muumlhlsteinovaacute et al2014) and alkaline and serpentinising springs (Blank et al 2009Suzuki et al 2013) Our preliminary study documented thatcyanoprokaryotes and algae are found on mafic and ultramaficsoils in SouthAfrica and that slope position plays a role in speciescomposition Leptolyngbya foveolarumMicrocoleus vaginatusPhormidium ambiguum Chlamydomonas ChlorococcumEustigmatos magnus as well as Hantzschia amphioxys werepresent on all three foot-slope sites at Ohrigstad BurgersfortandMalelane (Sites 1 6 and7) Environmental conditions such as

Table 3 (continued )

Species Abbreviations Siliciclastic rocks Mafic and ultramafic rocks Serpentineused in Fig 3 Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Site 7 Site 8

Chlorosarcinopsis aggregata Arceet Bold

Chls1 +

Chlorosarcinopsis minor (Gerneck)Herndon

Chls2 sdom + +

Chlorotetraedron sp Chlt +Hormotilopsis gelatinosa Trainoret Bold

Hormo +

Interfilum sp Inter + +Klebsormidium flaccidum (Kuumltz)PCSilva Mattox et WHBlackwell

Kleb +

Myrmecia biatorellae (Tschermak-Woess) JBPetersen

Myrm +

Pleurococcus Pleur +Scenedesmus sp Scene +Scotiellopsis rubescens Vinatzer Scot1 +Scotiellopsis terrestris (Reisigl)Puncochaacuterovaacute et Kalina

Scot2 + + +

Spongiochloris sp1 Spong1 + +Spongiochloris sp2 Spong2 + + +Tetracystis aggregata Brown et Bold Tetra1 + +Tetracystis elliptica Nakano Tetra2 +Tetracystis sp Tetra3 + +Total Chlorophyceae 12 7 1 7 5 8 10 9

EustigmatophyceaeEustigmatos magnus (JBPetersen)Hibberd

Eust + + + sdom +

Monodopsis subterranea (JBPetersen)Hibberd

Mono + +

Total Eustigmatophyceae 1 1 0 0 1 1 1 2

XantophyceaeBotrydiopsis arhiza Borzi Botry1 sdomBotrydiopsis sp Botry2 +Bumilleriopsis filiformis Vischer Bum1 sdomTotal Xantophyceae 0 1 0 0 0 1 1 0

BacillariophyceaeAmphora veneta Kuumltzing Amph1 +Pinnularia borealis Ehrenberg Pin + + sdomNavicula mutica Kuumltzing Nav1 + +Navicula pelliculosa (Kuumltzing) Hilse Nav2 + +Navicula veneta Kuumltzing Nav3 +Hantzschia amphioxys (Ehrenberg)Grunow

Hant + sdom dom sdom sdom + + +

Total Bacillariophyceae 4 1 1 3 4 1 2 1

Total species richness 24 14 6 19 14 16 22 20

H Australian Journal of Botany A Venter et al

an increase in soil moisture as a result of runoff and theaccumulation of organic matter (Chen et al 2007) could haveplayed a role in the assembly of certain species on foot slopes Thecyanoprokaryotes Leptolyngbya foveolarum and Phormidiumambiguum were also present at the foot slope of the Mg-richpyroxenite soil (Site 3) whereas Microcoleus vaginatusChlamydomonas Chlorococcum and Eustigmatos magnus didnot occur at this site The absence of these algae as well as the lowdiversity observed at Site 3 could be the result of the highconcentrations of Cd (001mg kgndash1) and Hg (00046mg kgndash1)measured at this site According to Fernandez-Pintildeas et al (1995)Cd induces progressive disorganisation and degradation ofthylakoid membranes as well as a decrease in the mobilisationof the polyphosphate granules leading to phosphorus starvationPinto et al (2003) showed that Cd andHg inhibit photosynthesisHowever further experimentation is clearly needed to determinewhether Cd and Hg were responsible for the reduced diversityobserved at this site

Most of the cyanoprokaryotes were represented by filamentousspecies such as Leptolyngbya foveolarumMicrocoleus vaginatusand Phormidium ambiquum and were found at all eight samplingsites These filamentous cyanoprokaryotes can wind throughoutthe uppermost soil layers forming a net-like structure that bindstogether soil particles (Rosentreter et al 2007) This formssoil aggregates that create pathways for water infiltration andsurfaces for nutrient transformations while also increasing thesoil resistance to wind and water erosion Once the filamentouscyanoprokaryotes stabilise thesoil single-celledcyanoprokaryotessuch as Synechocystis sp are able to colonise the substrate(Rosentreter et al 2007) The Chlorophyceae representedby mainly coccoid species such as Chlamydomonas andChlorococcum were found on all the sites except at Site 3Chlorococcum echinozygotum and Chlamydomonas macrostellawere subdominant on the siliciclastic-derived soils of Sites 1 and 2respectively Bacillariophyceae such as Hantzschia amphioxyswas found on all the sites and was subdominant at Sites 2 4 and 5

Legend Δ species and sites rarr environmental variables

Axis

ndash06

ndash10 15

10

1 2 3 4

Eigen values 052 0411 0388 0368

Cumulative percentage variance

Of species 202 362 513 656

Of environmental relations 202 362 513 656

Test of significance of first canonical axes p = 10

Test of significance of all canonical axes p = 10

Fig 3 Canonical correspondence analysis (CCA) of metal concentrations and algal and cyanoprokaryote species of the different sampling sitesAbbreviations of species are given in Table 3

Soil algae and cyanoprokaryotes Australian Journal of Botany I

and dominant at Site 3 According to Metting (1981)Leptolyngbya Microcoleus Phormidium ChlamydomonasChlorococcum and Hantzschia species are all typicalcosmopolitan soil algae

Shields and Durell (1964) and Starks and Shubert (1982)suggested that the species composition of soil algal populationsis affected less by the chemical nature of the substrate than bycertain physical properties that influence soil moisture levelsDuring February 2012 when sampling took place the study areahad received between 50 and 200mm rain which accounts formore than 75 of the expected average summer rainfall for thearea (South African Weather Service 2012) Moisture wastherefore not a limiting factor and the chemical nature of thesoils along with other abiotic habitat features appear to haveinfluenced species richness and composition In addition tothe abiotic conditions such as edaphic features biotic factorsespecially the type of vegetation can influence diversity andcommunity composition of cryptogamic biota For exampleRosentreter et al (2007) documented that vagrant populationsofDermatocarponoccur on poorly drained basaltflats dominatedby Artemisia rigida A papposa Antennaria flagellaris andPoa sandbergii The ecological conditions found at such sitescan support completely or partially vagrant life forms inDermatocarpon and other lichen genera The differences invegetation cover of the plant communities at the sampling sites(Sites 1ndash8) could have created microenvironments that favourspecific algal and cyanoprokaryote species assemblages Sites1ndash7 had moderate cover whereas Site 8 was an open savannacharacterised by numerous bare soil patches (serpentine effect)

Twenty different species were identified at Site 8 (serpentine)despite this site showing elevated concentrations of heavymetalsincluding Co Cr Fe Mn and Ni (Table 2) Chroococcus spScytonema ocellatum Nostoc linckia Chlorotetraedron spHormotilopsis gelatinosa Klebsormidium flaccidiumPleurococcus sp and Tetracystis elliptica were unique to thissite Metals can be divided into those that are required byorganisms in small quantities such as Fe Cu and Zn whichare essential in some biochemical reactions (Raven et al 1999)andnon-essentialmetals such asAs Pb andHgwhichmaycausesevere harm to organisms even at very low concentrationsbecause they do not provide any known biochemical function(Monteiro et al 2012) However the toxic effects of metallicelements on microalgae are complex and differ markedly amongspecies depending on the element itself and the prevailingenvironmental conditions (Monteiro et al 2012) Stark andShubert (1979) reported a positive correlation between Mn NP silica (Si) Al zinc (Zn) and Pb concentrations and algalabundance and a negative correlation with Na Cd Culithium (Li) Mo and strontium (Sr) Cyanoprokaryotes havedeveloped efficient strategies for metal uptake andaccumulation (Shcolnick and Keren 2006) According toKeren et al (2002) cyanoprokaryotes are able to accumulatehigh concentrations of Mn in the envelope layers of their cellsWhether the highMn supports the growth of cyanoprokaryotes orreduces competition by other groups that are less tolerant of Mnwas unclear Soil pH plays a critical role in the bioavailability ofheavy metals (Rajakaruna and Boyd 2008) and althoughserpentine Site 7 had high metal concentrations the relativelyhigher pH at this site may have made those metals less

bioavailable (Neilson and Rajakaruna 2012) Hence it wasunclear whether the higher algal diversity at this site resultedfrom metal tolerance or reduced metal toxicity owing to lowermetal availabilityHowever algae are able togrow in thepresenceof heavy metals as a result of a variety of tolerance mechanismsfor example binding to cell wall precipitation in vacuole andsynthesis of heavy metal-binding compounds such as proteinsorganic acids and phenolic compounds (Metha and Gaur 2005)Serpentine soils are renowned for low Ca Mg ratio howeverDeGrood et al (2005) found that low Ca Mg ratio does notsignificantly explain variation in microbial community patternsalso our study did not show a strong correlation between algaldiversity and Ca Mg Microbes may therefore be respondingto increased soil organicmatter concentrations probably becauseof the associated increases in nutrient availability and water-holding capacity

Algae as a group are physiologically heterogeneous makingany generalisation about their soil relations difficult (OrsquoKelly1974) However favourable soil pH moisture conditions andnutrient content contribute to species diversity and communitycomposition (Shields andDurell 1964)Themultivariate analysesdid not suggest a distinct species association for serpentine sites(Sites 7 and8) Thiswas also true for themafic andultramafic sites(Sites 3 4 5 and 6) as well as the siliciclastic sites (Sites 1 and 2)The lack of a strong correlation with lithic type and a strongercorrelation with slope position suggests that the type of slopemay bemore important in influencing diversity Site pairs 1 and 23 and 4 as well as 5 and 6 were sampled at different slopepositions and at times shared similar species associationsbased on slope position (Fig 3)

Conclusions

It seems unlikely that soil chemistry alone was responsiblefor determining species diversity and no unique algal florafor serpentine soils was confirmed However the serpentinesoil at Kaapschehoop (Site 8) did have a unique speciesassemblage comprising Chroococcus sp Chlorotetraedronsp Hormotilopsis gelatinosa Klebsormidium flaccidiumPleurococcus sp and Tetracystis elliptica However thisrequires further investigation to determine whether it was alithic or climatic effect

Soil features alongwith other biotic (vegetation composition)and abiotic (slope exposure) habitat characteristics mayinfluence the presence and dominance of some algal andcyanoprokaryote species in harsh edaphic settings Our resultssuggested that topography (ie slope position) rather than thechemistry of the lithic class (ie rock type) wasmost important ininfluencing species diversity However high concentrations ofheavy metals also influenced species richness as well ascommunity composition

The characterisation of microbial communities using anisolation approach can be biased because this approach tendsto favour some groups of microbes over others Therefore in thefuture we plan to incorporate molecular approaches (Bjellandet al 2011 Daae et al 2013) to characterise the diversity ofcyanoprokaryotes and soil algae at our sites Additionally theecological heterogeneity within each site should be taken intoconsideration because our sampling strategy (ie pooling of soil

J Australian Journal of Botany A Venter et al

samples) did not allow for detecting species that may berestricted to distinct microhabitats found within our sites Acomposite sample does not allow for a correlation with bioticand abiotic factors but gives a general and preliminary view onthe diversity at each site Although our findings are preliminarythe study sets the stage for detailed investigations on the relativeimportance of edaphic versus other habitat features on algal andcyanoprokaryote diversity and community assembly

Acknowledgements

We thank the staff at Eko-Analitika North-West University Potchefstroomfor the soil analyses Christel Pretorius for drawing the map of the study areaand two anonymous reviewers for their useful suggestions to improve thequality of our manuscript

References

Alexander EB Coleman RG Keeler-Wolf T Harrison SP (2007)lsquoSerpentine geoecology of western North America geology soilsand vegetationrsquo (Oxford University Press New York)

ATSDR (Agency for Toxic Substances and Disease Registry) (2008)lsquoToxicological profile for aluminiumrsquo (US Department of Health andHuman Services Public Health Service Atlanta GA) Available at httpwwwatsdrcdcgovToxProfilestp22pdf [Verified 19 August 2014]

Belnap J (2003) The world at your feet desert biological soil crustsFrontiers in Ecology and the Environment 1 181ndash189doi1018901540-9295(2003)001[0181TWAYFD]20CO2

Belnap J Lange OL (2001) lsquoBiological soil crusts structure function andmanagementrsquo (Springer Berlin)

Bjelland T Grube M Hoem S Jorgensen SL Daae FL Thorseth IH OvrearingsL (2011) Microbial metacommunities in the lichen-rock habitatEnvironmental Microbiology Reports 3 434ndash442doi101111j1758-2229201000206x

Blank JG Green SJ Blake D Valley JW Kita NT Treiman A Dobson PF(2009) An alkaline spring system within the Del Puerto Ophiolite(California USA) a Mars analog site Planetary and Space Science57 533ndash540 doi101016jpss200811018

Bramwell D Caujape-Castells J (2011) lsquoThe biology of island florarsquo(Cambridge Press Cambridge UK)

Brown RM Larson DA Bold HC (1964) Airborne algae their abundanceand heterogeneity Science 143 583ndash585doi101126science1433606583

Cabala J Rahmonov O Jablonska M Teper E (2011) Soil algal colonizationand its ecological role in an environment polluted by past ZnndashPb miningand smelting activity Water Air and Soil Pollution 215 339ndash348doi101007s11270-010-0482-1

Cardace D Meyer-Dombard DR Olsen A Parenteau MN (2014) Bedrockand geochemical controls on extremophile habitats In lsquoPlant ecologyand evolution in harsh environmentsrsquo (Eds N Rajakaruna RS BoydTB Harris) pp 1ndash32 (Nova Science Publishers New York)

Chen YN Wang Q Li WH Ruan X (2007) Microbiotic crusts andthere interrelations with environmental factors in the Gurbantonggutdesert western China Environmental Geology 52 691ndash700doi101007s00254-006-0505-9

Clarke KR Gorley RN (2001) lsquoPRIMER v5 user manualtutorialrsquo(PRIMER-E Plymouth UK)

Clarke B Uken R Reinhardt J (2009) The geometry and emplacementmechanics of a Bushveld complex peridotite body South Africa SouthAfrican Journal of Geology 112 141ndash162 doi102113gssajg1122141

Couteacute A Tell G Therezien Y (1999) Aerophytic Cyanophyceae fromNew Caledonia Cryptogamie Algologie 20 301ndash344doi101016S0181-1568(00)88147-7

Daae FLOslashkland I DahleH Joslashrgensen SL Thorseth IH Pedersen RB (2013)Microbial life associated with low-temperature alteration of ultramafic

rocks in the Leka ophiolite complex Geobiology 11 318ndash339doi101111gbi12035

Daghino S Murat C Sizzano E Girlanda M Perotto S (2012) Fungaldiversity is not determined by mineral and chemical differences inserpentine substrates PLoS ONE 7(9) e44233doi101371journalpone0044233

DeGrood SH Claassen VP Scow KM (2005) Microbial communitycomposition on native and drastically disturbed serpentine soils SoilBiology amp Biochemistry 37 1427ndash1435doi101016jsoilbio200412013

Ettl H Gaumlrtner G Heynig H Mollenhauer D Komaacuterek J Anagnostidis K(1999) lsquoSuumlsswasser von Mittleeuropa Cyanoprokaryota Teil 1Chroococcalesrsquo (Gustav Fisher Verlag Stuttgart Germany)

Favero-Longo SE Isocrono D Piervittori R (2004) Lichens and ultramaficrocks a review Lichenologist (London England) 36 391ndash404doi101017S0024282904014215

Fernandez-Pintildeas F Mateo P Bonilla I (1995) Ultrastructural changesinduced by selected cadmium concentrations in the cyanobacteriumNostoc UAM208 Journal of Plant Physiology 147 452ndash456doi101016S0176-1617(11)82182-6

Hahs AK McDonnell MJ (2006) Selecting independent measures toquantify Melbournersquos urbanndashrural gradient Landscape and UrbanPlanning 78 435ndash448 doi101016jlandurbplan200512005

Harrison SP Rajakaruna N (2011) lsquoSerpentine evolution and ecology in amodel systemrsquo (University of California Press Berkeley CA)

Hauer T (2008) Epilithic cyanobacterial flora of Mohelenskaacute Hadcovaacutesteppe nature reseve (western Moravia Czech Republic) 70 years agoand now Fottea 8 129ndash132

Hindak F (2008) lsquoAtlas of cyanophytesrsquo (VEDA Bratislava Slovakia)Hofmann A Wilson A Kroumlner A Guy B Beukes N (2012) lsquoWorkshop

on craton formation and destruction post-conference excursionguidebook early craton formation stabilisation plume-relatedmagmatism and meteorite impact (Vredefort Witwatersrand Bushveldand Barberton)rsquo (Burlington House Piccadilly London) Availableat httpwwwgssaorgzaDocumentsCraton20Formation20and20Destruction20Excursion20Guidepdf [Verified 27November 2013]

Huumlber-Pestalozzi G (1961) lsquoDas Phytoplankton des Suumlsswassers SystematikundBiologie Tiel 5 Chlorophyceae (Gruumlnalgen) OrdnungVolvocalesrsquo(Verlagbuchhandlung Stuttgart Germany)

John DM Whitton BA Brook AJ (2002) lsquoFreshwater algal flora of theBritish Isles a guide to freshwater and terrestrial algaersquo (CambridgeUniversity Press Cambridge UK)

Keren N Kidd MJ Penner-Hahn JE Pakrasi HB (2002) A light-dependentmechanism formassive accumulation ofmanganese in the photosyntheticbacterium Synechocystis sp PCC 6803Biochemistry 41 15 085ndash15 092doi101021bi026892s

Komaacuterek J Anagnostidis K (2005) lsquoCyanoprokaryota Oscillatorialesrsquo(Spektrum Akademischer Verlag Heidelberg Germany)

Kruckeberg AR (2002) lsquoGeology and plant life the effects oflandforms and rock type on plantsrsquo (University Washington PressSeattle WA)

Kruumlger GHJ (1978) lsquoThe effect of physico-chemical factors on growthrelevant to the mass culture of Microcystis under sterile conditionsrsquo(University of the Orange Free State Bloemfontein South Africa)

Langhans TM Storm C Schwabe A (2009) Community assembly ofbiological soil crusts of different successional stages in a temperatesand ecosystem as assessed by direct determination and enrichmenttechniques Microbial Ecology 58 394ndash407doi101007s00248-009-9532-x

Lukesova A (2001) Soil algae in brown coal lignite post-mining areas incentral Europe (Czech Republic and Germany) Restoration Ecology 9341ndash350 doi101046j1526-100X200194002x

Lund JWG (1945) Observations on soil algae I The ecology size andtaxonomy of British soil diatoms Part 1 New Phytologist 44 196ndash219doi101111j1469-81371945tb05033x

Soil algae and cyanoprokaryotes Australian Journal of Botany K

an increase in soil moisture as a result of runoff and theaccumulation of organic matter (Chen et al 2007) could haveplayed a role in the assembly of certain species on foot slopes Thecyanoprokaryotes Leptolyngbya foveolarum and Phormidiumambiguum were also present at the foot slope of the Mg-richpyroxenite soil (Site 3) whereas Microcoleus vaginatusChlamydomonas Chlorococcum and Eustigmatos magnus didnot occur at this site The absence of these algae as well as the lowdiversity observed at Site 3 could be the result of the highconcentrations of Cd (001mg kgndash1) and Hg (00046mg kgndash1)measured at this site According to Fernandez-Pintildeas et al (1995)Cd induces progressive disorganisation and degradation ofthylakoid membranes as well as a decrease in the mobilisationof the polyphosphate granules leading to phosphorus starvationPinto et al (2003) showed that Cd andHg inhibit photosynthesisHowever further experimentation is clearly needed to determinewhether Cd and Hg were responsible for the reduced diversityobserved at this site

Most of the cyanoprokaryotes were represented by filamentousspecies such as Leptolyngbya foveolarumMicrocoleus vaginatusand Phormidium ambiquum and were found at all eight samplingsites These filamentous cyanoprokaryotes can wind throughoutthe uppermost soil layers forming a net-like structure that bindstogether soil particles (Rosentreter et al 2007) This formssoil aggregates that create pathways for water infiltration andsurfaces for nutrient transformations while also increasing thesoil resistance to wind and water erosion Once the filamentouscyanoprokaryotes stabilise thesoil single-celledcyanoprokaryotessuch as Synechocystis sp are able to colonise the substrate(Rosentreter et al 2007) The Chlorophyceae representedby mainly coccoid species such as Chlamydomonas andChlorococcum were found on all the sites except at Site 3Chlorococcum echinozygotum and Chlamydomonas macrostellawere subdominant on the siliciclastic-derived soils of Sites 1 and 2respectively Bacillariophyceae such as Hantzschia amphioxyswas found on all the sites and was subdominant at Sites 2 4 and 5

Legend Δ species and sites rarr environmental variables

Axis

ndash06

ndash10 15

10

1 2 3 4

Eigen values 052 0411 0388 0368

Cumulative percentage variance

Of species 202 362 513 656

Of environmental relations 202 362 513 656

Test of significance of first canonical axes p = 10

Test of significance of all canonical axes p = 10

Fig 3 Canonical correspondence analysis (CCA) of metal concentrations and algal and cyanoprokaryote species of the different sampling sitesAbbreviations of species are given in Table 3

Soil algae and cyanoprokaryotes Australian Journal of Botany I

and dominant at Site 3 According to Metting (1981)Leptolyngbya Microcoleus Phormidium ChlamydomonasChlorococcum and Hantzschia species are all typicalcosmopolitan soil algae

Shields and Durell (1964) and Starks and Shubert (1982)suggested that the species composition of soil algal populationsis affected less by the chemical nature of the substrate than bycertain physical properties that influence soil moisture levelsDuring February 2012 when sampling took place the study areahad received between 50 and 200mm rain which accounts formore than 75 of the expected average summer rainfall for thearea (South African Weather Service 2012) Moisture wastherefore not a limiting factor and the chemical nature of thesoils along with other abiotic habitat features appear to haveinfluenced species richness and composition In addition tothe abiotic conditions such as edaphic features biotic factorsespecially the type of vegetation can influence diversity andcommunity composition of cryptogamic biota For exampleRosentreter et al (2007) documented that vagrant populationsofDermatocarponoccur on poorly drained basaltflats dominatedby Artemisia rigida A papposa Antennaria flagellaris andPoa sandbergii The ecological conditions found at such sitescan support completely or partially vagrant life forms inDermatocarpon and other lichen genera The differences invegetation cover of the plant communities at the sampling sites(Sites 1ndash8) could have created microenvironments that favourspecific algal and cyanoprokaryote species assemblages Sites1ndash7 had moderate cover whereas Site 8 was an open savannacharacterised by numerous bare soil patches (serpentine effect)

Twenty different species were identified at Site 8 (serpentine)despite this site showing elevated concentrations of heavymetalsincluding Co Cr Fe Mn and Ni (Table 2) Chroococcus spScytonema ocellatum Nostoc linckia Chlorotetraedron spHormotilopsis gelatinosa Klebsormidium flaccidiumPleurococcus sp and Tetracystis elliptica were unique to thissite Metals can be divided into those that are required byorganisms in small quantities such as Fe Cu and Zn whichare essential in some biochemical reactions (Raven et al 1999)andnon-essentialmetals such asAs Pb andHgwhichmaycausesevere harm to organisms even at very low concentrationsbecause they do not provide any known biochemical function(Monteiro et al 2012) However the toxic effects of metallicelements on microalgae are complex and differ markedly amongspecies depending on the element itself and the prevailingenvironmental conditions (Monteiro et al 2012) Stark andShubert (1979) reported a positive correlation between Mn NP silica (Si) Al zinc (Zn) and Pb concentrations and algalabundance and a negative correlation with Na Cd Culithium (Li) Mo and strontium (Sr) Cyanoprokaryotes havedeveloped efficient strategies for metal uptake andaccumulation (Shcolnick and Keren 2006) According toKeren et al (2002) cyanoprokaryotes are able to accumulatehigh concentrations of Mn in the envelope layers of their cellsWhether the highMn supports the growth of cyanoprokaryotes orreduces competition by other groups that are less tolerant of Mnwas unclear Soil pH plays a critical role in the bioavailability ofheavy metals (Rajakaruna and Boyd 2008) and althoughserpentine Site 7 had high metal concentrations the relativelyhigher pH at this site may have made those metals less

bioavailable (Neilson and Rajakaruna 2012) Hence it wasunclear whether the higher algal diversity at this site resultedfrom metal tolerance or reduced metal toxicity owing to lowermetal availabilityHowever algae are able togrow in thepresenceof heavy metals as a result of a variety of tolerance mechanismsfor example binding to cell wall precipitation in vacuole andsynthesis of heavy metal-binding compounds such as proteinsorganic acids and phenolic compounds (Metha and Gaur 2005)Serpentine soils are renowned for low Ca Mg ratio howeverDeGrood et al (2005) found that low Ca Mg ratio does notsignificantly explain variation in microbial community patternsalso our study did not show a strong correlation between algaldiversity and Ca Mg Microbes may therefore be respondingto increased soil organicmatter concentrations probably becauseof the associated increases in nutrient availability and water-holding capacity

Algae as a group are physiologically heterogeneous makingany generalisation about their soil relations difficult (OrsquoKelly1974) However favourable soil pH moisture conditions andnutrient content contribute to species diversity and communitycomposition (Shields andDurell 1964)Themultivariate analysesdid not suggest a distinct species association for serpentine sites(Sites 7 and8) Thiswas also true for themafic andultramafic sites(Sites 3 4 5 and 6) as well as the siliciclastic sites (Sites 1 and 2)The lack of a strong correlation with lithic type and a strongercorrelation with slope position suggests that the type of slopemay bemore important in influencing diversity Site pairs 1 and 23 and 4 as well as 5 and 6 were sampled at different slopepositions and at times shared similar species associationsbased on slope position (Fig 3)

Conclusions

It seems unlikely that soil chemistry alone was responsiblefor determining species diversity and no unique algal florafor serpentine soils was confirmed However the serpentinesoil at Kaapschehoop (Site 8) did have a unique speciesassemblage comprising Chroococcus sp Chlorotetraedronsp Hormotilopsis gelatinosa Klebsormidium flaccidiumPleurococcus sp and Tetracystis elliptica However thisrequires further investigation to determine whether it was alithic or climatic effect

Soil features alongwith other biotic (vegetation composition)and abiotic (slope exposure) habitat characteristics mayinfluence the presence and dominance of some algal andcyanoprokaryote species in harsh edaphic settings Our resultssuggested that topography (ie slope position) rather than thechemistry of the lithic class (ie rock type) wasmost important ininfluencing species diversity However high concentrations ofheavy metals also influenced species richness as well ascommunity composition

The characterisation of microbial communities using anisolation approach can be biased because this approach tendsto favour some groups of microbes over others Therefore in thefuture we plan to incorporate molecular approaches (Bjellandet al 2011 Daae et al 2013) to characterise the diversity ofcyanoprokaryotes and soil algae at our sites Additionally theecological heterogeneity within each site should be taken intoconsideration because our sampling strategy (ie pooling of soil

J Australian Journal of Botany A Venter et al

samples) did not allow for detecting species that may berestricted to distinct microhabitats found within our sites Acomposite sample does not allow for a correlation with bioticand abiotic factors but gives a general and preliminary view onthe diversity at each site Although our findings are preliminarythe study sets the stage for detailed investigations on the relativeimportance of edaphic versus other habitat features on algal andcyanoprokaryote diversity and community assembly

Acknowledgements

We thank the staff at Eko-Analitika North-West University Potchefstroomfor the soil analyses Christel Pretorius for drawing the map of the study areaand two anonymous reviewers for their useful suggestions to improve thequality of our manuscript

References

Alexander EB Coleman RG Keeler-Wolf T Harrison SP (2007)lsquoSerpentine geoecology of western North America geology soilsand vegetationrsquo (Oxford University Press New York)

ATSDR (Agency for Toxic Substances and Disease Registry) (2008)lsquoToxicological profile for aluminiumrsquo (US Department of Health andHuman Services Public Health Service Atlanta GA) Available at httpwwwatsdrcdcgovToxProfilestp22pdf [Verified 19 August 2014]

Belnap J (2003) The world at your feet desert biological soil crustsFrontiers in Ecology and the Environment 1 181ndash189doi1018901540-9295(2003)001[0181TWAYFD]20CO2

Belnap J Lange OL (2001) lsquoBiological soil crusts structure function andmanagementrsquo (Springer Berlin)

Bjelland T Grube M Hoem S Jorgensen SL Daae FL Thorseth IH OvrearingsL (2011) Microbial metacommunities in the lichen-rock habitatEnvironmental Microbiology Reports 3 434ndash442doi101111j1758-2229201000206x

Blank JG Green SJ Blake D Valley JW Kita NT Treiman A Dobson PF(2009) An alkaline spring system within the Del Puerto Ophiolite(California USA) a Mars analog site Planetary and Space Science57 533ndash540 doi101016jpss200811018

Bramwell D Caujape-Castells J (2011) lsquoThe biology of island florarsquo(Cambridge Press Cambridge UK)

Brown RM Larson DA Bold HC (1964) Airborne algae their abundanceand heterogeneity Science 143 583ndash585doi101126science1433606583

Cabala J Rahmonov O Jablonska M Teper E (2011) Soil algal colonizationand its ecological role in an environment polluted by past ZnndashPb miningand smelting activity Water Air and Soil Pollution 215 339ndash348doi101007s11270-010-0482-1

Cardace D Meyer-Dombard DR Olsen A Parenteau MN (2014) Bedrockand geochemical controls on extremophile habitats In lsquoPlant ecologyand evolution in harsh environmentsrsquo (Eds N Rajakaruna RS BoydTB Harris) pp 1ndash32 (Nova Science Publishers New York)

Chen YN Wang Q Li WH Ruan X (2007) Microbiotic crusts andthere interrelations with environmental factors in the Gurbantonggutdesert western China Environmental Geology 52 691ndash700doi101007s00254-006-0505-9

Clarke KR Gorley RN (2001) lsquoPRIMER v5 user manualtutorialrsquo(PRIMER-E Plymouth UK)

Clarke B Uken R Reinhardt J (2009) The geometry and emplacementmechanics of a Bushveld complex peridotite body South Africa SouthAfrican Journal of Geology 112 141ndash162 doi102113gssajg1122141

Couteacute A Tell G Therezien Y (1999) Aerophytic Cyanophyceae fromNew Caledonia Cryptogamie Algologie 20 301ndash344doi101016S0181-1568(00)88147-7

Daae FLOslashkland I DahleH Joslashrgensen SL Thorseth IH Pedersen RB (2013)Microbial life associated with low-temperature alteration of ultramafic

rocks in the Leka ophiolite complex Geobiology 11 318ndash339doi101111gbi12035

Daghino S Murat C Sizzano E Girlanda M Perotto S (2012) Fungaldiversity is not determined by mineral and chemical differences inserpentine substrates PLoS ONE 7(9) e44233doi101371journalpone0044233

DeGrood SH Claassen VP Scow KM (2005) Microbial communitycomposition on native and drastically disturbed serpentine soils SoilBiology amp Biochemistry 37 1427ndash1435doi101016jsoilbio200412013

Ettl H Gaumlrtner G Heynig H Mollenhauer D Komaacuterek J Anagnostidis K(1999) lsquoSuumlsswasser von Mittleeuropa Cyanoprokaryota Teil 1Chroococcalesrsquo (Gustav Fisher Verlag Stuttgart Germany)

Favero-Longo SE Isocrono D Piervittori R (2004) Lichens and ultramaficrocks a review Lichenologist (London England) 36 391ndash404doi101017S0024282904014215

Fernandez-Pintildeas F Mateo P Bonilla I (1995) Ultrastructural changesinduced by selected cadmium concentrations in the cyanobacteriumNostoc UAM208 Journal of Plant Physiology 147 452ndash456doi101016S0176-1617(11)82182-6

Hahs AK McDonnell MJ (2006) Selecting independent measures toquantify Melbournersquos urbanndashrural gradient Landscape and UrbanPlanning 78 435ndash448 doi101016jlandurbplan200512005

Harrison SP Rajakaruna N (2011) lsquoSerpentine evolution and ecology in amodel systemrsquo (University of California Press Berkeley CA)

Hauer T (2008) Epilithic cyanobacterial flora of Mohelenskaacute Hadcovaacutesteppe nature reseve (western Moravia Czech Republic) 70 years agoand now Fottea 8 129ndash132

Hindak F (2008) lsquoAtlas of cyanophytesrsquo (VEDA Bratislava Slovakia)Hofmann A Wilson A Kroumlner A Guy B Beukes N (2012) lsquoWorkshop

on craton formation and destruction post-conference excursionguidebook early craton formation stabilisation plume-relatedmagmatism and meteorite impact (Vredefort Witwatersrand Bushveldand Barberton)rsquo (Burlington House Piccadilly London) Availableat httpwwwgssaorgzaDocumentsCraton20Formation20and20Destruction20Excursion20Guidepdf [Verified 27November 2013]

Huumlber-Pestalozzi G (1961) lsquoDas Phytoplankton des Suumlsswassers SystematikundBiologie Tiel 5 Chlorophyceae (Gruumlnalgen) OrdnungVolvocalesrsquo(Verlagbuchhandlung Stuttgart Germany)

John DM Whitton BA Brook AJ (2002) lsquoFreshwater algal flora of theBritish Isles a guide to freshwater and terrestrial algaersquo (CambridgeUniversity Press Cambridge UK)

Keren N Kidd MJ Penner-Hahn JE Pakrasi HB (2002) A light-dependentmechanism formassive accumulation ofmanganese in the photosyntheticbacterium Synechocystis sp PCC 6803Biochemistry 41 15 085ndash15 092doi101021bi026892s

Komaacuterek J Anagnostidis K (2005) lsquoCyanoprokaryota Oscillatorialesrsquo(Spektrum Akademischer Verlag Heidelberg Germany)

Kruckeberg AR (2002) lsquoGeology and plant life the effects oflandforms and rock type on plantsrsquo (University Washington PressSeattle WA)

Kruumlger GHJ (1978) lsquoThe effect of physico-chemical factors on growthrelevant to the mass culture of Microcystis under sterile conditionsrsquo(University of the Orange Free State Bloemfontein South Africa)

Langhans TM Storm C Schwabe A (2009) Community assembly ofbiological soil crusts of different successional stages in a temperatesand ecosystem as assessed by direct determination and enrichmenttechniques Microbial Ecology 58 394ndash407doi101007s00248-009-9532-x

Lukesova A (2001) Soil algae in brown coal lignite post-mining areas incentral Europe (Czech Republic and Germany) Restoration Ecology 9341ndash350 doi101046j1526-100X200194002x

Lund JWG (1945) Observations on soil algae I The ecology size andtaxonomy of British soil diatoms Part 1 New Phytologist 44 196ndash219doi101111j1469-81371945tb05033x

Soil algae and cyanoprokaryotes Australian Journal of Botany K

and dominant at Site 3 According to Metting (1981)Leptolyngbya Microcoleus Phormidium ChlamydomonasChlorococcum and Hantzschia species are all typicalcosmopolitan soil algae

Shields and Durell (1964) and Starks and Shubert (1982)suggested that the species composition of soil algal populationsis affected less by the chemical nature of the substrate than bycertain physical properties that influence soil moisture levelsDuring February 2012 when sampling took place the study areahad received between 50 and 200mm rain which accounts formore than 75 of the expected average summer rainfall for thearea (South African Weather Service 2012) Moisture wastherefore not a limiting factor and the chemical nature of thesoils along with other abiotic habitat features appear to haveinfluenced species richness and composition In addition tothe abiotic conditions such as edaphic features biotic factorsespecially the type of vegetation can influence diversity andcommunity composition of cryptogamic biota For exampleRosentreter et al (2007) documented that vagrant populationsofDermatocarponoccur on poorly drained basaltflats dominatedby Artemisia rigida A papposa Antennaria flagellaris andPoa sandbergii The ecological conditions found at such sitescan support completely or partially vagrant life forms inDermatocarpon and other lichen genera The differences invegetation cover of the plant communities at the sampling sites(Sites 1ndash8) could have created microenvironments that favourspecific algal and cyanoprokaryote species assemblages Sites1ndash7 had moderate cover whereas Site 8 was an open savannacharacterised by numerous bare soil patches (serpentine effect)

Twenty different species were identified at Site 8 (serpentine)despite this site showing elevated concentrations of heavymetalsincluding Co Cr Fe Mn and Ni (Table 2) Chroococcus spScytonema ocellatum Nostoc linckia Chlorotetraedron spHormotilopsis gelatinosa Klebsormidium flaccidiumPleurococcus sp and Tetracystis elliptica were unique to thissite Metals can be divided into those that are required byorganisms in small quantities such as Fe Cu and Zn whichare essential in some biochemical reactions (Raven et al 1999)andnon-essentialmetals such asAs Pb andHgwhichmaycausesevere harm to organisms even at very low concentrationsbecause they do not provide any known biochemical function(Monteiro et al 2012) However the toxic effects of metallicelements on microalgae are complex and differ markedly amongspecies depending on the element itself and the prevailingenvironmental conditions (Monteiro et al 2012) Stark andShubert (1979) reported a positive correlation between Mn NP silica (Si) Al zinc (Zn) and Pb concentrations and algalabundance and a negative correlation with Na Cd Culithium (Li) Mo and strontium (Sr) Cyanoprokaryotes havedeveloped efficient strategies for metal uptake andaccumulation (Shcolnick and Keren 2006) According toKeren et al (2002) cyanoprokaryotes are able to accumulatehigh concentrations of Mn in the envelope layers of their cellsWhether the highMn supports the growth of cyanoprokaryotes orreduces competition by other groups that are less tolerant of Mnwas unclear Soil pH plays a critical role in the bioavailability ofheavy metals (Rajakaruna and Boyd 2008) and althoughserpentine Site 7 had high metal concentrations the relativelyhigher pH at this site may have made those metals less

bioavailable (Neilson and Rajakaruna 2012) Hence it wasunclear whether the higher algal diversity at this site resultedfrom metal tolerance or reduced metal toxicity owing to lowermetal availabilityHowever algae are able togrow in thepresenceof heavy metals as a result of a variety of tolerance mechanismsfor example binding to cell wall precipitation in vacuole andsynthesis of heavy metal-binding compounds such as proteinsorganic acids and phenolic compounds (Metha and Gaur 2005)Serpentine soils are renowned for low Ca Mg ratio howeverDeGrood et al (2005) found that low Ca Mg ratio does notsignificantly explain variation in microbial community patternsalso our study did not show a strong correlation between algaldiversity and Ca Mg Microbes may therefore be respondingto increased soil organicmatter concentrations probably becauseof the associated increases in nutrient availability and water-holding capacity

Algae as a group are physiologically heterogeneous makingany generalisation about their soil relations difficult (OrsquoKelly1974) However favourable soil pH moisture conditions andnutrient content contribute to species diversity and communitycomposition (Shields andDurell 1964)Themultivariate analysesdid not suggest a distinct species association for serpentine sites(Sites 7 and8) Thiswas also true for themafic andultramafic sites(Sites 3 4 5 and 6) as well as the siliciclastic sites (Sites 1 and 2)The lack of a strong correlation with lithic type and a strongercorrelation with slope position suggests that the type of slopemay bemore important in influencing diversity Site pairs 1 and 23 and 4 as well as 5 and 6 were sampled at different slopepositions and at times shared similar species associationsbased on slope position (Fig 3)

Conclusions

It seems unlikely that soil chemistry alone was responsiblefor determining species diversity and no unique algal florafor serpentine soils was confirmed However the serpentinesoil at Kaapschehoop (Site 8) did have a unique speciesassemblage comprising Chroococcus sp Chlorotetraedronsp Hormotilopsis gelatinosa Klebsormidium flaccidiumPleurococcus sp and Tetracystis elliptica However thisrequires further investigation to determine whether it was alithic or climatic effect

Soil features alongwith other biotic (vegetation composition)and abiotic (slope exposure) habitat characteristics mayinfluence the presence and dominance of some algal andcyanoprokaryote species in harsh edaphic settings Our resultssuggested that topography (ie slope position) rather than thechemistry of the lithic class (ie rock type) wasmost important ininfluencing species diversity However high concentrations ofheavy metals also influenced species richness as well ascommunity composition

The characterisation of microbial communities using anisolation approach can be biased because this approach tendsto favour some groups of microbes over others Therefore in thefuture we plan to incorporate molecular approaches (Bjellandet al 2011 Daae et al 2013) to characterise the diversity ofcyanoprokaryotes and soil algae at our sites Additionally theecological heterogeneity within each site should be taken intoconsideration because our sampling strategy (ie pooling of soil

J Australian Journal of Botany A Venter et al

samples) did not allow for detecting species that may berestricted to distinct microhabitats found within our sites Acomposite sample does not allow for a correlation with bioticand abiotic factors but gives a general and preliminary view onthe diversity at each site Although our findings are preliminarythe study sets the stage for detailed investigations on the relativeimportance of edaphic versus other habitat features on algal andcyanoprokaryote diversity and community assembly

Acknowledgements

We thank the staff at Eko-Analitika North-West University Potchefstroomfor the soil analyses Christel Pretorius for drawing the map of the study areaand two anonymous reviewers for their useful suggestions to improve thequality of our manuscript

References

Alexander EB Coleman RG Keeler-Wolf T Harrison SP (2007)lsquoSerpentine geoecology of western North America geology soilsand vegetationrsquo (Oxford University Press New York)

ATSDR (Agency for Toxic Substances and Disease Registry) (2008)lsquoToxicological profile for aluminiumrsquo (US Department of Health andHuman Services Public Health Service Atlanta GA) Available at httpwwwatsdrcdcgovToxProfilestp22pdf [Verified 19 August 2014]

Belnap J (2003) The world at your feet desert biological soil crustsFrontiers in Ecology and the Environment 1 181ndash189doi1018901540-9295(2003)001[0181TWAYFD]20CO2

Belnap J Lange OL (2001) lsquoBiological soil crusts structure function andmanagementrsquo (Springer Berlin)

Bjelland T Grube M Hoem S Jorgensen SL Daae FL Thorseth IH OvrearingsL (2011) Microbial metacommunities in the lichen-rock habitatEnvironmental Microbiology Reports 3 434ndash442doi101111j1758-2229201000206x

Blank JG Green SJ Blake D Valley JW Kita NT Treiman A Dobson PF(2009) An alkaline spring system within the Del Puerto Ophiolite(California USA) a Mars analog site Planetary and Space Science57 533ndash540 doi101016jpss200811018

Bramwell D Caujape-Castells J (2011) lsquoThe biology of island florarsquo(Cambridge Press Cambridge UK)

Brown RM Larson DA Bold HC (1964) Airborne algae their abundanceand heterogeneity Science 143 583ndash585doi101126science1433606583

Cabala J Rahmonov O Jablonska M Teper E (2011) Soil algal colonizationand its ecological role in an environment polluted by past ZnndashPb miningand smelting activity Water Air and Soil Pollution 215 339ndash348doi101007s11270-010-0482-1

Cardace D Meyer-Dombard DR Olsen A Parenteau MN (2014) Bedrockand geochemical controls on extremophile habitats In lsquoPlant ecologyand evolution in harsh environmentsrsquo (Eds N Rajakaruna RS BoydTB Harris) pp 1ndash32 (Nova Science Publishers New York)

Chen YN Wang Q Li WH Ruan X (2007) Microbiotic crusts andthere interrelations with environmental factors in the Gurbantonggutdesert western China Environmental Geology 52 691ndash700doi101007s00254-006-0505-9

Clarke KR Gorley RN (2001) lsquoPRIMER v5 user manualtutorialrsquo(PRIMER-E Plymouth UK)

Clarke B Uken R Reinhardt J (2009) The geometry and emplacementmechanics of a Bushveld complex peridotite body South Africa SouthAfrican Journal of Geology 112 141ndash162 doi102113gssajg1122141

Couteacute A Tell G Therezien Y (1999) Aerophytic Cyanophyceae fromNew Caledonia Cryptogamie Algologie 20 301ndash344doi101016S0181-1568(00)88147-7

Daae FLOslashkland I DahleH Joslashrgensen SL Thorseth IH Pedersen RB (2013)Microbial life associated with low-temperature alteration of ultramafic

rocks in the Leka ophiolite complex Geobiology 11 318ndash339doi101111gbi12035

Daghino S Murat C Sizzano E Girlanda M Perotto S (2012) Fungaldiversity is not determined by mineral and chemical differences inserpentine substrates PLoS ONE 7(9) e44233doi101371journalpone0044233

DeGrood SH Claassen VP Scow KM (2005) Microbial communitycomposition on native and drastically disturbed serpentine soils SoilBiology amp Biochemistry 37 1427ndash1435doi101016jsoilbio200412013

Ettl H Gaumlrtner G Heynig H Mollenhauer D Komaacuterek J Anagnostidis K(1999) lsquoSuumlsswasser von Mittleeuropa Cyanoprokaryota Teil 1Chroococcalesrsquo (Gustav Fisher Verlag Stuttgart Germany)

Favero-Longo SE Isocrono D Piervittori R (2004) Lichens and ultramaficrocks a review Lichenologist (London England) 36 391ndash404doi101017S0024282904014215

Fernandez-Pintildeas F Mateo P Bonilla I (1995) Ultrastructural changesinduced by selected cadmium concentrations in the cyanobacteriumNostoc UAM208 Journal of Plant Physiology 147 452ndash456doi101016S0176-1617(11)82182-6

Hahs AK McDonnell MJ (2006) Selecting independent measures toquantify Melbournersquos urbanndashrural gradient Landscape and UrbanPlanning 78 435ndash448 doi101016jlandurbplan200512005

Harrison SP Rajakaruna N (2011) lsquoSerpentine evolution and ecology in amodel systemrsquo (University of California Press Berkeley CA)

Hauer T (2008) Epilithic cyanobacterial flora of Mohelenskaacute Hadcovaacutesteppe nature reseve (western Moravia Czech Republic) 70 years agoand now Fottea 8 129ndash132

Hindak F (2008) lsquoAtlas of cyanophytesrsquo (VEDA Bratislava Slovakia)Hofmann A Wilson A Kroumlner A Guy B Beukes N (2012) lsquoWorkshop

on craton formation and destruction post-conference excursionguidebook early craton formation stabilisation plume-relatedmagmatism and meteorite impact (Vredefort Witwatersrand Bushveldand Barberton)rsquo (Burlington House Piccadilly London) Availableat httpwwwgssaorgzaDocumentsCraton20Formation20and20Destruction20Excursion20Guidepdf [Verified 27November 2013]

Huumlber-Pestalozzi G (1961) lsquoDas Phytoplankton des Suumlsswassers SystematikundBiologie Tiel 5 Chlorophyceae (Gruumlnalgen) OrdnungVolvocalesrsquo(Verlagbuchhandlung Stuttgart Germany)

John DM Whitton BA Brook AJ (2002) lsquoFreshwater algal flora of theBritish Isles a guide to freshwater and terrestrial algaersquo (CambridgeUniversity Press Cambridge UK)

Keren N Kidd MJ Penner-Hahn JE Pakrasi HB (2002) A light-dependentmechanism formassive accumulation ofmanganese in the photosyntheticbacterium Synechocystis sp PCC 6803Biochemistry 41 15 085ndash15 092doi101021bi026892s

Komaacuterek J Anagnostidis K (2005) lsquoCyanoprokaryota Oscillatorialesrsquo(Spektrum Akademischer Verlag Heidelberg Germany)

Kruckeberg AR (2002) lsquoGeology and plant life the effects oflandforms and rock type on plantsrsquo (University Washington PressSeattle WA)

Kruumlger GHJ (1978) lsquoThe effect of physico-chemical factors on growthrelevant to the mass culture of Microcystis under sterile conditionsrsquo(University of the Orange Free State Bloemfontein South Africa)

Langhans TM Storm C Schwabe A (2009) Community assembly ofbiological soil crusts of different successional stages in a temperatesand ecosystem as assessed by direct determination and enrichmenttechniques Microbial Ecology 58 394ndash407doi101007s00248-009-9532-x

Lukesova A (2001) Soil algae in brown coal lignite post-mining areas incentral Europe (Czech Republic and Germany) Restoration Ecology 9341ndash350 doi101046j1526-100X200194002x

Lund JWG (1945) Observations on soil algae I The ecology size andtaxonomy of British soil diatoms Part 1 New Phytologist 44 196ndash219doi101111j1469-81371945tb05033x

Soil algae and cyanoprokaryotes Australian Journal of Botany K

samples) did not allow for detecting species that may berestricted to distinct microhabitats found within our sites Acomposite sample does not allow for a correlation with bioticand abiotic factors but gives a general and preliminary view onthe diversity at each site Although our findings are preliminarythe study sets the stage for detailed investigations on the relativeimportance of edaphic versus other habitat features on algal andcyanoprokaryote diversity and community assembly

Acknowledgements

We thank the staff at Eko-Analitika North-West University Potchefstroomfor the soil analyses Christel Pretorius for drawing the map of the study areaand two anonymous reviewers for their useful suggestions to improve thequality of our manuscript

References

Alexander EB Coleman RG Keeler-Wolf T Harrison SP (2007)lsquoSerpentine geoecology of western North America geology soilsand vegetationrsquo (Oxford University Press New York)

ATSDR (Agency for Toxic Substances and Disease Registry) (2008)lsquoToxicological profile for aluminiumrsquo (US Department of Health andHuman Services Public Health Service Atlanta GA) Available at httpwwwatsdrcdcgovToxProfilestp22pdf [Verified 19 August 2014]

Belnap J (2003) The world at your feet desert biological soil crustsFrontiers in Ecology and the Environment 1 181ndash189doi1018901540-9295(2003)001[0181TWAYFD]20CO2

Belnap J Lange OL (2001) lsquoBiological soil crusts structure function andmanagementrsquo (Springer Berlin)

Bjelland T Grube M Hoem S Jorgensen SL Daae FL Thorseth IH OvrearingsL (2011) Microbial metacommunities in the lichen-rock habitatEnvironmental Microbiology Reports 3 434ndash442doi101111j1758-2229201000206x

Blank JG Green SJ Blake D Valley JW Kita NT Treiman A Dobson PF(2009) An alkaline spring system within the Del Puerto Ophiolite(California USA) a Mars analog site Planetary and Space Science57 533ndash540 doi101016jpss200811018

Bramwell D Caujape-Castells J (2011) lsquoThe biology of island florarsquo(Cambridge Press Cambridge UK)

Brown RM Larson DA Bold HC (1964) Airborne algae their abundanceand heterogeneity Science 143 583ndash585doi101126science1433606583

Cabala J Rahmonov O Jablonska M Teper E (2011) Soil algal colonizationand its ecological role in an environment polluted by past ZnndashPb miningand smelting activity Water Air and Soil Pollution 215 339ndash348doi101007s11270-010-0482-1

Cardace D Meyer-Dombard DR Olsen A Parenteau MN (2014) Bedrockand geochemical controls on extremophile habitats In lsquoPlant ecologyand evolution in harsh environmentsrsquo (Eds N Rajakaruna RS BoydTB Harris) pp 1ndash32 (Nova Science Publishers New York)

Chen YN Wang Q Li WH Ruan X (2007) Microbiotic crusts andthere interrelations with environmental factors in the Gurbantonggutdesert western China Environmental Geology 52 691ndash700doi101007s00254-006-0505-9

Clarke KR Gorley RN (2001) lsquoPRIMER v5 user manualtutorialrsquo(PRIMER-E Plymouth UK)

Clarke B Uken R Reinhardt J (2009) The geometry and emplacementmechanics of a Bushveld complex peridotite body South Africa SouthAfrican Journal of Geology 112 141ndash162 doi102113gssajg1122141

Couteacute A Tell G Therezien Y (1999) Aerophytic Cyanophyceae fromNew Caledonia Cryptogamie Algologie 20 301ndash344doi101016S0181-1568(00)88147-7

Daae FLOslashkland I DahleH Joslashrgensen SL Thorseth IH Pedersen RB (2013)Microbial life associated with low-temperature alteration of ultramafic

rocks in the Leka ophiolite complex Geobiology 11 318ndash339doi101111gbi12035

Daghino S Murat C Sizzano E Girlanda M Perotto S (2012) Fungaldiversity is not determined by mineral and chemical differences inserpentine substrates PLoS ONE 7(9) e44233doi101371journalpone0044233

DeGrood SH Claassen VP Scow KM (2005) Microbial communitycomposition on native and drastically disturbed serpentine soils SoilBiology amp Biochemistry 37 1427ndash1435doi101016jsoilbio200412013

Ettl H Gaumlrtner G Heynig H Mollenhauer D Komaacuterek J Anagnostidis K(1999) lsquoSuumlsswasser von Mittleeuropa Cyanoprokaryota Teil 1Chroococcalesrsquo (Gustav Fisher Verlag Stuttgart Germany)

Favero-Longo SE Isocrono D Piervittori R (2004) Lichens and ultramaficrocks a review Lichenologist (London England) 36 391ndash404doi101017S0024282904014215

Fernandez-Pintildeas F Mateo P Bonilla I (1995) Ultrastructural changesinduced by selected cadmium concentrations in the cyanobacteriumNostoc UAM208 Journal of Plant Physiology 147 452ndash456doi101016S0176-1617(11)82182-6

Hahs AK McDonnell MJ (2006) Selecting independent measures toquantify Melbournersquos urbanndashrural gradient Landscape and UrbanPlanning 78 435ndash448 doi101016jlandurbplan200512005

Harrison SP Rajakaruna N (2011) lsquoSerpentine evolution and ecology in amodel systemrsquo (University of California Press Berkeley CA)

Hauer T (2008) Epilithic cyanobacterial flora of Mohelenskaacute Hadcovaacutesteppe nature reseve (western Moravia Czech Republic) 70 years agoand now Fottea 8 129ndash132

Hindak F (2008) lsquoAtlas of cyanophytesrsquo (VEDA Bratislava Slovakia)Hofmann A Wilson A Kroumlner A Guy B Beukes N (2012) lsquoWorkshop

on craton formation and destruction post-conference excursionguidebook early craton formation stabilisation plume-relatedmagmatism and meteorite impact (Vredefort Witwatersrand Bushveldand Barberton)rsquo (Burlington House Piccadilly London) Availableat httpwwwgssaorgzaDocumentsCraton20Formation20and20Destruction20Excursion20Guidepdf [Verified 27November 2013]

Huumlber-Pestalozzi G (1961) lsquoDas Phytoplankton des Suumlsswassers SystematikundBiologie Tiel 5 Chlorophyceae (Gruumlnalgen) OrdnungVolvocalesrsquo(Verlagbuchhandlung Stuttgart Germany)

John DM Whitton BA Brook AJ (2002) lsquoFreshwater algal flora of theBritish Isles a guide to freshwater and terrestrial algaersquo (CambridgeUniversity Press Cambridge UK)

Keren N Kidd MJ Penner-Hahn JE Pakrasi HB (2002) A light-dependentmechanism formassive accumulation ofmanganese in the photosyntheticbacterium Synechocystis sp PCC 6803Biochemistry 41 15 085ndash15 092doi101021bi026892s

Komaacuterek J Anagnostidis K (2005) lsquoCyanoprokaryota Oscillatorialesrsquo(Spektrum Akademischer Verlag Heidelberg Germany)

Kruckeberg AR (2002) lsquoGeology and plant life the effects oflandforms and rock type on plantsrsquo (University Washington PressSeattle WA)

Kruumlger GHJ (1978) lsquoThe effect of physico-chemical factors on growthrelevant to the mass culture of Microcystis under sterile conditionsrsquo(University of the Orange Free State Bloemfontein South Africa)

Langhans TM Storm C Schwabe A (2009) Community assembly ofbiological soil crusts of different successional stages in a temperatesand ecosystem as assessed by direct determination and enrichmenttechniques Microbial Ecology 58 394ndash407doi101007s00248-009-9532-x

Lukesova A (2001) Soil algae in brown coal lignite post-mining areas incentral Europe (Czech Republic and Germany) Restoration Ecology 9341ndash350 doi101046j1526-100X200194002x

Lund JWG (1945) Observations on soil algae I The ecology size andtaxonomy of British soil diatoms Part 1 New Phytologist 44 196ndash219doi101111j1469-81371945tb05033x

Soil algae and cyanoprokaryotes Australian Journal of Botany K

Metha SK Gaur JP (2005) Use of algae for removing heavy metal ions fromwastewater progress and prospects Critical Reviews in Biotechnology25 113ndash152

MettingB (1981)The systematics and ecologyof soil algaeBotanical Review47 195ndash312 doi101007BF02868854

Michaud AB Šabackaacute M Priscu JC (2012) Cyanobacterial diversity acrosslandscape units in a polar desert Taylor Valley Antarctica FEMSMicrobiology Ecology 82 268ndash278doi101111j1574-6941201201297x

Monteiro MN Castro PML Macata FX (2012) Metal uptake by microalgaeunderlyingmechanism and practical applicationBiotechnologyProgress28 299ndash311 doi101002btpr1504

Mucina L Rutherford MC (2006) lsquoThe vegetation of South Africa Lesothoand Swaziland Strelitzia 19rsquo (South African National BiodiversityInstitute Pretoria South Africa)

Muumlhlsteinovaacute R Johansen JR Pietrasiak N Martin MP (2014) Polyphasiccharacterization of Kastovskya adunca gen nov et comb nov(Cyanobacteria Oscillatoriales) from desert soils of the AtacamaDesert Chile Phytotaxa 163 216ndash228 doi1011646phytotaxa16342

Neilson S Rajakaruna N (2012) Roles of rhizospheric processes and plantphysiology in phytoremediation of contaminated sites using oilseedBrassicas In lsquoThe plant family Brassicaceae contribution towardsphytoremediation Environmental pollution book series Vol 21rsquo (EdsNA Anjum I Ahmad ME Pereira AC Duarte S Umar NA Khan)pp 313ndash330 (Springer Dordrecht The Netherlands)

Norman N Whitfield G (2006) lsquoGeological journeys a travellerrsquos guide toSouth Africarsquos rocks and landformsrsquo (Struik Cape Town South Africa)

NSSSA (Non-Affiliated Soil Analysis Work Committee Soil ScienceSociety of South Africa) (1990) lsquoHandbook of standard soil testingmethods for advisory purposesrsquo (Soil Science Society of South AfricaPretoria South Africa)

OrsquoDell RE Rajakaruna N (2011) Intraspecific variation adaptation andevolution In lsquoSerpentine evolution and ecology in a model systemrsquo(Eds SP Harrison N Rajakaruna) pp 97ndash137 (University of CaliforniaPress Berkeley CA)

OrsquoKelly JC (1974) Inorganic nutrients In lsquoAlgal physiology andbiochemistryrsquo (Ed WDP Stewart) pp 610ndash635 (Blackwell London)

Oline DK (2006) Phylogenetic comparisons of bacterial communities fromserpentine and non-serpentine soils Applied and EnvironmentalMicrobiology 72 6965ndash6971 doi101128AEM00690-06

Orlekowsky T Venter A VanWyk F Levanets A (2013) Cyanobacteria andalgae of gold mine tailings in the Northwest Province of South AfricaNova Hedwigia 97 281ndash294 doi1011270029-503520130117

Pinto E Sigaud-Kutner TCS Leitao MAS Okamoto OK Morse DColepicolo P (2003) Heavy metal-induced oxidative stress in algaeJournal of Phycology 39 1008ndash1018doi101111j0022-3646200302-193x

Proctor J Woodell SRJ (1975) The ecology of serpentine soils Advances inEcological Research 9 255ndash366 doi101016S0065-2504(08)60291-3

Rajakaruna N Boyd RS (2008) The edaphic factor In lsquoThe encyclopedia ofecology Vol 2rsquo (Eds SE Jorgensen B Fath) pp 1201ndash1207 (ElsevierOxford UK)

Rajakaruna NB Harris TB Alexander EB (2009) Serpentine geoecology ofeastern North America a review Rhodora 111 21ndash108doi10311907-231

Rajakaruna N Knudsen KA Fryday RE OrsquoDell N Pope F Olday CWoolhouse S (2012) Investigation of the importance of rock chemistryfor saxicolous lichen communities of the New Idria serpentinite massSan Benito County California USA Lichenologist 44 695ndash714doi101017S0024282912000205

RajkumarMMaY FreitasH (2008)Characterization ofmetal-resistant plantgrowth promoting Bacillus weihenstephanensis isolated from serpentinesoil in Portugal Journal of Basic Microbiology 48 500ndash508doi101002jobm200800073

Raven JA Evans MCW Korb RE (1999) The role of trace metals inphotosynthetic electron transport in O2 evolving organismsPhotosynthesis Research 60 111ndash150 doi101023A1006282714942

Reddy RA Balkwill K Mclellan T (2001) Is there a unique serpentine floraon the Witwatersrand South African Journal of Science 97 485ndash495

Rehaacutekovaacute K Chlumskaacute Z Doležal J (2011) Soil cyanobacterial andmicroalgal diversity in dry mountains of Ladakh NW Himalaya asrelated to site altitude and vegetation Microbial Ecology 62 337ndash346doi101007s00248-011-9878-8

Rosentreter R McCune B (1992) Vagrant Dermatocarpon in western NorthAmerica The Bryologist 95 15ndash19 doi1023073243779

Rosentreter R Bowker M Belnap J (2007) A field guide to biologicalsoil crusts of western US drylandsrsquo (US Government Printing OfficeDenver CO)

Schuster RM (1983) Phytogeography of the bryophytes In lsquoNew manual ofbryologyrsquo (Ed RM Schuster) pp 463ndash626 (Hattori BotanicalLaboratory Nichinan Japan)

Shcolnick S Keren N (2006) Metal homeostasis in cyanobacteria andchloroplasts Balancing benefits and risks to the photosyntheticapparatus 1Plant Physiology141 805ndash810 doi101104pp106079251

Shields LM Durell LW (1964) Algae in relation to soil fertility BotanicalReview 30 92ndash128 doi101007BF02858614

Siebert SJ Van Wyk AE Bredenkamp GJ (2002) The physical environmentand major vegetation types of Sekhukhuneland South Africa SouthAfrican Journal of Botany 68 127ndash142

Soil Classification Working Group (1991) lsquoSoil classification a taxonomicsystem for South Africarsquo (Department of Agricultural DevelopmentPretoria South Africa)

South African Weather Service (2012) Rainfall maps Climate Summary ofSouth Africa 23 4ndash5

Southworth D Tackaberry LE Massicotte HB (2014) Mycorrhizal ecologyon serpentine soils Plant Ecology amp Diversity 7 445ndash455doi101080175508742013848950

Stark TL Shubert LE (1979) Algal colonization on reclaimed surface minedarea inwesternNorthDakota In lsquoEcologyandcoal resourcedevelopmentVol 2rsquo (Ed MK Wali) pp 652ndash660 (Pergamon Press New York)

Starks TL Shubert LE (1982) Colonization and succession of algae andsoilndashalgal interactions associated with disturbed areas Journal ofPhycology 18 99ndash107 doi101111j1529-88171982tb03162x

Starmach K (1963) lsquoFlora Slodkowodna Polski Rosliny SlodkovodneWstep Ogoacutelny I Zarys Method Badaniarsquo (Panstwowe Wyd-wo NaukWarszawa Poland)

Stein JR (1973) lsquoHandbook of phycological methods and culture methodsand growth measurementsrsquo (Cambridge University Press CambridgeUK)

Suzuki S Ishii S Wu A Cheung A Tenney A Wanger G Kuenen JGNealson KH (2013) Microbial diversity in the cedars an ultrabasicultrareducing and low salinity serpentinizing ecosystem Proceedingsof the National Academy of Sciences USA 110 15 336ndash15 341doi101073pnas1302426110

Taylor JC HardingWRArchibald CGM (2007) An illustrated guide to somecommon diatom species from South Africa WRC Report TT28207(Water Research Commission Pretoria South Africa)

Ter Braak CJF Smilauer P (1998) lsquoCANOCO reference manual and userrsquosguide to Canoco for Windows software for canonical communityordination (version 4)rsquo (Microcomputer Power Ithaca NY)

Terlizzi DE Karlander EP (1979) Soil algae from a Maryland serpentineformation Soil Biology amp Biochemistry 11 205ndash207doi1010160038-0717(79)90102-0

US EPA (1996) lsquoMethod 3050B Acid digestion of sediments sludges andsoilsrsquo Available at httpwwwepagovwasteshazardtestmethodssw846online3_serieshtm [Verified 1 August 2014]

Wehr JD Sheath RG (2003) lsquoFreshwater algae of North America ecologyand classificationrsquo (Academic Press London)

L Australian Journal of Botany A Venter et al

wwwpublishcsiroaujournalsajb