I<.e1nrd.-; nftlle Western A IIstraIllliI Mllselllll 24: 395-410 (200S).

A new genus and species of stygobitic paramelitid amphipodfrom the Pilbara, Western Australia

Terrie L. Finston, Michael S. Johnson and Brenton Knott

School of Animal Biology (M092), University of Western Australia, 35 Stirling Highway,Crawley, Western Australia 6009, Australia

Abstract - The Pilbara region of Western Australia contains a diverseand endemic ground water fauna dominated by crustaceans, withamphipods being a conspicuous component of many communities. Previousmorphological and molecular research has demonstrated the need forrevision of the current amphipod taxonomy. Specifically, examples of crypticspeciation and of morphological variation within single species suggest thata combined approach is needed to address the taxonomic gap. In this paperwe use molecular and morphological evidence to show the distinctiveness ofa new genus and species of stygobitic amphipod, Kruptlls lil1l1l1ei, known fromSpearhole Creek in the Pilbara.

Key words: allozymes, crangonyctoid, morphology, Neoniphargidae,taxonomv

INTRODUCTIONThe Pilbara is a large (178 000 km 2) semi­

arid bio-region in the northwest of WesternAustralia. The region is rich in iron ore, hence itis an area of significant mining activity. Belowthe arid landscape, the Pilbara hosts a richground water fauna (Humphreys 1999, 2001). Arecent survey suggested the presence of more than150 groundwater species, many of which are newto science, and of which nearly 60(/;, are endemicto the area (Eberhard et Ill. 2005a). Amphipods arean abundant component of the groundwater faunaof the Pilbara. So far, 17 species of amphipodswithin the family Paramelitidae Bousfield 1977have been described from the Pilbara (Bradburyand Williams 1997a, Bradbury 20(0), and thereare records of at least two additional familiespresent. Specimens of Melitidae Bousfield 1973and Neoniphargidae Bousfield 1977 were collectedduring a survey of the groundwater fauna ofthe Pilbara conducted by the Department ofEnvironment and Conservation (DEC; S. HaIse,pers. comm.).

The taxonomy of the amphipods of the PiIbara ispoorly resolved. Allozymes suggest the presenceof many undescribed species (Finston and Johnson20(4). Further, morphological interpretationrarely coincides with molecular diversity. Forexample, the 14 species of Cllljdl1eklltll Bradbury2000, described from Ethel Corge in the upperj:ortescue River basin northwest of Newman,W.A., suggest morphological diversity that is notmatched by molecular diversity; neither allozymes

nor mtDNA indicate the presence of more thanone species of Chydaekata in the area (Finston etal. 2004; Finston et Ill. 2007). In contrast, moleculardiversity in PilbllfllS Bradbury and WiIliams1997a and Chydaekllta is associated with separatetributaries, but no morphological charactershave been found to differentiate the distinctmtDNA lineages (Finston et al. 2007). Lefebure etal. (2006) attribute the preponderance of crypticspecies in the groundwater to constraints onform or convergence of characters in extremeenvironments. The potential for cryptic diversity,in combination with historical difficulties withmorphological taxonomy of the amphipods ofthe Pilbara, indicates the need for molecularassistance to guide the placement of taxonomicboundaries. This paper utilises a combinedapproach towards documentation of the diversityof amphipod species in the groundwater of thePilbara.

The Ciles Mini site (23°17'06"S, 119°09'54"E) lieson Spearhole Creek, in the upper Fortescue Rivercatchment, at the western end of the OphthalmiaRange. Uplift of shale layers and bedrock alongthe Ophthalmia fault has resulted in the presenceof multiple, confined aquifers in the region, whichare overlain with a shallow laver of calcrete(Ilarmsworth cl Ill. 1990; Jason Pepper, pers.comm.). The presence of confined aquifers couldprovide the isolation necessary for the evolutionof multiple species, each restricted to a differentaquifer. Indeed, allozyme comparisons revealedthe presence of four genetic groups of amphipods

396

in the area, two in the shale, and two in thecalcrete (Finston and Johnson 2004). According tothe allozymes, all four groups were paramelitids,and one showed both morphological andmolecular affinities to Chydaekata, a Widespreadgenus in the upper Fortescue River basin. Theother three groups were not identified. One group,found in six bores, was relatively widespread inthe shale, suggesting that movement does occurbetween formations; perhaps through fissures inthe rock (Finston and Johnson 2004). The secondgroup was found in a single bore, also in shale,and the third unidentified group occurred in asingle bore in a calcrete aquifer. The present studyutilizes morphology to assess the distinctivenessof the Widespread allozyme group found in sixbores. We use the combined results to describea new genus and species of amphipod fromSpearhole Creek.

METHODS

Collections and allozyme analysis

Groundwater amphipods were collected fromeight bores from the Giles Mini site in 2001and 2002, using standard methods (Eberhardet al. 2005b; see Table 1, Figure 1). The boreswere sampled along a 3 km transect, and thesa mples were included in a regional study ofallozyme diversity in the amphipod fauna inthe Pilbara (Finston and Johnson 2004). Twoshallow bores into calcretes were sampled, andthe other six bores sampled aqui fers in shale(Dale's Gorge, Whaleback and Joffre formations;Table 1), at depths ranging from 54 to 112 m.Due to the difficulties involved with samplingstygofauna (limited access to the groundwater,temporal variation in recharge and spatialvariation in aquifer yield; Eberhard et al. 2005a)sample sizes were small, ranging from 3 to 30amphipods per bore. In total, 100 individualswere analysed for variation at between nine and

T.L. Finston, M.S. Johnson, B. Knott

fifteen allozyme loci, as described in Finston andJohnson (2004). For the present paper, we havere-analysed the data for the nine loci for whichwe have population data (due to their small size,individuals could not be analysed for all 15 loci;a smaller subset of individuals was analysed forvariation at the additional six loci) from Finstonand Johnson (2004) for the Spearhole Creeksamples. We also included a single multilocusgenotype from an individual collected at theSeven Mile Creek, which showed genetic affinitiesto one of the allozyme groups from SpearholeCreek. We quantified the divergence betweengenetically distinct groups in two ways, Nei'sgenetic distance (0; Nei 1972) and the proportionof loci that were diagnostic, i.e. those loci sharingno alleles between a pair of groups. We usedmultidimensional scaling (MOS) of the matrix ofNei's 0 to illustrate the genetic relationships.

Morphological assessment

A two-step approach was used to assess thetaxonomic status of the widespread geneticgroup. First, existing keys and descriptions ofthe Australian amphipod fauna (Williams andBarnard 1988; Bradbury and Williams 1999;Lowry and Springthorpe 2001) were used toidentify the specimens to the lowest taxonomiclevel possible. Second, to assess the genericand familial affinities of the specimens, wecompiled 26 diagnostic characters from thegeneric descriptions and taxonomic keys ofAustralian freshwater amphipods (Williams andBarnard 1988; Bradbury and Williams 1996, 1997,1999; Bradbury 20(0) and recorded characterstates for representatives of each genus describedfrom the Pilbara (Pilbarus Bradbury and Williams1997, Chydaekata, Molina Bradbury 2(00) and thespecimens from Spearhole Creek. In additionto the described genera, we also included twopotential sister taxa in the analysis. Specimens ofNeoniphargidae from the Robe and OeGrey River

Table 1 Bores sampled for the present study, lithology (geological formation and depth targeted by the bore), allozymegroup and number (n) analysed, and Western Australian Museum voucher numbers for the material used.

Bore

GM019GM027GM039GM049GM069GM118GMI'22GMP77

Target geology

Multiple Whaleback ShaleJoffreMultiple Whaleback ShaleDale's GorgeDale's GorgeMultiple Whaleback Shalecalcretecalcrete

Depth (m)

104627084112544023

AIlozyme group* (n)

H (13)H(3)H (12)H (8)G (5), H (25)H(4)1(24)F (6)

Voucher number

WAM C40034

WAM C40035WAM C40036WAM C40037WAM C40038, WAM C40039WAM C40040

* groups as assigned in Finston and Johnson (2004).

A new stygobitic amphipod of the Pilbara 397

CapeRange

1. Molina*2. Pilbarus'"3. Chydaekata*4. Spearhole Creek5. nr. Wesniphargus6. Seven Mile Creek

Indian Ocean

Barrow Island

N

/, 100 km

Figure 1 Map of sampling locations or type localities (*) for specimens used in the present study. Names and outlinedareas indicate drainage basins.

basins in the Pilbara, most closely resemblingWesniphargus Williams and Barnard 1988 (hereinreferred to as 'nL Wcslliplwrglls'), were includedas was ProtocrallgolllJx fOlltillalis Nicholls 1926from the Paramelitidae. Two species of the genusNcdsia Barnard and Williams 1995 from the familyMelitidae were included as outgroups. Characterstates for Pilbarus lI/i1lsi Bradbury and Williams1997, Cllljdackata aCllll/illata Bradbury 2000, C.brachybasis Bradbury 2000, C. Illldllla Bradbury2000, C. scopllla Bradbury 2000, Molilla plcobrallcllOsBradbury 2000, ProtocrallgollYx fOlltillalis, Nedsiadouglasi Barnard and Williams 1995, and N.straskra/Ja Bradbury and Williams 1996 weredetermined from the original descriptions.Character states for the specimens from SpearholeCreek were obtained from examination ofpreserved material as follows: we examined 14specimens of the widespread genetic group fromfive bores (five males, five females, four juveniles).Three specimens belonging to two presumptivetaxa of nr. Wcslliphllrglls, nr. Wcslliphllrgll.s (Robe),n=2 and nr. Wcsllipharglls (DeGrey), n=l, wereloaned bv the DEC and dissection was notpermissible. Externally, the specimens matched thedescription of the genus Wcslliplwrglls (Williamsand Barnard 1988), with one major exception ourspecimens lacked eyes. Of the 26 characters, most(21) were observable without dissection. As these

characters matched the description of Wcslliphllrglls,the remaining features (presence of inner lobes onthe lower lip, rugosity on the maxilliped palps,and three characteristics of the mandibular palp)were assumed also to match the description of thegenus and were scored as per the description. Inaddition, we had to make assumptions about thegender of these specimens. All three specimens hadasthetascs on antenna 1 and calceoli on antenna2, hence we assumed that these specimens weremales, as per the description. In total, 11 speciesfrom six genera and three families were included inthe comparison with the specimens from SpearholeCreek. We did not include all species of CllljdllCklltlland Ncdsia because the analysis was intendedsimply to assess morphological affinities at thegeneric and familial levels.

A data matrix of the scores of 12 taxa for the26 characters was constructed using NDE 5.0(Page 2(01), and all characters were included asunordered and unweighted. Phylogenetic analysisof this matrix involved a heuristic search usingparsimony and tree bisection reconnection (TBR)branch svvapping, performed in PAUl' (Swofford2(01), with the starting tree obtained by step-Wiseaddition. A 50(;/0 majority rule consensus tree wasobtained from lOO bootstrap replicates. The twospecies of Ncdsill were used as the outgroup.

T.L. Finston, M.S. Johnson, B. Knott

Axis 1 - Genetic Distance

RESULTS

Table 2 Measures of genetic differentiation amongallozyme groups. Above diagonal: Nei's (1972)genetic distance. Below diagonal: proportionof loci showing allelic substitutions betweengroups.

Morphological assessment

The specimens from group H could not beidentified to species using existing keys anddescriptions. They fit the definition of WiIliamsand Barnard (1988) for crangonyctoid amphipods,namely having a prominent third uropod,which is bi-ramous and with the inner ramusreduced, prominent mandibular palps, accessoryflagellum present on antenna 1, and the presenceof simple sternaI gills. If we follow strictly thekey of Bradbury and Williams (1999), we fail todifferentiate our specimens as either Corophiidaeor Crangonyctoidea, due to the presence of anaccessory flagellum on antenna 1 (Crangoncytoid)in combination with an entire telson, lacking anynotching (Corophiidae). However, if we acceptthat crangonyctoids may have an entire telson(e.g. Protocrangonyx Nicholls 1926), then ourspecimens key to the family Neoniphargidae, dueto the presence of rugosities on the gnathopods.Within the Neoniphargidae, the key fails at thefirst couplet, because the sternal gills in ourspecimens are small and bud-shaped, not sausage­shaped, nor dendritic. Further, all neoniphargidsspecies so far described have rugosities on theprodopus and the maxillipedal palp, whereas

distances of 0.71 to 1.98, and in proportions ofdiagnostic loci from 0.22 to 0.56 (Table 2). Thesmallest distance between groups occurredbetween groups G and H, where D was 0.71 andthe proportion of diagnostic loci was 0.22 (Table2). The genetic distance between group Handthe individual from Seven Mile Creek, whichshowed some affinities to group H in a previousstudy (Finston and Johnson 2004) was 0.78, andthe proportion of diagnostic loci between the twogroups was 0.56.

Table 3 Characteristics of specimens from Group Hand the family to which the character belongs.N = Neoniphargidae, P = Paramelitidae.

Character Family

Urosomes nearly smooth NCoxae reduced, narrow PAntennal sinus shallow P*Lower lip with weak inner lobes NOuter plate of maxilla 1 with -10 apical robust setae PPereopod 7 longest PTelson entire NApices of telson with one seta PUropod 3 terminal article lacking P*Palmar setae of gnathopods simple P*Sternal gills present on peraeon 7 PDistal article of uropod 3 absent P*Rugosities present on gnathopods N

* indicates character is within acceptable range, but is notthe usual state.

o

S L

-1

10

D= 1.0

-2

F OChydaekata

A

Q>()c:t1linis.~

IDc:Q>

C}I

C\lCl>

f·x«

J-3

Figure 2 MDS of Nei's (1972) genetic distance Dbetween multilocus genotypes pooled for eachbore at Spearhole Creek. Circles = genotypesfrom Spearhole Creek, triangle = genotypefrom Seven Mile Creek. Genetic groups F-l asin Finston and Johnson (2004); S = Seven MileCreek.

AllozymesFour genetic groups were detected in the

samples from Giles Mini (Table 1; groupdesignations as in Finston and Johnson (2004);Figure 2). The most common group, H, wascomposed of 65 individuals from six bores (Table1), and represents the new genus and speciesdescribed in this paper. Groups F, G, and Iwere found in single bores (Table 1). Group F,represented by six individuals from bore GM077,showed both morphological and molecularaffinities to Chydaekata (Finston and Johnson2004). This genus differed from groups G, HandI by genetic distances ranging from 1.08 to 3.53(Table 2) and in the high proportion of diagnosticloci, which ranged from 0.44 to 0.89 (Table 2).Group H differed from all other groups by genetic

Group F G H S

F x 3.53 1.98 1.08 1.49G 0.71 x 0.71 1.04 1.06H 0.33 0.50 x 1.09 0.78I 0.47 0.79 0.27 x 1.44S 0.57 0.79 0.53 0.36 x

A new stygobitic amphipod of the Pilbara

A

399

N. douglasi

N. straskraba

100

} Melitidae

---- M. pleobranchos

P. millsi

C. acuminataB

C. brachybasis91

C. nudula

c. dolichodactyla

L..- K. linnaei (group H)

c1------ P. fontinalis

Paramelitidae

1..-_0 1 nr. Wesniphargus (DeGrey)

100 nr. Wesniphargus (Robe)} Neoniphargidae

Figure 3 50'/', majority rule consensus tree for morphological character state data for known species of paramelitidsfrom the Pilbara, and two representatives of neoniphargids from the DeCrey and Robe River basins. lwospecies of the melitid genus Ncdsia were used as the outgroup. Clades are labelled A D, and bootstrap valuesshown below branches.

rugosities are limited to the ischium and merusin the Ciles group H specimens. In addition, thespecimens from group 11 lack the body type ofthe neoniphargids, such as a short and deep body,deep coxae, and lobate carpi of the gnathopods.Although keying to the neoniphargids, severalcharacters place the Ciles group H specimenswith the Paramelitidae, such as reduced coxae, thepresence of sterna I gills on the 7'h pereopod, andthe absence of well-developed inner lobes of thelower lip (Table 3). If we assume our specimens areparamelitids, despite the rugose gnathopods, thenour specimens key to Protocmllgollljx

due to the presence of simple sternal gills andthe telson being entire. Despite this superficialsimilarity, our specimens clearly are not P.fontinalis, which has many characteristics thatdiffer from the group II specimens, including thelack of calceoli, an accessory f1agellum on antenna1 of only two articles, the presence of a coxalgill on gnathopod 2, the lack of a coxal gill onpereopd 7, the presence of large, strongly obliquegnathopods, and short carpi on gnathopod 2, the

proportions of article 1 of the mandibular palpbeing approximately as long as wide, and havinglong posterior setae on perepods 3 and 4.

A heuristic phylogenetic analysis of 26 diagnosticcharacters (Table 4) showed four clades (Figure 3).Clack A contained the melitid outgroup (Ncdsill),clade B contained the paramelitids exceptingProtOCI"IIllgOIlIjX, clade C contained Protocmllgolll/X,

and clade D contained the neoniphargids (Figure3). The specimens from group II occurred as along branch in the paramelitid clade (clade B).The taxonomic position of ProtocmllgollIjx wasunresolved, falling outside of the paramelitidclade (Figure 3). Bootstrap support was high forboth the paramelitid and the neoniphargid(100"0) clades. The group II specimens share manycharacteristics vvith the other paramelitid genera,and in particular, with lv10lillll. The two taxa sharesuch traits as the coxal gills extending to segment7. and possessing a full set of sternal gills onsternites 2-7. Nevertheless, group H individualsdiffered from Molillll in eight cha racll'rs (Table

400 T.L. Finston, M.S. Johnson, B. Knott

Table 4 Characters and character states used for classification of known species of paramelitids and undescribedneoniphargids from the Pilbara. Two species of Nedsia (melitid) were used as outgroups.

1. Number of articles on accessory flagellum AI:1. <3 (short)2. ~3 (long)

2. Inner ramus V3:O. short or absent1. moderately elongate; ~ 113 length outer

3. Mandibular palp:O. reduced (2 articles)1. prominent (3 articles)

4. Calceoli on A2 (male):O. absent1. present

5. A2 length of flagellum:O. ~ length of peduncle1. > length of peduncle

6. Coxae:O. reduced1. intermediate2. expanded

7. First location of coxal gills:O. 21. 3

8. Last location of coxal gills:O. 61. 7

9. Sternal gills:O. absent1. partial set (6-7)2. near full set (2-6 or 3-6)3. full set (2-7)

10. Palmar angle Gl:O. strongly oblique1. weakly oblique2. transverse

11. Palmar angle G2:O. strongly oblique1. weakly oblique2. transverse

12. Carpus of G2:O. short; ~ 50'%, prodopus1. long; "" prodopus

13. Telson cleftness:O. 0% (entire)1. <50'%,2. ~50%

14. Basio-facial seta VI:O. always absent1. sometimes or always present

15. Inner lobes lower lip:O. absent1. present

16. Calceoli on peduncle:O. absent1. present

17. Rugosities on gnathopods:O. absent1. present

18. Rugosities on maxilliped palp:O. absent1. present

19. Type of sternal gills:O. simple or absent1. dendritic or lumpy

20. Number of articles in pleopod rami:O. short (3-7)1. long (8-11)

21. Distal article of outer ramus of uropod 3:O. absent1. present

22. Article 1 of mandibular palp:O. length"" width1. length noticeably longer than width

23. Article 2 of manibular palp:O. with few setae1. moderately setose

24. Posterior setae on article 2 of pereopods 3 and 4:O. short1. long

25. Distal margin of coxa 4:O. not excavate1. excavate

26. Mandibular palp article 3:O. lacking C setae1. bearing C setae

A new stygobitic amphipod of the Pilbara 401

,~

A1

A2

LL

/ .--e,,--/ J

~,RMD GuJ

Figure 4 Kruptlls lillllaci sp. nov., holotype male 2.4 mm, whole animal (top), antennae (A), calceolus (Cl, mandible(MD), lower lip (LL), R", right, L '" ]dt Scale lines 200 pm.

including the shape of the sternal gills (Figure 4;simple buds versus sausage-shaped, respectively),the number of articles in the accessory tlagellum(Figure 4; three versus six, respectively), thepresence (group H) or absence (Molilll7) of calceolion antenna 2 (Figure 4), cleftness of the telson(Figure 5; 0% and respectively), and thepresence (group 11) or absence (Molilll7) of rugosityon the gnathopods (Figure In comparison,the number of character differences betweenother pairs of paramelitid genera range from twobetween Pilbarus and Clzydl7ckatll (the absence

or presence of sterna I gills and the shape ofgnathopod 2) to four betwl'en Pilbllrlls and fvlolillll.While the specimens from group H share thecharacter of rugosities on the gnathopods withthe specimens of nr. thev differin 19 other characters, including the number ofarticles in the accessory flagellum of antenna 1,the relative length of the flagellum to the peduncleof antenna 2, the location of the coxal and sternalgills, the shape of the gnathopods and relativesize of the carpus, the cleftness of the telson,type of sterna I gills, and size and setation of the

402 T.L. Finston, M.S. Johnson, B. Knoll

Table 5 Character matrix for known species of Pilbara paramelitids and undescribed neoniphargids. Nedsia (melitid)is used as an outgroup. Character states that are inapplicable are scored as in the matrix. nr. Wes = nr.Wesnipharglls. DeG = DeGrey River, Rob = Robe River.

1 2 3 4 5 6 7 8 9 10 11 12

N. dOllglasi 1 0 0 0 0 0 0 0 0 0 0 0N. straskraba 1 0 0 0 0 0 0 0 0 0 0 0M. pleobranchos 2 1 ] 0 1 1 0 1 3 2 ] 1P millsi 2 ] 1 ] 1 1 0 0 0 2 1 1C. aCllminata 2 1 ] 1 1 ] 0 0 ] 2 1 1C. brachybasis 2 1 1 1 ] 1 0 0 1 2 2 1C. nudula 2 1 1 1 1 1 0 0 1 2 1 1C. scopula 2 1 1 1 1 1 0 0 1 2 1 1Group H 2 1 ] 1 1 0 ] 1 3 2 2 1P. fontinalis 1 1 ] 0 0 ] 0 0 2 0 0 0nr. Wes (DeG) 1 1 1 1 0 2 0 0 2 1 1 0nr. Wes (Rob) 1 1 1 1 0 2 0 0 2 1 1 0

Characters 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Species

N. douglasi 2 1 1 0 0 0 0 0 1 0 1 0 0N. straskraba 2 1 1 0 0 0 0 0 1 0 1 0 0M. pleobranchos 2 0 ] 1 0 0 0 1 1 ] 0 1 1 0P. millsi 2 0 1 1 0 0 0 1 1 1 0 1 1 1C. acuminata 2 0 1 1 0 0 0 1 1 1 0 1 1 0C. brachybasis 2 0 1 1 0 0 0 1 1 1 0 1 0 0C. nlldula 2 0 1 1 0 0 0 1 1 1 0 1 1 0C. scopula 2 0 1 1 0 0 0 1 1 1 0 1 1 0Group H 0 0 ] 1 1 0 0 0 0 1 0 0 0 0P. fmltinalis 0 0 0 0 1 0 0 0 0 0 0 1 0 0nr. Wes (DeG) 2 0 0 0 ] 1 1 0 1 0 ] 0 1 1nr. Wes (Rob) 2 0 0 0 1 1 1 0 1 0 ] 0 1 1

articles of the mandibular palp (Table 5). Despitesome similarities to Protacranganyx fantinalis, suchas an entire telson and lack of a distal article onthe outer ramus of uropod 3 (Figure 5), rugositieson the gnathopods but not on the maxillipedalpalps (Figure 6), and short pleopodal rami (Figure7), the two taxa differ in 14 other characteristics,such as the number of articles in the accessoryflagellum of antenna 1, the presence (group H) orabsence (P. fantinalis) of calceoli on antenna 2, therelative length of the flagellum to the peduncleof antenna 2, the location of the coxal and sternaIgills, the shape of the sternaI gills, the shape ofthe gnathopods and relative size of the carpus,the relative proportion of the articles of themandibular palp, and setation on pereopods 3and 4.

DISCUSSIONMolecular and morphological data confirmed

the distinctiveness of the specimens from groupH, and verified their status as a new genusand species. Analysis of allozymes from 100

individuals from Spearhole Creek revealed fourhighly divergent genetic groups, which werecharacterised by allelic substitutions at a largeproportion of loci and high genetic distances.The low sample size of some of these groups,such as F (Chydaekata) and G precludes detailedanalysis of genetic diversity within groups, anderror associated with small sample sizes and alimited number of loci may bias between-groupvariation. Nevertheless, these results providethe basis for seeking congruent morphologicalvariation. Hence morphological evidence, whichsupports the genetic findings, provides a robustframework for identifying unique taxa. Group Fwas identified as Chydaekata, and group I differedfrom group H in a number of characters, includinga telson that was nearly fully cleft and that wasornamented with both setae and plumose setae,very small, highly oblique gnathopods, and theouter ramus of uropod 3, which possessed a distalarticle and had setal tufts along the margin. Onlygroup G has not been confirmed morphologicallybecause only five individuals were present in

A new stygobitic amphipod of the Pilbara 403

LG2

T

Figure 5 Kruptlls 1i1l1li7ci sp. nov., holotype, appendages: gnathopods (C), telson (T), uropods (U). 1\= right, L = left.Scale lines= 200 pm.

our collections and they were consumed duringelectrophoresis. On a broader geographical scale,we have analysed genetic diversity in amphipodsfrom 35 bores at eight sites in the Pilbara(Finston and Johnson 2004; Finston et 171. 2(04),and identified only a single specimen showinggenetic affinities to the group H specimens. Thisspecimen, from Seven Mile Creek, differed fromgroup H by a genetic distance of 0.78, and in ahigh proportion of diagnostic loci (0.56). Yet thisspecies is perhaps the closest relative to groupH, and may represent a congener. The specimen

was consumed for allozyme electrophoresis,so future collections should focus on findingmore representatives of this species, in order todetermine its morphological relationship to group11.

The distribution of species at Spearhole Creeksheds some light on dispersal capabilities of thegenetic groups and on hyd rological connectionsbetween geological formations at small and largescales. While discrete aquifers were predictedto be contained within the shale formations, thepresence of group H in multiple shale formations

404

indicates that movement is possible over theseshort distances, perhaps through cracks andfissures. The presence of C/zydaekata, a widespreadgenus comprising multiple distinct lineagesthat occupy separate creek catchments overhundreds of kilometres in the upper FortescueRiver basin (Finston et al. 2007), is unlikely to bedue to recent migratory events. Such widespreaddistributions are hypothesised to occur as aresult of extended periods of drying, duringwhich time invasions of the groundwater bysurface ancestors were followed by isolation andgenetic divergence (Bradbury and Williams 1997b;Humphreys 1999, 2001; Finston et al. 2007). Therestriction of Chydaekata to the calcrete aquiferat Spearhole Creek and its exclusion from theshale deposits may reflect its need for largervoid spaces. We conclude that habitat type andconnections in groundwater flow are importantfactors in determining the local movement anddistribution of species, while hydrological eventsand historical processes play a role in shapingbroader distributions.

Morphological analysis upheld thedistinctiveness of the specimens from group H.The specimens do not fit the descriptions of anyspecies, and differ in a number of diagnosticcharacters at a level greater than that betweenthe other genera in this study. Hence bothmolecular and morphological evidence supportthe description of a new genus and speciesfrom group H from Spearhole Creek. While themajority of the evidence suggests the speciesbelongs in the family Paramelitidae, the speciesfrom group H also possesses some charactersdiagnostic of the neoniphargids, and charactersthat should exclude it from the paramelitids.Similarly, Protocrangonyx possesses characters ofboth families, however the weight of evidenceplaces group H in the paramelitids, whilst thetaxonomic position of Protocrangonyx could not beresolved. The characters considered to define thesefamilies have changed since their inception in1977 (Bousfield 1977), with the addition of newlyexamined material and critical evaluation of therange of acceptable character states (Will iamsand Barnard 1988, Bradbury and Williams 1997a,Bradbury and Williams 1999). Currently, onlytwo characters appear to separate the families: 1)the presence of rugosities on the gnathopods andpalp of maxillipeds in the neoniphargids, and theabsence of these structures in the paramelitids;and 2) the presence of sterna I gills on segment 7 inthe paramelitids, whilst they terminate at segment6 in the neoniphargids (Bradbury and Williams1999). Many other characters have overlappingdistributions between the two families. Forexample, the inner lobes of the lower lip aredescribed as being absent in the paramelitids

T.L. Finston, M.S. Johnson, B. Knott

and "poorly to moderately developed" in theneoniphargids (Bousfied 1977), but are absent orindistinct in several neoniphargid genera, such asNeoniphargus Stebbing 1899, Wesniphargus Williamsand Barnard 1988, Yulia Williams and Barnard1988 and Tasniphargus Williams and Barnard1988 (Williams and Barnard 1988). Similarly,the sterna I gills may be absent or simple in theparamelitids, but range from simple to dendriticin the neoniphargids (Bousfield 1977). Animportant feature of the neoniphargid body planis the lengthening of the coxae, especially coxae1-4 (Bousfield 1977), but the coxae are describedas "moderately elongate" in such paramelitidgenera as Austrogammarus Barnard and Karaman1983, An tipodeus Williams and Barnard 1988and Hurleya Straskraba 1966 (Williams andBarnard 1988). Williams and Barnard (1988),while amending the diagnostic characters of theParamelitidae and Neoniphargidae, highlightedthe need for a formal re-diagnosis of thesefamilies. The inability to resolve the placementof Protocrangonyx, coupled with the discoveryof group H, a new genus and species possessingcharacters from both families, confirms the needfor such a revision, in which rugosities on thegnathopods, which may be present or absent,should be considered a diagnostic character ofthe paramelitids.

Family Paramelitidae Bousfield 1977

Kruptus gen. novo

Type speciesKruptus linl1aei sp. novo

Diagnosis

Body subvermiform; pereonites and urosomiteslacking dorsal setation; coxae short, coxa 4 notexcavate, coxal gill lacking on coxa 7; epimera withfew dorsal setae. SternaI gills small, simple, bud­shaped. Accessory flagellum of antenna 1 of threearticles, the first and third being markedly short,and the second being markedly long, about 3 to 4xas great as the third article. Antenna 2 with type9 (crangonyctoid) calceoli. Article 1 of mandibularpalp markedly longer than wide; article 2 withfew setae; article 3 with DE setae; inner lobes oflower lip asymmetrical, poorly developed. Palmsof gnathopods weakly oblique to transverse. Rowof long setae on medial surface of prodopus ofgnathopod 2; rugosities on merus of gnathopod2; carpi of gnathopod 2 long. Posterior setae onarticle 2 of pereopods 3 and 4 short. Inner ramusof uropod ~ relatively long, about Y2 the length ofouter ramus; outer ramus lacking the distal article;urosomes of uropods 1 and 2 short, approximatelyequal in length to peduncles. Telson entire.

A new stygobitic amphipod of the Pilbara

RMX1

405

LMX2

RMP

RMX2

Figure 6 Kruptlls linnill'i sp. nov., holotype, mouthparts: maxilla (MX), maxillipeds (MP). R= right, L = left. Scale lines200 pm.

Distribution

Krllptlls limlllci is known only from SpearholeCreek in the upper Fortescue River drainagebasin, Pilbara, Western Australia

Relationships

Krllptlls resembles Pilbarlls, Clll/dackata, andMo/ilia in having weakly oblique ·or transversegnathopods. In addition, as in Clll/dackata,asthetascs are present on antenna 1 and calceoliare present on antenna 2 in the male. Krllptlls canbe distinguished from these genera by its telson,which is entire, while in the former genera the

telson is deeply cleft, ranging from 70 to 90%.KrlIptlls shares several characteristics with Mo/illo,such as having a complete set of sternal gills7), but it can be distinguished from Mohlla by thepresence of calceoli on the second antenna in themale, which are absent in Mo li 1111 , the number ofarticles in the accessory flagellum, which is threein Krllptlls, but six in Mohl/a, and the shape of thesternal gills in Krllptlls, which are small buds,while they are sausage-shaped in Mohllo. KrlIptllSalso shares some traits with If\",such as the lack of the distal article on the outerramus of uropod 3, and an entire telson, but can

406

be distinguished from that genus by its smaller·gnathopods, coxal gill 7, which is present inKrllptlls but absent in Protocrangonyx, the numberof articles in the accessory flagellum of antenna1, which is two in Protocrangonyx and three inKrllptlls, the lack of calceoli on antenna 2 inProtocrangonyx, the proportion of article 1 ofthe mandibular palp, which is approximatelyas long as wide in Protocrangonyx but markedl~

longer than wide in KruptllS, the proportion ofthe carpus of gnathopod 2, which is short inProtocrangonyx, but long in KruptllS, and the typeof posterior setae on pereopods 3 and 4, whichare long in Protocrangonyx and short in Kruptus.Kruptus also shares some characteristics withneonipargids such as Wesnipharglls, namelypossessing rugosities on the gnathopods, butalso in having three articles in the accessoryflagell um of antenna 1, possessing calceoli onantenna 2 in the male, having short pleopodalrami with few articles, and possessing shortposterior setae on article 2 of pereopods 3 and4. It also shares traits with Ginipharglls in havingsmall, nearly transverse gnathopods, and thecarpus of gnathopod 2 elongate and lacking inlobes, however, Krllptlls can be distinguishedfrom Wesnipharglls by the accessory flagellumof antenna 1, which has three articles, therelatively longer flagellum of antenna 2, thelack of a coxal gill on pereopod 7, the small andnearly transverse shape of the gnathopods andrelatively long carpus of gnathopod 2, the lack ofa cleft in the telson, the small bud-shaped sternalgills, and the long first article of the mandibularpalp, and from Ginipharglls by its less vermiformbody, and lack of the second article on theouter ramus of uropod 3, which is present andmarkedly long in Giniphargus.

Etymology

The genus name comes from the Greek word for'hidden' (kruptos), to highlight its subterraneandistribution and to indicate the presence ofcryptic characters belonging to other groups ofamphipods. The species name is in recognitionof the 300th anniversary of the birth of CarlLinnaeus.

Kruptus linnaei sp. novoFigures 4-7

Material examined

Holotype

Australia: Western Australia: G (2.4 mm), Pilbararegion, 5pearhole Creek, bore GM069, 23°16'57"5,119°11'20"E, October 2001, T. Finston, G. Humphreys,MS Johnson (WAM C40037, 2 slides).

T.L. Finston, M.S. Johnson, B. Knott

Allotype

Australia: Western Australia: ¥ (2.0 mm),Pilbara region, 5pearhole Creek, bore GM019,23°16'55"5, 119°11'20"E, October 2001, T. Finston,G. Humphreys, MS Johnson (WAM C40034, 2slides).

Paratypes

Australia: Western Australia: 3 specimens,Pilbara region, 5pearhole Creek, bore GM019,23°16'55"5, 119°11'37"E (WAM C40034); 2 specimens,5pearhole Creek, bore GM039, 23°16'57"5,119°11'4S"E (WAM C40035); 1 specimen, 5pearholeCreek, bore GM049, 23°16'50"5, 119°12'23"E (WAMC40036); 7 specimens, 5pearhole Creek, boreGM069, 23°16'57"5, 119°11'20"E (WAM C40037);2 specimens, 5pearhole Creek, bore GMIIS,23°16'53"5, 119°12'6"E (WAM C4003S, WAMC40039).

Description

Male (WAM C40037)

Head: sinus shallow; antenna 1 having 11 articleson left and 12 on right, peduncle longest article,asthetascs present on articles 3-11. Accessoryflagellum short, of three articles, extending tomiddle of article 2 of the flagellum. Articles 1and 3 of the accessory flagellum are markedlyshort, article 2 being markedly long, about 4xgreater than article 3. Antenna 2 of six articleson left and five articles on right, possessing long,feather-shaped calceoli on articles 1-4, plus onecalceolus on the peduncle. Right mandible: palpwith three articles, article 2 longest. Article 1nude; article 2 with small protuberance or notchon ventral surface, followed by two setae; article 3with four setae on ventral margin, and two setaeapically. One seta originates on dorsal surface,nearly apically. Incisor with five teeth; lacinia aflat, lightly serrated plate with a small thumb-likeprojection and an underlying flat plate with aserrated edge. Molar raised, with a flat grindingsurface and one long seta, setal row of veryshort setae. Two serrated setae arise from base oflacinia. Left mandible: missing. Right maxilla 1:Palp with two articles; article 2 with one apicalrobust seta plus three very short, blunt projectionsand one long seta. Inner plate with ten bifid robustsetae, some serrate. Outer plate short, conical,about 1h the length of the inner plate, and withtwo plumose setae and fine short setae on outermargin. Left maxilla 1: Palp with two articles;article 2 with three apical robust setae and onelong seta. Inner plate with ten bifid robust setae,some serrate. Outer plate short, about 1h length ofinner plate, conical, and with two apical plumosesetae. Right maxilla 2: Inner and outer plates

A new stygobitic amphipod of the Pilbara 407

P3

Figure 7 Krupl1l5 lillllaei sp. nov., holotype, appendages: pereopod 3 (1'3), pereopod 7 (1'7), pleopod 3 (I'L3) andenlargement of epimera (FP). Scale lines 200 pm.

both long, thin, sausage-like. Outer plate witheight long setae, outer margin with few, fine shortsetae. Inner plate with seven apical setae, innermargin with many fine setae along full length,surface also sparsely covered with fine setae. Leftmaxilla 2: Inner and outer plates both long, verythin, sausage-like. Inner plate approximately 5/6the length of outer plate. Outer plate with finesetae on outer margin and nine apical setae, somecurved at tip. Inner plate with two plumose apicalsetae; inner margin with many fine setae. Rightmaxilliped: palp of four articles. Article 1 witha single seta on inner surface near the base ofarticle 2. Article 2 with ten setae on inner margin.

Article 3 with three setae on inner margin, nearbase of dactyl; dactyl with one short seta on innermargin, near base of claw. Outer plate with long,fine setae on outer margin and eight apical broad,flat, setae and one apical seta. Inner plate with sixrobust setae at apex plus one sub-marginal setathat originates at the approximate midpoint. Leftmaxilhped: palp with four articles. Article I withsingle seta, sub-apical, at inner corner near base ofarticle 2; article 2 with 11 setae in a row on innermargin; article 3 with a single apical seta on outercorner and three on inner margin at base of article4. Outer plate bulbous, with seven fine setae onouter margin and one apical seta; inner margin

408

with six broad, curved robust setae. Inner platewith four apical setae, three fine, sub-marginalsetae on outer margin and inner margin withthree setae. Upper lip: relatively symmetrical, withtwo patches of dense short setae at apex corners.Lower lip: outer lobes with short setae at apices;inner lobes weakly developed, asymmetrical,nearly absent on right side and covered with shortsetae. Body: subvermiform in shape; coxae 2-4displaced from their corresponding pereonites;coxa 4 not excavate. Cuticle relatively smooth withlittle setation. Coxal gills present on coxae 3-7;sternaI gills present on pereonites 2-7, increasingin size posteriorly. Gnathopod 1: prodopus nearlymittenform, palm slightly oblique, convex, andwith four sub-marginal setae in a row at base ofpalm, ending in one robust seta. Carpus short,about 1;2 length of prodopus, and with four sub­marginal setae. Merus with one marginal seta andsmall rugose patch on ventral surface. Ischiumwith short seta near base. Basis with one setanear base. Coxa extended anteriorly with twosetae on anterior margin. Gnathopod 2: prodopusmittenform, slightly smaller than gnathopod 1,palm nearly transverse, with four setae at corner.Base of dactyl with one seta; prodopus with onelong seta on dorsal margin, near base of dactyl,and three setal rows on surface of prodopus,two on medial surface, each with two setae andone near ventral margin, composed of five setae.Carpus extended, approximately equal in lengthto prodopus and bearing two setal rows, theanterior with four and the posterior with threesetae. Merus with one seta and small rugosepatch on ventral surface, and one seta on medialsurface. Ischium with one seta and small rugosepatch on ventral surface. Basis with one setanear apical corner and one long seta on posteriormargin. Coxa expanded anteriorly, and with twoanterior setae. Pereopods 3-4: with sparse, shortsetation (one to two setae per article) on posteriormargin, merus of each slightly lobate anteriorally,dactyls with single short seta. Pereopods 5-7: withthree to four robust setae at base of each article,dactyls with single seta; pereopod 7 longest.Pleopods 1-3: peduncle with one retinacula andone accessory retinacula, rami with three tofour articles. Uropod 1: Peduncle with two longsetae on dorsal margin near apex; rami relativelyshort; inner ramus equal in length to peduncleand outer ramus approximately 0.9 x the lengthof the peduncle, both rami terminating in twoshort and one long setae. Inner ramus also withone long sub-apical seta. Uropod 2: pedunclewith three short robust setae, one sub-marginalabout halfway along outer margin, and two sub­apically, one on inner margin near base of innerramus and one medially. Inner and outer ramiclearly shorter than peduncle, both terminating

T.L. Finston, M.S. Johnson, B. Knott

111 three apical and one sub-apical seta. Innerramus 0.85 x the length of the peduncle, outerramus approximately 0.65 x the length of thepeduncle. Uropod 3: outer ramus 2.0 x the lengthof peduncle, peduncle bearing one robust seta atouter distal corner. Outer ramus with one robustseta on outer margin, and one long seta on innermargin, and terminating in two apical robustsetae and one short and two long apical setae;distal article of uropod 3 absent. Inner ramusleaf-shaped, 1.15 x the length of peduncle, tip isrecurved, with one tiny seta at sub-apex. Telson:entire, with one apical seta at each corner.

Fetnale (WAM C40034)

Similar to male except in the followingcharacteristics:

Head: antenna 1 having ten articles on both leftand right, with asthetascs on articles 5-9. Antenna2 having five articles on both left and right andbearing no calceoli. Right mandible; article 2 long,rectangular, lacking notch on ventral surface;margin straight with one long seta; article 3with three setae near margin on inner and outersurfaces, and one long seta and two short setaeapically. Left mandible: palp with three articles;article 2 longest. Article 1 with one marginal seta,medially, and one short seta sub-apically, nearbase of article 2. Article 2 with two setae on thedorsal surface and with two setal rows on innermargin, the distal row containing four setae andthe proximal row containing three setae. Article 3with two rows of two setae on the dorsal surfaceand with a setal row on inner margin containingthree setae, and three apical and three sub-apicalsetae. Incisor with five teeth; lacinia a flat platewith five teeth. Molar raised with a flat grindingsurface and one short seta, and a setal row of veryshort setae. Three serrate setae arise from baseof lacinia, plus one plumose seta. Right maxilla1: Article 2 of palp with five apical robust setae,article 1 nude. Left maxilla 1: Article 2 with fourapical setae of similar length, and one sub-apical,sub-marginal seta. Right maxilliped: pedunclewith long seta instead of a robust seta. Leftmaxilliped: peduncle with long seta instead of arobust seta. Gnathopod 1: prodopus with threesub-marginal setae in row near base of palm,merus with three marginal setae. Gnathopod 2:distinctly smaller than gnathopod 1; prodopuslacking dorsal setae on margin, ischium lackingrugose patch. Uropod 1: lacking two long setaeon peduncle; instead with three short setae oneach side dorsally, plus one very large, robust setasub-apically near base of outer ramus. Uropod 2:peduncle with a single robust seta sub-apicallynear base of inner ramus. Uropod 3: pedunclenude, outer ramus with two setae on inner margin

A new stygobitic amphipod of the Pilbara

and terminating in three robust setal' and onelong seta.

Character variation

The number of articles in the flagella ofantennae 1 and 2 varies from 10-11 and 5-6,respectively among individuals, and sometimesbetween left and right of the same individual.The prodopus and carpus of the gnathopodsare slightly lobate post-ventrally in someindividuals, and the degree of lobateness ofthe merus of pereopods 3 and 4 differs amongindividuaJs. The angle of the palm of gnathopod1 varies among individuals from slightly obliqueto moderately oblique. Setation differs on thepeduncle and rami of the uropods and on thearticles of gnathopods 1 and 2, as well as on themouthparts. Most differences occur as changesin the number of setae, however, changes inthe structure of the setae sometimes occur,particularly on the peduncle of uropod 1 and themaxillipeds.

ACKNOWLEDGEMENTS

This project was funded by BHP-Billiton Pty.Ltd, Pilbara Iron Pty. Ltd., and ARC Linkagegrant LP0349199. Samples were collected with theassistance of Biota Environmental Sciences. Manythanks to Susan Johnson for the illustrations,Garth Humphreys for overall coordination of thesampling, and Jason Pepper and Sally Maddenfor information about the geological structure ofSpearhole Creek. Thanks to Adrian Pinder of theDepartment of the Environment and Conservationfor providing samples of neoniphargids. Twoanonymous reviewers gave helpful comments onan earlier draft of the manuscript.

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Manuscript received 31 March 2008, accepted 11 July 2008.