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A polysaccharide from Lichina pygmaea and L. confinissupports the recognition of Lichinomycetes

Alicia PRIETOa,*, J. Antonio LEALa, Manuel BERNABEb, David L. HAWKSWORTHc,d

aCentro de Investigaciones Biologicas, Consejo Superior de Investigaciones Cientıficas, Ramiro de Maeztu 9, ES-28040 Madrid, SpainbDepartamento de Quımica Organica Biologica, Instituto de Quımica Organica, Consejo Superior de Investigaciones Cientıficas,

Juan de la Cierva 3, ES-28006 Madrid, SpaincDepartamento de Biologıa Vegetal II, Facultad de Farmacia, Universidad Complutense, Plaza Ramon y Cajal, ES-28040 Madrid, SpaindDepartment of Botany, The Natural History Museum, Cromwell Road, London SW7 5BD, UK

a r t i c l e i n f o

Article history:

Received 5 October 2006

Received in revised form

21 September 2007

Accepted 25 October 2007

Corresponding Editor: Martin Grube

Keywords:

Ascomycota

Eurotiomycetes

Lecanoromycetes

Lichen

NMR spectroscopy

* Corresponding author.E-mail address: aliprieto@cib.csic.es

0953-7562/$ – see front matter ª 2007 The Bdoi:10.1016/j.mycres.2007.10.013

a b s t r a c t

The lichen-forming order Lichinales, generally characterized by prototunicate asci and the

development of thalli with cyanobacteria, has recently been recognized as a separate class

of ascomycetes, Lichinomycetes, as a result of molecular phylogenetic studies. As alkali and

water-soluble (F1SS) polysaccharides reflect phylogeny in other ascomycetes, a polysaccha-

ride from Lichina pygmaea and L. confinis was purified and characterized to investigate

whether these F1SS compounds in the Lichinomycetes were distinctive. Nuclear magnetic

resonance (NMR) spectroscopy and chemical analyses revealed this as a galactomannan

comprising a repeating unit consisting of an a-(1/6)-mannan backbone, mainly

substituted by single a-galactofuranose residues at the O-2- or the O-2,4- positions linked

to a small mannan core. With the exception of the trisubstituted mannopyranose residues

previously described in polysaccharides from other lichens belonging to orders now placed

in Lecanoromycetes, the structure of this galactomannan most closely resembles those

found in several members of the Onygenales in Eurotiomycetes. Our polysaccharide data sup-

port molecular studies showing that Lichina species are remote from Lecanoromycetes as the

galactofuranose residues are in the a-configuration. That the Lichinomycetes were part of an

ancestral lichenized group can not be established from the present data because the ex-

tracted polysaccharide does not have the galactofuranose residue in the b configuration;

however, the data does suggest that an ancestor of the Lichinomycetes contained a mannan

and was part of an early radiation in the ascomycetes.

ª 2007 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction (Hibbett et al. 2007). The representatives of the order are

The lichen-forming ascomycete order Lichinales comprises

around 250 species placed in 52 genera and four families

(Eriksson 2006). Its phylogenetic relationships were uncertain

in earlier molecular studies (Wedin et al. 2005), but the order is

now treated as belonging to a separate class, Lichinomycetes

ritish Mycological Society

unusual amongst lichen-forming groups in that the asci are

generally thin-walled and ‘prototunicate’ (without separating

wall layers nor any specialized apical apparatus), have a dis-

tinctive ascoma ontogeny, a relative non-specialized thallus

structure, form lichens exclusively with cyanobacteria, and

also mainly live on rocks or compacted soils in extreme arid

. Published by Elsevier Ltd. All rights reserved.

382 A. Prieto et al.

conditions or aquatic/marine habitats (Henssen & Jahns 1973;

Moreno & Egea 1991; Schultz et al. 2001). The representatives

of the eight genera so far studied by molecular approaches

form a clade with strong support (Schultz et al. 2001).

Eriksson et al. (2004) placed the order in the class Lecanoro-

mycetes. However, SSU nuDNA data analysed by Persoh et al.

(2004) showed them as a sister group to the Sordariomycetes

and remote from Lecanoromycetes, and combined data from

RPB2 and nuSSU and nuLSU sequences placed Lichinales as

a sister group to Lecanoromycetes–Eurotiomycetes (Reeb et al.

2004). This later placement was confirmed by the four-locus

analyses of Lutzoni et al. (2004), using nuSSU, nuLSU, mtSSU

rDNA and RPB2 sequence data, although with less support.

Reeb et al. (2004) studied the RPB2 coding gene, as well as

nuSSU and nuLSU, and introduced the new class name Lichino-

mycetes. However, trees based only on nuLSU rDNA and

mtSSU rDNA generated by Wedin et al. (2005) positioned

them in a broad Chaetothyriomycetes–Eurotiomycetes clade,

rather than as a sister group to such a grouping. Eriksson

(2005a) adopted the class name Lichinomycetes, but also noted

that this was not supported by Wedin et al.’s data. James

et al. (2006), using a six-gene phylogeny, confirmed the class

Lichinomycetes as described by Reeb et al. (2004). This has

been endorsed in the overall Assembling the Fungal Tree of

Life (AFTOL) classification, a scheme now recommended for

general use (Hibbett et al. 2007).

As alkali extractable and water soluble polysaccharides

(F1SS) reflect phylogeny in other ascomycetes (Prieto et al.

2004), we hypothesized that these compounds should be dis-

tinctive if these fungi merit recognition as a separate class.

The alkali-extractable and water-soluble F1SS polysaccha-

rides from free-living fungi are minor components of the

cell wall (2–8 %), and differ in composition and structure

between genera and, in certain cases, amongst groups of spe-

cies of a genus. These molecules have been demonstrated to

be phylogenetically informative not only at the generic but

also at suprageneric levels (Leal et al. 2001; Prieto et al.

2004). Polysaccharide moieties similar to the F1SS polysac-

charides occur in fungal cell wall glycoproteins (Gander

1974; Jikibara et al. 1992; Leal et al. 2001). The complex carbo-

hydrates of these molecules are antigenically relevant (e.g.

(De Ruiter et al. 1991; Domenech et al. 1999; Latge et al.

1991) and serve different biological functions, most impor-

tantly in cell–cell and/or cell–host recognition (Albersheim

et al. 1984).

Ecophysiologically, Lichina is interesting owing to its inter-

tidal habitat, which is inhabited by a rather limited number

of mostly crustose, pyrenocarpous lichens. There it is the

only shrubby lichen and associated with a cyanobacterial

photobiont (it is currently unclear whether this is Calothrix

or Scytonema), which allows both acquisition of inorganic car-

bon and nitrogen fixation. Moreover, the osmolyte mannosyl

mannose, assisting in salt-tolerance, was found in L. confinis

(Feige 1972, 1973). This compound is rapidly produced during

exposure to seawater, presumably by the photobiont partner

(Feige 1975).

Several polysaccharides have been reported in different

lichens (Common 1991; Teixeira et al. 1995; Woranovicz-

Barreira et al. 1999), and have been considered to have

potential as phylogenetic markers (Carbonero et al. 2001;

Common 1991). Correlations with phylogenetic lineages

have also been established for some of the more complex

polysaccharides in Lecanorales (Blanco et al. 2004). Further,

the F1SS polysaccharides in Lasallia pustulata (Umbilicariales)

have a mannan core with galactofuranosidic side-chains,

a result compatible with molecular data that indicate that

Umbilicariales merit recognition as distinct from other

lichen-forming orders (Miadlikowska et al. 2007; Pereyra

et al. 2003). In order to ascertain whether the nature of

the F1SS polysaccharides also would support the class

status of Lichinales, we analysed the F1SS in two marine

species of Lichina, the type species L. pygmaea and also

L. confinis.

Materials and methods

Lichen material

Lichina pygmaea: UK: South Devon: Dunscombe, Hook Ebb,

national grid reference 30/155878, on the tops and sea-facing

side of calcareous boulders just below high-water-mark, 12

Aug 2002, B. Benfield 0852/2.

L. confinis: UK: South Devon: Start Point, Peartree Point, 50�

13.070N, 03� 39.365 W, on schist in the littoral zone, 15 Oct

2006, D. L. Hawksworth.

Voucher material has been deposited in the reference col-

lection of the Departamento de Biologıa Vegetal II, Facultad de

Farmacia, Universidad Complutense de Madrid (MAF).

Polysaccharide extraction

Thalli were washed with water (three times) and air-dried

at 60 �C; 50 g were then extracted according to Pereyra

et al. (2003). In addition, the alkali and water-soluble

fraction was suspended in 50 % ethanol in water, and the

insoluble material discarded in order to purify the F1SS

polysaccharide.

Chemical analysis

For analysis of neutral sugars, the polysaccharide was hydro-

lysed with 3 M trifluoroacetic acid (TFA) for 1 h at 121 �C. The

resulting monosaccharides were converted into their corre-

sponding alditol acetates (Laine et al. 1972) and identified

and quantified by glc using an SP-2380 (Supelco, Bellefonte,

PA) fused silica column (30 m� 0.25 mm I.D.� 0.2 mm film

thickness) with a temperature program (210–240 �C, initial

time 3 min, ramp rate 15 �C min�1, final time 7 min), and

a flame ionization detector.

Absolute configuration of the monosaccharides released

after hydrolysis was determined as devised by Gerwig

et al. (1979) by glc-mass spectrometry (glc-ms) of the

tetra-O-trimethylsilyl-(þ)-2-butylglycosides using an SPB-1

(Supelco) fused silica column (30 m� 0.25 mm I.D.� 0.2 mm

film thickness) with a temperature program (150–210 �C,

initial time 1 min, ramp rate 3 �C min�1). The components

of the sample were identified on the basis of their retention

times and mass spectra.

Fig 1 – Anomeric region of the 1H-NMR spectra (D2O, 40 �C,

500 MHz) of the F1SS polysaccharides from (A) Lichina

pygmaea and (B) L. confinis.

Polysaccharides and the recognition of Lichinomycetes 383

Methylation analysis

The polysaccharide (1–5 mg) was methylated according to

Ciucanu & Kerek (1984), and the methylated material was

then treated and analysed following Ahrazem et al. (2000).

Partial hydrolysis of the F1SS polysaccharide

A 50 mg sample of the polysaccharide was hydrolysed with

5 ml of 0.15 M TFA for 5 h at 100 �C. The degraded polysaccha-

ride was then recovered by dialysis (molecular weight cut-off

ca 3 kDa) and lyophilization.

NMR analysis

The F1SS polysaccharide and the degraded polysaccharide (ca

20 mg) were dissolved in D2O (1 ml) followed by centrifugation

(10 000 g, 20 min) and lyophilization. The process was re-

peated twice, and the final sample was dissolved in D2O

(0.7 ml, 99.98 % D).

1D and 2D 1H- and 13C-NMR experiments were carried out

at 40 �C on a Varian Unity 500 (500/125 MHz, H/C) spectrome-

ter (Varian, Palo Alto, CA) with a reverse probe and a gradient

unit. Proton chemical shifts refer to residual HDO at d

4.61 ppm. Carbon chemical shifts were measured relative to

internal acetone at d 31.07 ppm. The 2D-NMR experiments

were performed by using the standard Varian software.

Fig 2 – 1H-NMR spectra (D2O, 40 �C, 500 MHz) of the anomeric

region of: (A) the mannan backbone obtained by mild acid

hydrolysis of the F1SS polysaccharide; and (B) the intact

F1SS polysaccharide from Lichina pygmaea.

Results

Chemical analysis of the F1SS polysaccharides gave mannose

and galactose in a proportion ca 1:1, as shown by glc of their

alditol acetates. The absolute configuration was shown to be

D for both sugars.

The 1H-NMR spectra of polysaccharides from both species

(Fig 1) contained, inter alia, two main anomeric sharp signals at

5.2 and 5.19 ppm, and at least five minor broad signals be-

tween 5.3 and 4.9 ppm. This last signal, assigned to un-

branched a-(1/6)-mannopyranose residues, is more intense

in the spectrum of Lichina confinis. The sharp peak (a ‘singlet’)

at 5.19 ppm was later found to constitute one of the two com-

ponents of a double peak (a ‘doublet’) partially overlapping

with the signal at 5.20 ppm; the residues were labelled A–G

(Fig 2B). As both polysaccharides displayed similar chemical

composition and 1H-NMR spectra, further analysis were car-

ried out only with the polysaccharide of L. pygmaea.

Methylation analysis of the polysaccharide indicated that

it was mainly composed of terminal galactofuranose (51.8 %)

and 2,6-di-O-substituted mannopyranose (27.7 %), although

minor amounts of 2-O-substituted (9.7 %), 6-O-substituted

(3.4 %), and 2,4,6-tri-O-substituted (7.4 %) mannopyranoses

were also detected. The 13C-NMR spectrum (Fig 3) also showed

two main and several small anomeric singlets. 2D homo- and

hetero-NMR experiments (Fig 4) led to the complete assign-

ment of the proton and carbon chemical shifts of residues

A and B, and partial assignment of the rest of the residues

(Table 1). Comparison of the data with standard values (Bock

& Pedersen 1983; Jimenez-Barbero et al. 1993) led to the con-

clusion that: A and E were 2,6-di-O-substituted-a-Manp; B, C,

and D, terminal a-Galf, F, terminal Manp; and G, 6-O-

substituted Manp. The different chemical shifts of identical

residues, like A and E, and also B, C, and D, are indicative of

different linkage positions or different neighbourhoods

around those residues.

The anomeric coupling constant of B was J1,2¼ 4.7 Hz,

which suggested a galactofuranose residue with a-

configuration (cfr the b-anomeric coupling constants around

2 Hz (Cyr & Perlin 1979). The anomeric carbon chemical

Fig 3 – 13C-NMR (D2O, 40 �C, 125 MHz) spectrum of the F1SS

polysaccharide from Lichina pygmaea.

Table 1 – 1H- and 13C-NMR chemical shifts (d)* for thealkali-extractable water-soluble polysaccharide F1SSisolated from Lichina pygmaea

Units 1 2 3 4 5 6a 6b

A H 5.2 4.07 3.94 3.8 3.84 4 3.76

C 99.3a 79.8 71.5b 67.7 72 66.7

B H 5.19 4.15 4.2 3.83 3.76 3.72 3.64

C 103.6 77.4 74.8 82c 72.7 63.4

C H 5.31 4.17 4.12 3.77

C 103.7 77.4 75 81.8c 72.6 63.1

D H 5.27 4.14 4.17 3.83

C 103 77.4 74.8 81.9c 72.4

E H 5.11 4.03 3.89 3.75

C 99.2a 79.4 71 73.4 66.1

F H 5.05 4.07 3.68 ca 3.75 3.89 3.76

C 103 70.8 71.4b 67.8 74 61.8

G H 4.91 4.02 3.81 3.95 3.77

C 100.3 70.9 71.6 66.5

Underlined bold numbers represent glycosylation sites.

a,b,c These values may have to be interchanged.

384 A. Prieto et al.

shifts of A and B at 99.3 and 103.6 ppm, respectively, are

also indicative of an a-configuration for both units (Bock &

Pedersen 1983).

Although the anomeric signal of the 2,4,6-tri-O-substituted

Manp (H) deduced from the methylation results was not

detected in the NMR spectra, probably due to it being both

a small proportion and also overlapping with peaks around

5.2 ppm, a model of a 2,4,6-tri-O-substituted Manp tetrasac-

charide (Takeda et al. 1981) gave values of 80.3, 75.2, and

67.4 ppm for the chemical shifts of carbons C-2, C-4, and

C-6, respectively. Therefore, similar values have to be

expected for analogous carbons of the 2,4,6-tri-O-substituted

Manp residue contained in the F1SS polysaccharide of L.

confinis and L. pygmaea. Small signals around 79.5, 75, and

66.5 ppm were observed in the 13C-NMR spectrum. In addition,

Fig 4 – Partial heteronuclear multiple quantum coherence-total

the F1SS polysaccharide of Lichina pygmaea. Relevant cross-pea

cross-peaks of H-1C with a carbon at 79.5 ppm, and of H-1D

with another at 75 ppm, can also be seen in the heteronuclear

multiple bond correlation (HMBC) spectrum (Fig 5); this

suggests connections of those terminal Galf units with posi-

tions 2 and 4 of the 2,4,6-tri-O-substituted residue of Manp.

Concerning the connections of the different units,

a nuclear Overhauser enhancement spectroscopy (NOESY)

ly correlated spectroscopy (HSQC-TOCSY) NMR spectrum of

ks of selected residues have been labelled.

Fig 5 – Partial HMBC NMR spectrum of the F1SS polysac-

charide of Lichina pygmaea. Relevant cross-peaks of the

anomeric peaks have been labelled.

Polysaccharides and the recognition of Lichinomycetes 385

experiment (not shown) enabled, among others, cross-peaks

H-1A/ H-6aþ 6bA (G), H-1 G/H-6aþ 6bA (G), H-1B/H-2A to be

observed. In addition, an HMBC spectrum (Fig 5) showed

cross-peaks H-1A/C-6A (H, G), H-1B/C-2A, H-1E/C-6A (H, G),

and H-1 G/C-6A (E, H, G), where A and G represent a second

molecule of A and G. This strongly suggests the presence of

a backbone of (1/6)-Manp containing a high proportion of

terminal residues of Galf linked to position 2 in almost each

Manp unit.

The small amounts (<10 %) of 2-O-substituted mannopyr-

anose observed in the methylation analyses were poorly

detected in the NMR spectra. Therefore, in order to further

investigate the minor components of the mannan backbone,

the F1SS polysaccharide was treated with diluted acid, which

selectively hydrolysed the furanosidic side residues, yielding

a new polysaccharide composed exclusively of mannose.

Methylation analysis gave terminal mannopyranose, 2-O-

substituted, 6-O-substituted and 2,6-di-O-substituted man-

nopyranoses. The 1H-NMR spectrum of the mannan core

was in accordance with the methylation results, showing, in-

ter alia, a main anomeric signal at 4.9 ppm corresponding to

6-O-substituted-Manp (z60 %), and six minor signals be-

tween 5.3 and 5.05 ppm (Fig 2B) attributed to 2-O-substituted

(z22 %), 2,6-di-O-substituted mannopyranoses (z9 %) and

terminal mannopyranose (z10 %). The absence of galactofur-

anose and 2,4,6-tri-O-substituted Manp, the drastic reduction

of the 2,6-di-O-substituted Manp, and the increase of

6-O-substituted-Manp in an analogous proportion constitutes

an indirect chemical evidence of those two residues being

substituted by terminal units of Galf.

Comparison of the chemical shift values with those of

analogous mannan derivatives (Gomez-Miranda et al. 2004)

strongly suggests the presence of a small proportion of short

chains of (1/2) linked mannopyranoses, connected to posi-

tion 2 in the (1/6)-a-Manp backbone; the backbone has

a major proportion of unbranched residues, mostly produced

by the hydrolysis of the Galf side units.

A coupled heteronuclear multiple quantum coherence

(HMQC) experiment allowed the measurement of anomeric

coupling constants in one 1H–13C bond. The values obtained

for all the residues in both species were in the range 1JH-1-C-1¼173� 0.6 Hz, which are demonstrative of a-configuration for

all of them (Bock & Pedersen 1974).

Combining all the different data elements, the galacto-

mannan F1SS of L. pygmaea and L. confinis appears to have

the following idealized structure:

D-D-Galf

1 A H

4 [ 6)- -D-Manp-(1 6)]n [- -D-Manp-(1 6)]m

Rest of the mannanbackbone2 2

-D-Galf -D-Galf

B C

being n z 30, and m z 8.

With respect to the mannan backbone, the analysis and

the 1H-NMR spectrum (Fig 2A) indicate a structure very similar

to those of the cores already found in several fungal polysac-

charides (Gomez-Miranda et al. 2004). That means that the

mannan obtained from mild hydrolysis of the polysaccharide

from L. pygmaea and L. confinis has the structure:

G E E E E 6)-Manp-(1 6)-Manp-(1 6)-Manp-(1 6)-Manp-(1 6)-Manp-(1

2 2 2 2

1 1 1 1 F Manp Manp Manp Manp I

2 2 2

1 1 1 F Manp Manp Manp I

2 2

1 1 F Manp Manp I

2

1 F Manp

For the sake of simplification, all the residues of 2-O-

substituted mannopyranoses have been labelled ‘I’, although

their chemical shifts depend on the position occupied along

the short lateral chains. Unfortunately, it is not possible to de-

termine the positions of each side-chain along the mannan

backbone in the intact F1SS polysaccharide.

386 A. Prieto et al.

Discussion

An alkali and water-soluble polysaccharide was purified

from thalli of Lichina pygmaea and L. confinis. The mycobiont

usually constitutes the bulk of the thallus (Rai et al. 2000),

and in the case of gelatinous lichens with cyanobacteria,

the cyanobacteria produce a polysaccharidic sheath that

contributes to water retention. The characteristics of cyano-

bacterial exopolysaccharides are different to that described

for bacteria, algae, and fungi (Morvan et al. 1997). Most cya-

nobacteria produce anionic extracellular polysaccharides

with at least one uronic acid and several neutral sugars

(Moreno et al. 2000), as occurs in Nostoc (Brull et al. 2000;

Rainer et al. 2007) or Anabaena (Moreno et al. 2000). Unfortu-

nately, nothing has been described for the exopolysaccharide

from the photobiont of Lichina, although its structure would

be expected to be similar to those reported for other cyano-

bacteria. In the present work, the polysaccharide purified

from the two Lichina species was established as comprising

a repeating unit made up of a galactomannan with

a a (1/6)-mannan backbone mainly substituted by single

a-galactofuranose residues at the O-2- or the O-2,4- positions

and a small proportion of short chains of a-(1/2)-manno-

pyranose. The presence of a a-(1/6) mannan backbone or

mannan core is a common characteristic of the F1SS poly-

saccharides from the cell walls of Ascomycota (Prieto et al.

2004), including several lichenized fungi from other orders

associated with green algal photobionts (Gorin et al. 1988;

Gorin & Iacomini 1985; Pereyra et al. 2003; Teixeira et al.

1995; Woranovicz-Barreira et al. 1999). The chemical and

structural characteristics of the polysaccharide isolated

from the two Lichina species are similar to those reported

for F1SS cell wall polysaccharides of Ascomycota, while simi-

lar structures have not been described as cyanobacterial con-

stituents. Thus, we are confident that the polysaccharide

reported in the present study is a component of the cell

wall of the mycobiont and not of the included cyanobacte-

rium. The structures of 37 different repeating units in F1SS

polysaccharides known in non-lichenized ascomycetes

were compiled by Prieto et al. (2004), who also listed the gen-

era in which their occurrence has been confirmed. With the

exception of the trisubstituted mannopyranose residues,

previously described in polysaccharides from lichenized

fungi belonging to other orders now placed in Lecanoromy-

cetes (Gorin et al. 1988; Gorin & Iacomini 1985; Pereyra et al.

2003; Teixeira et al. 1995; Woranovicz-Barreira et al. 1999),

the structure of this galactomannan, although distinctive,

most closely resembles those found in several members of

Onygenales in the class Eurotiomycetes. In particular, the re-

peating unit is closest to structures 10–13 in Prieto et al.

(2004), and so far found mainly in dermatophytic and allied

genera.

Although mannose-containing polysaccharides of a few

other lichen fungi have been characterized (see above), all

are from orders in Lecanoromycetes and none have a-D-Galf

residues. The (1/6)-mannan backbone can have attach-

ments in different positions, notably single residues of termi-

nal a-Galp, b-Galp, b-Glcp, a-Manp or b-Galf (Gorin & Iacomini

1985; Gorin et al. 1988; Teixeira et al. 1995; Woranovicz et al.

1999). These polysaccharides resemble those found in the

onygenalean radiation (Prieto et al. 2004), and therefore,

such lichenized groups may be related to ancestral ascomy-

cetes. The differences of the galactose residues regarding

ring size, configuration, and the linkage to different positions

of the mannose residues show different states of the F1SS

polysaccharide character. On the basis of the F1SS data,

these structures appear to be included in a radiation whose

ancestor is a mannan (Bernabe et al. 2002; Prieto et al. 2004).

Most of these structures belong to evolutionary dead-ends

as only the b-D-Galf-(1/ lineage seems to have led to the

F1SS polysaccharides found in most of the non-lichenized

cleistothecial and perithecial ascomycetes so far investi-

gated. Therefore, although the simple structure of the F1SS

polysaccharide from Lichina confirms that it is an ancestral

ascomycete and occupies an isolated position, it cannot itself

be regarded as the ancestor of Eurotiomycetes and Sordariomy-

cetes, as it does not have the galactofuranose residues in the

b-configuration, the building block of the galactofuranose

chains linked to the mannan core in these fungi (Prieto

et al. 2004).

The polysaccharide data support indications from recent

molecular studies that the Lichinales are distinct from other

lichen-forming, and indeed other ascomycete groups, and

merit treatment as a separate order in the separate class Lichi-

nomycetes (Reeb et al. 2004; Eriksson 2005a; James et al. 2006;

Hibbett et al. 2007). However, when so few representatives of

the order have yet been investigated by molecular methods,

and the polysaccharides have now been examined only in

two, the data must be interpreted with caution. Unfortunately

no Lichina species were included in the AFTOL analyses, only

two species of Peltula (Peltulaceae) which grouped with Geoglos-

saceae (Miadlikowska et al. 2007; Spatafora et al. 2007). As Pel-

tula belongs in a separate family and has lecanoralean not

prototunicate asci, it is evident that too much emphasis

should not be placed on this result and that a wider taxon

sampling is required, as noted by Spatafora et al. (2007).

The hypothesis that the ancestral state of the ascomycetes

was lichenized, and that the ability to form lichens has subse-

quently been lost in many families (Eriksson 2005b; Lutzoni

et al. 2001) is not sufficiently confirmed by the Lichina data.

However, the relatively basal position of Lichina with respect

to F1SS polysaccharides is consistent with the suggestion

that ancestral lichens had cyanobacteria as their photosyn-

thetic partners, an hypothesis canvassed since the 1980s

(e.g. Hawksworth 1982, 1988; Eriksson 2005b). Intriguingly

the earliest fossil lichen known, discovered in Lower

Devonian deposits, is associated with cyanobacteria (Taylor

et al. 1997).

Acknowledgements

We thank Frank S. Dobson and Barbara Benfield for collecting

fresh material of Lichina species for use in our studies, and

Jesus Lopez for technical assistance. This work was supported

by Grant MEC CTQ-2006-10874-C02-01 from the Direccion

General de Investigacion, and partially undertaken while

Polysaccharides and the recognition of Lichinomycetes 387

D.L.H. was in receipt of a Programa Ramon y Cajal award of the

Ministerio de Ciencia y Tecnologıa de Espana.

Supplementary material

Supplementary material associated with this article can be

found, in the online version, at doi:10.1016/j.mycres.2007.

10.013.

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