of and Diplotomma, with Diploicia subcanescens Diplotomma...

Lichenologist 34(6): 509-519 (2002)doi:10.1006/lich.2002.0420, available online at http://www.idealibrary.com on

Molecular phylogeny and status of Diploicia and Diplotomma, withobservations on Diploicia subcanescens and Diplotomma

rivas-martinezii

Maria del Carmen MOLINA, Ana CRESPO, Oscar BLANCO, Nestor HLADUNand David L. HAWKSWORTH

Abstract: ITS rDNA sequence data shows that Diploicia and Diplotomma species form a mono-phyletic clade distinct from other Buellia species. This indicates that Diplotomma merits acceptance asa genus, and suggests that Diploicia should be treated as a synonym of Diplotomma, the earlier name.The data also shows Diploicia subcanescens, considered the fertile counterpart in a species pair withD. canescens, is nested within D. canescens and should be treated as a synonym despite reportedchemical differences. In addition, the molecular data support the distinctness of Diplotommarivas-martinezii, a species restricted to gypsum rocks in Spain, from the widespread D. venustum,which grows on calcareous rocks. Aposymbiotic cultures suggest that D. rivas-martinezii also differsfrom D. venustum in its germination and isolation success rates. One new combination is made:Diplotomma pulverulenta (Anzi) D. Hawksw. (syn. Abrothallus pulverulentus Anzi) for the lichenicolousspecies previously known as Buellia pulverulenta.

V 2002 The British Lichen Society. Published by Elsevier Science Ltd. All rights reserved.

Key words: Buellia, Lecanorales, lichens, Physciaceae, species pairs, Spain.

Introduction

Since the generic name Diplotomma Flotow(Lecanorales, Physciaceae) was resurrectedas distinct from Buellia De Not. byHawksworth et al. (1980), it has beenadopted in numerous checklists and regionalstudies (e.g. Wei 1991; Purvis et al. 1992;Nimis 1993; Esslinger & Egan 1995; Filson1996; Malcolm & Galloway 1997; Marbach,2000; Eriksson et al. 2001; Llimona &Hladun 2001; Hafellner & Turk 2001;

M. del C. Molina (corresponding author): Departa-mento de Ciencias Experimentales e Ingenieria, EscuelaSuperior de Ciencias Experimentales y Tecnologia,Universidad Rey Juan Carlos, Madrid 28933, Spain.A. Crespo, O. Blanco & D. L Hawksworth: Departa-mento de Biologia Vegetal II, Facultad de Farmacia,Universidad Complutense, Madrid 28040, Spain.N. Hladun: Departament de Biologia Vegetal(Botanica), Universitat de Barcelona, Av. Diagonal645, Barcelona 08028, Spain.

Coppins 2002). However, the name has notbeen universally accepted (e.g. Santesson1993; Wirth 1995; Nordin 1996; Vitikainenet al. 1997; Diederich & Serusiaux 2000;Scholz 2000) and the status of the genus hasremained controversial.

However, in a study of the Buellia s. lat.species with pluriseptate ascospores, Nordin(2000) noted that Diplotomma 'might beused in a more narrow sense', as has beenthe case in the checklists accepting thegenus referred to above, for species withcalcium oxalate in the thallus, often pruinoseapothecial discs, and a thick perispore;Purvis et al. (1992) also stressed the initiallyimmersed ascomata, and the distinctiveascospores which they interpreted as dis-toseptate. However, the term 'distoseptate'has been used in different senses, as dis-cussed by Nordin (1997), and is perhapsbest avoided, but nevertheless the spores ofDiploicia also have similarly thickened spore

0024-2829/02/060509+11 $35.00/0 2002 The British Lichen Society. Published by Elsevier Science Ltd. All rights reserved.

510 THE LICHENOLOGIST Vol. 34

walls and septa developing secondarilyrecalling the polarilocular Dirinaria-type(Matzer et al. 1997). Nordin noted that theDiplotomma s. str. group was also distinctbiologically, in that it included obligately orfacultatively lichenicolous species. Interest-ingly, Diploicia Massal., which has a similarthallus structure and ascospores but a placo-dioid habit, has been unhestitatingly ac-cepted by all authors in recent decades.However, the type species of the latter gen-us, D. canescens (Dickson) Massal., was in-cluded in Diplotomma when the genericname was first used (Flotow 1849, 1850). Inorder to check the affinities of Diploicia withrespect to Diplotomma and also Buellia s. str.,we examined several collections of the typespecies of these genera and further its sup-posed primary, generally fertile species Dc.subcanescens (Werner) Hafellner & Poelt.The latter has also been reported to differchemically from the sorediate and generallyinfertile Dc. canescens (Elix et al. 1988). Thistopic is of particular interest since somepresumed species pairs are proving to besingle species when studied using molecularmethods (Articus et al. 2002).

Buellia rivas-martinezii Barreno & A.Crespo (Follmann & Crespo 1975), aspecies name not mentioned by Nordin(2000), was transferred to Diplotomma byMolina & Crespo (2000). This species is afrequent and moderately polymorphic taxonthat appears to be confined to crystallinegypsum rocks in dry conditions in theMeso-mediterranean bioclimatical level(Rivas-Martinez 1982). Diplotomma rivas-martinezii has a thin, pale grey or ochra-ceous, rimose or granular, K - thallus andlacks a dark prothallus. The apothecia areinnate, abundant, rounded (0-8-1-2 mmdiam.), and have an ephemeral thalline ex-ciple which is normally K+ red; the apoth-ecia later become slightly substipitate andsomewhat convex (Barreno & Crespo 1975;sub Buellia follmannii Barreno & A. Crespo,non Dodge 1967). Probably due to therareity of the biotopes where it grows, thespecies is not represented even in many largelichen herbaria; consequently it has not beenincluded in revisionary studies of buellioid

lichens (Scheidegger 1993; Nordin 1996,2000). The species is related to Din. ve-nnstum Korb. (Barreno & Crespo 1975; asBuellia venusta), a species discussed in detailby Nordin (2000), which has innate apoth-ecia and usually a smooth or rimose, oftensubeffigurate thallus and frequently showinga peripheral black prothallus. Although insome collections the morphological andchemical differences between the two taxaare not unequivocal, the particular habitat ofDm. rivas-martinezii facilitates its identifi-cation. Based on chemical and morphologi-cal characters, Clauzade & Roux (1985),accepted B. venusta as an independentspecies with a K+ yellow thallus, butincluded B. rivas-martinezii as a synonym ofB. epipolia (Ach.) Mong. with a K~ thallus.Nordin (1996) noted that B. epipolia s. str.was a synonym of B. alboatra (Hoffm.) Th.Fr. and used the name B. venusta to includeK+ and K — specimens.

The taxonomic level to be used for par-ticular taxa is always open to different inter-pretations, but new and independent datasets, and especially molecular characters,may support one interpretation better thananother. Additionally, molecular charactersmay give information about evolutionarypathways that can be used in hypotheses forthe process of speciation. Features of thesubstratum have been considered as oneimportant selective determinant of the pres-ence of most lichen species. Lopez-Bilbaoet al. (1996) demonstrated a differentialgene expression depending on the sub-stratum. However, there are no data relatingthis feature to the process of speciation.Although vegetative (production of soredia,isidia, or other dual propagules) versussexual reproduction (production of basi-diospores or ascospores) has been proposedas one evolutionary trend in lichens (Poelt1970, 1972; Tehler 1982) no other hypoth-esis has been vindicated. Paraphyletic evolu-tion is considered to be one importantprocess by which the delimitation of newplant species may be explained (Rieseberg &Brouillet 1994; Vargas et al. 1998) althoughno model of paraphyletic speciation has beenproposed amongst lichen-forming fungi.

2002 Molecular phylogeny and status of Diploicia and Diplotomma—Molina et al. 511

TABLE 1. Species included in the analysis, locations and reference collection codes for newly sequenced specimens, presence ofintron products, and GenBank accession numbers

Species Locality (reference collection codef)Intron

present (+) GenBank no.

1. Diplotomma rivas-martinezii2. Dm. rivas-martinezii*3. Dm. rivas-martinezii4. Dm. venustum5. Dm. venustum6. Dm. venustum7. Dm. alboatrum8. Diploicia canescens9. Dc. canescens

10. Dc. canescens*11. Dc. subcanescens12. Dc. subcanescens*13. Buellia frigida14. B. disciformis15. B. elegans16. B. zoharyi17. B. georgei18. B. dijiana19. B. epigaea20. B. schaereri21. B capitis-regum22. B. insignisX23. B. erubescens24. B. subdisciformis25. B. lindigeri25. Physconia grisea21. Ph. grisea

Morata-Titulcia, Madrid (MAF 7028)Segarra, Cataluna (BCC 13365)

Santos de la Humosa, Madrid (MAF 7029)Villalazan, Zamora (MAF 6227)Montblanc, Cataluna (BCC 13749)

Cap de Creus, Cataluna (BCC 13752)

Castello d'Empuries, Gerona (MAF 8657)Cap de Creus, Cataluna (BCC 13571)Port de Selva, Gerona (MAF 8656)

Cap de Creus, Cataluna (BCC 13750)

Casa de Campo, Madrid (MAF-Cub GPAR2)

AF352316AF352317AF101286AF352319AF352318AF352320AF224351AF352321AF250793AF532168AF352322AF532167AF276070AF250784AJ421415AJ421418AF250787AF250788AF250785AF250871AF250783AF250790AF250786AF352323AF250789AF542506AF224368

*DNA extraction using DNeasy Plant Mini Kit (QIAGEN).fBCC, Departament de Botanica, Facultat de Biologia, Universitat de Barcelona; MAF, Departamento de

Botanica, Facultad de Farmacia, Universidad Complutense de Madrid.^Incorrectly identified as Buellia papilla in Grube & Arup (2001) and GenBank.

This study had three main objectives, totest: (1) the robustness of the generic statusof Diplotomma s. str. in relation to Buelliaand Diploicia; (2) whether Diploicia canescensand D. subcanescens are a species pair as hasoften been presumed; and (3) if Diplotommarivas-martinezii is genetically distinct fromD. venustum.

temperature. Physconia grisea (Lam.) Poelt was used asthe outgroup.

DNA extraction

Total fungal DNA was extracted from the thalliaccording to the CTAB extraction method (Crespoet al. 1997; Cubero et al. 1999). Occasions when DNAextraction was carried out using the DNeasy Plant MiniKit (QIAGEN) are indicated in Table 1.

Material and MethodsLichen material

Details of the material, area of collection, andlocation of reference specimens, are presented inTable 1. Specimens were air-dried and stored at room

PCR amplification

Amplification of the internally transcribed spacerregion of the rDNA gene cluster was undertaken withthe primer ITS1 F, as described by Gardes & Bruns(1993), or ITS5 and ITS4, as described by White et al.(1990). ITS,p was designed to be specific for fungalsequences at the 3' end of the small subunit gene of the


rDNA and overlaps with ITS5, whereas ITS4 has beendescribed as a 'universal' primer and hybridizes at the 5'end of the large subunit gene (Gardes & Bruns 1993).

Amplification was carried out in 50 (jl volumesconsisting of 5 JJ.1 of 10 x DNA polymerase buffer(Biotools), 5 ul of 25 mM MgCl2, 1 ul of a dinucleotidetriphosphate (dNTP) containing 10 mM of each base,2-5 nl of each primer 10 uM, 1-25 ul of a DNA polym-erase (Biotools) and 8 ul of a 50-fold dilution of stockDNA. The PCR conditions were: denaturation 1 min at94°C; annealing 1 min at 54°C; extension 2 min at 72°C(35 cycles); final extension 5 min at 72°C.

PCR products were purified with Biotools' PCRpurification kit and sequenced, using the same primers,by the PRISM dRhodamine Terminator Candlesequencing Ready Reaction kit (Applied Biosystems)with detection on a 373A stretch automatic sequencingapparatus (Applied Biosystems). The total fragmentwas sequenced, comprising the 3' end of the smallsubunit gene, ITS 1, the 5-8S gene, ITS2 and the 5'terminus of the large subunit gene.

Sequence analysis

Complementary strands were compared with theassistance of Windows SeqMan (DNAStar) to check forreading errors and when possible, resolving ambiguities.Sequences were then aligned using Clustal X version65b (Jeanmougin et al. 1998) and visually adjusted.Ambiguous aligned sites were removed, and the finalalignment was used for the analysis. Maximumparsimony was applied to the data using the PAUP 4.0program (Swofford 1999) with gaps being interpretedas missing data. The heuristic search option was usedemploying random addition of sequences and 100replicates were generated with the tree bisection-reconnection (TBR) method of branch swapping, andthe MulTrees options in effect. A neighbour-joiningtree based on Kimura 2-parameter distances wasalso calculated using PAUP 4.0. Node reliability wasassessed from 1000 bootstraps in both cases.

Isolation and culture of mycobionts

Isolated multispores were obtained from apotheciaof Diplotomma rivas-martinezii (MAF 7028) and D.venustum (MAF 7029). The fungi were grown fromdischarged ascospores following Ahmadjian (1993).After germination, uncontaminated multispore isolateswere maintained on BBM, pH 5-85 (Deason & Bold1960) or transferred to 4% glucose LBM, pH 5-30(Lilly & Barnett 1951) or malt yeast extract, pH 5-43(11 distilled water, containing malt extract 5'00 g,glucose 2-00 g, yeast extract 0-25 g). The mediumcontained 1-5% w/v agar. Simultaneously, enrichedmalt yeast extract was prepared by adding 40 ml of soilextract, at pH 5-80 (Esser 1976). Soil (500g) from thecollection area were suspended in 1 1 of bi-distilledwater and boiled for 1 h. The supernatant wasdecanted, filtered, and made up to 1 1. The pH waschecked and adjusted to pH 5-7 when necessary. As astock solution, 100 ml of this extract were made up to

1 1 with bi-distilled water and then sterilized. Theinoculated plates were incubated at 18 ± 2°C, followingMolina & Crespo (2000).

Results

DNA was extracted from all specimens andthe rDNA target sequence was amplified.These PCR products were sequenced andtheir length differences (Table 1) werefound to be due to a group I intron ofapproximately 200 bp inserted at the 3' endof the small subunit (1516). Previous reportsof introns within lichen-forming fungi haveidentified a number of insertion sites withinthe ribosomal small subunit gene (DePriest1994; Gargas et al. 1995).

The final data matrix contained 399nucleotides after removing the intron,ambiguous sequences and alignments;30-8% (123 nucleotides) were informative.Six trees which were equally parsimoniouswere obtained, each of 408 steps. The con-sensus parsimony tree obtained using thismatrix (Fig. 1) had a consistency index (CI)of 0-822 and a retention index (RI) of 0-667and reliably distinguished two mono-phyletic groups. One of them corresponds toBuellia lindigeri and the other one groups allremaining samples into two clades; one ofthese has two sister branches. The first(Fig. 1A) groups the Diplotomma s. str.species included (i.e. Dm. rivas-martinezii,Dm. venustum, and Dm. alboatrum) andalso the species of Diploicia studied (i.e.Dc. canescens, and Dc. subcanescens). Theother clade (Fig. IB) groups all the remain-ing Buellia species, including the gener-ally accepted type species B. disciformis.Diplotomma and Diploicia species are re-spectively monophyletic and sister groupswith a reasonably robust bootstrap value(80%) in Fig. 1.

The distance tree (Fig. 2) shows a similartopology to the parsimony one. The Diploi-cia canescens and Dc. subcanescens collectionswere paraphyletic, and combined into asingle clade with high bootstrap support(92%). In contrast, the specimens ofDiplotomma rivas-martinezii formed amonophyletic clade with strong support

2002 Molecular phylogeny and status of. Diploida and Diplotomma—Molina et al. 513

701

84 p—

65

95

80

95

100

77

92

74

100

57

100

68

100

2. Dm.rivas-martinezii


I. Dm.rivas-martinezii

5. Dm.venustum

4. Dm.venustum

6. Dm.venustum

7. Dm.venustum

II. Dc.subcanescens

12. Dc.subcanescens

9. Dc.canescens

— 8. Dc.canescens

— 10. Dc.canescens

17. B.georgei

19. B.epigaea

18. B.dijiana

— 15. B.elegans

— 16. B.zoharyi

C20. B.schaereri

21. B.capitis-regum

r— 13. B.frigida

'— 22. B.insignis

24. B.subdisciformis

23. B.erubescens

14. B.disciformis

25. B.lindigeri

26. Ph.grisea

27. Ph.grisea

FIG. 1. Strict parsimony consensus tree. Branches are collapsed for bootstrap values of less than 50%. Physconiadistorta was selected as the outgroup. B. =Buellia; Dc. = Diploida; Dm. ^Diplotomma.

B

(84%) and separate from collections of Dm. on BBM for 4 months, a clear degenerationvenustum which was not resolved as a of the germinated hyphae was observed,monophyletic group itself. These alterations, detected by optical

When the cultured multisporic group of microscopy, resulted in cell death on thisisolates from Dm. venustum was maintained medium—probably as a consequence of the


60

100

82

98

100




— 6. Dm.venustum

— 4. Dm.venustum

5. Dm.venustum

7. Dm.venustum

75

87

93

11. Dc.subcanescens

12. Dc.subcanescens

9. Dc.canescens

53

8. Dc.canescens

10. Dc.canescens

17. B.georgei

100 | I 18. B.dijiana

19. B.epigaea

100 15. B.elegans

16. B.zoharyi

75 • 24. B.subdisciformis

52

23. B.erubescens

- 13. B.frigida

— 22. B.insignis

- 14. B.disciformis

20. B.schaereri

- 21. B.capitis-regum

B

25. B.lindigeri

— 26. Ph.grisea

— 27.Ph.grisea

0.1

FIG. 2. Neighbour-joining distance tree. Bootstrap values over 50% are indicated. Scale bar indicates 0-1 Kimura-2distance units. B. -Buellia; Dc. -Diphkia; Dm. -Diplotomma.

absence of a carbon source (Fig. 3A), sincegerminated isolated multispores transferredto 4% glucose LBM generated a thallinemycelium after 3 months of 2-3 mm diam-eter (Fig. 3B). Diplotomma rivas-martineziideveloped through the first stages on BBM

only. However, transfer of the multisporesof the latter species to 4% glucose LBMresulted in collapse of the hyphae with struc-tural alterations culminating in cell death(Fig. 3C). Surprisingly, a mycelium with a95% success rate on inorganic medium was


FIG. 3. Developmental stages of Diplotomma grown under axenic conditions. A, Dm. epipolium on BBM after3 months; B, Dm. epipolium grown on 4% glucose LBM after 3 months (white arrow shows white superficial hyphaeand black arrow indicates dark internal hyphae); C, Dm. rivas-maninezii on 4% glucose on BBM after 3 months;

D, Dm. rivas-martinezi grown on BBM after 3 months. Scales: A, C & D= 15 urn; B = 0-2 mm.

observed 3 months after initial germinationon that medium. These hyphae had a longinterseptum distance and a low number oflipid drops, but structural alterations werenot observed (Fig. 3D). Neither Dm. rivas-martinezii nor Dm. venustum grew on maltextract, with or without soil extract.

Discussion

Generic conceptsA phylogenetic hypothesis for evolution in

the Physciaceae by Grube & Arup (2001),also based on ITS sequences, showed aclade where Diploicia canescens clustered


as the sister clade of Buellia capitis-regnumW. A. Weber, an endemic North Americanspecies with thick-walled muriformascospores and a non-placodioid thallus(Nordin 2000: fig. 13). However, whileGrube & Arup (2001) labelled this as having'Diplotomma' ascospores, these were notdefined and no Diplotomma s. str. specieswas included in their analysis. However, theresults from our study (Figs 1 & 2) show thatthe Diploicia and Diplotomma s. str. speciesstudied form a well-supported monophyleticgroup, a sister group of the clade groupingmost of the other species of Buellia s. lat.(Fig. 1). Although a multigenic study wouldbe ideal to confirm that Diploicia andDiplotomma are congeneric, the morphologi-cal and anatomical evidence and the highbootstrap levels support this decision. In anycase, the retention of Diploicia at the genericlevel and the keeping of Diplotomma s. str.species in Buellia are indefensible. Bothgroups do, however, form separate mono-phyletic clades, and it could be argued thatboth should therefore be retained. However,differences in the thallus alone do not justifythe recognition of separate genera in otherlichen groups, for example Caloplaca, and inthe absence of other supporting features wesuggest extending the circumscription ofDiplotomma s. str. to include Diploiciaspecies with a placodioid thallus, unitingthem under the oldest generic name,Diplotomma* Interestingly, this idea is farfrom new as Flotow (1849, 1850) alreadyincluded the type species of Diploicia withinDiplotomma when introducing the genericname.

Almost all species in this expandedconcept of Diplotomma already have validly

*The author citation given to the genus varies. It wasfirst mentioned by Flotow (1849: 129) 'ad int.' But nodescription was provided. However, Flotow (1850)provided a detailed account with diagnostic features(: 380) and inclusion in a generic key (: 381-2) and thename was clearly validly published in that work.The generic name has sometimes been attributed toMassalongo (1852), as by Greuter et al. (1993), butFlotow has been cited as author by several recentworkers (e.g. Nordin, 2000; Eriksson et al. 2001; Kirket al. 2001) and we see no reason to dissent from thatview.

published binomials under that genericname (i.e. Dm. alboatrum (Hoffm.) Flotow,Dm. canescens (Dicks.) Flotow, Dm. lutosumA. Massal.f, Dm. pharcidium (Ach.) M.Choisy, Dm. rivas-martinezii, Dm. scheideg-geriana (Bricaud & Cl. Roux) Nimis, andDm. venustum). We do not include B. capitis-regum here in view of its grouping in the'Buellia' clade in our trees, and the lack ofcalcium oxalate in the thallus; the structureof the ascospores merits further study. Onename change remains necessary, for Buelliapulverulenta which Nordin (2000) found tobelong to the Diplotomma s. str. clade in hiscladistic analysis based on non-molecularfeatures^. Whether some other lichenicolousspecies merit treatment in the genus requiresfurther study.

The status of 'Diploicia' subcanescensTwo of the three species (Hawksworth et al.1995) of Diploicia were used in our analysis:D. canescens (secondary sorediate species),and D. subcanescens (fertile primary species)which constitute a typical species pair (Poelt1970). First, the topology of the parsimonytree indicates that, at least for this DNAcharacter, genetically the taxon does notmerit recognition as two separate species,specimens of D. canescens being nested in aclade along with D. subcanescens. This resultis similar to that found in some otherexamples of 'species pairs' where the taxa arealso not monophyletic (Lohtander et al.1998; Cubero & Crespo 1999; Articus et al.2002). Elix et al. (1988) reported that D.subcanescens also differed from D. canescensin the presence of gyrophoric acid, but we donot consider that such a single unreplacedchemical warrants the recognition of sucha chemotype as a species (Hawksworth1976; Lumbsch 1998). Matzer et al. (1997)illustrated the ascospores and conidia of thetwo taxa which do not appear to differ

fThe correct name for Buellia subdispersa [syn.Diplotomma subdispersum (Krempelh.) Arnold] whenplaced in Diplotomma (Nordin 2000: 91).^Diplotomma pulverulentum (Anzi) D. Hawksw.,comb. nov. (basionym: Abrothallus pulverulentus Anzi,Cat. Lich. Sander.: 116, 1860; syn.: Buellia pulverulenta(Anzi) Jatta, Syll. Lieh. Ital: 400, 1900).


significantly. We therefore propose thatD. subcanescens is treated as a synonymof Diplotomma canescens.

The status of Diplotommarivas-martinezii

Some of the morphological differencesreported between Diplotomma rivas-martinezii and Dm. venustum, for instancethe colour or texture and habit of the thallussurface, may be due to different ecologicalconditions of the substrata or to localadaptations. However, the isolated fungalcomponents of the two species differedand behaved oppositely when cultured oninorganic and organic media. While Dm.venustum grew and developed a three-dimensional 'thallus-like' mycelium onthe enriched medium (4% glucose LBM;Fig. 3B), Dm. rivas-martinezii was com-pletely unable to grow on such a medium(Fig. 3C). The contrary occurred on basalmedium (BBM), where Dm. venustum didnot grow (Fig. 3A) but Dm. rivas-martineziideveloped an extensive superficial mycelium(Fig. 3D). In the latter case, three-dimensional structures were not observedand hyphal diameter and the number of lipiddrops were smaller (Molina et al. 1997).Additionally, the isolates from both speciesdiffered with respect to ejection, develop-ment rates, and the manner of germination(Molina & Crespo 2000). However, sincecultures were obtained from only one collec-tion of each species, nothing is known of anypossible infraspecific variation and too muchemphasis should not be placed on theseobservations. Nevertheless, such distinct be-haviours suggest that different geneticallydetermined biological or physiological pat-terns occur at least between the two samplescultured.

The molecular analysis of ITS sequences(Figs 1 & 2) showed that the three samplesof Dm. rivas-martinezii from gypsaceoussubstrata in different geographical regionswere monophyletic (Fig. IB). Samples ofDm. venustum collected near Dm. rivas-martinezii localities were placed further apartin the phylogenetic tree (for example, thetwo sites of Dm. rivas-martinezii and Dm.

venustum from Madrid are separated by nomore than 30 km). In evolutionary terms(Figs 1 & 2) Dm. rivas-martinezii could be amonophyletic species more recently derivedfrom a population of Dm. venustum. Al-though the bootstrap values of 65% (maxi-mum parsimony) and 60% (neighbourjoining) are too low to have the highestconfidence in this result, a paraphyleticrelationship is suggested in relationship toDm. alboatrum. However, the three samplesof Dm. venustum studied were not mono-phyletic. A number of taxa have been namedmorphologically as close to Dm. venustumand/or Dm. alboatrum (cf. B. lainea inClauzade & Roux 1985; synonymized withDm. alboatrum by Nordin 2000) and anyfurther taxonomic conclusions require aseparate molecular study of additionalpopulations. Genetic isolation leading to ap-parently cryptic species has been reported inan increasing number of cases in free-livingand lichen-forming fungi (Crespo et al.1999; Hawksworth 2001). Even in the eventof Dm. venustum being considered anheterogeneous taxon, the topology of thephylogenetic tree nevertheless supports theretention of Dm. rivas-martinezii as an inde-pendent taxon. The results from the prelimi-nary culture experiments suggest thatphysiological characteristics may reinforcethis interpretation. Moreover, the ITS re-gion has proved to be a good species-levelmarker for lichens (Groner & LaGreca1997; Crespo & Cubero 1998). Conse-quently these results justify the retention ofDm. rivas-martinezii at the level of species.

We are indebted to two anonymous referees forconstructive remarks on our original submission. Thiswork was supported by a grant from the DirectionGeneral de Investigation of the Consejeria deEducation, Cultura y Deporte (07B/0014/1997) of theComunidad Autonoma de Madrid and the projects(PB98 0774, and REN 2001-1272/GLO) of theMinisterio de Ciencia y Tecnologia of Spain. The workwas completed while D.L.H. was supported by thePrograma Ramon y Cajal.

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