303 Mycological Progress 4(4): 303–316, November 2005 A new circumscription of the lichen genus Nephromopsis (Parmeliaceae, lichenized Ascomycetes) Arne THELL1, Tiina RANDLANE2, Andres SAAG2, and Ingvar KÄRNEFELT3 The phylogeny of the cetrarioid lichens with bifusiform spermatia and dorsiventral thalli which contain usnic acid is reanalysed using three parts of the genome, ITS rDNA, β-tubulin and GAPDH sequences. Molecular data from five cetrarioid species are presented for the first time, and 13 new sequences are combined with sequences from the gene bank to delimit the genus Nephromopsis. A monophyletic clade of Nephromopsis, Tuckneraria, ’Cetraria’ leucostigma and ’C.’ melaloma is identified and circumscribed as one genus, Nephromopsis, which now includes 19 species. Four new combinations are presented. A key to the species is provided. Taxonomical novelties: Nephromopsis ahtii (Randlane & Saag) Randlane & Saag, Nephromopsis leucostigma (Lév.) A. Thell & Randlane, Nephromopsis melaloma (Nyl.) A. Thell & Randlane, Nephromopsis togashii (Asahina) A. Thell & Kärnefelt Keywords: Nephromopsis, cetrarioid lichens, Parmeliaceae, DNA-sequences, phylogeny, lichen taxonomy, eastern Asia istory of the genus Nephromopsis. Nephromopsis Müll. Arg. is one of the oldest cetrarioid genera (MÜLLER ARGOVIENSIS 1891). In the original description it included merely one species, Nephromopsis stracheyi (Bab.) Müll. Arg. which was said to have a thallus similar to Cetraria but the position of apothecia as in Nephroma (RANDLANE, THELL & SAAG 1995). Except for HUE (1899, 1900), ZAHLBRUCKNER (1928, 1930), ASAHINA (1935), GYELNIK (1931), SATÔ (1938) and RÄSÄNEN (1952), who accepted Nephromopsis, the genus was not in general use until LAI (1980) revived the name for three additional species in a survey of cetrarioid lichens of East Asia. RÄSÄNEN (1952), who summarized the genus, included 11 species and one variety; six of those taxa belong to Nephromopsis as circumscribed in this study. LAI (1980) combined several species in Nephromopsis, one of which was new, N. morrisonicola M. J. Lai (Tab. 1). Two of the 10 listed Nephromopsis-species were later accomodated in the genera Allocetraria Kurok. & M. J. Lai and Cetreliopsis M. J. Lai (RANDLANE, THELL & SAAG 1995; THELL et al. 1995). Further combinations were proposed by KUROKAWA (1991), Nephromopsis kurokawae (Shibuichi H & Yoshida) Kurok. and Nephromopsis laureri (Kremp.) Kurok., whereas the combination Nephromopsis isidioidea (Räsänen) Randlane & Saag was proposed the following year (RANDLANE & SAAG 1992). All species included in Nephromopsis by RANDLANE, THELL & SAAG (1995) and RANDLANE & SAAG (1998) still belong to the genus. Four of eight species positioned in Nephromopsis earlier are currently accomodated in Tuckermanopsis (’Tuckermannopsis’) Gyeln., namely T. ciliaris (Ach.) Gyeln., T. orbata (Nyl.) M. J. Lai (earlier usually named Nephromopsis californica Gyeln.), T. platyphylla (Tuck.) Hale, and T. platyphylloides (Asahina) M. J. Lai (KÄRNEFELT & THELL 2001; RANDLANE et al. 1997). Of the two most recently described Nephromopsis species, N. hengduanensis L. H. Chen and N. weii X. Q. Gao & L. H. Chen (CHEN & GAO 2001), the latter has been transferred to Tuckermanopsis because of its subglobose spores and absence of usnic acid (RANDLANE & SAAG 2003). The generic position of N. hengduanensis has yet to be confirmed by molecular data; the spores of this taxon are extremely large and supplied with a thick epispore, reminding of the spores of Parmelaria or Cetrelia, but otherwise unusual among cetrarioid lichens. Nephromopsis and allied groups Botanical Museum, Department of the Biological Museums, Lund University, Östra Vallgatan 18, S-22361 Lund, Sweden. 2 Institute of Botany and Ecology, University of Tartu, Lai Street 38, 51005 Tartu, Estonia. e-mail: randlane@ut.ee 3 Botanical Museum, Department of the Biological Museums, Lund University, Östra Vallgatan 18, S-22361 Lund, Sweden. e-mail Ingvar.Karnefelt@botmus.lu.se * Corresponding author: Arne Thell, Phone + 46 46 2228978; Fax + 46 46 2224234; e-mail: Arne.Thell@botmus.lu.se 1 A revision of Nephromopsis, Cetrariopsis and Cetreliopsis was published by RANDLANE, THELL & SAAG (1995), where the presence of pseudocyphellae on the lower side, the marginal, usually nephromoid apothecia, and the oblong ascospores were highlighted as being among the most characteristic features of Nephromopsis. Two originally monotypic genera, Cetrariopsis Kurok. and Cetreliopsis M. J. Lai (KUROKAWA 1980; LAI 1980), were thought to be the two genera most clo© DGfM 2005 304 THELL, RANDLANE, SAAG & KÄRNEFELT: A new circumscription of the lichen genus Nephromopsis Tab. 1: Two earlier circumscriptions of Nephromopsis compared with the present, or in this study proposed, taxonomic positions of the species. Species proposed as belonging to Nephromopsis in the present circumscription are written in bold style. Nephromopsis sensu RÄSÄNEN (1952) Nephromopsis sensu LAI (1980)* 1. N. ciliaris (Ach.) Hue = Tuckermanopsis ciliaris (Ach.) Gyeln. 2. N. daibuensis (Räsänen) Räsänen = N. laxa M. Satô 3. N. delavayi Hue = N. ornata (Müll. Arg.) Hue (Lai 1980: 224) 4. N. endocrocea Asahina 5. N. ornata (Müll. Arg.) Hue 6. N. pallidula (Riddle) Herre = Ahtiana pallidula (Riddle) Goward & A. Thell 7. N. platyphylla (Tuck.) Herre = Tuckermanopsis platyphylla (Tuck.) Hale 8. N. platyphylloides Asahina = Tuckermanopsis platyphylloides (Asahina) M. J. Lai 9. N. rhytidocarpa (Mont. & Bosch) Zahlbr. = Cetreliopsis rhytidocarpa (Mont. & Bosch) M. J. Lai 10. N. rugosa Asahina 11. N. stracheyi (Bab.) Müll. Arg. 12. N. stracheyi var. nephromoides (Nyl.) Räsänen = N. nephromoides (Nyl.) Ahti & Randlane 1. N. asahinae (Satô) Räs. = Cetreliopsis asahinae (M. Satô) Randlane & A. Thell 2. N. endocrocea Asahina 3. N. globulans (Nyl.) M. J. Lai = Allocetraria globulans (Nyl.) A. Thell & Randlane 4. N. laxa (Zahlbr.) M. Satô 5. N. morrisonicola M. J. Lai 6. N. nipponensis (Asahina) M. J. Lai = Nephromopsis pseudocomplicata (Asahina) M. J. Lai 7. N. ornata (Müll. Arg.) Hue 8. N. pseudocomplicata (Asahina) M. J. Lai 9. N. rugosa Asahina 10. N. stracheyi (Bab.) Müll. Arg. * LAI (1980) treated exclusively East Asian species. sely related to Nephromopsis. Cetrariopsis, with the single species C. wallichiana (Taylor) Kurok., was preliminarily characterized by laminal apothecia (KUROKAWA 1980). A second species was described later, C. laii A. Thell & Randlane (RANDLANE, THELL & SAAG 1995). Cetrariopsis was synonymized with Nephromopsis when laminal apothecia had been observed in Nephromopsis as well as marginal ones in Cetrariopsis (RANDLANE, SAAG & THELL 1997), and the combination Nephromopsis pallescens (Schaer.) Y. S. Park (PARK 1990) turned out to be the correct name for the Cetrariopsis wallichiana. The genus Cetreliopsis was described by LAI (1980) to accommodate the Cetraria rhytidocarpa-complex which occurs mainly in southeastern Asia. The group included three different species from Java, Philippines and Taiwan with a broadlobed morphology similar to several Nephromopsis species. Lai synonymized all three species on morphological and chemical grounds; he also pointed out the affinities to Cetrelia and Nephromopsis, combining the names of these genera for the new monotypic genus. According to our present knowledge, Cetreliopsis includes seven species (RANDLANE, THELL & SAAG 1995; LAI & ELIX 2002; RANDLANE & SAAG 2003). The genus is morphologically differentiated by pseudocyphellae on both sides; furthermore, it has a characteristic secondary chemistry, distinguished by the presence of a complex of β-orcinol depsidones which demonstrate chemosyndromic variation analogous to that which was identified in the genus Cetrelia (CULBERSON & CULBERSON 1976). Earlier interpretations of phylogenetic affinities of Cetreliopsis to Cetrelia or Nephromopsis have not been confirmed. A preliminary DNA-study, based on one sequence only, placed the type spe© DGfM 2005 cies of Cetreliopsis, C. rhytidocarpa (Mont. & Bosch) M. J. Lai, close to Cetraria s. str. (THELL et al. 2002). Another new genus, Tuckneraria Randlane & A. Thell, was proposed in the 1990s (RANDLANE et al. 1994). Three of the species included were earlier accomodated in Nephromopsis, i. e., T. laureri (Kremp.) Randlane & A. Thell, T. laxa (Zahlbr.) Randlane & A. Thell and T. pseudocomplicata (Asahina) Randlane & Saag, and one new species, Tuckneraria ahtii Randlane & Saag, was described. Tuckneraria togashii (Asahina) Randlane & A. Thell, a rare Japanese endemic (ASAHINA 1953), was transferred from Cetraria one year later (THELL, KÄRNEFELT & RANDLANE 1995). One more species, Tuckneraria sikkimensis Divakar and Upreti has recently been described from India (DIVAKAR & UPRETI, unpublished). The genus Tuckneraria differed from Nephromopsis by the presence of cilia and subglobose ascospores, and was at that time believed to belong to a separate evloutionary line of cetrarioid lichens with globose or subglobose spores (THELL 1996; SAAG et al. 2002). A very close relationship between Nephromopsis and Tuckneraria was evident after a phylogeny analysis inferred from ITS rDNA and nuclear β-tubulin sequences (THELL et al. 2002). The three investigated Tuckneraria species, T. ahtii, T. laureri and the type species of the genus, T. pseudocomplicata, constituted a monophyletic group, together with ’Cetraria’ leucostigma Lév. and ’C.’ melaloma (Nyl.) Kremp., nested within the Nephromopsis clade. The inclusion of the former Cetrariopsis was confirmed by its type species, now known as Nephromopsis pallescens. The systematic position of two rare Himalayan cetrarioid lichens, ‘Cetraria’ leucostigma Lév. and ’C.’ melaloma (Nyl.) Kremp. has stayed unclear for a long time. The two spe- 305 Mycological Progress 4(4) / 2005 cies are only distantly related with Cetraria in a strict sense and do not belong to this genus in its revised form (KÄRNEFELT, MATTSSON & THELL 1993). Nevertheless, ’C.’ leucostigma and ’C.’ melaloma were never combined elsewhere because of the sparse information on reproductive characters. Ascospores have never been reported for ’C.’ melaloma and for ’C.’ leucostigma only once. The spores were ellipsoid (AWASTHI 1982) as in many other cetrarioid genera, e.g., Cetreliopsis, Flavocetraria or Nephromopsis. Spermatia have never been observed in either species, and apothecia are still unknown for ’C.’ melaloma. Both species were described in detail by RANDLANE, SAAG & OBERMAYER (2001). One of the taxa, ‘C.’ melaloma’, has specific pseudocyphellae (surrounded by a dark rim) on both surfaces like species belonging to Cetreliopsis. The secondary chemistry, characterized by usnic acid in the upper cortex and lichesterinic-protolichesterinic type fatty acids in the medulla, and the terricolous habit are features in common with Allocetraria and Flavocetraria. Their distribution, restricted to high altitudes and in very limited area in the Himalayas, is similar to that of Allocetraria species. From these observations it has been speculated that ’C.’ leucostigma and ’C.’ melaloma have affinities to Cetreliopsis or Allocetraria. A preliminary position was clarified by DNAdata (ITS and β-tubulin sequences) – both taxa were nested within a clade containing the species of Nephromopsis and Tuckneraria (THELL et al. 2002). Still, new combinations were not proposed in that study. The monophyletic core of cetrarioid lichens Cetrarioid lichens used to be a vaguely defined group of the Parmeliaceae, usually recognized by an erect foliose habit and marginal or submarginal position of apothecia and pycnidia. Well aware of the polyphyletic origin of this form group, RANDLANE, SAAG & THELL (1997) listed 135 species within 23 genera. Phylogenetic analyses based on ITS and β-tubulin sequences combined with morphology, anatomy and secondary chemistry, revealed a monophyletic core of cetrarioid lichens, composed of 13 genera (THELL et al. 2002), composed of ca. 85 species. The delimitation of the group was confirmed later when GAPDH sequences supplemented earlier investigations (THELL et al. 2004). The cetrarioid genera Asahinea W. L. Culb. & C. F. Culb, Cetrelia W. L. Culb. & C. F. Culb., Coelopogon Brusse & Kärnefelt, Cornicularia Hoffm., Dactylina Nyl., Esslingeriana Hale & M. J. Lai, and Platismatia W. L. Culb. & C. F. Culb. were positioned outside the monophyletic clade of cetrarioid lichens at different places of the family (THELL et al. 2002). Parmelaria D. D. Awasthi is probably closely related to Parmotrema A. Massal. The monotypic genus Nimisia, endemic to southern South America, has so far not been investigated using DNA-characters. The Tuckermanopsis-group The cetrarioid clade was divided into two large subclades, a division that to a large degree correlates with conidial shape (THELL et al. 2002). One of the subclades exclusively includes taxa with bifusiform conidia: Ahtiana Goward, Arctocetraria Kärnefelt & A. Thell, Flavocetraria Kärnefelt & A. Thell, Kaernefeltia A. Thell & Goward, Nephromopsis Müll. Arg., Tuckermanopsis Gyelnik, Tuckneraria Randlane & A. Thell, the ’Cetraria’ fendleri group, later described as a separate genus, Tuckermanella Essl. (ESSLINGER 2003), and ’Cetraria’ leucostigma and ’C.’ melaloma. The second large subclade, centered around Cetraria s. str. (KÄRNEFELT, MATTSSON & THELL 1993), is mainly composed of taxa characterized by conidia with one swelling, i.e. filiform, sublageniform or citriform shapes. This subclade includes Allocetraria Kurok. & M. J. Lai, Cetraria Ach., Cetrariella Kärnefelt & A. Thell, Cetreliopsis M. J. Lai, ’Tuckermanopsis’ subalpina (Imshaug) Kärnefelt, ’T.’ platyphylla (Tuck.) Hale and Vulpicida J.-E. Mattsson & M. J. Lai. Cetraria sepincola (Ehrh.) Ach., having typically bifusiform spermatia, is a striking exception within this single-fusiform subclade (THELL et al. 2004). The monotypic genus Masonhalea Kärnefelt was positioned in a separate clade as a sister group of either of the large subclades (THELL et al. 2002, 2004). It is characterized by short bacillariform conidia, unique for cetrarioid lichens (KÄRNEFELT 1977). The DNA data matrix of the formerly described subclade is complemented, preferably with GAPDH sequences, and phylogenetically reanalysed to present a circumscription of Nephromopsis complemented with the new data. Some representatives from the other subclades (species of Allocetraria, Cetraria, Masonhalea and Vulpicida) are also included in the analysis. Two species of Melanelia Essl., in earlier analyses identified as the sister group of the cetrarioid clade, are selected as the outgroup. Material and methods Selected material Thirteen new sequences were produced from fresh collections from Bhutan, Greenland, Italy, Philippines and U. S. A. (Tab. 2). Molecular data from five cetrarioid species, Arctocetraria andrejevii, Kaernefeltia californica, Nephromopsis nephromoides, Tuckermanopsis orbata and Vulpicida viridis, are presented for the first time. The matrix was complemented with additional sequences downloaded from the GenBank (NCBI: http://www.ncbi.nlm.nih.gov) published earlier. Extraction and amplification Minute fragments of the fresh collections were ground with sterile plastic pestels. Total DNA was extracted using the DNEasy Plant Mini Kit from Qiagen following the accompanying protocol. Three different DNA-fragments, the complete ITS1-5.8S-ITS2 region, partial β-tubulin and glyceraldehyde phosphate dehydrogenase (GAPDH) regions, were © DGfM 2005 306 THELL, RANDLANE, SAAG & KÄRNEFELT: A new circumscription of the lichen genus Nephromopsis Tab. 2: Material used in the study. GenBank accession numbers for sequences produced within this study start with DQ. Species Extr. No. Sample-ID GenBank accession numbers ITS1-5.8S-ITS2, beta-tubulin, GAPDH Ahtiana pallidula Ahtiana sphaerosporella Allocetraria stracheyi Arctocetraria andrejevii Arctocetraria nigricascens Cetraria islandica Cetrariella delisei Flavocetraria cucullata Flavocetraria nivalis Kaernefeltia californica Kaernefeltia merrillii Masonhalea richardsonii Nephromopsis ahtii Nephromopsis komarovii Nephromopsis laureri Nephromopsis laureri Nephromopsis leucostigma Nephromopsis melaloma Nephromopsis morrisonicola Nephromopsis morrisonicola Nephromopsis nephromoides Nephromopsis ornata Nephromopsis ornata Nephromopsis pallescens Nephrom. pseudocomplicata Nephromopsis stracheyi Tuckermanella coralligera Tuckermanella fendleri Tuckermanella weberi Tuckermanopsis americana Tuckermanopsis chlorophylla Tuckermanopsis orbata Tuckermanopsis platyphylla Vulpicida viridis Melanelia hepatizon Melanelia stygia 1268 73 875 1364 793 548 234 932 700 1704 190 794 607 621 610 938 604 430 903 904 1693 620 624 618 907 606 1158 612 614 148 1022 1267 75 1291 934 922 USA, Montana, Hauck s. n (priv. herb.) Canada, B.C., Miao & Taylor (TDI 211) China, Sichuan, Obermayer 8139 (GZU) SW Greenland, Hansen, exs. 836 (LD) Canada, N.W.T., Westberg 1614 (LD) Estonia, Tartumaa, Thell 9901 (LD) Iceland, N. Mulasysla, Thell 9714 (LD) Austria, Feuerer & Thell 64185 (HBG) Iceland, Frödén s. n. (LD) USA, Oregon, McCune 27703 (LD) Canada, B.C., Thell 9698 (LD) Canada, Yukon T., Westberg 1246 (LD) Bhutan, Paro Distr., Søchting 8489 (LD) Russia, Promorie, Skirina 10972 (LD) Bhutan, Søchting 8124 (LD) Italy, Feuerer & Thell 64288 (HBG) Bhutan, Thimpu, Søchting 9151 (LD) Bhutan, Thimpu, Søchting 9181 (LD) China, Sichuan, Obermayer 8279 (GZU) China, Sichuan, Obermayer 8282 (GZU) Philippines, Mindanao, Ejem s. n. (H) Russia, Primorie, Skirina 10967 (LD) Russia, Pri., Kudryavtseva 10980 (LD) Bhutan, Søchting 8206 (LD) China, Sichuan, Obermayer 8276 (GZU) Bhutan, Søchting 8095 (LD) USA., N.M., Worthington 28821 (ASU) USA, Arizona, Westberg 543 (LD) USA, Arizona, Westberg 548 (LD) Canada, B.C., Goward 961350 (LD) S Africa, Feuerer & Thell s. n. (HBG) USA, Montana, Hauck s. n. (priv. herb.) Canada, B.C., Thell 75 (LD) USA, Connecticut, Feuerer s. n. (HBG) Italy, Feuerer & Thell 64248 (HBG) Italy, Feuerer & Thell 64247 (HBG) AY353709* AF072224* AF404129* DQ004575 AF254628* AF228296* AF228305* AF451739* AF451794* DQ004571 AF072230* AF254617* AF404122* AF451779* AF404123* AF451786* AF451777* AF451778* AF451780* AF451781 DQ004574 AF451782 AF451783* AF451784* AF404131* AF451785* AF457924* AF451791* AF451792* AF072233* AF451789* DQ004572 AF072236* DQ004573 AF451776* AF451775* – – AF449733* – AF449728* AF449740* AF449737* AF449719* – – – AF449730* – AF449722* AF449723* AF449724* AF449716* AF449720* – – – – AF449721* – AF449725* – – – – AF449726* AF449727* – AF449741* – DQ004576 AY074778* AY249602* – – – AY249599* AY249594* AY249595* AY249601* – – AY249598* – – – DQ004579 DQ004583 – – DQ004578 AY249603* AY249605* DQ004582 – – – AY249600* – DQ004581 DQ004580 DQ004577 AY249607* * Earlier published sequences (SAAG et al. 2002; THELL 1998; THELL et al. 2000, 2002, 2004), accessible in the GenBank, http://www.ncbi.nlm.nih.gov amplified using the following primer pairs: ITS 4 and ITS 5 for the ITS region (WHITE et al. 1990), bt3LM and bt10LM for the partial β-tubulin gene (MYLLYS, LOHTANDER & TEHLER 2001; MYLLYS, STENROOS & THELL 2002), and gpd1 and gpd2 for the partial GAPDH gene (MYLLYS, STENROOS & THELL 2002). Ready To Go PCR beads (in 0.2 ml tubes) from Pharmacia Biotec Inc. were dissolved in 11.8 µl distilled water, 0.35 µl of a 16 µM concentration of each of the primers. The ITS fragments were amplified with a Perkin-Elmer Gene Amp PCR System 9700 thermal cycler. 12.5 µl of the concentrated DNA extractions were added to the solution, resulting in final reaction volumes of ca. 25 µl. The PCR started with 2 min. at 95 °C, followed by a 30–35 cycle schedule using a denaturation temperature of 95 °C for 1 min., an annealing temperature of 60 °C for 1 min., and an extension temperature of 72 °C for 1 min. © DGfM 2005 The PCR products were purified with QIAquick PCR purification kit, and diluted in 30 µl of the enclosed elution buffer. A 25-cycle sequencing PCR with a denaturation temperature of 96 °C for 10 sec., an annealing temperature of 50 °C for 5 sec., and an extension time of 60 °C for 4 min. was performed to amplify the DNA-fragments prior to the sequencing procedure. 12 µl deionized water including 30–90 ng of the purified PCR-product and 3.2 pmol of the same primers used for amplification except for ITS 5 which were replaced by ITS1LM (MYLLYS et al. 1999). The primers were used in separate reactions, each mixed with 8 µl BigDye Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq Polymerase FS from Perkin Elmer according to the accompanying protocol. The sequences were produced using an automatic sequencer, ABI Prism 377 from Perkin-Elmer. 307 Mycological Progress 4(4) / 2005 Sequence alignment and phylogeny methods All introns were removed and the sequences were aligned manually. All new sequences, including their introns, were submitted to the GenBank (http//ncbi.nlm.nih.gov). Analyses were carried out using the programs PAUP 4.0b (SWOFFORD 1998) and MrBAYES (RONQUIST & HUELSENBECK 2003) version 3.0b4, based on a combined matrix of the ITS, β-tubulin and GAPDH sequences. The matrix was composed of 33 species and 36 samples, including the outgroup Melanelia, the sister group of the monophyletic cetrarioid clade, according to earlier analyses (THELL et al. 2002, 2004). Phylogenetic analyses were performed using a Bayesian approach (MrBayes 3.0b4), together with maximum parsimony and maximum likelihood as optimization criteria, as implemented in PAUP 4.0b. In the parsimony analysis, a bootstrap consensus tree based on 1000 replicates was calculated using the full heuristic search option and the TBR branch swapping method. Gaps were treated as missing characters. Groups with a bootstrap support of 50 % or higher were retained. In the likelihood analysis, the follwing settings were used: heuristic search; random addition sequence; TBR branch swapping; settings from the best-fit model (GTR+I+G) selected by AIC in MrModeltest 2.2; multrees option in effect. Branch support was estimated by bootstrap analysis of 20 replicates. The data were also analysed using a Bayesian approach (LARGET & SIMON 1999). Posterior probabilities were approximated by sampling trees using a variant of Markov Chain Monte Carlo (MCMC) method called Metropolis-coupled Markov Chain Monte Carlo (MCMCMC). Settings applied: the bestfit model (GTR+I+G) selected by AIC in MrModeltest 2.2; number of generations = 1,000,000; number of simultaneous chains = 4; sample frequency = 100; ‘temperature’ = 0.2. No molecular clock was assumed. The initial 1000 trees were discarded as burn-in before stationarity was reached. Using sumt command of MrBayes, a majority-rule consensus tree was calculated from 9001 trees sampled after reaching likelihood convergence to calculate the posterior probabilities of the tree nodes. Phylogenetic trees were drawn using TreeView. Results and discussion Data matrix The data aligned set included a total of 2082 characters, including gaps, of which 286 offered parsimony information. The maximum parsimony (MP) analysis resulted in 19 shortest trees with a length of 1074, CI = 0.6034, RI = 0.5761, RC = 0.3476. Support values of 50 % or above are indicated in the bootsrap consensus tree (Fig. 1). In the maximum likelihood (ML) analysis, the total number of rearrangements tried was 40866, and the score of the best tree found – 8546.88072. Bootstrap support of 50% or more is shown in the tree (Fig. 2). By performing MrBayes multiple chains starting from random trees, the inferences for independent chains were the same. Credible sets of trees for one chain (7646 trees sampled) are as follows: 50 % credible set contains 3146 trees, 90 % credible set contains 6746 trees, 95 % credible set contains 7196 trees, 99 % credible set contains 7556 trees. Posterior probability values equal or above 50 % are indicated in Fig 3. Phylogenetic trees More than half of all cetrarioid species have been molecularly investigated, and a large amount of sequences have been published (SAAG et al. 2002; THELL 1998; THELL, STENROOS & MYLLYS 2000, THELL et al. 2002, 2004). Five additional species are sequenced within this study for the first time and the group on which this study is focussed is one of the most intensively treated in this respect among lichens (Tab. 2). Three phylogenetic trees, based on different anaylses of the same data matrix are presented. All three trees (Figs 1-3) are divided into two main clades which correlate well with the groups defined by phylogeny and using the spermatial shape according to THELL et al. (2002). Clade I, with 78 % bootstrap support in MP analysis (Fig. 1.) and 100 % in ML analysis (Fig. 2), includes cetrarioid taxa with fusiform spermatia with one swelling, here represented by four species representing the genera Allocetraria, Cetraria, Cetrariella and Vulpicida. A strongly supported clade II (86 % and 89 % bootstrap support, respectively) is composed of two distinct sister groups, the moderately supported Masonhalea-Tuckermanopsis platyphylla subclade III, and the strongly supported subclade IV, composed of cetrarioid taxa with bifusiform spermatia and a pseudobifacial thallus symmetry. The latter group consists of ten branches (V–XV on Figs 1–2) with unresolved positions in both parsimony and likelihood bootstrap trees. The genera Flavocetraria, clade VI, and Kaernefeltia, clade VII, are composed of five species of which four are represented in this analysis. For Kaernefeltia, DNA-information from the type species, K. californica (Tuck.) A. Thell & Goward, is presented for the first time. The genus Tuckermanella, clade VIII, is represented by three of six species (ESSLINGER 2003). All three genera have considerable or moderate bootstrap support in both MP and ML analyses (Flavocetraria – 66 % in MP, 84 % in ML; Kaernefeltia – 81 % in MP, 75 % in ML; Tuckermanella – 80 % in MP, 100 % in ML). Tuckermanopsis and Ahtiana, clades III, IX–XII, however, appear as paraphyletic genera. The position of Tuckermanopsis platyphylla, together with Masonhalea richardsonii, is unexpected, although the two species are gathered by a very moderate support (58 % in MP, 63 % in ML). The same species had independent positions in a previous analysis based on ITS and β-tubulin sequences (THELL at al. 2002, Clade A). The topology of the tree received by the Bayesian analysis (Fig. 3) is the same regarding clades I–IV, with high posterior probability values (93–100 %). Minor differences are in the position of clades V–XII indicating that the genera Kaernefeltia and Tuckermanella might be phylogenetically related. © DGfM 2005 308 THELL, RANDLANE, SAAG & KÄRNEFELT: A new circumscription of the lichen genus Nephromopsis Fig. 1: Parsimony analysis: bootstrap consensus tree from 1000 replicates based on a matrix of ITS, β-tubulin and GAPDHsequences. Support values of 50 ore more are indicated above branches; clade numbers (see text) are below branches. Nevertheless, the genus Flavocetraria is supported, and the genera Ahtiana and Tuckermanopsis appear paraphyletic also according to this analysis. Clade XIII, on which this study is focused, delimits Nephromopsis as proposed here. It includes 11 species of 19 known in the genus. This Nephromopsis clade has indeed a weak bootstrap support, 55 % in MP tree and posterior probability 79 % in the Baeysian analysis. In the ML analysis, N. ornata (Müll. Arg.) Hue (branch XV) is not included in the clade and the position of N. ornata remains unresolved. However, the position of this species [and its suggested relatives © DGfM 2005 N. endocrocea Asahina and N. isidioidea (Räsänen) Randlane & Saag, not sequenced in this study] in the genus Nephromopsis is maintained since no better positions have been found. See further comments on N. ornata below. Clade XIV, having a much stronger support (81 % bootstrap and 100 % posterior probability, accordingly) covers all other analysed Nephromopsis taxa, incl. the former type species of Cetrariopsis, now known as Nephromopsis pallescens, three species until now accomodated in Tuckneraria, T. ahtii, T. laureri & T. pseudocomplicata, and ’Cetraria’ leucostigma and ’C.’ melaloma. 309 Mycological Progress 4(4) / 2005 Fig. 2: Likelihood analysis: bootstrap consensus tree from 20 replicates based on a matrix of ITS, β-tubulin and GAPDHsequences. Support values of 50 ore more are indicated above branches; clade numbers (see text) are below branches. Taxonomic conclusions Nephromopsis Müll. Arg., Flora 74: 374. 1891. Based on these results, it is proposed that the genus Tuckneraria, ’C.’ leucostigma, and ’C.’ melaloma are included in Nephromopsis. Four new combinations are presented. Nephromopsis is thereby a genus of 19 species, being the largest among the cetrarioid lichens. Type species: Nephromopsis stracheyi (Bab.) Müll. Arg. – Cetrariopsis Kurok., Mem. Natl. Sci. Mus. Tokyo 13: 140. 1980. – Tuckneraria Randlane & A. Thell, Acta Bot. Fenn. 150: 144-146. 1994. Nephromopsis is a genus of 19 foliose, usnic acid containing species, of which 18 are distributed in eastern or southeastern Asia; the exception being the disjunct N. laureri which occurs in central European, Asian and South American mountains (see distribution map in RANDLANE & SAAG 2004). However, material of European and Asian origin may represent separate © DGfM 2005 310 THELL, RANDLANE, SAAG & KÄRNEFELT: A new circumscription of the lichen genus Nephromopsis Fig. 3: Bayesian analysis: 50 %-majority-rule consensus tree of 9001 trees from MCMC tree sampling procedure based on a matrix of ITS, beta-tubulin and GAPDH-sequences. The numbers above branches correspond to the Bayesian posterior probability (%) of the node to which a branch points; the numbers below branches are those of clades (see text). species according to this and also earlier DNA-investigations (THELL et al. 2002). The diagnostic traits of the genus are as follows: thallus foliose; bifacial with similar upper and lower cortex anatomy, pseudocyphellae present at least on the lower surface; cilia, isidia and secondary lobules occur in several species, soredia known only in a single taxon (N. laureri); apothecia marginal on the lower side of the thallus or submarginal, rarely laminal on the upper side, from small (1–2,5 mm) to very large (to 32 © DGfM 2005 mm in diameter); ascospores one-celled, 6–12 × 2,5–6 µm, oblong, subglobose or globose; spermatia bifusiform, 4–5 × 1–1,5 µm; usnic acid in the upper and lower cortex; various fatty acids and/or some orcinol or β-orcinol depsides/depsidones in the medulla. All species except for N. kurokawae (SHIBUISHI & YOSHIDA 1982) have recently been characterized and their distribution mapped (Tab. 3). 311 Mycological Progress 4(4) / 2005 New combinations Nephromopsis ahtii (Randlane & Saag) Randlane & Saag comb. nov. Basionym: Tuckneraria ahtii Randlane & Saag, Acta Bot. Fenn. 150. 147–148, 1994. Type: China. Prov. Yunnan, Lijiang County, Mt. Yulongshan, lower central E slope, Ganheba, 3200-3300 m, 27°06’N, 100°14’E, on Abies, 23 April 1987, Teuvo Ahti, JianBin Chen & Li-Song Wang 46649 (H!, holotype; TU!, isotype). Herbarium specimens of this species have frequently been labelled as Nephromopsis delavayi Hue, although several characters do not correspond with the original description (HUE 1899, 1900). However, LAI (1980) showed that N. delavayi is a synonym of N. ornata. N. ahtii differs from the latter species by such characteristic features as white medulla, presence of marginal cilia, shape of ascospores (subglobose to globose). It occurs in southern provinces of China, Taiwan, Nepal and Bhutan (RANDLANE & SAAG 2004). Nephromopsis togashii (Asahina) A. Thell & Kärnefelt comb. nov. Basionym: Cetraria (Platysma) togashii Asahina. J. Jap. Bot. 28: 136, 1953. Type: Japan. Honshu, Prov. Suruga, Gotemba-machi, 14 April 1952, Y. Asahina & M. Togashii no. 28810 (TNS!, lectotype). – Tuckneraria togashii (Asahina) Randlane & A. Thell, J. Hatt. Bot. Lab. 78: 238–242, 1995. Shape of ascospores (globose to subglobose), presence of cilia, although sparse, and pseudocyphellae on the lower side were the main reasons for placing this isidiate species in Tuckneraria (THELL, KARNEFELT & RANDLANE 1995). However, a position in the proposed wider circumscription of Nephromopsis is presumably the best destination for the species. Originally it was described as a Japanese endemic, but it has since been found in China and Taiwan (WEI 1991). Comments on some other Nephromopsis species Nephromopsis leucostigma (Lév.) A. Thell & Randlane comb. nov Nephromopsis kurokawae (Shibuichi & K. Yoshida) Kurok. Basionym: Cetraria leucostigma Lév., in Jacquemont, Voyage dans l’Inde Bot.: 180, 1841–1844. Type: India orientalis, dédit J. H. Léveillé (H-NYL 36 083!, neotype, proposed by Awasthi 1982). – Platysma leucostigmum (Lév.) Nyl., Mém. Soc. Sci. Nat. Cherbourg 5: 100, 1858. – Cetraria sikkimensis Räsänen, Arch. Soc. Zool. Bot. Fenn. Vanamo 5: 25, 1950. Type: India. Sikkim, Changgu, alt. ca. 3900 m, on branches of Rhododendron shrub, May 1947, D. D. Awasthi 355 (H, holotype!; herb. Awasthi, isotype). A close relationship of Cetraria kurokawae with Cetraria togashii, two isidiate Japanese species, was proposed when describing C. kurokawae (SHIBUISHI & YOSHIDA 1982). The species was later transferred to Nephromopsis (KUROKAWA 1991). However, this position was rejected by RANDLANE, SAAG & THELL (1997) because of the presence of fumarprotocetraric acid in the medulla, a characteristic substance for the genus Cetreliopsis. Furthermore, a combination in Tuckneraria was never proposed for N. kurokawae because of the occurrence of pseudocyphellae on the upper surface and the absence of cilia. Neither apothecia nor spermatia have been reported for N. kurokawae. It remains the single species within Nephromopsis which has pseudocyphellae on the upper, rather than the lower, surface, and contains β-orcinol depsidones (fumarprotocetraric and succinprotocetraric acids). This species has a very limited distribution range, being known only from the Himalayas (Bhutan, India and Nepal). N. leucostigma differs from its closest relative N. melaloma by the colour of the lower surface, which is always brown, and by the presence of pseudocyphellae exclusively on the lower side. The spores are 8–9 × 6 µm (AWASTHI 1982), a common size in Nephromopsis. The species very rarely fruits, and spermatia have never been observed in the rare pycnidia. Nephromopsis melaloma (Nyl.) A. Thell & Randlane comb. nov. Basionym: Platysma melalomum Nyl., Syn. Lich. 1: 303, 1860. Type: India, Sikkim, Jongri, regione alpina, supra mare alt. 13 000 ft, inter Cladonias et muscos, J. D. Hooker 2665 (BM!, lectotype, H-NYL 36072!, isolectotype). – Cetraria melaloma (Nyl.) Kremp. Verh. Zool.-Bot. Ges. Wien 18: 315, 1868. – Cetraria pallida D. D. Awasthi, Proc. Indian Acad. Sci. 45: 139, 1957. Type: Nepal, Topkegola, Thaglabhanjyang, alt. 4200 m, 29 May 1953, D. D. Awasthi 2347 (herb. Awasthi, holotype). Nephromopsis melaloma has a similar, restricted distribution as N. leucostigma but has, in addition, been collected in some provinces of China (WEI 1991). Both taxa grow mainly on soil in alpine areas at high altidudes (3500–4800 m) (RANDLANE et al. 2001). Nephromopsis melaloma differs from other Nephromopsis species by the presence of pseudocyphellae on both sides (RANDLANE & SAAG 2003). Neither spores nor spermatia have so far been reported for N. melaloma. Nephromopsis laureri (Kremp.) Kurok. This is the only sorediate taxon in Nephromopsis, and the only one which is extensively distributed on two continents, covering Central Europe, Asia and northern part of South America (RANDLANE & SAAG 2004). THELL et al. (2002) investigated ITS rDNA and β-tubulin of one Asian and one European population. The two populations did not form a monophyletic group, indicating that this disjunct taxon may include at least two separate species. Here they appear as separate but parallel branches in the parsimony analysis and are situated clearly in different clades in both maximum likelihood and MCMC analyses. South American material has not been investigated. Nephromopsis ornata (Müll. Arg.) Hue All three phylogeny analyses reveal N. ornata as the sister group to other Nephromopsis species. The whole Nephromopsis clade is rather weakly supported (55 % bootstrap support) in the parsimony analysis (Fig. 1) while credibility of the same © DGfM 2005 312 THELL, RANDLANE, SAAG & KÄRNEFELT: A new circumscription of the lichen genus Nephromopsis Tab. 3: Nephromopsis as circumscribed in the present study. Species Basionym Type 1. N. ahtii (Randlane & Saag) Tuckneraria ahtii Randlane China, Yunnan, Lijiang, T. Randlane & Saag, comb. & Saag, Acta Bot. Fenn. Ahti, J. B. Chen & L. S. nov. 150: 147. 1994. Wang 46649 (H, holotype; TU, isotype). Additional descriptions and maps Descr.: Randlane et al. 1994, p. 147–148. Maps: Randlane et al. 2001, p. 409; Randlane & Saag 2004, p. 373. 2. N. endocrocea Asahina, J. Jap. Bot. 11: 24. 1935. Japan, Honshu, Nasuzan, Descr. and map: Randlane & Saag Faurie 339 (KY). 1998, p. 178–179. 3. N. isidioidea (Räsänen) Cetraria wallichiana var. isiRandlane & Saag, Mycota- dioidea Räsänen, Arch. xon 44: 487. 1992. Soc. Zool. Bot. Fenn. Vanamo 5, 1: 25. 1950. India, East Himalayas Descr. and map: Randlane & Saag (West Bengal), Darjeeling, 1998, p. 178–179. D. D. Awasthi 179 (H, holotype). 4. N. komarovii (Elenkin) J. C. Cetraria komarovii Elenkin, Russia, Irkutsk, Nilova Ou- Descr. and map: Randlane & Saag Wei, Enumer. Lich. China: Izv. Imp. S.- Peterburgsk. styn, A. Elenkin 155 (LE, 1998, p. 179–180. 158. 1991. Bot. Sada 3: 51. 1903. holotype). Add. data: Randlane et al. 2001, p. 414. 5. N. kurokawae (Shibuichi & Cetraria kurokawae Shibui- Japan, Honshu, Musasi, Mt. Yoshida) Kurok., J. Jap. chi & Yoshida, J. Jap. Bot. Mitsumine, S. Kurokawa Bot. 66: 156. 1991. 57: 296. 1982. 64315 (TNS, holotype). 6. N. laii (A. Thell & Randlane) Cetrariopsis laii A. Thell & Russia, Primorye, KedroSaag & A. Thell, Bryologist Randlane, Cryptog. Bryol. vaya Padj Nature Reserve, 100: 111. 1997. Lichénol. 16: 46. 1995. 17.09.1961, Pärn (TU, holotype; LD, isotype). Descr.: Randlane et al. 1995, p. 46–47. Map: Randlane & Saag 1998, p. 181. Add. data: Randlane et al. 2001, p. 414–415. 7. N. laureri (Kremp.) Kurok., Cetraria laureri Kremp., Germany, Bavaria, Inzell, J. Jap. Bot. 66: 156. 1991. Flora 34: 673. 1851. Appichl, an Fichten, c. ap. Krempelhuber (WU, lectotype, see Obermayer, Biblioth. Lichenol. 88: 513. 2004] Descr.: Randlane et al. 1994, p. 149. Maps: Randlane et al. 2001, p. 409; Randlane & Saag 2004, p. 373. Add. data: Randlane et al. 2001, p. 420–421. 8. N. laxa (Zahlbr.) M. Satô, J. Nephromopsis ciliaris var. Taiwan, Mt. Arisan, Niman- Descr.: Randlane et al. 1994, p. Jap. Bot. 14: 783. 1938. laxa Zahlbr., Feddes Re- daira, Y. Asahina 88 (H, iso- 149–150. pert. 33: 61. 1933. type). Map: Randlane & Saag 2004, p. 373. 9. N. leucostigma (Lév.) A. Cetraria leucostigma Lév. in India orientalis, J. H. Descr. and map: Randlane et al. 2001, Thell & Randlane, comb. Jacquemont, Voyage dans Léveillé (H-NYL 36 083, p. 408–409. nov. l’Inde Bot.: 180: neotype). 1841–1844. 10. N. melaloma (Nyl.) A. Thell Platysma melalomum Nyl., India, Sikkim, Jongri, J. D. Descr. and map: Randlane et al. 2001, & Randlane, comb. nov. Syn. Lich. 1: 303. 1860. Hooker 2665 (BM, lectoty- p. 408–411. pe) 11. N. morrisonicola M. J. Lai, Quart. J. Taiwan Mus. 33: 223. 1980. Taiwan, Nanton, Mt. Morri- Descr.: Randlane et al. 1995, p. 38–39. son, M. J. Lai 10438 (TAIM, Map: Randlane & Saag 1998, p. 182. holotype) Add. data: Randlane et al. 2001, p. 415–416. 12. N. nephromoides (Nyl.) Ahti Platysma nephromoides India, W. Bengal, Hooker fil. Descr. and map: Randlane & Saag & Randlane, Cryptog. Bryol. Nyl., Flora 52: 442–443. & T. Thomson 2080 (H- 1998, p. 183–184. Lichénol. 19: 183. 1998. 1869. NYL 36 068, lectotype; B, Add. data: Randlane et al. 2001, p. 416. PC, UPS isolectotypes) 13. N. ornata (Müll. Arg.) Hue, Cetraria ornata Müll. Arg., Japan, Mt. Ontake No. 109. Descr. and map: Randlane & Saag Nouv. Arch. Mus. Hist. Nat. Nuovo Giorn. Bot. Ital. 23: 1998, p. 184–185. Ser. 4, 2: 90. 1900. 122. 1891. Add. data: Randlane et al. 2001, p. 417. 14. N. pallescens (Schaer.) Cetraria pallescens Scha- Indonesia, Java, Mt. Pan- Descr.: Randlane et al. 1995, p. 42–44. Park, Bryologist 93: 122. er., in Moritzi, Syst. Verzei- gerango, H. Zollinger 449 Map: Randlane & Saag 1998, p. 186. 1990. chn.: 129. 1845–1846. (G, holotype) Add. data: Randlane et al. 2001, p. 417–418. 15. N. rugosa Asahina, J. Jap. Bot. 11: 12. 1935. © DGfM 2005 Japan, Honshu, Musasi, Mt. Descr. and map: Randlane & Saag Kobusi, 22.07.1933, Y. Asa- 1998, p. 187–188. hina (DUKE, isotype). 313 Mycological Progress 4(4) / 2005 Tab. 3: Continued Species Basionym Type Additional descriptions and maps 16. N. pseudocomplicata (Asahina) M. J. Lai, Quart. J. Taiwan Mus. 33: 224. 1980. Cetraria pseudocomplicata Asahina, J. Jap. Bot. 12: 804. 1936 Japan, Shikoku, Iyo Pref., Mt. Ishizuchi, Fujikawa (DUKE, lectotype) Descr.: Randlane et al. 1994, p. 150. Map: Randlane & Saag 2004, p. 373. 17. N. stracheyi (Bab.) Müll. Arg., Flora 74: 374. 1891. Cetraria stracheyi Bab., Hooker’s J. Bot. Kew Gard. Misc. 4: 245. 1852. India, Himalayas, Kathi, R. Strachey & E. Winterbottom (BM, holotype) Descr. and map: Randlane & Saag 1998, p. 189–190. Add. data: Randlane et al. 2001, p. 418–419. 18. N. togashii (Asahina) Saag & A. Thell, comb. nov. Cetraria (Platysma) togashii Asahina, J. Jap. Bot 28: 136. 1953. Japan, Honshu, Suruga, Y. Asahina & M. Togashi 28810 (TNS, lectotype). Descr.: Thell et al. 1995a, p. 238–242 Map: Randlane & Saag 2004, p. 373. 19. N. yunnanensis (Nyl.) Randlane & Saag, Mycotaxon 44: 488. 1992. Platysma yunnanense Nyl., Lich. Nov. Zeland.: 150. 1888. China, Yunnan, R. P. Delavay 1602 (H-NYL 36 134, lectotype) Descr.: Randlane et al. 1995, p. 39–40. Map: Randlane & Saag 1998, p. 190. Add. data: Randlane et al. 2001, p. 419. group is 79 % in MCMC analysis (Fig. 3). Presumed relatives of N. ornata, N. endocrocea and N. isidioidea, were not included in the analysis. N. ornata differs from all other Nephromopsis species included in this study by having Cetraria-type asci, i.e., characterized by a narrow axial body surrounded by a strongly amyloid zone, ”ring structure”, in the tholus, an ascus type typical for the distantly related genera Cetraria and Flavocetraria (KÄRNEFELT, MATTSSON & THELL 1993, 1994; THELL, MATTSSON & KÄRNEFELT 1995). A tendency of such a ring structure is present also in asci of N. morrisonicola, a species distinguished from N. endocrocea and N. ornata by minor morphological characters (black lower surface instead of brown) but mainly chemically (absence of secalonic acids and endocrocin in medulla), visible as a white instead of a yellow medulla (RANDLANE & SAAG 1998). Nephromopsis pallescens (Schaer.) Park The type species of the synonymized genus Cetrariopsis Kurok. was divided into two distinct varieties of which Nephromopsis pallescens var. pallescens is included in the present analysis (RANDLANE, THELL & SAAG 1995). It constitutes, like all species, except for N. leucostigma and N. melaloma, a separate clade in the present parsimony analysis (Fig. 1). However, in THELL et al. (2002) N. pallescens was positioned, although rather weeky supported, on a branch together with N. stracheyi, the type species of the genus Nephromopsis. ML analysis of this data matrix showed a similar result (Fig. 2), while in the MCMC tree N. pallescens formed a separate clade together with N. stracheyi, N. laureri (from Asia) and N. komarovii (Fig. 3). Excluded taxa ‘Nephromopsis’ hengduanensis L. H. Chen The taxon was recently described from China (CHEN & GAO 2001). It is characterized by a yellowish foliose thallus; numerous pseudocyphellae on the lower surface and sparse pseudocyphellae on the upper surface; nephromoid apothecia with large, broadly clavate asci (80–88 × 32–37 µm) and extremly large ascospores; presence of usnic, lichesterinic, protolichesterinic, caperatic and olivetoric acids. The large ascospores (27–30 × 16–18 µm) with a thick epispore (2–3 µm) are unusual for cetrarioid lichens, and are more reminiscent of Parmelaria or Cetrelia spores (RANDLANE & SAAG 2003). Species belonging to Nephromopsis have much smaller, oblong, subglobose or globose spores (6–12 × 2,5–6 µm) borne in narrowly clavate or cylindrical asci (30–50 (70) × 10–15 µm). The generic position of N. hengduanensis has yet to be confirmed by molecular data. It is known from only two localities (in the counties Weixi and Lushui) in the province of Yunnan. The collections originate from the 1980s (CHEN & GAO 2001), and are presumably too old for DNA sequencing by standard procedures. Tuckneraria sikkimensis Divakar & Upreti (nom. inval.) The official description of this taxon is not published yet. The species is characterized by a pale yellow upper surface and black lower surface with pale brown margins; white pseudocyphellae on the lower side of the thallus; presence of marginal cilia and dorsiventral, marginal to submarginal lobules; verruciform marginal pycnidia and citriform (3 × 1 µm) spermatia; containing usnic and lichesterinic acids (DIVAKAR & UPRETI, in press). Apothecia have not been reported. The species is corticolous on Rhododendron and is known only from the type locality in India, Sikkim. The authors of the © DGfM 2005 314 THELL, RANDLANE, SAAG & KÄRNEFELT: A new circumscription of the lichen genus Nephromopsis Key to Nephromopsis species Terricolous or saxicolous . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2 Saxicolous; thallus foliose, adnate to the substratum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N. koarovii 2* Terricolous; thallus laciniate, suberect to erect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3 Upper surface yellow, lower surface brown. Pseudocyphellae white and very distinct on the lower side of the thallus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N. leucostigma 3* Upper surface yellow, lower surface yellow to brown. Pseudocyphellae grey, surrounded by a distinct dark line, on the lower side, positioned on the lower, rarely on the upper, side . . . . . . . . . . . . . . . . . . . . . . . . . . . . N. melaloma 1* Corticolous or lignicolous . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 4 Thallus sorediate or isidiate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 5 Thallus sorediate, soredia marginal, whitish; isidia absent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N. laureri 5* Thallus isidiate, isidia marginal or laminal, simple or coralloid; soredia absent . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 6 Medulla Pd – . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N. togashii 6* Medulla Pd + red; fumarprotocetraric acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N. kurokawae 4* Soredia or isidia absent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 7 Medulla yellow or orange, K+ deep yellow, reddish or lilac . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 8 Lower side of the thallus strongly rugose and reticulate; pseudocyphellae laminally on ridges and plug-like outgrowths . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N. isidioidea 8* Lower side moderately reticulate; pseudocyphellae on ridges or laminally mainly in the marginal zone of the lower surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 9 Medulla pale yellow, K + deep yellow; secalonic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N. ornata 9* Medulla orange, K+ lilac; endocrocin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N. endocrocea 7* Medulla white, K– . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 10 Apothecia small and numerous, mainly laminal . . . . . . . . . . . . . . . . . . . . . . . . . . N. pallescens var. pallescens 10*Apothecia of various size and number, rarely absent, mainly marginal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 11 Thallus light or bright yellow on both surfaces; lower side more or less smooth . . . . . . . . . . . . . . . . . . 12 12 Lobes elongate and narrow, to 2 mm broad; marginal cilia present . . . . . . . . . . . . . . . . . . . . . . N. laxa 12*Lobes wider; marginal cilia absent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N. pallescens var. citrina 11*Upper and lower surfaces of different colour, not uniformly yellow; lower side smooth to strongly rugose 13 13 Lower surface black, only margins brown to pale brown . . . . . . . . . . . . . . . . . . . . . N. morrisonicola 13*Lower surface brown to whitish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 14 Medulla C + red or KC + red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 15 Medulla C + red; olivetoric or anziaic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 16 Thallus regularly reticulate; pseudocyphellae on the lower side small, flat, and developed mainly on ridges; pycnidia on emergent projections; olivetoric acid in medulla N. rugosa 16*Thallus smooth or slightly wrinkled; pseudocyphellae on the lower side medium to large, flat or concave, developed on the surface; pycnidia immersed; anziaic acid in medulla . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N. stracheyi 15*Medulla C–, KC + red; alectoronic or physodic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 17 Ascospores subglobose, 5–6 × 4–5 µm . . . . . . . . . . . . . . . . . . . . . . N. pseudocomplicata 17*Ascospores oblong, 7–10 × 3–5 µm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 18 Thallus regularly and distinctly reticulate; physodic acid in medulla . . . . N. rugosa 18*Thallus smooth or only slightly rugose; alectoronic acid in medulla . . . . . . . . N. laii 14*Medulla C – and KC – . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 19 Lower surface highly rugose; pseudocyphellae on ridges and plug-like outgrowths . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N. yunnanensis 19*Lower surface smooth or moderately rugose; pseudocyphellae either on the surface or on ridges but not on special outgrowths . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 20 Numerous or occasional marginal cilia present; pycnidia on emergent projections; ascospores subglobose, 5–7 × 4–5 µm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N. ahtii 20*Cilia absent; pycnidia absent or immersed; ascospores oblong, 7–10 × 3–4 µm . . . . . 21 21 Thallus moderately rugose, often with secondary marginal lobules; pseudocyphellae on the lower side small and flat, mainly developed on ridges . . . . . . . . . . . . . . . . N. laii 21*Thallus smooth or slightly wrinkled, without secondary marginal lobes; pseudocyphellae on the lower side medium to large, flat or concave, developed on the surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N. nephromoides 1 © DGfM 2005 Mycological Progress 4(4) / 2005 taxon find resemblances between it and Tuckneraria ahtii, and point out marginal cilia, presence of pseudocyphellae on the lower surface and chemical constituents as characteristic features to the genus Tuckneraria. They also recognise that citriform spermatia provide some doubt regarding the generic position of the new species. According to the new circumscription of the genus Nephromopsis, this new species would also belong here. 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