The Lichenologist 41(5): 489–511 (2009) © 2009 British Lichen Society doi:10.1017/S0024282909990090 Printed in the United Kingdom Phylogeny of the cetrarioid core (Parmeliaceae) based on five genetic markers Arne THELL, Filip HÖGNABBA, John A. ELIX, Tassilo FEUERER, Ingvar KÄRNEFELT, Leena MYLLYS, Tiina RANDLANE, Andres SAAG, Soili STENROOS, Teuvo AHTI and Mark R. D. SEAWARD Abstract: Fourteen genera belong to a monophyletic core of cetrarioid lichens, Ahtiana, Allocetraria, Arctocetraria, Cetraria, Cetrariella, Cetreliopsis, Flavocetraria, Kaernefeltia, Masonhalea, Nephromopsis, Tuckermanella, Tuckermannopsis, Usnocetraria and Vulpicida. A total of 71 samples representing 65 species (of 90 worldwide) and all type species of the genera are included in phylogentic analyses based on a complete ITS matrix and incomplete sets of group I intron,
-tubulin, GAPDH and mtSSU sequences. Eleven of the species included in the study are analysed phylogenetically for the first time, and of the 178 sequences, 67 are newly constructed. Two phylogenetic trees, one based solely on the complete ITS-matrix and a second based on total information, are similar, but not entirely identical. About half of the species are gathered in a strongly supported clade composed of the genera Allocetraria, Cetraria s. str., Cetrariella and Vulpicida. Arctocetraria, Cetreliopsis, Kaernefeltia and Tuckermanella are monophyletic genera, whereas Cetraria, Flavocetraria and Tuckermannopsis are polyphyletic. The taxonomy in current use is compared with the phylogenetic results, and future, probable or potential adjustments to the phylogeny are discussed. The single non-DNA character with a strong correlation to phylogeny based on DNA-sequences is conidial shape. The secondary chemistry of the poorly known species Cetraria annae is analyzed for the first time; the cortex contains usnic acid and atranorin, whereas isonephrosterinic, nephrosterinic, lichesterinic, protolichesterinic and squamatic acids occur in the medulla. Notes on the anatomy of Cetraria annae and Flavocetraria minuscula are also provided. Key words: Cetraria s. lat., conidial shape, DNA sequences, lichen phylogeny, taxonomy Introduction The delimitation of the Parmeliaceae A. Thell (corresponding author) and I. Kärnefelt: The Biological Museums, Lund University, Östra Vallgatan 18-20, SE-223 61 Lund, Sweden. Email: arne.thell@botmus.lu.se F. Högnabba, L. Myllys, S. Stenroos and T. Ahti: Botanical Museum, Finnish Museum of Natural History, P.O. Box 7, FI-000 14 University of Helsinki, Finland. J. A. Elix: Research School of Chemistry, Building 33, Australian National University, Canberra, ACT 0200, Australia. T. Feuerer: Hamburg Univesity, Biozentrum Klein Flottbek, Department of Botany and Botanical Garden, Ohnhorststrasse 18, D-22609 Germany. T. Randlane and A. Saag: Institute of Ecology and Earth Sciences, University of Tartu, Lai Street 38, 51005 Tartu, Estonia. M. R. D. Seaward: Department of Archaeological, Geographical & Environmental Sciences, University of Bradford, Bradford, BD7 1DP, UK. The Parmeliaceae is the largest (c. 2300 species) and most intensely studied of all lichen families. It has a characteristic ascoma ontogeny, a cupular exciple and forms a monophyletic clade, including recent segregates such as Alectoriaceae, Hypogymniaceae and Usneaceae (Crespo et al. 2007). The number of genera in the family increased from c. 20 in the early 1970s to c. 90 at the end of the last century (Elix 1993; Henssen & Jahns 1974; Kärnefelt & Thell 1992; Thell et al. 2004). Phylogenetic studies based on DNA sequences resulted in both synonymization and the creation of new genera. However, the number of genera has decreased over the last decade, but the taxonomy is still 490 THE LICHENOLOGIST far from settled, as shown by adjustments made as a result of several large phylogenetic investigations. Thallus-form groups, alectorioid, cetrarioid, hypogymnioid, parmelioid and usneoid lichens, have often been discerned in the family, which to some degree correspond to the family names previously segregated from the Parmeliaceae (Thell et al. 2004). The mainly foliose parmelioid group constitutes the major part of the family, comprising c. 1500 species, of which 800 belong to the largest genus in the family, Xanthoparmelia (Vain.) Hale (Crespo et al. 2007). Most of the fruticose members, mainly alectorioid and usneoid, are gathered in the genera Alectoria Ach., Bryoria Brodo & D, Hawksw. and Usnea Adans., the last perhaps comprising as many as 600 species (Wirtz et al. 2006). Monophyletic cores of the thallusform groups have been identified with the aid of different molecular markers, but all of them nested within the Parmeliaceae (Blanco et al. 2004a, 2005; Crespo et al. 2007; Divakar et al. 2006; Ohmura 2002; Ohmura & Kanda 2004). The sister family of the Parmeliaceae is the Lecanoraceae wherein the genus Protoparmelia M. Choisy is probably most closely related to the Parmeliaceae (Arup et al. 2007; Crespo et al. 2007). The cetrarioid core The morphological group ‘cetrarioid lichens’ contains c. 135 species spread over 20 genera, of which c. 90 species and 14 genera form a monophyletic clade (Randlane et al. 1997; Thell et al. 2002; 2004) (Table 1). Thus, eight genera, Asahinea W. L. Culb. & C. F. Culb., Bryocaulon Kärnefelt, Cetrelia W. L. Culb. & C. F. Culb., Coelopogon Brusse & Kärnefelt, Cornicularia (Schreb.) Hoffm., Dactylina Nyl., Esslingeriana Hale & M. J. Lai, Parmelaria D. D. Awasthi and Platismatia W. L. Culb. & C. F. Culb., traditionally known as cetrarioid, are spread within different clades in the Parmeliaceae, without having any close affinities to each other. Some cetrarioid species are combined in non-cetrarioid genera, such as Himantormia deusta (Hook. f.) A. Thell & Søchting, Melanelia agnata (Nyl.) A. Thell, M. culber- Vol. 41 sonii (Hale) A. Thell and M. hepatizon (Ach.) A. Thell. The genus Parmelaria is closely related to Parmotrema A. Massal. s. lat. (Blanco et al. 2005). The genera Esslingeriana and Melanelia are sister groups to the core of cetarioid lichens and, according to a phylogenetic study of the Parmeliaceae (A. Crespo et al., unpublished), Dactylina is an additional genus that is closely related to the cetrarioid core. Molecular phylogeny vs. taxonomy Before the era of DNA sequencing, the systematics of macrolichens was based on morphology, anatomy and secondary chemistry, with an increased emphasis on reproductive structures in the 1980s and 1990s, frequently with a focus on ascus apex characters (Hafellner 1984; Kärnefelt & Thell 1994; Thell et al. 1995c), which show little correlation with DNA sequences, both in the Parmeliaceae and in other lichen families (Thell et al. 2004; Lumbsch et al. 2007). Cetrarioid lichens probably show the greatest variation of asci and conidia in comparison with the other morphological groups of the Parmeliaceae. Somewhat unexpectedly, DNA based phylogeny of cetrarioid lichens shows a clear correlation with conidial shape (Thell et al. 2002). Hitherto, taxonomic changes based on DNA sequences have not been peformed on a large scale, and have mainly been responsible for reducing the number of polyphyletic genera (Blanco et al. 2004b) or for lumping rather than splitting genera (Blanco et al. 2004a). Among cetrarioid lichens, Tuckneraria Randlane & A. Thell was synonymized with Nephromopsis Müll. Arg., and Nimisia Kärnefelt & A. Thell with Himantormia I. M. Lamb (Thell et al. 2007). A detailed history of the pre-DNA sequencing taxonomy of cetrararioid lichens is provided by Thell et al. (2002). The aim of this paper is to present a complementary phylogeny of the monophyletic core of cetrarioid lichens and discuss the relationship of the taxonomy to phylogeny within the group. Eleven species are investigated for the first time (Table 2), Ahtiana 2009 Phylogeny of the cetrarioid core (Parmeliaceae)—Thell et al. 491 T 1. Genera and type species of the cetrarioid core of the Parmeliaceae Genus Type species Ahtiana Goward, Bryologist 88: 370, 1985. Allocetraria Kurok. & M. J. Lai, Bull. Nat. Sci. Mus. Tokyo, ser. B 17: 60, 1991. Arctocetraria Kärnefelt & A. Thell, Bryologist 96: 402, 1993. Cetraria Ach., Meth. Lich.: 292, 1803. Cetrariella Kärnefelt & A. Thell, Bryologist 96: 402, 1993. Cetreliopsis M. J. Lai, Quart. J. Taiwan Mus. 33: 218, 1980. Flavocetraria Kärnefelt & A. Thell, Acta Bot. Fennica 150: 81, 1994. Kaernefeltia A. Thell & Goward, Bryologist 99: 125, 1996. Masonhalea Kärnefelt, Bot. Notiser 130: 102, 1977. Nephromopsis Müll. Arg., Flora 74: 374, 1891. Tuckermanella Essl. Mycotaxon 85: 135–136. 2003. Tuckermannopsis Gyeln., Acta Fauna Fl. Univ., ser. 2 (Bot.), 1 (5/6): 6, 1933. Usnocetraria M. J. Lai & J. C. Wei, J. Nat. Taiwan Mus. 60: 45–61, 2007. Vulpicida Mattsson & M. J. Lai, Mycotaxon 46: 427, 1993. aurescens (Tuck.) Randlane & A. Thell, ‘Cetraria’ annae Zahlbr., Cetraria australiensis Kärnefelt, Cetraria crespoae (Barreno & Vázquez) Kärnefelt, Cetraria laevigata Rass., Cetraria kamczatica Savicz, Cetreliopsis laeteflava (Zahlbr.) Randlane & A. Saag, ‘Flavocetraria’ minuscula (Elenk. & Savicz) Ahti, Poryadina & Zhurb., ‘Melanelia’ sorediella (Lettau) V. J. Rico, van den Boom & Barrasa, Tuckermannopsis ciliaris (Ach.) Gyeln. and Tuckermannopsis inermis (Nyl.) Kärnefelt. Ahtiana sphaerosporella (Müll. Arg.) Goward Allocetraria stracheyi (Bab.) Kurok. & M. J. Lai Arctocetraria andrejevii (Oxner) Kärnefelt & A. Thell Cetraria islandica (L.) Ach Cetrariella delisei (Schaer.) Kärnefelt & A. Thell Cetreliopsis rhytidocarpa (Mont. & Bosch) M. J. Lai Flavocetraria cucullata (Bellardi) Kärnefelt & A. Thell Kaernefeltia californica (Tuck.) A. Thell & Goward Masonhalea richardsonii (Hook.) Kärnefelt Nephromopsis stracheyi (Bab.) Müll. Arg. Tuckermanella weberi (Essl.) Essl. Tuckermannopsis ciliaris (Ach.) Gyelnik Usnocetraria oakesiana (Tuck.) M. J. Lai & J. C. Wei Vulpicida juniperinus (L.) Mattsson & M. J. Lai cetrarioid core, and not closely related with each other, were selected as an external outgroup. Earlier molecular phylogenetic studies on the cetrarioid group constituted a base for the combined, complementary data set. Amplification of ITS sequences were easily performed for all species, whereas
-tubulin, GAPDH and mtSSU sequences were much more difficult to amplify, the latter marker sequenced for the cetrarioid core on a large scale for the first time. Group I intron sequences are absent from more than half of the species investigated (Thell 1999; Thell et al. 2000, 2002, 2004). DNA analysis Material and Methods Selected material and genetic markers The matrix composed of 77 samples, representing 71 species, including the outgroups, were selected to investigate the phylogeny within the monophyletic core Cetraria s. lat., represented by 65 of the 90 species worldwide. Eleven of the species have not been previously analyzed for their DNA, 67 of the 178 sequences are new, and mitochondrial DNA is used in this group for the first time (Table 2). Representatives from five genera were selected as outgroups. Melanelia and Esslingeriana are sister groups to the monophyletic core of cetrarioid lichens according to earlier investigations (Blanco et al. 2005, 2006; Thell et al. 2002, 2004). The three genera, Alectoria, Cetrelia and Platismatia, all rather distantly related to the Extraction MagAttract 96 DNA Plant Extraction Kit from Qiagen (08/2003) was used. The samples were rigorously shaken in a vortex machine in 300 µl extraction buffer incorporating one steel bead into each microtube; otherwise, the enclosed protocol for manual DNA purification was followed. Amplification 25 µl PCR-reactions were prepared to amplify the nuclear ITS1-5.8S-ITS2 ribosomal DNA region. The primers ITS1F (Gardes & Bruns 1993), ITS4 (White et al. 1991), bt3LM, bt10LM (Myllys et al. 2001), gpd1LM, gpd2LM (Myllys et al. 2002), and mrSSU1, mrSSU3 (Zoller et al. 1999) were used. 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 16µM concentration of each of the primers. The ITS Species Alectoria ochroleuca Cetrelia olivetorum Platismatia glauca Esslingeriana idahoensis Melanelia hepatizon Ahtiana aurescens A. pallidula A. sphaerosporella Allocetraria ambigua A. flavonigrescens A. globulans A. madreporiformis A. sinensis A. stracheyi Arctocetraria andrejevii A. nigricascens Cetraria aculeata C. annae Intron ITS bt Gpd mtSSU AT976 AT913 – – AF457926 AF451763 AF457926 AF449716 AY249638 AY249611 – AT550 AT146 – – AF451758 AF227513 AF457925 – AY249593 – – AT934 AF451776* AF451776 DQ004576 DQ004577 EU435364 AT1917 AT922 EU401769 – EU401769 AF451775 – AY074778 EU423865 AY249607 EU435369 EU435365 FH195 – EU401756 – – – AT1268 – AY353709 – AY249602 – AT73 AF141859 AF141859 – AY249604 – AT874 AT873 AT870 AT973 AT868 AT875 – – – – – – AF404128 AF404127 AF404126 AF416460 AF404125 AF404129 – – – – – AF449733 – – – – – – – – – – – EU435368 AT1364 – DQ004575 – – – AT793 AF254628* AF254628 AF449728 AY249599 – AT1922 – EU401758 – – – FH194 – EU401759 – – EU435375 Vol. 41 Austria, Tirol, Feuerer & Thell s. n. (HBG) Austria, Tirol, Feuerer & Thell 64372 (HBG) Estonia, Tartumaa, Thell 9903 (TUR) Canada, British Columbia, Goward 961348 (UBC) Italy, Trentino Alto Adige, Feuerer & Thell 64248 (HBG) Andorra, Ordino, Rico (MAF–Lich 10592) Italy, Trentino Alto Adige, Feuerer & Thell 64247 (HBG) USA, Michigan, Gogebic Co., Wetmore 882794 (MIN) USA, Montana, Sanders Co., Hauck (private hb.) Canada, British Columbia, Miao & Taylor (TDI–211) China, Sichuan, Obermayer 08141 (GZU) China, Sichuan, Obermayer 08140 (GZU) China, Sichuan, Obermayer 08137 (GZU) Austria, Tyrol, Obermayer 7746 (M) China, Sichuan, Obermayer 08148 (GZU) China, Sichuan, Hengduan Shan, Obermayer 8139 (GZU) Greenland, Qeqertannguit, Hansen, exs. 836 (LD-1001631) Canada, N. W. T., Melville Isl., Westberg 1614 (LD) Spain, Castlla & León, Feuerer s. n. (LD-1196893) Russia, Baikal region, Urbanavicius (LD-1271346) Extr. THE LICHENOLOGIST M. sorediella M. stygia Specimen–ID 492 T 2. Lichen material and sequences used in the analyses of the cetratoid core of the Parmeliaceae. Sequences with accession-numbers beginning with EU were produced in this study. 2009 T 2. Continued Species C. crespoae C. ericetorum C. islandica C. kamczatica C. laevigata C. muricata C. nigricans C. oakesiana C. oakesiana C. obtusata C. odontella C. sepincola Cetrariella commixta C. delisei C. delisei C. fastigiata Cetreliopsis asahinae C. laeteflava Extr. Intron ITS Canada, Ontario (TDI–220) Australia, N. S. W., Kosciusko State Park, Feuerer (HBG) Spain, Castilla Y Léon, Feuerer (LD– 1199338) Sweden, Skåne, Åhus, Thell & Marth 9928 (TUR) Estonia, Tartumaa, Taevaskoja, Thell 9901 (TUR) USA, Alaska, Noatak Nat. Preserve, Ahti 63296 (H) Russia, Sakha Republic, Ahti 64755 (H) Spain, Castilla Y Léon, Feuerer (LD– 1197733) Canada, Baffin Island, Westberg 2377 (LD) Slovenia, Kärnefelt 960306 (LD-1077935) Germany, Bavaria, Oberbayern, v. Brackel (IVL) Austria, Tyrol, Feuerer & Thell s. n. (HBG) Finland, E. H., Sysmä, Haikonen 23297 (H) Finland, Varsinais–Suomi, Puolasma & Thell 0202 (HBG) Finland, Tavastia australis, Haikonen 19093 (H) Iceland, S. Mulasysla, Thell 9714 (LD1016978) USA, Alaska, Noatak, Holt 23063 (LD1190098) Finland, KiL, Haikonen 24443 (H) South Korea (Hur 040500, unpubl.) Taiwan, Kaoshiung County, Lai (private hb.) AT173 AT1696 AF115758 EU401760 AF115758 EU401760 AT1920 – AT544 bt Gpd mtSSU – – – EU423860 – EU435366 EU401761 – EU423861 EU435379 AF228296 AF228296 AF449740 AY249594 – AT548 AF228290 AF228290 AF449739 – – AT1950 EU401763 EU401763 – – – AT1952 AT1921 EU401764 – EU401764 EU410409 – – – – – – AT791 AT136 AT1915 – – – AF457922 AF116179 EU401757 – AF449731 – – – EU423859 – – EU435374 AT990 AT1694 AF449739 EU401765 AF449739 EU401765 – EU410411 – EU423863 EU435378 EU435367 AT1248 – EU401766 – EU423864 EU435371 AT720 – AF451796 AF449735 AY249596 – AT234 – AF228305 AF449737 AY249595 EU435368 AT1913 – EU401767 – – – AT1951 – – – EU435370 FH124 – EU401768 DQ394386 EU401770 – – EU435372 Phylogeny of the cetrarioid core (Parmeliaceae)—Thell et al. C. arenaria C. australiensis Specimen–ID 493 494 T 2. Continued Species C. rhytidocarpa Flavocetraria cucullata F. cucullata Kaernefeltia merrillii K. californica Masonhalea richardsonii Nephromopsis ahtii N. komarovii N. laureri N. leucostigma N. melaloma N. morrisonicola N. nephromoides N. ornata Intron ITS bt Gpd mtSSU DQ980008 FH89 AT932 – – EU401771 AF451793 – – – AY249601 EU435382 – FH90 FH91 FH93 AT700 – – – – EU401772 EU401773 EU401774 AF451794 – – – – – – – – EU435381 – – EU435383 AT1918 DQ395292* DQ395292 EU410412 EU423866 EU435380 AT1703 – DQ004571 – – – AT792 – AF254617 AF449730 AY249598 – AT607 AF404123 AF404123 – – – AF451779 AF449722 – – DQ004529 – AT621 AT938 AF451786 AF451786 AF449724 AT604 AF451777 AF449716 DQ004583 AT430 AF451778 AF449720 AT903 AT1693 AT624 – – – AF451780 DQ004574 AF451783 – – AF449721 – – – – – – AT618 AT907 AT606 – – AF451785* AF451784 AF404131 AF451785 – AF449725 – DQ004578 AY249603 AY249605 – – EU435373 – – Vol. 41 N. pallescens N. pseudocomplicata N. stracheyi Philippines (Bawingan CL0582, Mol. Phyl. Evol. 44) Russia, Sakha Republic, Ahti 61793 (H) Austria, Tyrol, Feuerer & Thell 64185 (HBG) Russia, Sakha Republic, Ahti 61573 (H) Russia, Sakha Republic, Ahti 61682 (H) USA, Alaska, Zhurbenko 04204 (H) Iceland, S. Thingeyar sysla, Frödén 643 (LD-1025426) Spain, Madrid, El Berrueco, Thell 0501 et al. (LD-1038537) USA, Oregon, Lincoln Co., McCune 27703 (LD-1045103) Canada, Yukon Territory, Westberg 1246 (LD) Bhutan, Paro Distr., Søchting 8489 (LD1061438) Russia, Primorie, Skirina 10972 (LD1000141) Italy, Trentino-Alto Adige, Feuerer & Thell 64288 (HBG) Bhutan, Thimpu Distr., Søchting 9151 (LD-1003792) Bhutan, Thimpu Distr., Søchting 9181 (LD-1056669) China, Sichuan, Obermayer 8279 (GZU) The Philippines, Ejem s. n. (H). Russia, Primorye, Kudryavtseva 10980 (LD-1022832) Bhutan, Søchting 8206 (C) China, Sichuan, Obermayer 08276a (GZU) Bhutan, Thimpu, Søchting 8095 (C) Extr. THE LICHENOLOGIST F. minuscula F. minuscula F. minuscula F. nivalis Specimen–ID 2009 T 2. Continued Species T. chlorophylla T. ciliaris T. inermis T. orbata T. platyphylla T. platyphylla T. subalpina Tuckermanella coralligera T. fendleri T. weberi Vulpicida canadensis V. juniperinus V. pinastri V. tubulosus V. viridis Canada, British Columbia, Goward 961350 (UBC) South Africa, Western Cape, Feuerer & Thell s. n. (HBG) USA, N. Carolina, 2004-10-17, Fraker et al. (AFTOL proj.) USA, Alaska, Noatak Nat. Pr., Holt 23441 (LD-1190038) USA, Montana, Hauck s. n. (private hb.) Canada, British Columbia, Thell & Veer 9643 (LD-1058556) Canada, British Columbia, Thell 9675 (LD-1096447) Canada, British Columbia, Thell 9606 (LD) USA, New Mexico, Worthington 28821 (ASU) USA, Arizona, Westberg 543 (LD1059952) USA, Arizona, Westberg 548 (LD1012687) Canada, British Columbia, Thell & Veer 96250 (LD) Finland, Varsinais–Suomi, Lohja, Pykälä 21426 (H) Sweden, Scania, Thell 9604 (LD-1056353) Austria, Tyrol, Innsbruck, Feuerer & Thell s. n. (HBG) USA, Connecticut, Tolland Co., Feuerer s. n. (HBG) Extr. Intron ITS bt Gpd mtSSU AT148 AF072233 AF072233 AF449726 – – AT1022 – AF451789 AF449727 AY249600 – – FJ005090 – – – AT1912 – EU401762 EU410410 EU423862 EU435376 AT1067 AT43 DQ004572* AF072235 DQ004572 AF072235 – – – – – – AT75 – AF072236 AF449741 DQ004581 – AT109 AF072237 AF072237 – – – AT1158 AF457924* AF457924 – DQ004582 – AT612 AF451791 AF451791 – – – AT614 – AF451792 – – – AT36 – AF072238 – – – AT1695 – EU401775 – – – AT02 AT933 AF139031 AF404132* AF139031AF404132 – AF449736 – – – – AT1291 DQ004573* DQ004573 – DQ004580 – Phylogeny of the cetrarioid core (Parmeliaceae)—Thell et al. Tuckermannopsis americana Specimen–ID *Unpublished intron-sequences obtained from old ITS sequences. 495 496 THE LICHENOLOGIST 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. The PCR program described by Ekman (2001) was employed: after a 2 min. hold at 94°C, six cycles followed with denaturation at 94°C for 60 sec., annealing at 62°–56°C for 60 sec. (decreasing 1°C per cycle), and an extension at 72°C for 105 sec., followed by 34 cycles with denaturation at 94°C for 30 sec., annealing at 56°C for 30 sec. and extension at 72°C for 105 sec.; finally, a 10 min. hold at 72°C was performed before the PCR products were cooled to 4°C. Purification and sequencing The PCR products were cleaned with PCR clean-up NucleoFast 96 PCR Purification Kit from MachereyNagel following the user manual (2002/3/Rev. 01. p.12). 50 µl TE buffer was applied to each sample prior to the standard procedure for purification of PCR products under vacuum, which was followed, except for the second, optional, washing step, with purified water. The purified samples were collected in 50 µl water and the amount of DNA was measured in an Eppendorf BioPhotometer. The concentrations ranged between 18 and 54 ng/µl. The amount of DNA for sequencing, 1 ng/base pair, was dried for 1 hr at 65°C. Finally, the DNA–fragments were sent to Macrogen to be sequenced, using the same primers as for the amplification. Phylogeny The phylogenetic analyses of the manually aligned sequences were done with PAUP version 4.0b (Swofford 1998). Trees were calculated using the general heuristic search option and the TBR branch swapping method, whereas gaps were treated as missing characters. Bootstrap analyses with 1000 replicates were performed using the same settings. Support values of 50 or above are marked in the consensus trees (Figs. 1 & 2). Two phylogenies are presented, the first based on the complete ITS matrix and and a second using all the DNA sequences. To complement the parsimony analysis, data were analysed using a Bayesian approach (Larget & Simon 1999), MrBayes 3.1 program. Posterior probabilities were approximated by sampling trees using a variant of the Markov Chain Monte Carlo (MCMC) method called Metropolis-coupled Markov Chain Monte Carlo (MCMCMC). The following priors were used: flat Dirichlets for the substitution rate matrix and nucleotide frequencies, uniform (0, 200) and (0, 1) distributions for the gamma curve shape parameter and proportion of invariable sites, an exponential distribution with mean 0·1 for the branch lengths, and a uniform distribution across tree topologies. The following settings were applied: the best-fit model (GTR+I+G) selected by AIC in MrModeltest 2.2; number of generations = 1100 000; number of simultaneous independent analyses = 2; number of simultaneous chains = 4; sample frequency = 100; ‘temperature’ = 0·2. No molecular clock was assumed. The initial 2500 trees were discarded as Vol. 41 burn-in before stability was reached. Using sumt option of MrBayes, a majority-rule consensus tree was calculated from 8500 trees sampled after reaching likelihood convergence to calculate the posterior probabilities of the tree nodes. Phylogenetic trees were drawn using TreeView. Anatomy Complementary anatomical studies were performed on two rare cetrarioid species, ‘Cetraria’ annae and ‘Flavocetraria’ minuscula. Anatomical sections, 15 µm thick, produced using a Kryomate, Leitz freezing microtome, were mounted in lactophenol cotton-blue. The sections were studied with a Zeiss Axioscope light microscope. Secondary chemistry Secondary compounds of Cetraria annae were detected by means of high performance liquid chromatography (Elix et al. 2004). Taxonomy The taxonomy according to the latest world list of cetrarioid lichens (Randlane & Saag 2002) is strictly used, with the addition of two recently added genera in the cetrarioid core, Tuckermanella and Usnocetraria (Esslinger 2003; Lai et al. 2007) and two species in other genera Cetrariella commixta (Nyl.) A. Thell & Kärnefelt (Thell et al. 2004: 309) and ‘Melanelia’ sorediella (Rico et al. 2005: 205). Several existing, more natural combinations than the ones used here are discussed below. New combinations are needed for a few species, after careful evaluation of the genus concept. Taxonomic changes are postponed for the third updated world list of cetrarioid lichens (T. Randlane, A. Saag, A. Thell & T. Ahti, unpublished). Results Results of the phylogenetic analyses Phylogenetic analyses were performed on two data sets, the first on the complete ITS matrix, and the second based on the total matrix. The ITS analysis resulted in eight most parsimonious trees, all with a length of 1086 changes, CI=0·4171, RI=0·6463. Including gaps, the matrix was composed of 517 characters, of which 101 provided parsimony information. The analysis based on the combined data set resulted in 23 most parsimonious trees, having a length of 2923 changes, CI=0·5279, RI=0·6067. Including 2009 Phylogeny of the cetrarioid core (Parmeliaceae)—Thell et al. 497 F. 1. Consensus tree from eight most parsimonious trees using the heuristic search option in PAUP 4.0b, based on ITS sequences. Bootstrap support values R 50 from 1000 replicates are indicated above the branches. Posterior probability values R 50% from the Bayesian analysis are indicated below the branches. Strongly supported clades (bootstrap support R 70% and posterior probabilities R 95% in the Bayesian analysis) are marked in bold. 498 THE LICHENOLOGIST Vol. 41 F. 2. Consensus tree from 23 most parsimonious trees using the heuristic search option in PAUP 4.0b, based on the combined data matrix (Table 2). Bootstrap support values R 50 from 1000 replicates are indicated above the branches. Posterior probability values R 50% from the Bayesian analysis are indicated below the branches. Strongly supported clades (bootstrap support R 70% and posterior probabilities R 95% in the Bayesian analysis) are marked in bold. 2009 Phylogeny of the cetrarioid core (Parmeliaceae)—Thell et al. gaps, the matrix was extended to 3314 characters, of which 644 proved to be parsimony informative. The two consensus trees, calculated by PAUP, based on the ITS and combined data set, are similar, but not congruent, in their topologies (Figs 1 & 2). All groups supported by the bootstrap analysis are present in the PAUP consensus trees, whereas a few groups with support in the Bayesian analysis are not. By performing MrBayes multiple runs starting from random trees, the inferences for independent runs were the same. Credible sets of trees for one run (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 on the consensus trees. Supported clades Most clades of the consensus trees calculated with PAUP were supported either by the bootstrap or Bayesian analyses. The clades that received bootstrap support equal or above 70% and/or posterior probabilities equal or above 95% in the Bayesian analysis were considered as strongly supported. The position of the species within the clades A1–4 and B9 and B12 differ slightly between the PAUP and Bayesian analyses. Generally, more clades were supported by the Bayesian analyses than by the bootstrap analyses. The cetrarioid core – ingroup The study group selected, comprising 14 genera (Table 1), was divided into two subgroups, the Cetraria and Nephromopsis clades, both with maximum posterior probabilities in the Bayesian analysis of the combined DNA-matrix (Figs. 1 & 2, clades A & B). A. The Cetraria clade This clade contains about half of the cetrarioid core and is divided mainly between five genera, Allocetraria, Cetraria, Cetrariella, Usnocetraria and Vulpicida, characterized by conidia with one swelling (apical or central, 499 i.e. citriform, sublageniform or filiform), with Cetraria sepincola (Ehrh.) Ach., having dumb-bell shaped conidia, as the single known exception. The variation in morphology, anatomy and secondary chemistry is large and no key character for the clade can be presented. The species included are mainly from the Northern Hemisphere (often Himalayan) in their distribution ranges. The clade is strongly supported in the bootstrap analysis, 79 in both the ITS and combined DNA matrix analyses, as well as by the Bayesian analysis of the combined DNA matrix (maximum posterior probability). A1. The Allocetraria clade The bootstrap support value for Allocetraria is 87 in both the ITS and in the total analysis, although the genus is nested within Vulpicida in the PAUP analysis based on the entire matrix (Fig. 2). Six of the nine accepted species were investigated, and they all belong to this clade. The additional taxon, Usnocetraria oakesiana (Ach.) M. J. Lai & J. C. Wei (formerly treated in Allocetraria) is absent from this clade in all our analyses, appearing as one of several singular species. A2. The Cetrariella clade Cetrariella is composed of a core of two closely related species, C. delisei (Schaer.) Kärnefelt & A. Thell and C. fastigiata (Nyl.) Kärnefelt & A. Thell, which are not separated by ITS data. The presence of Cetrariella commixta and ‘Melanelia’ sorediella in Cetrariella is weakly supported in this study. Cetraria sepincola was positioned in the same clade by the analysis based on the total matrix. A3. The Cetraria s. str. clade The genus Cetraria s. str. received moderate bootstrap support, 78 in both analyses, but is recognized in its entirety by the Bayesian analyses. Cetraria s. str. is divided into two species groups, centred around C. aculeata (Schreb.) Fr. and C. islandica (L.) Ach. All known members of the group, except for C. steppae (Savicz) Kärnefelt, were included in the study. Cetraria laevigata, for which only group I intron and ITS sequences were available, appears as a sister group to 500 THE LICHENOLOGIST other Cetraria s. str. in the PAUP analysis based on the total evidence matrix, but is strongly supported as a member of the C. islandica group using the complete ITS matrix. A4. The Vulpicida clade Vulpicida is a chemically distinct genus and is supported as monophyletic by the Bayesian analysis of the ITS data, with posterior probability of 94 (Fig. 1). However, the genus appears to be paraphyletic in the PAUP analysis based on the entire matrix (Fig. 2) as Allocetraria is nested within the same clade. The positions of Vulpicida species remain unresolved in the PAUP analysis of the combined data set (Fig. 2). Five of the six species known worldwide were included in the analyses. A5. Usnocetraria oakesiana and Cetraria obtusata These two species form a separate clade according to three out of four analyses, but the support for the clade is weak (e.g. 51 bootstrap support and 80 posterior probability in the analyses of the combined matrix) and it is not definitely delimited from the other groups. The clade has a few nonmolecular characters in common, i.e. presence of sublageniform conidia and secalonic acid. B. The Nephromopsis clade This clade, including the rest of the cetrarioid core, the genera Ahtiana, Arctocetraria, Cetreliopsis, Flavocetraria, Kaernefeltia, Masonhalea, Nephromopsis, Tuckermanella and Tuckermannopsis, lacks bootstrap support but is maximally supported by the Bayesian analysis based on the total matrix (Figs. 1 & 2). The group is characterized by conidia with two apical or subapical swellings, dumb-bell to disc-bar shaped conidia. However, conidia of different types have been observed in two of the species, Ahtiana pallidula (Riddle) Goward & A. Thell and Arctocetraria andrejevii (Oxner) Kärnefelt & A. Thell (Kärnefelt 1979: 59; Thell et al. 1995a: 602, fig. 15), and no additional nonDNA character that characterizes the clade is Vol. 41 known. Furthermore, some species included in the Masonhalea clade (see below) still have conidia without swellings or with one apical swelling. The position of this clade varies, being excluded from the Nephromopsis clade according to the Bayesian analysis of the ITS matrix and appearing as a sister clade to the remaining part of the cetrarioid core (such topology having, however, posterior probability of 89). The species included in the Nephromopsis clade often have a western North American or South-east Asian distribution. The genera Arctocetraria, Cetreliopsis, Kaernefeltia, Nephromopsis s. str. and Tuckermanella form monophyletic clades according to the present taxonomy, whereas Nephromopsis ornata (Müll. Arg.) Hue is more closely related to Cetreliopsis than Nephromopsis, thus not belonging to Nephromopsis s. str. The genera Ahtiana, Flavocetraria and Tuckermannopsis appear to be polyphyletic. B6. The Arctocetraria clade Arctocetraria, a genus of only two species with an arctic distribution, is moderately supported by bootstrap analyses and strongly supported by Bayesian analyses. The inclusion of Arctocetraria within the genus Flavocetraria based exclusively on ITS sequences is only weakly supported. B7. The Flavocetraria clade All three accepted species were investigated. Two ‘old’ taxa, Flavocetraria cucullata (Bell.) Kärnefelt & A. Thell and F. nivalis (L.) Kärnefelt & A. Thell formed a separate clade together with the rare and recently rediscovered species ‘Cetraria’ annae. The third Flavocetraria species, the recently proposed ‘F.’ minuscula, is included in another major clade of the cetrarioid core – the Cetraria clade. B8. The Tuckermanella clade The bootstrap support of 76 for the North American genus Tuckermanella is weaker in the bootstrap analysis when based exclusively on ITS sequences, but when compared with the analysis inferred from the total matrix, the support value reached 85. Maximum 2009 Phylogeny of the cetrarioid core (Parmeliaceae)—Thell et al. support was received from the Bayesian analyses. Three of six accepted species were included. B9. The Kaernefeltia clade Kaernefeltia is an additional genus composed of two species. Like Arctocetraria, the genus received strong support by both bootstrap and Bayesian analyses. 501 types of conidia, bacillariform and sublageniform, differ from that observed in the major Nephromopsis clade (clade B on Figs 1 & 2). The third taxon, Tuckermannopsis platyphylla (Tuck.) Hale, is related to these two taxa in three out of four analyses, but only with weak support. Discussion B10. The Tuckermannopsis clade Altogether 25 species have been combined in the genus, only eight are included in the genus in the strict sense (Randlane & Saag 2003). Four ‘true’ Tuckermannopsis species, T. americana (Spreng.) Hale, T. chlorophylla (Willd.) Hale, T. ciliaris and T. orbata (Nyl.) M. J. Lai, were investigated in this study. Three species (in various combinations) are included in the Tuckermannopsis clade in different analyses (Figs. 1–2). The clade is, however, only weakly supported. B11. The Cetreliopsis clade The monophyletic status of this East Asian genus is strongly supported by all the analyses. The genus could be seen as nested within Nephromopsis s. lat. because N. ornata is positioned as a sister group to Cetreliopsis. However, the support for the common clade of Cetreliopsis-Nephromopsis (B11 & B12) is lower than acceptable. Three species of seven worldwide were included in the analyses. B12. The Nephromopsis s. str. clade Eleven of the 19 species were analysed. The clade, including 10 taxa (N. ornata excepted), received weak support, slightly above 60, in both bootstrap analyses, but high posterior probabilities (99 and 100) in the Bayesian analyses. B13. Masonhalea richardsonii and Tuckermannopsis inermis Masonhalea richardsonii (Hook.) Kärnefelt, a species characterized by bacillariform conidia, and Tuckermannopsis inermis, with sublageniform conidia, form a clade supported by all analyses. However, the two species have no non-DNA characters in common except for their arctic distribution. Both Conidial shape and phylogeny The variation in conidial shape is greater in the cetrarioid core than in any other corresponding clade in the Parmeliaceae (Thell 1995b) and is the character that shows strongest correlation with DNA based phylogeny (Thell et al. 2002), as illustrated by the different colours in Figs. 1 & 2. The main difference between the conidia is the number of swellings: none, one or two. Conidia with two swellings are dominant among species of the major Nephromopsis clade (B). There are slight differences between them, sometimes discerned as dumb-bell and disc-bar shaped, but the swellings are always more or less apical and are treated as one type in this study. This type is characteristic of the genera Flavocetraria, Kaernefeltia, Nephromopsis, Tuckermanella and Tuckermannopsis. The second type of conidium has one swelling that may be situated either apically or centrally. Conidia with one apical swelling are either sublageniform (bottle-shaped) if they are shorter than 12 µm or filiform if they are longer than 12 µm. Sublageniform conidia occur in three genera of the Cetraria clade (A): Cetrariella, Usnocetraria and Vulpicida, as well as in Cetraria obtusata (Schaer.) v. d. Boom & Sipman and C. subalpina Imshaug. Filiform conidia is a key character for distinguishing the genus Allocetraria. Conidia with a central swelling are fusiform, varying from short to oblong citriform; the latter is typical for Cetraria s. str., whereas the short citriform type is known from Cetrariella commixta, Vulpicida canadensis (Räsänen) J.-E. Mattsson and V. viridis (Swein.) J.-E. Mattsson. The third type of conidium, without swellings (bacillariform), is rare in cetrarioid 502 THE LICHENOLOGIST lichens, being restricted to Masonhalea richardsonii. In spite of the strong correlation between conidial shape and DNA, there are notable exceptions, such as Cetraria sepincola, the only species with dumb-bell shaped conidia in the major Cetraria clade, Arctocetraria andrejevii, where both dumb-bell and oblong citriform conidia have been found, and Ahtiana pallidula, where different conidial shapes have been observed within the same pycnidium (Thell et al. 1995a). Obermayer (2008) recently reported variation in conidial shape within Cetraria islandica. Notes on the genera Ahtiana Goward Ahtiana was originally proposed as a monotypic genus for A. sphaerosporella (Müll. Arg.) Goward, a species segregated from Parmelia Ach. (Goward 1985). The inclusion of two additional North American species, A. aurescens and A. pallidula (Thell et al. 1995a), based on their similar spherical ascospores and the presence of caperatic acid in the medulla, is not supported by cladistic analyses (Figs 1 & 2); however, accommodating these taxa into any other genera has no support either. Returning to a monotypic Ahtiana, including only A. sphaerosporella, would seem attractive to lower the number of polyphyletic genera, but is not confirmed in the present study. Hale (in Egan 1987) combined several species in Tuckermanopsis including Ahtiana pallidula but not A. aurescens, and the only alternative combination for A. aurescens, namely Cetraria aurescens Tuck., would not be tenable if one preserves a multigenus concept within the cetrarioid core. Allocetraria Kurok. & M. J. Lai This genus includes nine species, mainly occurring at high altitudes, seven of which are endemic to the Himalayas (Kurokawa & Lai 1991; Randlane & Saag 2004). The type species, A. stracheyi (Bab.) Kurok. & M. J. Lai, also occurs infrequently in North America, and A. madreporiformis (Ach.) Kärnefelt & A. Thell is widespread in arctic and alpine areas of the Northern Hemisphere Vol. 41 (Randlane & Saag 2004). Some species originally described in the genera Cetraria and Dactylina have been transferred to this genus on two occasions since the original circumscription of the genus (Thell et al. 1995b; Kärnefelt & Thell 1996). One such species, A. oakesiana (Tuck.) Randlane & A. Thell, differs in having shorter conidia (7–12 µm long) when compared with Allocetraria s. str. (12–21 µm long). The exclusion of A. oakesiana from the genus results in a strongly supported Allocetraria-clade (Figs 1 & 2). As presently delimited, Allocetraria is characterized by filiform conidia, slightly thicker at one end, and longer than in any other genus of the Parmeliaceae, except for Parmeliopsis, which has conidia of similar length Lai et al. (2007) proposed the genus Usnocetraria to include 11 species formerly accommodated in Allocetraria, Cetraria, Nephromopsis and Tuckermannopsis, and returned A. madreporiformis to Dactylina. However, the position of A. madreporiformis close to the type species of the genus Allocetraria, A. stracheyi, was confirmed in a phylogenetic analysis by Saag et al. (2002). Therefore, the genus Usnocetraria is proposed here as being monotypic for U. oakesiana (see Usnocetraria). Arctocetraria Kärnefelt & A. Thell Arctocetraria was segregated from Cetraria because of differences in ascus structure, mainly the broader axial body, as well as the presence of bifusiform conidia (although the conidia are usually citriform in A. andrejevii), and the occurrence of norrangiformic and rangiformic acids in the medulla (Kärnefelt et al. 1993). The consensus tree based on ITS sequences suggests a relationship between Arctocetraria and Flavocetraria, but without bootstrap support. Both of these Cetraria-segregates are positioned outside the rather well supported Cetraria s. lat. clade in the phylogenetic trees (Fig. 1). Cetraria Ach. This traditional, well-known genus is characterized by the fatty acids, lichesterinic and protolichesterinic acids, in the medulla, and the fusiform, oblong citriform conidia, 2009 Phylogeny of the cetrarioid core (Parmeliaceae)—Thell et al. 6–10 × 1 µm (Kärnefelt 1979, 1986; Kärnefelt et al. 1992), often clavate, according to Obermayer (2008). The Cetraria s. str. clade (A3) is exclusively composed of the C. aculeata and C. islandica groups. However, in order to avoid the creation of a large number of new genera, Cetraria is considered to include some additional species, C. kamczatica, C. obtusata and C. sepincola, from the Cetraria s. lat. clade (A). They differ from Cetraria s. str. in both secondary chemistry and conidial shape, with C. sepincola being the most divergent in having dumb-bell shaped conidia. The name C. subalpina should be used instead of Tuckermannopsis subalpina (Imshaug) Kärnefelt, and ‘Flavocetraria’ minuscula is probably more appropriately accommodated in Cetraria than in any of the other genera from clade A or in Flavocetraria from clade B according to the results presented here. Cetrariella Kärnefelt & A. Thell This Cetraria segregate was proposed for the closely related species, Cetrariella delisei and C. fastigiata, characterized by broader asci and axial bodies than in Cetraria s. str., sublageniform conidia, and presence of gyrophoric and hiascic acids in the medulla (Kärnefelt et al. 1993). Subsequently, the former Cetraria commixta (Nyl.) Th. Fr. was shown to be in the same clade as Cetrariella delisei and C. fastigiata following a cladistic analysis based on DNA sequences, and the new combination in Cetrariella was proposed (Thell et al. 2004). Cetrariella commixta had earlier been transferred to Melanelia together with the superficially similar Melanelia agnata (Nyl.) A. Thell, M. culbersonii (Hale) A. Thell and M. hepatizon (Ach.) A. Thell (Thell 1995a). Another taxon, formerly known as Cetraria commixta f. sorediella Lettau, was raised to species level as Melanelia sorediella (Rico et al. 2005). This species was subjected to DNA analysis in the present study and shown to be the closest relative of Cetrariella commixta, which would suggest that the two taxa evidently belong to the same genus. However, the position of both Cetrariella commixta and ‘Melanelia’ sorediella in the Cetrariella-clade 503 has lower support than accepted values, and furthermore these locations are not supported by any non-DNA characters. If Cetrariella survives as a separate genus, it could be delimited either to include additionally ‘M.’ sorediella or retained exclusively for C. delisei and C. fastigiata. The present results support the latter solution as the phylogenetic position of Cetrariella commixta and ‘M.’ sorediella remains unresolved. The consensus tree based on ITS sequences places Cetraria subalpina as a sister group to Cetrariella, a position which is not supported in the consensus tree based on the complete matrix. Cetreliopsis M. J. Lai Cetreliopsis is a genus of large foliose species which occur in the eastern and southeastern Asia (Randlane et al. 2001; Randlane & Saag 2004). The genus was originally described as monotypic (Lai 1980), but was later re-evaluated and distinguished from Nephromopsis by the presence of pseudocyphellae on both sides of the thallus in combination with the presence of fumarprotocetaric acid in the medulla. The genus currently includes seven species with this combination of characters (Randlane et al. 1995; Lai & Elix 2002; Randlane & Saag 2003). Relatively few species, three out of a possible seven, were included in the present study but the genus has rather strong bootstrap support as it is currently circumscribed. Flavocetraria Kärnefelt & A. Thell Hitherto, this genus was composed of three arctic-alpine species which occur mainly in the Northern Hemisphere. Two widely known taxa, F. cucullata and F. nivalis, are presumed to be closest relatives, with strong bootstrap support in earlier analyses (Kärnefelt et al. 1994; Thell & Miao 1998; Thell et al. 2002; 2004; 2005). However, in this study, the situation is different because of the inclusion of two rare species, an amphi-Beringian ‘F.’ minuscula, and the recently rediscovered terricolous ‘Cetraria’ annae from Siberia. The latter is most closely related to F. cucullata with which it forms 504 THE LICHENOLOGIST a weakly supported clade, together with F. nivalis. Following the results based on the combined data set, the most natural position for ‘Cetraria’ annae would be in Flavocetraria (for detailed information on ‘C.’ annae see below). According to the present analyses, ‘Flavocetraria’ minuscula has no affinities with the genus Flavocetraria, but belongs to another major clade of the cetrarioid core, the Cetraria s. lat. clade (Fig 1 & 2); for further details on ‘F.’ minuscula see below. Thus as it currently stands, the genus Flavocetraria appears to be polyphyletic. Kaernefeltia A. Thell & Goward Kaernefeltia is a genus of two closely related species which are characterized by conidia with two swellings (disc-bar shaped) and the presence of several distinctive fatty acids. Kaernefeltia merrilli (Du Rietz) A. Thell & Goward is the more common species, with a remarkable disjunct distribution, occurring in western North America, from Baja California to northern British Columbia and eastwards to Saskatchewan, as well as in central Spain, whereas K. californica (Tuck.) A. Thell & Goward is maritime along the Pacific coast in North America (Thell & Goward 1996). The genus has moderate support in the present study. Masonhalea Kärnefelt Compared with the cetrarioid core, this monotypic genus has several unique characters, i.e. a freely-rolling (vagrant) habit, decorticate patches on the lower side, a thick, prosoplectenchymatous upper cortex composed of pachydermatous hyphae and bacillariform conidia (Kärnefelt 1977a, 1979). The presence of alectoronic acid is rare in the cetrarioid core, but is found in some genera and species, such as Cetrariella commixta, Nephromopsis pallescens (Schaer.) Park and Tuckermannopsis s. str. (Kärnefelt & Thell 1993). The lateral position of the apothecia is a character in common with the closely related Tuckermannopsis inermis and some additional Tuckermannopsis species, as well as with the genera Cetreliopsis and Nephromopsis. The monotypic status of the genus is not Vol. 41 supported phylogenetically according to the present study. However, another member of the well-supported Masonhalea clade, T. inermis, shares no non-molecular characters with M. richardsonii, except for the similar arctic, northern-Beringian distribution. The pseudocyphellae are conspicuous in both species, forming unique, large decorticate patches on the lower side of M. richardsonii, and a continuous line close to the margin of the lower side of T. inermis. Nephromopsis Müll. Arg. This genus is the second oldest of the group, originally described as monotypic (Müller 1891). The generic name was rarely used until re-established by Lai (1980) and re-delimited by Randlane et al. (1995). An outline of the genus, including distribution maps and keys is presented by Randlane & Saag (1998). At that time Nephromopsis included the genus Cetrariopsis Kurok. More recently, the genus Tuckneraria Randlane & A. Thell was synonymized with Nephromopsis and two new combinations were made, Nephromopsis leucostigma (Lév.) A. Thell & Randlane and N. melaloma (Nyl.) A. Thell & Randlane following a molecular analysis (Thell et al. 2005) so that Nephromopsis then included 19 species. The most closely related genus is Cetreliopsis, which is distinguished by the presence of pseudocyphellae on both upper and lower surfaces, and fumarprotocetraric acid in the medulla. Tuckermanella Essl. The genus Tuckermanella was recently described to accommodate six rather small, brown, adnate foliose species, apparently endemic to southern and western inland areas of North America (Esslinger 2003). The presence of mostly ellipsoid ascospores, submarginal and laminal pycnidia and continuous marginal pseudocyphellae distinguish this genus from Tuckermannopsis where most of these species had previously been accommodated. Three of the six species have been analyzed phylogenetically and form a wellsupported clade nested within the large Nephromopsis clade. 2009 Phylogeny of the cetrarioid core (Parmeliaceae)—Thell et al. Tuckermannopsis Gyeln. This genus was little used until resurrected by Lai (1981). Since then it has been used as a ‘dustbin’ where various cetrarioid species (25 in total) of dubious generic position have been placed (Randlane & Saag 2003). Kärnefelt & Thell (2001) attempted to delimit the genus based on morphology and DNA sequences and listed seven species in Tuckermannopsis s. str., four of which have been investigated in this study: T. americana, T. chlorophylla, T. ciliaris and T. orbata. The three species excluded from our analyses are endemic to Japan, namely T. gilva (Asahina) M. J. Lai, T. microphyllica (W. L. Culb. & C. F. Culb.) M. J. Lai and T. ulophylloides (Asahina) M. J. Lai. In addition, the DNA of two further rare species combined in Tuckermannopsis but evidently not belonging to the genus in a strict sense remain to be studied, the Japanese endemic T. platyphylloides (Asahina) M. J. Lai and the Chinese endemic, T. weii (X. Q. Gao & L. H. Chen) Randlane & Saag. Two additional species, T. inermis and T. platyphylla, were included in the present study but their generic positions still remains unclear. The delimitation of Tuckermannopsis s. str. is difficult to determine. The Tuckermannopsis clade, including T. americana, T. chlorophylla and T. orbata according to analyses based on the ITS matrix, and T. americana, T. ciliaris and T. chlorophylla according the combined DNA matrix, has no acceptable support. The species are dispersed in the different analyses, among Ahtiana, Kaernefeltia and Tuckermanella (Figs 1 & 2). Tuckermannopsis inermis forms a supported clade together with Masonhalea richardsonii, while T. platyphylla is related to these taxa in three out of four analyses, but with only weak support. As with Cetraria, the polyphyly of Tuckermannopsis is currently accepted in order to avoid erecting several new genera. Usnocetraria M. J. Lai & C. J. Wei In a synopsis of Chinese cetrarioid lichens in a broad sense, the genus Allocetraria was re-delimited and the new genus Usnocetraria proposed (Lai et al. 2007). Usnocetraria oakesiana was selected as the type species of the 505 genus. This species was not closely related to the three genera where it has previously been combined, i.e. Allocetraria, Cetraria s. str. and Tuckermanopsis. Consequently, Usnocetraria is proposed here as being monotypic for U. oakesiana, a disjunct alpine species in the Northern Hemisphere (Randlane & Saag 2004). A second proposed species which was transferred from Nephromopsis, U. kurokawae (Shibuichi & Yoshida) M. J. Lai & J. C. Wei, endemic in Japan, was not supported by the present study. Nine additional taxa of different phylogenetic origins were listed under Usnocetraria as new combinations (Lai et al. 2007: 45), but are invalidly published as they lacked basionym citations. None of these species are closely related to U. oakesiana, whose closest relative is most likely Cetraria obtusata. The latter taxon occurs in the Alps and is very different from U. oakesiana in gross morphology, being erect fruticose, but it does have similar sublageniform conidia and contains secalonic acid in the medulla (v.d. Boom & Sipman 1994). Vulpicida J.-E. Mattsson & M. J. Lai This genus is characterized by the secondary chemistry, namely the presence of pulvinic and vulpinic acids, compounds derived from the shikimic acid pathway. The presence of these substances together with usnic acid give the thallus and medulla a shiny yellow or greenish yellow colour (Mattsson & Lai 1993; Mattsson 1993). Five of the six known species were included in the present analyses. Two of these species, V. canadensis and V. viridis, are endemic to different parts of North America and characterized by short citriform conidia. The Eurasian V. juniperinus (L.) J.-E. Mattsson & M. J. Lai and the disjunct European V. tubulosus (Schaerer) J.-E. Mattsson & M. J. Lai are closely related, whereas the widespread V. pinastri (Scop.) J.-E. Mattsson & M. J. Lai has a more isolated position within the genus. The sixth species, the Eurasian arctic-alpine V. tilesii (Ach.) J.-E. Mattsson & M. J. Lai (Randlane & Saag 2005), was not included. The genus is supported as being monophyletic by the Bayesian analysis of the ITS data (Fig. 1), whereas the positions of the 506 THE LICHENOLOGIST Vol. 41 F. 3. Cetraria annae, Okinskii District, Russia, 18 viii 2007 Urbanavichus (LD-1271346, dupl. ex TU), × 4. species remained unresolved in the PAUP analysis of the combined data set because Allocetraria was nested within Vulpicida (Fig. 2). Comments on some rare and phylogenetically interesting species Cetraria annae Oxner Cetraria annae was forgotten soon after its description by Oxner (1933). However, this species was recently rediscovered after being collected again from the same area. The species is pale yellow, terricolous, and closely attached to the substratum (Fig. 3). The thallus is foliose, to 3 cm wide, with narrow, rounded lobes, 1–3 mm wide and c. 300 µm thick. Lobules are present, and are often constricted at the base. The lobe margins are somewhat crenulate. The soralia are conspicuous along margins and weakly labri- form; soredia are white, granular. Both upper and lower cortices are composed of mesodermatous paraplechtenchyma, with both layers c. 50 µm thick. The medulla is white, and almost as compact as the cortical layers. Algal cells occur in clusters, 5–20 together, in the upper part of the medulla. No apothecia were seen, but the scattered, marginal, black pycnidia were pronounced, but lacked conidia. Cetraria annae is positioned phylogenetically close to Flavocetraria cucullata. Chemical analysis of two specimens (Urbanavichus 1996, 2001; TU) revealed the presence of usnic acid [major], isonephrosterinic acid [major], lichesterinic acid [minor], atranorin [minor], squamatic acid [minor], protolichesterinic acid [trace] and nephrosterinic acid [trace]. Usnic, lichesterinic and protolichesterinic acids also occur in F. cucullata. However, the anatomical studies provided 2009 Phylogeny of the cetrarioid core (Parmeliaceae)—Thell et al. 507 no confirmation of the weakly supported phylogenetic position of this species in Flavocetraria. Specimens examined. Russia: Baikal region, River Levaya Anosovka, on riverbank rocks, 1996, Urbanavicius (TU, det. Randlane); Baikal Nature Reserve, in the vicinity of Lake Tschornaya, on mossy rocks, 2001, Urbanavicius, (TU, det. Randlane); Republic of Buriat, Okinskii district, River Zhom-Bolok, 52°42' 42.3$N 99°17' 37.3$E, on mossy rocks, 2007, Urbanavicius (TU, LD-1271346). Cetraria australiensis Kärnefelt This Australian endemic is morphologically very similar to C. odontella (Nyl.) Nyl. However, these two closely related species differ in their morphology, substratum and distribution, with C. australiensis being dorsiventral and growing on twigs or on soil in alpine habitats of Australia, whereas the lobes of the widely distributed, tuft-forming C. odontella are more or less isodiametric (Kärnefelt 1977b, 1986). One report of C. odontella from Australia, based on Group I and ITS sequences (Thell et al. 2000: AF228285), proved to be C. australiensis after comparison with the newly determined but identical ITS sequence performed in the present study, as well as by re-evaluating the morphology. The ITS sequences of the two species differ in two positions. Flavocetraria minuscula (Elenk. & Savicz) Ahti, Poryadina & Zhurb. Flavocetraria minuscula, known earlier as a form or variety within F. cucullata, was raised to species status by Zhurbenko et al. (2005) because of its distinctive morphological characters, being smaller than F. cucullata with tube-like lobes, a pruinose surface and characteristic helmet-shaped, hooked lobe tips (Fig. 4). The anatomy of F. minuscula was studied in the present work but no differences were found between the amphi-Beringian F. minuscula and the more widely distributed F. cucullata. The cortical layers of each are c. 50 µm wide and are composed of mesodermatous paraplectenchyma, while the medulla is composed of densely interwoven, thick-walled hyphae and the algal cells are scattered in the upper part of the medulla. F. 4. Flavocetraria minuscula, Verkhoyansk District, Russia, 31 vii 2006, Ahti 64946 (LD-1154094, dupl. ex H), × 4. Flavocetraria minuscula is included in a separate clade within the Cetraria s. lat. clade, composed of taxa having, with one exception, conidia with one thickening. Unfortunately the conidia of F. minuscula have yet to be observed. Ultimately, the conidial shape may confirm the unexpected phylogenetic position of F. minuscula distant from F. cucullata, a species characterized by dumb-bell shaped conidia. ‘Melanelia’ sorediella (Lettau) V. J. Rico, van den Boom & Barrasa ‘Melanelia’ sorediella, formerly known as Cetraria commixta f. sorediella, was raised to 508 THE LICHENOLOGIST Vol. 41 F. 5. Tuckermanopsis inermis, Noatak National Preserve, Alaska, 12 vii 2005, Holt 23441 (LD-1190038, dupl. ex OSU), × 7. species level because of the presence of pycnoisidia and the absence of pseudocyphellae and apothecia (Rico et al. 2005). Cetraria commixta was previously transferred to Melanelia because of similarities in reproductive structures and other anatomical characters (Thell 1995a), and later to Cetrariella based on DNA analyses (Thell et al. 2004). The conidia of ‘M’. sorediella are citriform like those of the closely related Cetrariella commixta. Rico et al. (2005) hesitated about the taxonomic position of ‘M’. sorediella, and the present placement in Melanelia remains tentative due to the large phylogenetic distance between Melanelia and Cetrariella. Results of this study demonstrate that Cetrariella commixta and ‘Melanelia’ sorediella should belong to the same genus while the exact phylogenetic position of these two taxa remains unresolved. Tuckermannopsis inermis (Nyl.) Kärnefelt Except for their similar arctic, northernBeringian distribution, the most closely related species, Masonhalea richardsonii, shares no structural or chemical characters with Tuckermannopsis inermis (Kärnefelt 1977a; 1979). The pseudocyphellae are conspicuous in both species, forming unique, large decorticate patches on the lower side of M. richardsonii, and a continuous line close to the margin of the lower side of T. inermis (Fig. 5). Conclusions The interpretation of molecular phylogenetic studies in taxonomy is still in an initial phase. In Parmeliaceae, some new monophyletic groups have been detected and some superfluous genera have been synonymized 2009 Phylogeny of the cetrarioid core (Parmeliaceae)—Thell et al. (Blanco et al. 2004a; b). However, after four decades of splitting, a trend towards larger genera has more recently been adopted in the Parmeliaceae (Divakar et al. 2005, 2006; Wirtz et al. 2006). The morphological, anatomical and chemical variation in the cetrarioid core is probably as large as in the Xanthoparmelia clade, although the conidial variation is greater among the cetrarioid lichens. Today, Xanthoparmelia (Vain.) Hale is a genus of approximately 800 species comprising eight previously accepted genera, a clade supported by the presence of Xanthoparmeliatype lichenan in the cell walls (Prado et al. 2007; Thell et al. 2006). The second largest genus of the family, Usnea Adans., is characterized by a dense cord in the medulla and comprises up to 600 species after the inclusion of Neuropogon and Protousnea (Wirtz et al. 2006). Polyphyletic genera in the cetrarioid core could, in some cases, be eliminated or adjusted by using alternative, previously published combinations. However, neither the whole cetrarioid core nor either of the main clades (A & B) can be defined by any presently known non-molecular characters. The general shape of conidia (with one swelling versus two swellings) corresponds, with some exceptions (Fig 1 & 2), to the division of the cetrarioid core into two major groups, Cetraria s. lat. and Nephromopsis s. lat. The only alternative to the multi-generic concept proposed here, where smaller monophyletic clades are accepted as genera (albeit nested within larger, paraphyletic genera), would be to name the whole cetraroid core Cetraria, despite the lack of any known correlating characters. 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