REMOTOTRACHYNA, A NEWLY RECOGNIZED TROPICAL LINEAGE

Transcription

1 American Journal of Botany 97(4): REMOTOTRACHYNA, A NEWLY RECOGNIZED TROPICAL LINEAGE OF LICHENS IN THE HYPOTRACHYNA CLADE (PARMELIACEAE, ASCOMYCOTA), ORIGINATED IN THE INDIAN SUBCONTINENT 1 Pradeep K. Divakar2, H. Thorsten Lumbsch 3, Zuzana Ferencova 2, Ruth Del Prado 2, and Ana Crespo2, 4 2 Departamento de Biolog í a Vegetal II, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid Spain; and 3 Department of Botany, The Field Museum, 1400 S. Lake Shore Drive, Chicago, Illinois USA Biogeographical studies of lichens used to be complicated because of the large distribution ranges of many species. Molecular systematics has revitalized lichen biogeography by improving species delimitation and providing better information about species range limitations. This study focuses on the major clade of tropical parmelioid lichens, which share a chemical feature, the presence of isolichenan in the cell wall, and a morphological feature, microscopic pores in the uppermost layer. Our previous phylogenetic studies revealed that the largest genus in this clade, Hypotrachyna, is polyphyletic with a clade mainly distributed in South and East Asia clustering distant from the core of the genus. To divide the Hypotrachyna clade into monophyletic groups and to reevaluate morphological and chemical characters in a phylogenetic context, we sampled ITS, nuclear large subunit (nulsu) and mitochondrial small subunit (mtssu) rdna sequences from 77 species. We are erecting the new genus Remototrachyna for a core group of 15 former Hypotrachyna species. The segregation of Remototrachyna from Hypotrachyna receives support from morphological and chemical data, as well from maximum parsimony, maximum likelihood, and Bayesian phylogenetic analyses of the DNA. We used a likelihood approach to study the geographic range evolution of Remototrachyna and Bulbothrix, which are sister groups. This analysis suggests that the ancestral range of Remototrachyna was restricted to India and that subsequent long-distance dispersal is responsible for the pantropical occurrence of two species of Remototrachyna. Key words: Ascomycota; Hypotrachyna ; Indian flora; molecular phylogeny; multiple gene; new lineage; Parmeliaceae; Remototrachyna. Lichen biogeography has recently become a dynamic field of study in which distribution patterns are analyzed in a phylogenetic framework ( Printzen and Lumbsch, 2000 ; Crespo et al., 2002 ; H ö gberg et al., 2002 ; Printzen and Ekman, 2002 ; Arnerup et al., 2004 ), using statistical methods to address ancestral range evolution of clades ( L ü cking et al., 2008a ). Lichen-forming fungi have generally larger distribution ranges than most vascular plants with numerous cosmopolitan or pantropical species ( Culberson, 1972b ; Crespo et al., 2002 ; Printzen and Ekman, 2002 ; Feuerer and Hawksworth, 2007 ). This has led to a widespread notion that lichen distribution is primarily shaped by ecological conditions rather than by historical events. Before the molecular era, the few biogeographical studies of lichens addressed specific hypotheses, including (1) the impact of glaciations on the European lichen flora as an explanation of Eastern North American Eastern Asian disjunctions in several genera ( Poelt, 1963 ; Yoshimura, 1968 ) and (2) the invocation 1 Manuscript received 18 May 2009; revision accepted 28 January The authors thank Maria Isabel Garc í a Saez (Madrid) and Fabian Ernemann (Chicago) for assistance in the molecular laboratory. This work was supported by the Spanish Ministerio de Ciencia e Innovaci ó n (CGL /BOS, CGL E/BOS) and Ram ó n y Cajal grant (RYC ) to P.K.D. and FPU grant to Z.F. Most of the sequencing was performed at the Centro de Gen ó mica y Prote ó mica del Parque Científico de Madrid, while some sequences were obtained from the Pritzker Laboratory for Molecular Systematics at The Field Museum (Chicago). The authors thank Rick Ree (Chicago) for help with the program LaGrange. 4 Author for correspondence ( acrespo@farm.ucm.es) doi: /ajb of continental drift to explain current distribution patters of some tropical and southern hemisphere genera ( Culberson, 1972b ; Galloway, 1987, 1988 ). Molecular data have challenged the current largely morphology-based species concept in lichenology. An increasing number of studies reveal that some of the widely distributed, morphologically circumscribed species in fact consist of several distinct lineages ( Goffinet et al., 2003 ; Molina et al., 2004 ; Divakar et al., 2005a, b ; Arg ü ello et al., 2007 ; Blaha and Grube, 2007 ; L ü cking et al., 2008b ; Muggia et al., 2008 ; Wirtz et al., 2008 ; Elix et al., 2009 ; Wedin et al., 2009 ). Additionally, phylogenetic studies showed that some genera, circumscribed on the basis of a few morphological and/or chemical characters, were polyphyletic. The smaller, monophyletic groups, however, tend to have more distinct distribution patterns. The Hypotrachyna clade is an excellent example of this. The Hypotrachyna clade is the major clade of Parmeliaceae from the humid tropics ( Blanco et al., 2006 ; Divakar et al., 2006 ; Lumbsch et al., 2008 ). As a group, these lichens are distinguished from others in Parmeliaceae by having isolichenan in their cell walls and pores in their epicortex. Like other members of Parmeliaceae, most have a foliose thallus, thread-like rhizines on their lower surface, and cup-shaped apothecia with Lecanora-type asci on their upper surface ( Crespo et al., 2007 ). The Hypotrachyna clade includes the genera Bulbothrix, Cetrariastrum, Everniastrum, Hypotrachyna s.l., Parmelinella, and Parmelinopsis and the genera Myelochroa and Parmelina that were previously regarded as parmelinioid ( Blanco et al., 2006 ; Divakar et al., 2006 ). The largest genus in the Hypotrachyna clade, the pantropical genus Hypotrachyna includes ca. 190 species ( Crespo et al., 2007 ). Its description was based on American Journal of Botany 97(4): , 2010; Botanical Society of America 579

2 580 American Journal of Botany [Vol. 97 Table 1. Specimens used in the study, with location, reference collection detail, and GenBank accession numbers. Newly obtained sequences for this study are in boldface. Herbarium acronym according to Index Herbariorum and accession number of herbarium, if applicable. Species Locality Collector(s) Voucher specimen GenBank no. ITS mtssu nulsu Bulbothrix apophysata Costa Rica: San Jose L ü cking 16650b F DQ DQ EU B. coronata South Africa: W Cape Crespo & al. s/n MAF-Lich DQ DQ EU B. decurtata South Africa: W Cape Crespo & al. s/n MAF-Lich DQ DQ EU B. goebelii South Africa: W Cape Lumbsch s/n MAF-Lich DQ DQ EU B. aff. goebelii 1 Fiji: Taveuni Island Lumbsch 19817e F GQ GQ GQ B. aff. goebelii 2 Fiji: Taveuni Island Lumbsch 19817g F GQ GQ GQ B. isidiza 1 Congo: Kahuri-Biega National Mamush s/n MAF-Lich GQ GQ GQ Park B. isidiza 2 Madagascar: Col de Tapia Ertz BR GQ GQ GQ N Ambositra B. hypochraea Madagascar: Col de Tapia Ertz BR GQ GQ N Ambositra B. aff. hypochraea Madagascar: Col de Tapia Ertz BR GQ GQ N Ambositra B. aff. klementii Costa Rica: Puntarenas L ü cking 15170a F DQ DQ B. laevigatula Costa Rica: Puntarenas L ü cking 15045b F GQ GQ B. meizospora India: Uttaranchal Divakar s/n GUH AY AY AY B. sensibilis Rwanda: W. Province Ertz BR GQ GQ B. setschwanensis China: Yunnan Crespo, Blanco & Arguello s/n MAF-Lich AY AY B. suffi xa Madagascar: Col de Tapia Ertz BR GQ GQ GQ N Ambositra B. tabacina 1 Congo: Kahuri-Biega National Mamush s/n MAF-Lich GQ GQ Park B. tabacina 2 Kenya: Kakamega district Crespo, Lumbsch & Divakar s/n MAF-Lich16112 GQ GQ GQ Cetrariastrum andense Peru: Ancash Lumbsch, Wirtz & Ramirez F (MAF-Lich GQ GQ GQ ) C. dubitans Peru: Ancash Lumbsch, Wirtz & Ramirez F (MAF-Lich GQ GQ GQ ) Everniastrum cirrhatum 1 Costa Rica: San Jose Trest 149 MAF-Lich 7465 AY AY AY E. cirrhatum 2 Peru: Quebrada Paron Lumbsch 19342r MAF-Lich DQ DQ EU E. lipidiferum Peru: Quebrada Cojup Lumbsch 19309b MAF-Lich DQ DQ EU E. nepalense India: Uttaranchal Divakar s/n GUH AY AY AY E. rhizodendroideum China: Yunnan Aptroot ABL DQ DQ EU E. sorocheilum China: Yunnan Crespo, Blanco & Arguello s/n MAF-Lich DQ DQ EU E. vexans China: Yunnan Aptroot ABL DQ DQ EU Hypotrachyna booralensis Australia: Queensland Lumbsch s/n MAF -Lich DQ DQ EU H. brasiliana Brazil: Municipio de Piraquara Sanders s/n DQ H. britannica Ireland: Kerry Crespo & Gavilan s/n MAF-Lich GQ GQ GQ H. caraccensis DQ DQ H. degelii DQ DQ H. endochlora Great Britain: Scotland Coppins s/n MAF-Lich AY AY AY H. exsecta China: Yunnan Crespo, Blanco & Arguello s/n MAF-Lich DQ DQ EU H. fi ssicarpa South Africa: W Cape Crespo & al. s/n MAF -Lich DQ DQ H. imbricatula 1 Costa Rica: Manzanillo Molina s/n MAF-Lich DQ DQ GQ H. imbricatula 2 South Africa: W Cape Crespo & al. s/n MAF-Lich DQ DQ EU H. immaculata Australia: Queensland Louwhoff, Molina & Elix s/n MAF-Lich 7462 AY AY AY H. aff. immaculata China: Yunnan Crespo, Blanco & Arguello s/n MAF-Lich DQ DQ EU H. laevigata Great Britain: Scotland Coppins s/n MAF-Lich AY AY AY H. livida Argentina: Salta Arg ü ello s/n MAF-Lich GQ GQ H. neodissecta South Africa: W Cape Crespo & al. s/n MAF-Lich DQ DQ EU H. osseoalba 1 China: Yunnan Crespo, Blanco & Arguello s/n MAF-Lich DQ DQ EU H. osseoalba 2 South Africa: W. Cape Crespo & al. s/n MAF-Lich GQ GQ H. physcioides China: Yunnan Crespo, Blanco & Arguello s/n MAF-Lich DQ DQ EU H. polydactyla Kenya: W province Divakar &. Lumbsch s/n MAF-Lich GQ GQ GQ H. pseudosinuosa 1 China: Yunnan Crespo, Blanco & Arguello s/n MAF-Lich DQ DQ EU H. pseudosinuosa 2 China: Yunnan Crespo, Blanco & Arguello s/n MAF-Lich DQ DQ GQ H. reducens Costa Rica: Nat. Park Lücking F DQ DQ Irazu H. revoluta Spain: Puerto Urkiola, Vizcaya Noya & Olea s/n MAF-Lich 6047 AY AF AY H. rockii Peru: Quebrada Paron Lumbsch 19342l MAF -Lich DQ DQ EU H. sinuosa Great Britain: Scotland Coppins s/n MAF-Lich AY AY AY H. taylorensis Great Britain: Scotland Hawksworth s/n MAF-Lich 9921 AY AY AY Myelochroa aurulenta India: North Sikkim Divakar s/n MAF-Lich DQ EF EF M. irrugans China: Yunnan Crespo & al s/n. MAF-Lich AY AY AY M. metarevoluta China: Yunnan Crespo & al. s/n MAF-Lich AY AY AY Parmelina carporrhizans Spain: Madrid Crespo s/n MAF-Lich 6057 AY AY AY607818

3 April 2010] Divakar et al. REMOTOTRACHYNA, a new tropical lineage in Parmeliaceae 581 Table 1. Continued. Species Locality Collector(s) Voucher specimen GenBank no. ITS mtssu nulsu P. pastillifera Spain: C á diz Crespo s/n MAF-Lich 6058 AY EU AY P. tiliacea Spain: Teruel Crespo & al. s/n MAF-Lich 6056 AY AF AY Parmelinella wallichiana India: Sikkim Chatterjee & Divakar s/n MAF-Lich 7653 AY AY AY Parmelinopsis afrorevoluta 1 Australia: New South Wales Elix Elix (MAF-Lich 15619) GQ GQ GQ P. afrorevoluta 2 Canary Island: Tenerife Crespo s/n MAF-Lich DQ DQ EU P. cryptochlora China: Yunnan Crespo, Blanco & Arguello s/n MAF-Lich DQ DQ EU P. horrescens Spain: La Coru ñ a Carvallal s/n MAF-Lich 9913 AY AY AY P. minarum Spain: C á diz Crespo & al. s/n MAF-Lich 7639 AY AY AY P. neodamaziana Australia: Queensland Louwhoff, Molina & Elix s/n MAF-Lich AY AY AY P. spumosa USA: Pennsylvania Lendemer & Macklin s/n HB. LENDEMER GQ GQ (MAF-Lich 15618) P. subfatiscens Australia: Queensland Louwhoff, Molina & Elix s/n MAF-Lich 6878 AY AF AY Parmeliopsis ambigua AF EU AY Parmeliopsis hyperopta Spain: Madrid Blanco s/n MAF -Lich AY AY AY Remototrachyna adducta 1 China: Yunnan Crespo, Blanco & Arguello s/n MAF-Lich DQ DQ R. adducta 2 China: Yunnan Crespo, Blanco & Arguello s/n MAF-Lich AY AY AY R. awasthii 1 India: Tamil Nadu Divakar, Lumbsch & Upreti s/n MAF-Lich GQ GQ GQ R.. awasthii 2 India: Tamil Nadu Divakar, Lumbsch & Upreti s/n MAF-Lich GQ GQ GQ R. ciliata China: Yunnan Crespo, Blanco & Arguello s/n MAF-Lich AY AY AY R. costaricensis 1 Costa Rica: Molina s/n MAF-Lich AY AY AY Volcán Arenal R. costaricensis 2 Cuba: Granma P é rez Ortega s/n MAF-Lich GQ GQ R. crenata China: Yunnan Crespo, Blanco & Arguello s/n MAF-Lich DQ DQ EU R. aff. crenata India: Karnatka Divakar, Lumbsch & Upreti s/n MAF-Lich GQ GQ GQ R. dodapetta 1 India: Tamil Nadu Divakar, Lumbsch & Upreti s/n MAF-Lich GQ GQ GQ R. dodapetta 2 India: Tamil Nadu Divakar, Lumbsch & Upreti s/n MAF-Lich GQ GQ GQ R. fl exilis 1 India: North Sikkim Divakar s/n MAF -Lich DQ DQ R. fl exilis 2 India: North Sikkim Divakar s/n MAF-Lich DQ DQ EU R. incognita 1 China: Yunnan Crespo, Blanco & Arguello s/n MAF-Lich DQ DQ EU R. incognita 2 China: Yunnan Crespo, Blanco & Arguello s/n MAF-Lich DQ DQ R. infi rma 1 China: Yunnan Crespo, Blanco & Arguello s/n MAF-Lich DQ DQ R. infi rma 2 China: Yunnan Crespo, Blanco & Arguello s/n MAF-Lich AY AY AY R. aff. infi rma India: Karnataka Divakar, Lumbsch & Upreti s/n MAF-Lich GQ GQ GQ R. kingii 1 India: Tamil Nadu Divakar, Lumbsch & Upreti s/n MAF-Lich GQ GQ GQ R. kingii 2 India: Tamil Nadu Divakar, Lumbsch & Upreti s/n MAF-Lich GQ GQ GQ R. koyaensis China: Yunnan Crespo, Blanco & Arguello s/n MAF-Lich DQ DQ EU R. rhabdiformis India: North Sikkim Divakar s/n MAF-Lich GQ R. scytophylla 1 China: Yunnan Crespo, Blanco & Arguello s/n MAF-Lich DQ DQ EU R. scytophylla 2 India: Uttaranchal Divakar s/n MAF-Lich GQ GQ the presence of a combination of morphological characters, such as a pored epicortex; narrow, sublinear to linear lobes with truncate apices; dichotomously branched rhizines; oval-ellipsoid ascospores; and bifusiform conidia ( Hale, 1974a ; Elix, 1993 ). Within the genus, a group of Asian species was found to be distinct by having broader lobes ( Hale, 1975 ; Divakar and Upreti, 2003, 2005 ). Subsequent molecular studies showed that Hypotrachyna is polyphyletic and that some morphological characters (e.g., lobe morphology) are nonrandomly distributed among the major clades, which were classified in Hypotrachyna s.l. ( Divakar et al., 2006 ). The study focuses on a clade of 15 broad-lobed species that is currently classified within the polyphyletic genus Hypotrachyna. Most of these species occur in South and Southeast Asia. We previously showed that this clade was unrelated to Hypotrachyna s.s. ( Divakar et al., 2006 ). We are now extending our taxon sampling to include all but two known species from the clade and expanding our sampling of ITS, nuclear large subunit (nulsu), and mitochondrial small subunit (mtssu) rdna. With this new data set, we will (1) test the monophyly of the broad-lobed Asian Hypotrachyna clade and elucidate their rela- tionships with other genera, (2) relate the distribution of morphological and chemical features to the phylogeny, characterizing the Asian species and distinguishing Hypotrachyna s.s. from the closely related Bulbothrix, and (3) study the ancestral range evolution of this clade in a likelihood framework. MATERIALS AND METHODS Taxon sampling Data matrices of 94 samples (77 species) of the Hypotrachyna and parmelinoid clades ( Blanco et al., 2006 ) were assembled using sequences of nuclear ITS and LSU, and mitochondrial SSU rdna. Of 243 described species within the genera of the Hypotrachyna clade, 69 taxa are included in this study. This study mainly addresses Asian, broad-lobed Hypotrachyna species, and we included 13 of 15 described species and also 15 taxa out of 58 described species of the closely related genus Bulbothrix s.l. Two species of Parmeliopsis were used as the outgroup because this genus has previously been shown to be closely related to these clades ( Blanco et al., 2006 ; Crespo et al., 2007 ; Lumbsch et al., 2008 ). Details of the studied material, including GenBank accession numbers are shown in Table 1. The data sets include 173 sequences from previous publications by our group ( Blanco et al., 2004a, b ; Divakar et al., 2006 ; Crespo et al., 2007 ; Lumbsch et al., 2008 ), five downloaded from GenBank and 80 newly generated sequences.

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5 April 2010] Divakar et al. REMOTOTRACHYNA, a new tropical lineage in Parmeliaceae 583 Molecular methods Small samples (2 mm 2 ) prepared from freshly collected and frozen specimens were ground with sterile plastic pestles. Total genomic DNA was extracted using the DNeasy Plant Mini Kit (Qiagen,) according to the manufacturer s instructions but with slight modifications ( Crespo et al., 2001 ). Genomic DNA (5 25 ng) was used for PCR amplifications of the nuits, nulsu rdna, and mtssu rdna regions. Primers, PCR, and cycle sequencing conditions were the same as described previously ( Divakar et al., 2005a ; Crespo et al., 2007 ). Sequence fragments obtained were assembled with the program SeqMan 4.03 (DNAStar) and manually adjusted. Sequence alignments We used the program MUSCLE ( Edgar, 2004 ) to align DNA sequences of 94 specimens ( Table 1 ) for each data set separately. The ITS and mtssu rdna data sets contained variable regions where alignment among distantly related taxa was ambiguous. We excluded 152 bp in the mtssu and 54 bp in the ITS (33 bp in ITS1 and 21 bp in ITS2 regions) data set that could not be aligned with statistical confidence from the phylogenetic analysis. Ambiguously aligned region were delimited manually ( Lutzoni et al., 2000 ). Phylogenetic analyses The alignments were analyzed using maximum parsimony (MP), maximum likelihood (ML), and a Bayesian Markov chain Monte Carlo approach (B/MCMC). Maximum parsimony analyses were performed using the program PAUP* ( Swofford, 2003 ). Heuristic searches with 1000 random taxon addition replicates were conducted with tree-bisectionreconnection (TBR) branch swapping and MulTrees option in effect, equally weighted characters, and gaps treated as missing data. Bootstrapping ( Felsenstein, 1985 ) was performed based on 2000 pseudoreplicates with random sequence additions. To assess homoplasy levels, we calculated the consistency index (CI) and retention index (RI) from each parsimony search. The ML analysis of the combined data set was performed using an online version of the program RaxML ( Stamatakis et al., 2005, 2008 ), assuming a general time-reversible model of nucleotide substitution ( Rodriguez et al., 1990 ) and a discrete gamma distribution with six rate categories. Bootstrap analysis was run with 2000 pseudoreplicates. The MRBAYES program ( Huelsenbeck and Ronquist, 2001 ) was employed to sample trees using a MCMC method. The analyses were performed assuming the general time-reversible model of nucleotide substitution ( Rodriguez et al., 1990 ) including estimation of invariant sites, assuming a discrete gamma distribution with six rate categories and allowing site-specific rates (GTR+I+G) for the single-gene and the combined analyses. A nucleotide substitution model was selected using the program jmodeltest ( Posada, 2008 ). The combined data set was apportioned into the three parts (ITS, nulsu, and mtssu), and each partition was allowed to have its own parameters as suggested by Nylander et al. (2004). No molecular clock was assumed. Two parallel runs of 2 million generations were made, starting with a random tree and employing 12 simultaneous chains each. Every 100th tree was saved into a file. The first generations (i.e., 2000 trees) were deleted as the burn in of the chains. We plotted the log-likelihood scores of sample points against generation time using the program TRACER 1.0 ( html?id=tracer ; Rambaut and Drummond, 2003 ) to ensure that stationarity was achieved after the first generations by checking whether the log-likelihood values of the sample points reached a stable equilibrium ( Huelsenbeck and Ronquist, 2001 ). Additionally, we used the program AWTY ( Nylander et al., 2007 ) to compare split frequencies in the different runs and to plot cumulative split frequencies to ensure that stationarity was reached. Of the remaining trees ( from each parallel run), a majority-rule consensus tree with average branch lengths was calculated using the sumt option of MrBayes. Recent studies have shown that the utility of the incongruence length difference test (ILD; Farris et al., 1994 ) is limited as a measure of the incongruence among data partitions ( Barker and Lutzoni, 2002 ; Dowton and Austin, 2002 ). Therefore, we used an MP approach to examine the heterogeneity in phylogenetic signal among the three data partitions ( DeQueiroz, 1993 ; Wiens, 1998 ; Buckley et al., 2002 ). The level of bootstrap was used to detect significance levels of localized incongruence among the three gene partitions. For the three genes and the concatenated analyses, 2000 bootstrap replicates with random se- quence additions were performed using the program PAUP* ( Swofford, 2003 ), and the set of topologies reaching 70% bootstrap under parsimony was estimated. We interpreted this bootstrap value as strong support for a particular node and identified the conflicted nodes by comparing each gene partition with a threshold between conflicting ( 70% bootstrap) and nonconflicting ( 70% bootstrap) nodes ( Hillis and Bull, 1993 ; Wiens, 1998 ). If no conflict was evident, it was assumed that the two data sets were congruent and could be combined. Only clades that received bootstrap support 70% in MP and ML analyses and posterior probabilities equal or above 0.95 were considered as strongly supported. Phylogenetic trees were drawn using the program TREEVIEW ( Page, 1996 ). Hypothesis testing Because the results of the phylogenetic analyses were incongruent with the current classification of the genus Hypotrachyna, we tested whether our data were sufficient to reject the monophyly of Hypotrachyna s.l. For hypothesis testing, we compared the ML tree constrained to have Hypotrachyna monophyletic and the unconstrained ML tree. These trees were inferred in the program Tree-PUZZLE 5.2 ( Schmidt et al., 2002 ) employing the GTR+I+G nucleotide substitution model. We used two methods to compare the different topologies: the Shimodaira Hasegawa (SH) test ( Shimodaira and Hasegawa, 1999 ) and the expected likelihood weight (ELW) test ( Strimmer and Rambaut, 2002 ). The SH and ELW tests were performed using Tree-PUZZLE 5.2 with the combined data set on a sample of 200 unique trees, the best trees agreeing with the null hypotheses and the unconstrained ML tree. Ancestral area reconstruction We used the likelihood approach of ancestral range reconstruction developed by Ree and colleagues ( Ree et al., 2005 ; Ree and Smith, 2008 ) to test our hypothesis of an Indian origin of Remototrachyna. Analyses were conducted on an ultrametric tree estimated using the lognormal relaxed clock model ( Drummond et al., 2006 ) implemented in an MCMC framework in the program BEAST v1.4.5 ( Drummond and Rambaut, 2007 ), using the ML tree as the start tree and a GTR+I+G model of nucleotide substitution, with a total run of 10 million generations. This tree was imported into the LaGrange 2.0 program ( ; Ree and Smith, 2008 ) using the LaGrange configuration module ( lagrange/configurator/index). Presence in six different areas was coded for all species (Australasia [i.e., Australia, New Zealand, and New Guinea], South and Central Africa, North America, Neotropics, India, Central and SE Asia) with no restrictions on the number of allowed areas in which ancestral species may have been present ( Sanmart í n and Ronquist, 2004 ). Geographically adjacent areas (e.g., North America and neotropics) were allowed to be contiguous ancestral distribution ranges. Morphological and chemical studies Thallus morphology was studied using a Leica Wild M 8 dissecting microscope to measure lobe shape, size, and width. All specimens of clades A, B, C, E, and H included in the molecular analysis were studied (see Table 1 ). Vertical sections of apothecia were cut using a freezing microtome or razor blade. Ascospore size and exciple anatomy were observed in water and lactophenol cotton blue (methyl blue [0.05 g/ml] 50 mg, phenol 25 g, lactic acid 20.8 ml, glycerol 39.5 ml, water 100 ml). Sections obtained were observed and photographed by light microscopy (Nikon Eclipse 80i) using Nomarski differential interference contrast optics. Chemical constituents were identified by thin-layer chromatography using standardized methods ( Culberson, 1972a ; Culberson and Johnson, 1982 ). RESULTS Phylogenetic studies A total of 28 new nuclear ITS, 25 new nulsu rdna and 27 new mtssu rdna sequences were generated ( Table 1 ). These were aligned with 173 sequences that we previously published and five downloaded from Gen- Bank ( Table 1 ). The aligned matrix contained 447 unambiguous Fig. 1. The 50% majority rule consensus tree of phylogenetic relationships in the Hypotrachyna clade. This tree is based on trees from a B/MCMC tree-sampling procedure from a combined data set of ITS, nulsu, and mtssu rdna. Posterior probabilities 0.95 are mentioned below the branches and values indicated above the branches are MP/ML bootstrap 70%. Branches that received strong support in all three analyses (MP, RaxML and B/MCMC) are in boldface.

6 584 American Journal of Botany [Vol. 97 Table 2. Major diagnostic characters and geography of Remotorachyna, Hypotrachyna s.s., and Bulbothrix s.s. Character Hypotrachyna s.s. (clade E) Bulbothrix s.s. (clade B) Remototrachyna (clade A) Lobes Narrow, sublinear to linear-elongate, truncate, subdichotomously to dichotomously branched Long, sparse to richly dichotomously Narrow, sublinear-elongate, truncate, subdichotomously branched Broad, subirregular, rotund, irregularly branched Rhizines Long or short, sparse or richly dichotomously Short, richly dichotomously branched branched branched Marginal cilia Absent Bulbate Absent or rarely present, then simple and in lobe axils Hymenium µm high µm high µm high Outer exciple layer Plectenchyma with thin cell walls Plectenchyma with thin and thick cell walls Plectenchyma with very thick cell walls Ascospore size µm µm µm Conidia Bifusiform Cylindrical Bifusiform Medullary chemistry Orcinol depsidones, beta-orcinol Orcinol depsides Orcinol depsides, beta-orcinol depsidones depsidones, aliphatic acids Distribution Pantropical Neotropical (two species pantropical) Southeast Asia (one species pantropical) nucleotide position characters in the ITS, 849 in the nulsu and 784 in the mtssu. The final alignment of the combined data set was 2080 positions long, with 764 variable characters. The ITS PCR product obtained ranged between 600 to 800 bp. Differences in size were due to the presence or absence of insertions of ~200 bp identified as group I introns ( DePriest and Been, 1992 ; Gutierrez et al., 2007 ) at the 3 end of the mtssu rdna. We excluded introns, 152 bp of the mtssu, 33 bp of the ITS1, and 21 bp of the ITS2 from the analysis. Testing for topological incongruence showed no supported conflicts (results not shown); hence, single-gene data sets were combined and analyzed. Because the topologies of the MP, ML, and B/MCMC analyses did not show any supported conflict (i.e., PP 0.95 in B/MCMC analysis; MP and ML bootstrap 70%), only the 50% majority-rule consensus tree of the Bayesian tree sampling is shown ( Fig. 1 ). The MP analysis of the combined data matrix resulted in 540 most parsimonious trees (tree length = 2714 steps, CI = , RI = ). The taxa of the Hypotrachyna clade fell into two well-supported groups ( Fig. 1 ). Group I was comprised of three strongly supported clades: clade A, the Asian Hypotrachyna clade with 13 species (including the pantropical H. costaricensis ); clade B with seven of the Bulbothrix spp.; and clade C with the type species of Parmelinella and eight Bulbothrix spp. Based on morphological similarity with the type species B. semilunata, clade B corresponded to Bulbothrix s.s. Group II contained Hypotrachyna s.s. (including the type species H. brasiliana ), Everniastrum, and Parmelinopsis species. A sister-group relationship of group I with the monophyletic genera Myelochroa and Parmelina is unsupported here, but this relation was strongly supported in a previous study ( Crespo et al., 2007 ). Within group II, five supported clades can be distinguished: (1) clade D, containing seven species of Parmelinopsis, including the type species ( P. horrescens ), and four Hypotrachyna species; (2) clade E with eight Hypotrachyna species ( Hypotrachyna s.s.), including the type species ( H. brasiliana ); (3) clade F including five Everniastrum spp.; (4) clade G with Cetrariastrum and Everniastrum lipidiferum ; and (5) clade H with 11 Hypotrachyna taxa. Sister to these five clades is Hypotrachyna fi ssicarpa from South Africa. The relationships between the clades largely lack strong support, with the exception of a sistergroup relationship of clade D and E. The SH and ELW tests both significantly reject monophyly of Hypotrachyna s.l. ( P < in both tests). Morphological and chemical studies The new genus Remototrachyna (= the Asian group, clade A) differs from Hypotrachyna s.s. (clade E) in which it was previously placed by lobe morphology, rhizine length, hymenium height, exciple structure, and ascospore size ( Table 2 ). Remototrachyna can be distinguished from its sister group, Bulbothrix s.s. (clade B), by having broader lobes, a thicker hymenium, larger ascospores, and bifusiform conidia and in lacking bulbate cilia. The chemistry of the three genera overlaps and does not conclusively distinguish these monophyletic clades. Bulbothrix s.s. and Remototrachyna also have different centers of distribution. While the former is predominantly neotropical, the latter has its center of distribution in Southeast Asia ( Table 2 ). A major, previously overlooked character in this group is found in the ascoma anatomy. Species in clade A have a cupulate exciple ( Fig. 2B ) consisting of plectenchyma with thick cell walls ( Fig. 2D ), while the exciple of taxa in clade E is composed of plectenchyma with thin cell walls ( Fig. 2A, C ). Ancestral area reconstruction The results of the ancestral range evolution analyses are summarized in Table 3 and Fig. 3. For node 1, South and East Africa, Southeast Asia, or the Indian subcontinent were recovered as the most likely ancestral ranges. The fact that three different ancestral ranges are statistically plausible indicates localized uncertainty. The ancestral range reconstructions for most of the other nodes revealed only a single most likely ancestral range within the confidence window of two log-likelihood units ( Edwards, 1972 ) with the exception of node 4 (base of Bulbothrix clade B). In the latter case, the reconstruction revealed Southeast Asia and South and East Africa as the most likely ancestral ranges. For nodes 2 and 3, the base of the new genus Remototrachyna and the base of the Asian clade of Remototrchyna spp. the analyses strongly support ancestral ranges in the Indian subcontinent. At the base of clade 5, the analyses support an ancestral range in South and East Africa. Taxonomy The results of the phylogenetic analyses and alternative hypothesis testing coupled with evidence from morphological and chemical characters ( Table 2 ) support the distinction of the Asian Hypotrachyna clade as a separate genus, which is formally described here. Genus Remototrachyna Divakar & A. Crespo gen. nov. MycoBank no. MB515249

7 April 2010] Divakar et al. REMOTOTRACHYNA, a new tropical lineage in Parmeliaceae 585 Fig. 2. Exciple anatomy of lichens in the Hypotrachyna clade. (A, C) Hypotrachyna osseoalba; (B, D) Remototrachyna flexilis. (A, B) Overviews of cross-section through apothecium. (C, D) Details showing type of tissue in cupulate exciple, indicated by arrows. Bars = 25 µm in A, B; 10 µm in C, D. Hym = hymenium, Sub = subhymenium, Exc = cupulate exciple, Alg = algal layer, Med = medulla. Etymology Name derived from remoto (far apart), referring to the phylogenetic distance of the new genus from Hypotrachyna; in other words, the name means that the new genus is morphologically similar to Hypotrachyna but genetically very distant. rounded (Fig. 4), margins eciliate or rarely ciliate. Upper surface, whitish-gray to gray, smooth, emaculate or rarely maculate, lacking pseudocyphellae. Upper cortex palisade plectenchymatous, covered by a pored ( µm diam. in SEM) epicortex. Medulla white. Lower surface smooth, black with brown margins, rhizinate. Rhizines short, often densely dichotomously branched. Ascomata apothecial, laminal, sessile to subpedicellate; discs imperforate, brown; epihymenium 9 20 µm high; hymenium µm high. Cupulate exciple consisting of plectenchyma with thick cell walls (Fig. 2B, D). Asci Lecanora-type, 8-spored. Ascospores ellipsoid, µm. Conidiomata pycnidial, immersed, laminal. Conidia bifusiform, 6 1 µm long. Description Thallus foliose, loosely to moderately adnate, irregularly lobate. Lobes subirregular, broad, 2 10 mm wide, apices Chemistry Cortex containing atranorin; medulla containing orcinol depsides (gyrophoric acid), β-orcinol depsidones Diagnosis Thallus foliosus, laxus vel modice adnatus, lobis 2 10 mm latus, apices rotundatae; rhizinae brevae, dense bifurcate ramosae. Apothecia adnata vel substipitata, hymenium µm altum, excipulum cupulatum, scleroplectenchymaticum, ascosporae ellipsoideae. Type species Remototrachyna flexilis (Fig. 4).

8 586 American Journal of Botany [Vol. 97 Table 3. Inferences about the ancestral area and range evolution parameters of the genus Remototrachyna at selected nodes as indicated in Fig. 3. Only inferences inside the confidence window of two log-likelihood units ( Edwards, 1972 ) listed. Global ML at root node: (dispersal = 17.76, extinction = 6.41) Node Taxa included Area ln L Relative probability 1 Remototrachyna + Bulbothrix South and East p. pt. Africa Southeast Asia Indian subcontinent 2 Remototrachyna (clade A) Indian subcontinent 3 Asian Remototrachyna spp. Indian subcontinent 4 Bulbothrix clade I (clade B) Southeast Asia South and East Africa 5 Bulbothrix clade II (clade C) South and East Africa (protocetraric, salazinic, norstictic, and stictic acids) and aliphatic acids (protolichesterinic and caperatic acid). Observations Remototrachyna is characterized by broad, subirregular lobes with rounded apices, short, mostly dichotomously branched rhizines, and in having scleroplectenchymatous exciple, and large ellipsoid ascospores. The genus as it is now circumscribed includes 15 species that grow on bark and rocks in higher elevations of tropical regions ( m a.s.l.), mostly in humid and open areas of Southeast Asia. Only a single species ( R. costaricensis ) is pantropical. New combinations Remototrachyna adducta (Nyl.) Divakar & A. Crespo Basionym Parmelia adducta Nyl., Flora 68: 610 (1885). Synonym Hypotrachyna adducta (Nyl.) Hale, Phytologia 28: 340 (1974). Remototrachyna awasthii (Hale & Patw.) Divakar & A. Crespo Basionym Hypotrachyna awasthii Hale & Patw., Bryologist 77: 637 (1974). Remototrachyna ciliata (Sheng L. Wang, J.B. Chen & Elix) Divakar & A. Crespo Basionym Hypotrachyna ciliata Sheng L. Wang, J.B. Chen & Elix, Mycotaxon 76: 296 (2000). Remototrachyna costaricensis (Nyl.) Divakar & A. Crespo Basionym Parmelia costaricensis Nyl. in Polakowsky, J. Bot. Brit. and Fore. 13: 225 (1887). Synonym Hypotrachyna costaricensis (Nyl.) Hale, Smithson. Contr. Bot. 25: 29 (1975). Remototrachyna crenata (Kurok.) Divakar & A. Crespo comb. nov. Basionym Parmelia crenata Kurok. in Hale & Kurok., Contr. U.S. Nat. Herb. 36: 168 (1964). Synonym Hypotrachyna crenata (Kurok.) Hale, Phytologia 28: 341 (1974). Remototrachyna dodapetta (Hale & Patw.) Divakar & A. Crespo Basionym Hypotrachyna dodapetta Hale & Patw., Bryologist 77: 637 (1974). Remototrachyna fl exilis (Kurok.) Divakar & A. Crespo comb. nov. Basionym Parmelia fl exilis Kurok. in Hara, Flora E. Himalaya 607 (1966). Synonym Hypotrachyna fl exilis (Kurok.) Hale, Phytologia 28: 341 (1974). Remototrachyna incognita (Kurok.) Divakar & A. Crespo Basionym Parmelia incognita Kurok. in Hara, Flora E. Himalaya 608 (1966). Synonym Hypotrachyna incognita (Kurok.) Hale, Phytologia 28: 341 (1974). Remototrachyna infi rma (Kurok.) Divakar & A. Crespo comb. nov. Basionym Parmelia infi rma Kurok. in Hale & Kurok., Contr. U.S. Nat. Herb. 36: 179 (1964). Synonym Hypotrachyna infi rma (Kurok.) Hale, Phytologia 28: 341 (1974). Remototrachyna kingii (Hale) Divakar & A. Crespo comb. nov. Basionym Parmelia kingii Hale, J. Jap. Bot. 43: 324 (1968). Synonym Hypotrachyna kingii (Hale) Hale, Phytologia 28: 341 (1974). Remototrachyna koyaensis (Asahina) Divakar & A. Crespo Basionym Parmelia koyaensis Asahina, J. Jap. Bot. 28: 67 (1953). Synonym Hypotrachyna koyaensis (Asahina) Hale, Smithson. Contr. Bot. 25: 44 (1975). Remototrachyna rhabdiformis (Kurok.) Divakar & A. Crespo Basionym Parmelia rhabdiformis Kurok., in Hale & Kurok., Contr. U.S. Nat. Herb. 36: 183 (1964). Synonym Hypotrachyna rhabdiformis (Kurok.) Hale, Smithson. Contr. Bot. 25: 62 (1975). Remototrachyna rigidula (Kurok.) Divakar & A. Crespo comb. nov. Basionym Parmelia rigidula Kurok. in Hale & Kurok., Contr. U.S. Nat. Herb. 36: 184 (1964). Synonym Hypotrachyna rigidula (Kurok.) Hale, Phytologia 28: 341 (1974). Remototrachyna scytophylla (Kurok.) Divakar & A. Crespo Basionym Parmelia scytophylla Kurok. in Hale & Kurok., Contr. U.S. Nat. Herb. 36: 185 (1964). Synonym Hypotrachyna scytophylla (Kurok.) Hale, Phytologia 28: 342 (1974). Remototrachyna thryptica (Hale) Divakar & A. Crespo comb. nov. Basionym Parmelia thryptica Hale, Bryologist 75: 99 (1972). Synonym Hypotrachyna thryptica (Hale) Hale, Phytologia 28: 342 (1974). DISCUSSION We used our molecular phylogeny and a likelihood framework to infer that the Indian subcontinent was the most likely ancestral range of our newly described genus Remototrachyna. Subsequent range extension gave rise to the pantropical Remototrachyna species. The ancestral range of the Bulbotrix species

9 April 2010] Divakar et al. REMOTOTRACHYNA, a new tropical lineage in Parmeliaceae 587 Fig. 3. Maximum likelihood reconstructions of geographic range evolution in Remototrachyna and Bulbothrix using LaGrange (see text for explanation). This is an ultrametric tree estimated as described in Materials and Methods. Only ingroup taxa are included in this tree. Actual distribution of species marked in boxes to the left of the species name (black = present, white = absent) (boxes from left to right: Australasia, South and Central Africa, North America, South and Central America, Indian subcontinent, Southeast Asia). When two samples of a species formed a monophyletic species, the terminal lineage is shown only once. The most likely ancestral area is indicated in gray for selected clades. Reconstruction analyses that yielded inconclusive results are marked with?.

10 588 American Journal of Botany [Vol. 97 Fig. 4. Remototrachyna fl exilis habit (bar = 0.5 cm). in clade II (clade C) was South and East Africa. These conclusions must be viewed with some caution, however, because simulation studies showed that the number of accurately reconstructed ancestral ranges decreased when the number of speciation and extinction events was as high as the 17 dispersals/ extinctions inferred in our analysis ( Ree and Smith, 2008 ). Further, the ancestral range reconstruction did not yield conclusive results regarding the ancestral range of whole group I (including Bulbothrix s.l. clades B and C, and Remototrachyna clade A). Our analyses cannot rule out an origin of the whole group I either in Southeast Asia or South and East Africa. Indeed, species of Remototrachyna differ morphologically from Cetrariastrum, Everniastrum, Hypotrachyna, and Parmelinopsis species clustered in group II by having broad, subirregular lobes with rotund apices, and short, densely branched rhizines ( Divakar and Upreti, 2005 ). Micromorphological characters of the ascomata have been largely ignored in parmelioid lichens, based on the assumption that they are uniform within the group, with the exception of ascospore size. However, we have shown here that Remototrachyna differs from Hypotrachyna in the anatomy of the cupular exciple (cell wall thickness, Fig. 2 ). Previously, we demonstrated that another clade of parmelioid lichens (Xanthoparmelia clade) is characterized by a certain type of ascospore morphology ( Del Prado et al., 2007 ). These observations show that ascomatal characters need more attention than has previously been paid in these lichenized fungi. The new genus Remototrachyna including broad-lobed Southeast Asian species was previously placed in Hypotrachyna (Hale, 1975 ; Divakar and Upreti, 2003, 2005 ). Currently, 15 described species are known in this group, and we included 13 species in the present work. Species of Hypotrachyna s.s. species with narrower lobes ( Hale, 1975 ; Divakar and Upreti, 2005 ) clustered in the distant group II. Several species in clade A, such as R. crenata, R. incognita, R. infi rma, and R. scytophylla are not monophyletic, indicating that additional studies are necessary to clarify the current species concept in this clade, which is largely based on macromorphological and chemical characters. The genus Bulbothrix falls into two distinct lineages: clade B (with seven taxa), which is sister to Remototrachyna and eight taxa forming a sister group with Parmelinella wallichiana, making up clade C. The two genera Bulbothrix and Parmelinella are mainly distinguished on the basis of the presence

11 April 2010] Divakar et al. REMOTOTRACHYNA, a new tropical lineage in Parmeliaceae 589 or absence of bulbate, marginal cilia ( Hale, 1974b, 1976 ; Elix and Hale, 1987 ). Our studies suggest that this character is of minor phylogenetic significance. The question of whether Bulbothrix is monophyletic needs to be addressed with a larger taxon sampling of Bulbothrix spp. and taxa of related groups. Further, inclusion of the type species ( B. semilunata ) is necessary before nomenclatural changes can be proposed. We also corroborated previous phylogenetic analyses in showing that the generic concept in group II needs revision with poly- and paraphyly of most of the current genera Everniastrum, Hypotrachyna, and Parmelinopsis ( Divakar et al., 2006 ; Crespo et al., 2007 ). 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