Who's getting around? Assessing species diversity and phylogeography in the widely distributed lichen-forming fungal genus Montanelia (Parmeliaceae, Ascomycota).

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Contents lists available at ScienceDirect

Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev 6 7 3 4 5 8 9 10 11 12 13 14 15 17 16 18 2 4 0 3 21 22 23 24 25 26 27 28 29 30 31 32 33

Who’s getting around? Assessing species diversity and phylogeography in the widely distributed lichen-forming fungal genus Montanelia (Parmeliaceae, Ascomycota) q Steven D. Leavitt a,b,⇑, Pradeep K. Divakar c, Yoshihito Ohmura d, Li-song Wang e, Theodore L. Esslinger f, H. Thorsten Lumbsch b a

Committee on Evolutionary Biology, University of Chicago, Chicago, IL, USA Science & Education, The Field Museum, Chicago, IL, USA c Departamento de Biología Vegetal II, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid E-28040, Spain d Department of Botany, National Museum of Nature and Science, 4-1-1 Amaklubo, Tsukuba, Ibaraki 305-0005, Japan e Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Yunnan 650201, China f Department of Biological Sciences #2715, North Dakota State University, PO Box 6050, Stevens Hall, Fargo, ND 58108-6050, USA b

a r t i c l e

i n f o

Article history: Received 27 March 2014 Revised 13 February 2015 Accepted 30 April 2015 Available online xxxx Keywords: ABGD Cosmopolitan species Fungi GYMC PTP Species delimitation

a b s t r a c t Brown parmelioid lichens comprise a number of distinct genera in one of the most species-rich families of lichen-forming fungi, Parmeliaceae (Ascomycota). In spite of their superficial similarity, a number of studies on brown parmelioids have provided important insight into diversification in lichen-forming fungi with cosmopolitan distributions. In this study we assess species diversity, biogeography and diversification of the genus Montanelia, which includes alpine to temperate saxicolous species. We sampled each of the five known species, four of which are known from broad, intercontinental distributions. In order to identify potential biogeographical patterns, each broadly distributed species was represented by individuals collected across their intercontinental distributions. Molecular sequence data were generated for six loci, including four nuclear protein-coding markers, the internal transcribed spacer region, and a fragment of the mitochondrial small subunit. We used three sequence-based species delimitations methods to validate traditional, phenotype-based species and circumscribe previously unrecognized species-level lineages in Montanelia. Relationships among putative lineages and divergence times were estimated within a coalescent-based multi-locus species tree framework. Based on the results of the species delimitation analyses, we propose that the genus Montanelia is likely comprised of six to nine species-level lineages, including previously unrecognized species-level diversity in the nominal taxa M. panniformis and M. tominii. In contrast, molecular sequence data suggest that M. predisjuncta may be conspecific with the widespread taxon M. disjuncta in spite of distinct morphological differences. The rate-based age estimation of the most recent common ancestor of Montanelia (ca. 23.1 Ma) was similar to previous estimates based on the fossil record. Furthermore, our data suggest that diversification in Montanelia occurred largely during the Neogene. At least three Montanelia species are broadly distributed throughout Asia, Europe, and North America with no evidence of phylogeographic substructure. In contrast to broadly distributed Montanelia species, our study suggests Pleistocene-dominated diversification and complex biogeographic history of the M. tominii group. Our analyses provide additional insight for understanding diversification and uncovering cryptic diversity in cosmopolitan species of lichen-forming fungi. Ó 2015 Elsevier Inc. All rights reserved.

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63

q

This paper has been recommended for acceptance by J.-M. Moncalvo.

⇑ Corresponding author at: Committee on Evolutionary Biology, University of Chicago, Chicago, IL, USA. E-mail address: sleavitt@fieldmuseum.org (S.D. Leavitt).

1. Introduction

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Understanding diversification histories and speciation in cosmopolitan lineages can offer valuable insight into evolutionary factors operating at broad geographic scales (Bossuyt et al., 2006;

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http://dx.doi.org/10.1016/j.ympev.2015.04.029 1055-7903/Ó 2015 Elsevier Inc. All rights reserved.

Please cite this article in press as: Leavitt, S.D., et al. Who’s getting around? Assessing species diversity and phylogeography in the widely distributed lichen-forming fungal genus Montanelia (Parmeliaceae, Ascomycota). Mol. Phylogenet. Evol. (2015), http://dx.doi.org/10.1016/j.ympev.2015.04.029

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Martiny et al., 2006; Richardson et al., 2004; Roelants et al., 2007). However, some species traditionally believed to have widespread or cosmopolitan distributions may harbor previously unrecognized diversity and biogeographic patterns (Pringle et al., 2005; Salgado-Salazar et al., 2013; Suatoni et al., 2006). Therefore, re-examinations of species boundaries in putatively cosmopolitan species can provide novel insights into factors driving diversification and distributions at broad scales, particularly in groups with sparse or subtle diagnostic morphological characters. Studies of lichen-forming fungi in the species-rich family Parmeliaceae (Ascomycota) have provided in-depth insight into diversification, diversity, and biogeography at multiple scales (Amo de Paz et al., 2012; Amo de Paz et al., 2011; Crespo et al., 2010; Crespo and Pérez-Ortega, 2009; Del-Prado et al., 2013; Fernández-Mendoza and Printzen, 2013; Leavitt et al., 2011). Studies in Parmeliaceae have revealed a common pattern of presence of cryptic species (see definition in Bickford et al., 2007) in traditional, phenotype-based species circumscriptions (Crespo and Pérez-Ortega, 2009). While a number of species in Parmeliaceae have been shown to be truly widespread (e.g., Crespo et al., 2002; Geml et al., 2010; Leavitt et al., 2013b), distinct biogeographic patterns have been observed for other species (e.g., Argüello et al., 2007; Divakar et al., 2010; Molina et al., 2011a, 2004). Diversification of most lineages in Parmeliaceae occurred during the Tertiary Period (Amo de Paz et al., 2011), with many genera showing patterns of Miocene- or Pliocene-dominated species-level diversification (Amo de Paz et al., 2012; Divakar et al., 2012; Leavitt et al., 2012a, 2012b, 2012c). However, climatic fluctuations during the Pleistocene appear to have played a lesser role in driving diversification in Parmeliaceae (Fernández-Mendoza and Printzen, 2013; Leavitt et al., 2012a, 2013d). Brown parmelioid lichens comprise a number of distinct genera in Parmeliaceae (Ascomycota) (Blanco et al., 2004; Divakar et al., 2012; Esslinger, 1977). The majority of brown parmelioid species are found in four major genera: Melanelia Essl., Melanelixia O. Blanco, A. Crespo, Divakar, Essl., D. Hawksw. & Lumbsch, Melanohalea O. Blanco, A. Crespo, Divakar, Essl., D. Hawksw. & Lumbsch, and Montanelia Divakar, A. Crespo, Wedin & Essl. Many species have broad, intercontinental distributions and commonly occur across a wide range of habitats (Esslinger, 1977; Otte et al., 2005), and a number of previous studies have provided important insight into diversification in lichen-forming fungi with cosmopolitan distributions (Divakar et al., 2012, 2010; Leavitt et al., 2012a, 2012b, 2013a, 2013b). The genus Montanelia was recently segregated from Melanelia, with five currently accepted species: M. disjuncta (Erichsen) Divakar, A. Crespo, Wedin & Essl., M. panniformis (Nyl.) Divakar, A. Crespo, Wedin & Essl., M. predisjuncta (Essl.) Divakar, A. Crespo, Wedin & Essl., M. sorediata (Ach.) Divakar, A. Crespo, Wedin & Essl., and M. tominii (Oxner) Divakar, A. Crespo, Wedin & Essl. (Fig. 1; Divakar et al., 2012). All Montanelia species occur exclusively on rock and have broad geographic distributions in montane regions throughout the Northern Hemisphere (Supplementary files S1–S4), with the exception of M. predisjuncta, which is known exclusively from Asia (Divakar et al., 2012; Esslinger, 1977; Kashiwadani et al., 1998; Wang et al., 2009). Previous studies of widespread lichen-forming fungi have revealed contrasting patterns of truly cosmopolitan species (e.g., Crespo et al., 2002; Fernández-Mendoza et al., 2011; Lindblom and Søchting, 2008), contrasted with other nominal species masking striking biogeographical patterns (Divakar et al., 2007, 2010; Elix et al., 2009; Hodkinson and Lendemer, 2011; Otálora et al., 2010). In other genera, closely related species may have contrasting distribution patterns, with some showing broad geographic distributions and others having restricted distributions (Leavitt et al., 2012a, 2012b, 2013c). However, traditional

phenotype-based species circumscriptions may misrepresent diversity in lichen-forming fungi and may confound our interpretation of biogeographic patterns (Crespo and Lumbsch, 2010; Crespo and Pérez-Ortega, 2009; Lumbsch and Leavitt, 2011; Molina et al., 2011a, 2011b). The intercontinental distribution patterns for the majority of Montanelia species provide an interesting group for assessing phylogeographic patterns in commonly occurring lichens. In this study, we assessed diversity, distributions, and the timing of diversification in Montanelia. Specifically, our objectives were to (i) validate traditional morphology-based species circumscriptions and identify previously unrecognized lineages hidden under similar morphologies using sequenced-based species delimitation methods (SDM); (ii) investigate phylogeographic patterns in broadly distributed Montanelia species using multilocus phylogenetic reconstructions; and (iii) estimate a species tree and divergence times for Montanelia. Our results, reported below, provide valuable insight into understanding diversification in parmelioid lichens with broad, intercontinental distributions.

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2. Materials and methods

153

2.1. Specimen sampling

154

In this study, we sampled a total of 64 specimens representing the five known Montanelia species (Divakar et al., 2012), including: M. disjuncta (22 specimens), M. panniformis (12), M. predisjuncta (1), M. sorediata (8), and M. tominii (21). In order to identify potential phylogeographical patterns each broadly distributed species was represented by individuals collected across their intercontinental distributions (Table 1; Supplementary files S1–S4). Specimens identification followed Esslinger (1977). While M. tominii specimens generally produce soredia, vegetative reproductive propagules containing both fungal hyphae and algal cells, four esorediate individuals were collected from western North America that otherwise matched M. tominii. Similarly, a single esorediate specimen representing M. disjuncta was also collected from northwestern Canada. The sister-group to Montanelia remains uncertain, hence Melanelixia californica, Melanohalea subolivacea, Xanthoparmelia loxodes, and X. wyomingica were used as outgroups (Blanco et al., 2006; Crespo et al., 2010; Divakar et al., 2012).

155

2.2. DNA extraction, amplification and sequencing

172

DNA was extracted from a small piece of thallus material free from visible damage or contamination using the USB PrepEase Genomic DNA Isolation Kit (USB, Cleveland, OH) and following the manufacturer’s recommendations. For each specimen, we attempted to generate sequence data from a total of six markers: the complete nuclear ribosomal internal transcribed spacer region (ITS), and fragments of the nuclear ribosomal large subunit (nrLSU), mitochondrial small subunit (mtSSU), and three protein-coding markers, the mini-chromosome maintenance complex 7 (MCM7), RNA polymerase II largest subunit (RPB1), and RNA polymerase II 2nd largest subunit (RPB2). Primers and annealing temperatures used for polymerase chain reaction (PCR) amplifications are reported in Leavitt et al. (2012a). PCR amplifications were performed using Ready-To-Go PCR Beads (GE Healthcare, Pittsburgh, PA, USA) using primers and annealing temperatures as described in Leavitt et al. (2012a). Products were visualized on 1% agarose gel and cleaned using ExoSAP-IT (USB, Cleveland, OH, USA). We sequenced complementary strands using BigDye v3.1 (Applied Biosystems, Foster City, CA, USA) and the same primers used for amplifications. Sequenced PCR products were run on an ABI 3730 automated sequencer (Applied Biosystems) at the

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Fig. 1. The maximum likelihood (ML) tree estimated from a concatenated data set of ribosomal (ITS and nrLSU), nuclear protein-coding (MCM7, RPB1, and RPB2), and mitochondrial (mtSSU) markers representing Montanelia species. Thickened branches indicate ML bootstrap values P70% from the RAxML analysis and Bayesian posterior probabilities P0.95 from the BEAST analysis. Results of species delimitation analyses are summarized to the right of the terminal labels of the ML tree, including from left to right: Automatic Barcode Gap Discovery – ‘ABGD’ – analysis of the ITS alignment; Poisson tree processes – ‘PTP⁄’ – analysis of a maximum likelihood (ML) ITS topology; ‘PTP⁄’ analysis of the concatenated six-gene ML topology; and a Bayesian general mixed Yule-coalescent model – ‘bGMYC’ – analysis of a chronogram inferred from the ITS alignment. Habit shots for: A. M. tominii. B. M. sorediata. C. M. panniformis. D. M. disjuncta (M. predisjuncta is not shown but was recovered with M. disjuncta). Photo credits: J. Hollinger (A); Curtis Björk (B); and Leif Stridvall (C and D). 194 195

Pritzker Laboratory for Molecular Systematics and Evolution at the Field Museum, Chicago, IL, USA.

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2.3. Sequence data analyses

197

New sequences were assembled and edited using Sequencher v4.10 (Gene Codes Corporation, Ann Arbor, MI). Multiple sequence alignments for each locus were performed using the program MAFFT v7 (Katoh et al., 2005; Katoh and Toh, 2008). For the protein-coding (MCM7, RPB1 and RPB2) and nrLSU markers, we used the G-INS-i alignment algorithm and ‘1PAM/K = 2’ scoring matrix, with an offset value of 0.9, and the remaining parameters were set to default values. For the ribosomal ITS and mtSSU loci, we used the same parameters, with the exception of an offset value set to 0.0 rather than 0.9. While alignments for other loci, including

198 199 200 201 202 203 204 205 206

the ITS, were straightforward and did not require manual corrections, exploratory analyses revealed a number of ambiguous regions in the mtSSU alignment. Therefore, we used the program Gblocks v0.91b (Talavera and Castresana, 2007) to delimit and remove ambiguous alignment nucleotide positions from the final mtSSU alignment using the online web server (http://molevol. cmima.csic.es/castresana/Gblocks_server.html) and implementing the options for a less stringent selection of ambiguous nucleotide positions.

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2.4. Species delimitation analyses

216

In lichen-forming fungi morphology-based species circumscriptions may mask multiple species-level lineages within a single nominal taxon (Crespo and Lumbsch, 2010; Lumbsch and Leavitt,

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208 209 210 211 212 213 214 215

218 219

4

ABGD/PTP Clade

Geographic Origin

Voucher

ITS

nrLSU

mtSSU

MCM7

RPB1

RBP2

M. M. M. M. M. M. M. M. M. M. M. M. M. M. M. M. M. M. M. M. M. M. M.

‘disjuncta’ ‘disjuncta’ ‘disjuncta’ ‘disjuncta’ ‘disjuncta’ ‘disjuncta’ ‘disjuncta’ ‘disjuncta’ ‘disjuncta’ ‘disjuncta’ ‘disjuncta’ ‘disjuncta’ ‘disjuncta’ ‘disjuncta’ ‘disjuncta’ ‘disjuncta’ ‘disjuncta’ ‘disjuncta’ ‘disjuncta’ ‘disjuncta’ ‘disjuncta’ ‘disjuncta’ ‘disjuncta’

Canada, Yukon Territory Denmark, Greenland, Northwest Denmark, Greenland, South Denmark, Greenland, Northwest USA, Maine, Knox County Norway, Tromso Canada, Alberta Canada, British Columbia Canada, British Columbia Canada, British Columbia Canada, Yukon Territory Canada, Yukon Territory USA, Alaska Canada, New Brunswick Denmark, Greenland, South, Igaliku Denmark, Greenland, Northwest, Siorapaluk Austria, Steiermark GenBank Sweden: Lycksele Lappmark Sweden: Lycksele Lappmark U.K., Scotland Canada, British Columbia India, Uttaranchal

KP771824 KP771825 KP771826 KP771827 KP771828 KP771829 KF257962 KF257963 KF257964 KF257965 KF257966 KP771830 KP771831 KF257969 KP771832 KP771833 AY611077 DQ980015 DQ980015 KP771834 JX974654 JX974658 GU994556

– KM386143 KM386144 – – KM386145 KM386146 KM386147 KM386148 KM386149 KM386150 KM386151 KM386152 KM386153 – – – – DQ923666 KM386154 – KM386155 KM386156

– KM386234 KM386240 – KM386239 KM386243 KM386241 KM386238 KM386246 KM386248 KM386235 KM386236 KM386237 KM386242 – – – – – KM386247 KM386245 KM386249 KM386282

– KM386185 KM386186 – – KM386187 KM386188 KM386189 KM386190 KM386191 KM386192 KM386193 KM386194 KM386195 – – – – – KM386196 KM386197 KM386198 KM386199

– KM386283 KM386290 – – KM386287 KM386284 KM386285 KM386286 – – KM386291 KM386292 KM386288 – – – – – – – – –

KM386103 KM386107 KM386114 – – KM386109 KM386110 KM386112 KM386104 KM386111 KM386113 KM386108 KM386105 KM386106 – – – – – – – – –

5971 5990 3891

M. panniformis M. panniformis M. panniformis

KM386100 – KP771838

KM386162 – KM386157

KM386258 KM386259 KM386250

KM386206 KM386207 KM386200

KM386301 – KM386293

KM386141 – KM386129

3998

M. panniformis

3999

M. panniformis

4000

M. panniformis

4843

M. panniformis

8598

M. panniformis

8599

M. panniformis

MEPA1075

M. panniformis

MEPA1080

M. panniformis

MEPA1086

M. panniformis

8597

M. predisjuncta

‘panniformis ‘panniformis ‘panniformis str’. ‘panniformis str’. ‘panniformis str’. ‘panniformis str’. ‘panniformis str’. ‘panniformis str’. ‘panniformis str’. ‘panniformis str’. ‘panniformis str’. ‘panniformis str’. ‘disjuncta’

4001

M. sorediata

‘sorediata’

disjuncta disjuncta disjuncta disjuncta disjuncta disjuncta disjuncta disjuncta disjuncta disjuncta disjuncta disjuncta disjuncta disjuncta disjuncta disjuncta disjuncta disjuncta disjuncta disjuncta disjuncta disjuncta disjuncta

A’ A’ s.

Russia, Khabarovskiy Krai Alaska, Denali N.P. USA, Maine, Washington County

Spribille s.n. ESH-09B.363 (C) ESH-08.304 (C) ESH-09B.051 (C) Harris 52938 (NY) Bjerke WP286-2 (TLE) Hollinger 1061 (UBC) Goward 10–19 (UBC) Esslinger BP109-1 (TLE) Esslinger BP97-1 (TLE) Esslinger BP94-2 Esslinger BP94-3 (TLE) Esslinger 19403 (TLE) McMullin 7483 (TLE) Hansen ESH-08.216 (C) Hansen ESH-09B.323 (C) Mayrhofer 13743 NA Wedin 7143 (UPS) Wedin 7143 (UPS) Coppins s.n (MAF) Goward 2008 (MAF) Divakar, 2007 (MAF-Lich 15512) Spribille 31832 (GZU) Spribille 27965 (GZU) Lumbsch 2010/M8

s.

USA, Maine, Knox County

Harris 55589 (NY)

KP771839

KM386158

KM386251

KM386201

KM386294

KM386130

s.

USA, Maine, Washington County

Harris 54728 (NY)

KP771840

KM386159

KM386257

KM386202

KM386295

KM386131

s.

USA, Maine, Washington County

Harris 52834(NY)

KP771841

KM386160

KM386253

KM386203

–

KM386132

s.

Canada, British Columbia

Esslinger BP97-3 (TLE)

KP771842

–

KM386255

KM386204

KM386296

KM386133

s.

Ohmura 9673

–

–

–

KM386205

–

–

Ohmura 9654

KM386099

KM386161

KM386254

–

–

–

s.

Japan, Azusayama, Kawakami-mura, Minamisaku-gun Japan, Azusayama, Kawakami-mura, Minamisaku-gun Canada, British Columbia

Goward 2007 (MAF)

JX974655

JX974668

KM386281

KM386208

–

–

s.

Sweden, HŠlsingland

Wedin 8285 (MAF)

JX974656

KM386163

KM386256

KM386209

–

–

s.

USA, Washington

Esslinger 18643 (TLE)

JX974657

KM386164

KM386252

KM386210

–

–

Japan, Azusayama, Kawakami-mura, Minamisaku-gun USA, Pennsylvania. Lackawanna Co

Ohmura 9653

KM386098

KM386165

KM386244

KM386211

KM386289

–

Lendemer 13329 (NY)

KP771846

KM386168

KM386277

KM386214

–

KM386136

s.


Taxon

3921 3956 3957 3963 3995 4503 4667 4668 4826 4841 4850 4851 5970 6374 6574 6575 AY611077 DQ980015 MDISJMATS MDISJUNCT MEDI637 MESO1076 MESO773

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ID



Table 1 Collection information for all specimens included in the present study: ‘ID’, individual code; ‘Taxon’, morphological/chemical species identification; ‘AMBD/PTP Clade’, putative species inferred from the Automatic Barcode Gap Discovery and Poisson tree processes species delimitation analyses; ‘Geographic Origin’; ‘Voucher’; and GenBank accession numbers for the six sample markers (ITS, nrLSU, mtSSU, MCM7, RPB1, and RPB2).

M. sorediata M. tominii M. tominii M. tominii M. tominii M. tominii M. tominii M. tominii M. tominii M. tominii M. tominii M. tominii M. tominii M. tominii M. tominii M. tominii M. tominii M. tominii M. tominii M. tominii M. tominii M. tominii Melanelixia californica Melanohalea subolivacea Xanthoparmelia verruculifera Xanthoparmelia wyomingica

‘sorediata’ ‘sorediata’ ‘sorediata’ ‘sorediata’ ‘sorediata’ ‘sorediata’

Bjork 15153 (UBC) Esslinger BP111-1 (TLE) Esslinger BP73-6 (TLE) Spribille 31972 (GZU) McMullin 8139 (TLE) Ohmura 9666

KM386102 KF257979 KF257980 KP771847 KF257982 KM386101

– KM386171 KM386166 KM386169 KM386172 KM386170

– KM386279 KM386275 KM386276 KM386280 KM386278

KM386217 KM386218 KM386212 KM386215 KM386219 KM386216

– KM386299 KM386297 – KM386300 KM386298

KM386139 KM386140 KM386134 KM386137 KM386135 KM386138

‘sorediata’ ‘tominii A’ ‘tominii A’ ‘tominii A’ ‘tominii A’ ‘tominii A’ ‘tominii A’ ‘tominii A’ ‘tominii A’ ‘tominii A’ ‘tominii A’ ‘tominii B’ ‘tominii C’ ‘tominii C’ ‘tominii C’ ‘tominii C’ ‘tominii D’ ‘tominii D’ ‘tominii D’ ‘tominii D’ ‘tominii D’ ‘tominii E’ outgroup outgroup outgroup

Canada, British Columbia Canada, British Columbia USA, Alaska Russia, Khabarovskiy Krai Canada, Ontario Japan, Mt. Ohyama, Azusayama, Kawakami-mura, Minamisaku-gun Sweden, VŠsterbotten ÊUSA, California ÊUSA, Montana USA, New Mexico, Catron County USA, Colorado USA, UT, Wayne County USA, UT, Wayne County USA, California, San Diego County USA, California, Tuolumne Co Canada, British Columbia Canada, British Columbia China, Heilongjiang USA, Alaska Canada, British Columbia Canada, Yukon Territory Canada, Yukon Territory China, Hebei, Mt, Wulingshan China, Inner Mongolia, Mt. Arxan China, Inner Mongolia, Bairin Youqi India, Uttaranchal India, Uttaranchal China, Heilongjiang USA, California USA, Washington, Spokane Co. Canada, British Columbia

Wedin 6862 (UPS) Hutten 14162 (YOSE) ÊEsslinger 16627 (TLE) BRY-C32307 Esslinger (TLE) Leavitt 1021 (F) Leavitt 1022 (F) Lendemer 4494 (NY) Lendemer 19701 (NY) Esslinger BP109-4 (TLE) Esslinger BP105-4 (TLE) Ren, Qiang s.n. (HMAS-L) McCune 30793 (OSU) Esslinger BP105-2 (TLE) Esslinger BP93-1 (TLE) Esslinger BP20-1 (TLE) 114000 (HMAS-L) 36389 (HMAS-L) 71058 (HMAS-L) Divakar s.n MAF-Lich 15516 Ren, Qiang Esslinger 18915 Leavitt 2010-976 (F) Goward 09-358 (UBC)

GU994557 KP771848 KP771849 KP771850 KP771851 KP771852 KP771853 KP771854 KP771855 KF257994 KF257996 KP771856 KF257993 KF257995 KF257997 KP771857 EU784154 EU784155 EU784156 METO402 GU994559 KP771858 EU761210 JQ813752 KM386097

KM386167 – – – KM386173 KM386174 KM386175 – KM386176 – KM386177 KM386178 KM386179 KM386180 KM386181 KM386182 – – – – – KM386183 JQ812998 JQ813103 KM386184

– KM386260 KM386261 KM386262 KM386263 KM386264 KM386265 – KM386267 KM386268 KM386266 KM386269 KM386271 KM386273 KM386272 KM386274 – – – – GU994647 KM386270 – JQ813194 –

KM386213 KM386220 KM386221 – KM386222 KM386223 KM386224 KM386225 KM386226 KM386227 KM386228 KM386229 KM386230 KM386231 KM386232 KM386233 – – – – JX974707 – JQ813899 JQ813975 –

– KM386302 KM386303 – KM386304 KM386305 KM386306 – KM386308 KM386314 KM386307 KM386309 KM386310 KM386311 KM386312 KM386313 – – – – GU994708 KM386315 JQ813985 JQ814058 –

– KM386115 KM386121 – KM386116 KM386122 KM386117 – KM386123 KM386118 KM386119 KM386120 KM386124 KM386125 KM386126 KM386127 – – – – – KM386128 JQ813200 JQ813294 KM386142

outgroup

USA, Wyoming, Big Horn Mountains

Leavitt 826 (BRY-C)

HM578953

HM579360

–

HM579746

KF908576

KF908755

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MESO778 3752 3771 3925 3954 3967 3982 4004 4005 4829 4835 3653 4390 4833 4852 5217 EU784154 EU784155 EU784156 METO402 METO776 3658 3742 4341 4669 826

sorediata sorediata sorediata sorediata sorediata sorediata

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M. M. M. M. M. M.



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2011). Therefore, we used three sequence-based SDMs to validate traditional, morphology-based species delimitations and circumscribed any previously unrecognized species-level lineages in Montanelia. Specifically, we used the automatic barcode gap discovery method (ABGD; Puillandre et al., 2012); the General Mixed Yule Coalescent approach (GMYC; Fujisawa and Barraclough, 2013; Pons et al., 2006); and a recently described Poison tree processes model to infer putative species boundaries (PTP; Zhang et al., 2013). The ITS region has recently been formally adopted as the barcoding marker for fungi (Schoch et al., 2012), and we used this locus for species delimitation analyses. ABGD employs a genetic distance-based approach to detect a ‘‘barcode gap’’ separating candidate species based on non-overlapping values of intra- and interspecific genetic distances and is independent of any topology (Hebert et al., 2003; Puillandre et al., 2012). This method uses a range of prior intraspecific divergence to infer a model-based confidence limit for intraspecific divergence, and then detects the barcode gap as a first significant gap beyond this limit to infer primary partitions. The primary data partitions are then recursively split to get finer partitions using the same approach until no further gaps can be detected (Puillandre et al., 2012). The ITS marker has been adopted as the primary barcode marker for fungi (Schoch et al., 2012), and we used the ABGD web server (http://wwwabi.snv.jussieu.fr/public/abgd/abgdweb. html) to identify a barcode gap in the Montanelia ITS data matrix. Based on empirical comparisons of groupings inferred using ABGD with data from previous studies where species groups are well-characterize, Puillandre et al. (2012) suggested that implementing a Pmax value of 0.01 provides the most accurate estimate for the number of groups. Genetic distances were calculated using the JC69 model (default parameter), and model parameters were set using default parameter values as follows, with the exception of the Pmax value: Pmin = 0.001, Pmax = 0.01, steps = 10, and Nb bins = 20. We implement a range of values for the gap width (X), between 0.1 and 1.5 to assess the consistency of the inferred groups under varying gap width values. The original GMYC method is a likelihood approach for delimiting species by fitting within- and between-species branching models to gene topologies (Fujisawa and Barraclough, 2013; Monaghan et al., 2009; Pons et al., 2006). In order to incorporate gene tree uncertainty, we used a Bayesian implementation of the GMYC (bGMYC) that samples over the posterior distribution of trees (Reid and Carstens, 2012). Ultrametric ITS gene trees were estimated from the complete ITS data matrix using the program BEAST v1.8.0 (Drummond and Rambaut, 2007; Drummond et al., 2012). We ran two independent Markov Chain Monte Carlo (MCMC) chains for 25 million generations, implementing a relaxed lognormal clock and a constant coalescent speciation process prior. Default values were used for the remaining priors. Chain mixing and convergence were evaluated in Tracer v1.5 (Rambaut and Drummond, 2003), considering effective sample size (ESS) values > 200 as a good indicator; and the first 25 million generations were discarded as burn-in. As input for the bGMYC analysis, a total of 100 trees were evenly sampled from the throughout the posterior distribution of trees (50 trees from each independent run). The starting number of species was set to half the total number of tips, and the remaining settings were left at their default values. The bGMYC package was run in in R v3.0.2 (R Core Team, 2014) for 50,000 generations, sampling every 200th generation, and discarding the first 40,000 trees as burn-in. The PTP model for species delimitation is intended for delimiting species in single-locus molecular phylogenies, and provides an objective approach for delimiting putative species boundaries that are consistent with the phylogenetic species criteria (Taylor et al., 2000; Zhang et al., 2013). In contrast to the GMYC model, PTP does not require an ultrametic input tree, rather PTP models speciation in terms of

the number of substitutions (Zhang et al., 2013). PTP has been shown to outperform the GMYC method, particularly when evolutionary distances between species are small (Zhang et al., 2013). We used the program RAxML v8.0.0 (Stamatakis, 2006; Stamatakis et al., 2008) to reconstruct a maximum likelihood (ML) ITS locus-tree using 500 ML tree searches and implementing the ‘GTRGAMMA’ model on the CIPRES Science Gateway server (http://www.phylo.org/portal2/). We used the PTP web server (http://species.h-its.org) to delimit putative species groups using the ITS gene topology as the input tree, with the P-value set to 0.001. Although the PTP model is intended for delimiting species from single gene topologies, we also used PTP to delimit species from the concatenated six-gene topology (see Section 2.4.1) and compared putative species with those resulting other species delimitation methods.

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2.4.1. Phylogenetic relationships within Montanelia Phylogenetic relationships were inferred using ML, and Bayesian inference (BI), in addition to a multispecies coalescent approach (see Section 2.4.2). Exploratory phylogenetic analyses of individual gene topologies showed no evidence of well-supported (P70% bootstrap values) topological conflict, and relationships were estimated from the concatenated, six-locus data matrices using a total-evidence approach (Kluge, 1989; Wiens, 1998). Although our combined data matrix consisted of approximately 30% of missing data (see results), phylogenetic analyses can accurately infer relationships, particularly if many characters are sampled overall (Wiens and Morrill, 2011). We used the program RAxML v8.0.0 (Stamatakis, 2006; Stamatakis et al., 2008) to reconstruct the concatenated ML gene-tree using the CIPRES Science Gateway server (http://www.phylo.org/portal2/). We implemented the ‘GTRGAMMA’ model, used locus-specific model partitions treating all loci as separate partitions, and evaluated nodal support using 1000 bootstrap pseudoreplicates. An alternative partition strategy was inferred via PartitionFinder v1.1.1 (Lanfear et al., 2012). The best-fitting partition scheme was selected from a total of 20 initial pre-defined partitions, corresponding to: the ITS1, 5.8S, and ITS2 (from the ITS region); the complete LSU region; the complete mtSSU region; the first, second and third codon positions of the MCM7; the first, second and third codon positions of two coding region in the RPB1; two introns in the RPB1; two intronic regions in the PRB1; the first, second and third codon positions of the coding region in the RPB2; and an intron in the RPB2. We also reconstructed phylogenetic relationships from the concatenated multi-locus data matrices under BI using the program BEAST v1.8.0 (Drummond and Rambaut, 2007; Drummond et al., 2012). We ran two independent Markov Chain Monte Carlo (MCMC) chains for 50 million generations, implementing a relaxed lognormal clock, a constant coalescent speciation process prior, and using the partitioning strategy inferred from PartitionFinder. The first 12.5 million generation were discarded as burn-in. Chain mixing and convergence were evaluated in Tracer v1.5 (Rambaut and Drummond, 2003), considering ESS values > 200 as a good indicator. Posterior trees from the two independent runs were combined using the program LogCombiner v1.8.0 (Drummond et al., 2012), and the final maximum clade credibility (MCC) tree was estimated from the combined posterior distribution of trees.

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2.4.2. Species trees and divergence dating By incorporating the coalescent histories of individual markers, multi-locus species tree methods can provide more accurate hypotheses of evolutionary relationships (Degnan and Rosenberg, 2006; Degnan and Rosenberg, 2009; Heled and Drummond, 2010; Leaché, 2009) and more biologically realistic estimates of divergence times (Edwards and Beerli, 2000; McCormack et al.,

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2011), relative to concatenated gene tree approaches. Therefore, we used the coalescent-based hierarchical Bayesian model ⁄BEAST implemented in BEAST v1.8.0 (Drummond and Rambaut, 2007; Heled and Drummond, 2010) to estimate a species tree and divergence times for Montanelia. *BEAST incorporates the coalescent process and the uncertainty associated with gene trees and nucleotide substitution model parameters and estimates the species tree directly from the sequence data (Heled and Drummond, 2010). Population assignments are required a priori for *BEAST analyses, and we used the candidate species inferred from the ABGD and PTP analyses. These species delimitation approaches resulted in consistent estimates of species boundaries and were further corroborated to varying degrees by phenotypic characters and/or biogeographic patterns (see Section 3.2). A number of previous studies indicate that estimated substitution rates for the ITS and nrLSU markers can provide divergence estimates similar to those from fossil-calibrated divergence times in Parmeliaceae (Amo de Paz et al., 2011; Divakar et al., 2012; Leavitt et al., 2012a, 2012b, 2012c). In this study, we used 2.43 10 9 substitution/site/year (s/s/y) for the ITS marker (Leavitt et al., 2012b). This rate is similar to other estimates of ITS substitution rates for lichen-forming fungi (2.38 10 9 s/s/y Oropogon, Parmeliaceae, Lecanorales; Leavitt et al., 2012c) and a non-lichenized fungus (2.52 10 9 s/s/y, Erysiphales; Takamatsu and Matsuda, 2004). For the nrLSU we used the Parmeliaceae-specific rate of 0.70 10 9 s/s/y for the nrLSU (Amo de Paz et al., 2011). The substitution rates for other loci were co-estimated under a uniform prior between 0 and 100. We estimated divergence times under an uncorrelated relaxed lognormal molecular clock (Drummond et al., 2006), implementing a Yule process and gamma-distributed population sizes for the species tree prior and a piecewise linear population size model with a constant root. Two independent MCMC runs of 100 million generations were performed, sampling every 2000 steps. Chain mixing and convergence were evaluated in Tracer v1.5 (Rambaut and Drummond, 2003) considering ESS values > 200 as a good indicator. After excluding the first 25% of sampled trees as burn-in, trees from the two independent runs were combined using the program LogCombiner v1.8.0 (Drummond et al., 2012), and the final MCC tree was estimated from the combined posterior distribution of trees.using TreeAnnotator v1.8.0 (Rambaut and Drummond, 2009).

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3. Results

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3.1. Molecular data

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All new sequences generated in association with this study were deposited in GenBank under accession numbers KM386097–KM386315 and KP771824–KP771858. Genetic variability of sampled markers used in this study and models of evolution selected using the program jModeltest are reported in Table 2. Twenty-five ambiguously aligned nucleotide positions were removed from the mtSSU alignment using GBlocks. The final concatenated six-gene data matrix was comprised of 3988 aligned nucleotide positions, and both the final, trimmed data matrix and a data matrix including the ambiguous mtSSU characters are deposited in TreeBase (#16237). Overall, approximately 30% of sequences were missing from the concatenated data matrix, including ITS sequences from four specimens (Table 2).

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3.2. Species delimitation analyses

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Results of the three sequence-based methods for species delimitation are summarized in Fig. 1. Using a Pmax value = 0.01, the ABGD analysis resulted in estimates from four to eight distinct

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Table 2 Genetic variability of sampled markers for the complete dataset used in this study, including the total number of samples, N (number of haplotypes); aligned length; variable and parsimony-informative (PI) sites for each sampled locus; and model of evolution selected using the program PartitionFinder. Locus

N (haplotypes)

Aligned bp

No. variable sites (PI sites)

Model

ITS nrLSU mtSSU MCM7 RPB1 RPB2 All loci

65 45 51 53 36 43

517 569 847 566 749 740 3988

157 (109) 72 (39) 61 (29) 146 (77) 186 (89) 177 (117) 799 (460)

TrNef + I + G TrNef + G HKY + I TrNef + G TrNef + G TrNef + G NA

(43) (24) (29) (34) (20) (26) 69

groups, depending on the gap width parameter. For gap width values ranging from 0.7 to 1.5, the ABGD analyses inferred four distinct groups, lumping all specimens identified as M. sorediata and M. tominii into a single group. Gap width values between 0.1 and 0.6 resulted in five to eight groups, with M. tominii specimens grouped into one to four putative species (results not shown). In all cases, M. predisjuncta was recovered in the M. disjuncta group. The ABGD analysis implementing a gap width value of 0.5 resulted in eight groups consistent with those inferred from the PTP species delimitation method (see below), and these groups are indicated in Fig. 1. The posterior distribution of the bGMYC species delimitation analysis is shown in Supplementary File S5 and is summarized in Fig. 1). Putative unrecognized species-lineages were recovered in both M. panniformis s. lat. and M. tominii s. lat. (Fig. 1). A total of eight putative species were identified in Montanelia from the PTP analyses of the ITS topology, with a speciation rate = 31.940; coalescent rate = 261.131; null logl = 208.568; max logl = 241.371; and P-value < 0.001. A PTP analysis of the concatenated six-gene topology resulted in six putative species, with a speciation rate = 43.480; coalescent rate = 819.620; null logl = 412.924; max logl = 503.672; and P-value < 0.001. The PTP analysis of both the ITS and concatenated ML topologies (see Section 3.3) resulted in putative species boundaries similar to those delimited by the ABGD analysis, including previously unrecognized species-level lineages in M. panniformis and M. tominii, with slight differences in the number of putative species in the M. tominii clade (Fig. 1; Supplementary File S6 and S7).

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3.3. Phylogenetic analyses

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The ML phylogeny estimated from the concatenated 6-locus data matrix is shown in Fig. 1. Montanelia was recovered as monophyletic with strong statistical support, and relationships among clades within Montanelia were supported with bootstrap (BS) values P 70%. Three major, well-supported clades were recovered in the total evidence phylogeny: the ‘M. disjuncta clade’, which included the single sampled M. predisjuncta specimen; the ‘M. panniformis/sorediata clade’, and the ‘M. tominii clade’; (Fig. 1). Well-supported phylogenetic substructure was recovered in the ‘M. panniformis/sorediata’ and ‘M. tominii’ clades. While, specimens identified as M. sorediata were recovered as monophyletic, M. panniformis was recovered as paraphyletic, with two specimens from the Russian Far East and Alaska, USA recovered as sister to M. sorediata (Fig. 1). Three well-supported sub-clades were recovered within the M. tominii clade, in addition to two specimens that fell outside of these clades (Fig. 1). Sorediate and esorediate individuals from North America were recovered in two separate, well-supported clades (Fig. 1). The third sub-clade within the M. tominii complex was comprised of individuals collected from Asia

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(China and India). The two remaining sub-clades in the M. tominii clade, both represented by a single specimen, were collected from the Heilongjiang Province in northeastern China. The BI topology inferred from the concatenated six-locus dataset in BEAST was congruent with the ML topology (Supplementary File S8). The alternative partition strategy inferred via PartitionFinder resulted in four partitions: (i) ITS1 and ITS2; (ii) 5.8S, LSU, first codon positions for the MCM7, RPB1, and RPB2; (iii) mtSSU and second codon positions for the MCM7, RPB1, and RPB2; and (iv) introns in the RPB1 and RPB2 and third codon positions for the MCM7, RPB1, and RPB2. The resulting topology inferred from this partitioning strategy was identical to that presented in Fig. 1, and nodal support values were highly similar to those estimated for the gene-specific partition strategy (data not shown).

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3.4. Species tree and divergence estimates

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In the *BEAST analyses, run lengths were sufficient to generate ESS > 200, and topologies and divergence estimates were congruent across independent runs. A MCC species tree based on the results of the GMYC species delimitation analysis is shown in Fig. 2. The diversification of the extant members of Montanelia was estimated to have begun 23.1 Ma (HPD = 16.8–30.4 Ma),

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consistent with a previous fossil-calibrated estimate (Divakar et al., 2012). Our divergence time estimates support a Neogene-dominated diversification history in Montanelia (Fig. 2). Diversification of the M. panniformis/sorediata clade began during the Miocene, ca. 7.1 Ma (HPD = 4.6–9.9 Ma). Our data suggest the split between M. sorediata and ‘M. panniformis C’ occurred during near the Miocene/Pliocene boundary (ca. 5 Ma, HPD = 2.7–7.5 Ma; Fig. 2). However, diversification among all putative within M. disjuncta s. l., some lineages within M. panniformis s. l. (including M. panniformis lineages designated as ‘A’ and ‘B’) and M. tominii s. l. was estimated to have occurred, at least in part, during the Pleistocene (Fig. 2). In the case of the M. tominii complex, our divergence estimates suggest that the initial diversification of the M. tominii species complex began during the late Miocene (7.5 Ma, HPD = 4.7–10.6 Ma) and continued into the Pleistocene (Fig 2). M. predisjuncta was nested in the M. disjuncta clade, and, molecular sequence data did not support the former as a distinct lineage.

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4. Discussion

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In this study, we sampled the five known species in the brown parmelioid lichen-forming fungal genus Montanelia, four of which have broad intercontinental distributions. Using sequence-based

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Fig. 2. Rate-calibrated species trees for Montanelia inferred from six loci (ITS, nrLSU, mtSSU, MCM7, RPB1, and RPB2) using the program *BEAST. Putative species used in the * BEAST analysis were inferred from ITS sequence data using the ABGD and PTP species delimitation methods (inferences were identical between these two methods). Posterior probabilities (PP) are shown above branches, with thickened branches indicating PP P 0.95; and numbers at nodes indicate estimated node ages (millions of years ago [Ma]).


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SDMs, we identified previously unrecognized species-level lineages in the nominal taxa M. panniformis and M. tominii (Fig. 1) and validated the traditional, phenotype based species circumscription of M. sorediata. Specimens identified as M. disjuncta were also consistently recovered in a single OTU in all analyses, which also included the morphologically distinct species M. predisjuncta. No evidence of phylogenetic substructure corresponding to distinct geographic regions was identified for the majority of OTUs circumscribed in this study, including M. disjuncta, M. panniformis s. str., and M. sorediata. In contrast to other Montanelia species, our results revealed complex biogeographic patterns in the M. tominii group. Below we compare sequence-based SDMs and discuss species diversity, distributions and the timing of diversification in Montanelia.

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4.1. Comparisons of sequence-based species delimitation methods

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Molecular data provide a valuable resource for preliminary species delimitations or validating traditional phenotype-based species circumscriptions (Fujisawa and Barraclough, 2013; Kelly et al., 2011; Puillandre et al., 2012; Schoch et al., 2012). Employing multiple SDMs can increase confidence by identifying congruence in putative species across analytical methods operating under distinct assumptions (Carstens et al., 2013; Jörger et al., 2012), and a careful examination of discordance among SDMs may facilitate an improved perspective of species boundaries (Kekkonen and Hebert, 2014). In our empirical comparison of the ABGD, PTP, and GMYC species delimitation methods using data from all known Montanelia species, the ABGD analysis of the ITS alignment and PTP analysis of the ITS gene tree provided congruent estimates of species diversity for the genus (Fig. 1). While the ABGD method formulates species boundaries by detecting a gap in the distribution of pairwise genetic distances (Puillandre et al., 2012), PTP delimits species based on a phylogenetic species criterion by modeling speciation in terms of the number of substitutions (Zhang et al., 2013). Coupled with a general corroboration of traditional morphology-based species circumscription, biogeographic and morphological patterns in M. tominii s. lat., the complete congruence between these two distinct SDMs offers an additional level of confidence in the inferred OTUs in Montanelia (Fig. 1). However, in exploratory ABGD analyses, input parameters for the maximum intraspecific distance had a significant impact on the estimated number of clusters, ranging from two distinct groups using default parameters values to nine groups when implementing Pmax = 0.001 and X = 0.5 (data not shown). While discrepancies in the number of putative species when using different Pmax parameter values is expected, our results also indicated the gap width parameter can also have a significant impact on the number of groups inferred using ABGD under an identical Pmax value. Ultimately, objectively defining the appropriate parameter values for ABGD remains challenging and has practical implications for assessing species-level diversity, particularly when results vary widely across a range of parameter values and phenotypic characters for OTUs are unobservable (e.g., environmental sampling or assessing diversity in microbial communities). Our study offers additional support that PTP can provide an objective and reasonable starting point for species delimitation using single gene topologies. Compared to the GMYC approaches, PTP has been shown to be more accurate for preliminary species delimitation (Zhang et al., 2013). Furthermore, relative to the GYMC methods, PTP offers a more straightforward implementation, requiring a simple phylogenetic tree, rather than a time-calibrated chronogram. Our empirical study of Montanelia indicated that the PTP species delimitation method provided reasonable results, consistent with inferences obtained using ABGD,

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and generally congruent with either traditional morphology-based species circumscriptions and or biogeographic and morphological patterns in the M. tominii complex. In our empirical study of the genus Montanelia, we show that using a topology generated from concatenated data for the PTP analysis resulted in significant discrepancies in the number of circumscribed OTUs when compared to results from single gene topology. Putative species-level lineages can be validated within an integrative taxonomic framework by incorporating a careful examination of morphology, behavior, ecology, or other factors potentially corroborating the evolutionary independence of putative species (Dayrat, 2005; Fujita et al., 2012). The coalescent-based model for species delimitation, Bayesian Phylogenetics and Phylogeography (BP&P), provides an objective approach for validating putative species using multilocus sequence data by calculating posterior probabilities of different species delimitation models using a reversible-jump Markov chain Monte Carlo (Yang and Rannala, 2010). While this method has been shown to perform accurately under a wide range of scenarios, BP&P has lower accuracy when fewer loci and individuals are sampled per species (Camargo et al., 2012b; Zhang et al., 2011), as was the case for a number of putative Montanelia OTUs circumscribed in this study (i.e. M. ‘panniformis C’, M. ‘tominii B’, and M. ‘tominii E’). Exploratory analyses using BP&P failed to provide high speciation probabilities (>0.95) for any of the species within Montanelia under a number of scenarios (data not shown), in spite of the fact that these were supported in using other single-locus SDMs. This discrepancy is most likely due to the fact the lineages M. panniformis ‘A’, M. tominii ‘B’, and M. tominii ‘E’ were represented by very few individuals and were missing data for a number of loci. Our limited sampling of individuals and genetic markers for a number of OTUs suggests that the current species tree must be interpreted with some degree of caution. Additional sampling of M. panniformis s. lat. and M. tominii s. lat. populations will be crucial to validating species boundaries in these two groups.

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4.2. Diversity, distributions and phylogeography in Montanelia

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Based on the results of this study, we propose that the genus Montanelia is likely comprised of six to nine species-level lineages, including previously unrecognized species-level diversity in M. panniformis s.lat. and M. tominii s. lat. (Fig. 1). Our data also suggest that M. predisjuncta is likely synonymous with M. disjuncta. According to Esslinger (1977), Montanelia predisjuncta differs from M. disjuncta by the absence of soredia, narrow and somewhat thinner lobes, subpulvinate or pulvinate habit, and more distinctive and more numerous pseudocyphellae in the former. However, the author also discussed the great variability in number of soralia produced by M. disjucta. In addition to the original report from Japan, esorediate specimens identified as M. predisjuncta have also been reported from Russia (Makryi, 1981) and China (Wang et al., 2009). Our study included only a single specimen representing M. predisjuncta collected at the type locality in central Japan. Another esorediate specimen identified as M. disjuncta (ID #4850) from the Yukon Territory, Canada was also recovered within the M. disjuncta clade, further indicating that M. disjuncta specimens may not consistently produce soredia. In contrast to M. disjuncta, esorediate specimens in the M. tominii complex corresponded to a distinct, well-supported clade that was also recovered as a distinct OTU in three sequence-based SDMs (Fig. 1). Based on the current sampling, the production of soredia serves as a diagnostic character for distinguishing the two M. tominii s. lat. OTUs occurring in North America. However, additional sampling of Asian material representing M. tominii s. lat. will be required to adequately assess diversity and biogeography in the M. tominii complex. Our data indicate that M. panniformis s.lat. consists of at least two

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species-level lineages, including M. panniformis s. str. and M. ‘panniformis A’. Our dating analysis suggested that M. panniformis s. str. split from the M. ‘panniformis A’/M. sorediata clade during the Miocene ca. 7.2 Ma (Fig. 2). M. ‘panniformis A’ is morphologically similar to other M. panniformis s. lat. specimens, rather than its sister OTU, M. sorediata (Fig. 1). However, preliminary morphological comparisons of the lineage circumscribed as M. ‘panniformis A’ with other M. panniformis collections failed to reveal ecological differences or diagnostic phenotypic traits corroborating the separation of M. ‘panniformis A’ from M. panniformis s. str. Montanelia. ‘panniformis A’ is so far known from northwestern North America and eastern Asia, while both M. panniformis s. str. and M. sorediata have broad, intercontinental distributions throughout the Northern Hemisphere. At least three Montanelia species – M. disjuncta, M. panniformis s. str., and M. sorediata – have broad, intercontinental distributions, with no evidence of phylogeographic substructure (Fig. 1). Each of these species is broadly distributed throughout Asia, Europe, and North America. In contrast, striking biogeographic patterns were observed in the M. tominii species complex (Fig. 1). While M. tominii has rarely been reported from Europe (Hawksworth et al., 2008; Kristinsson et al., 2010), it occurs commonly throughout western North America and Asia. Our study reveals a diversification history of the M. tominii group beginning in the late Miocene and continuing into the Pleistocene (Fig. 2). Based on our current sampling, two putative lineages in the M. tominii complex appear to be restricted to Asia, while two other lineages appear to occur exclusively in North America (Fig. 1). While the two candidate species in North America can be distinguished morphologically – one sorediate ‘M. tominii A’ and the other lacking soredia ‘M. tominii C’, at this point we have not identified diagnostic phenotypic characters distinguishing the sorediate OTUs. It is worth noting that the Asian and North American OTUs from the GYMC analysis were not recovered as monophyletic groups. Rather, it appears that OTUs from Asia and North America, respectively, are each other’s closest relative for the four OTUs estimated to have diversified during the Pleistocene (Fig. 2). These results provide circumstantial evidence supporting dispersal between the Asian highlands and the New World through Beringia during the Pleistocene. Similar biogeographic patterns have been identified previously for some clades within another genus of brown parmelioid lichens, the epiphytic genus Melanohalea, which also appear to have diversified during the Pleistocene (Leavitt et al., 2012a). This pattern has also been observed for a number other groups (Dixon, 2013; Vila et al., 2011). Future studies investigating the role of glacial refugia in Beringia for intercontinental dispersal will be essential to provide additional insight in promoting diversification in Parmeliaceae, and lichen-forming fungi in general. The contrasting pattern of broad, intercontinental distributions for most Montanelia species vs. more geographically restricted species distributions in the M. tominii complex highlights the importance of additional research investigating dispersal and reproductive barriers among closely related species in the M. tominii complex.

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4.3. Rate-calibrated divergence estimates

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Similar to previous studies (Amo de Paz et al., 2012; Amo de Paz et al., 2011; Divakar et al., 2012; Leavitt et al., 2012a; Leavitt et al., 2012b; Leavitt et al., 2012c), the results of this study highlight the importance of the Miocene and Pliocene in diversification in Parmeliaceae, but also support the role of Pleistocene climatic fluctuations in driving speciation in some groups. The estimated age of the most recent common ancestor (MRCA) for Montanelia (ca. 23.1 Ma; Fig. 2) is similar to estimates for other genera in Parmeliaceae, including Parmelia (ca. 23.6 Ma), Remototrachyna

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(ca. 23.5 Ma), and Xanthoparmelia (ca. 21.6 Ma). In spite of the similar ages of the MRCA for each of these genera, species richness varies widely among each genus, ranging from ca. six to nine species in Montanelia, ca. 15 species in Remototrachyna, ca. 57 in Parmelia, to over 820 species in Xanthoparmelia (Thell et al., 2012). Disparity in species diversity among clades of similar ages are the result of differences in extinction and/or speciation rates (Ricklefs, 2007; Wertheim and Sanderson, 2011), and future studies investigating factors influencing the tempo and mode of species diversification through time will be crucial to understanding evolution in the species-rich lichen-forming fungal family Parmeliaceae. In our rate-calibrated species tree, the estimated origin of Montanelia was very similar to a previous fossil-calibrated estimate for the genus (Divakar et al., 2012), 23.1 Ma vs. 23.2 Ma, respectively. Previous studies investigating divergence times in other genera in Parmeliaceae have also revealed similar estimates between fossil- and rate-calibrated trees (Leavitt et al., 2012a; Leavitt et al., 2012b). For example, divergence times for the origin of the Melanohalea clade were estimated at ca. 35.2 Ma in a rate-calibrated species-tree of the genus Melanohalea using Parmeliaceae-specific rates for the RPB1, nrLSU, and mtSSU (Leavitt et al., 2012a), relative to an estimate of 32.7 Ma in a fossil-calibrated chronogram of parmelioid lichens (Amo de Paz et al., 2011). Previous studies for Parmeliaceae have reported an estimated substitution rate for the ITS marker ranging from 2.43 10 9 s/s/y (Melanelixia, Leavitt et al., 2012b) to 3.41 9 s/s/y (Melanohalea; Leavitt et al., 2012a). These rates are similar to an estimated substitution rate of 2.52 10 9 s/s/y for a non-lichenized fungus in Erysiphales (Takamatsu and Matsuda, 2004). A number of previous studies using similar ITS mutation rates to estimate the temporal context of diversification in a number of genera in Parmeliaceae have resulted in divergence estimates largely consistent with fossil-calibrated dates (Fernández-Mendoza and Printzen, 2013; Leavitt et al., 2012c; Leavitt et al., 2013c), and similar ITS mutation rates have also been applied to other lineages of fungi (Leavitt et al., 2013c; Werth et al., 2013). However, a recent study of the specie-rich lichen-forming fungal genus Xanthoparmelia indicates that using taxon-specific mutation rates from different loci may result in significant differences in divergence estimates (Leavitt et al., 2013d). Therefore, rate-calibrated divergence times for Parmeliaceae must be interpreted with a high degree of caution, particularly with the absence of fossil calibrations or other independent support. In addition, our species tree and divergence time inferences are somewhat limited by small sample sizes and missing molecular data for a number of OTUs. The estimated recent diversification history for M. tominii s. lat., coupled with lineages with restricted geographic distributions, highlights a contrasting pattern of diversification between the M. tominii complex and other Montanelia species. Montanelia tominii s. lat. generally occurs on exposed rock in arid continental habitats. Cyclical climatic fluctuations during the Pleistocene caused major shifts in the distributions of suitable habitats for many species, and we assume changing habitat conditions due to climatic fluctuations during the Pleistocene also played a role in diversification in the M. tominii complex. In spite of the relatively recent and rapid speciation in the M. tominii complex, relationships among putative species were generally recovered with strong support in both the concatenated gene tree and coalescent-based species tree (Figs. 1 and 2). This differs from other species complexes that have undergone Pleistocene diversification, such as the genera Letharia (Altermann et al., 2014) or Xanthoparmelia (Leavitt et al., 2013d).

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Our results provide additional evidence supporting the Miocene a major period of diversification in the species-rich


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lichen-forming fungal family Parmeliaceae and highlight role of the Pleistocene in speciation in some lineages. While the majority of Montanelia species distributed across Asia, Europe, and North America show no evidence of phylogeographic substructure, our study reveals a complex diversification and biogeography history of the M. tominii group beginning in the late Miocene and continuing into the Pleistocene. Our analyses provide additional insight into understanding diversification and uncovering cryptic diversity in cosmopolitan species of lichen-forming fungi. Pending additional sampling of M. tominii populations in Europe and Asia, we aim to complete a formal taxonomic revision for the M. tominii complex. Other formal taxonomic revisions in Montanelia, including the potential synonymy of M. predisjuncta with M. disjuncta and a putative previously unrecognized species in M. panniformis (‘M. panniformis C’) will require sampling additional populations to confirm the validity of the inferences made in this study.

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Camargo et al. (2012a), Hamilton et al. (2014), Lanier and Knowles (2012), Miralles and Vences (2013), Posada (2008), Posada and Buckley (2004) and Talavera et al. (2013).

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We gratefully acknowledge two anonymous reviewers, whose comments and suggestions greatly improved the manuscript. We also thank the late Bradley Loomis for his invaluable contribution in the lab. This study was supported by grants from the University of Chicago, NSF (http://nsf.gov/; ‘‘Hidden diversity in parmelioid lichens’’, DEB-0949147) and Ministerio de Ciencia e Innovación, Spain (GLCL2010-21646/BOS). Y. Ohmura was partly supported by JSPS KAKENHI Grant No. 24300314. The Chinese collections were supported by The National Natural Science Foundation of China (Nos. 31170023 and 31370069) and Flora Lichenum Sinicorum (KSCX2-EW-Z-9).

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Appendix A. Supplementary material

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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ympev.2015.04. 029.

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Species delimitation in the lichenized fungal genus Vulpicida (Parmeliaceae, Ascomycota) using gene concatenation and coalescent-based species tree approaches.

An Integrative Approach for Understanding Diversity in the Punctelia rudecta Species Complex (Parmeliaceae, Ascomycota).

Coalescent-based species delimitation approach uncovers high cryptic diversity in the cosmopolitan lichen-forming fungal genus Protoparmelia (Lecanorales, Ascomycota).

Lichen-Associated Fungal Community in Hypogymnia hypotrypa (Parmeliaceae, Ascomycota) Affected by Geographic Distribution and Altitude.

Phylogeography of Ramalina menziesii, a widely distributed lichen-forming fungus in western North America.

Characterization of microsatellite loci in the lichen-forming fungus Cetraria aculeata (Parmeliaceae, Ascomycota).

Fungus-specific SSR markers in the Antarctic lichens Usnea antarctica and U. aurantiacoatra (Parmeliaceae, Ascomycota).

Phylogeography above the species level for perennial species in a composite genus.

Biogeography and Genetic Structure in Populations of a Widespread Lichen (Parmelina tiliacea, Parmeliaceae, Ascomycota).

Seven wood-inhabiting new species of the genus Trichoderma (Fungi, Ascomycota) in Viride clade.

Corrigendum: Seven wood-inhabiting new species of the genus Trichoderma (Fungi, Ascomycota) in Viride clade.

Human phylogeography and diversity.

Fungal specificity and selectivity for algae play a major role in determining lichen partnerships across diverse ecogeographic regions in the lichen-forming family Parmeliaceae (Ascomycota).

Terpene synthases are widely distributed in bacteria.

A brief chronicle of the genus cordyceps fr., the oldest valid genus in cordycipitaceae (hypocreales, ascomycota).

Comparative phylogeography of co-distributed Phrygilus species (Aves, Thraupidae) from the Central Andes.

The Genus Letrouitia (Letrouitiaceae: Lichenized Ascomycota) New to Cambodia.

Biogeography and evolutionary diversification in one of the most widely distributed and species rich genera of the Pacific.

Strong reproductive isolation despite occasional hybridization between a widely distributed and a narrow endemic Rhododendron species.

Rethinking biogeographic patterns: high local variation in relation to latitudinal clines for a widely distributed species.

Hidden relationships and genetic diversity: Molecular phylogeny and phylogeography of the Levantine lizards of the genus Phoenicolacerta (Squamata: Lacertidae).

Genetic diversity and structuring across the range of a widely distributed ladybird: focus on rear-edge populations phenotypically divergent.

Pukalide, a widely distributed octocoral diterpenoid, induces vomiting in fish.

Importance of Resolving Fungal Nomenclature: the Case of Multiple Pathogenic Species in the Cryptococcus Genus.