© 2018 W. Szafer Institute of Botany Polish Academy of Sciences

Plant and Fungal Systematics 63(2): 45–64, 2018 ISSN 2544-7459 (print) DOI: 10.2478/pfs-2018-0007

Species in section Peltidea (aphthosa group) of the genus Peltigera remain cryptic after molecular phylogenetic revision

Jolanta Miadlikowska1*, Nicolas Magain1,2, Carlos J. Pardo-De la Hoz1, Dongling Niu3, Trevor Goward4, Emmanuël Sérusiaux2 & François Lutzoni1

Abstract. Closely related lichen-forming fungal species circumscribed using phenotypic Article info traits (morphospecies) do not always align well with phylogenetic inferences based on Received: 2 Dec. 2018 molecular data. Using multilocus data obtained from a worldwide sampling, we inferred Revision received: 5 Dec. 2018 phylogenetic relationships among five currently accepted morphospecies ofPeltigera section Accepted: 6 Dec. 2018 Published: 14 Dec. 2018 Peltidea (P. aphthosa group). Monophyletic circumscription of all currently recognized morphospecies (P. britannica, P. chionophila, P. frippii and P. malacea) except P. aphthosa, Corresponding Editor which contained P. britannica, was confirmed with high bootstrap support. Following their Martin Kukwa re-delimitation using bGMYC and Structurama, BPP validated 14 putative species includ- ing nine previously unrecognized potential species (five within P. malacea, five within P. aphthosa, and two within P. britannica). Because none of the undescribed potential species are corroborated morphologically, chemically, geographically or ecologically, we concluded that these monophyletic entities represent intraspecific phylogenetic structure, and, therefore, should not be recognized as new species. Cyanobionts associated with Peltidea mycobionts (51 individuals) represented 22 unique rbcLX haplotypes from five phylogroups in Clade II subclades 2 and 3. With rare exceptions, Nostoc taxa involved in trimembered and bimembered associations are phylogenetically closely related (subclade 2) or identical, suggesting a mostly shared cyanobiont pool with infrequent switches. Based on a broad geographical sampling, we confirm a high specificity ofNostoc subclade 2 with their mycobionts, including a mutualistically exclusive association between phylogroup III and specific lineages of P. malacea. Key words: cyanolichen, molecular systematics, morphospecies, Nostoc, phylogeny, species delimitation, specificity, symbiosis

Introduction

The lichen-forming genus Peltigera (Peltigerales, Asco- cephalodia that sometimes can grow and form bimem- mycota) has been divided into eight sections based on bered thalli, also referred to as photomorph). Currently, morpho-chemical data and the large subunit ribosomal this section includes five accepted morphospecies (Hol- RNA gene (Miadlikowska & Lutzoni 2000). Section tan-Hartwig 1993; O’Brien et al. 2009; Miadlikowska Peltidea (P. aphthosa group) is unique in containing both et al. 2014; Magain et al. 2017a) of which three are tri- bimembered and trimembered species in which, respec- membered: P. aphthosa, the type species of the section and tively, the mycobiont associates either with a cyanobiont one of the earliest described lichens (as Lichen aphthosus; (Nostoc spp.) or with a chlorobiont (Coccomyxa spp.) Linneus 1753), P. britannica (Tønsberg & Holtan-Hartwig and a cyanobiont (Nostoc spp., encapsulated in external 1983), and P. chionophila (Goward & Goffinet 2000); and two are bimembered: P. frippii (Holtan-Hartwig 1988) and P. malacea (Funck 1827). Phylogenetically, 1 Department of Biology, Duke University, Durham, NC 27708-0338, section Peltidea clusters (with low bootstrap support; USA 2 Evolution and Conservation Biology, University of Liège, B-4000 Miadlikowska et al. 2014; Magain et al. 2017a) with the Liège, Belgium remaining trimembered sections in the genus, i.e., section 3 Life Science School, Ningxia University, Yinchuan, Ningxia Hui Au- Chloropeltigera (P. latiloba, P. leucophlebia and P. nigri- tonomous Region 750021, China punctata) and section Phlebia (P. venosa) (Miadlikowska 4 UBC Herbarium, Beaty Museum, University of British Columbia, Vancouver, BC V6T 1Z4, Canada & Lutzoni 2000). Members of section Peltidea are wide- * Corresponding author e-mail: [email protected] spread and relatively common in the arctic and boreal

This work is licensed under the Creative Commons BY-NC-ND 4.0 License 46 Plant and Fungal Systematics 63(2): 45–64, 2018

biomes, as well as in mountainous regions of Eurasia and present in more than one morphotype) in P. aphthosa North America. They are found across a broad ecological and P. malacea. Although overall secondary chemistry spectrum, ranging from hygrophytic to xerophytic hab- was shown to be useful and supportive of species cir- itats, but they are most abundant in habitats with mesic cumscriptions across Peltigera (e.g., Holtan-Hartwig conditions. They are almost absent from southern tem- 1993; Vitikainen 1994; Kotarska 1999; Miadlikowska perate lowland areas (except rare records of P. malacea: & Lutzoni 2000; Magain et al. unpubl.), their recogni- Vitikainen 1994; Martínez et al. 2003). Section Peltidea tion in sections Peltidea and Chloropeltigera cannot rely can be easily distinguished from other Peltigera sections solely on chemotypic variation because the same set of based on ITS sequences from the mycobiont (e.g., Goff- triterpenoids was reported from different species (e.g., inet & Bayer 1997). Although rarely tested, no evidence chemotype III of P. aphthosa is identical to chemotype of gene flow or hybridization has been observed between I of P. malacea; ­Holtan-Hartwig 1993), which can be the recognized species (P. frippi was not examined), and morphologically similar (e.g., P. leucophlebia chemotype fixed differences in nucleotide substitutions were found is similar to P. latiloba chemotype II; Holtan-Hartwig for at least two of the three loci of the collections studied 1993), but phylo­genetically unrelated (e.g., P. leucophle- from British Columbia (O’Brien et al. 2009). bia chemotype is similar to P. aphthosa chemotype III; In response to environmental factors, P. aphthosa and Vitikainen 1994). P. britannica may form photomorphs (Brodo & Rich- Molecular phylogenies, sometimes in combination ardson 1978; Armaleo & Clerc 1991; Stocker-Wörgötter with phenotypic data, allowed the evaluation of the & Türk 1994; Stocker-Wörgötter 1995; Goffinet & Bayer alleged monophyly of described morphospecies. In some 1997), i.e., morphologically distinct thalli with different cases, this led to the discovery of new species in many photobionts (where cephalodia of trimembered thalli with lichen-forming groups of fungi, including the genus Pelti- Coccomyxa and Nostoc can develop into bimembered gera (e.g., Goffinet & Miadlikowska 1999; Miadlikowska thalli with Nostoc only) – a phenomenon reported also in et al. 2003; Goffinet et al. 2003; Sérusiaux et al. 2009; Han section Phlebia (P. venosa; Tønsberg & Holtan-Hartwig et al. 2013; Han et al. 2015; Manoharan-Basil et al. 2016; 1983; Ott 1988; Miadlikowska 1998) and in other genera Magain et al. 2016; Jüriado et al. 2017). O’Brien et al. within the order Peltigerales (e.g., Stenroos et al. 2003; (2009) used ITS data (in combination with population Magain et al. 2012; Moncada et al. 2013), as well as from genetic methods) to show that P. leucophlebia (section lichenized Arthoniomycetes (Ertz et al. 2018). Except Chloropeltigera) in British Columbia contains at least P. venosa (section Phlebia), which has a very distinctive three well-supported monophyletic groups, which might thallus appearance, trimembered Peltigera with broad represent putative species. More recently, Magain et al. and leafy thalli (the P. aphthosa group sensu lato includ- (2017a, b) reported a high level of seemingly cryptic ing members of section Chloropeltigera; Holtan-Hartwig diversity in section Polydactylon, which was discovered 1993; Miadlikowska & Lutzoni 2000) can have similar by implementing multiple species delimitation and vali- morphology and chemistry, and therefore are sometimes dation methods on multilocus data including three newly difficult to identify without sequence data. Presence developed Collinear Orthologous Regions (containing of a continuous cortex on the lower side of apothecia intergenic spacers): COR1b, COR3, and COR16 that allows the identification of fertile thalli of section Pelti- provided higher levels of phylogenetic signal than the dea (P. aphthosa, P. britannica and P. chionophila) in fungal barcode ITS (Schoch et al. 2012). Furthermore, the comparison with often co-occurring P. leucophlebia and authors showed that each of the three iconic and broadly P. latiloba, which have ecorticate or patchily corticate recognized species-specific morphotypes: P. scabrosa, apothecia (Holtan-Hartwig 1993; Goward et al. 1995) P. dolichorhiza and P. neopolydactyla represented mul- and belong to section Chloropeltigera (P. nigripunctata is tiple and sometimes unrelated phylogenetic lineages, and restricted to Southeast Asia; Goward et al. 1995; Martinez therefore questioning the current delimitation of common et al. 2003). However, some species such as P. britannica morphospecies in section Polydactylon. and P. chionophila are rarely found with apothecia and Regardless of the focus group and sequenced loci, overall many collections of trimembered species are ster- most studies implementing species delimitation and val- ile. Although typical specimens can be identified based idation methods revealed multiple undescribed species on other diagnostic features, including the thallus habit with much narrower geographic ranges than traditionally and lobe margin, degree of venation (evenly rounded boat circumscribed morphospecies, whereas an overestima- shape lobes with fused veins in P. aphthosa), or the shape tion of species richness based on phenotypic traits was of the cephalodia (peltate in P. britannica), a tremen- rarely reported. Most importantly, molecular phyloge- dous morphological plasticity that has been documented netics enables the reevaluation of characters regarded as in all trimembered species obscures species boundaries. taxonomically important (e.g., Leavitt et al. 2011a, b, c, Their overlapping geographical ranges and field ecology 2013; Lücking et al. 2014; Saag et al. 2014; Singh et al. further contribute to difficulties in species recognition 2015; Kirika et al. 2016 and references therein; but see (e.g., P. aphthosa and P. chionophila inhabit similar snow Wei et al. 2016). For example, in the scabrosoid clade of prolonged sites in British Columbia, Canada; Goward section Polydactylon, seven of ten delimited species were et al. 1995). For example, in Norway, Holtan-Hartwig putatively new, whereas in the dolichorhizoid clade 22 (1993) distinguished three morphotypes and the corre- potentially new species were delimited for a total of 27 sponding sets of chemotypes (with the same chemotype (Magain et al. 2017a, b). For Peltigera hydrothyria s.l., J. Miadlikowska et al. Species in section Peltidea remain cryptic after molecular phylogenetic revision 47 the only aquatic member of the genus (section Hydro- (O’Brien et al. 2013); and 5) compare patterns of sym- thyriae), the ITS region alone provided sufficient genetic biont specificity in section Peltidea to other sections of information to distinguish three cryptic species (Lendemer the genus Peltigera. & O’Brien 2011), which were later confirmed by analyzing We sampled members of section Peltidea across their additional loci (Miadlikowska et al. 2014). Phylogenetic ranges, but with special emphasis on North America (from inferences based on multilocus data including intergenic arctic regions southward through the Rocky Mountains spacers (COR) and the implementation of different spe- and in the southern Appalachians) where trimembered cies delimitation methods (bGMYC, bPTP, BPP) sug- species of Peltidea co-occur with somewhat phenotypi- gested the presence of 88 species in section Peltigera, cally similar and morphologically variable species from including 50 species potentially new to science, hence section Chloropeltigera, P. leucophlebia s.l. and P. lati- uncovering a surprisingly high proportion of previously loba. We identified the mycobiont using four markers unnoticed, but mostly cryptic biodiversity (Magain et al. (β-tubulin, COR1b, COR3, and ITS) and the cyanobiont 2018). However, poor sampling, i.e., restricted to few with rbcLX, i.e., the last 82 amino acids of the RUBISCO localities that are separated by long distances, can also large subunit (rbcL), a putative chaperone gene (rbcX), mislead species recognition and validation methods in and two intergenic spacers (Li & Tabita 1997). We inferred detecting an artificially high number of species. Collec- phylogenetic relationships for each partner, i.e., Peltigera tions for areas between these isolated sampling sites can and Nostoc. We delimited Peltigera species using multiple reveal intermediary patterns of variation and signatures species discovery and validation methods and discussed of gene flow, which led to the circumscription of two the results within a phylogenetic framework. subspecies (ssp. udeghe and ssp. polydactylon) in the broadly distributed P. polydactylon, which better reflects Materials and methods the evolution of these populations and speciation mecha- Taxon sampling and data acquisition nisms than describing new species (Magain et al. 2016). A wide range of specificity has been reported forPel - Based on examined collections from various fungaria tigera-Nostoc associations depending on the phylogenetic (AMNH, B, BG, CONN, DUKE, GZU, H, LG, MIN, NY, breadth of the mycobiont and the spatial scale. Viewed OSU, QFA, UBC, UPS) and freshly collected specimens at an intercontinental (global) scale, for example, most (e.g., fieldtrips to Norway, Canada: Québec, USA: Alaska species in section Peltigera show a relatively low level in 2011; Canada: Alberta; USA: Michigan in 2013), we of specificity, such that widely distributed species usu- selected 196 specimens representing a wide morpho- ally recruit a broader selection of Nostoc phylogroups logical spectrum and broad geographical ranges within (up to eight phylogroups) than species with more limited each species currently classified in section Peltidea: distributions (usually with a single phylogroup: Magain P. aphthosa (83), P. britannica (21), P. chionophila (11), et al. 2018). At an intrabiome scale, more specifically P. frippii (5), and P. malacea (76). For each specimen, within the boreal zone, Peltigera species mostly associ- the internal transcribed spacer (ITS) region, which is the ate with one or sometimes two cyanobiont phylogroups, universal fungal barcode marker (Schoch et al. 2012) was which themselves have broader distribution than their sequenced. We also sequenced three other loci: β-tubulin mycobiont partners and associate with many Peltigera for 173 specimens, and one intergenic spacer from each of species (Lu et al. 2018). In general, reciprocal mutually the two CORs (Magain et al. 2017b): COR1b for 125 and exclusive specificity at the species level seems to be a rare COR3 for 70 specimens (Appendix 1). We supplemented phenomenon in cyanolichens, demonstrated to date in the newly acquired sequences with ITS and β-tubulin data the isidiated (producing vegetative propagules contain- from GenBank (mostly from O’Brien et al. 2009) for an ing both symbionts) species of Leptogium and Collema additional 78 individuals: 34 for P. aphthosa, 21 for P. bri- (Collemataceae, Peltigerales; Otálora et al. 2010), and for tannica, two for P. chionophila, and 21 for P. malacea. a few species of Peltigera including P. malacea, which Four specimens from the closely related section Chlo- was found to associate predominantly with Nostoc from ropeltigera (two from P. leucophlebia s.l. and two from phylogroup III of subclade 2 (mostly records from British P. latiloba) were chosen as the outgroup (Miadlikowska Columbia and a few collections from Europe and Asia; et al. 2014; Magain et al. 2017a) in subsequent phyloge- O’Brien et al. 2013), P. neopolydactyla 5, P. sp. 11 and netic analyses. These four markers were sequenced for P. vainioi associated with phylogroups XIb, IX, XXVIa these outgroup specimens. Overall, we obtained 568 new respectively (Magain et al. 2017a, 2018). sequences, specifically 196 of ITS, 177 of β-tubulin, 125 For practical purposes, this study constitutes Part 3 of COR1b and 70 of COR3. The data matrix contains (after section Polydactylon and Peltigera) in our ongoing 274 OTUs (including the outgroup) of which 53 were worldwide phylogenetic revision of Peltigera. Its purpose represented by all markers, 79 by three markers (mostly is to: 1) provide a comprehensive multilocus phylogeny ITS, β-tubulin, and COR1b), 106 by two markers (mostly for section Peltidea; 2) evaluate the currently recognized ITS and β-tubulin) and 36 by the ITS locus only. COR3 species in a phylogenetic context using multiple discovery was the least represented marker among the four loci. and validation methods; 3) reassess the validity of spe- In addition, we sequenced rbcLX for the cyanobacterial cies-specific phenotypic characters including secondary partners associated with 51 of the 196 selected Peltigera chemistry; 4) reevaluate the high mutually exclusive spec- specimens and obtained sequences from GenBank for 21 ificity reported for P. malacea and their Nostoc partners of the 78 individuals from section Peltidea included in the 48 Plant and Fungal Systematics 63(2): 45–64, 2018

study of O’Brien et al. (2009) for a total of 72 specimens two subsets were defined for the ITS (ITS1+ITS2 vs. for which we had sequences from the mycobiont and 5.8S) and no partition for each, COR1b and COR3 locus associated Nostoc (Appendix 1). Information about the analyses. For the concatenated analyses, a partition of extraction protocol, amplification conditions, primers and four and three subsets was estimated in ML1 and ML2 reagents used, as well as Sanger sequencing are provided data sets, respectively by implementing PartitionFinder in Magain et al. (2017a, b). v. 1.1.0 (Lanfear et al. 2012) greedy search and using the All newly acquired sequences were subjected to Bayesian information criterion (BIC) for model selection BLAST searches to confirm their fungal or cyanobacterial (ML1: ITS1 + ITS2, 5.8S + β-tubulin 1st, β-tubulin 2nd, and origin. They were assembled and edited using the software β-tubulin 3rd + β-tubulin introns + COR1b + COR3; ML2: package Sequencher™ 4.1 (Gene Codes Corporation, Ann ITS1 + ITS2, 5.8S + β-tubulin 1st and 2nd, and β-tubulin Arbor, MI, USA) and aligned manually with MacClade 3rd + β-tubulin introns + COR1b + COR3). Relationships 4.08 (Maddison & Maddison 2005). The “Nucleotide receiving bootstrap support above 70% were considered with AA color” option was used for guiding (delimiting strongly supported. exons and introns) all alignments of protein-coding genes. Ambiguously-aligned regions (sensu Lutzoni et al. 2000) Species delimitation and validation analyses were delimited manually and excluded from subsequent Sequences from 88 individuals representing section Pelti- analyses. dea (ML1 data set excluding the outgroup) were subjected to two species discovery methods, Structurama (Huelsen- Data sets and phylogenetic analyses beck et al. 2011), and bGMYC (Reid & Carstens 2012). For the mycobionts, we assembled a separate data set The delimited species were validated using BPP (Yang for each locus: β-tubulin (228 OTUs), ITS (274 OTUs), & Rannala 2010). Structurama analyses were performed COR1b (125 OTUs), and COR3 (70 OTUs). Single locus separately on two data sets (the P. malacea clade: 36 alignments were concatenated and reduced to 125 unique specimens, and the P. aphthosa clade: 52 specimens). multilocus sequence types (H; Appendix 1) where each In preparation for the Structurama analyses, we used an OTU was represented by at least one locus (1- to 4-locus in-house PERL script (prepstructurama.pl; Magain 2018) data set; ML1) using in-house PERL scripts (combine. to code the alleles represented in each sequenced locus pl and collapse_multi.pl, respectively; Magain 2018). In (ITS, β-tubulin, COR1b, COR3) for each individual using addition to this most inclusive data set for 125 OTUs, we 100% similarity as the criterion for collapsing haplotypes compiled a reduced data set for 92 OTUs where each OTU into a single allele. We ran Structurama on each data was represented by at least three loci (3- to 4-locus data set for two million generations, sampling every 1000th set; ML2) using in-house PERL scripts (compare_and_ generation and tested several gamma hyperpriors on the choose.pl; Magain 2018). For cyanobionts from species expected number of populations (a gamma scale varying of section Peltidea, the 51 new rbcLX sequences were from 1 to 3 and a gamma shape varying from 1 to 6). We filtered to 23 unique haplotypes (using collapse_multi. selected a gamma scale value of 1 and a gamma shape pl) including one haplotype from P. latiloba, a member value of 3 for the final analyses on theP. malacea and the of section Chloropeltigera (part of the outgroup). We P. aphthosa clades, respectively. We performed bGMYC assembled rbcLX data set (ML3) containing the 23 newly analyses on chronograms derived from BEAST v. 1.8 added and 11 previously published Nostoc haplotypes (Drummond & Rambaut 2007) analyses (as implemented (HT; O’Brien et al. 2013) from section Peltidea, in addi- on the CIPRES portal; Miller et al. 2010) on each of the tion to free-living, non-lichen-associated, and lichen-asso- four following loci individually (ITS, β-tubulin, COR1b, ciated Nostoc (from the remaining sections of Peltigera COR3). We ran BEAST for each locus for 10,000,000 and from other cyanolichens, mainly Peltigerales) for generations, sampling every 1000th generation. We used a total of 318 OTUs (see Appendix 1 for rbcLX sequences a strict molecular clock and the models GTR+G for ITS, from section Peltidea). GTR for β-tubulin, HKY+G for COR1b, and HKY for Maximum likelihood analyses using RAxMLH- COR3, respectively (as determined by a MrModelTest PC-MPI-SSE3 (Stamatakis 2006; Stamatakis et al. 2008) v2.3 analysis; Nylander 2004). From each single-locus were performed (at the nucleotide level) on each locus BEAST analysis, we selected 200 chronograms from the separately and each concatenated data set (ML1 and ML2) posterior tree distribution. Each file initially contained for the mycobiont, and on the cyanobiont rbcLX (ML3) 10,000 trees, of which we kept one out of every 50th (Appendix 1). Optimal tree and bootstrap searches were sampled trees using an in-house PERL script (lignes.pl; conducted with the rapid hill-climbing algorithm for 1000 Magain 2018) to generate a final file of 200 trees. We replicates with GTR substitution model (Rodríguez et al. ran bGMYC on each set of 200 trees for 50,000 genera- 1990) and gamma distribution parameter as implemented tions per chronogram. We discarded 40,000 generations in the Mobyle SNAP Workbench version 1.0.5, a portal as burn-in with a thinning value of 100 and threshold for evolutionary and population genetics analyses (North values (corresponding to the interval of possible number Carolina State University online facilities) developed as of species) from 2 to 30. We considered a species to be part of the Dimensions of Biodiversity project (DoB; well-delimited by bGMYC when the probability of group- Monacell & Carbone 2014). For the analyses of rbcLX ing haplotypes together was higher than the probability and β-tubulin, the 1st, 2nd, and 3rd codon positions, and of any alternative groupings that included at least one introns in the latter locus were treated as different subsets; haplotype from this putative species. We ran BPP v. 2.2 J. Miadlikowska et al. Species in section Peltidea remain cryptic after molecular phylogenetic revision 49 on four single-locus alignments implementing a consen- was detected within P. malacea, which comprises five sus species delimitation (numbered clades in Fig. 2) and strongly supported monophyletic groups. a guide-tree corresponding to the ML2 topology (Fig. 2). Altogether, the sequenced cyanobionts (51 indi- We used the species delimitation algorithm and kept all viduals) associated with species from section Peltidea sites containing missing data. We estimated the relative represented 22 unique haplotypes (with one additional rates of evolution for single loci based on substitution haplotype detected in P. latiloba section Chloropeltig- rates from the ML analyses. For the τ prior we set the era; H1-23; Appendix 1) representing four phylogroups gamma shape to 1 and the gamma scale to 1000, esti- (III-VI) and one undetermined lineage (Fig. 3; Appen- mating the height of the root based on the branch lengths dix 1). The most frequent haplotype is H5, which was in the RAxML single locus trees. For the θ prior, we found in 13 specimens of P. malacea collected in several set the gamma shape to 1000, and tested shape values localities in North America and Norway. Four other hap- of 0.5, 2, 5 and 10, so that the mean of the θ prior was lotypes were encountered multiple times (H3, H6, H8, and 0.0005, 0.002, 0.005 and 0.01, respectively. We run the H9; N = 3–5) in different species, including P. aphthosa, analyses for 50,000 generations, with a burn-in of 2000 P. britannica, P. frippii and P. malacea in North America, generations, sampling every 2nd generation. We selected northeastern Asia and Norway. The remaining 17 hap- a θ mean of 0.005 for the final analysis and run it for lotypes were rare, i.e., recorded from one or two lichen 1 million generations, sampling every 100th generation, thalli. All Nostoc strains associated with section Peltidea and discarding 200,000 generations as burn-in. represent Clade II, subclades 2 (BS = 90%) and 3 (BS = 57%). All cyanobionts from P. malacea are grouped in Chromatography subclade 2, mostly phylogroup III (BS = 71%), which A total of 101 specimens (Appendix 1) were subjected to exclusively contains Nostoc from this species. A few thin-layer chromatography (TLC) following the protocol Nostoc strains from P. malacea including a haplotype by Orange et al. (2001). The lichen extracts were eluted in (H8) shared with P. aphthosa and P. frippii were placed the solvent systems C (referred to as TA in Holtan-Hartwig in phylogroup IV (BS < 70%), together with Nostoc asso- 1993) and G (Culberson et al. 1981). Identification of ciated with P. aphthosa and P. britannica, as well as with substances was made in comparison with the results of P. neopolydactyla 4 and other taxa from Peltigerineae Holtan-Hartwig (1993), Vitikainen (1994), and Kotarska including Sticta and Nephroma arcticum (sequences from (1999). Identified terpenoids (only well visible spots were GenBank). Subclade 2 contains a third poorly supported considered) and chemotypes were numbered following clade with Nostoc from other members of the order Pel- Holtan-Hartwig (1993) and Kotarska (1999). tigerales (Scytinium lichenoides [formerly Leptogium lichenoides] from Collemataceae and Protopannaria pezizoides from Pannariaceae). The remaining sequences Results of Nostoc (mostly from P. aphthosa and P. britannica) were found in subclade 3, phylogroups V (BS = 68%) Phylogenetic relationships and identity of the and VI (BS < 60%) together with many cyanobionts from mycobiont and cyanobiont other tri- and bimembered sections of Peltigera. A single Among the 196 sequenced representatives of Peltigera representative of Nostoc from P. aphthosa (P1330) was section Peltidea, we identified 125 unique multilocus placed outside of the delimited phylogroups in the part sequence types across the four molecular markers (ML1 of the tree where the phylogenetic relationships are very data set), with 92 of them being represented by at least unstable (possibly part of phylogroup I; Fig. 3). three loci (ML2 data set). ML1 and ML2 phylogenies pro- vided concordant relationships. Bootstrap support went up Species delimited and validated especially for the internal groupings (relationships among The number of species delimited in Peltigera section individuals) within P. malacea and P. aphthosa (Figs. 1 Peltidea by bGMYC analyses performed on each locus and 2) when we reduced the number of missing sequences separately varied from 12 (β-tubulin and COR3) to 13 by keeping only specimens with three and four loci. All (COR1b) and 15 (ITS), whereas Structurama grouped presently recognized species in the section, except P. aph- all individuals into 12 species based on the concatenated thosa, represent monophyletic highly supported lineages 3- to 4-locus data set (ML2). Of the 14 species tested, (Figs. 1 and 2; all specimens of P. frippii were represented all were validated by BPP with high posterior probabil- by a single sequence type). In both phylogenies, P. britan- ities (Fig. 2; Appendix 2). The overlap among species nica, a highly-supported clade, was found nested within boundaries established on each locus and by different P. aphthosa (one of the branches was so short that their methods varied across the section. We observed complete relationship is shown as a polytomy in Fig. 1). Therefore, agreement in delimitation of P. frippii and P. chionophila their sister relationship cannot be ruled out because the (except the ITS data, which supported the recognition of main divisions in the P. aphthosa s.l. clade received low P1140 as an additional species), some degree of similarity bootstrap support (Figs. 1 and 2). The first phylogenetic in species circumscriptions within P. malacea s.l. (four split within section Peltidea separates the bimembered to seven putative species, many of which were delimited species (P. frippii and P. malacea) from trimembered by multiple inferences, but only M2 was consistently rec- species (P. chionophila sister to P. aphthosa including ognized); and a noticeable discrepancy in species assign- P. britannica). Robust intraspecific phylogenetic structure ment within P. aphthosa and P. britannica (although the 50 Plant and Fungal Systematics 63(2): 45–64, 2018

P. latiloba P294 P. britannica P1049/H55 N=5 [1,2] P. latiloba P244 P. britannica P1065/H71 [3] P. leucophlebia P1115 91 P. britannica HOB040605-4-3/H58 N=2 [1] P. leucophlebia P1064 P. britannica P1324/H60 [1] P. frippii P1345/H80 N=5 [4,5,7,8] 81 P. britannica P234/H66 N=2 [1]

P. malacea P703/H110 [1] C l a d e 100 P. britannica P229/H65 [1] P. malacea P1364/H113 [8] P. britannica P1050/H57 N=7 [1] Clade 2 P. malacea P1130/H111 N=6 [1,3,8] 1 P. britannica HOB040605-6-3/H59 N=2 [1] P. malacea P783/H114 [8] P. britannica P1325/H61 [1] P. malacea P4042/H109 N=5 [1,4,6,8] P. britannica P747/H70 [10] C H L O R P E T I G A P. malacea P4050/H112 [3] 84 P. britannica P1033/H69 N=2 [8] P. malacea P275/H89 [1] P. britannica Canada Tonsberg7672/H67 b r i t a n c 97 P. malacea P1337/H100 N=3 [4,7] C l a d e

P. britannica HOB020708-66-5-7/H56 N=2 [1] P . P. malacea P4019/H84 [3] P. britannica P227/H63 N=2 [1] P. malacea P754/H104 [7]

1 P. britannica P200/H62 [1] P. malacea P4051/H83 N=3 [1,4] P. britannica P228/H64 [1] P. malacea P1350/H101 [4] 80 P. britannica P1043/H68 [2] P. malacea P1163/H105 N=2 [2] P. britannica P285/H53 N=3 [1,3]

P. malacea P1160/H106 [2] C l a d e 98 P. britannica P1078/H73 [1] C l a d e P. malacea P1161/H107 [2] P. britannica P779/H54 N=4 [1,9] P. malacea P1166/H108 [2] 2 P. britannica P1066/H72 [2]

3 P. malacea P1076/H120 [2]

m a l c e P. aphthosa P776 /H21 N=2 [4,7] P. malacea P274/H117 [1] 85 P. aphthosa P1037/H28 N=2 [4] P. malacea P1073/H118 N=2 [2] P . Clade 3 P. aphthosa P1019/H31 [8] P. malacea P1075/H116 N=3 [1,2] P. aphthosa P1010/H30 [8] P. malacea P217/H115 [1] 73 P. aphthosa P4045/H5 [1] P. malacea P1074/H119 [2] 80 P. aphthosa P1336/H42 N=2 [4] P. malacea P1008/H96 [8] 91 P. aphthosa P4043 /H4 N=7 [1,4,8] C l a d e

C l a d e P. malacea P1014/H98 [8] P. aphthosa P745/H8 N=2 [1] P. malacea P1027/H99 [8] P. aphthosa P718/H10 [1]

P. malacea P753/H102 [7] 4 4 P. aphthosa P1323/H1 [1] P. malacea P743/H81 [1] 85 P. aphthosa P773/H22 [7] P. malacea P1361/H91 N=4 [8,10] P. aphthosa P1032/H12 N=2 [3,8] 100 P. malacea P4028/H90 [10] P. aphthosa P790/H36 [8] 89 P. malacea P4018/H82 N=31 [1-4,8,10]

C l a d e P. aphthosa P788/H35 [8] P. malacea P1382/H85 [10] P. aphthosa P734/H121 [4] P. malacea P1127/H103 [3] s . l

5 P. aphthosa HOB020708-66-9-7/H16 [1] P. malacea P273/H88 [1] P. aphthosa P1058/H25 [3] P. malacea P1128/H95 [2] 73 96 C l a d e P. aphthosa P1026/H32 [8] P E L T I D A P. malacea P4029/H94 [3] 98 P. aphthosa P1029/H33 N=3 [6,8] P. malacea P216/H87 [1]

5 P. aphthosa P787/H34 [8] P. malacea P1028/H97 N=5 [8]

P. aphthosa HOB020708-39-1-9/H15 [1] a p h t o s P. malacea HOB020708-31-1-3/H86 [1] P. aphthosa P1187/H50 [1] P. malacea P781/H92 [9] P . P. aphthosa P1189/H51 [1] P. malacea P750/H93 [3] P. aphthosa HOB020708-31-1-7/H13 N=2 [1] P. chionophila P1140/H75 N=4 [1] P. aphthosa HOB020708-35-5-5/H14 [1] 100 P. chionophila P237/H78 [1] P. aphthosa HOB020708-8-5-9/H17 [1] P. chionophila P252/H79 [1] P. aphthosa P4049/H44 [3] P. chionophila P1139/H76 N=6 [1]

C l a d e P. aphthosa P1369/H52 [8] P. chionophila P1142/H77 [1] c h i o n p l a P. aphthosa P5005/H48 [4] P. chionophila TG96-494/H74 [1] P . P. aphthosa P4048/H43 [3] 1 P. aphthosa P1332/H41 [4] P. aphthosa P706/H6 N=14 [1,3,4] P. aphthosa P1330/H40 N=3 [4] P. aphthosa P791/H37 [8] P. aphthosa P4002/H3 N=2 [1] P. aphthosa P4017/H11 N=8 [1,3,4] s . l

C l a d e P. aphthosa P4000/H2 [1] P. aphthosa P1059/H26 [3] P. aphthosa P1312/H29 [1] P. aphthosa P201/H19 N=2 [1,3]

2 P. aphthosa P281/H20 [1] 76 P. aphthosa P1114/H7 N=24 [1,3,8,9] P. aphthosa P4021/H38 N=3 [4] P. aphthosa P717/H9 [1] P. aphthosa P755/H39 [4] P. aphthosa P1172/H27 N=2 [3,4] P. aphthosa P1035/H18 [4] a p h t o s P. aphthosa P4022/H23 [4] P. aphthosa P4023/H45 [4] P . P. aphthosa P759/H24 [3] P. aphthosa P4026/H46 [4] P. aphthosa P725/H49 [4] P. aphthosa P4027/H47 [4] 0.001 length units

Figure 1. Phylogenetic relationships among members of section Peltidea as revealed by a maximum likelihood analysis of the concatenated 4- to1-locus data set (ML1: ITS + β-tubulin + COR1b + COR3; 2706 nucleotides; taxa represented by one, two, three and four loci) for 125 OTUs including four outgroup taxa from section Chloropeltigera (used to root the tree; Miadlikowska et al. 2014). Support values that resulted from the bootstrap analysis of the ML1 data set are provided on thick internodes when values are ≥ 70 %. The remaining bootstrap values provided for thin internodes, which were poorly supported in ML1 (ML-BP below 70%), are derived from bootstrap analyses of the ML2 data set (92 taxa with 3- to 4-locus data set; Fig. 2). Grey bars show current circumscription of species in section Peltidea. Clades within each recognized species are numbered in accordance to species delimitations validated by BPP (Fig. 2; Appendix 2). Each terminal represents a distinct mycobiont sequence type (based on four combined loci). For each terminal we provide current species name, DNA extraction number (P) or voucher id (for specimens represented by GenBank sequences), mycobiont sequence type designation (H), the number of individuals represented by each sequence type (N), and their geographical origin (in square brackets) (see Appendix 1 and Fig. 2). J. Miadlikowska et al. Species in section Peltidea remain cryptic after molecular phylogenetic revision 51 S t r u c a m C h e m o t y p P h y l o g r u p C o n s e u bGMYC b e t a - u C O R 1 b C O R 3

CHLOROPELTIGERA (Outgroup) B P I T S P. frippii 1 2 3 4 5 6 7 8 9 10 fri I P. frippii P1345/H80 N=5 IV F1 F1 F1 F1 F1 F1 Clade 1 mal I P. malacea P275/H89 P275 96 mal I P. malacea P1337/H100 N=3 III M1c 71 mal I P. malacea P4051/H83 N=3 III M1 M1a M1 M1 M1 51 ? P. malacea P1350/H101 M1b 55 53 mal I P. malacea P754/H104 III 84 ? P. malacea P4019/H84 mal I P. malacea P703/H110 100 IV mal V P. malacea P1364/H113 Clade 2 mal I, V P. malacea P1130/H111 N=6 IV M2 M2 M2 M2 M2 M2 mal I P. malacea P783/H114 69 mal I, V P. malacea P4042/H109 N=5 IV 79 ? P. malacea P4050/H112 Clade 3 aph Ia P. malacea P1163/H105 N=2 III P. malacea 99 ? P. malacea P1160/H106 III ? P. malacea P1161/H107 III 72 ? P. malacea P1166/H108 III M3a mal I M3 M3 M3 M3+ M3 83 P. malacea P274/H117 mal I P. malacea P1076/H120 III M3b +P1361 77 ? P. malacea P1073/H118 N=2 III 76 ? P. malacea P1074/H119 III mal I P. malacea P1075/H116 N=3 III Clade 4 III 93 mal II P. malacea P1008/H96 ? P. malacea P1014/H98 III P E L T I D A 51 ? P. malacea P1027/H99 M4 M4 M4 M4 100 85 67 mal II P. malacea P743/H81 mal II P. malacea P753/H102 III mal I P. malacea P1361/H91 N=4* M6- 89 M5a mal I P. malacea P4028/H90 -P1361 Clade 5 69 55 mal I, II P. malacea P4018/H82 N=31 III,IV 55 mal I P. malacea P1128/H95 M5 M5 96 ? P. malacea P273/H88 M5b ? P. malacea P4029/H94 III 54 mal II P. malacea P750/H93 53 mal III P. malacea P216/H87 P. chionophila mal I, II P. malacea P1028/H97 N=5 III ? P. chionophila P1140/H75 N=4 P1140 100 90 chio I P. chionophila P1139/H76 N=6 63 chio I P. chionophila P1142/H77 CH1 CH1 CH1 CH1a CH1 CH1 55 ? P. chionophila P237/H78 ? P. chionophila P252/H79 Clade 1 85 aph II P. aphthosa P1332/H41 A1d aph II P. aphthosa P1330/H40 N=3 VI,U A1b A1b s . l ? P. aphthosa P4002/H3 N=2 P4002 P4002 75 aph I P. aphthosa P4000/H2 87 A1c+ Clade 2 ? P. aphthosa P1312/H29 A1 aph I P. aphthosa P4021/H38 N=3 +P1037 A1d 68 A1c 80 aph I P. aphthosa P1035/H18 +P776 ? P. aphthosa P4023/H45 a p h t o s 92 A1a A1e+ 88 ? P. aphthosa P4026/H46 +P734 P . ? P. aphthosa P4027/H47 91 ? P. britannica P227/H63 N=2* IV 64 bri I P. britannica P1324/H60* IV P. britannica 82 ? P. britannica P1049/H55 N=5 IV B1- 86 Clade 1 bri I P. britannica P1065/H71 -P227 B1d bri II P. britannica P229/H65* IV B1b -P1324 bri I, II P. britannica P1050/H57 N=7 IV,VI B1 -P229 bri I P. britannica P1078/H73 IV -P1043 77 bri I P. britannica P779/H54 N=4 Clade 2 ? P. britannica P1066/H72 B1a B1a 90 bri I P. britannica P1043/H68* P1043 B1c aph Ia P. britannica P285/H53 N=3 Clade 3 85 aph I, II P. aphthosa P1037/H28 N=2* A2 A2 A1d A2 aph I P. aphthosa P776/H21 N=2* aph II P. aphthosa P1019/H31 P1019 P1019 P1019 Clade 4 38 80 ? P. aphthosa P4045/H5 65 aph III P. aphthosa P1336/H42 N=2 VI A3e 58 55 ? P. aphthosa P745/H8 N=2 aph I,Ia,III P. aphthosa P4043/H4 N=7 A3c A3d A3c aph III P. aphthosa P718/H10 42 64 aph II, III P. aphthosa P1032/H12 N=2 64 aph III P. aphthosa P788/H35 A3f ? P. aphthosa P790/H36 aph III P. aphthosa P734/H121* A3- 73 60 aph I P. aphthosa P1058/H25 69 aph I, II P. aphthosa P1029/H33 N=3 -P734 Clade 5 98 ? P. aphthosa P1026/H32 ? P. aphthosa P4049/H44 62 aph III P. aphthosa P4017/H11 N=8 IV ? P. aphthosa P1059/H26 ? P. aphthosa P791/H37 A3a A3a A3a A3b A3b aph I, III P. aphthosa P706/H6 N=14 IV,VI aph III P. aphthosa P201/H19 N=2 V aph I, III P. aphthosa P1114/H7 N=24 IV ? P. aphthosa P1172/H27 N=2 62 ? P. aphthosa P717/H9 aph III P. aphthosa P759/H24 ? P. aphthosa P4022/H23

Figure 2. Summary of species delimitation (bGMYC and Structurama) and validation (BPP), chemotypic variation, cyanobiont phylogroup, and geographic origin (1-10) of the examined specimens within a phylogenetic framework (ML2 cladogram) for Peltigera section Peltidea. Phylogenetic relationships were inferred based on maximum likelihood analyses of a concatenated 3- to 4-locus data set (ML2: ITS + β-tubulin + COR1b + COR3; 2706 nucleotides; taxa represented by three and four loci) for 92 OTUs including four outgroup taxa from section Chloropeltigera (used to root the tree; Miadlikowska et al. 2014). Bootstrap values > 50% are provided. Internodes with ML bootstrap support (ML-BP ≥ 70%) are shown with thick branches. Within each recognized species, clades were numbered in accordance with species delimitations validated by BPP (see also Fig. 1; Appendix 2). Panels next to the cladogram (from left to right) represent: chemotype designation; taxonomic name according to the consensus species recognition and associated information; Nostoc phylogroup designation; geographic origin of sampled specimens; species delimitation by bGMYC based on β-tubulin, COR1b, COR3, and ITS loci; species delimitation by Structurama (with gamma shape of 1 and 3 for the P. aphthosa and the P. malacea clades, respectively); and species validation by BPP. Each terminal represents a distinct mycobiont mul- tilocus sequence type. Chemotype designation (Fig. 4; Appendix 1) based on Thin Layer Chromatography (TLC) is provided for all individuals examined within each multilocus sequence type. Question marks indicate terminals for which TLC data are not available. Each terminal name 52 Plant and Fungal Systematics 63(2): 45–64, 2018

Figure 2. Continued. contains the DNA extraction number (P) or voucher id (GenBank sequences) for the representative individual, mycobiont multilocus sequence type designation (H; after a slash), and the number of individuals represented by each sequence type (N) (see Fig. 1; Appendix 1). Asterisks in- dicate terminals assigned to polyphyletic or paraphyletic species delimited by bGMYC based on ITS and Structurama. Nostoc phylogroups were defined following O’Brien et al. (2013), Magain et al. (2017a), and this study (U indicated the placement outside of defined phylogroups; Fig. 3; Appendix 1). For the chemotype designations, aph refers to P. aphthosa, bri to P. britannica, chio to P. chionophila, fri to P. frippii, and mal to P. malacea, followed by the chemotype number (Fig. 4; see also Holtan-Hartwig 1993 and Vitikainen 1994). Black dots represent (single or multiple) specimens sampled in this study from the following geographic regions: 1) West Coast: Oregon, Washington, British Columbia, Alaska, Yukon; 2) Rocky Mountain Region: Montana, Wyoming, Colorado; 3) Midwest Region: Minnesota, South Dakota, Michigan, Alberta, Manitoba, Ontario; 4) Northeastern Region: Newfoundland, New Brunswick, Nova Scotia, Québec, Labrador; 5) Nunavut; 6) Central Northern Asia (Siberia): Krasnoyarsk Territory; 7) Eastern Northern Asia (Far Eastern District): Khabarovsk Territory, Kamchatka, Yakutia; 8) Fennoscandia; 9) Iceland; 10) remaining Europe including North Caucasus. Gray boxes show species delimited by bGMYC and Structurama and validated by BPP (for species abbreviations and posterior probability values see Appendix 2). White boxes within grey ones indicate nested species (e.g., species B1c is nested within species B1b as delimited by bGMYC based on ITS locus alone). Empty space indicates specimens that could not be assigned to any species because they were included in an alternative species delimitation with a higher posterior probability value or were not represented in a single gene matrix (missing data; Appendix 1). Singletons (species containing a single representative) are indicated by a unique DNA number (e.g., P275). Consensus species delimitations reflecting current species circumscriptions are indicated by black frames (notice that P. britannica as currently recognized, is nested within P. aphthosa s.l., but without strong bootstrap support).

number of recognized species was in overall agreement, specimens examined, we detected seven chemotypes their delimitations varied drastically; Fig. 2; Appendix 2). previously reported from members of Peltidea by Hol- A high level of uncertainty (low likelihood and proba- tan-Hartwig (1993) (Fig. 4): P. aphthosa I (aph I) from bility values) in species delimitation occurred in selected P. aphthosa, P. chionophila (chio I), and P. frippii (fri I); clades of P. malacea s.l. based on ITS and β-tubulin and P. aphthosa II (aph II) from P. aphthosa and P. britannica in general across P. aphthosa/britannica complex (based (bri II); P. aphthosa III (aph III) from P. aphthosa, and on β-tubulin, COR1b and COR3). bGMYC on the ITS P. malacea (mal I); P. britannica I (bri I) from P. bri- and Structurama analyses resulted in a few polyphyletic tannica; P. malacea II (mal II), P. malacea III (mal III), species (e.g., A1c+P1037+P776, M3+P1361 in P. malacea and P. malacea V (mal V) from P. malacea. Addition- s.l., A1e+P734 in P. aphthosa s.l.) and species nested ally, we detected a potentially new chemotype (aph Ia) within broadly delimited ones (e.g., B1c within B1b, from P. aphthosa, P. britannica and P. malacea, which A1e+P734 within A1d) (Fig. 2). Depending on the data resembles aph I with the addition of terpenoid 12. All sets used, several singletons were recognized as inde- examined specimens of P. chionophila and P. frippii pendent species (e.g., P275, P1140, P4002, P1043, and were represented by a single chemotype (chio I and fri P1019). According to BPP, the following species were val- I, respectively), while three chemotypes (aph Ia, bri I, idated with high probability: P. frippii, P. chionophila, five bri II) were detected in P. britannica, four chemotypes putative species within each, P. malacea and P. aphthosa (aph I, aph Ia, aph II, aph III) in P. aphthosa, and four (all of them are monophyletic and well supported except chemotypes (mal I, mal II, mal III, mal V) were found in two of P. aphthosa, which received bootstrap support P. malacea (Figs. 2 and 4; Appendix 1). For three species, below 70%), and two potential species in P. britannica specimens representing the same multilocus sequence (one paraphyletic and both poorly supported) (Figs. 1 type contained more than one (up to three) chemotypes. and 2). For example, chemotypes mal I and mal V were detected among individuals of P. malacea multilocus sequence Chemotypic diversity types 111 (H111) and 109 (H109), and chemotypes aph I, All specimens subjected to TLC analyses contained aph Ia, and aph III were detected in P. aphthosa multilo- secondary metabolites. We focused on conspicuous and cus sequence type 4 (H4) (Fig. 2; Appendix 2). Depsides tractable spots corresponding to terpenoids identified by aggregate and methyl gyrophorate were clearly visible on Holtan-Hartwig (1993). In many cases, a direct com- plates across most samples, especially in the G solvent. parison of our results with published chemical profiles was difficult or impossible because we did not use EHF Discussion solvent (contains diethyl ether, highly volatile and flam- mable), which was developed especially for Peltigera Molecularly defined species remain phenotypically inconsistent (Tønsberg & Holtan-Hartwig 1983). Although the EHF solvent resembles system G of Culberson et al. (1981), This is the most comprehensive phylogenetic study of which we used in this study, it gives a better separation Peltigera section Peltidea, where all five recognized of terpenoids and enhances the visibility of terpenoids morphospecies are represented by multiple individuals present in relatively low concentrations. Among the 101 from distinct geographic localities (Figs. 1 and 2). In all

Figure 3. Placement of 23 haplotypes of Nostoc (recognized among 51 newly sequenced cyanobionts) found in association with mycobionts from section Peltidea (plus one from section Chloropeltigera) within a broad phylogenetic context as revealed by maximum likelihood analysis of the rbcLX locus (ML3) for 318 OTUs. Fischerella muscicola (Stigonematales) was used to root the tree. Names for published sequences of free-living, non-lichen-associated, and lichen-associated symbionts are preceded by their GenBank accession numbers and followed by their geographic origin (when available). Names for new sequences (shown in bold) contain information about their geographic origin, haplotype designation (H), and number of individuals representing each haplotype (Appendix 1). Delimitation of clades, subclades, and phylogroups (gray boxes) follow Otálora et al. (2010), O’Brien et al. (2013), and Magain et al. (2017a). Bootstrap support values are provided above internodes. J. Miadlikowska et al. Species in section Peltidea remain cryptic after molecular phylogenetic revision 53 previously published phylogenies (Miadlikowska & Lut- Leavitt et al. 2011c, 2016; Lücking et al. 2014), including zoni 2000, 2004; O’Brien et al. 2009; Magain et al. 2017a, Peltigera sections Polydactylon and Peltigera, where the b, 2018), section Peltidea has been poorly sampled owing proportion of undiscovered biodiversity was unexpectedly in part to different taxonomic and geographical focusses. high (half or more of the recovered species were poten- Similar to other lichen genera and species complexes (e.g., tially new; Magain et al. 2017a, b, 2018), molecular data

DQ185299 Fischerella muscicola NZ DQ266029 Stereocaulon exutum Japan 100 DQ266032 Protopannaria pezizoides Finland DQ266030 Stereocaulon fronduliferum NZ AJ632066 Nostoc ellipsosporum Czech Republic AJ632057 Anabaena augstumalis Germany EU151928 Nostoc sp. 74 AJ632070 Trichormus variabilis Russia Nostoc Clade I AJ293165 Anabaena cf. cylindrica 75 DQ185301 Nostoc sp. (soil) Senegal 92 DQ185297 Nostoc sp. (soil) Indonesia 100 BA000019 Nostoc sp. Z94888 Nostoc sp. DQ266033 Vahliella leucophaea Finland 96 Subclade 1 DQ185264 P. didactyla 94 EU877497 Leptogium cyanescens 1 Spain EU877487 Leptogium azureum 2 Chile 93 AB075918 Nostoc sp. 83 EU151929 Nostoc sp. EU672829 “Parmotrema tinctorumÓ EU672828 “Parmotrema tinctorumÓ EU672830 “Parmotrema tinctorumÓ P1163 Peltigera malacea 3 USA (WY) H19 84 P781 Peltigera malacea 1 Iceland H21 P1011 Peltigera malacea 5 Norway, Canada (MB, QC)/malacea 3 USA (WY) H5 N=13 60 KC437901 Peltigera malacea 71 KC437896 Peltigera malacea HT28 Canada (BC) KC437917 Peltigera malacea KC437686 Peltigera malacea HT7 Canada (BC) P1160 Peltigera malacea 3 USA (WY) H18 P271 Peltigera malacea 3 Canada (BC) H20 Phylogroup III 94 KC437902 Peltigera malacea KC437690 Peltigera malacea HT9 Canada (BC) KC437906 Peltigera malacea KC437905 Peltigera malacea KC437904 Peltigera malacea KC437903 Peltigera malacea Subclade 2 KC437691 Peltigera malacea HT22 Canada (BC) 90 P1007 Peltigera malacea 5, 4 Norway, Kamchatka/malacea 1 Canada (QC) H6 N=5 EF102333 Peltigera malacea Finland P754 Peltigera malacea 1 Yakutia H14 85 C29081998 Peltigera malacea Europe 48 KC437885 Nephroma arcticum HT26 Canada (BC) EF102321 Protopannaria pezizoides Finland EU877508 Scytinium lichenoides Spain Nostoc Clade II EU877511 Scytinium lichenoides Spain EU877510 Scytinium lichenoides Portugal EU877507 Scytinium lichenoides Spain GQ184605 Scytinium lichenoides Sweden EU877509 Scytinium lichenoides Spain DQ266027 Sticta gaudichaudii Argentina DQ266028 Sticta hypochra Argentina 100 DQ266023 Sticta hypochra Argentina Phylogroup IV DQ266024 Sticta hypochra Argentina DQ266026 Sticta hypochra Argentina P1324 Peltigera britannica Canada (BC), USA (WA, OR) H9 N=5 DQ266025 Sticta hypochra Argentina P5003 Peltigera aphthosa Canada (QC) H11 N=2 KC437879 Peltigera aphthosa/leucophlebia HT21 Canada (BC) KC437651 Peltigera aphthosa/leucophlebia/britannica HT19 Canada (BC) KC437653 Peltigera aphthosa HT18 Canada (BC) P5007 Peltigera aphthosa Canada (QC) H4 N=3 P1033a Peltigera britannica Norway, Canada (BC)/aphthosa Canada (BC, QC) H3 N=5 KC437880 Peltigera aphthosa HT23 Canada (BC) P1185 Peltigera aphthosa Sweden H7 P276 Peltigera malacea 2 USA (AK) H15 KC437883 Peltigera malacea HT25 Canada (BC) KC437916 Peltigera malacea KC437886 Nephroma arcticum HT27 Canada (BC) P1078 Peltigera britannica Canada (YT) H22 P1033b Peltigera malacea 2 Norway/frippii Norway/aphthosa USA (AK) H8 N=3 76 P279 Peltigera malacea 2 Canada (BC) H16 P703 Peltigera malacea 2 USA (AK) H17 EU877483 Collema tenax Spain AJ632065 Nostoc edaphicum Czech Republic EU877462 Lathagrium auriforme Spain DQ185281 Peltigera neopolydactyla USA DQ266035 Peltigera neopolydacyla Finland KC437910 Peltigera neopolydacyla KF142684 Gunnera magellanica KF142708 Gunnera magellanica KF142688 Gunnera magellanica KF142705 Gunnera magellanica EU877529 Scytinium schraderi Spain EU877476 Enchylium polycarpon Spain RAxML phylogeny; EU877520 Leptogium pseudofurfuraceum USA EU877521 Leptogium pseudofurfuraceum Argentina EU877523 Scytinium pulvinatum Spain rbcLX ; 318 OTUs; EU877524 Scytinium pulvinatum Spain EU877461 Lathagrium auriforme Spain 636 nucleotides EU877522 Leptogium pseudofurfuraceum Argentina EU877492 Obryzum corniculatum Spain DQ185309 Cycas circinalis Brazil EU877506 Scutinium gelatinosum Spain EU877491 Obryzum corniculatum Spain EU877488 Leptogium azureum Argentina DQ266042 Pseudocyphellaria lechleri Argentina DQ266004 Pseudocyphellaria mallota Argentina DQ266014 Pseudocyphellaria hirsuta Argentina DQ266041 Pseudocyphellaria scabrosa Argentina DQ266010 Pseudocyphellaria crocata Argentina EF102305 Lobaria scrobiculata Finland DQ266002 Pseudocyphellaria crocata Canada DQ266017 Pseudocyphellaria pilosella Argentina DQ266000 Lobaria amplissima Norway 54 Plant and Fungal Systematics 63(2): 45–64, 2018

DQ266000 Lobaria amplissima Norway 57 EF102312 Nephroma laevigatum Finland DQ266009 Pseudocyphellaria crocata Argentina Subclade 3 DQ266012 Pseudocyphellaria crocata Argentina DQ266016 Pseudocyphellaria coriifolia Argentina DQ266008 Pseudocyphellaria scabrosa Argentina DQ266015 Pseudocyphellaria crocata Argentina DQ266005 Pseudocyphellaria coriifolia Argentina DQ185295 Pannaria conoplea Austria DQ266007 Pseudocyphellaria hirsuta Argentina DQ266006 Pseudocyphellaria intricata Argentina KC437912 Peltigera neopolydactyla DQ266022 Pseudocyphellaria coriifolia Argentina DQ185286 Sticta beauvoisii USA DQ266020 Pseudocyphellaria intricata Reunion Island DQ266013 Pseudocyphellaria intricata Argentina KC437913 Lobaria kurokawae KC437915 Lobaria kurokawae DQ266001 Lobaria amplissima Norway EF102320 Nephroma tangeriense Madeira EF102295 Pectenia plumbea Madeira EF102347 Pseudocyphellaria crocata Madeira DQ266018 Pseudocyphellaria mallota Argentina DQ185294 Nephroma helveticum Canada DQ266003 Pseudocyphellaria clathrata Brazil DQ266021 Pseudocyphellaria crocata Mauritius DQ185290 Lobaria amplissima Austria DQ185318 Sticta fuliginosa USA EF102304 Lobaria pulmonaria Finland EF102303 Lobaria pulmonaria Finland EF102302 Lobaria pulmonaria Finland KC437844 Nephroma parile HT05 BC EF102314 Nephroma parile Finland EF102315 Nephroma parile Finland EF102298 Lobaria pulmonaria Finland EU877480 Collema subnigrescens Portugal EF102300 Lobaria pulmonaria Finland EF102299 Lobaria pulmonaria Finland EU877464 Enchylium conglomeratum Spain EU877474 Collema nigrescens Spain EU877495 Leptogium cyanescens Panama DQ266019 Pseudocyphellaria crocata Thailand KC437909 Nephroma resupinatum T EF102323 Parmeliella triptophylla Finland KC437843 Nephroma resupinatum/N. bellum HT12 Canada (BC) EF102296 Lobaria pulmonaria Finland EF102322 Parmeliella triptophylla Finland EF102306 Nephroma bellum Finland T EF102318 Nephroma resupinatum Finland DQ185293 Nephroma bellum Austria DQ185291 Lobaria hallii USA EU877494 Leptogium corticola USA EU877493 Leptogium corticola CostaRica EU877478 Collema subnigrescens Portugal EU877463 Enchylium conglomeratum Spain EF102301 Lobaria pulmonaria Finland EU877475 Collema nigrescens Spain EU877472 Collema furfuraceum USA EU877471 Collema furfuraceum Portugal EU877469 Collema furfuraceum Spain EU877489 Leptogium brebissonii Spain 97 DQ185307 Peltigera membranacea Canada KC437835 Peltigera neopolydactyla/degenii/membranacea HT14 Canada (BC) IJX876603 Peltigera membranacea Iceland 68 EF102336 Peltigera neopolydactyla Finland P201 Peltigera aphthosa Canada (BC) H13 Phylogroup V 98 KC437874 Peltigera membranacea HT15 Canada (BC) 100 DQ266034 Peltigera degenii Finland EF102337 Peltigera neopolydactyla Finland DQ185303 Peltigera degenii Canada EF102334 Peltigera membranacea Finland AJ632063 Nostoc calcicola Czech Republic AJ632064 Nostoc calcicola Czech Republic Z94893 Nostoc flagelliforme KC437908 Peltigera neopolydactyla KC437907 Nephroma arcticum DQ185292 Massalongia carnosa USA DQ266031 Stereocaulon tomentosum Argentina DQ266036 Massalongia carnosa Finland DQ185289 Scytinium gelatinosum USA DQ185275 Peltigera rufescens Germany KJ413214 Peltigera praetextata Iceland KC437699 Peltigera canina/cin/poly/leu/fusc HT06 Canada (BC) KC437665 Peltigera aphthosa HT08 Canada (BC) KC437911 Peltigera leucophlebia Canada (BC) Phylogroup VI KC437676 Peltigera leucophlebia/venosa/britannica HT10 Canada (BC) KC437826 Peltigera leucophlebia HT30 Canada (BC) KC437876 Peltigera neopolydactyla HT24 Canada (BC) KC437789 Peltigera leuc/can/ven/pon/kris/fusc/prae/sp./aphthosa/britannica HT02 Canada (BC) KC437711 Peltigera praetextata/kris/leu/fusc/sp./britannica HT03 Canada (BC) DQ185312 Peltigera aphthosa Switzerland P200 Peltigera britannica Canada (BC) H10 N=2 P1340 Peltigera aphthosa Canada (QC) H2 P1336 Peltigera aphthosa Canada (QC) H12 N=2 EF102332 Peltigera leucophlebia Finland 89 EF102330 Peltigera leucophlebia Finland EF102345 Peltigera praetextata Finland DQ185306 Peltigera membranacea Russia EF102327 Peltigera leucophlebia Finland DQ185284 Peltigera membranacea USA KC437745 Peltigera horizontalis HT11 Canada (BC) DQ185260 Peltigera membranacea Canada KC437741 Peltigera membranacea/neoca/hor/poly/cinn/can HT01 Canada (BC) DQ185269 Blasia pusilla Germany DQ185315 Gunnera manicata Germany DQ185316 Geosiphon pyriforme Germany DQ185268 Blasia pusilla Germany KM006024 Peltigera aquatica USA (OR)

Figure 3. Continued. Peltigera aquatica HV12 J. Miadlikowska et al. Species in section Peltidea remain cryptic after molecular phylogenetic revision 55

KM006011 Peltigera hydrothyria Ê(PA) KM006022 Peltigera aquatica USAÊ(OR) KM005733 Peltigera gowardii USAÊ(WA) KM006007 Peltigera hydrothyria USAÊ(PA) KM005996 Peltigera gowardii USAÊ(MT) EU877486 Leptogium azureum Brazil 73 EU877484 Leptogium austroamericanum Colombia EU877485 Leptogium austroamericanum Argentina EU877505 Scytinium gelatinosum Spain EU877496 Leptogium cyanescens USA EU877498 Leptogium furfuraceum Spain EU877502 Leptogium furfuraceum Croatia EU877499 Leptogium furfuraceum Spain EU877504 Leptogium furfuraceum Portugal EU877503 Leptogium furfuraceum Portugal Z94890 Nostoc sp. EU877519 Leptogium pseudofurfuraceum USA EU877518 Leptogium pseudofurfuraceum USA EU877468 Collema undulatum Spain EU877470 Collema furfuraceum Croatia KC437638 Peltigera collina HT13 Canada (BC) EU877490 Leptogium brebissonii Spain KJ413204 Peltigera monticola Iceland EU877473 Collema nigrescens Spain KF142698 Gunnera magellanica KC437914 Peltigera neopolydactyla P1330 Peltigera aphthosa Canada (QC) H1 DQ185270 Anthoceros sp. Germany JX876594 Peltigera membranacea Iceland JX876602 Peltigera membranacea Iceland JX876592 Peltigera membranacea Iceland EF102335 Peltigera membranacea Finland EF102346 Peltigera sp. Finland JX876599 Peltigera membranacea Iceland KJ413213 Peltigera membranacea Iceland JX876588 Peltigera membranacea Iceland JX876596 Peltigera membranacea Iceland JX876584 Peltigera membranacea Iceland KC437817 Peltigera leucophlebia/neoca/neck/britannica HT04 Canada (BC) KC437743 Peltigera neckeri HT16 Canada (BC) DQ185276 Peltigera rufescens Germany DQ185274 Peltigera rufescens Germany Peltigera rufescens SSM44-4 Iceland KJ413218 Peltigera islandica Iceland KJ413217 Peltigera islandica Iceland KC437900 Peltigera neocanina HT29 Canada (BC) KJ413219 Peltigera islandica Iceland KJ413207 Peltigera ”neorufescens” Iceland 139773 Peltigera islandica Iceland DQ185271 Peltigera rufescens EU877460 Blennothallia crispa Spain EU877530 Scytinium schraderi Spain DQ185283 Peltigera membranacea USA DQ185282 Peltigera canina USA KJ413212 Peltigera canina Iceland KT875357 Peltigera “neorufescens” Iceland KJ413210 Peltigera neckeri Iceland DQ185308 Peltigera rufescens Poland DQ185279 Peltigera canina Germany Z94891 Nostoc sp. KJ413209 Peltigera neckeri Iceland KC437801 Peltigera leucophlebia HT20 Canada (BC) DQ185272 Peltigera rufescens Germany DQ185277 Peltigera didactyla Germany DQ185278 Peltigera rufescens Germany KJ413208 Peltigera islandica Iceland KJ413211 Peltigera rufescens Iceland GQ184606 Collema flaccidum USA DQ266039 Collema flaccidum Finland DQ266040 Collema flaccidum Finland EU877466 Collema flaccidum Spain EU877465 Collema flaccidum Spain EU877481 Collema tenax Spain DQ185267 Encephalartos natalensis Italy DQ185273 Blennothallia crispa Germany DQ185280 Nostoc commune (soil) USA EU877482 Collema tenax Spain EU877467 Collema undulatum Spain EU877515 Leptogium magnusonii Spain EU877513 Leptogium magnusonii Denmark EU877514 Leptogium magnusonii Spain EU877512 Leptogium magnusonii Sweden EU877517 Leptogium magnusonii Sweden EU877516 Leptogium magnusonii Spain EU877477 Enchylium polycarpon Spain Z94889 Nostoc sp. DQ185287 Peltigera canina USA DQ185314 Blasia pusilla Germany Z94892 Nostoc commune DQ185266 Anthoceros sp. Italy P294 Peltigera latiloba Canada (BC) H23 EF102329 Peltigera leucophlebia Finland DQ185305 Peltigera lepidophora Canada KC437873 Peltigera praetextata HT17 Canada (BC) DQ185265 Geosiphon pyriforme Germany DQ185262 Peltigera rufescens England DQ185261 Peltigera canina AJ632030 Nostoc sp. Finland DQ185317 Nostoc punctiforme (soil) France DQ185310 Stangeria paradosa England DQ185313 Nostoc muscorum (soil) France EU877528 Leptogium saturninum USA EU877527 Leptogium saturninum Spain EU877526 Leptogium saturninum France DQ266038 Leptogium saturninum Finland DQ266037 Leptogium saturninum Finland Figure 3. Continued. 0.01 length units 56 Plant and Fungal Systematics 63(2): 45–64, 2018

support the recognition of 14 putative species for section analyses confirms that applying one species discovery Peltidea, of which nine are undescribed. method on a single locus, e.g., the ITS region does not The impact of the incongruence among loci and provide a reliable species assignment scheme across dif- implemented methods on currently recognized species ferent fungal groups (but see Lücking et al. 2014). Out of has previously been reported across various taxonomic the five traditional morphospecies currently accepted in groups, including fungi and, among them, the genus Pel- section Peltidea, only two, P. chionophila and P. frippii, tigera (e.g., Singh et al. 2014 and references therein; Wei were well circumscribed (with the exception of the ITS et al. 2016; Magain et al. 2017a, b, 2018 and references of P1140 in P. chionophila) and validated as such. The therein; Da Silva et al. 2018). In this study we detected remaining three species – P. aphthosa, P. britannica and a relatively high level of discrepancy and uncertainty in P. malacea – were split into multiple putative species the species number and species boundaries, including corresponding to separate (with the exception of Clade cases of paraphyletic/polyphyletic entities, as well as 1 of P. britannica), sometimes highly supported lineages nested and singleton species, especially in P. aphthosa (Figs. 1 and 2). Peltigera aphthosa is the only morphospe- (Fig. 2). This high level of inconsistency among loci and cies with uncertain monophyly unless we expand its

SOLVENT SYSTEM G

12 12 12 14 14 9 14 15 15 15 15 15 19 17 17 17 13

aph I aph Ia aph II aph III bri I mal II mal III mal V (fri I) (bri II) (mal I) (chio I)

14 14 14 12 12 9 12 15 15 15 15 13 15 17 17 17 19

35?

SOLVENT SYSTEM C (TA) Figure 4. Chemotypes recognized based on Thin Layer Chromatography (TLC) analyses using the G solvent (top row) and C solvent (bottom row) among the examined specimens from section Peltidea. The chemotypes shown were part of different TLC plates, and therefore the placement of terpenoids cannot be directly compared. Chemotype abbreviations and terpenoid numbering follow Holtan-Hartwig (1993). Only conspicuous and previously recognized terpenoids are annotated. The top orange spots represent depside aggregates (mostly gyrophoric acid) and methyl gyrophorate. Abbreviations are as follow: aph = P. aphthosa, bri = P. britannica, chio = P. chionophila, fri = P. frippii, mal = P. malacea. Chemotypes: aph I/fri I/chio I (P. aphthosa I/P. frippii I/P. chionophila I): 15, 17, 14; aph Ia (P. aphthosa Ia/P. britannica): 15, 17, 14, 12; aph II/bri II (P. aphthosa II/P. britannica II): 15, 12 (other spots: 24, 35, 28 not visible); aph III/mal I (P. aphthosa III/P. malacea I): 19, 9; bri I (P. britannica I): 17, 14; mal II (P. malacea II): 15, 12 (14 not visible); mal III (P. malacea III): 13; mal V (P. malacea V): 15. 12 = dolichorrhizin, 15= zeorin, and 17 = phlebic acid B. J. Miadlikowska et al. Species in section Peltidea remain cryptic after molecular phylogenetic revision 57 circumscription to include P. britannica, which is nested (Fig. 5N). In P. aphthosa 5, cephalodia were usually in P. aphthosa (but with low bootstrap support). The sis- warty near the margin of the thallus and become flat ter relationship of these two species cannot be ruled out towards the thallus center (Fig. 5N, O). because of the high instability of the internal branching The combination of almost veinless lower surface, within the P. aphthosa s.l. clade in our phylogenies (Figs. flat, adnate cephalodia, and corticated underneath of 1 and 2). “At its best P. britannica is very dissimilar to apothecia was used to distinguish P. aphthosa from P. leu- P. aphthosa in morphology (…)” (Vitikainen 1994) due cophlebia (Vitikainen 1994). However, sterile specimens to the presence of squamulose cephalodia and its wrin- often cannot be accurately assigned to either species kled and pitted surface, however, these characters are not because of the overlapping interspecies morphological consistently present (e.g., specimens from Rocky Mts. variation, which seems to be a common phenomenon of P. britannica 2) and therefore, it could also represent observed in the material collected in North America. an intraspecific taxon (e.g., subspecies as suggested by Morphotypes of P. leucophlebia and P. aphthosa are Vitikainen 1994) or morphotype within a highly variable more distant and therefore easily distinguished in Europe P. aphthosa. The status of P. britannica requires further (Holtan-Hartwig 1993; Vitikainen 1994). Similar exten- investigation (more than four loci seem to be necessary). sive morphological variation in North American collec- Both species need to be sampled extensively to test their tions (perhaps due to a wider range of available habitats) monophyly and to evaluate possible interbreeding. contrary to the European material has been reported for We were unsuccessful in establishing morphological, sections Polydactylon (e.g., P. polydactylon and P. neo- chemical or geographic markers for our nine putative polydactyla; Magain et al. 2017a, b) and Peltigera (e.g., species (Fig. 2). We compared the three distinct mor- P. ponojensis/monticola and P. austroamericana com- photypes (with corresponding chemotypes) recognized plexes; Magain et al. 2018) as well as other groups of by Holtan-Hartwig (1993) in P. aphthosa and P. malacea lichens (Gueidan & Lendemer 2015). However, it is pos- from Norway with the morphological variation across the sible that in some cases it might be indicative of ongoing new species re-delimited in our geographically broader gene flow among lineages recognized as species based sampling. None of these phenotypic patterns were con- on selected molecular markers and analytical methods, sistent with the new proposed species boundaries (Fig. 2). similar to the potential gene flow signature discovered Most morphological features usually accepted as diag- in Eurasia between two subspecies of P. polydactylon, nostic in section Peltidea (e.g., Vitikainen 1994; Goward which were initially considered as independent species et al. 1995; Brodo et al. 2001) were observed across mul- (Magain et al. 2016). tiple species. For example, we observed a broad range Peltigera britannica is often distinguished from its of variation in several traits including thallus thickness, close relatives (P. aphthosa and P. chionophila) by the cephalodia morphology and vein distinctiveness across presence of peltate (to squamulose), loosely attached P. aphthosa s.l. (Fig. 5A–O). Although combinations of cephalodia (Vitikainen 1994; Fig. 6A, G). However, we some features were predominantly associated with certain examined some specimens of P. britannica 1 with both lineages, overall these morphological differences were adnate and peltate cephalodia (Fig. 6B) on the same thal- not consistently correlated with the species delimitation lus. Such variation might be indicative of a recent or scheme unveiled by our analyses. incipient speciation event resulting in P. britannica s.l. Most specimens of P. aphthosa 1 are medium to small and P. aphthosa, which could explain why their reciprocal (up to 5 cm) with ascending, thick (1 mm), tomentous monophyly was not recovered by phylogenetic inference margin and boat shaped lobes (Fig. 5B), and have flat, based on four highly variable loci (Figs. 1 and 2). We adnate cephalodia (Fig. 5C). The lower surface is vein- also observed broadly overlapping morphological varia- less or with pale veins in the margins abruptly becoming tion in P. britannica 1 and 2, in which the pilema ranges dark and fused towards the thallus center (Fig. 5A). from dark and veinless in both lineages (Fig. 6C, H) to This morphology is consistent with the description veinless with pale margin (Fig. 6C) or conspicuously of European material by Vitikainen (1994) (potential veined (Fig. 6D) in P. britannica 1. Both lineages within P. aphthosa s.str. clade), however, it is also found in the P. britannica s.l. have warty to fully corticated apothe- remaining four lineages of P. aphthosa s.l. Specimens cial reverses (Fig. 6E, F). Peltigera chionophila can be from P. aphthosa 2 have usually relatively thin thalli recognized by the presence of appressed, cerebriform (0.3–0.6 mm) with a conspicuous dark venation in the cephalodia (Fig. 6I, J) and dark, well defined, rhizinate lower surface extending all the way to the margin, and veins (Fig. 6K). It also has a narrow geographic distribu- fasciculate rhizines arranged along the veins (Fig. 5D). tion, being restricted to mountainous regions with heavy, Individuals with varying degree of conspicuous pale prolonged snow cover (Goward & Goffinet 2000). to dark veins in the lower surfaces, that become fused Holtan-Hartwig (1993) reported from Norway three towards the center of the thallus, occur in addition to morphotypes in P. malacea, which differed mainly in the distinct venation pattern in P. aphthosa 3 (Fig. 5G), thallus size and thickness, as well as abundance of hairs P. aphthosa 4 (Fig. 5J) and P. aphthosa 5 (Fig. 5M). All on the upper surface. While the morphological varia- lineages within P. aphthosa s.l. have flat, adnate cepha- tion captured in these morphotypes was observed in our lodia (Fig. 5C, F, H, K, L, O). However, smaller wart- material (Fig. 7A–J), some features were not consistently shaped cephalodia were also observed in P. aphthosa associated with the phylogenetic lineages revealed by our 2 (Fig. 5E), P. aphthosa 4 (Fig. 5I) and P. aphthosa 5 analyses (Figs. 1 and 2). Nevertheless, we did detect some 58 Plant and Fungal Systematics 63(2): 45–64, 2018

A B C

D E F

G H I

J K L

M N O

Figure 5. Morphological variation in P. aphthosa s.l. A – veinless margin on the lower surface of P. aphthosa 1; B – boat shaped lobe of P. aph- thosa 1; C – adnate cephalodia of P. aphthosa 1; D – well-defined, narrow veins in lower surface of P. aphthosa 2; E – warty cephalodia on the upper surface of P. aphthosa 2; F – adnate cephalodia on the upper surface of P. aphthosa 2; G – pale veins on the margin of the lower surface of P. aphthosa 3; H – adnate cephalodia on the upper surface of P. aphthosa 3; I – warty cephalodia on the upper surface of P. aphthosa 4; J – veins on the lower surface of P. aphthosa 4; K, L – elongated, adnate cephalodia on the upper surface of P. aphthosa 4; M – pale veins on the margin of the lower surface of P. aphthosa 5; N – warty cephalodia on the margin of the upper surface of P. aphthosa 5; O – adnate cephalodia in the center of the upper surface of the thallus in P. aphthosa 5. Scale bars = 5 mm. J. Miadlikowska et al. Species in section Peltidea remain cryptic after molecular phylogenetic revision 59 general trends. Most specimens of P. malacea 2 matched were shared by P. aphthosa, P. frippi and P. chionoph- morphotype B sensu Holtan-Hatwig (1993), with thick ila, and by P. aphthosa and P. malacea), 2) geography (1 mm) and exceptionally long (up to 10 cm) lobes (e.g., mal I was reported from P. malacea specimens (Fig. 7C) and hairs towards the lobe margin (Fig. 7D). collected in Europe and North America), and 3) Nostoc Peltigera malacea 5 was the only clade where almost phylogroups (e.g., thalli of P. britannica containing Nos- every examined specimen had a white pruina near the toc phylogroup IV displayed two different chemotypes) lobe margin (Fig. 7H). Less morphological consistency (Figs. 2 and 4). Contrary to some lichen groups where was present in the remaining lineages, where for exam- secondary metabolites continue to play a primary or sec- ple the lobes of P. malacea 4 are furnished with a dense ondary diagnostic role in species recognition (e.g., in erect tomentum inward almost to the thallus center in Cladonia, Graphidaceae, Haematomma, Heterodermia, some specimens while in others they are almost entirely Lecanora, Parmeliopsis, Pertusariaceae, Rhizoplaca, and glabrous throughout (Fig. 7F, G). Although most speci- Xanthoparmelia; Tehler & Källersjö 2001; Staiger 2002; mens of P. frippii have a shiny and glabrous upper cortex Nash & Elix 2004; Ryan et al. 2004; Smith & Lumbsch (Fig. 7I), some specimens have distinct marginal hairs 2004; Lumbsch et al. 2008; Lücking et al. 2008; Leavitt (Fig. 7J). Marginally tomentose specimens of P. frippii et al. 2011c; Papong & Lumbsch 2011), including rare can be distinguished from P. malacea s.l. by the pres- cases in the genus Peltigera (e.g., presence of unique ence of well-defined, rather narrow veins below, i.e., in chemotypes in P. venosa and selected newly delimited contrast to the lower surface of P. malacea s.l., which is species in section Polydactylon), chemotypic varia- either veinless or furnished with a few diffuse veins. The tion (presence/absence and the quantity of terpenoids) observed morphological variation among and within the across section Peltidea seems to be driven by other fac- putative species of section Peltidea has limited taxonomic tors (environment, habitat, thallus developmental stage, significance. lichen microbiome; e.g., Culberson et al. 1977; Bialonska With the exception of aph Ia (from P. aphthosa, P. bri- & Dayan 2005; Toni & Piercey-Normore 2013; Calcott tannica, and P. malacea), all chemotypes noted by us et al. 2018) than phylogenetic relatedness of mycobionts. have previously been reported from Peltigera in Norway Although within a broader phylogenetic context certain (Holtan-Hartwig 1993), Europe in general (Vitikainen degree of similarity in triterpenoids composition occurs 1994), and Québec, Canada (Kotarska 1999) (Fig. 4). We within selected sections (e.g., Horizontales, Polydac- did not observe any apparent association between chemo- tylon), shared chemotypes are reported from distantly typic variation and 1) species identity (e.g., chemotypes related lineages of the genus (e.g., chemotype with only

A B C D

E F G H

I J K

Figure 6. Morphological variation in P. britannica s.l. and P. chionophila. A, B – cephalodia on the upper surface of P. britannica 1; C, D – lower surface of P. britannica 1; E – underneath of apothecia of P. britannica 1; F – underneath of apothecia of P. britannica 2; G – cephalodia on the upper surface of P. britannica 2; H – lower surface of P. britannica 2; I, J – appressed cephalodia on the upper surface of P. chionophila; K – veins and rhizines of P. chionophila. Scale bars; A–G, I–K = 2 mm; H = 5 mm. 60 Plant and Fungal Systematics 63(2): 45–64, 2018

A B C D

E F G

H I J

Figure 7. Morphological variation in P. malacea s.l. A, B – upper surface of P. malacea 1; C – lobes of P. malacea; D – hairs on the margin of the upper surface of P. malacea 2; E – lobes of P. malacea 3; F – upper surface margin with dense tomentum in P. malacea 4; G – lobes of P. malacea 4; H – pruina on the margin of the upper surface of P. aphthosa 5; I – glabrescent lobes of P. frippii; J – hairs on the margin of the upper surface of P. frippii. Scale bars = 5 mm.

zeorin visible was detected in P. aphthosa, P. phyllidiosa, the next (Chagnon et al. 2018). One-to-one reciprocal P. scabrosa, P. horizontalis, and P. neopolydactyla s.l.; specificity is extremely rare in sections Polydactylon Holtan-Hartwig 1993; Kotarska 1999). (phylogroups IX and XIb paired with P. sp. 11 and P. neopolydactyla 5, respectively; Magain et al. 2017a) High specificity of both symbionts in section Peltidea and Peltigera (two symbiont pairs, P. patagonica, and All cyanobionts found in lichen thalli from section Pelti- P. vainioi pairing with Nostoc phylogroup XXVIII and dea belong to Nostoc Clade II, mostly subclade 2 (phy- XXVIa, respectively; Magain et al. 2018), and a rare logroups III and IV); only a few cyanobionts represented phenomenon in cyanolichens in general at a global scale subclade 3 (phylogroups V and VI; Figs. 2 and 3). Based (Otálora et al. 2010). This is consistent with the hypothesis on a broad sampling (Europe, North America, and Far East that “…some form of genetically determined specificity is Russia), our results confirmed a high and nearly exclusive operating…” (O’Brien et al. 2013) and warrants further specificity of Nostoc phylogroup III with P. malacea s.l. study. Mycobionts from the remaining species of section (P. malacea 1, 3, 4, and 5; Figs. 2 and 3) reported earlier Peltidea were found mostly in association with Nostoc by O’Brien et al. (2013) for specimens collected in Can- phylogroup IV (in subclade 2), which is closely related to ada (British Columbia) and Europe, though it should be phylogroup III (part of the same highly supported clade; noted that P. malacea s.l. can also associate with Nostoc Fig. 3) and somewhat also selective in Peltigera, interact- phylogroup IV (P. malacea 2). Such a degree of speci- ing almost exclusively with members of a single section ficity is highly unusual; in general, Peltigera species are (Peltidea), i.e., with the exception of P. neopolydactyla 4. more specialized than their Nostoc partners (Magain et al. Interestingly, subclade 2 contains another Nostoc lineage, 2017a). Possibly this asymmetric specialization helps to which is involved in an exclusive, reciprocally specific maintain the lichen symbiosis through time, especially interaction with Scytinium lichenoides, one of five Col- in a symbiotic system where the photobiont is transmit- lemataceae species which co-speciated with their cyano- ted predominantly horizontally from one generation to bacterial partner (Otálora et al. 2010). J. Miadlikowska et al. Species in section Peltidea remain cryptic after molecular phylogenetic revision 61

A few representatives of P. britannica s.l. and P. aph- Serusiaux (2014) proposed the same evolutionary scenario thosa s.l. are associated with Nostoc phylogroups from for the family Pannariaceae where subsequent diver- subclade 3, which includes other sections of the genus gence events resulted from a potential emancipation of Peltigera and other genera within Peltigerales. Contrary cephalodia. to local specialists (species associating with distinct Nostoc phylogroups in different geographic regions) Conclusions reported from section Polydactylon (e.g., P. occidentalis; Magain et al. 2017a) and species from other genera (e.g., We refrain from proposing formal changes to the current Nephroma parile; Fedorowitz et al. 2012), we did not taxonomy of Peltigera section Peltidea owing, first, to find any geographic evidence explaining the alternative the lack of reliable diagnostic (morphological, chemical pairings of P. aphthosa and P. britannica with their Nostoc and geographic) characters to circumscribe its molecular partners (phylogroup IV versus V and VI). diversity, and second, to a lack of convergence among loci Considering that the first split in the phylogeny of and analytical methods among most currently recognized section Peltidea divides bimembered and trimembered species. In the case of P. malacea, all newly recognized lichens (Figs. 1 and 2), it is interesting that most cyano- putative species represent lineages within a broadly cir- bionts in both types of symbioses (subclade 2) are phy- cumscribed, yet monophyletic morphospecies, which is logenetically closely related. One possible interpretation widely accepted and easily recognized. Trimembered spe- of this pattern is that mycobiont switches to alternative cies from sections Peltidea and Chloropeltigera (e.g., cyanobacterial partners have been evolutionarily infre- P. aphthosa, P. leucophlebia, P. britannica, P. latiloba, and quent in section Peltidea, in which partnerships formed P. chionophila) are often difficult to distinguish, especially instead from a pool of closely related Nostoc clades. in areas where they co-occur and because they frequently Alternatively, the low level of genetic variation in their display overlapping phenotypes. Unless shown to be cyanobacterial symbionts may simply reflect a case of reproductively isolated, a formal infraspecific status (e.g., coevolution. There are also instances where the same variety or subspecies) might be biologically more appro- Nostoc (phylogroup IV) occurs across broad geographic priate for accommodating the uncovered phylogenetic distances in both types of thalli: bimembered P. malacea structure within the current, monophyletic species within and P. frippii from Norway and trimembered P. aphthosa section Peltidea. The possible broader delimitation of in Alaska. This contrasts with phylogenetic relationships P. aphthosa (to include P. britannica) and the questionable within the genus Nephroma (trimembered species are phylogenetic status of P. britannica as a separate species polyphyletic), where Fedorowitz et al. (2012) suggested corroborated by morphological observations (specimens that repeated evolutionary transitions between the two from North America lacking its signature characteristic types of symbioses involved a concurrent switch from – the shape of cephalodia) should be confirmed based one lineage of Nostoc symbionts to another. on additional collections and loci before redefining their Considering that apothecia have not been frequently taxonomic boundaries. Careful reevaluation of morpho- observed in section Peltidea (however, they are locally logical traits and discovery of novel diagnostic characters, common, e.g., in coastal populations of P. britannica and as well as population genetic analyses to investigate gene P. chionophila in British Columbia, Canada), and vege- flow among putative species, should facilitate future tax- tative propagules (i.e., isidia and soredia) are absent, it onomic conclusions. Furthermore, the exclusive nature of is very likely that these species disperse mostly via thal- Nostoc phylogroup III and selected clades of P. malacea lus fragmentation (enabling vertical transmission of the s.l. deserves further attention. photobiont). However, rare events of sexual reproduction (horizontal transmission of the photobiont) might have led Acknowledgements to the acquisition of new Nostoc lineages from subclade 3 by trimembered species (Fig. 3). Three findings support We are very thankful to the curators of fungaria and collaborators who provided material for this study. We are also very grateful to the scenario that trimembered species in this section are all colleagues who helped us organize and successfully execute derived from bimembered associations by the acquisi- the field trips for this project. This study was supported by the tion of a green alga (Coccomyxa sp.), which became the National Science Foundation (NSF) DEB-1025930 REVSYS dominant photobiont, while Nostoc became restricted to award on the genus Peltigera and NSF SG DEB-1556995 award external cephalodia: 1) the most recent common ancestor on phylogenetic and network analyses using Peltigera as a model of Peltigera was reconstructed as a bimembered cyano- system to F. L. and J. M., as well as by the NSF award DEB- lichen (Miadlikowska & Lutzoni 2004), 2) the observed 1046065 Dimensions of Biodiversity to F. L. close relationships between Nostoc from bi- and trimem- bered species in Peltidea, and 3) the ontogenic similarity Supplementary electronic material in both symbiotic forms (Miadlikowska & Lutzoni 2004 Appendix 1. Specimens used in this study with their reference num- and references therein). This could have been followed bers (DNA extraction number), species assignment and clade number by a transition from a trimembered to a bimembered sym- (based on BPP validation; Fig. 2 and Appendix 2), voucher informa- biotic state according to O’Brien et al. (2013), through tion, mycobiont and cyanobiont (Nostoc) sequence type/haplotype assignment, Nostoc phylogroup (based on Magain et al. 2017a and a mechanism where cephalodia develop into indepen- this study; Fig. 3), chemotype (based on TLC analyses; Fig. 4), and dent cyanomorphs that led eventually to the speciation GenBank accession number (sequences obtained as part of this study of bimembered P. frippi and P. malacea. Magain and are in bold). Download file 62 Plant and Fungal Systematics 63(2): 45–64, 2018

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