Contrasting Symbiotic Patterns in Two Closely Related Lineages of Trimembered Lichens of the Genus Peltigera.

Species circumscription is key to the characterization of patterns of specificity in symbiotic systems at a macroevolutionary scale. Here, a worldwide phylogenetic framework was used to assess the biodiversity and symbiotic patterns of association among partners in trimembered lichens from the genus Peltigera, section Chloropeltigera. We sequenced six loci of the main fungal partner and performed species discovery and validation analyses to establish putative species boundaries. Single locus phylogenies were used to establish the identity of both photobionts, Nostoc (cyanobacterium) and Coccomyxa (green alga). Distribution and specificity patterns were compared to the closely related clade, section Peltidea, which includes mainly Peltigera species with trimembered thalli. For section Chloropeltigera, eight fungal species (including five newly delimited putative species) were found in association with nine Nostoc phylogroups and two Coccomyxa species. In contrast, eight fungal species (including three newly delimited putative species) in section Peltidea were found in association with only four Nostoc phylogroups and the same two Coccomyxa species as for section Chloropeltigera. This difference in cyanobiont biodiversity between these two sections can potentially be explained by a significantly higher frequency of sexual reproductive structures in species from section Chloropeltigera compared to section Peltidea. Therefore, horizontal transmission of the cyanobiont might be more prevalent in Chloropeltigera species, while vertical transmission might be more common in Peltidea species. All Peltigera species in section Chloropeltigera are generalists in their association with Nostoc compared to more specialized Peltigera species in section Peltidea. Constrained distributions of Peltigera species that associate strictly with one species of green algae (Coccomyxa subellipsoidea) indicate that the availability of the green alga and the specificity of the interaction might be important factors limiting geographic ranges of trimembered Peltigera, in addition to constraints imposed by their interaction with Nostoc partners and by climatic factors.

The phylogenetic analysis of fungi associated with lichenized ascomycete genus Bryoria reveals new lineages in the Tremellales including a new species Tremella huuskonenii hyperparasitic on Phacopsis huuskonenii.

The basidiomycete order Tremellales includes many species parasitic on or cohabiting with lichen-forming fungi. In this study, we examined the phylogenetic position of Tremellales obtained from Bryoria thalli using nSSU, 5.8S, and partial nLSU sequence data. Both Bayesian and maximum likelihood analyses revealed the presence of basidiomycetous fungi in three separate clades within Tremellales. Tremellales sp. A and Tremella sp. B exist asymptomatically in Bryoria thalli and should thus be regarded as endolichenic rather than lichenicolous fungi. The third lineage represents a new species and is described here as Tremella huuskonenii. It is hyperparasitic over galls induced by Phacopsis huuskonenii, a lichenicolous fungus growing in Bryoria thalli. We also examined the genetic diversity of Tremella sp. B and Tremella huuskonenii with an extended taxon sampling using ITS and partial nLSU sequence data. For comparison, ITS, GAPDH, and Mcm7 regions were used for phylogenetic analyses of the host lichen specimens. According to our results, phylogenetic structure within the two Tremella species does not appear to correlate with the geographic distribution nor with the phylogeny or the secondary chemistry of the host lichen. However, ITS haplotype analysis of T. huuskonenii revealed some genetic differences between European and North American populations as some haplotypes were more common in Europe than in North America and vice versa.

Evolution of complex symbiotic relationships in a morphologically derived family of lichen-forming fungi.

We studied the evolutionary history of the Parmeliaceae (Lecanoromycetes, Ascomycota), one of the largest families of lichen-forming fungi with complex and variable morphologies, also including several lichenicolous fungi. We assembled a six-locus data set including nuclear, mitochondrial and low-copy protein-coding genes from 293 operational taxonomic units (OTUs). The lichenicolous lifestyle originated independently three times in lichenized ancestors within Parmeliaceae, and a new generic name is introduced for one of these fungi. In all cases, the independent origins occurred c. 24 million yr ago. Further, we show that the Paleocene, Eocene and Oligocene were key periods when diversification of major lineages within Parmeliaceae occurred, with subsequent radiations occurring primarily during the Oligocene and Miocene. Our phylogenetic hypothesis supports the independent origin of lichenicolous fungi associated with climatic shifts at the Oligocene-Miocene boundary. Moreover, diversification bursts at different times may be crucial factors driving the diversification of Parmeliaceae. Additionally, our study provides novel insight into evolutionary relationships in this large and diverse family of lichen-forming ascomycetes.

How do you solve a problem like Letharia? A new look at cryptic species in lichen-forming fungi using Bayesian clustering and SNPs from multilocus sequence data.

The inclusion of molecular data is increasingly an integral part of studies assessing species boundaries. Analyses based on predefined groups may obscure patterns of differentiation, and population assignment tests provide an alternative for identifying population structure and barriers to gene flow. In this study, we apply population assignment tests implemented in the programs STRUCTURE and BAPS to single nucleotide polymorphisms from DNA sequence data generated for three previous studies of the lichenized fungal genus Letharia. Previous molecular work employing a gene genealogical approach circumscribed six species-level lineages within the genus, four putative lineages within the nominal taxon L. columbiana (Nutt.) J.W. Thomson and two sorediate lineages. We show that Bayesian clustering implemented in the program STRUCTURE was generally able to recover the same six putative Letharia lineages. Population assignments were largely consistent across a range of scenarios, including: extensive amounts of missing data, the exclusion of SNPs from variable markers, and inferences based on SNPs from as few as three gene regions. While our study provided additional evidence corroborating the six candidate Letharia species, the equivalence of these genetic clusters with species-level lineages is uncertain due, in part, to limited phylogenetic signal. Furthermore, both the BAPS analysis and the ad hoc ΔK statistic from results of the STRUCTURE analysis suggest that population structure can possibly be captured with fewer genetic groups. Our findings also suggest that uneven sampling across taxa may be responsible for the contrasting inferences of population substructure. Our results consistently supported two distinct sorediate groups, 'L. lupina' and L. vulpina, and subtle morphological differences support this distinction. Similarly, the putative apotheciate species 'L. lucida' was also consistently supported as a distinct genetic cluster. However, additional studies will be required to elucidate the relationships of other L. columbiana s.l. populations with the two sorediate genetic clusters.

A multigene phylogenetic synthesis for the class Lecanoromycetes (Ascomycota): 1307 fungi representing 1139 infrageneric taxa, 317 genera and 66 families.

The Lecanoromycetes is the largest class of lichenized Fungi, and one of the most species-rich classes in the kingdom. Here we provide a multigene phylogenetic synthesis (using three ribosomal RNA-coding and two protein-coding genes) of the Lecanoromycetes based on 642 newly generated and 3329 publicly available sequences representing 1139 taxa, 317 genera, 66 families, 17 orders and five subclasses (four currently recognized: Acarosporomycetidae, Lecanoromycetidae, Ostropomycetidae, Umbilicariomycetidae; and one provisionarily recognized, 'Candelariomycetidae'). Maximum likelihood phylogenetic analyses on four multigene datasets assembled using a cumulative supermatrix approach with a progressively higher number of species and missing data (5-gene, 5+4-gene, 5+4+3-gene and 5+4+3+2-gene datasets) show that the current classification includes non-monophyletic taxa at various ranks, which need to be recircumscribed and require revisionary treatments based on denser taxon sampling and more loci. Two newly circumscribed orders (Arctomiales and Hymeneliales in the Ostropomycetidae) and three families (Ramboldiaceae and Psilolechiaceae in the Lecanorales, and Strangosporaceae in the Lecanoromycetes inc. sed.) are introduced. The potential resurrection of the families Eigleraceae and Lopadiaceae is considered here to alleviate phylogenetic and classification disparities. An overview of the photobionts associated with the main fungal lineages in the Lecanoromycetes based on available published records is provided. A revised schematic classification at the family level in the phylogenetic context of widely accepted and newly revealed relationships across Lecanoromycetes is included. The cumulative addition of taxa with an increasing amount of missing data (i.e., a cumulative supermatrix approach, starting with taxa for which sequences were available for all five targeted genes and ending with the addition of taxa for which only two genes have been sequenced) revealed relatively stable relationships for many families and orders. However, the increasing number of taxa without the addition of more loci also resulted in an expected substantial loss of phylogenetic resolving power and support (especially for deep phylogenetic relationships), potentially including the misplacements of several taxa. Future phylogenetic analyses should include additional single copy protein-coding markers in order to improve the tree of the Lecanoromycetes. As part of this study, a new module ("Hypha") of the freely available Mesquite software was developed to compare and display the internodal support values derived from this cumulative supermatrix approach.

Species delimitation in taxonomically difficult lichen-forming fungi: an example from morphologically and chemically diverse Xanthoparmelia (Parmeliaceae) in North America.

Mounting evidence suggests many morphology-based species circumscriptions in lichenized ascomycetes misrepresent fungal diversity. The lichenized ascomycete genus Xanthoparmelia includes over 800 described species displaying a considerable range of morphological and chemical variation. Species circumscriptions in this genus have traditionally been based on thallus morphology, medullary chemistry, and the presence or absence of sexual or asexual reproductive structures. Notwithstanding concerted effort on the part of taxonomists to arrive at a natural classification, modern taxonomic concepts for the most part remain unclear. Here we assess the evolution of characters traditionally regarded as taxonomically important by reconstructing a phylogenetic hypothesis based on sequence data from four nuclear ribosomal markers as well as fragments from two protein-coding nuclear loci. A total of 414 individuals were tested, representing 19 currently accepted species. Most sampled species, as currently circumscribed, were recovered as polyphyletic, suggesting that major diagnostic characters have evolved in a homoplasious manner. The vagrant growth form, distinct medullary chemistries, and production of vegetative diaspores appear to have evolved independently multiple times. Application of a population assignment test resulted in the recognition of 21 species-level genetic clusters, each of which was supported by a comparison of genetic distances as well as a Bayesian species delimitation method calculating probabilities associated with speciation events. Inferred clusters are largely incongruent with traditionally circumscribed species due to the prevalence of cryptic diversity and, in some cases, high levels of intraspecific morphological and chemical variation. These results call for a major taxonomic revision of Xanthoparmelia species in western North America.

New approach to an old problem: Incorporating signal from gap-rich regions of ITS and rDNA large subunit into phylogenetic analyses to resolve the Peltigera canina species complex.

The Peltigera canina species complex consists of foliose lichenized bitunicate ascohymenial discomycetes forming section Peltigera within the genus Peltigera (Lecanoromycetes, lichen-forming Ascomycetes). To test the circumscription of highly polymorphic species and to resolve relationships among putative members of the P. canina complex, part of the nuclear ribosomal DNA large subunit (LSU rDNA) and the entire internal-transcribed spacer (ITS rDNA) were sequenced for 84 individuals representing 33 putative Peltigera taxa. Seventeen of the 25 taxa from the P. canina complex are well established and widely accepted. The remaining eight taxa have been proposed recently but are undescribed. A hypervariable region in ITS1 (ITS1-HR, sites 111-237 in our alignment) showed remarkable variation in length, especially in the P. canina complex, ranging from 8 to 126 bp, and contained several microsatellites. We describe here an alignment-free method to code such large gap-rich hypervariable regions for phylogenetic analyses. Variation among ITS1-HR sequences greatly contributed to species delimitation and species identification and can be a major asset to future population studies for specific species within section Peltigera. Sequences of ITS1-HR alone were sufficient to identify all existing species of Peltigera from the P. canina species complex and related sections Retifoveatae and Horizontales included in this study. However, only when INAASE (for short ambiguously aligned regions) and ITS1-HR coded characters were added to the combined analysis of nonambiguous LSU and ITS sites was it possible to reach the level of phylogenetic resolution and support necessary to disentangle the P. canina complex. We report here complete concordance between phylogenetically based and morphologically based species delimitation for 15 of the 17 species from the P. canina complex (P. canina, P. cinnamomea, P. degenii, P. evansiana, P. frigida, P. kristinssonii, P. laciniata, P. lambinonii, P. lepidophora, P. membranacea, P. monticola, P. ponojensis, P. praetextata, P. rufescens and P. ulcerata). Four of the eight newly proposed but undescribed taxa most likely represent new species (P. "fuscopraetextata", P. "neocanina", P. "neorufescens" and P. "scotteri") within the P. canina complex. We found that morphologically and chemically distinct P. didactyla s. str. and P. didactyla var. extenuata form two non-sister monophyletic entities, therefore the latter taxon should be recognized at the species level (P. extenuata). The North American and European populations of the morphologically uniform P. degenii might represent two sibling species because they were found to be genetically distinct and monophyletic. Two major monophyletic groups within the P. canina complex (CICADE = CInnamomea + CAnina + DEgenii group and PORUDI = POnojensis + RUfescens + DIdactyla group) seem to be correlated with different humidity preferences. Although some authors previously have suggested interspecies recombination within the P. canina complex, we did not find statistically significant evidence for this phenomenon based on LSU and ITS sequences.