Phylogenetic Classification at Generic Level in the Absence of Distinct Phylogenetic Patterns of Phenotypical Variation: A Case Study in Graphidaceae (Ascomycota)

<div><p>Molecular phylogenies often reveal that taxa circumscribed by phenotypical characters are not monophyletic. While re-examination of phenotypical characters often identifies the presence of characters characterizing clades, there is a growing number of studies that fail to identify diagnostic characters, especially in organismal groups lacking complex morphologies. Taxonomists then can either merge the groups or split taxa into smaller entities. Due to the nature of binomial nomenclature, this decision is of special importance at the generic level. Here we propose a new approach to choose among classification alternatives using a combination of morphology-based phylogenetic binning and a multiresponse permutation procedure to test for morphological differences among clades. We illustrate the use of this method in the tribe Thelotremateae focusing on the genus <em>Chapsa</em>, a group of lichenized fungi in which our phylogenetic estimate is in conflict with traditional classification and the morphological and chemical characters do not show a clear phylogenetic pattern. We generated 75 new DNA sequences of mitochondrial SSU rDNA, nuclear LSU rDNA and the protein-coding RPB2. This data set was used to infer phylogenetic estimates using maximum likelihood and Bayesian approaches. The genus <em>Chapsa</em> was found to be polyphyletic, forming four well-supported clades, three of which clustering into one unsupported clade, and the other, supported clade forming two supported subclades. While these clades cannot be readily separated morphologically, the combined binning/multiresponse permutation procedure showed that accepting the four clades as different genera each reflects the phenotypical pattern significantly better than accepting two genera (or five genera if splitting the first clade). Another species within the Thelotremateae, <em>Thelotrema petractoides</em>, a unique taxon with carbonized excipulum resembling <em>Schizotrema</em>, was shown to fall outside <em>Thelotrema</em>. Consequently, the new genera <em>Astrochapsa, Crutarndina, Pseudochapsa,</em> and <em>Pseudotopeliopsis</em> are described here and 39 new combinations are proposed.</p> </div

Species and specimens used in the present study, with location, reference collection details, and GenBank accession numbers. Newly obtained sequenced in bold.

<p>Species and specimens used in the present study, with location, reference collection details, and GenBank accession numbers. Newly obtained sequenced in bold.</p

Bayesian 50% majority-rule consensus tree depicting relationships among genera in the tribe Thelotrematae on the basis of a concatenated data set including mtSSU rDNA, nuLSU rDNA and protein-coding RPB2.

<p>Posterior probabilities equal or above 0.95 are indicated as bold branches. ML-bootstrap support equal or above 70% is shown as number at branches.</p

Placement of species within clades based on morphological characters using phylogenetic binning method under ML and MP weighting.

<p>Placement of species within clades based on morphological characters using phylogenetic binning method under ML and MP weighting.</p

Number of individual characters discriminating between groups using phylogenetic binning method under ML and MP weighting.

<p>Number of individual characters discriminating between groups using phylogenetic binning method under ML and MP weighting.</p

Discriminating characters as based on a Kruskal-Wallis ANOVA using the different clade solutions as grouping variables. P-values are indicated if below 0.1.

<p>Discriminating characters as based on a Kruskal-Wallis ANOVA using the different clade solutions as grouping variables. P-values are indicated if below 0.1.</p

Results of the multi-response permutation procedure (MRPP) analysis.

<p>p-values for significance of group distances based on morphological character matrix.</p

Hidden Genetic Diversity in an Asexually Reproducing Lichen Forming Fungal Group

<div><p>Asexual species with vegetative propagation of both symbiont partners (soredia) in lichens may harbor lower species diversity because they may indeed represent evolutionary dead ends or clones. In this study we aim to critically examine species boundaries in the sorediate lichen forming fungi <i>Parmotrema reticulatum–Parmotrema pseudoreticulatum</i> complex applying coalescent-based approaches and other recently developed DNA-based methods. To this end, we gathered 180 samples from Africa, Asia, Australasia, Europe, North and South America and generated sequences of internal transcribed spacer of nuclear ribosomal DNA (ITS) and DNA replication licensing factor MCM7 (MCM7). The dataset was analysed using different approaches such as traditional phylogeny–maximum likelihood and Bayesian–genetic distances, automatic barcode gap discovery and coalescent-based methods–PTP, GMYC, spedeSTEM and *Beast–in order to test congruence among results. Additionally, the divergence times were also estimated to elucidate diversification events. Delimitations inferred from the different analyses are comparable with only minor differences, and following a conservative approach we propose that the sampled specimens of the <i>P</i>. <i>reticulatum</i>–<i>P</i>. <i>pseudoreticulatum</i> complex belong to at least eight distinct species-level lineages. Seven are currently classified under <i>P</i>. <i>reticulatum</i> and one as <i>P</i>. <i>pseudoreticulatum</i>. In this work we discuss one of only few examples of cryptic species that have so far been found in sorediate reproducing lichen forming fungi. Additionally our estimates suggest a recent origin of the species complex–during the Miocene. Consequently, the wide distribution of several of the cryptic species has to be explained by intercontinental long-distance dispersal events.</p></div

*BEAST species time tree of <i>P</i>. <i>reticulatum</i>–<i>P</i>. <i>pseudoreticulatum</i> complex diversification.

<p>The chronogram was estimated from a two-locus, (ITS and MCM7), coalescent-based species tree in *BEAST. Mean ages in millions of years (Ma), and their 95% highest posterior density (HPD) bars are shown above nodes. Clades A-E represents putative species recovered in *BEAST analysis. J = out group.</p

Time-calibrated species trees for <i>Xanthoparmelia</i> clades ‘D’ and ‘E’.

<p>Divergence estimates were based on (A) genetic population clusters and (B) traditional, morphology-based species circumscriptions. Posterior probabilities at nodes indicate support from the *BEAST analyses and speciation probabilities. Inset tables include divergence times for specified nodes, including the highest posterior density interval (HPD), using fixed substitution rates in four different scenarios: dates estimated using the ITS, nrLSU, RPB1 loci, independently; and the forth using rates from both the ITS nrLSU loci (Units: substitution/site/10⁹ years). </p