Ancient Rapid Radiation Explains Most Conflicts Among Gene Trees and Well-Supported Phylogenomic Trees of Nostocalean Cyanobacteria.

Prokaryotic genomes are often considered to be mosaics of genes that do not necessarily share the same evolutionary history due to widespread horizontal gene transfers (HGTs). Consequently, representing evolutionary relationships of prokaryotes as bifurcating trees has long been controversial. However, studies reporting conflicts among gene trees derived from phylogenomic data sets have shown that these conflicts can be the result of artifacts or evolutionary processes other than HGT, such as incomplete lineage sorting, low phylogenetic signal, and systematic errors due to substitution model misspecification. Here, we present the results of an extensive exploration of phylogenetic conflicts in the cyanobacterial order Nostocales, for which previous studies have inferred strongly supported conflicting relationships when using different concatenated phylogenomic data sets. We found that most of these conflicts are concentrated in deep clusters of short internodes of the Nostocales phylogeny, where the great majority of individual genes have low resolving power. We then inferred phylogenetic networks to detect HGT events while also accounting for incomplete lineage sorting. Our results indicate that most conflicts among gene trees are likely due to incomplete lineage sorting linked to an ancient rapid radiation, rather than to HGTs. Moreover, the short internodes of this radiation fit the expectations of the anomaly zone, i.e., a region of the tree parameter space where a species tree is discordant with its most likely gene tree. We demonstrated that concatenation of different sets of loci can recover up to 17 distinct and well-supported relationships within the putative anomaly zone of Nostocales, corresponding to the observed conflicts among well-supported trees based on concatenated data sets from previous studies. Our findings highlight the important role of rapid radiations as a potential cause of strongly conflicting phylogenetic relationships when using phylogenomic data sets of bacteria. We propose that polytomies may be the most appropriate phylogenetic representation of these rapid radiations that are part of anomaly zones, especially when all possible genomic markers have been considered to infer these phylogenies. [Anomaly zone; bacteria; horizontal gene transfer; incomplete lineage sorting; Nostocales; phylogenomic conflict; rapid radiation; Rhizonema.].

Extensive photobiont sharing in a rapidly radiating cyanolichen clade.

Recent studies have uncovered remarkable diversity in Dictyonema s.lat. basidiolichens, here recognized as subtribe Dictyonemateae. This group includes five genera and 148 species, but hundreds more await description. The photobionts of these lichens belong to Rhizonema, a recently resurrected cyanobacterial genus known by a single species. To further investigate photobiont diversity within Dictyonemateae, we generated 765 new cyanobacterial sequences from 635 specimens collected from 18 countries. The ITS barcoding locus supported the recognition of 200 mycobiont (fungal) species among these samples, but the photobiont diversity was comparatively low. Our analyses revealed three main divisions of Rhizonema, with two repeatedly recovered as monophyletic (proposed as new species), and the third mostly paraphyletic. The paraphyletic lineage corresponds to R. interruptum and partnered with mycobionts from all five genera in Dictyonemateae. There was no evidence of photobiont-mycobiont co-speciation, but one of the monophyletic lineages of Rhizonema appears to partner predominantly with one of the two major clades of Cora (mycobiont) with samples collected largely from the northern Andes. Molecular clock estimations indicate the Rhizonema species are much older than the fungal species in the Dictyonemateae, suggesting that these basidiolichens obtained their photobionts from older ascolichen lineages and the photobiont variation in extant lineages of Dictyonemateae is the result of multiple photobiont switches. These results support the hypothesis of lichens representing "fungal farmers," in which diverse mycobiont lineages associate with a substantially lower diversity of photobionts by sharing those photobionts best suited for the lichen symbiosis among multiple and often unrelated mycobiont lineages.

From one to six: unrecognized species diversity in the genus Acantholichen (lichenized Basidiomycota: Hygrophoraceae).

We present a taxonomic revision of the lichenized basidiomycete genus Acantholichen, species of which produce a characteristic blue-gray, microsquamulose thallus with spiny apical hyphal cells known as acanthohyphidia. Since its discovery, the genus was thought to be monospecific, only including the generic type, A. pannarioides. However, a detailed morphological and anatomical study of recently collected specimens from the Galápagos, Costa Rica, Brazil and Colombia, combined with a molecular phylogenetic analysis of the internal transcribed spacer (ITS1-5.8S-ITS2) region and 28S of the nuc rDNA and RPB2 sequences, revealed a much more diverse and widespread species assemblage. Based on the results of these analyses, we describe five new species in the genus: A. albomarginatus, A. campestris, A. galapagoensis, A. sorediatus and A. variabilis. We also provide an identification key to all species, anatomical and morphological descriptions, photographs and a table comparing main characters of each species.

A single macrolichen constitutes hundreds of unrecognized species.

The number of Fungi is estimated at between 1.5 and 3 million. Lichenized species are thought to make up a comparatively small portion of this figure, with unrecognized species richness hidden among little-studied, tropical microlichens. Recent findings, however, suggest that some macrolichens contain a large number of unrecognized taxa, increasing known species richness by an order of magnitude or more. Here we report the existence of at least 126 species in what until recently was believed to be a single taxon: the basidiolichen fungus Dictyonema glabratum, also known as Cora pavonia. Notably, these species are not cryptic but morphologically distinct. A predictive model suggests an even larger number, with more than 400 species. These results call into question species concepts in presumably well-known macrolichens and demonstrate the need for accurately documenting such species richness, given the importance of these lichens in endangered ecosystems such as paramos and the alarming potential for species losses throughout the tropics.

Multiple ITS haplotypes in the genome of the lichenized basidiomycete Cora inversa (Hygrophoraceae): fact or artifact?

The internal transcribed spacer region (ITS) of the nuclear rDNA cistron represents the barcoding locus for Fungi. Intragenomic variation of this multicopy gene can interfere with accurate phylogenetic reconstruction of biological entities. We investigated the amount and nature of this variation for the lichenized fungus Cora inversa in the Hygrophoraceae (Basidiomycota: Agaricales), analyzing base call and length variation in ITS1 454 pyrosequencing data of three samples of the target mycobiont, for a total of 16,665 reads obtained from three separate repeats of the same samples under different conditions. Using multiple fixed alignment methods (PaPaRa) and maximum likelihood phylogenetic analysis (RAxML), we assessed phylogenetic relationships of the obtained reads, together with Sanger ITS sequences from the same samples. Phylogenetic analysis showed that all ITS1 reads belonged to a single species, C. inversa. Pyrosequencing data showed 266 insertion sites in addition to the 325 sites expected from Sanger sequences, for a total of 15,654 insertions (0.94 insertions per read). An additional 3,279 substitutions relative to the Sanger sequences were detected in the dataset, out of 5,461,125 bases to be called. Up to 99.3% of the observed indels in the dataset could be interpreted as 454 pyrosequencing errors, approximately 65% corresponding to incorrectly recovered homopolymer segments, and 35% to carry-forward-incomplete-extension errors. Comparison of automated clustering and alignment-based phylogenetic analysis demonstrated that clustering of these reads produced a 35-fold overestimation of biological diversity in the dataset at the 95% similarity threshold level, whereas phylogenetic analysis using a maximum likelihood approach accurately recovered a single biological entity. We conclude that variation detected in 454 pyrosequencing data must be interpreted with great care and that a combination of a sufficiently large number of reads per taxon, a set of Sanger references for the same taxon, and at least two runs under different emulsion PCR and sequencing conditions, are necessary to reliably separate biological variation from 454 sequencing errors. Our study shows that clustering methods are highly sensitive to artifactual sequence variation and inadequate to properly recover biological diversity in a dataset, if sequencing errors are substantial and not removed prior to clustering analysis.

Starting from scratch: Evolution of the lichen thallus in the basidiolichen Dictyonema (Agaricales: Hygrophoraceae).

Phylogenetic studies indicate that the basidiolichen genus Dictyonema s.lat., often thought to represent only a single genus with few species, includes several well-supported genus-level clades, all of which form associations with a unique lineage of obligately lichenized cyanobacteria (Rhizonema). In an attempt to elucidate the evolution and genus- and species-level diversification in Dictyonema s.lat., we generated 68 new sequences of the nuclear large subunit rDNA (nuLSU), the internal transcribed spacer (ITS), and the RNA polymerase II subunit (RPB2), for 29 species-level lineages representing all major clades of Dictyonema s.lat. and most of the species currently known. The multilocus phylogeny obtained via maximum likelihood and Bayesian approaches indicates the presence of five genus-level groups: a basal clade, Cyphellostereum, that is sister to the rest of the species, a paraphyletic grade representing Dictyonema s.str., and three clades representing the genera Acantholichen, Cora, and Corella. To determine the evolutionary transformations of the lichenized thallus in the group, ancestral character state reconstruction was done using six characters (lichenisation, thallus type, cortex type, hyphal sheath and haustorial type, photobiont morphology, and basidiocarp type). Our analysis indicates a progressive development of the lichenized thallus from loosely organized filamentous crusts with separate, cyphelloid basidiocarps in Cyphellostereum, to filamentous crusts with derived hyphal sheath and cyphelloid-stereoid basidiocarps partially incorporated into the lichen thallus in Dictyonema, to squamulose-foliose thalli with corticioid basidiocarps entirely supported by the lichen thallus in Cora. These results indicate a remarkable evolutionary integration of lichenized and reproductive tissues in Dictyonema s.lat., supporting the hypothesis that, at least in this case, lichenized thalli may have evolved from reproductive structures in their nonlichenized ancestors.