Myrmecia, Not Asterochloris, Is the Main Photobiont of Cladonia subturgida (Cladoniaceae, Lecanoromycetes).

This study explores the diversity of photobionts associated with the Mediterranean lichen-forming fungus Cladonia subturgida. For this purpose, we sequenced the whole ITS rDNA region by Sanger using a metabarcoding method for ITS2. A total of 41 specimens from Greece, Italy, France, Portugal, and Spain were studied. Additionally, two specimens from Spain were used to generate four cultures. Our molecular studies showed that the genus Myrmecia is the main photobiont of C. subturgida throughout its geographic distribution. This result contrasts with previous studies, which indicated that the main photobiont for most Cladonia species is Asterochloris. The identity of Myrmecia was also confirmed by ultrastructural studies of photobionts within the lichen thalli and cultures. Photobiont cells showed a parietal chloroplast lacking a pyrenoid, which characterizes the species in this genus. Phylogenetic analyses indicate hidden diversity within this genus. The results of amplicon sequencing showed the presence of multiple ASVs in 58.3% of the specimens studied.

Global Biodiversity Patterns of the Photobionts Associated with the Genus Cladonia (Lecanorales, Ascomycota).

The diversity of lichen photobionts is not fully known. We studied here the diversity of the photobionts associated with Cladonia, a sub-cosmopolitan genus ecologically important, whose photobionts belong to the green algae genus Asterochloris. The genetic diversity of Asterochloris was screened by using the ITS rDNA and actin type I regions in 223 specimens and 135 species of Cladonia collected all over the world. These data, added to those available in GenBank, were compiled in a dataset of altogether 545 Asterochloris sequences occurring in 172 species of Cladonia. A high diversity of Asterochloris associated with Cladonia was found. The commonest photobiont lineages associated with this genus are A. glomerata, A. italiana, and A. mediterranea. Analyses of partitioned variation were carried out in order to elucidate the relative influence on the photobiont genetic variation of the following factors: mycobiont identity, geographic distribution, climate, and mycobiont phylogeny. The mycobiont identity and climate were found to be the main drivers for the genetic variation of Asterochloris. The geographical distribution of the different Asterochloris lineages was described. Some lineages showed a clear dominance in one or several climatic regions. In addition, the specificity and the selectivity were studied for 18 species of Cladonia. Potentially specialist and generalist species of Cladonia were identified. A correlation was found between the sexual reproduction frequency of the host and the frequency of certain Asterochloris OTUs. Some Asterochloris lineages co-occur with higher frequency than randomly expected in the Cladonia species.

Phylogeny of the family Cladoniaceae (Lecanoromycetes, Ascomycota) based on sequences of multiple loci.

Cladoniaceae is a family of lichenized fungi that belongs to the Lecanorales, Ascomycota. The family is distributed widely, although several genera are restricted to the Southern Hemisphere. The circumscriptions of the genera and species in the family have traditionally been based on thallus morphology, the type of vegetative propagules and the secondary metabolites. However, numerous species are highly variable phenotypically, making their delimitation problematic. In the present study a new phylogeny of Cladoniaceae is constructed using five loci (ITS rDNA, IGS rDNA, RPB2, RPB1, EF-1a) from a worldwide sample of 643 specimens representing 304 species. Cladoniaceae was resolved as a monophyletic group. The circumscription of the genera and the relationships among them are discussed. Pycnothelia, Carassea and Metus are closely related, forming a sister clade to the larger genus Cladonia. Cladia in its recent wide sense turned out to be paraphyletic, including species that have been recognized in Thysanothecium and Notocladonia. Cladonia was resolved as monophyletic, with C. wainioi as the earliest diverging lineage. Eleven major clades were resolved in Cladonia. No synapomorphies were found for most of them. We propose the new genera Pulchrocladia and Rexia, as segregates of Cladia, five new combinations, and the resurrection of the genus Heterodea.

Genetic variation and factors affecting the genetic structure of the lichenicolous fungus Heterocephalacria bachmannii (Filobasidiales, Basidiomycota).

Heterocephalacria bachmannii is a lichenicolous fungus that takes as hosts numerous lichen species of the genus Cladonia. In the present study we analyze whether the geographical distance, the host species or the host secondary metabolites determine the genetic structure of this parasite. To address the question, populations mainly from the Southern Europe, Southern Finland and the Azores were sampled. The specimens were collected from 20 different host species representing ten chemotypes. Three loci, ITS rDNA, LSU rDNA and mtSSU, were sequenced. The genetic structure was assessed by AMOVA, redundance analyses and Bayesian clustering methods. The results indicated that the host species and the host secondary metabolites are the most influential factors over the genetic structure of this lichenicolous fungus. In addition, the genetic structure of H. bachmannii was compared with that of one of its hosts, Cladonia rangiformis. The population structure of parasite and host were discordant. The contents in phenolic compounds and fatty acids of C. rangiformis were quantified in order to test whether it had some influence on the genetic structure of the species. But no correlation was found with the genetic clusters of H. bachmannii.

Three common bryophilous fungi with meristematic anamorphs and phylogenetic alliance to Teratosphaeriaceae, Capnodiales.

Bryophilous ascomycetes are an overlooked and poorly known fungal group. In this study, the extreme and small-sized niche of Polytrichum piliferum hyaline leaf tips was screened for the presence of these fungi in Finland. Three closely related species were found. Bryochiton perpusillus and Bryochiton monascus were identified from several samples, and DNA isolations revealed a third closely related species, Bryochiton sp. In addition, melanised hyphae, typical to the Bryochiton species, were present in all the samples. According to phylogenetic analyses consisting of combined small subunit (SSU), large subunit (LSU), and 5.8S rDNA sequences, and internal transcribed spacer (ITS) rDNA sequences, the species showed affinity with Teratosphaeriaceae within Capnodiales, and especially with black, meristematic species often inhabiting rock substrate in extreme environments. The connection was supported by meristematic growth of the Bryochiton species in culture. Bryochiton is the second sexual genus associated within the family Teratosphaeriaceae, and B. perpusillus, and B. monascus constitute examples of teleomorphs within a group of meristematic anamorphs. These findings emphasize the multiform diversity underlying poorly researched fungal groups, such as the bryophilous fungi.

Multilocus approach to species recognition in the Cladonia humilis complex (Cladoniaceae, Ascomycota).

PREMISE OF THE STUDY: The members of the Cladonia humilis complex are characterized by a well-developed primary thallus and broadly scyphose podetia. In the present study, this complex was phylogenetically analyzed to test the boundaries between the species and to determine the usefulness of the phenotypic characters to distinguish them. The species C. conista, C. cyathomorpha, C. hammeri, C. humilis, C. kurokawae, C. nashii, C. pulvinella, and C. subconistea were examined. METHODS: Four DNA loci were sequenced and analyzed to test the monophyly of the species. For the phylogenetic reconstructions, maximum parsimony, maximum likelihood, and Bayesian methods were employed. The genealogical sorting index was used to quantify the exclusive ancestry of the nonmonophyletic species on the tree. KEY RESULTS: The performed phylogenetic analyses showed that the C. humilis complex is not monophyletic. Cladonia nashii is not closely related to the remaining taxa within the complex. Seven monophyletic lineages were identified, most of which comprise specimens belonging to more than one chemotype. Cladonia hammeri and C. pulvinella are conspecific, and this taxon is not present in Europe. CONCLUSIONS: This study suggests that morphological characters and secondary metabolites have less taxonomical value than thought in the Cladonia humilis complex. Use of multilocus phylogeny is recommended to delimit species.

Species delimitation in Cladonia (Ascomycota): a challenge to the DNA barcoding philosophy.

The lichen-forming fungal genus Cladonia is species-rich with approximately 500 described species. The accepted barcode for fungi (ITS rDNA) often fails in identifying Cladonia spp. In order to find other markers that, in combination with the ITS rDNA region can be used for species identification in Cladonia, we studied the loci IGS rDNA, ef1α, rpb2 and cox1. A total of 782 sequences from 36 species have been analyzed. PCR amplification success rate, intraspecific and interspecific genetic distance variation, calculated using the K2P model, and the correct identification percentage (PCI) were taken into account to assess possible barcode regions. The marker showing the least intraspecific genetic distance range was cox1, followed by ITS rDNA and ef1α. Of the five studied markers only cox1 showed a barcoding gap. The rpb2 locus showed the highest PCI values, but it was the most difficult to amplify. The highest correct identification rates using blast method were obtained with rpb2.