Ana Crespo

DNA extraction and PCR amplification method suitable for fresh, herbarium-stored, lichenized, and other fungi

This paper presents a DNA extraction method suitable for fresh, herbarium-stored, lichenized and other fungal specimens. The method is fast and reliable, comparatively inexpensive and is suitable for obtaining PCR amplification quality DNA from large numbers of samples in a short time. The method has been tested with over 300 samples ofAscochyta, Phyllosticta, Ramalina, Parmelia andPhysconia. Amplifiable fungal DNA was extracted from pure cultures, leaf lesions, whole thalli and dissected “only-fungal” sections of lichenized fungi. In addition, the method has proved suitable for use with herbarium specimens of both lichenized and non-lichenized fungi, stored as dried pure cultures or dried infected plant material.

New primers for promising single-copy genes in fungal phylogenetics and systematics.

Developing powerful phylogenetic markers is a key concern in fungal phylogenetics. Here we report degenerate primers that amplify the single-copy genes Mcm7 (MS456) and Tsr1 (MS277) across a wide range of Pezizomycotina (Ascomycota). Phylogenetic analyses of 59 taxa belonging to the Eurotiomycetes, Lecanoromycetes, Leotiomycetes, Lichinomycetes and Sordariomycetes, indicate the utility of these loci for fungal phylogenetics at taxonomic levels ranging from genus to class. We also tested the new primers in silico using sequences of Saccharomycotina, Taphrinomycotina and Basidiomycota to predict their potential of amplifying widely across the Fungi. The analyses suggest that the new primers will need no, or only minor sequence modifications to amplify Saccharomycotina, Taphrinomycotina and Basidiomycota.

A molecular phylogeny and a new classification of parmelioid lichens containing Xanthoparmelia -type lichenan (Ascomycota: Lecanorales)

Generic concepts in the parmelioid lichens have been discussed intensively over the past three decades without reaching a broad consensus. We have now employed molecular data from three genes to provide a basis for a revised generic concept of the parmelioid lichens containing Xanthoparmelia-type lichenan. The phylogeny of the parmelioid lichens containing Xanthoparmelia-type lichenan was reconstructed using a combined Bayesian analysis of nuclear ITS, LSU rDNA and mitochondrial SSU rDNA sequences, and a maximum parsimony analysis was also made for comparison. 179 new partial sequences of 58 taxa were generated and 12 sequences were downloaded from GenBank. Our results indicate that the lichens containing Xanthoparmelia-type lichenan form a monophyletic group. However, the segregates proposed earlier do not form distinct clades within the group. Alternative hypotheses of monophyletic Karoowia and Neofuscelia that are not nested within Xanthoparmelia were rejected with our dataset; Karoowia is polyphyletic, and Neofuscelia is reduced to synonymy under Xanthoparmelia. Xanthomaculina convoluta also belongs to Xanthoparmelia. Since we were unable to sequence the umbilicate type species ofXanthomaculina, we refrain from synonymizing that genus with Xanthoparmelia here. The synonymy of Chondropsis and Paraparmelia under Xanthoparmelia already proposed is supported. The revised and enlarged genus Xanthoparmelia includes species that have cell walls with Xanthoparmelia-type lichenan, a palisade plectenchyma with a pored epicortex, lack pseudocyphellae, with usually simple rhizines, generally bifusiform conidia, and medullary chemical diversity. Ten new names are proposed, and 129 new combinations are made into Xanthoparmelia.

Genetic distances within and among species in monophyletic lineages of Parmeliaceae (Ascomycota) as a tool for taxon delimitation

The species delimitation in fungi is currently in flux. A growing body of evidence shows that the morphology-based species circumscription underestimates the number of existing species. The large and ever growing number of DNA sequence data of fungi makes it possible to use these to identify potential cases of hidden species, which then need to be studied with extensive taxon samplings. We used Parmeliaceae, one of the largest families of lichenized fungi as a model. Intra- and interspecific distances derived from maximum-likelihood phylogenetic trees inferred from 491 nuclear ITS rDNA sequences were examined for five major clades of parmelioid lichens. The intra- and interspecific distances were well separated in most cases allowing the calculation of a threshold, with exceptions of highly deviating distances in a few cases. These situations are shown to be taxa in which the current delimitation needs revision. Thus the analysis of the distance distributions is shown to be a powerful tool for identifying species complexes.

Phylogenetic relationships and species concepts in Parmelia s. str. ( Parmeliaceae ) inferred from nuclear ITS rDNA and β-tubulin sequences

The phylogenetic relationships of 16 species of Parmelia s. str. are presented based on sequences of nuITS rDNA from 56 specimens, and β-tubulin gene sequences from 29 collections. Parmelia serrana sp. nov. a Mediterranean species morphologically very close to P. saxatilis is described. Parmelia ernstiae is the sister-group to P. saxatilis s. str., and a further undescribed North American species of the P. saxatilis complex may require recognition. The isidiate P. squarrosa is closely allied to the sorediate P. sulcata , which is paraphyletic. Japanese samples of P. cochleata form a monophyletic group but too few collections of these were studied to reach firm conclusions regarding their relationships. An epitype is selected for the lectotype of Lichen saxatilis to unequivocally fix the application of that epithet.

Origin and Diversification of Major Clades in Parmelioid Lichens (Parmeliaceae, Ascomycota) during the Paleogene Inferred by Bayesian Analysis

There is a long-standing debate on the extent of vicariance and long-distance dispersal events to explain the current distribution of organisms, especially in those with small diaspores potentially prone to long-distance dispersal. Age estimates of clades play a crucial role in evaluating the impact of these processes. The aim of this study is to understand the evolutionary history of the largest clade of macrolichens, the parmelioid lichens (Parmeliaceae, Lecanoromycetes, Ascomycota) by dating the origin of the group and its major lineages. They have a worldwide distribution with centers of distribution in the Neo- and Paleotropics, and semi-arid subtropical regions of the Southern Hemisphere. Phylogenetic analyses were performed using DNA sequences of nuLSU and mtSSU rDNA, and the protein-coding RPB1 gene. The three DNA regions had different evolutionary rates: RPB1 gave a rate two to four times higher than nuLSU and mtSSU. Divergence times of the major clades were estimated with partitioned BEAST analyses allowing different rates for each DNA region and using a relaxed clock model. Three calibrations points were used to date the tree: an inferred age at the stem of Lecanoromycetes, and two dated fossils: Parmelia in the parmelioid group, and Alectoria. Palaeoclimatic conditions and the palaeogeological area cladogram were compared to the dated phylogeny of parmelioid. The parmelioid group diversified around the K/T boundary, and the major clades diverged during the Eocene and Oligocene. The radiation of the genera occurred through globally changing climatic condition of the early Oligocene, Miocene and early Pliocene. The estimated divergence times are consistent with long-distance dispersal events being the major factor to explain the biogeographical distribution patterns of Southern Hemisphere parmelioids, especially for Africa-Australia disjunctions, because the sequential break-up of Gondwana started much earlier than the origin of these clades. However, our data cannot reject vicariance to explain South America-Australia disjunctions.

Parmelia barrenoae, a new lichen species related to Parmelia sulcata (Parmeliaceae) based on molecular and morphological data

Parmelia barrenoae is described as new to science in the P. sulcata complex on the basis of morphological and molecular data. The new species is superficially similar to P. sulcata but differs in having simple rhizines whereas the other species of the complex have squarrose rhizines. Nuclear ITS rDNA and partial -tubulin gene sequences have been used as molecular markers. In the phylogenetic analysis, P. sulcata falls into four well supported clades, one of them corresponds to the morphotype that is described here as a new taxon. Six samples of the new taxon from different locations on the Iberian Peninsula form a strongly supported monophyletic group.

Remototrachyna, a newly recognized tropical lineage of lichens in the Hypotrachyna clade (Parmeliaceae, Ascomycota), originated in the Indian subcontinent

Biogeographical studies of lichens used to be complicated because of the large distribution ranges of many species. Molecular systematics has revitalized lichen biogeography by improving species delimitation and providing better information about species range limitations. This study focuses on the major clade of tropical parmelioid lichens, which share a chemical feature, the presence of isolichenan in the cell wall, and a morphological feature, microscopic pores in the uppermost layer. Our previous phylogenetic studies revealed that the largest genus in this clade, Hypotrachyna, is polyphyletic with a clade mainly distributed in South and East Asia clustering distant from the core of the genus. To divide the Hypotrachyna clade into monophyletic groups and to reevaluate morphological and chemical characters in a phylogenetic context, we sampled ITS, nuclear large subunit (nuLSU) and mitochondrial small subunit (mtSSU) rDNA sequences from 77 species. We are erecting the new genus Remototrachyna for a core group of 15 former Hypotrachyna species. The segregation of Remototrachyna from Hypotrachyna receives support from morphological and chemical data, as well from maximum parsimony, maximum likelihood, and Bayesian phylogenetic analyses of the DNA. We used a likelihood approach to study the geographic range evolution of Remototrachyna and Bulbothrix, which are sister groups. This analysis suggests that the ancestral range of Remototrachyna was restricted to India and that subsequent long-distance dispersal is responsible for the pantropical occurrence of two species of Remototrachyna.

rDNA ITS and β-tubulin gene sequence analyses reveal two monophyletic groups within the cosmopolitan lichen Parmelia saxatilis

A considerable number of species of lichen-forming fungi have wide geographical distributions, but studies of their genetic variability are minimal. ITS rDNA sequences of 32 populations of Parmelia saxatilis from five continents revealed two monophyletic groups. β-tublin gene sequences from a subset of nine collections supported these conclusions. While the number of collections sequenced is limited, one monophyletic group (the Atlantic Population. AtP) was recognized as occurring in Arctic and Antarctic regions and also included collections from more atlantic sites. Samples from more mesic environments in the Mediterranean region belonged to a second monophyletic group (the Mediterranean Population, MeP). In addition, four subgroups were distinguishable within the Atlantic Population. Norstictic and protocetraric acids are reported from the species for the first time, the norstictic acid only being found in the Atlantic Population. Living thalli from the Atlantic Population were provenance-tested; specimens transported from the UK to central Spain where the Mediterranean Population occurs showed adverse symptoms after six months. These results demonstrate that there can be substantial large-scale genotypic variability within widespread lichen phenospecies, something which has implications for comparative ecological, physiological, and air pollution sensitivity studies as well as for lichen conservation.

Accelerated evolutionary rates in tropical and oceanic parmelioid lichens (Ascomycota).

BackgroundThe rate of nucleotide substitutions is not constant across the Tree of Life, and departures from a molecular clock have been commonly reported. Within parmelioid lichens, the largest group of macrolichens, large discrepancies in branch lengths between clades were found in previous studies. Using an extended taxon sampling, we test for presence of significant rate discrepancies within and between these clades and test our a priori hypothesis that such rate discrepancies may be explained by shifts in moisture regime or other environmental conditions.ResultsIn this paper, the first statistical evidence for accelerated evolutionary rate in lichenized ascomycetes is presented. Our results give clear evidence for a faster rate of evolution in two Hypotrachyna clades that includes species occurring in tropical and oceanic habitats in comparison with clades consisting of species occurring in semi-arid and temperate habitats. Further we explore potential links between evolutionary rates and shifts in habitat by comparing alternative Ornstein-Uhlenbeck models.ConclusionAlthough there was only weak support for a shift at the base of a second tropical clade, where the observed nucleotide substitution rate is high, overall support for a shift in environmental conditions at cladogenesis is very strong. This suggests that speciation in some lichen clades has proceeded by dispersal into a novel environment, followed by radiation within that environment. We found moderate support for a shift in moisture regime at the base of one tropical clade and a clade occurring in semi-arid regions and a shift in minimum temperature at the base of a boreal-temperate clade.