Reference-Based Restriction-Site-Associated DNA Sequencing Data Are Useful for Species Delineation in a Recently Diverged Asexually Reproducing Species Complex (Parmeliaceae, Ascomycota).

Cryptic species are common in lichen-forming fungi and have been reported from different genera in the most speciose family, Parmeliaceae. Herein, we address species delimitation in a group of mainly asexually reproducing Parmelina species. The morphologically distinct P. pastillifera was previously found nested within a morphologically circumscribed P. tiliacea based on several loci. However, these studies demonstrated a relatively high genetic diversity within P. tiliacea sensu lato. Here, we revisit the species delimitation in the group by analyzing single-nucleotide polymorphisms (SNPs) through genome-wide assessment using Restriction-Site-Associated sequencing and population genomic methods. Our data support previous studies and provide further insight into the phylogenetic relationships of the four clades found within the complex. Based on the evidence suggesting a lack of gene flow among the clades, we recognize the four clades as distinct species, P. pastillifera and P. tiliacea sensu stricto, and two new species, P. clandestina sp. nov. and P. mediterranea sp. nov.

Molecular phylogeny and evolution of inflorescence types in Eperua.

PREMISE: The Amazonian hyperdominant genus Eperua (Fabaceae) currently holds 20 described species and has two strongly different inflorescence and flower types, with corresponding different pollination syndrome. The evolution of these vastly different inflorescence types within this genus was unknown and the main topic in this study. METHODS: We constructed a molecular phylogeny, based on the full nuclear ribosomal DNA and partial plastome, using Bayesian inference and maximum likelihood methods, to test whether the genus is monophyletic, whether all species are monophyletic and if the shift from bat to bee pollination (or vice versa) occurred once in this genus. RESULTS: All but two species are well supported by the nuclear ribosomal phylogeny. The plastome phylogeny, however, shows a strong geographic signal suggesting strong local hybridization or chloroplast capture, rendering chloroplast barcodes meaningless in this genus. CONCLUSIONS: With our data, we cannot fully resolve the backbone of the tree to clarify sister genera relationships and confirm monophyly of the genus Eperua. Within the genus, the shift from bat to bee and bee to bat pollination has occurred several times but, with the bee to bat not always leading to a pendant inflorescence.

Reference-Based RADseq Unravels the Evolutionary History of Polar Species in 'the Crux Lichenologorum' Genus Usnea (Parmeliaceae, Ascomycota).

Nearly 90% of fungal diversity, one of the most speciose branches in the tree of life, remains undescribed. Lichenized fungi as symbiotic associations are still a challenge for species delimitation, and current species diversity is vastly underestimated. The ongoing democratization of Next-Generation Sequencing is turning the tables. Particularly, reference-based RADseq allows for metagenomic filtering of the symbiont sequence and yields robust phylogenomic trees of closely related species. We implemented reference-based RADseq to disentangle the evolution of neuropogonoid lichens, which inhabit harsh environments and belong to Usnea (Parmeliaceae, Ascomycota), one of the most taxonomically intriguing genera within lichenized fungi. Full taxon coverage of neuropogonoid lichens was sampled for the first time, coupled with phenotype characterizations. More than 20,000 loci of 126 specimens were analyzed through concatenated and coalescent-based methods, including time calibrations. Our analysis addressed the major taxonomic discussions over recent decades. Subsequently, two species are newly described, namely U. aymondiana and U. fibriloides, and three species names are resurrected. The late Miocene and Pliocene-Pleistocene boundary is inferred as the timeframe for neuropogonoid lichen diversification. Ultimately, this study helped fill the gap of fungal diversity by setting a solid backbone phylogeny which raises new questions about which factors may trigger complex evolutionary scenarios.

Interpreting phylogenetic conflict: Hybridization in the most speciose genus of lichen-forming fungi.

While advances in sequencing technologies have been invaluable for understanding evolutionary relationships, increasingly large genomic data sets may result in conflicting evolutionary signals that are often caused by biological processes, including hybridization. Hybridization has been detected in a variety of organisms, influencing evolutionary processes such as generating reproductive barriers and mixing standing genetic variation. Here, we investigate the potential role of hybridization in the diversification of the most speciose genus of lichen-forming fungi, Xanthoparmelia. As Xanthoparmelia is projected to have gone through recent, rapid diversification, this genus is particularly suitable for investigating and interpreting the origins of phylogenomic conflict. Focusing on a clade of Xanthoparmelia largely restricted to the Holarctic region, we used a genome skimming approach to generate 962 single-copy gene regions representing over 2 Mbp of the mycobiont genome. From this genome-scale dataset, we inferred evolutionary relationships using both concatenation and coalescent-based species tree approaches. We also used three independent tests for hybridization. Although different species tree reconstruction methods recovered largely consistent and well-supported trees, there was widespread incongruence among individual gene trees. Despite challenges in differentiating hybridization from ILS in situations of recent rapid radiations, our genome-wide analyses detected multiple potential hybridization events in the Holarctic clade, suggesting one possible source of trait variability in this hyperdiverse genus. This study highlights the value in using a pluralistic approach for characterizing genome-scale conflict, even in groups with well-resolved phylogenies, while highlighting current challenges in detecting the specific impacts of hybridization.

Early stages of speciation with gene flow in the Amazilia Hummingbird (Amazilis amazilia) subspecies complex of Western South America.

Disentangling the factors underlying the diversification of geographically variable species with a wide geographical range is essential to understanding the initial stages and drivers of the speciation process. The Amazilia Hummingbird, Amazilis amazilia, is found along the Pacific coast from northern Ecuador down to the Nazca Valley of Peru, and is currently classified as six phenotypically differentiated subspecies. We aimed to resolve the evolutionary relationships of the six subspecies, to assess the geographical pattern and extent of evolutionary divergence, and to test for introgression using both a mtDNA marker and a genome-by-sequencing dataset from 86 individuals from across the species range. The consensus phylogenetic tree separated the six subspecies into three distinct clades, corresponding with the Ecuador lowlands (A. amazilia dumerilii), the Ecuador highlands (A. amazilia alticola and A. amazilia azuay), and the Peruvian coast (A. amazilia leucophoea, A. amazilia amazilia, and A. amazilia caeruleigularis). However, an unresolved mtDNA network suggests that the diversification of the subspecies was recent and rapid. We found evidence of gene flow among the subspecies A. amazilia dumerilii, A. amazilia alticola, and A. amazilia leucophoea, with strong genetic isolation of the subspecies A. amazilia azuay in the isolated Yunguilla Valley of Ecuador. Finally, environmental data from each subspecies' capture locations were concordant with the three distinct clades. Overall, our results suggest that both expansions into new habitats and geographic isolation shaped the present-day phylogeny and range of the A. amazilia subspecies, and that A. amazilia azuay may be genetically divergent enough to be considered a separate species.

Should we hail the Red King? Evolutionary consequences of a mutualistic lifestyle in genomes of lichenized ascomycetes.

The Red Queen dynamic is often brought into play for antagonistic relationships. However, the coevolutionary effects of mutualistic interactions, which predict slower evolution for interacting organisms (Red King), have been investigated to a lesser extent. Lichens are a stable, mutualistic relationship of fungi and cyanobacteria and/or algae, which originated several times independently during the evolution of fungi. Therefore, they represent a suitable system to investigate the coevolutionary effect of mutualism on the fungal genome. We measured substitution rates and selective pressure of about 2000 protein-coding genes (plus the rDNA region) in two different classes of Ascomycota, each consisting of closely related lineages of lichenized and non-lichenized fungi. Our results show that independent lichenized clades are characterized by significantly slower rates for both synonymous and non-synonymous substitutions. We hypothesize that this evolutionary pattern is connected to the lichen life cycle (longer generation time of lichenized fungi) rather than a result of different selection strengths, which is described as the main driver for the Red Kind dynamic. This first empirical evidence of slower evolution in lichens provides an important insight on how biotic cooperative interactions are able to shape the evolution of symbiotic organisms.

The genomic basis of army ant chemosensory adaptations.

The evolution of mass raiding has allowed army ants to become dominant arthropod predators in the tropics. Although a century of research has led to many discoveries about behavioural, morphological and physiological adaptations in army ants, almost nothing is known about the molecular basis of army ant biology. Here we report the genome of the iconic New World army ant Eciton burchellii, and show that it is unusually compact, with a reduced gene complement relative to other ants. In contrast to this overall reduction, a particular gene subfamily (9-exon ORs) expressed predominantly in female antennae is expanded. This subfamily has previously been linked to the recognition of hydrocarbons, key olfactory cues used in insect communication and prey discrimination. Confocal microscopy of the brain showed a corresponding expansion in a putative hydrocarbon response centre within the antennal lobe, while scanning electron microscopy of the antenna revealed a particularly high density of hydrocarbon-sensitive sensory hairs. E. burchellii shares these features with its predatory and more cryptic relative, the clonal raider ant. By integrating genomic, transcriptomic and anatomical analyses in a comparative context, our work thus provides evidence that army ants and their relatives possess a suite of modifications in the chemosensory system that may be involved in behavioural coordination and prey selection during social predation. It also lays the groundwork for future studies of army ant biology at the molecular level.

Museum genomics reveals the Xerces blue butterfly (Glaucopsyche xerces) was a distinct species driven to extinction.

The last Xerces blue butterfly was seen in the early 1940s, and its extinction is credited to human urban development. This butterfly has become a North American icon for insect conservation, but some have questioned whether it was truly a distinct species, or simply an isolated population of another living species. To address this question, we leveraged next-generation sequencing using a 93-year-old museum specimen. We applied a genome skimming strategy that aimed for the organellar genome and high-copy fractions of the nuclear genome by a shallow sequencing approach. From these data, we were able to recover over 200 million nucleotides, which assembled into several phylogenetically informative markers and the near-complete mitochondrial genome. From our phylogenetic analyses and haplotype network analysis we conclude that the Xerces blue butterfly was a distinct species driven to extinction.

Population genomics of a reindeer lichen species from North American lichen woodlands.

PREMISE: Lichens are one of the main structural components of plant communities in the North American boreal biome. They play a pivotal role in lichen woodlands, a large ecosystem situated north of the closed-crown forest zone, and south of the forest-tundra zone. In Eastern Canada (Quebec), there is a remnant LW found 500 km south of its usual distribution range, in the Parc National des Grands-Jardins, originated mainly because of wildfires. We inferred the origin of the lichen Cladonia stellaris from this LW and assessed its genetic diversity in a postfire succession. METHODS: We genotyped 122 individuals collected across a latitudinal gradient in Quebec. Using the software Stacks, we compared four different approaches of locus selection and single-nucleotide polymorphism calling. We identified the best fitting approach to investigate population structure and estimate genetic diversity of C. stellaris. RESULTS: Populations in southern Quebec are not genetically different from those of northern LWs. The species consists of at least four phylogenetic lineages with elevated levels of genetic diversity and low co-ancestry. In Parc National des Grands-Jardins, we reported high values of genetic diversity not related with time since fire disturbance and low genetic differentiation among populations with different fire histories. CONCLUSIONS: This first population genomic study of C. stellaris is an important step forward to understand the origin and biogeographic patterns of lichen woodlands in North America. Our findings also contribute to the understanding of the effect of postfire succession on the genetic structure of the species.

Species boundaries in the messy middle-A genome-scale validation of species delimitation in a recently diverged lineage of coastal fog desert lichen fungi.

Species delimitation among closely related species is challenging because traditional phenotype-based approaches, for example, using morphology, ecological, or chemical characteristics, may not coincide with natural groupings. With the advent of high-throughput sequencing, it has become increasingly cost-effective to acquire genome-scale data which can resolve previously ambiguous species boundaries. As the availability of genome-scale data has increased, numerous species delimitation analyses, such as BPP and SNAPP+Bayes factor delimitation (BFD*), have been developed to delimit species boundaries. However, even empirical molecular species delimitation approaches can be biased by confounding evolutionary factors, for example, hybridization/introgression and incomplete lineage sorting, and computational limitations. Here, we investigate species boundaries and the potential for micro-endemism in a lineage of lichen-forming fungi, Niebla Rundel & Bowler, in the family Ramalinaceae by analyzing single-locus and genome-scale data consisting of (a) single-locus species delimitation analysis using ASAP, (b) maximum likelihood-based phylogenetic tree inference, (c) genome-scale species delimitation models, e.g., BPP and SNAPP+BFD, and (d) species validation using the genealogical divergence index (gdi). We specifically use these methods to cross-validate results between genome-scale and single-locus datasets, differently sampled subsets of genomic data and to control for population-level genetic divergence. Our species delimitation models tend to support more speciose groupings that were inconsistent with traditional taxonomy, supporting a hypothesis of micro-endemism, which may include morphologically cryptic species. However, the models did not converge on robust, consistent species delimitations. While the results of our analysis are somewhat ambiguous in terms of species boundaries, they provide a valuable perspective on how to use these empirical species delimitation methods in a nonmodel system. This study thus highlights the challenges inherent in delimiting species, particularly in groups such as Niebla, with complex, relatively recent phylogeographic histories.

Using target enrichment sequencing to study the higher-level phylogeny of the largest lichen-forming fungi family: Parmeliaceae (Ascomycota).

Parmeliaceae is the largest family of lichen-forming fungi with a worldwide distribution. We used a target enrichment data set and a qualitative selection method for 250 out of 350 genes to infer the phylogeny of the major clades in this family including 81 taxa, with both subfamilies and all seven major clades previously recognized in the subfamily Parmelioideae. The reduced genome-scale data set was analyzed using concatenated-based Bayesian inference and two different Maximum Likelihood analyses, and a coalescent-based species tree method. The resulting topology was strongly supported with the majority of nodes being fully supported in all three concatenated-based analyses. The two subfamilies and each of the seven major clades in Parmelioideae were strongly supported as monophyletic. In addition, most backbone relationships in the topology were recovered with high nodal support. The genus Parmotrema was found to be polyphyletic and consequently, it is suggested to accept the genus Crespoa to accommodate the species previously placed in Parmotrema subgen. Crespoa. This study demonstrates the power of reduced genome-scale data sets to resolve phylogenetic relationships with high support. Due to lower costs, target enrichment methods provide a promising avenue for phylogenetic studies including larger taxonomic/specimen sampling than whole genome data would allow.

Genome-Wide Analysis of Biosynthetic Gene Cluster Reveals Correlated Gene Loss with Absence of Usnic Acid in Lichen-Forming Fungi.

Lichen-forming fungi are known to produce a large number of secondary metabolites. Some metabolites are deposited in the cortical layer of the lichen thallus where they exert important ecological functions, such as UV filtering. The fact that closely related lineages of lichen-forming fungi can differ in cortical chemistry suggests that natural product biosynthesis in lichens can evolve independent from phylogenetic constraints. Usnic acid is one of the major cortical pigments in lichens. Here we used a comparative genomic approach on 46 lichen-forming fungal species of the Lecanoromycetes to elucidate the biosynthetic gene content and evolution of the gene cluster putatively responsible for the biosynthesis of usnic acid. Whole-genome sequences were gathered from taxa belonging to different orders and families of Lecanoromycetes, where Parmeliaceae is the most well-represented taxon, and analyzed with a variety of genomic tools. The highest number of biosynthetic gene clusters was found in Evernia prunastri, Pannoparmelia angustata, and Parmotrema austrosinense, respectively, and lowest in Canoparmelia nairobiensis, Bulbothrix sensibilis, and Hypotrachyna scytodes. We found that all studied species producing usnic acid contain the putative usnic acid biosynthetic gene cluster, whereas the cluster was absent in all genomes of species lacking usnic acid. The absence of the gene cluster was supported by an additional unsuccessful search for ß-ketoacylsynthase, the most conserved domain of the gene cluster, in the genomes of species lacking usnic acid. The domain architecture of this PKS cluster-homologous to the already known usnic acid PKS cluster (MPAS) and CYT450 (MPAO)-varies within the studied species, whereas the gene arrangement is highly similar in closely related taxa. We hypothesize that the ancestor of these lichen-forming fungi contained the putative usnic acid producing PKS cluster and that the gene cluster was lost repeatedly during the evolution of these groups. Our study provides insight into the genomic adaptations to the evolutionary success of these lichen-forming fungal species and sets a baseline for further exploration of biosynthetic gene content and its evolutionary significance.

IMA Genome - F13: Draft genome sequences of Ambrosiella cleistominuta, Cercospora brassicicola, C. citrullina, Physcia stellaris, and Teratosphaeria pseudoeucalypti.

Draft genomes of the fungal species Ambrosiella cleistominuta, Cercospora brassicicola, C. citrullina, Physcia stellaris, and Teratosphaeria pseudoeucalypti are presented. Physcia stellaris is an important lichen forming fungus and Ambrosiella cleistominuta is an ambrosia beetle symbiont. Cercospora brassicicola and C. citrullina are agriculturally relevant plant pathogens that cause leaf-spots in brassicaceous vegetables and cucurbits respectively. Teratosphaeria pseudoeucalypti causes severe leaf blight and defoliation of Eucalyptus trees. These genomes provide a valuable resource for understanding the molecular processes in these economically important fungi.

Genome-scale data reveal the role of hybridization in lichen-forming fungi.

Advancements in molecular genetics have revealed that hybridization may be common among plants, animals, and fungi, playing a role in evolutionary dynamics and speciation. While hybridization has been well-documented in pathogenic fungi, the effects of these processes on speciation in fungal lineages with different life histories and ecological niches are largely unexplored. Here we investigated the potential influence of hybridization on the emergence of morphologically and reproductively distinct asexual lichens. We focused on vagrant forms (growing obligately unattached to substrates) within a clade of rock-dwelling, sexually reproducing species in the Rhizoplaca melanophthalma (Lecanoraceae, Ascomycota) species complex. We used phylogenomic data from both mitochondrial and nuclear genomes to infer evolutionary relationships and potential patterns of introgression. We observed multiple instances of discordance between the mitochondrial and nuclear trees, including the clade comprising the asexual vagrant species R. arbuscula, R. haydenii, R. idahoensis, and a closely related rock-dwelling lineage. Despite well-supported phylogenies, we recovered strong evidence of a reticulated evolutionary history using a network approach that incorporates both incomplete lineage sorting and hybridization. These data suggest that the rock-dwelling western North American subalpine endemic R. shushanii is potentially the result of a hybrid speciation event, and introgression may have also played a role in other taxa, including vagrant species R. arbuscula, R. haydenii and R. idahoensis. We discuss the potential roles of hybridization in terms of generating asexuality and novel morphological traits in lichens. Furthermore, our results highlight the need for additional study of reticulate phylogenies when investigating species boundaries and evolutionary history, even in cases with well-supported topologies inferred from genome-scale data.

Characterizing the ribosomal tandem repeat and its utility as a DNA barcode in lichen-forming fungi.

BACKGROUND: Regions within the nuclear ribosomal operon are a major tool for inferring evolutionary relationships and investigating diversity in fungi. In spite of the prevalent use of ribosomal markers in fungal research, central features of nuclear ribosomal DNA (nrDNA) evolution are poorly characterized for fungi in general, including lichenized fungi. The internal transcribed spacer (ITS) region of the nrDNA has been adopted as the primary DNA barcode identification marker for fungi. However, little is known about intragenomic variation in the nrDNA in symbiotic fungi. In order to better understand evolution of nrDNA and the utility of the ITS region for barcode identification of lichen-forming fungal species, we generated nearly complete nuclear ribosomal operon sequences from nine species in the Rhizoplaca melanophthalma species complex using short reads from high-throughput sequencing. RESULTS: We estimated copy numbers for the nrDNA operon, ranging from nine to 48 copies for members of this complex, and found low levels of intragenomic variation in the standard barcode region (ITS). Monophyly of currently described species in this complex was supported in phylogenetic inferences based on the ITS, 28S, intergenic spacer region, and some intronic regions, independently; however, a phylogenetic inference based on the 18S provided much lower resolution. Phylogenetic analysis of concatenated ITS and intergenic spacer sequence data generated from 496 specimens collected worldwide revealed previously unrecognized lineages in the nrDNA phylogeny. CONCLUSIONS: The results from our study support the general assumption that the ITS region of the nrDNA is an effective barcoding marker for fungi. For the R. melanophthalma group, the limited amount of potential intragenomic variability in the ITS region did not correspond to fixed diagnostic nucleotide position characters separating taxa within this species complex. Previously unrecognized lineages inferred from ITS sequence data may represent undescribed species-level lineages or reflect uncharacterized aspects of nrDNA evolution in the R. melanophthalma species complex.

Multiple historical processes obscure phylogenetic relationships in a taxonomically difficult group (Lobariaceae, Ascomycota).

In the age of next-generation sequencing, the number of loci available for phylogenetic analyses has increased by orders of magnitude. But despite this dramatic increase in the amount of data, some phylogenomic studies have revealed rampant gene-tree discordance that can be caused by many historical processes, such as rapid diversification, gene duplication, or reticulate evolution. We used a target enrichment approach to sample 400 single-copy nuclear genes and estimate the phylogenetic relationships of 13 genera in the lichen-forming family Lobariaceae to address the effect of data type (nucleotides and amino acids) and phylogenetic reconstruction method (concatenation and species tree approaches). Furthermore, we examined datasets for evidence of historical processes, such as rapid diversification and reticulate evolution. We found incongruence associated with sequence data types (nucleotide vs. amino acid sequences) and with different methods of phylogenetic reconstruction (species tree vs. concatenation). The resulting phylogenetic trees provided evidence for rapid and reticulate evolution based on extremely short branches in the backbone of the phylogenies. The observed rapid and reticulate diversifications may explain conflicts among gene trees and the challenges to resolving evolutionary relationships. Based on divergence times, the diversification at the backbone occurred near the Cretaceous-Paleogene (K-Pg) boundary (65 Mya) which is consistent with other rapid diversifications in the tree of life. Although some phylogenetic relationships within the Lobariaceae family remain with low support, even with our powerful phylogenomic dataset of up to 376 genes, our use of target-capturing data allowed for the novel exploration of the mechanisms underlying phylogenetic and systematic incongruence.

Genome-scale data resolve ancestral rock-inhabiting lifestyle in Dothideomycetes (Ascomycota).

Dothideomycetes is the most diverse fungal class in Ascomycota and includes species with a wide range of lifestyles. Previous multilocus studies have investigated the taxonomic and evolutionary relationships of these taxa but often failed to resolve early diverging nodes and frequently generated inconsistent placements of some clades. Here, we use a phylogenomic approach to resolve relationships in Dothideomycetes, focusing on two genera of melanized, extremotolerant rock-inhabiting fungi, Lichenothelia and Saxomyces, that have been suggested to be early diverging lineages. We assembled phylogenomic datasets from newly sequenced (4) and previously available genomes (238) of 242 taxa. We explored the influence of tree inference methods, supermatrix vs. coalescent-based species tree, and the impact of varying amounts of genomic data. Overall, our phylogenetic reconstructions provide consistent and well-supported topologies for Dothideomycetes, recovering Lichenothelia and Saxomyces among the earliest diverging lineages in the class. In addition, many of the major lineages within Dothideomycetes are recovered as monophyletic, and the phylogenomic approach implemented strongly supports their relationships. Ancestral character state reconstruction suggest that the rock-inhabiting lifestyle is ancestral within the class.

Lycophyte plastid genomics: extreme variation in GC, gene and intron content and multiple inversions between a direct and inverted orientation of the rRNA repeat.

Lycophytes are a key group for understanding vascular plant evolution. Lycophyte plastomes are highly distinct, indicating a dynamic evolutionary history, but detailed evaluation is hindered by the limited availability of sequences. Eight diverse plastomes were sequenced to assess variation in structure and functional content across lycophytes. Lycopodiaceae plastomes have remained largely unchanged compared with the common ancestor of land plants, whereas plastome evolution in Isoetes and especially Selaginella is highly dynamic. Selaginella plastomes have the highest GC content and fewest genes and introns of any photosynthetic land plant. Uniquely, the canonical inverted repeat was converted into a direct repeat (DR) via large-scale inversion in some Selaginella species. Ancestral reconstruction identified additional putative transitions between an inverted and DR orientation in Selaginella and Isoetes plastomes. A DR orientation does not disrupt the activity of copy-dependent repair to suppress substitution rates within repeats. Lycophyte plastomes include the most archaic examples among vascular plants and the most reconfigured among land plants. These evolutionary trends correlate with the mitochondrial genome, suggesting shared underlying mechanisms. Copy-dependent repair for DR-localized genes indicates that recombination and gene conversion are not inhibited by the DR orientation. Gene relocation in lycophyte plastomes occurs via overlapping inversions rather than transposase/recombinase-mediated processes.

Population genomic analyses of RAD sequences resolves the phylogenetic relationship of the lichen-forming fungal species Usneaantarctica and Usneaaurantiacoatra.

Neuropogonoid species in the lichen-forming fungal genus Usnea exhibit great morphological variation that can be misleading for delimitation of species. We specifically focused on the species delimitation of two closely-related, predominantly Antarctic species differing in the reproductive mode and representing a so-called species pair: the asexual U.antarctica and the sexual U.aurantiacoatra. Previous studies have revealed contradicting results. While multi-locus studies based on DNA sequence data provided evidence that these two taxa might be conspecific, microsatellite data suggested they represent distinct lineages. By using RADseq, we generated thousands of homologous markers to build a robust phylogeny of the two species. Furthermore, we successfully implemented these data in fine-scale population genomic analyses such as DAPC and fineRADstructure. Both Usnea species are readily delimited in phylogenetic inferences and, therefore, the hypothesis that both species are conspecific was rejected. Population genomic analyses also strongly confirmed separated genomes and, additionally, showed different levels of co-ancestry and substructure within each species. Lower co-ancestry in the asexual U.antarctica than in the sexual U.aurantiacoatra may be derived from a wider distributional range of the former species. Our results demonstrate the utility of this RADseq method in tracing population dynamics of lichens in future analyses.