Mitogenomic phylogeny, biogeography, and cryptic divergence of the genus Silurus (Siluriformes: Siluridae).

The genus Silurus, an important group of catfish, exhibits heterogeneous distribution in Eurasian freshwater systems. This group includes economically important and endangered species, thereby attracting considerable scientific interest. Despite this interest, the lack of a comprehensive phylogenetic framework impedes our understanding of the mechanisms underlying the extensive diversity found within this genus. Herein, we analyzed 89 newly sequenced and 20 previously published mitochondrial genomes (mitogenomes) from 13 morphological species to reconstruct the phylogenetic relationships, biogeographic history, and species diversity of Silurus. Our phylogenetic reconstructions identified eight clades, supported by both maximum-likelihood and Bayesian inference. Sequence-based species delimitation analyses yielded multiple molecular operational taxonomic units (MOTUs) in several taxa, including the Silurus asotus complex (four MOTUs) and Silurus microdorsalis (two MOTUs), suggesting that species diversity is underestimated in the genus. A reconstructed time-calibrated tree of Silurus species provided an age estimate of the most recent common ancestor of approximately 37.61 million years ago (Ma), with divergences among clades within the genus occurring between 11.56 Ma and 29.44 Ma, and divergences among MOTUs within species occurring between 3.71 Ma and 11.56 Ma. Biogeographic reconstructions suggested that the ancestral area for the genus likely encompassed China and the Korean Peninsula, with multiple inferred dispersal events to Europe and Central and Western Asia between 21.78 Ma and 26.67 Ma and to Japan between 2.51 Ma and 18.42 Ma. Key factors such as the Eocene-Oligocene extinction event, onset and intensification of the monsoon system, and glacial cycles associated with sea-level fluctuations have likely played significant roles in shaping the evolutionary history of the genus Silurus.

Exploring hidden diversity: Molecular insights into the Leporinus species of the rivers of the Brazilian states of Maranhão and Piauí.

The present study delved into the world of hidden diversity by examining specimens identified as Leporinus piau from the river basins of the northern Brazilian states of Maranhão and Piauí. Using genetic analyses that combined data from three mitochondrial markers and one nuclear marker, the study identified two well-supported groups, reinforcing the findings of previous publications. The first group, found in samples from the Itapecuru, Mearim, Turiaçu, and Pericumã basins, in Maranhão, appears to represent a relatively ancient diversification and the possibility of concealed cryptic diversity. The second group, comprising specimens from the Parnaíba (Piauí) and Mearim (Maranhão) basins, appears to have resulted from a more recent process of diversification and has a close relationship with Leporinus friderici from the type locality. Our findings not only confirm the existence of a complex scenario of cryptic diversity in the genus Leporinus from the study basins but also underscore the taxonomic inconsistencies within this group of fish. This study offers a comprehensive analysis of the species diversity of the Maranhão and Piauí basins, which are critical regions for the conservation of Amazonian fish, providing valuable insights for the sustainable management and conservation of these fish.

Identification of fish specimens of the Tocantins River, Brazil, using DNA barcoding.

The fish fauna of the Tocantins River possesses many endemic species; however, it is little studied in molecular terms and is quite threatened by the construction of several hydroelectric dams. Therefore, the objective of this study was to identify the ichthyofauna of the Tocantins River using DNA barcoding. For this, collections were carried out in five points of this river, which resulted in the capture of 725 individuals from which partial sequences of the cytochrome oxidase subunit I (COI) gene were obtained for genetic analysis. A total of 443 haplotypes were recovered with the mean intraspecific K2P genetic distance of 1.82%. Altogether, 138 species were identified based on morphological criteria, which was a quantity that was much lower than that indicated by the four molecular methods (assemble species by automatic partitioning [ASAP], barcode index number [BIN], generalized mixed Yule coalescent (GMYC), and Bayesian Poisson tree processes [bPTP]) through which 152-157 molecular entities were identified. In all, 41 unique BINs were obtained based on the data generated in the BOLDSystems platform. According to the result indicated by ASAP (species delimitation approach considered the most appropriate in the present study), there was an increase of 17 molecular entities (12.32%), when compared to the number of species identified through their morphological criteria, as it can show cryptic diversity, candidates for new species, and misidentifications. There were 21 incongruities indicated between the different identification approaches for species. Therefore, it is suggested that these taxonomic problems be cautiously evaluated by experts to solve such taxonomic issues.

The global genetic diversity of planktonic foraminifera reveals the structure of cryptic speciation in plankton.

The nature and extent of diversity in the plankton has fascinated scientists for over a century. Initially, the discovery of many new species in the remarkably uniform and unstructured pelagic environment appeared to challenge the concept of ecological niches. Later, it became obvious that only a fraction of plankton diversity had been formally described, because plankton assemblages are dominated by understudied eukaryotic lineages with small size that lack clearly distinguishable morphological features. The high diversity of the plankton has been confirmed by comprehensive metabarcoding surveys, but interpretation of the underlying molecular taxonomies is hindered by insufficient integration of genetic diversity with morphological taxonomy and ecological observations. Here we use planktonic foraminifera as a study model and reveal the full extent of their genetic diversity and investigate geographical and ecological patterns in their distribution. To this end, we assembled a global data set of ~7600 ribosomal DNA sequences obtained from morphologically characterised individual foraminifera, established a robust molecular taxonomic framework for the observed diversity, and used it to query a global metabarcoding data set covering ~1700 samples with ~2.48 billion reads. This allowed us to extract and assign 1 million reads, enabling characterisation of the structure of the genetic diversity of the group across ~1100 oceanic stations worldwide. Our sampling revealed the existence of, at most, 94 distinct molecular operational taxonomic units (MOTUs) at a level of divergence indicative of biological species. The genetic diversity only doubles the number of formally described species identified by morphological features. Furthermore, we observed that the allocation of genetic diversity to morphospecies is uneven. Only 16 morphospecies disguise evolutionarily significant genetic diversity, and the proportion of morphospecies that show genetic diversity increases poleward. Finally, we observe that MOTUs have a narrower geographic distribution than morphospecies and that in some cases the MOTUs belonging to the same morphospecies (cryptic species) have different environmental preferences. Overall, our analysis reveals that even in the light of global genetic sampling, planktonic foraminifera diversity is modest and finite. However, the extent and structure of the cryptic diversity reveals that genetic diversification is decoupled from morphological diversification, hinting at different mechanisms acting at different levels of divergence.

Integrative Taxonomy Reveals Hidden Diversity in the Aloina catillum Complex (Pottiaceae, Bryophyta).

Aloina catillum is a variable moss typical of xerophytic environments in the Neotropics, characterized against other closely allied Aloina species with well-differentiated leaf border by its setae twisted to the left throughout. In order to clarify its variability and its relationships with the allied species with differentiated leaf border A. brevirostris, A. obliquifolia, and A. rigida, we performed an integrative study including sequence data from four markers (nuclear ITS, plastid atpB-rbcL, trnG, trnL-F), morphometry, and species assembling by automatic partitioning (ASAP) algorithm. Our data suggest that A. catillum consists of at least three species: A. calceolifolia (an earlier name for A. catillum), and two species described here as a new, A. bracteata sp. nov. and A. limbata sp. nov. This latter species includes the specimens previously identified as A. obliquifolia from South America. Additionally, some morphological and molecular variability was also detected in A. limbata, but was not consistent enough to be recognized taxonomically. The study supports the presence of A. brevirostris in the Neotropics and A. rigida is tentatively excluded from South America. Full descriptions of the A. catillum s.l. species and a diagnostic key to this complex in South America are provided.

Microgeographic speciation in a complex of Anatolian bush crickets facilitated by fast evolution of reproductive isolation.

Identifying the drivers of microgeographic speciation (i.e., speciation over small, local geographic scales) is key to understand the origin of speciose groups. Here, we use genomic data to infer the demographic processes underlying diversification in Poecilimon luschani (Orthoptera: Tettigoniidae), a species complex belonging to the most diverse genus of bush crickets from the Mediterranean region (>170 taxa) that comprises three recognized subspecies with small allopatric distributions in the topographically complex Teke Peninsula, southwestern Anatolia. Phylogenomic reconstructions that include all other taxa within the species group confirmed that subspecies of P. luschani originated from a common ancestor during the Pleistocene, supporting recent (<1 Ma) diversification within a small geographical area (ca. 120 × 80 km). Genetic clustering analyses corroborated the distinctiveness of each subspecies and the cohesiveness of their respective populations, with abrupt genetic discontinuities coinciding with contemporary range boundaries. Indeed, our analyses uncovered the presence of two sympatric cryptic sister lineages that diverged <300 ka ago and do not admix despite being co-distributed. Collectively, these results support that all lineages within the complex represent independently evolving entities corresponding to full-fledged species. Statistical evaluation of alternative models of speciation strongly supports a scenario of divergence in isolation followed by a period of limited gene flow during the last glacial period, when all lineages experienced marked expansions according to demographic reconstructions. Our study exemplifies how localized allopatric divergence and fast evolution of reproductive isolation can promote microgeographic speciation and explain the high rates of endemism characterizing biodiversity hotspots.

Resolving the taxonomy of the Polysiphonia scopulorum complex and the Bryocladia lineage (Rhodomelaceae, Rhodophyta).

Cryptic diversity is common among marine macroalgae, with molecular tools leading to the discovery of many new species. To assign names to these morphologically similar species, the type and synonyms have to be examined, and if appropriate, new species must be described. The turf-forming red alga Polysiphonia scopulorum was originally described from Rottnest Island, Australia, and subsequently widely reported in tropical and temperate coasts based on morphological identifications. A recent study of molecular species delineation revealed a complex of 12 species in Australia, South Africa, and Europe. These species are placed in a taxonomically unresolved lineage of the tribe Polysiphonieae. The aim of this study was to resolve the genus- and species-level taxonomy of this complex and related species using molecular and morphological information. Three morphologically indistinguishable species of the complex were found at the type locality of P. scopulorum, preventing a straightforward assignment of the name to any of the molecular lineages. Therefore, we propose a molecularly characterized epitype. Polysiphonia caespitosa is reinstated for the only species found in its type locality in South Africa. We describe seven new species. Only one species of the complex can be morphologically recognized, with the other eight species indistinguishable based on morphometric analysis. The studied complex, together with another seven species currently placed in Polysiphonia and two Bryocladia species, formed a clade distinct from Polysiphonia sensu stricto. Based on observations of Bryocladia cervicornis (the generitype), we describe our seven new species in the genus Bryocladia and transfer another nine species from Polysiphonia to Bryocladia.

Amblyceps crassioris, a new sisoroid catfish from Odisha, India (Siluriformes: Amblycipitidae).

Amblyceps crassioris, a new species of amblycipitid catfish, is described from the Mahanadi River basin in Odisha, India. The new species can be distinguished from its congeners in having a combination of the following characters: a deeply forked caudal fin, centrally projecting hooks on proximal lepidotrichia of median caudal-fin rays absent, jaws equal in length, lateral line absent, body depth at anus 15.1%-19.5% standard length (SL), caudal peduncle depth 13.0%-18.3% SL, adipose-fin base length 21.1%-27.1% SL, eye diameter 7.35%-14.1% head length and 38 total vertebrae.

New species based on the biological species concept within the complex of Lariophagus distinguendus (Hymenoptera, Chalcidoidea, Pteromalidae), a parasitoid of household pests.

The pteromalid parasitoid Lariophagus distinguendus (Foerster) belongs to the Hymenoptera, a megadiverse insect order with high cryptic diversity. It attacks stored product pest beetles in human storage facilities. Recently, it has been shown to consist of two separate species. To further study its cryptic diversity, strains were collected to compare their relatedness using barcoding and nuclear genes. Nuclear genes identified two clusters which agree with the known two species, whereas the barcode fragment determined an additional third Clade. Total reproductive isolation (RI) according to the biological species concept (BSC) was investigated in crossing experiments within and between clusters using representative strains. Sexual isolation exists between all studied pairs, increasing from slight to strong with genetic distance. Postzygotic barriers mostly affected hybrid males, pointing to Haldane's rule. Hybrid females were only affected by unidirectional Spiroplasma-induced cytoplasmic incompatibility and behavioural sterility, each in one specific strain combination. RI was virtually absent between strains separated by up to 2.8% COI difference, but strong or complete in three pairs from one Clade each, separated by at least 7.2%. Apparently, each of these clusters represents one separate species according to the BSC, highlighting cryptic diversity in direct vicinity to humans. In addition, these results challenge the recent 'turbo-taxonomy' practice of using 2% COI differences to delimitate species, especially within parasitic Hymenoptera. The gradual increase in number and strength of reproductive barriers between strains with increasing genetic distance also sheds light on the emergence of barriers during the speciation process in L. distinguendus.

Ghost introgression facilitates genomic divergence of a sympatric cryptic lineage in Cycas revoluta.

A cryptic lineage is a genetically diverged but morphologically unrecognized variant of a known species. Clarifying cryptic lineage evolution is essential for quantifying species diversity. In sympatric cryptic lineage divergence compared with allopatric divergence, the forces of divergent selection and mating patterns override geographical isolation. Introgression, by supplying preadapted or neutral standing genetic variations, can promote sympatric cryptic lineage divergence via selection. However, most studies concentrated on extant species introgression, ignoring the genetic legacy of introgression from extinct or unsampled lineages ("ghost introgression"). Cycads are an ideal plant for studying the influence of ghost introgression because of their common interspecific gene flow and past high extinction rate. Here, we utilized reference-based ddRADseq to clarify the role of ghost introgression in the evolution of a previously identified sympatric cryptic lineage in Cycas revoluta. After re-evaluating the evolutionary independency of cryptic lineages, the group-wise diverged single-nucleotide polymorphisms among sympatric and allopatric lineages were compared and functionally annotated. Next, we employed an approximate Bayesian computation method for hypothesis testing to clarify the cryptic lineage evolution and ghost introgression effect. SNPs with the genomic signatures of ghost introgression were further annotated. Our results reconfirmed the evolutionary independency of cryptic lineage among C. revoluta and demonstrated that ghost introgression to the noncryptic lineage facilitated their divergence. Gene function related to heat stress and disease resistance implied ecological adaptation of the main extant populations of C. revoluta.

The Fergana Valley Is an Isolate of Biodiversity: A Discussion of the Endemic Herpetofauna and Description of Two New Species of Alsophylax (Sauria: Gekkonidae) from Eastern Uzbekistan.

The high level of endemism in Fergana Valley has been well documented in numerous studies for various groups of animals and plants. In a relatively small area, there are 45 endemic plant species, five endemic insect species, and five endemic reptile species. In surveying this area for data on distribution, abundance, acoustics, and genetic samples for species of reptiles, we discovered two new species of gecko from the genus Alsophylax. Phylogenetic analyses of mitochondrial DNA sequences indicate the relatives of these new species are the even-fingered gecko, Alsophylax pipiens, and the southern even-fingered gecko, Alsophylax laevis, located hundreds of kilometers to the northwest and southwest of the Fergana Valley. The threats to these new endemic species are significant given the amount of continued agricultural development that involves new territories previously considered "unsuitable" for any species of significance that is leading to the further reduction in, fragmentation of, and degradation of the remaining natural ecosystems in the Fergana Valley. The conservation of these rare and locally endemic species depends directly on the readiness of the state to create areas with IUCN I and II protection. The many studies documenting levels of endemism, along with the data published in this study, are the basis for the justification for state-protected areas in the Fergana Valley.

Cryptic Species in Ecotoxicology.

The advent of genetic methods has led to the discovery of an increasing number of species that previously could not be distinguished from each other on the basis of morphological characteristics. Even though there has been an exponential growth of publications on cryptic species, such species are rarely considered in ecotoxicology. Thus, the particular question of ecological differentiation and the sensitivity of closely related cryptic species is rarely addressed. Tackling this question, however, is of key importance for evolutionary ecology, conservation biology, and, in particular, regulatory ecotoxicology. At the same time, the use of species with (known or unknown) cryptic diversity might be a reason for the lack of reproducibility of ecotoxicological experiments and implies a false extrapolation of the findings. Our critical review includes a database and literature search through which we investigated how many of the species most frequently used in ecotoxicological assessments show evidence of cryptic diversity. We found a high proportion of reports indicating overlooked species diversity, especially in invertebrates. In terrestrial and aquatic realms, at least 67% and 54% of commonly used species, respectively, were identified as cryptic species complexes. The issue is less prominent in vertebrates, in which we found evidence for cryptic species complexes in 27% of aquatic and 6.7% of terrestrial vertebrates. We further exemplified why different evolutionary histories may significantly determine cryptic species' ecology and sensitivity to pollutants. This in turn may have a major impact on the results of ecotoxicological tests and, consequently, the outcome of environmental risk assessments. Finally, we provide a brief guideline on how to deal practically with cryptic diversity in ecotoxicological studies in general and its implementation in risk assessment procedures in particular. Environ Toxicol Chem 2023;42:1889-1914. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Evidence using morphology, molecules, and biogeography clarifies the taxonomic status of mole crabs of the genus Emerita Scopoli, 1777 (Anomura, Hippidae) and reveals a new species from the western Atlantic.

Uncertainties regarding the taxonomic status and biogeographical distribution of some species of the genus Emerita from the western Atlantic led to thorough examination of the subtle morphological differences between two coexistent species (E.brasiliensis Schmitt, 1935 and E.portoricensis Schmitt, 1935) along the Brazilian coast and compare them using two genetic markers. The molecular phylogenetic analysis based on sequences of the 16S rRNA and COI genes showed that individuals identified as E.portoricensis were clustered into two clades: one containing representatives from the Brazilian coast and another containing specimens distributed in Central America. Our molecular-based phylogeny, combined with a detailed morphological analysis, revealed the Brazilian population as a new species, which is described here as Emeritaalmeidai Mantelatto & Balbino, sp. nov. The number of species in the genus Emerita is now raised to 12, with five of them occurring in the western Atlantic, five in the Indo-Pacific, and two in the eastern Pacific.

MHC class II genes mediate susceptibility and resistance to coronavirus infections in bats.

Understanding the immunogenetic basis of coronavirus (CoV) susceptibility in major pathogen reservoirs, such as bats, is central to inferring their zoonotic potential. Members of the cryptic Hipposideros bat species complex differ in CoV susceptibility, but the underlying mechanisms remain unclear. The genes of the major histocompatibility complex (MHC) are the best understood genetic basis of pathogen resistance, and differences in MHC diversity are one possible reason for asymmetrical infection patterns among closely related species. Here, we aimed to link asymmetries in observed CoV (CoV-229E, CoV-2B and CoV-2Bbasal) susceptibility to immunogenetic differences amongst four Hipposideros bat species. From the 2072 bats assigned to their respective species using the mtDNA cytochrome b gene, members of the most numerous and ubiquitous species, Hipposideros caffer D, were most infected with CoV-229E and SARS-related CoV-2B. Using a subset of 569 bats, we determined that much of the existent allelic and functional (i.e. supertype) MHC DRB class II diversity originated from common ancestry. One MHC supertype shared amongst all species, ST12, was consistently linked to susceptibility with CoV-229E, which is closely related to the common cold agent HCoV-229E, and infected bats and those carrying ST12 had a lower body condition. The same MHC supertype was connected to resistance to CoV-2B, and bats with ST12 were less likely be co-infected with CoV-229E and CoV-2B. Our work suggests a role of immunogenetics in determining CoV susceptibility in bats. We advocate for the preservation of functional genetic and species diversity in reservoirs as a means of mitigating the risk of disease spillover.

Genome-wide data reveal cryptic diversity and hybridization in a group of tree ferns.

Discovery of cryptic diversity is essential to understanding both the process of speciation and the conservation of species. Determining species boundaries in fern lineages represents a major challenge due to lack of morphologically diagnostic characters and frequent hybridization. Genomic data has substantially enhanced our understanding of the speciation process, increased the resolution of species delimitation studies, and led to the discovery of cryptic diversity. Here, we employed restriction-site-associated DNA sequencing (RAD-seq) and integrated phylogenomic and population genomic analyses to investigate phylogenetic relationships and evolutionary history of 16 tree ferns with marginate scales (Cyatheaceae) from China and Vietnam. We conducted multiple species delimitation analyses using the multispecies coalescent (MSC) model and novel approaches based on genealogical divergence index (gdi) and isolation by distance (IBD). In addition, we inferred species trees using concatenation and several coalescent-based methods, and assessed hybridization patterns and rate of gene flow across the phylogeny. We obtained highly supported and generally congruent phylogenies inferred from concatenated and summary-coalescent methods, and the monophyly of all currently recognized species were strongly supported. Our results revealed substantial evidence of cryptic diversity in three widely distributed Gymnosphaera species, each of which was composite of two highly structure lineages that may correspond to cryptic species. We found that hybridization was fairly common between not only closely related species, but also distantly related species. Collectively, it appears that scaly tree ferns may contain cryptic diversity and hybridization has played an important role throughout the evolutionary history of this group.

Multilocus approach reveals distinct evolutionary units of the South American apapa Pellona flavipinnis (Valenciennes, 1837) (Clupeiformes, Pristigasteridae).

The discovery and characterization of cryptic diversity is important for conservation and management, especially for ichthyofauna, whose diversity is underestimated and understudied. Cryptic diversity is especially common in widely distributed species, and Pellona flavipinnis is one such species. Thus, the aim of the present study was to investigate and test whether P. flavipinnis harbours cryptic diversity. In this study we used the COI and control region sequences and microsatellite loci of 86-114 specimens from 11-12 locations throughout the Amazon basin, depending on the molecular marker used. We also included two COI GenBank sequences from the type locality of the species, the Paraná River. The results from COI sequences showed that P. flavipinnis from the Amazon basin presented two spatially structured lineages differentiated from P. flavipinnis from the Paraná River by 10.6%-9.8% (depending on the lineages) and 45 mutational steps. The genetic distance between the Amazon lineages was 2.4% using COI, with high population differentiation values (ФST = 0.8686 and ФST = 0.8483 for COI and control region, respectively). Among the five species delimitation methods employed, three indicated two lineages in P. flavipinnis in the Amazon basin, and all five methods indicated that the Amazonian lineages are different from that of Paraná. Results from microsatellite loci also showed that P. flavipinnis from the Amazon basin is composed of two evolutionary units. The results of 13 morphometric measurements indicated that there are no differences in shape between the P. flavipinnis lineages in the Amazon basin. The present findings suggest that there are two sympatric lineages of P. flavipinnis in the Amazon basin.

Phyllosphere eukaryotic microalgal communities in rainforests: Drivers and diversity.

Phyllosphere algae are common in tropical rainforests, forming visible biofilms or spots on plant leaf surfaces. However, knowledge of phyllosphere algal diversity and the environmental factors that drive that diversity is limited. The aim of this study is to identify the environmental factors that drive phyllosphere algal community composition and diversity in rainforests. For this purpose, we used single molecule real-time sequencing of full-length 18S rDNA to characterize the composition of phyllosphere microalgal communities growing on four host tree species (Ficus tikoua, Caryota mitis, Arenga pinnata, and Musa acuminata) common to three types of forest over four months at the Xishuangbanna Tropical Botanical Garden, Yunnan Province, China. Environmental 18S rDNA sequences revealed that the green algae orders Watanabeales and Trentepohliales were dominant in almost all algal communities and that phyllosphere algal species richness and biomass were lower in planted forest than in primeval and reserve rainforest. In addition, algal community composition differed significantly between planted forest and primeval rainforest. We also found that algal communities were affected by soluble reactive phosphorous, total nitrogen, and ammonium contents. Our findings indicate that algal community structure is significantly related to forest type and host tree species. Furthermore, this study is the first to identify environmental factors that affect phyllosphere algal communities, significantly contributing to future taxonomic research, especially for the green algae orders Watanabeales and Trentepohliales. This research also serves as an important reference for molecular diversity analysis of algae in other specific habitats, such as epiphytic algae and soil algae.

Unveiling the evolutionary relationships and the high cryptic diversity in Andean rainfrogs (Craugastoridae: Pristimantis myersi group).

Background: Pristimantis is the most diverse genus of terrestrial frogs. Historically, it has been divided into several phenetic groups in order to facilitate species identification. However, in light of phylogenetic analysis, many of these groups have been shown to be non-monophyletic, denoting a high degree of morphological convergence and limited number of diagnostic traits. In this study, we focus on the Pristimantis myersi group, an assemblage of small rainfrogs distributed throughout the Andes of Ecuador and Colombia, whose external morphology is highly conserved, and its species diversity and evolutionary relationships largely unknown. Methods: We inferred a new phylogenetic hypothesis for the frog genus Pristimantis, including all available sequences of the mtDNA 16S rRNA, as well as new DNA sequences from 175 specimens. Our sampling included 19 of the 24 species currently recognized as part of the Pristimantis myersi group. Results: Our new evolutionary hypothesis recovered the P. myersi group as non-monophyletic and composed of 16 species. Therefore, we exclude P. albujai, P. bicantus, P. sambalan, and P. nelsongalloi in order to preserve the monophyly of the group. We discovered at least eight candidate species, most of them hidden under the names of P. leoni, P. hectus, P. festae, P. gladiator, and P. ocreatus. Discussion: Our results reveal the occurrence of a high level of cryptic diversity to the species level within the P. myersi group and highlight the need to redefine some of its species and reassess their conservation status. We suggest that the conservation status of six species within the group need to be re-evaluated because they exhibit smaller distributions than previously thought; these species are: P. festae, P. gladiator, P. hectus, P. leoni, P. ocreatus, and P. pyrrhomerus. Finally, given that the Pristimantis myersi group, as defined in this work, is monophyletic and morphologically diagnosable, and that Trachyphrynus is an available name for the clade containing P. myersi, we implement Trachyphrynus as a formal subgenus name for the Pristimantis myersi group.

Freshwater Lichens, Including New Species in the Genera Verrucaria, Placopyrenium and Circinaria, Associated with Lobothallia hydrocharis (Poelt & Nimis) Sohrabi & Nimis from Watercourses of Sardinia.

This work summarizes the results of the exploration of freshwater lichen biota on the island of Sardinia associated with the regional flagship species Lobothallia hydrocharis, a large-sized crustose lichen from the splash zone along mountain streams, so far known from Sardinia only. Molecular data were used to confirm its distinctiveness from other taxa and its systematic placement and to identify critical taxa among its associated lichen biota. We found 25 species of lichenized fungi, including three species new to science in the genera Verrucaria, Placopyrenium, and Circinaria, and seven species new to Sardinia (Hydropunctaria rheithrophila, Ionaspis chrysophana, I. odora, Verrucaria aquatilis, V. collematodes, V. pseudovirescens), or new to Southern Europe (V. devensis). Specific traits for the freshwater lichen biota of Sardinia were identified and compared to those reported from freshwater sites in the Alps and Carpathian mountains, e.g., a relative scarcity of subgelatinous lichens. Parasitic or epilichenic interactions were found frequently but only in the splash zone and not in the permanently submerged zone, i.e., two parasitic Placopyrenium species, and clearly lichenicolous thalli of Kuettlingeria atroflava and Lobothallia hydrocharis. Due to its specific trait profile and the great potential for the discovery of new species, we recommend the inclusion of Sardinian and further Mediterranean sites in continental-scale monitoring programs for freshwater lichens.