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A recently published molecular phylogenetic analysis, focusing on selected Western Atlantic subspecies of Acetesamericanus Ortmann, 1893 and allies, was inconclusive about relationships among these members. This previous study found three groups that split into two distinct lineages: Acetesamericanus (Brazil 1) (= A.americanus sensu stricto) and Acetesamericanus (Brazil 2) + A.americanus (USA). Combined morphometry and molecular analyses applied to members of the group Acetesamericanus (Brazil 2) revealed a new unidentified species genetically related to the A.americanus representatives. However, at that time, no conclusive morphological characters were found to identify it. In the present study, following an in-depth morphological analysis of specimens from the three groups, including data on the type series and consideration of the subtle distinctions of members of each lineage, morphological features of the reproductive structures (petasma and genital sternite) were found to characterize the new species, which is formally described and named herein.

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Trombiculid mites were collected from the edible dormouse (Glis glis) within the Carpathian-Balkan distribution gradient of host species. Representatives of five genera (Leptotrombidium, Neotrombicula, Brunehaldia, Hirsutiella, Schoutedenichia) and 10 species of chiggers were discovered in the material, based on morphological and/or molecular data. Brunehaldia, new to the fauna of Greece, was recorded for the first time from the edible dormouse. Neotrombicula talmiensis was new to the fauna of Greece and Neotrombicula vulgaris was new to the fauna of North Macedonia. Successful amplification and sequencing of COI was carried out in relation to three genera and six species. The intraspecific variation of taxa hitherto distinguished based on morphological criteria was juxtaposed with molecular data, using the distance method and the phylogenetic approach. The molecular methods indicated wider than hitherto recognized, intraspecific morphological variation for Leptotrombidium europaeum and N. talmiensis. On the other hand, an inference limited to morphology proved to be insufficient for species delineation, which was confirmed by the relatively low identity (%) of examined COI sequences as well as the size of inter-/intraspecific K2P distance threshold. Our study provides support for integrative taxonomy that combines different sources of evidence and contributes to recognition of the scope of intraspecific variation. The high degree of hidden diversity revealed with the application of molecular tools, votes for a careful approach to the identification of chiggers. The confirmed cases of co-invasion, including the representatives of various genera (Leptotrombidium and Neotrombicula, Brunehaldia and Neotrombicula, Neotrombicula and Schoutedenichia, Hirsutiella and Schoutedenichia) additionally support the need to include all larvae found on a given host specimen in the identification process.

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Filamentous green algae of the genus Zygnema are an essential part of hydro-terrestrial ecosystems. Despite several studies on their resistance to natural stresses, little is known about the composition of their assemblages and the changes they undergo over time. Two sites at altitudes above 2200 m a.s.l. in the Austrian Alps were selected for a 2-year observation period and sampled five times. Molecular phylogenetic analysis of the 152 isolated strains of Zygnema sp. was performed based on the rbcL and trnG sequences. Seven genotypes were found at these sites during the samplings, but their proportion varied throughout the seasons. The site with a more stable water regime also had a more stable representation of genotypes, in contrast to the site with fluctuating water availability. The mats formed resistant pre-akinetes at the end of the season with reduced photosynthetic activity. Contrary to expectations, the mats were not exposed to extremely cold temperatures in winter due to snow cover. Some genotypes have been previously observed at this site, indicating that the population composition is stable. This work highlights the importance of resistant pre-akinetes in surviving winter conditions, the ability of algae to re-establish mats, and the need to address the hidden diversity of the genus Zygnema.

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A new species, Chrysosphaerella septentrionalis, is described from a peat bog located on the bank of the Paz River (Pasvik Nature Reserve, Murmansk Region, Russia). Scale ultrastructure was studied using a scanning electron microscope. Morphologically, C. septentrionalis seems to closely resemble C. longispina. However, C. septentrionalis possesses subcircular scales in addition to the oval scales, and they are much smaller than in C. longispina. We suppose that C. septentrionalis is the first pseudocryptic species within the C. longispina complex. Additionally, we proposed an infrageneric classification of Chrysosphaerella based on the scale structure and divided the genus into three sections: Chrysosphaerella, Brevispinae sect. nov. and Septispinae sect. nov. The formal description of a new family Chrysosphaerellaceae fam. nov. is also provided.

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Photosynthetic microorganisms are important components of most ecosystems and have important roles regarding biogeochemical cycles and the basis of the trophic chain. However, they sometimes are present in low abundance compared to other heterotrophic organisms. The Chapada das Mesas National Park (PNCM) is a Conservation Unit in Brazilian Cerrado biome, which is considered a hotspot for biodiversity conservation and possesses important rivers, waterfalls, and springs with economical and touristic importance. The aim of this study was to perform a comparative analysis of enriched and total microbiome of sediments to understand the impact of pre-cultivation in discovery of underrepresented groups like photosynthesizers. All sediment samples were cultivated in BG-11 medium under illumination to enrich for photosynthetic microorganisms and both the raw samples and the enriched ones were submitted to DNA extraction and sequencing of the V3-V4 hypervariable region of the 16S rRNA gene on the Ion Torrent platform. The reads were analyzed using QIIME2 software and the Phyloseq package. The enrichment allowed detection and identification of many genera of cyanobacteria in the Chapada das Mesas National Park (PNCM), which would probably not be possible without the combination of approaches. A total of 58 groups of photosynthetic microorganisms were classified in the samples from the enrichments and their relative abundance based on amplified 16S rRNA sequences were estimated, highlighting the genus Synechocystis which represented 10.10% of the abundance of the phylum Cyanobacteria and the genus Dunaliella, which represented 45.66% of the abundance of algae as the most abundant groups at the PNCM. In the enrichments, microorganisms from the phyla Proteobacteria (45.2%), Bacteroidetes (18%), and Planctomycetes (3.3%) were also identified, since there are ecological associations between the photosynthetic community and other groups of heterotrophic microorganisms. As for the functional analysis, metabolic functions associated with methanotrophy and methylotrophy, hydrocarbon degradation, phototrophy, and nitrogen fixation were predicted. The results highlight a great diversity of photosynthetic microorganisms in Cerrado and the importance of using a combination of approaches when analyzing target groups which are usually underrepresented such as cyanobacteria and microalgae.

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Despite its importance in intertidal and shallow-water marine ecosystems, Caprella penantis continues to be one of the most taxonomically challenging amphipods in the world. A recent molecular study focusing on C. penantis sensu stricto pointed out the existence of three highly divergent lineages, indicating the possible existence of a process of ongoing speciation and, thus, casting doubt on the taxonomic status of this species. In the present study, we used an integrative approach to continue to shed light on the taxonomy and distribution of this caprellid. To this end, we combined morphological and genetic data (COI and 18S) and included, for the first time, populations from its type locality. Our analyses provide strong evidence of the existence of potentially three distinct species, genetically and geographically restricted, within C. penantis sensu stricto, with the distribution of the true C. penantis sensu stricto restricted to the UK (type locality), the northern coast of the Iberian Peninsula, and the Azores. Results show the co-occurrence of two of these species in a locality of northern Portugal and indicate the existence of distinct evolutionary and diversification patterns along the eastern Atlantic region. Overall, our study highlights the use of an integrative approach to properly assess species boundaries and unravel hidden biodiversity in amphipods.

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The Atacama Desert at its margins harbors a unique biodiversity that is still very poorly known, especially in coastal fog oases spanning from Perú towards the Atacama coast. An outstanding species-rich fog oasis is the latitudinal fringe Paposo-Taltal, that is considered an iconic site of the Lomas formation. This contribution is the first to reveal the knowledge on arthropods of this emblematic coastal section. We used pitfall traps to study the taxonomic composition, richness, and abundance of terrestrial arthropods in 17 sample sites along a 100 km section of the coast between 24.5 and 25.5 southern latitude, in a variety of characteristic habitats. From a total of 9154 individuals, we were able to identify 173 arthropod species grouped into 118 genera and 57 families. The most diverse group were insects, with 146 species grouped in 97 genera and 43 families, while arachnids were represented by 27 species grouped into 21 genera and 14 families. Current conservation challenges on a global scale are driving the creation and evaluation of potential conservation sites in regions with few protected areas, such as the margins of the Atacama Desert. Better taxonomic, distributional, and population knowledge is urgently needed to perform concrete conservation actions in a biodiversity hotspot at a desert edge.

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This paper is a contribution to the current knowledge of taxonomy, ecology and distribution of South American Cortinarius (Pers.) Gray. Cortinarius is among the most widely distributed and species-rich basidiomycete genera occurring with South American Nothofagaceae and species are found in many distinct habitats, including shrublands and forests. Due to their ectomycorrhizal role, Cortinarius species are critical for nutrient cycling in forests, especially at higher latitudes. Some species have also been reported as edible fungi with high nutritional quality. Our aim is to unravel the taxonomy of selected Cortinarius belonging to phlegmacioid and myxotelamonioid species based on morphological and molecular data. After widely sampling Cortinarius specimens in Patagonian Nothofagaceae forests and comparing them to reference collections (including holotypes), we propose five new species of Cortinarius in this work. Phylogenetic analyses of concatenated rDNA ITS-LSU and RPB1 sequences failed to place these new species into known Cortinarius sections or lineages. These findings highlight our knowledge gaps regarding the fungal diversity of South American Nothofagaceae forests. Due to the high diversity of endemic Patagonian taxa, it is clear that the South American Cortinarius diversity needs to be discovered and described in order to understand the evolutionary history of Cortinarius on a global scale.

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The chubbyhead barb, Enteromius anoplus, as currently described, is the most widely distributed freshwater fish in South Africa. The species occurs in almost all the major river systems across the country, with the exception of the small coastal drainages on the south coast. The use of a comprehensive data set of mitochondrial (mtDNA) cytochrome b (cyt b) sequences uncovered the presence of four distinct lineages or operational taxonomic units (OTUs) within E. anoplus: (a) the Gouritz lineage endemic to the Gouritz River system, (b) the Olifants lineage endemic to the Olifants-Doring River system, (c) the Orange lineage endemic to the Orange River system and (d) the Eastern Cape lineage which is distributed across six river systems from the Great Fish to the Buffalo rivers in the Eastern Cape Province of South Africa. The present study provides a new diagnosis for E. anoplus s.s. (the Gouritz lineage), revalidates Enteromius cernuus (the Olifants lineage) and Enteromius oraniensis (the Orange lineage) as distinct species. The study also provides a description for a new species, Enteromius mandelai sp. nov (the Eastern Cape lineage). E. cernuus and E. mandelai differ from both E. anoplus and E. oraniensis by having a complete lateral line (vs. an incomplete lateral line in the latter species). E. cernuus further differs from the other three species by having long maxillary barbels which reach or exceed the vertical through the middle of the eye and the lowest number of circumpendicular scales (10-12 vs. 12-16 for the other three species). E. oraniensis is distinctive from the other three species by having inconspicuous barbels. These findings add to the growing body of literature that shows that the freshwater fishes of the Cape Fold and adjacent freshwater ecoregions have narrow geographic ranges. This has ramifications for conservation planning and management, as well as the understanding of the evolutionary history of the stream fishes in these global endemic hotspots.

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The present study used molecular and morphological approaches to investigate hidden diversity within the Hippopotamyrus ansorgii species complex in southern Africa. Phylogenetic reconstructions and three species delimitation methods based on two mitochondrial markers (cytochrome b and cytochrome oxidase I) and one nuclear marker (S7) revealed 12 Molecular Operational Taxonomic Units (MOTUs), with two of them representing two recently described species, Hippopotamyrus longilateralis and Hippopotamyrus szaboi. The highest diversity occurred in the Kwanza River system, which contained five MOTUs, and the Upper Zambezi River system that had two MOTUs. Five other river systems contained a single MOTU each. A major impediment to the review of this complex has been the uncertainty surrounding the type locality of the specimens that were used for the description of H. ansorgii. The present study has, through a careful examination of published literature and synthesis of information on the travel activities of Dr. William Ansorge who collected the specimens, identified the Kwanza River system as the most plausible source of the syntypes. The resolution of the type locality of H. ansorgii facilitates future work on the review of this complex which is critical for providing reliable biodiversity estimates, identifying effective conservation management strategies and understanding the evolutionary history and biogeographic patterns of the fishes of this region.

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BACKGROUND: Climatic and topographic changes function as key drivers in shaping genetic structure and cladogenic radiation in many organisms. Southern Africa has an exceptionally diverse tortoise fauna, harbouring one-third of the world's tortoise genera. The distribution of Psammobates tentorius (Kuhl, 1820) covers two of the 25 biodiversity hotspots in the world, the Succulent Karoo and Cape Floristic Region. The highly diverged P. tentorius represents an excellent model species for exploring biogeographic and radiation patterns of reptiles in Southern Africa. RESULTS: We investigated genetic structure and radiation patterns against temporal and spatial dimensions since the Miocene in the Psammobates tentorius species complex, using multiple types of DNA markers and niche modelling analyses. Cladogenesis in P. tentorius started in the late Miocene (11.63-5.33 Ma) when populations dispersed from north to south to form two geographically isolated groups. The northern group diverged into a clade north of the Orange River (OR), followed by the splitting of the group south of the OR into a western and an interior clade. The latter divergence corresponded to the intensification of the cold Benguela current, which caused western aridification and rainfall seasonality. In the south, tectonic uplift and subsequent exhumation, together with climatic fluctuations seemed responsible for radiations among the four southern clades since the late Miocene. We found that each clade occurred in a habitat shaped by different climatic parameters, and that the niches differed substantially among the clades of the northern group but were similar among clades of the southern group. CONCLUSION: Climatic shifts, and biome and geographic changes were possibly the three major driving forces shaping cladogenesis and genetic structure in Southern African tortoise species. Our results revealed that the cladogenesis of the P. tentorius species complex was probably shaped by environmental cooling, biome shifts and topographic uplift in Southern Africa since the late Miocene. The Last Glacial Maximum (LGM) may have impacted the distribution of P. tentorius substantially. We found the taxonomic diversify of the P. tentorius species complex to be highest in the Greater Cape Floristic Region. All seven clades discovered warrant conservation attention, particularly Ptt-B-Ptr, Ptt-A and Pv-A.

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The genus Schizodon is part of a group of headstanders and relatives (Family Anostomidae) that are widespread and ecologically important fishes in South American rivers. Schizodon includes 15 nominal species but their taxonomy has been challenging due to paucity of decisive characters to diagnose species. We present new molecular data to assess species boundaries or molecular operational taxonomic units (MOTUs), and to infer phylogenetic relationships among species. Evidence from two mitochondrial and three nuclear genes was used in these analyses. Mitochondrial DNA data for 112 specimens (from 11 nominal species) supported 13 consensus MOTUs, six of which matched valid nominal species (Schizodon borellii, S. fasciatus, S. intermedius, S. isognathus, S. knerii and S. scotorhabdotus). The nominal species Schizodon vittatus, S. nasutus, and S. dissimilis were subdivided into two MOTUs each, revealing either cryptic species or strong population structuring. In contrast, S. platae and S. jacuiensis constituted a single MOTU, indicating a possible case of synonymy. Our phylogenetic analysis subdivided the genus Schizodon into two large clades that are compatible with observed color patterns and biogeographic distribution. The first clade includes species with three to four conspicuous dark vertical bars on the flanks that originated in the Amazonas region (S. borellii, S. dissimilis, S. intermedius, S. fasciatus, S. scotorhabdotus, S. vittatus, and a cryptic species, Schizodon aff. vittatus). The second clade includes species with a conspicuous dark caudal blotch on the caudal peduncle, with vertical bars absent or inconspicuous, with a biogeographic origin in the La Plata drainage (S. isognathus, S. jacuiensis, S. knerii, S. nasutus and S. platae). Our results reinforce the importance of using molecular analyses to accelerate the study of diversity, particularly in groups with a wide distribution, few variable meristic characters, and high morphological plasticity, which may hide still unknown or underestimated diversity.

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Cryptic diversity (CD), the presence of highly divergent phylogenetic lineages within closed morphological species, has been documented for many taxa. Great arachnid orders such as Araneae or Scorpiones are well studied and many cases of CD have been described therein; to date, however, related research on smaller arachnid orders, such as whip spiders (Amblypygi), remains lacking. In the current study, we investigated CD based on cytochrome oxidase 1 ( COI) in three nominal species of the genus Heterophrynus ( H. alces, H. batesii, and H. longicornis), represented by 65 specimens. The sequences were compared using three different methods. All three species showed geographically structured CD. Thus, given its existence in this genus, it is important that CD and its spatial distribution be considered in future studies and possible conservation projects.

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With 149 currently recognized species, Hypostomus is one of the most species-rich catfish genera in the world, widely distributed over most of the Neotropical region. To clarify the evolutionary history of this genus, we reconstructed a comprehensive phylogeny of Hypostomus based on four nuclear and two mitochondrial markers. A total of 206 specimens collected from the main Neotropical rivers were included in the present study. Combining morphology and a Bayesian multispecies coalescent (MSC) approach, we recovered 85 previously recognized species plus 23 putative new species, organized into 118 'clusters'. We presented the Cluster Credibility (CC) index that provides numerical support for every hypothesis of cluster delimitation, facilitating delimitation decisions. We then examined the correspondence between the morphologically identified species and their inter-specific COI barcode pairwise divergence. The mean COI barcode divergence between morphological sisters species was 1.3 ± 1.2%, and only in 11% of the comparisons the divergence was ≥2%. This indicates that the COI barcode threshold of 2% classically used to delimit fish species would seriously underestimate the number of species in Hypostomus, advocating for a taxon-specific COI-based inter-specific divergence threshold to be used only when approximations of species richness are needed. The phylogeny of the 108 Hypostomus species, together with 35 additional outgroup species, confirms the monophyly of the genus. Four well-supported main lineages were retrieved, hereinafter called super-groups: Hypostomus cochliodon, H. hemiurus, H. auroguttatus, and H. plecostomus super-groups. We present a compilation of diagnostic characters for each super-group. Our phylogeny lays the foundation for future studies on biogeography and on macroevolution to better understand the successful radiation of this Neotropical fish genus.

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Zearaja chilensis has been reported from Southern Brazil in the Southwest Atlantic (SWA) to northern Chile in the Southeast Pacific (SEP), and it was listed as vulnerable by the IUCN. Recent molecular studies have called into question the conspecificity between specimens from these opposite coasts of South America, which can have implications for the conservation status of the species. To verify the identity of specimens identified as Z. chilensis, 47 individuals from SWA and 22 from SEP were examined. By comparing external morphology, spinulation pattern, clasper, egg cases, and mitochondrial cytochrome c oxidase I (COI) sequence data, differences between groups were found. Adults from SWA presented longer snout length and shorter tail than those from SEP. Dermal denticles were restricted to the rostral area in SWA skates, whereas in SEP skates most of the dorsal surface was covered with denticles. Marked differences in the morphology of several components of clasper were noticeable. Egg cases of SWA skates had thinner lateral keels than those of SEP. Molecular analysis revealed two well-defined cohesive clusters, corresponding to SWA and SEP specimens, respectively. Average K2P distance between groups was 3.4%, higher than expected for intraspecific differences, and sequences were assigned to different BINs. These integrative approaches strongly support that specimens from SWA known as Z. chilensis correspond to a different nominal species than those from SEP. Herein, Z. brevicaudata (Marini 1933) is resurrected from synonymy with Z. chilensis.

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Unicellular, eukaryotic organisms (protists) play a key role in soil food webs as major predators of microorganisms. However, due to the polyphyletic nature of protists, no single universal barcode can be established for this group, and the structure of many protistean communities remains unresolved. Plasmodial slime moulds (Myxogastria or Myxomycetes) stand out among protists by their formation of fruit bodies, which allow for a morphological species concept. By Sanger sequencing of a large collection of morphospecies, this study presents the largest database to date of dark-spored myxomycetes and evaluate a partial 18S SSU gene marker for species annotation. We identify and discuss the use of an intraspecific sequence similarity threshold of 99.1% for species differentiation (OTU picking) in environmental PCR studies (ePCR) and estimate a hidden diversity of putative species, exceeding those of described morphospecies by 99%. When applying the identified threshold to an ePCR data set (including sequences from both NGS and cloning), we find 64 OTUs of which 21.9% had a direct match (>99.1% similarity) to the database and the remaining had on average 90.2 ± 0.8% similarity to their best match, thus thought to represent undiscovered diversity of dark-spored myxomycetes.

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Molecular surveys of eukaryotic microbial communities employing high-throughput sequencing (HTS) techniques are rapidly supplanting traditional morphological approaches due to their larger data output and reduced bench work time. Here, we directly compare morphological and Illumina data obtained from the same samples, in an effort to characterize ciliate faunas from sediments in freshwater environments. We show how in silico processing affects the final outcome of our HTS analysis, providing evidence that quality filtering protocols strongly impact the number of predicted taxa, but not downstream conclusions such as biogeography patterns. We determine the abundance distribution of ciliates, showing that a small fraction of abundant taxa dominates read counts. At the same time, we advance reasons to believe that biases affecting HTS abundances may be significant enough to blur part of the underlying biological picture. We confirmed that the HTS approach detects many more taxa than morphological inspections, and highlight how the difference varies among taxonomic groups. Finally, we hypothesize that the two datasets actually correspond to different conceptions of "diversity," and consequently that neither is entirely superior to the other when investigating environmental protists.

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A large-scale comprehensive reference library of DNA barcodes for European marine fishes was assembled, allowing the evaluation of taxonomic uncertainties and species genetic diversity that were otherwise hidden in geographically restricted studies. A total of 4118 DNA barcodes were assigned to 358 species generating 366 Barcode Index Numbers (BIN). Initial examination revealed as much as 141 BIN discordances (more than one species in each BIN). After implementing an auditing and five-grade (A-E) annotation protocol, the number of discordant species BINs was reduced to 44 (13% grade E), while concordant species BINs amounted to 271 (78% grades A and B) and 14 other had insufficient data (grade D). Fifteen species displayed comparatively high intraspecific divergences ranging from 2·6 to 18·5% (grade C), which is biologically paramount information to be considered in fish species monitoring and stock assessment. On balance, this compilation contributed to the detection of 59 European fish species probably in need of taxonomic clarification or re-evaluation. The generalized implementation of an auditing and annotation protocol for reference libraries of DNA barcodes is recommended.

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