Species delimitation with limited sampling: An example from rare trapdoor spider genus Cyclocosmia (Mygalomorphae, Halonoproctidae).

The outcome of species delimitation depends on many factors, including conceptual framework, study design, data availability, methodology employed and subjective decision making. Obtaining sufficient taxon sampling in endangered or rare taxa might be difficult, particularly when non-lethal tissue collection cannot be utilized. The need to avoid overexploitation of the natural populations may thus limit methodological framework available for downstream data analyses and bias the results. We test species boundaries in rare North American trapdoor spider genus Cyclocosmia Ausserer (1871) inhabiting the Southern Coastal Plain biodiversity hotspot with the use of genomic data and two multispecies coalescent model methods. We evaluate the performance of each methodology within a limited sampling framework. To mitigate the risk of species over splitting, common in taxa with highly structured populations, we subsequently implement a species validation step via genealogical diversification index (gdi), which accounts for both genetic isolation and gene flow. We delimited eight geographically restricted lineages within sampled North American Cyclocosmia, suggesting that major river drainages in the region are likely barriers to dispersal. Our results suggest that utilizing BPP in the species discovery step might be a good option for datasets comprising hundreds of loci, but fewer individuals, which may be a common scenario for rare taxa. However, we also show that such results should be validated via gdi, in order to avoid over splitting.

Integrative Taxonomy Approach Reveals Cryptic Diversity within the Phoretic Pseudoscorpion Genus Lamprochernes (Pseudoscorpiones: Chernetidae).

Pseudoscorpions represent an ancient, but homogeneous group of arachnids. The genus Lamprochernes comprises several morphologically similar species with wide and overlapping distributions. We implemented an integrative approach combining molecular barcoding (cox1), with cytogenetic and morphological analyses in order to assess species boundaries in European Lamprochernes populations. The results suggest ancient origins of Lamprochernes species accompanied by morphological stasis within the genus. Our integrative approach delimited three nominal Lamprochernes species and one cryptic lineage Lamprochernes abditus sp. nov. Despite its Oligocene origin, L. abditus sp. nov. can be distinguished from its closest relative only by molecular and cytogenetic differences, or alternatively, by a complex multivariate morphometric analysis involving other Lamprochernes species. The population structure and common haplotype sharing across geographically distant populations in most Lamprochernes species suggest that a phoretic manner of dispersal is efficient in this group.

Molecular data do not support the traditional morphology-based groupings in the scorpion family Buthidae (Arachnida: Scorpiones).

The family Buthidae represents an early-diverging and most species-rich lineage of extant scorpions, but its internal phylogenetic relationships are still poorly understood. The family is traditionally divided into six morpho-groups; however, the monophyly of some of them remains unclear. We combined multilocus sequence data with extensive taxon sampling to reconstruct the phylogenetic relationships among Buthidae and assess the validity of the morphology-based groupings. We recovered a monophyletic Buthus group as a sister clade to all the remaining Buthidae. We also found support for the monophyly of the Tityus group, but the remaining morpho-groups were recovered as para-/polyphyletic. Our results also suggest that some genera are in need of a taxonomic revision.

Phylogenomic analysis, reclassification, and evolution of South American nemesioid burrowing mygalomorph spiders.

The family Nemesiidae was once among the most species-rich of mygalomorph spider families. However, over the past few decades both morphological and molecular studies focusing on mygalomorph phylogeny have recovered the group as paraphyletic. Hence, the systematics of the family Nemesiidae has more recently been controversial, with numerous changes at the family-group level and the recognition of the supra-familial clade Nemesioidina. Indeed, in a recent study by Opatova and collaborators, six nemesiid genera were transferred to the newly re-established family Pycnothelidae. Despite these changes, 12 South American nemesiid genera remained unplaced, and classified as incertae sedis due to shortcomings in taxon sampling. Accordingly, we evaluate the phylogenetic relationships of South American nemesioid species and genera with the principle aim of resolving their family level placement. Our work represents the most exhaustive phylogenomic sampling for South American Nemesiidae by including nine of the 12 genera described for the continent. Phylogenetic relationships were reconstructed using 457 loci obtained using the spider Anchored Hybrid Enrichment probe set. Based on these results Nemesiidae, Pycnothelidae, Microstigmatidae and Cyrtaucheniidae are not considered monophyletic. Our study also indicates that the lineage including the genus Fufius requires elevation to the family level (Rhytidicolidae Simon, 1903 (NEW RANK)). In Pycnothelidae, we recognize/delimit five subfamilies (Diplothelopsinae, Pionothelinae (NEW SUBFAMILY), Prorachiinae (NEW SUBFAMILY), Pselligminae (NEW RANK), Pycnothelinae). We also transfer all the 12 South American nemesiid genera to Pycnothelidae: Chaco, Chilelopsis, Diplothelopsis, Flamencopsis, Hermachura, Longistylus, Lycinus, Neostothis, Prorachias, Psalistopoides, Pselligmus, Rachias. Additionally, we transferred the microstigmatid genus Xenonemesia to Pycnothelidae, and we propose the following generic synonymies and species transfers: Neostothis and Bayana are junior synonyms of Pycnothele (NEW SYNONYMY), as P. gigas and P. labordai, respectively (NEW COMBINATIONS); Hermachura is a junior synonym of Stenoterommata (NEW SYNONYMY), as S. luederwaldti (NEW COMBINATION); Flamencopsis is a junior synonym of Chilelopsis (NEW SYNONYMY), as C. minima (NEW COMBINATION); and Diplothelopsis is a junior synonym of Lycinus (NEW SYNONYMY), as L. ornatus and L. bonariensis (NEW COMBINATIONS). Considering the transferred genera and synonymies, Pycnothelidae now includes 15 described genera and 137 species. Finally, these results provide a robust phylogenetic framework that includes enhanced taxonomic sampling, for further resolving the biogeography and evolutionary time scale for the family Pycnothelidae.

Phylogenomic analysis, reclassification, and evolution of South American nemesioid burrowing mygalomorph spiders

The family Nemesiidae was once among the most species-rich of mygalomorph spider families. However, over the past few decades both morphological and molecular studies focusing on mygalomorph phylogeny have recovered the group as paraphyletic. Hence, the systematics of the family Nemesiidae has more recently been controversial, with numerous changes at the family-group level and the recognition of the supra-familial clade Nemesioidina. Indeed, in a recent study by Opatova and collaborators, six nemesiid genera were transferred to the newly re-established family Pycnothelidae. Despite these changes, 12 South American nemesiid genera remained unplaced, and classified as incertae sedis due to shortcomings in taxon sampling. Accordingly, we evaluate the phylogenetic relationships of South American nemesioid species and genera with the principle aim of resolving their family level placement. Our work represents the most exhaustive phylogenomic sampling for South American Nemesiidae by including nine of the 12 genera described for the continent. Phylogenetic relationships were reconstructed using 457 loci obtained using the spider Anchored Hybrid Enrichment probe set. Based on these results Nemesiidae, Pycnothelidae, Microstigmatidae and Cyrtaucheniidae are not considered monophyletic. Our study also indicates that the lineage including the genus Fufius requires elevation to the family level (Rhytidicolidae Simon, 1903 (NEW RANK)). In Pycnothelidae, we recognize/delimit five subfamilies (Diplothelopsinae, Pionothelinae NEW SUBFAMILY, Prorachiinae NEW SUBFAMILY, Pselligminae NEW RANK, Pycnothelinae). We also transfer all the 12 South American nemesiid genera to Pycnothelidae: Chaco, Chilelopsis, Diplothelopsis, Flamencopsis, Hermachura, Longistylus, Lycinus, Neostothis, Prorachias, Psalistopoides, Pselligmus, Rachias. Additionally, we transferred the microstigmatid genus Xenonemesia to Pycnothelidae, and we propose the following generic synonymies and species transfers: Neostothis and Bayana are junior synonyms of Pycnothele (NEW SYNONYMY), as P. gigas and P. labordai, respectively (NEW COMBINATIONS); Hermachura is a junior synonym of Stenoterommata (NEW SYNONYMY), as S. luederwaldti (NEW COMBINATION); Flamencopsis is a junior synonym of Chilelopsis (NEW SYNONYMY), as C. minima (NEW COMBINATION); and Diplothelopsis is a junior synonym of Lycinus (NEW SYNONYMY), as L. ornatus and L. bonariensis (NEW COMBINATIONS). Considering the transferred genera and synonymies, Pycnothelidae now includes 15 described genera and 137 species. Finally, these results provide a robust phylogenetic framework that includes enhanced taxonomic sampling, for further resolving the biogeography and evolutionary time scale for the family Pycnothelidae.

Karyotype evolution and preliminary molecular assessment of genera in the family Scorpiopidae (Arachnida: Scorpiones).

The scorpions represent an ancient and morphologically conserved order of arachnids. Despite that, their karyotypes may differ considerably even among closely related species. In this study, we identify the trends of the karyotype evolution in the family Scorpiopidae based on integrating cytogenetic data and multi-locus molecular phylogenetic approaches. We detected considerable variability in diploid numbers of chromosomes (from 48 to 147), 18S rRNA gene cluster positions (from terminal to pericentromeric) at the interspecific level. Moreover, we identified independent fusions, fissions and inversions in the evolution of the family Scorpiopidae, leading to a remarkable diversification of the karyotypes. The dynamic system of the karyotype changes in this group is further documented by the presence of interstitial telomeric sequences (ITS) in two species. The cytogenetic differences observed among the analyzed species highlight the potential of this type of data for species-level taxonomy in scorpion lineages with monocentric chromosomes. Additionally, the results of our phylogenetic analyses support the monophyly of the family Scorpiopidae, but rendered several genera para- or polyphyletic.

Cytogenetics of entelegyne spiders (Arachnida, Araneae) from southern Africa.

Spiders represent one of the most studied arachnid orders. They are particularly intriguing from a cytogenetic point of view, due to their complex and dynamic sex chromosome determination systems. Despite intensive research on this group, cytogenetic data from African spiders are still mostly lacking. In this study, we describe the karyotypes of 38 species of spiders belonging to 16 entelegyne families from South Africa and Namibia. In the majority of analysed families, the observed chromosome numbers and morphology (mainly acrocentric) did not deviate from the family-level cytogenetic characteristics based on material from other continents: Tetragnathidae (2n♂ = 24), Ctenidae and Oxyopidae (2n♂ = 28), Sparassidae (2n♂ = 42), Gnaphosidae, Trachelidae and Trochanteriidae (2n♂ = 22), and Salticidae (2n♂ = 28). On the other hand, we identified interspecific variability within Hersiliidae (2n♂ = 33 and 35), Oecobiidae (2n♂ = 19 and 25), Selenopidae (2n♂ = 26 and 29) and Theridiidae (2n♂ = 21 and 22). We examined the karyotypes of Ammoxenidae and Gallieniellidae for the first time. Their diploid counts (2n♂ = 22) correspond to the superfamily Gnaphosoidea and support their placement in this lineage. On the other hand, the karyotypes of Prodidominae (2n♂ = 28 and 29) contrast with all other Gnaphosoidea. Similarly, the unusually high diploid number in Borboropactus sp. (2n♂ = 28) within the otherwise cytogenetically uniform family Thomisidae (mainly 2n♂ = 21-24) supports molecular data suggesting a basal position of the genus in the family. The implementation of FISH methods for visualisation of rDNA clusters facilitated the detection of complex dynamics of numbers of these loci. We identified up to five loci of the 18S rDNA clusters in our samples. Three different sex chromosome systems (X0, X1X20 and X1X2X30) were also detected among the studied taxa.

Phylogenetic Systematics and Evolution of the Spider Infraorder Mygalomorphae Using Genomic Scale Data.

The infraorder Mygalomorphae is one of the three main lineages of spiders comprising over 3000 nominal species. This ancient group has a worldwide distribution that includes among its ranks large and charismatic taxa such as tarantulas, trapdoor spiders, and highly venomous funnel-web spiders. Based on past molecular studies using Sanger-sequencing approaches, numerous mygalomorph families (e.g., Hexathelidae, Ctenizidae, Cyrtaucheniidae, Dipluridae, and Nemesiidae) have been identified as non-monophyletic. However, these data were unable to sufficiently resolve the higher-level (intra- and interfamilial) relationships such that the necessary changes in classification could be made with confidence. Here, we present a comprehensive phylogenomic treatment of the spider infraorder Mygalomorphae. We employ 472 loci obtained through anchored hybrid enrichment to reconstruct relationships among all the mygalomorph spider families and estimate the timeframe of their diversification. We sampled nearly all currently recognized families, which has allowed us to assess their status, and as a result, propose a new classification scheme. Our generic-level sampling has also provided an evolutionary framework for revisiting questions regarding silk use in mygalomorph spiders. The first such analysis for the group within a strict phylogenetic framework shows that a sheet web is likely the plesiomorphic condition for mygalomorphs, as well as providing insights to the ancestral foraging behavior for all spiders. Our divergence time estimates, concomitant with detailed biogeographic analysis, suggest that both ancient continental-level vicariance and more recent dispersal events have played an important role in shaping modern day distributional patterns. Based on our results, we relimit the generic composition of the Ctenizidae, Cyrtaucheniidae, Dipluridae, and Nemesiidae. We also elevate five subfamilies to family rank: Anamidae (NEW RANK), Euagridae (NEW RANK), Ischnothelidae (NEW RANK), Pycnothelidae (NEW RANK), and Bemmeridae (NEW RANK). Three families Entypesidae (NEW FAMILY), Microhexuridae (NEW FAMILY), and Stasimopidae (NEW FAMILY), and one subfamily Australothelinae (NEW SUBFAMILY) are newly proposed. Such a major rearrangement in classification, recognizing nine newly established family-level rank taxa, is the largest the group has seen in over three decades. [Biogeography; molecular clocks; phylogenomics; spider web foraging; taxonomy.].

A DNA barcode-assisted annotated checklist of the spider (Arachnida, Araneae) communities associated to white oak woodlands in Spanish National Parks.

BACKGROUND: A large scale semi-quantitative biodiversity assessment was conducted in white oak woodlands in areas included in the Spanish Network of National Parks, as part of a project aimed at revealing biogeographic patterns and identify biodiversity drivers. The semi-quantitative COBRA sampling protocol was conducted in sixteen 1-ha plots across six national parks using a nested design. All adult specimens were identified to species level based on morphology. Uncertain delimitations and identifications due to either limited information of diagnostic characters or conflicting taxonomy were further investigated using DNA barcode information. NEW INFORMATION: We identified 376 species belonging to 190 genera in 39 families, from the 8,521 adults found amongst the 20,539 collected specimens. Faunistic results include the discovery of 7 new species to the Iberian Peninsula, 3 new species to Spain and 11 putative new species to science. As largely expected by environmental features, the southern parks showed a higher proportion of Iberian and Mediterranean species than the northern parks, where the Palearctic elements were largely dominant. The analysis of approximately 3,200 DNA barcodes generated in the present study, corroborated and provided finer resolution to the morphologically based delimitation and identification of specimens in some taxonomically challenging families. Specifically, molecular data confirmed putative new species with diagnosable morphology, identified overlooked lineages that may constitute new species, confirmed assignment of specimens of unknown sexes to species and identified cases of misidentifications and phenotypic polymorphisms.

Molecular technique reveals high variability of 18S rDNA distribution in harvestmen (Opiliones, Phalangiidae) from South Africa.

The knowledge of cytogenetics in the harvestmen family Phalangiidae has been based on taxa from the Northern Hemisphere. We performed cytogenetic analysis on Guruia africana (Karsch, 1878) (2n=24) and four species of the genus Rhampsinitus Simon, 1879 (2n=24, 26, 34) from South Africa. Fluorescence in situ hybridization with an 18S rDNA probe was used to analyze the number and the distribution of this cluster in the family Phalangiidae for the first time. The results support the cytogenetic characteristics typical for the majority of harvestmen taxa, i.e. the predominance of small biarmed chromosomes and the absence of morphologically well-differentiated sex chromosomes as an ancestral state. We identified the number of 18S rDNA sites ranging from two in R. qachasneki Kauri, 1962 to seven in one population of R. leighi Pocock, 1903. Moreover, we found differences in the number and localization of 18S rDNA sites in R. leighi between populations from two localities and between sexes of R. capensis (Loman, 1898). The heterozygous states of the 18S rDNA sites in these species may indicate the presence of XX/XY and ZZ/ZW sex chromosomes, and the possible existence of these systems in harvestmen is discussed. The variability of the 18S rDNA sites indicates intensive chromosomal changes during the differentiation of the karyotypes, which is in contrast to the usual uniformity in chromosomal morphology known from harvestmen so far.

Phylogeny of a cosmopolitan family of morphologically conserved trapdoor spiders (Mygalomorphae, Ctenizidae) using Anchored Hybrid Enrichment, with a description of the family, Halonoproctidae Pocock 1901.

The mygalomorph family Ctenizidae has a world-wide distribution and currently contains nine genera and 135 species. However, the monophyly of this group has long been questioned on both morphological and molecular grounds. Here, we use Anchored Hybrid Enrichment (AHE) to gather hundreds of loci from across the genome for reconstructing the phylogenetic relationships among the nine genera and test the monophyly of the family. We also reconstruct the possible ancestral ranges of the most inclusive clade recovered. Using AHE, we generate a supermatrix of 565 loci and 115,209 bp for 27 individuals. For the first time, analyses using all nine genera produce results definitively establishing the non-monophyly of Ctenizidae. A lineage formed exclusively by representatives of South African Stasimopus was placed as the sister group to the remaining taxa in the tree, and the Mediterranean Cteniza and Cyrtocarenum were recovered with high support as sister to exemplars of Euctenizidae, Migidae, and Idiopidae. All the remaining genera-Bothriocyrtum, Conothele, Cyclocosmia, Hebestatis, Latouchia, and Ummidia-share a common ancestor. Based on these results, we formally elevate this clade to the level of family. Our results definitively establish both the non-monophyly of the Ctenizidae and non-validity of the subfamilies Ummidiinae and Ctenizinae. In order to establish the placement of the remaining three ctenizid genera, Cteniza, Cyrtocarenum, and Stasimopus, thorough analyses within the context of a complete mygalomorph phylogenetic framework are needed. We formally describe the family Halonoproctidae Pocock 1901 and infer that the family's most recent common ancestor was likely distributed in western North America and Asia.

Karyotype diversity of pseudoscorpions of the genus Chthonius (Pseudoscorpiones, Chthoniidae) in the Alps.

Pseudoscorpions are found in almost all terrestrial habitats. However, their uniform appearance presents a challenge for morphology-based taxonomy, which may underestimate the diversity of this order. We performed cytogenetic analyses on 11 pseudoscorpion species of the genus Chthonius C. L. Koch, 1843 from the Alps, including three subgenera: Chthonius (Chthonius) C. L. Koch, 1843, Chthonius (Ephippiochthonius) Beier, 1930 and Chthonius (Globochthonius) Beier, 1931 inhabiting this region. The results show that the male diploid number of chromosomes ranges from 21-35. The sex chromosome system X0 has been detected in all male specimens. The X sex chromosome is always metacentric and represents the largest chromosome in the nucleus. Achiasmatic meiosis, already known from the family Chthoniidae, was further confirmed in males of Chthonius. C-banding corroborated the localization of constitutive heterochromatin in the centromere region, which corresponds to heteropycnotic knobs on the standard chromosome preparations. Morphological types and size differentiation of chromosomes in the karyotype suggest that the main chromosomal rearrangements in the evolution of Chthonius are centric or tandem fusions resulting in a decrease in the number of chromosomes. Pericentric inversions, inducing the change of acrocentric chromosomes into biarmed chromosomes, could also be expected. Variability in chromosome morphology and number was detected in several species: Chthonius (Chthonius) ischnocheles (Hermann, 1804), Chthonius (Chthonius) raridentatus, Chthonius (Chthonius) rhodochelatus Hadzi, 1930, and Chthonius (Chthonius) tenuis L. Koch, 1873. We discuss the intraspecific variability within these species and the potential existence of cryptic species.

Spiders on a Hot Volcanic Roof: Colonisation Pathways and Phylogeography of the Canary Islands Endemic Trap-Door Spider Titanidiops canariensis (Araneae, Idiopidae).

Studies conducted on volcanic islands have greatly contributed to our current understanding of how organisms diversify. The Canary Islands archipelago, located northwest of the coast of northern Africa, harbours a large number of endemic taxa. Because of their low vagility, mygalomorph spiders are usually absent from oceanic islands. The spider Titanidiops canariensis, which inhabits the easternmost islands of the archipelago, constitutes an exception to this rule. Here, we use a multi-locus approach that combines three mitochondrial and four nuclear genes to investigate the origins and phylogeography of this remarkable trap-door spider. We provide a timeframe for the colonisation of the Canary Islands using two alternative approaches: concatenation and species tree inference in a Bayesian relaxed clock framework. Additionally, we investigate the existence of cryptic species on the islands by means of a Bayesian multi-locus species delimitation method. Our results indicate that T. canariensis colonised the Canary Islands once, most likely during the Miocene, although discrepancies between the timeframes from different approaches make the exact timing uncertain. A complex evolutionary history for the species in the archipelago is revealed, which involves two independent colonisations of Fuerteventura from the ancestral range of T. canariensis in northern Lanzarote and a possible back colonisation of southern Lanzarote. The data further corroborate a previously proposed volcanic refugium, highlighting the impact of the dynamic volcanic history of the island on the phylogeographic patterns of the endemic taxa. T. canariensis includes at least two different species, one inhabiting the Jandia peninsula and central Fuerteventura and one spanning from central Fuerteventura to Lanzarote. Our data suggest that the extant northern African Titanidiops lineages may have expanded to the region after the islands were colonised and, hence, are not the source of colonisation. In addition, T. maroccanus may harbour several cryptic species.

Ancient origins of the Mediterranean trap-door spiders of the family Ctenizidae (Araneae, Mygalomorphae).

The family Ctenizidae is a worldwide-distributed trapdoor spider group, with a modest number of genera and species but interesting biogeography. Its monophyly has been questioned on the basis of both morphological and molecular evidence. The family is represented in the Mediterranean Basin by three genera and nine species: Cteniza and Cyrtocarenum, mostly endemic to the region, and Ummidia, long considered an anthropogenic introduction to the Mediterranean because the bulk of its diversity is in the New World. The taxonomic status of some of the species and genera (e.g. Mediterranean Ummidia species or Cteniza and Cyrtocarenum) has been called into question due to their close morphological affinities. Here, we use a multilocus approach that combines DNA sequence data from three nuclear genes 28S rRNA, EF1γ and H3 to investigate the origins and phylogenetic position of the Mediterranean taxa within the context of ctenizid generic-level diversity. For the first time, all known ctenizid genera are included in a phylogenetic analysis. Additionally, Bayesian relaxed clock methods and specific substitution rates are used to infer the timing of the group's diversification. Our results disagree with the traditional division of the family Ctenizidae into two subfamilies finding them polyphyletic and stress the need for re-evaluating the morphological characters that have been used in the group's classification. Time estimates indicate an ancient origin and long history of Mediterranean ctenizids. The present day disjunct distribution of Ummidia seems to be the result of the opening of the Atlantic Ocean, suggesting a former Laurasian distribution of the genus that is further supported by Baltic amber fossils. Similarly, the opening of the western Mediterranean Basin has likely played a key role in the diversification of both Ummidia and Cteniza, whereas the origin of Cyrtocarenum species preceded the breakup of the former continuous landmass that formed the Aegean region. Deep divergence times and reciprocal monophyly support the status of Cteniza and Cyrtocarenum as independent evolutionary lineages. Alternatively, the taxonomic status of Ummidia with regard to the closely related genus Conothele remains unclear; a more thorough sampling of the latter is needed to evaluate whether the synonymy of the two genera is necessary.