Genetic diversity of P1/pathogenic Leptospira species hosted by bats worldwide.

INTRODUCTION: Bats are a diverse group of mammals that have unique features allowing them to act as reservoir hosts for several zoonotic pathogens such as Leptospira. Leptospires have been classified into pathogenic, intermediate, and saprophytic groups and more recently into clades P1, P2, S1, and S2, being all the most important pathogenic species related to leptospirosis included within the P1/pathogenic clade. Leptospira has been detected from bats in several regions worldwide; however, the diversity of leptospires harboured by bats is still unknown. AIM: The aim of the present study was to determine the genetic diversity of Leptospira spp. harboured by bats worldwide. METHODS: A systematic review was conducted on four databases to retrieve studies in which Leptospira was detected from bats. All studies were screened to retrieve all available Leptospira spp. 16S rRNA sequences from the GenBank database and data regarding their origin. Sequences obtained were compared with each other and reference sequences of Leptospira species and analysed through phylogenetic analysis. RESULTS: A total of 418 Leptospira spp. 16S rRNA sequences isolated from 55 bat species from 14 countries were retrieved from 15 selected manuscripts. From these, 417 sequences clustered within the P1/pathogenic group, and only one sequence clustered within the P2/intermediate group. Six major clades of P1/pathogenic Leptospira spp. were identified, three of them composed exclusively of sequences obtained from bats. CONCLUSION: We identified that bats harbour a great genetic diversity of Leptospira spp. that form part of the P1/pathogenic clade, some of which are closely related to leptospirosis-associated species. This finding contributes to the knowledge of the diversity of leptospires hosted by bats worldwide and reinforces the role of bats as reservoirs of P1/pathogenic Leptospira spp.

Neglected no longer: Phylogenomic resolution of higher-level relationships in Solifugae.

Advanced sequencing technologies have expedited resolution of higher-level arthropod relationships. Yet, dark branches persist, principally among groups occurring in cryptic habitats. Among chelicerates, Solifugae ("camel spiders") is the last order lacking a higher-level phylogeny and have thus been historically characterized as "neglected [arachnid] cousins". Though renowned for aggression, remarkable running speed, and xeric adaptation, inferring solifuge relationships has been hindered by inaccessibility of diagnostic morphological characters, whereas molecular investigations have been limited to one of 12 recognized families. Our phylogenomic dataset via capture of ultraconserved elements sampling all extant families recovered a well-resolved phylogeny, with two distinct groups of New World taxa nested within a broader Paleotropical radiation. Divergence times using fossil calibrations inferred that Solifugae radiated by the Permian, and most families diverged prior to the Paleogene-Cretaceous extinction, likely driven by continental breakup. We establish Boreosolifugae new suborder uniting five Laurasian families, and Australosolifugae new suborder uniting seven Gondwanan families using morphological and biogeographic signal.

DNA isolation and genome sequence of the 134-year-old holotype specimen of Boletus subvelutipes Peck.

Molecular characterization of type specimens is a powerful tool used in clarifying species identity/circumscription, as well as establishing the taxonomic and phylogenetic status of organisms in question. However, DNA sequencing of aged herbarium collections can be a challenge due to the quantity and quality of DNA still present in the specimens. Herein, we report a custom DNA isolation protocol suitable for processing minute quantities of old specimen tissue and its utilization via high-throughput sequencing technologies to obtain, for the first time, the genome assembly of the 134-year-old holotype of Boletus subvelutipes Peck, a North American fleshy pored mushroom of taxonomic and historical significance. A side-by-side evaluation of our DNA isolation method with that of a commercial "kit" by Qiagen is also presented. By relying on the type material, we have established the genetic identity of B. subvelutipes, as well as providing preliminary phylogenetic evidence for its generic affinities in Neoboletus within Boletaceae. The reference genome of the B. subvelutipes holotype provides a resource for future comparative genomic studies, taxonomic revisions in Boletaceae, and other evolutionary studies of fungi.

In the land of the blind: Exceptional subterranean speciation of cryptic troglobitic spiders of the genus Tegenaria (Araneae: Agelenidae) in Israel.

Caves have long been recognized as a window into the mechanisms of diversification and convergent evolution, due to the unique conditions of isolation and life in the dark. These lead to adaptations and reduce dispersal and gene flow, resulting in high levels of speciation and endemism. The Israeli cave arachnofauna remains poorly known, but likely represents a rich assemblage. In a recent survey, we found troglophilic funnel-web spiders of the genus Tegenaria in 26 caves, present mostly at the cave entrance ecological zone. In addition, we identified at least 14 caves inhabited by troglobitic Tegenaria, which are present mostly in the twilight and dark ecological zones. Ten of the caves, located in the north and center of Israel, are inhabited by both troglophilic and troglobitic Tegenaria. These spiders bear superficial phenotypic similarities but differ in the levels of eye reduction and pigmentation. To test whether these taxa constitute separate species, as well as understand their relationships to epigean counterparts, we conducted a broad geographic sampling of cave-dwelling Tegenaria in Israel and Palestine, using morphological and molecular evidence. Counterintuitively, our results show that the troglobitic Tegenaria we studied are distantly related to the troglophilic Tegenaria found at each of the cave entrances we sampled. Moreover, seven new troglobitic species can be identified based on genetic differences, eye reduction level, and features of the female and male genitalia. Our COI analysis suggest that the Israeli troglobitic Tegenaria species are more closely related to eastern-Mediterranean congeners than to the local sympatric troglophile Tegenaria species, suggesting a complex biogeographic history.

Recent speciation and phenotypic plasticity within a parthenogenetic lineage of levantine whip spiders (Chelicerata: Amblypygi: Charinidae).

Caves constitute ideal study systems for investigating adaptation and speciation, as the abiotic conditions shared by aphotic habitats exert a set of environmental filters on their communities. Arachnids constitute an important component of many cave ecosystems worldwide. We investigated the population genomics of two whip spider species: Sarax ioanniticus, a widely distributed parthenogenetic species found across the eastern Mediterranean; and S. israelensis, a recently described troglomorphic species that is endemic to caves in Israel. Here, we show that S. israelensis is completely genetically distinct from S. ioanniticus and most likely also a parthenogen. Counterintuitively, despite the lack of genetic variability within S. ioanniticus and S. israelensis, we discovered considerable variation in the degree of median eye reduction, particularly in the latter species. Natural history data from captive-bred specimens of S. israelensis validated the interpretation of parthenogenesis. Our results are most consistent with a scenario of a sexual ancestral species that underwent speciation, followed by independent transitions to apomictic parthenogenesis in each of the two daughter species. Moreover, the lack of genetic variability suggests that variation in eye morphology in S. israelensis is driven exclusively by epigenetic mechanisms.

Phylogenomics of Scorpions Reveal Contemporaneous Diversification of Scorpion Mammalian Predators and Mammal-Active Sodium Channel Toxins.

Scorpions constitute a charismatic lineage of arthropods and comprise more than 2500 described species. Found throughout various tropical and temperate habitats, these predatory arachnids have a long evolutionary history, with a fossil record that began in the Silurian. While all scorpions are venomous, the asymmetrically diverse family Buthidae harbors nearly half the diversity of extant scorpions, and all but one of the 58 species that are medically significant to humans. However, the lack of a densely sampled scorpion phylogeny has hindered broader inferences of the diversification dynamics of scorpion toxins. To redress this gap, we assembled a phylogenomic data set of 100 scorpion venom gland transcriptomes and genomes, emphasizing the sampling of highly toxic buthid genera. To infer divergence times of venom gene families, we applied a phylogenomic node dating approach for the species tree in tandem with phylostratigraphic bracketing to estimate the minimum ages of mammal-specific toxins. Our analyses establish a robustly supported phylogeny of scorpions, particularly with regard to relationships between medically significant taxa. Analysis of venom gene families shows that mammal-active sodium channel toxins (NaTx) have independently evolved in five lineages within Buthidae. Temporal windows of mammal-targeting toxin origins are correlated with the basal diversification of major scorpion mammal predators such as shrews, bats, and rodents. These results suggest an evolutionary model of relatively recent diversification of buthid NaTx homologs in response to the diversification of scorpion predators. [Adaptation; arachnids; phylogenomic dating; phylostratigraphy; venom.].

Comprehensive Species Sampling and Sophisticated Algorithmic Approaches Refute the Monophyly of Arachnida.

Deciphering the evolutionary relationships of Chelicerata (arachnids, horseshoe crabs, and allied taxa) has proven notoriously difficult, due to their ancient rapid radiation and the incidence of elevated evolutionary rates in several lineages. Although conflicting hypotheses prevail in morphological and molecular data sets alike, the monophyly of Arachnida is nearly universally accepted, despite historical lack of support in molecular data sets. Some phylotranscriptomic analyses have recovered arachnid monophyly, but these did not sample all living orders, whereas analyses including all orders have failed to recover Arachnida. To understand this conflict, we assembled a data set of 506 high-quality genomes and transcriptomes, sampling all living orders of Chelicerata with high occupancy and rigorous approaches to orthology inference. Our analyses consistently recovered the nested placement of horseshoe crabs within a paraphyletic Arachnida. This result was insensitive to variation in evolutionary rates of genes, complexity of the substitution models, and alternative algorithmic approaches to species tree inference. Investigation of sources of systematic bias showed that genes and sites that recover arachnid monophyly are enriched in noise and exhibit low information content. To test the impact of morphological data, we generated a 514-taxon morphological data matrix of extant and fossil Chelicerata, analyzed in tandem with the molecular matrix. Combined analyses recovered the clade Merostomata (the marine orders Xiphosura, Eurypterida, and Chasmataspidida), but merostomates appeared nested within Arachnida. Our results suggest that morphological convergence resulting from adaptations to life in terrestrial habitats has driven the historical perception of arachnid monophyly, paralleling the history of numerous other invertebrate terrestrial groups.

The genome of a daddy-long-legs (Opiliones) illuminates the evolution of arachnid appendages.

Chelicerate arthropods exhibit dynamic genome evolution, with ancient whole-genome duplication (WGD) events affecting several orders. Yet, genomes remain unavailable for a number of poorly studied orders, such as Opiliones (daddy-long-legs), which has hindered comparative study. We assembled the first harvestman draft genome for the species Phalangium opilio, which bears elongate, prehensile appendages, made possible by numerous distal articles called tarsomeres. Here, we show that the genome of P. opilio exhibits a single Hox cluster and no evidence of WGD. To investigate the developmental genetic basis for the quintessential trait of this group-the elongate legs-we interrogated the function of the Hox genes Deformed (Dfd) and Sex combs reduced (Scr), and a homologue of Epidermal growth factor receptor (Egfr). Knockdown of Dfd incurred homeotic transformation of two pairs of legs into pedipalps, with dramatic shortening of leg segments in the longest leg pair, whereas homeosis in L3 is only achieved upon double Dfd + Scr knockdown. Knockdown of Egfr incurred shortened appendages and the loss of tarsomeres. The similarity of Egfr loss-of-function phenotypic spectra in insects and this arachnid suggest that repeated cooption of EGFR signalling underlies the independent gains of supernumerary tarsomeres across the arthropod tree of life.

Taxonomic Sampling and Rare Genomic Changes Overcome Long-Branch Attraction in the Phylogenetic Placement of Pseudoscorpions.

Long-branch attraction is a systematic artifact that results in erroneous groupings of fast-evolving taxa. The combination of short, deep internodes in tandem with long-branch attraction artifacts has produced empirically intractable parts of the Tree of Life. One such group is the arthropod subphylum Chelicerata, whose backbone phylogeny has remained unstable despite improvements in phylogenetic methods and genome-scale data sets. Pseudoscorpion placement is particularly variable across data sets and analytical frameworks, with this group either clustering with other long-branch orders or with Arachnopulmonata (scorpions and tetrapulmonates). To surmount long-branch attraction, we investigated the effect of taxonomic sampling via sequential deletion of basally branching pseudoscorpion superfamilies, as well as varying gene occupancy thresholds in supermatrices. We show that concatenated supermatrices and coalescent-based summary species tree approaches support a sister group relationship of pseudoscorpions and scorpions, when more of the basally branching taxa are sampled. Matrix completeness had demonstrably less influence on tree topology. As an external arbiter of phylogenetic placement, we leveraged the recent discovery of an ancient genome duplication in the common ancestor of Arachnopulmonata as a litmus test for competing hypotheses of pseudoscorpion relationships. We generated a high-quality developmental transcriptome and the first genome for pseudoscorpions to assess the incidence of arachnopulmonate-specific duplications (e.g., homeobox genes and miRNAs). Our results support the inclusion of pseudoscorpions in Arachnopulmonata (new definition), as the sister group of scorpions. Panscorpiones (new name) is proposed for the clade uniting Scorpiones and Pseudoscorpiones.

Phylogenomic Resolution of Sea Spider Diversification through Integration of Multiple Data Classes.

Despite significant advances in invertebrate phylogenomics over the past decade, the higher-level phylogeny of Pycnogonida (sea spiders) remains elusive. Due to the inaccessibility of some small-bodied lineages, few phylogenetic studies have sampled all sea spider families. Previous efforts based on a handful of genes have yielded unstable tree topologies. Here, we inferred the relationships of 89 sea spider species using targeted capture of the mitochondrial genome, 56 conserved exons, 101 ultraconserved elements, and 3 nuclear ribosomal genes. We inferred molecular divergence times by integrating morphological data for fossil species to calibrate 15 nodes in the arthropod tree of life. This integration of data classes resolved the basal topology of sea spiders with high support. The enigmatic family Austrodecidae was resolved as the sister group to the remaining Pycnogonida and the small-bodied family Rhynchothoracidae as the sister group of the robust-bodied family Pycnogonidae. Molecular divergence time estimation recovered a basal divergence of crown group sea spiders in the Ordovician. Comparison of diversification dynamics with other marine invertebrate taxa that originated in the Paleozoic suggests that sea spiders and some crustacean groups exhibit resilience to mass extinction episodes, relative to mollusk and echinoderm lineages.

Systemic paralogy and function of retinal determination network homologs in arachnids.

BACKGROUND: Arachnids are important components of cave ecosystems and display many examples of troglomorphisms, such as blindness, depigmentation, and elongate appendages. Little is known about how the eyes of arachnids are specified genetically, let alone the mechanisms for eye reduction and loss in troglomorphic arachnids. Additionally, duplication of Retinal Determination Gene Network (RDGN) homologs in spiders has convoluted functional inferences extrapolated from single-copy homologs in pancrustacean models. RESULTS: We investigated a sister species pair of Israeli cave whip spiders, Charinus ioanniticus and C. israelensis (Arachnopulmonata, Amblypygi), of which one species has reduced eyes. We generated embryonic transcriptomes for both Amblypygi species, and discovered that several RDGN homologs exhibit duplications. We show that duplication of RDGN homologs is systemic across arachnopulmonates (arachnid orders that bear book lungs), rather than being a spider-specific phenomenon. A differential gene expression (DGE) analysis comparing the expression of RDGN genes in field-collected embryos of both species identified candidate RDGN genes involved in the formation and reduction of eyes in whip spiders. To ground bioinformatic inference of expression patterns with functional experiments, we interrogated the function of three candidate RDGN genes identified from DGE using RNAi in the spider Parasteatoda tepidariorum. We provide functional evidence that one of these paralogs, sine oculis/Six1 A (soA), is necessary for the development of all arachnid eye types. CONCLUSIONS: Our work establishes a foundation to investigate the genetics of troglomorphic adaptations in cave arachnids, and links differential gene expression to an arthropod eye phenotype for the first time outside of Pancrustacea. Our results support the conservation of at least one RDGN component across Arthropoda and provide a framework for identifying the role of gene duplications in generating arachnid eye diversity.

Ordered phylogenomic subsampling enables diagnosis of systematic errors in the placement of the enigmatic arachnid order Palpigradi.

The miniaturized arachnid order Palpigradi has ambiguous phylogenetic affinities owing to its odd combination of plesiomorphic and derived morphological traits. This lineage has never been sampled in phylogenomic datasets because of the small body size and fragility of most species, a sampling gap of immediate concern to recent disputes over arachnid monophyly. To redress this gap, we sampled a population of the cave-inhabiting species Eukoenenia spelaea from Slovakia and inferred its placement in the phylogeny of Chelicerata using dense phylogenomic matrices of up to 1450 loci, drawn from high-quality transcriptomic libraries and complete genomes. The complete matrix included exemplars of all extant orders of Chelicerata. Analyses of the complete matrix recovered palpigrades as the sister group of the long-branch order Parasitiformes (ticks) with high support. However, sequential deletion of long-branch taxa revealed that the position of palpigrades is prone to topological instability. Phylogenomic subsampling approaches that maximized taxon or dataset completeness recovered palpigrades as the sister group of camel spiders (Solifugae), with modest support. While this relationship is congruent with the location and architecture of the coxal glands, a long-forgotten character system that opens in the pedipalpal segments only in palpigrades and solifuges, we show that nodal support values in concatenated supermatrices can mask high levels of underlying topological conflict in the placement of the enigmatic Palpigradi.

A Critical Appraisal of the Placement of Xiphosura (Chelicerata) with Account of Known Sources of Phylogenetic Error.

Horseshoe crabs (Xiphosura) are traditionally regarded as sister group to the clade of terrestrial chelicerates (Arachnida). This hypothesis has been challenged by recent phylogenomic analyses, but the non-monophyly of Arachnida has consistently been disregarded as artifactual. We re-evaluated the placement of Xiphosura among chelicerates using the most complete phylogenetic data set to date, expanding outgroup sampling, and including data from whole genome sequencing projects. In spite of uncertainty in the placement of some arachnid clades, all analyses show Xiphosura consistently nested within Arachnida as the sister group to Ricinulei (hooded tick spiders). It is apparent that the radiation of arachnids is an old one and occurred over a brief period of time, resulting in several consecutive short internodes, and thus is a potential case for the confounding effects of incomplete lineage sorting (ILS). We simulated coalescent gene trees to explore the effects of increasing levels of ILS on the placement of horseshoe crabs. In addition, common sources of systematic error were evaluated, as well as the effects of fast-evolving partitions and the dynamics of problematic long branch orders. Our results indicated that the placement of horseshoe crabs cannot be explained by missing data, compositional biases, saturation, or ILS. Interrogation of the phylogenetic signal showed that the majority of loci favor the derived placement of Xiphosura over a monophyletic Arachnida. Our analyses support the inference that horseshoe crabs represent a group of aquatic arachnids, comparable to aquatic mites, breaking a long-standing paradigm in chelicerate evolution and altering previous interpretations of the ancestral transition to the terrestrial habitat. Future studies testing chelicerate relationships should approach the task with a sampling strategy where the monophyly of Arachnida is not held as the premise.

Integration of phylogenomics and molecular modeling reveals lineage-specific diversification of toxins in scorpions.

Scorpions have evolved a variety of toxins with a plethora of biological targets, but characterizing their evolution has been limited by the lack of a comprehensive phylogenetic hypothesis of scorpion relationships grounded in modern, genome-scale datasets. Disagreements over scorpion higher-level systematics have also incurred challenges to previous interpretations of venom families as ancestral or derived. To redress these gaps, we assessed the phylogenomic relationships of scorpions using the most comprehensive taxonomic sampling to date. We surveyed genomic resources for the incidence of calcins (a type of calcium channel toxin), which were previously known only from 16 scorpion species. Here, we show that calcins are diverse, but phylogenetically restricted only to parvorder Iurida, one of the two basal branches of scorpions. The other branch of scorpions, Buthida, bear the related LKTx toxins (absent in Iurida), but lack calcins entirely. Analysis of sequences and molecular models demonstrates remarkable phylogenetic inertia within both calcins and LKTx genes. These results provide the first synapomorphies (shared derived traits) for the recently redefined clades Buthida and Iurida, constituting the only known case of such traits defined from the morphology of molecules.

Phylogenomics, Diversification Dynamics, and Comparative Transcriptomics across the Spider Tree of Life.

Dating back to almost 400 mya, spiders are among the most diverse terrestrial predators [1]. However, despite considerable effort [1-9], their phylogenetic relationships and diversification dynamics remain poorly understood. Here, we use a synergistic approach to study spider evolution through phylogenomics, comparative transcriptomics, and lineage diversification analyses. Our analyses, based on ca. 2,500 genes from 159 spider species, reject a single origin of the orb web (the "ancient orb-web hypothesis") and suggest that orb webs evolved multiple times since the late Triassic-Jurassic. We find no significant association between the loss of foraging webs and increases in diversification rates, suggesting that other factors (e.g., habitat heterogeneity or biotic interactions) potentially played a key role in spider diversification. Finally, we report notable genomic differences in the main spider lineages: while araneoids (ecribellate orb-weavers and their allies) reveal an enrichment in genes related to behavior and sensory reception, the retrolateral tibial apophysis (RTA) clade-the most diverse araneomorph spider lineage-shows enrichment in genes related to immune responses and polyphenic determination. This study, one of the largest invertebrate phylogenomic analyses to date, highlights the usefulness of transcriptomic data not only to build a robust backbone for the Spider Tree of Life, but also to address the genetic basis of diversification in the spider evolutionary chronicle.

Species delimitation of the North American orchard-spider Leucauge venusta (Walckenaer, 1841) (Araneae, Tetragnathidae).

The orchard spider, Leucauge venusta (Walckenaer, 1841) is one of the most common and abundant orb-weavers in North America. This species has a broad geographic distribution extending across tropical and temperate regions of the Americas from Canada to Brazil. Guided by a preliminary observation of the barcode gap between sequences from specimens of L. venusta collected in Florida and other North American localities, we collected across a transect through the southeastern USA to investigate the observed genetic divide. The dataset, complemented with additional samples from Mexico, and Brazil was analyzed for species delimitation using STACEY and bGMYC based on sequences from one nuclear (ITS2) and one mitochondrial marker (COI). The analyses clearly separate USA samples into two deeply divergent and geographically structured groups (north-south) which we interpret as two different species. We generated ecological niche models for these two groups rejecting a niche equivalence hypothesis for these lineages. Taxonomic changes are proposed based on these findings, Leucauge venusta is restricted to denote the northern clade, and its known distribution restricted to the USA. Leucauge argyrobapta (White, 1841) is removed from synonymy to denote the populations in Florida, Mexico and Brazil. Although the delimitation analyses suggest each of these geographic clusters within the L. argyrobapta samples represent different species, more specimens from Central and South America are needed to properly test the cohesion of L. argyrobapta populations.

A New Orthology Assessment Method for Phylogenomic Data: Unrooted Phylogenetic Orthology.

Current sequencing technologies are making available unprecedented amounts of genetic data for a large variety of species including nonmodel organisms. Although many phylogenomic surveys spend considerable time finding orthologs from the wealth of sequence data, these results do not transcend the original study and after being processed for specific phylogenetic purposes these orthologs do not become stable orthology hypotheses. We describe a procedure to detect and document the phylogenetic distribution of orthologs allowing researchers to use this information to guide selection of loci best suited to test specific evolutionary questions. At the core of this pipeline is a new phylogenetic orthology method that is neither affected by the position of the root nor requires explicit assignment of outgroups. We discuss the properties of this new orthology assessment method and exemplify its utility for phylogenomics using a small insects dataset. In addition, we exemplify the pipeline to identify and document stable orthologs for the group of orb-weaving spiders (Araneoidea) using RNAseq data. The scripts used in this study, along with sample files and additional documentation, are available at https://github.com/ballesterus/UPhO.

Resolving the phylogeny of a speciose spider group, the family Linyphiidae (Araneae).

For high-level molecular phylogenies, a comprehensive sampling design is a key factor for not only improving inferential accuracy, but also for maximizing the explanatory power of the resulting phylogeny. Two standing problems in molecular phylogenies are the unstable placements of some deep and long branches, and the phylogenetic relationships shown by robust supported clades conflict with recognized knowledge. Empirical and theoretical studies suggest that increasing taxon sampling is expected to ameliorate, if not resolve, both problems; however, sometimes neither the current taxonomic system nor the established phylogeny can provide sufficient information to guide additional sampling design. We examined the phylogeny of the spider family Linyphiidae, and selected ingroup species based on epigynal morphology, which can be reconstructed in a phylogenetic context. Our analyses resulted in seven robustly supported clades within linyphiids. The placements of four deep and long branches are sensitive to variations in both outgroup and ingroup sampling, suggesting the possibility of long branch attraction artifacts. Results of ancestral state reconstruction indicate that successive state transformations of the epigynal plate are associated with early cladogenetic events in linyphiid diversification. Representatives of different subfamilies were mixed together within well supported clades and examination revealed that their defining characters, as per traditional taxonomy, are homoplastic. Furthermore, our results demonstrated that increasing taxon sampling produced a more informative framework, which in turn helps to study character evolution and interpret the relationships among linyphiid lineages. Additional defining characters are needed to revise the linyphiid taxonomic system based on our phylogenetic hypothesis.