Increasing Information Content and Diagnosability in Family-Level Classifications.

Higher-level classifications often must account for monotypic taxa representing depauperate evolutionary lineages and lacking synapomorphies of their better-known, well-defined sister clades. In a ranked (Linnean) or unranked (phylogenetic) classification system, discovering such a depauperate taxon does not necessarily invalidate the rank classification of sister clades. Named higher taxa must be monophyletic to be phylogenetically valid. Ranked taxa above the species level should also maximize information content, diagnosability, and utility (e.g., in biodiversity conservation). In spider classification, families are the highest rank that is systematically catalogued, and incertae sedis is not allowed. Consequently, it is important that family-level taxa be well defined and informative. We revisit the classification problem of Orbipurae, an unranked suprafamilial clade containing the spider families Nephilidae, Phonognathidae, and Araneidae sensu stricto. We argue that, to maximize diagnosability, information content, conservation utility, and practical taxonomic considerations, this "splitting" scheme is superior to its recently proposed alternative, which lumps these families together as Araneidae sensu lato. We propose to redefine Araneidae and recognize a monogeneric spider family, Paraplectanoididae fam. nov. to accommodate the depauperate lineage Paraplectanoides. We present new subgenomic data to stabilize Orbipurae topology which also supports our proposed family-level classification. Our example from spiders demonstrates why classifications must be able to accommodate depauperate evolutionary lineages, for example, Paraplectanoides. Finally, although clade age should not be a criterion to determine rank, other things being equal, comparable ages of similarly ranked taxa do benefit comparative biology. [Classification, family rank, phylogenomics, systematics, monophyly, spider phylogeny.].

Light Environment Interacts with Visual Displays in a Species-Specific Manner in Multimodal-Signaling Wolf Spiders.

AbstractLight availability is highly variable, yet predictable, over various timescales and is expected to play an important role in the evolution of visual signals. Courtship displays of the wolf spider genus Schizocosa always involve the use of substrate-borne vibrations; however, there is substantial variation in the presence and complexity of visual displays among species. To gain insight into the role the light environment plays in the evolution of courtship displays, we tested the function of visual courtship signaling across distinct light environments in four species of Schizocosa that vary in their degree of ornamentation and dynamic visual signals. We ran mating and courtship trials at three light intensities (bright, dim, and dark) and tested the hypothesis that ornamentation interacts with light environment. We also examined each species' circadian activity patterns. The effects of the light environment on courtship and mating varied between species, as did circadian activity patterns. Our results suggest that femur pigmentation may have evolved for diurnal signaling, whereas tibial brushes may function to increase signal efficacy under dim light. Additionally, we found evidence for light-dependent changes in selection on male traits, illustrating that short-term changes in light intensity have the potential for strong effects on the dynamics of sexual selection.

Phylogeny and secondary sexual trait evolution in Schizocosa wolf spiders (Araneae, Lycosidae) shows evidence for multiple gains and losses of ornamentation and species delimitation uncertainty.

Members of the Nearctic spider genus Schizocosa Chamberlin, 1904 have garnered much attention in behavioral studies and over many decades, a number of species have developed as model systems for investigating patterns of sexual selection and multimodal communication. Many of these studies have employed a comparative approach using putative, but not rigorously tested, sister species pairs that have distinctive morphological traits and attendant behaviors. Despite past emphasis on the efficacy of these presumably comparative-based studies of closely related species, generating a robust phylogenetic hypothesis for Schizocosa has been an ongoing challenge. Here, we apply a phylogenomic approach using anchored hybrid enrichment to generate a data set comprising over 400 loci representing a comprehensive taxonomic sample of 23 Nearctic Schizocosa. Our sampling also includes numerous outgroup lycosid genera that allow for a robust evaluation of genus monophyly. Based on analyses using concatenation and coalescent-based methods, we recover a well-supported phylogeny that infers the following: 1) The New World Schizocosa do not form a monophyletic group; 2) Previous hypotheses of North American species require reconsideration along with the composition of species groups; 3) Multiple longstanding model species are not genealogically exclusive and thus are not "good" species; 4) This updated phylogenetic framework establishes a new working paradigm for studying the evolution of characters associated with reproductive communication and mating. Ancestral character state reconstructions show a complex pattern of homoplasy that has likely obfuscated previous attempts to reconstruct relationships and delimit species. Important characters presumably related to sexual selection, such as foreleg pigmentation and dense bristle formation, have undergone repeated gain and loss events, many of which have led to increased morphological divergence between sister-species. Evaluation of these traits in a comparative framework illuminates how sexual selection and natural selection influence character evolution and provides a model for future studies of multimodal communication evolution and function.

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.

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.].

Shifting evolutionary sands: transcriptome characterization of the Aptostichus atomarius species complex.

BACKGROUND: Mygalomorph spiders represent a diverse, yet understudied lineage for which genomic level data has only recently become accessible through high-throughput genomic and transcriptomic sequencing methods. The Aptostichus atomarius species complex (family Euctenizidae) includes two coastal dune endemic members, each with inland sister species - affording exploration of dune adaptation associated patterns at the transcriptomic level. We apply an RNAseq approach to examine gene family conservation across the species complex and test for patterns of positive selection along branches leading to dune endemic species. RESULTS: An average of ~ 44,000 contigs were assembled for eight spiders representing dune (n = 2), inland (n = 4), and atomarius species complex outgroup taxa (n = 2). Transcriptomes were estimated to be 64% complete on average with 77 spider reference orthologs missing from all taxa. Over 18,000 orthologous gene clusters were identified within the atomarius complex members, > 5000 were detected in all species, and ~ 4700 were shared between species complex members and outgroup Aptostichus species. Gene family analysis with the FUSTr pipeline identified 47 gene families appearing to be under selection in the atomarius ingroup; four of the five top clusters include sequences strongly resembling other arthropod venom peptides. The COATS pipeline identified six gene clusters under positive selection on branches leading to dune species, three of which reflected the preferred species tree. Genes under selection were identified as Cytochrome P450 2c15 (also recovered in the FUSTr analysis), Niemann 2 Pick C1-like, and Kainate 2 isoform X1. CONCLUSIONS: We have generated eight draft transcriptomes for a closely related and ecologically diverse group of trapdoor spiders, identifying venom gene families potentially under selection across the Aptostichus atomarius complex and chemosensory-associated gene families under selection in dune endemic lineages.

Integrative species delimitation reveals cryptic diversity in the southern Appalachian Antrodiaetus unicolor (Araneae: Antrodiaetidae) species complex.

Although species delimitation can be highly contentious, the development of reliable methods to accurately ascertain species boundaries is an imperative step in cataloguing and describing Earth's quickly disappearing biodiversity. Spider species delimitation remains largely based on morphological characters; however, many mygalomorph spider populations are morphologically indistinguishable from each other yet have considerable molecular divergence. The focus of our study, the Antrodiaetus unicolor species complex containing two sympatric species, exhibits this pattern of relative morphological stasis with considerable genetic divergence across its distribution. A past study using two molecular markers, COI and 28S, revealed that A. unicolor is paraphyletic with respect to A. microunicolor. To better investigate species boundaries in the complex, we implement the cohesion species concept and use multiple lines of evidence for testing genetic exchangeability and ecological interchangeability. Our integrative approach includes extensively sampling homologous loci across the genome using a RADseq approach (3RAD), assessing population structure across their geographic range using multiple genetic clustering analyses that include structure, principal components analysis and a recently developed unsupervised machine learning approach (Variational Autoencoder). We evaluate ecological similarity by using large-scale ecological data for niche-based distribution modelling. Based on our analyses, we conclude that this complex has at least one additional species as well as confirm species delimitations based on previous less comprehensive approaches. Our study demonstrates the efficacy of genomic-scale data for recognizing cryptic species, suggesting that species delimitation with one data type, whether one mitochondrial gene or morphology, may underestimate true species diversity in morphologically homogenous taxa with low vagility.

Molecular species delimitation in the primitively segmented spider genus Heptathela endemic to Japanese islands.

Determining species boundaries forms an important foundation for biological research. However, the results of molecular species delimitation can vary with the data sets and methods that are used. Here we use a two-step approach to delimit species in the genus Heptathela, a group of primitively segmented trapdoor spiders that are endemic to Japanese islands. Morphological evidence suggests the existence of 19 species in the genus. We tested this initial species hypothesis by using six molecular species-delimitation methods to analyse 180 mitochondrial COI sequences of Heptathela sampled from across the known range of the genus. We then conducted a set of more focused analyses by sampling additional genetic markers from the subset of taxa that were inconsistently delimited by the single-locus analyses of mitochondrial DNA. Multilocus species delimitation was performed using two Bayesian approaches based on the multispecies coalescent. Our approach identified 20 putative species among the 180 sampled individuals of Heptathela. We suggest that our two-step approach provides an efficient strategy for delimiting species while minimizing costs and computational time.

Golden Orbweavers Ignore Biological Rules: Phylogenomic and Comparative Analyses Unravel a Complex Evolution of Sexual Size Dimorphism.

Instances of sexual size dimorphism (SSD) provide the context for rigorous tests of biological rules of size evolution, such as Cope's rule (phyletic size increase), Rensch's rule (allometric patterns of male and female size), as well as male and female body size optima. In certain spider groups, such as the golden orbweavers (Nephilidae), extreme female-biased SSD (eSSD, female:male body length $\ge$2) is the norm. Nephilid genera construct webs of exaggerated proportions, which can be aerial, arboricolous, or intermediate (hybrid). First, we established the backbone phylogeny of Nephilidae using 367 anchored hybrid enrichment markers, then combined these data with classical markers for a reference species-level phylogeny. Second, we used the phylogeny to test Cope and Rensch's rules, sex specific size optima, and the coevolution of web size, type, and features with female and male body size and their ratio, SSD. Male, but not female, size increases significantly over time, and refutes Cope's rule. Allometric analyses reject the converse, Rensch's rule. Male and female body sizes are uncorrelated. Female size evolution is random, but males evolve toward an optimum size (3.2-4.9 mm). Overall, female body size correlates positively with absolute web size. However, intermediate sized females build the largest webs (of the hybrid type), giant female Nephila and Trichonephila build smaller webs (of the aerial type), and the smallest females build the smallest webs (of the arboricolous type). We propose taxonomic changes based on the criteria of clade age, monophyly and exclusivity, classification information content, and diagnosability. Spider families, as currently defined, tend to be between 37 million years old and 98 million years old, and Nephilidae is estimated at 133 Ma (97-146), thus deserving family status. We, therefore, resurrect the family Nephilidae Simon 1894 that contains Clitaetra Simon 1889, the Cretaceous GeratonephilaPoinar and Buckley (2012), Herennia Thorell 1877, IndoetraKuntner 2006, new rank, Nephila Leach 1815, Nephilengys L. Koch 1872, Nephilingis Kuntner 2013, Palaeonephila Wunderlich 2004 from Tertiary Baltic amber, and TrichonephilaDahl 1911, new rank. We propose the new clade Orbipurae to contain Araneidae Clerck 1757, Phonognathidae Simon 1894, new rank, and Nephilidae. Nephilid female gigantism is a phylogenetically ancient phenotype (over 100 Ma), as is eSSD, though their magnitudes vary by lineage.

A tangle of forms and phylogeny: Extensive morphological homoplasy and molecular clock heterogeneity in Bonnetina and related tarantulas.

Tarantula spider systematics has long been considered problematic. Species diagnosis and phylogenetic hypotheses have historically relied on morphological features, which are known to be relatively conserved and/or highly homoplastic across the family. Morphology-based attempts to clarify the phylogeny of the highly diverse New World Theraphosinae, have only been moderately successful, and the time-frame of tarantulas' evolution is nearly terra incognita. Here we present a molecular phylogenetic analysis of the Theraphosinae genus Bonnetina and related lineages, employing one mitochondrial (COI) and five nuclear (ITS1, EF1G, MID1IP1, MRPL44, and I3568) loci. We also perform ancestral state reconstruction of a newly formulated morphological data matrix. Our analysis includes 47 species placed in 17 genera and other undetermined lineages. We obtained well resolved and supported topologies. COI and EF1G substitution rates were much lower than the values generally accepted for mygalomorph evolution, with substantial rate heterogeneity among lineages. The origin of Theraphosinae was dated during the Late Cretaceous, followed by rapid diversification into the three recently proposed Theraphosinae tribes. North and Central American Hapalopini (including Bonnetina) form a monophyletic group that likely originated during the Oligocene to a dispersing ancestor from the then isolated South America. A clade that includes all but one Bonnetina species is estimated to have originated in the early Miocene and is the sister group of two morphologically divergent undescribed species. Morphological homoplasy is extensive across the tree. The two features that diagnose Bonnetina are homoplastic, but in combination still define the genus. Finally, we establish three groups of species within Bonnetina. Our results challenge the reliability of morphological characters for phylogenetic reconstruction in Theraphosinae, and indicate caution when interpreting Theraphosidae supra-specific classification in absence of a solid phylogenetic framework. They also question the dependability of universal substitution rates of COI and EF1G to calibrate phylogenetic analyses across Mygalomorphae.

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.

The spider tree of life: phylogeny of Araneae based on target-gene analyses from an extensive taxon sampling.

We present a phylogenetic analysis of spiders using a dataset of 932 spider species, representing 115 families (only the family Synaphridae is unrepresented), 700 known genera, and additional representatives of 26 unidentified or undescribed genera. Eleven genera of the orders Amblypygi, Palpigradi, Schizomida and Uropygi are included as outgroups. The dataset includes six markers from the mitochondrial (12S, 16S, COI) and nuclear (histone H3, 18S, 28S) genomes, and was analysed by multiple methods, including constrained analyses using a highly supported backbone tree from transcriptomic data. We recover most of the higher-level structure of the spider tree with good support, including Mesothelae, Opisthothelae, Mygalomorphae and Araneomorphae. Several of our analyses recover Hypochilidae and Filistatidae as sister groups, as suggested by previous transcriptomic analyses. The Synspermiata are robustly supported, and the families Trogloraptoridae and Caponiidae are found as sister to the Dysderoidea. Our results support the Lost Tracheae clade, including Pholcidae, Tetrablemmidae, Diguetidae, Plectreuridae and the family Pacullidae (restored status) separate from Tetrablemmidae. The Scytodoidea include Ochyroceratidae along with Sicariidae, Scytodidae, Drymusidae and Periegopidae; our results are inconclusive about the separation of these last two families. We did not recover monophyletic Austrochiloidea and Leptonetidae, but our data suggest that both groups are more closely related to the Cylindrical Gland Spigot clade rather than to Synspermiata. Palpimanoidea is not recovered by our analyses, but also not strongly contradicted. We find support for Entelegynae and Oecobioidea (Oecobiidae plus Hersiliidae), and ambiguous placement of cribellate orb-weavers, compatible with their non-monophyly. Nicodamoidea (Nicodamidae plus Megadictynidae) and Araneoidea composition and relationships are consistent with recent analyses. We did not obtain resolution for the titanoecoids (Titanoecidae and Phyxelididae), but the Retrolateral Tibial Apophysis clade is well supported. Penestomidae, and probably Homalonychidae, are part of Zodarioidea, although the latter family was set apart by recent transcriptomic analyses. Our data support a large group that we call the marronoid clade (including the families Amaurobiidae, Desidae, Dictynidae, Hahniidae, Stiphidiidae, Agelenidae and Toxopidae). The circumscription of most marronoid families is redefined here. Amaurobiidae include the Amaurobiinae and provisionally Macrobuninae. We transfer Malenellinae (Malenella, from Anyphaenidae), Chummidae (Chumma) (new syn.) and Tasmarubriinae (Tasmarubrius, Tasmabrochus and Teeatta, from Amphinectidae) to Macrobuninae. Cybaeidae are redefined to include Calymmaria, Cryphoeca, Ethobuella and Willisius (transferred from Hahniidae), and Blabomma and Yorima (transferred from Dictynidae). Cycloctenidae are redefined to include Orepukia (transferred from Agelenidae) and Pakeha and Paravoca (transferred from Amaurobiidae). Desidae are redefined to include five subfamilies: Amphinectinae, with Amphinecta, Mamoea, Maniho, Paramamoea and Rangitata (transferred from Amphinectidae); Ischaleinae, with Bakala and Manjala (transferred from Amaurobiidae) and Ischalea (transferred from Stiphidiidae); Metaltellinae, with Austmusia, Buyina, Calacadia, Cunnawarra, Jalkaraburra, Keera, Magua, Metaltella, Penaoola and Quemusia; Porteriinae (new rank), with Baiami, Cambridgea, Corasoides and Nanocambridgea (transferred from Stiphidiidae); and Desinae, with Desis, and provisionally Poaka (transferred from Amaurobiidae) and Barahna (transferred from Stiphidiidae). Argyroneta is transferred from Cybaeidae to Dictynidae. Cicurina is transferred from Dictynidae to Hahniidae. The genera Neoramia (from Agelenidae) and Aorangia, Marplesia and Neolana (from Amphinectidae) are transferred to Stiphidiidae. The family Toxopidae (restored status) includes two subfamilies: Myroinae, with Gasparia, Gohia, Hulua, Neomyro, Myro, Ommatauxesis and Otagoa (transferred from Desidae); and Toxopinae, with Midgee and Jamara, formerly Midgeeinae, new syn. (transferred from Amaurobiidae) and Hapona, Laestrygones, Lamina, Toxops and Toxopsoides (transferred from Desidae). We obtain a monophyletic Oval Calamistrum clade and Dionycha; Sparassidae, however, are not dionychans, but probably the sister group of those two clades. The composition of the Oval Calamistrum clade is confirmed (including Zoropsidae, Udubidae, Ctenidae, Oxyopidae, Senoculidae, Pisauridae, Trechaleidae, Lycosidae, Psechridae and Thomisidae), affirming previous findings on the uncertain relationships of the "ctenids" Ancylometes and Cupiennius, although a core group of Ctenidae are well supported. Our data were ambiguous as to the monophyly of Oxyopidae. In Dionycha, we found a first split of core Prodidomidae, excluding the Australian Molycriinae, which fall distantly from core prodidomids, among gnaphosoids. The rest of the dionychans form two main groups, Dionycha part A and part B. The former includes much of the Oblique Median Tapetum clade (Trochanteriidae, Gnaphosidae, Gallieniellidae, Phrurolithidae, Trachelidae, Gnaphosidae, Ammoxenidae, Lamponidae and the Molycriinae), and also Anyphaenidae and Clubionidae. Orthobula is transferred from Phrurolithidae to Trachelidae. Our data did not allow for complete resolution for the gnaphosoid families. Dionycha part B includes the families Salticidae, Eutichuridae, Miturgidae, Philodromidae, Viridasiidae, Selenopidae, Corinnidae and Xenoctenidae (new fam., including Xenoctenus, Paravulsor and Odo, transferred from Miturgidae, as well as Incasoctenus from Ctenidae). We confirm the inclusion of Zora (formerly Zoridae) within Miturgidae.

Expanding anchored hybrid enrichment to resolve both deep and shallow relationships within the spider tree of life.

BACKGROUND: Despite considerable effort, progress in spider molecular systematics has lagged behind many other comparable arthropod groups, thereby hindering family-level resolution, classification, and testing of important macroevolutionary hypotheses. Recently, alternative targeted sequence capture techniques have provided molecular systematics a powerful tool for resolving relationships across the Tree of Life. One of these approaches, Anchored Hybrid Enrichment (AHE), is designed to recover hundreds of unique orthologous loci from across the genome, for resolving both shallow and deep-scale evolutionary relationships within non-model systems. Herein we present a modification of the AHE approach that expands its use for application in spiders, with a particular emphasis on the infraorder Mygalomorphae. RESULTS: Our aim was to design a set of probes that effectively capture loci informative at a diversity of phylogenetic timescales. Following identification of putative arthropod-wide loci, we utilized homologous transcriptome sequences from 17 species across all spiders to identify exon boundaries. Conserved regions with variable flanking regions were then sought across the tick genome, three published araneomorph spider genomes, and raw genomic reads of two mygalomorph taxa. Following development of the 585 target loci in the Spider Probe Kit, we applied AHE across three taxonomic depths to evaluate performance: deep-level spider family relationships (33 taxa, 327 loci); family and generic relationships within the mygalomorph family Euctenizidae (25 taxa, 403 loci); and species relationships in the North American tarantula genus Aphonopelma (83 taxa, 581 loci). At the deepest level, all three major spider lineages (the Mesothelae, Mygalomorphae, and Araneomorphae) were supported with high bootstrap support. Strong support was also found throughout the Euctenizidae, including generic relationships within the family and species relationships within the genus Aptostichus. As in the Euctenizidae, virtually identical topologies were inferred with high support throughout Aphonopelma. CONCLUSIONS: The Spider Probe Kit, the first implementation of AHE methodology in Class Arachnida, holds great promise for gathering the types and quantities of molecular data needed to accelerate an understanding of the spider Tree of Life by providing a mechanism whereby different researchers can confidently and effectively use the same loci for independent projects, yet allowing synthesis of data across independent research groups.

Phylogeography and evolution of the Red Salamander (Pseudotriton ruber).

Phylogeographic studies frequently result in the elevation of subspecific taxa to species given monophyly, or the synonymy of subspecies that are not monophyletic. However, given limited or incongruent datasets, retention of subspecies can be useful to describe hypothesized incipient species or to illustrate interesting biological phenomena driving morphological diversity. Four subspecific taxa have been used to describe largely allopatric geographic variation within the species Pseudotriton ruber, a plethodontid salamander occupying stream and spring habitats across eastern North America: P. r. vioscai occurs in lowland Coastal Plain habitats, while P. r. ruber, P. r. nitidus, and P. r. schencki occupy upland regions in and around the Appalachian Mountains. Pseudotriton ruber co-occurs through its distribution with the aposematic newt Notophthalmus viridescens, and both species are hypothesized to be part of a Müllerian mimicry complex. In this study, we sequenced regions of two mitochondrial (cytochrome b, NADH dehydrogenase subunit 2) and one single copy nuclear protein-coding gene (pro-opiomelanocortin) from individuals sampled across much of the distribution of P. ruber and then used maximum-likelihood and Bayesian phylogenetic inference to test the monophyly of subspecies, reconstruct biogeographic history, and make inferences about morphological evolution. Phylogeographic hypotheses from mitochondrial and nuclear datasets described structure among populations of P. ruber which separated Coastal Plain and upland Appalachian populations, but subspecies were not monophyletic. Biogeographic reconstruction estimated the ancestor of all populations to have occupied and initially diverged in the Coastal Plain during the Pliocene (∼3.6mya), before one lineage subsequently invaded upland areas of Appalachia. Bold bright coloration of high elevation subspecies P. r. nitidus and P. r. schencki appears to have evolved twice. We hypothesize that the Müllerian mimicry complex with N. viridescens and P. ruber may provide a selective mechanism driving the co-evolution of striking bright and dull morphological variation among populations of both species. While P. ruber subspecies were not consistent with our criteria for diagnosing species (monophyly) and therefore could not be elevated to species, we advocate for the retention of subspecies because they describe hypotheses about an incipient species (P. r. vioscai) and how Müllerian mimicry may shape morphological diversity of species.

Molecular phylogeny of Pompilinae (Hymenoptera: Pompilidae): Evidence for rapid diversification and host shifts in spider wasps.

Pompilinae is one of the largest subfamilies of spider wasps (Pompilidae). Most pompilines are generalist spider predators at the family level, but some taxa exhibit ecological specificity (i.e., to spider-host guild). Here we present the first molecular phylogenetic analysis of Pompilinae, toward the aim of evaluating the monophyly of tribes and genera. We further test whether changes in the rate of diversification are associated with host-guild shifts. Molecular data were collected from five nuclear loci (28S, EF1-F2, LWRh, Wg, Pol2) for 76 taxa in 39 genera. Data were analyzed using maximum likelihood (ML) and Bayesian inference (BI). The phylogenetic results were compared with previous hypotheses of subfamilial and tribal classification, as well as generic relationships in the subfamily. The classification of Pompilus and Agenioideus is also discussed. A Bayesian relaxed molecular clock analysis was used to examine divergence times. Diversification rate-shift tests accounted for taxon-sampling bias using ML and BI approaches. Ancestral host family and host guild were reconstructed using MP and ML methods. Ancestral host guild for all Pompilinae, for the ancestor at the node where a diversification rate-shift was detected, and two more nodes back in time was inferred using BI. In the resulting phylogenies, Aporini was the only previously proposed monophyletic tribe. Several genera (e.g., Pompilus, Microphadnus and Schistonyx) are also not monophyletic. Dating analyses produced a well-supported chronogram consistent with topologies from BI and ML results. The BI ancestral host-use reconstruction inferred the use of spiders belonging to the guild "other hunters" (frequenting the ground and vegetation) as the ancestral state for Pompilinae. This guild had the highest probability for the ML reconstruction and was equivocal for the MP reconstruction; various switching events to other guilds occurred throughout the evolution of the group. The diversification of Pompilinae shows one main rate-shift coinciding with a shift to ground-hunter spiders, as reconstructed by the BI ancestral character-state analysis.

An evaluation of sampling effects on multiple DNA barcoding methods leads to an integrative approach for delimiting species: a case study of the North American tarantula genus Aphonopelma (Araneae, Mygalomorphae, Theraphosidae).

The North American tarantula genus Aphonopelma provides one of the greatest challenges to species delimitation and downstream identification in spiders because traditional morphological characters appear ineffective for evaluating limits of intra- and interspecific variation in the group. We evaluated the efficacy of numerous molecular-based approaches to species delimitation within Aphonopelma based upon the most extensive sampling of theraphosids to date, while also investigating the sensitivity of randomized taxon sampling on the reproducibility of species boundaries. Mitochondrial DNA (cytochrome c oxidase subunit I) sequences were sampled from 682 specimens spanning the genetic, taxonomic, and geographic breadth of the genus within the United States. The effects of random taxon sampling compared traditional Neighbor-Joining with three modern quantitative species delimitation approaches (ABGD, P ID(Liberal), and GMYC). Our findings reveal remarkable consistency and congruence across various approaches and sampling regimes, while highlighting highly divergent outcomes in GMYC. Our investigation allowed us to integrate methodologies into an efficient, consistent, and more effective general methodological workflow for estimating species boundaries within the mygalomorph spider genus Aphonopelma. Taken alone, these approaches are not particularly useful - especially in the absence of prior knowledge of the focal taxa. Only through the incorporation of multiple lines of evidence, employed in a hypothesis-testing framework, can the identification and delimitation of confident species boundaries be determined. A key point in studying closely related species, and perhaps one of the most important aspects of DNA barcoding, is to combine a sampling strategy that broadly identifies the extent of genetic diversity across the distributions of the species of interest and incorporates previous knowledge into the "species equation" (morphology, molecules, and natural history).

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.

Microgeographic population structuring in a genus of California trapdoor spiders and discovery of an enigmatic new species (Euctenizidae: Promyrmekiaphila korematsui sp. nov.).

The recognition and delineation of cryptic species remains a perplexing problem in systematics, evolution, and species delimitation. Once recognized as such, cryptic species complexes provide fertile ground for studying genetic divergence within the context of phenotypic and ecological divergence (or lack thereof). Herein we document the discovery of a new cryptic species of trapdoor spider, Promyrmekiaphila korematsui sp. nov. Using subgenomic data obtained via target enrichment, we document the phylogeography of the California endemic genus Promyrmekiaphila and its constituent species, which also includes P. clathrata and P. winnemem. Based on these data we show a pattern of strong geographic structuring among populations but cannot entirely discount recent gene flow among populations that are parapatric, particularly for deeply diverged lineages within P. clathrata. The genetic data, in addition to revealing a new undescribed species, also allude to a pattern of potential phenotypic differentiation where species likely come into close contact. Alternatively, phenotypic cohesion among genetically divergent P. clathrata lineages suggests that some level of gene flow is ongoing or occurred in the recent past. Despite considerable field collection efforts over many years, additional sampling in potential zones of contact for both species and lineages is needed to completely resolve the dynamics of divergence in Promyrmekiaphila at the population-species interface.

A multilocus perspective on phylogenetic relationships in the Namib darkling beetle genus Onymacris (Tenebrionidae).

Tenebrionid beetles, common constituent faunae of arid ecosystems worldwide, are particularly abundant in Africa's Namib and Kalahari deserts. Within this region, flightless, diurnal members of the tribe Adesmiini are among the more intensively studied of all desert beetles, especially with regard to ecology. Much of this research centers on Onymacris, a psammophilous genus largely endemic to the Namib. Here we present the first molecular phylogenetic analysis conducted for Onymacris, emphasizing relationships among other adesmiines. Our multilocus phylogeny identifies a strongly supported clade containing Onymacris and two other genera, Eustolopus and Physadesmia-an assemblage recovered in earlier morphological analyses. However, Onymacris is not monophyletic; rather, we demonstrate its paraphyly with respect to the genus Physadesmia, identified as the sister taxon to the white-bodied species of Onymacris. In turn, the Physadesmia-'white'Onymacris clade is the sister group to the remaining (black-bodied) Onymacris. Non-monophyly of 'black' versus 'white'Onymacris is corroborated by distribution patterns and nodal age estimates, which suggest separate origins in different dune systems.

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.