The rediscovery of a relict unlocks the first global phylogeny of whip spiders (Amblypygi).

Asymmetrical rates of cladogenesis and extinction abound in the Tree of Life, resulting in numerous minute clades that are dwarfed by larger sister groups. Such taxa are commonly regarded as phylogenetic relicts or "living fossils" when they exhibit an ancient first appearance in the fossil record and prolonged external morphological stasis, particularly in comparison to their more diversified sister groups. Due to their special status, various phylogenetic relicts tend to be well-studied and prioritized for conservation. A notable exception to this trend is found within Amblypygi ("whip spiders"), a visually striking order of functionally hexapodous arachnids that are notable for their antenniform first walking leg pair (the eponymous "whips"). Paleoamblypygi, the putative sister group to the remaining Amblypygi, is known from Late Carboniferous and Eocene deposits, but is survived by a single living species, Paracharon caecus Hansen, 1921, that was last collected in 1899. Due to the absence of genomic sequence-grade tissue for this vital taxon, there is no global molecular phylogeny for Amblypygi to date, nor a fossil-calibrated estimation of divergences within the group. Here, we report a previously unknown species of Paleoamblypygi from a cave site in Colombia. Capitalizing upon this discovery, we generated the first molecular phylogeny of Amblypygi, integrating ultraconserved element sequencing with legacy Sanger datasets and including described extant genera. To quantify the impact of sampling Paleoamblypygi on divergence time estimation, we performed in silico experiments with pruning of Paracharon. We demonstrate that the omission of relicts has a significant impact on the accuracy of node dating approaches that outweighs the impact of excluding ingroup fossils, which bears upon the ancestral range reconstruction for the group. Our results underscore the imperative for biodiversity discovery efforts in elucidating the phylogenetic relationships of "dark taxa", and especially phylogenetic relicts in tropical and subtropical habitats. The lack of reciprocal monophyly for Charontidae and Charinidae leads us to subsume them into one family, Charontidae, new synonymy.

Dual Functions of labial Resolve the Hox Logic of Chelicerate Head Segments.

Despite an abundance of gene expression surveys, comparatively little is known about Hox gene function in Chelicerata. Previous investigations of paralogs of labial (lab) and Deformed (Dfd) in a spider have shown that these play a role in tissue maintenance of the pedipalp segment (lab-1) and in patterning the first walking leg identity (Dfd-1), respectively. However, extrapolations of these data across chelicerates are hindered by the existence of duplicated Hox genes in arachnopulmonates (e.g., spiders and scorpions), which have resulted from an ancient whole genome duplication (WGD) event. Here, we investigated the function of the single-copy ortholog of lab in the harvestman Phalangium opilio, an exemplar of a lineage that was not subject to this WGD. Embryonic RNA interference against lab resulted in two classes of phenotypes: homeotic transformations of pedipalps to chelicerae, as well as reduction and fusion of the pedipalp and leg 1 segments. To test for combinatorial function, we performed a double knockdown of lab and Dfd, which resulted in a homeotic transformation of both pedipalps and the first walking legs into cheliceral identity, whereas the second walking leg is transformed into a pedipalpal identity. Taken together, these results elucidate a model for the Hox logic of head segments in Chelicerata. To substantiate the validity of this model, we performed expression surveys for lab and Dfd paralogs in scorpions and horseshoe crabs. We show that repetition of morphologically similar appendages is correlated with uniform expression levels of the Hox genes lab and Dfd, irrespective of the number of gene copies.

A plurality of morphological characters need not equate with phylogenetic accuracy: A rare genomic change refutes the placement of Solifugae and Pseudoscorpiones in Haplocnemata.

Recent advances in higher-level invertebrate phylogeny have leveraged shared features of genomic architecture to resolve contentious nodes across the tree of life. Yet, the interordinal relationships within Chelicerata have remained recalcitrant given competing topologies in recent molecular analyses. As such, relationships between topologically unstable orders remain supported primarily by morphological cladistic analyses. Solifugae, one such unstable chelicerate order, has long been thought to be the sister group of Pseudoscorpiones, forming the clade Haplocnemata, on the basis of eight putative morphological synapomorphies. The discovery, however, of a shared whole genome duplication placing Pseudoscorpiones in Arachnopulmonata provides the opportunity for a simple litmus test evaluating the validity of Haplocnemata. Here, we present the first developmental transcriptome of a solifuge (Titanopuga salinarum) and survey copy numbers of the homeobox genes for evidence of systemic duplication. We find that over 70% of the identified homeobox genes in T. salinarum are retained in a single copy, while representatives of the arachnopulmonates retain orthologs of those genes as two or more copies. Our results refute the placement of Solifugae in Haplocnemata. Subsequent reevaluation of putative interordinal morphological synapomorphies among chelicerates reveals a high incidence of homoplasy, reversals, and inaccurate coding within Haplocnemata and other small clades, as well as Arachnida more broadly, suggesting existing morphological character matrices are insufficient to resolve chelicerate phylogeny.

Phylogenomic data reveal three new families of poorly studied Solifugae (camel spiders).

The systematics of the arachnid order Solifugae have been an enigma, owing to challenges in interpreting morphology, a paucity of molecular phylogenetic studies sampling across the group, and a dearth of taxonomic attention for many lineages. Recent work has suggested that solifuge families largely exhibit contiguous distributions and reflect patterns of vicariance, with the exception of three families: Melanoblossidae, Daesiidae and Gylippidae. Morphological studies have cast doubt on their existing circumscriptions and the present composition of these taxa renders their distributions as disjunct. We leveraged ultraconserved elements (UCEs) to test the phylogenetic placement of three key lineages of Solifugae that cause these anomalous distributions: Dinorhax rostrumpsittaci (putative melanoblossid), Namibesia (putative daesiid), and Trichotoma (putative gylippid). Phylogenetic placement of these three genera based on UCEs rendered the families that harbor them as para- or polyphyletic, recovering instead relationships that better accord with a biogeographic history driven by vicariance. Toward a stable and phylogenetically informed classification of Solifugae, we establish three new families, Dinorhaxidae new rank, Namibesiidae new rank and Lipophagidae new rank.

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.

Too early for the ferry: The biogeographic history of the Assamiidae of southeast Asia (Chelicerata: Opiliones, Laniatores).

Opiliones (harvestmen) have come to be regarded as an abundant source of model groups for study of historical biogeography, due to their ancient age, poor dispersal capability, and high fidelity to biogeographic terranes. One of the least understood harvestman groups is the Paleotropical Assamiidae, one of the more diverse families of Opiliones. Due to a labyrinthine taxonomy, poorly established generic and subfamilial boundaries, and the lack of taxonomic keys for the group, few efforts have been undertaken to decipher relationships within this arachnid lineage. Neither the monophyly of the family, nor its exact placement in the harvestman phylogeny, have been established. Here, we assessed the internal phylogeny of Assamiidae using a ten-locus Sanger dataset, sampling key lineages putatively ascribed to this family for five of the ten markers. Our analyses recovered Assamiidae as a monophyletic group, in a clade with the primarily Afrotropical Pyramidopidae and the southeast Asian Beloniscidae. Internal relationships of assamiids disfavored the systematic validity of subfamilies, with biogeography reflecting much better phylogenetic structure than the existing higher-level taxonomy. To assess whether the Asian assamiids came to occupy Indo-Pacific terranes via rafting on the Indian subcontinent, we performed divergence dating to infer the age of the family. Our results show that Indo-Pacific clades are ancient, originating well before the Cretaceous and therefore predate a vicariant mechanism commonly encountered for Paleotropical taxa.

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.

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.].

Congruence between ultraconserved element-based matrices and phylotranscriptomic datasets in the scorpion Tree of Life.

Scorpions are ancient and historically renowned for their potent venom. Traditionally, the systematics of this group of arthropods was supported by morphological characters, until recent phylogenomic analyses (using RNAseq data) revealed most of the higher-level taxa to be non-monophyletic. While these phylogenomic hypotheses are stable for almost all lineages, some nodes have been hard to resolve due to minimal taxonomic sampling (e.g. family Chactidae). In the same line, it has been shown that some nodes in the Arachnid Tree of Life show disagreement between hypotheses generated using transcritptomes and other genomic sources such as the ultraconserved elements (UCEs). Here, we compared the phylogenetic signal of transcriptomes vs. UCEs by retrieving UCEs from new and previously published scorpion transcriptomes and genomes, and reconstructed phylogenies using both datasets independently. We reexamined the monophyly and phylogenetic placement of Chactidae, sampling an additional chactid species using both datasets. Our results showed that both sets of genome-scale datasets recovered highly similar topologies, with Chactidae rendered paraphyletic owing to the placement of Nullibrotheas allenii. As a first step toward redressing the systematics of Chactidae, we establish the family Anuroctonidae (new family) to accommodate the genus Anuroctonus.

A conserved role for arrow in posterior axis patterning across Arthropoda.

Segmentation is a key characteristic of Arthropoda that is linked to the evolutionary success of this lineage. It has previously been shown in both vertebrates and short germ insects that posterior segmentation requires canonical Wnt (cWnt) signaling, which maintains the expression of Caudal and the posterior growth zone; disruption of cWnt signaling incurs posterior truncations in these lineages due to the loss of the tail bud. However, comparable datasets for Wnt signaling are limited outside of holometabolous insects, due to incomparable phenotypic spectra and inefficacy of gene misexpression methods in certain model species. We applied RNA interference (RNAi) against the Wnt co-receptor arrow (arr), a key member of the cWnt signaling pathway in holometabolous insects and vertebrates, to examine posterior axis elongation of the cobweb spider Parasteatoda tepidariorum (short germ embryogenesis; one Wnt8 homolog), the cricket Gryllus bimaculatus (intermediate germ; one Wnt8 homolog), and the milkweed bug Oncopeltus fasciatus (short germ; two Wnt8 homologs). Knockdown of arr in insects resulted in posterior truncations affecting the gnathos through the abdomen in O. fasciatus, whereas posterior truncations only affected the T3 segment through the abdomen in G. bimaculatus. Spider embryos with disrupted arr expression exhibited defects along the entire axis, including segmentation defects throughout the germband. RNA-Seq-based differential gene expression analysis of severe Ptep-arr loss-of-function phenotypes at two developmental stages was used to confirm that knockdown of Ptep-arr results in systemic disruption of the Wnt pathway. Intriguingly, we found that knockdown of arr did not abrogate Wnt8 expression in any of the three species, with cad expression additionally retained in severe loss-of-function phenotypes in the cricket and the spider. Together with data from a holometabolous insect, our results suggest that cWnt signaling is not required for maintenance of Wnt8 expression across Arthropoda. These outcomes underscore the diagnostic power of differential gene expression analyses in characterizing catastrophic phenotypes in emerging model species.

The Evolution of Sox Gene Repertoires and Regulation of Segmentation in Arachnids.

The Sox family of transcription factors regulates many processes during metazoan development, including stem cell maintenance and nervous system specification. Characterizing the repertoires and roles of these genes can therefore provide important insights into animal evolution and development. We further characterized the Sox repertoires of several arachnid species with and without an ancestral whole-genome duplication and compared their expression between the spider Parasteatoda tepidariorum and the harvestman Phalangium opilio. We found that most Sox families have been retained as ohnologs after whole-genome duplication and evidence for potential subfunctionalization and/or neofunctionalization events. Our results also suggest that Sox21b-1 likely regulated segmentation ancestrally in arachnids, playing a similar role to the closely related SoxB gene, Dichaete, in insects. We previously showed that Sox21b-1 is required for the simultaneous formation of prosomal segments and sequential addition of opisthosomal segments in P. tepidariorum. We studied the expression and function of Sox21b-1 further in this spider and found that although this gene regulates the generation of both prosomal and opisthosomal segments, it plays different roles in the formation of these tagmata reflecting their contrasting modes of segmentation and deployment of gene regulatory networks with different architectures.

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.

Phylogenetic assessment of the Metamasius hemipterus species complex (Coleoptera, Curculionidae, Dryophthorinae).

Metamasius is a large genus of dryophthorine weevils, with nearly 85 species. Among the economically important pests in the genus, M. hemipterus is currently separated in three subspecies, based largely on color patterns of the elytra, pronotum, and sternum. The tenuous limits of M. hemipterus subspecies were created over fifty years ago and never tested under a phylogenetic framework. Here, for the first time, we address the M. hemipterus species boundaries applying a molecular approach. We constructed a reduced genome representation of a few species using restriction site-associated DNA sequencing (RADseq). Phylogenetic analysis using either a complete supermatrix or only SNPs revealed a clear separation of Metamasius species. We suggest that M. h. carbonarius syn. nov. and M. h. sericeus be treated as the same species, M. sericeus (Oliver) stat. n., and elevate M. h. hemipterus as a separate species M. hemipterus (Linnaeus). We updated Vaurie's identification key to reflect the new species status. This systematic reassessment reflects a more natural classification for these remarkable and economically significant weevils.

How many long branch orders occur in Chelicerata? Opposing effects of Palpigradi and Opilioacariformes on phylogenetic stability.

Excepting a handful of nodes, phylogenetic relationships between chelicerate orders remain poorly resolved, due to both the incidence of long branch attraction artifacts and the limited sampling of key lineages. It has recently been shown that increasing representation of basal nodes plays an outsized role in resolving the higher-level placement of long-branch chelicerate orders. Two lineages have been consistently undersampled in chelicerate phylogeny. First, sampling of the miniaturized order Palpigradi has been restricted to a fragmentary transcriptome of a single species. Second, sampling of Opilioacariformes, a rarely encountered and key group of Parasitiformes, has been restricted to a single exemplar. These two lineages exhibit dissimilar properties with respect to branch length; Opilioacariformes shows relatively low evolutionary rate compared to other Parasitiformes, whereas Palpigradi possibly acts as another long-branch order (an effect that may be conflated with the degree of missing data). To assess these properties and their effects on tree stability, we constructed a phylogenomic dataset of Chelicerata wherein both lineages were sampled with three terminals, increasing the representation of these taxa per locus. We examined the effect of subsampling phylogenomic matrices using (1) taxon occupancy, (2) evolutionary rate, and (3) a principal components-based approach. We further explored the impact of taxon deletion experiments that mitigate the effect of long branches. Here, we show that Palpigradi constitutes a fourth long-branch chelicerate order (together with Acariformes, Parasitiformes, and Pseudoscorpiones), which further destabilizes the chelicerate backbone topology. By contrast, the slow-evolving Opilioacariformes were consistently recovered within Parasitiformes, with certain subsampling practices recovering their placement as the sister group to the remaining Parasitiformes. Whereas the inclusion of Opilioacariformes always resulted in the non-monophyly of Acari with support, deletion of Opilioacariformes from datasets consistently incurred the monophyly of Acari except in matrices constructed on the basis of evolutionary rate. Our results strongly suggest that Acari is an artifact of long- branch attraction.

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.

Eggs to long-legs: embryonic staging of the harvestman Phalangium opilio (Opiliones), an emerging model arachnid.

BACKGROUND: The comparative embryology of Chelicerata has greatly advanced in recent years with the integration of classical studies and genetics, prominently spearheaded by developmental genetic works in spiders. Nonetheless, the understanding of the evolution of development and polarization of embryological characters in Chelicerata is presently limited, as few non-spider species have been well studied. A promising focal species for chelicerate evo-devo is the daddy-long-legs (harvestman) Phalangium opilio, a member of the order Opiliones. Phalangium opilio, breeds prolifically and is easily accessible in many parts of the world, as well as tractable in a laboratory setting. Resources for this species include developmental transcriptomes, a draft genome, and protocols for RNA interference, but a modern staging system is critically missing for this emerging model system. RESULTS: We present a staging system of P. opilio embryogenesis that spans the most important morphogenetic events with respect to segment formation, appendage elongation and head development. Using time-lapse imaging, confocal microscopy, colorimetric in situ hybridization, and immunohistochemistry, we tracked the development of synchronous clutches from egg laying to adulthood. We describe key events in segmentation, myogenesis, neurogenesis, and germ cell formation. CONCLUSION: Considering the phylogenetic position of Opiliones and the unduplicated condition of its genome (in contrast to groups like spiders and scorpions), this species is poised to serve as a linchpin for comparative studies in arthropod development and genome evolution. The staging system presented herein provides a valuable reference for P. opilio that we anticipate being useful to the arthropod evo-devo community, with the goal of revitalizing research in the comparative development of non-spider arachnids.

Implications of a cheliceral axial duplication in Tetragnatha versicolor (Araneae: Tetragnathidae) for arachnid deuterocerebral appendage development.

The homology of the arachnid chelicera with respect to other head appendages in Panarthropoda has long been debated. Gene expression data and the re-interpretation of early transitional fossils have supported the homology of the deutocerebrum and its associated appendages, implying a homology between primary antennae (mandibulates), chelicerae (euchelicerates), and chelifores (sea spiders). Nevertheless, comparatively little is known about the mechanistic basis of proximo-distal (PD) axis induction in chelicerates, much less the basis for cheliceral fate specification. Here, we describe a new cheliceral teratology in the spider Tetragnatha versicolor Walckenaer, 1841, which consists on a duplication of the PD axis of the left chelicera associated with a terminal secondary schistomely on the fang of the lower axis. This duplication offers clues as to potential shared mechanisms of PD axis formation in the chelicera. We review the state of knowledge on PD axis induction mechanisms in arthropods and identify elements of gene regulatory networks that are key for future functional experiments of appendage development in non-insect model systems. Such investigations would allow a better understanding of PD axis induction of modified and poorly studied arthropod limbs (e.g., chelicerae, chelifores, and ovigers).

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.

Cooption of an appendage-patterning gene cassette in the head segmentation of arachnids.

The jointed appendages of arthropods have facilitated the spectacular diversity and success of this phylum. Key to the regulation of appendage outgrowth is the Krüppel-like factor (KLF)/specificity protein (Sp) family of zinc finger transcription factors. In the fruit fly, Drosophila melanogaster, the Sp6-9 homolog is activated by Wnt-1/wingless (wg) and establishes ventral appendage (leg) fate. Subsequently, Sp6-9 maintains expression of the axial patterning gene Distal-less (Dll), which promotes limb outgrowth. Intriguingly, in spiders, Dll has been reported to have a derived role as a segmentation gap gene, but the evolutionary origin and regulation of this function are not understood because functional investigations of the appendage-patterning regulatory network are restricted to insects. We tested the evolutionary conservation of the ancestral appendage-patterning network of arthropods with a functional approach in the spider. RNAi-mediated knockdown of the spider Sp6-9 ortholog resulted in diminution or loss of Dll expression and truncation of appendages, as well as loss of the two body segments specified by the early Dll function. In reciprocal experiments, Dll is shown not to be required for Sp6-9 expression. Knockdown of arrow (Wnt-1 coreceptor) disrupted segmentation and appendage development but did not affect the early Sp6-9 expression domain. Ectopic appendages generated in the spider "abdomen" by knockdown of the Hox gene Antennapedia-1 (Antp-1) expressed Sp6-9 comparably to wild-type walking legs. Our results support (i) the evolutionary conservation of an appendage-patterning regulatory network that includes canonical Wnt signaling, Sp6-9, and Dll and (ii) the cooption of the Sp6-9/Dll regulatory cassette in arachnid head segmentation.