Reproductive biology of harvestmen (Arachnida: Opiliones): a review of a rapidly evolving research field.

Harvestmen are a major arachnid order that has experienced a dramatic increase in biological knowledge in the 21st century. The publication of the book Harvestmen: The Biology of Opiliones in 2007 stimulated the development of many behavioral studies. Although the book is relatively recent, our understanding of the reproductive biology of harvestmen is already outdated due to the fast accumulation of new data. Our goal is to provide an updated review of the subject to serve as a benchmark for the following years. In the pre-copulatory phase, we explore the evolution of facultative parthenogenesis, the factors that may affect the types of mating system, and the role of nuptial gifts in courtship. Regarding the copulatory phase, harvestmen are unique arachnids because they have aflagellate spermatozoa and a penis with complex morphology. We discuss the implications of these two features for sperm competition and cryptic female choice. In the post-copulatory phase, we connect oviposition site selection and climate conditions to the widespread occurrence of resource defense polygyny, alternative reproductive tactics, and sexual dimorphism in several clades of tropical harvestmen. Finally, we present the different forms of parental care in the order, and discuss the benefits and costs of this behavior, which can be performed either by females or males. Throughout the review, we indicate gaps in our knowledge and subjects that deserve further studies. Hopefully, the information synthesized here will stimulate researchers worldwide to embrace harvestmen as a study system and to improve our effort to unravel the mysteries of their reproductive biology.

The Geography of Sexual Conflict: A Synthetic Review.

AbstractSexual conflict is a mechanism of selection driven by the divergent fitness interests between females and males. This disagreement can be great enough to promote antagonistic/defensive traits and behaviors. Although the existence of sexual conflict has been identified in many species, less research has explored the conditions that initially promote sexual conflict in animal mating systems. In previous work in Opiliones, we observed that morphological traits associated with sexual conflict occurred only in species from northern localities. We hypothesized that by shortening and compartmentalizing time periods optimal for reproduction, seasonality represents a geographic condition sufficient to promote sexual conflict. We conducted a systematic review of the literature on reproductive traits and behaviors. Using standardized criteria, we reviewed publications to identify whether subjects occurred in a temperate (high-seasonality) or tropical (low-seasonality) biome. After identifying and adjusting for a publication bias toward temperate research, we identified no significant difference in the strength of sexual conflict between temperate and tropical study systems. A comparison between the distribution of taxa studied in sexual conflict articles and articles focused on general biodiversity indicates that species with conflict-based mating systems more accurately represent the distribution of terrestrial animal species. These findings contribute to ongoing efforts to characterize the origins of sexual conflict as well as life history traits that covary with sexual conflict.

The Ties that Stick: Challenges and Future Promise in the Field of Bioadhesives.

Bioadhesives are wet or dry polymeric compounds that rely upon physical and chemical properties to generate characteristic sticky forces. The past decade has seen a rapidly evolving field of research around the functions, genetics, biochemistry, and mimetics of bioadhesives, but challenges unique to this research area continue to arise. We polled the presenters of SICB Symposium #8 to describe the "Ties that Stick:" challenges and exciting prospects that most resonated with their research pursuits. Themes that emerged from the poll included difficulties working with adhesive-producing organisms, field inherent knowledge gaps in theoretical modeling, molecular interactions, technology, and the interdisciplinary rigor of the bioadhesives field. We address each challenge with a discussion of the opportunities and applications presented by bioadhesives research.

Repeat Variation Resolves a Complete Aggregate Silk Sequence of Bolas Spider Mastophora phrynosoma.

Many species of spider use a modified silk adhesive, called aggregate glue, to aid in prey capture. Aggregate spidroins (spider fibroins) are modified members of the spider silk family; however, they are not spun into fibers as are their solid silk relatives. The genes that encode for aggregate spidroins are the largest of the known spidroin genes and are similarly highly repetitive. In this study, we used long read sequencing to discover the aggregate spidroin genes of the toad-like bolas spider, Mastophora phrynosoma, which employs the glue in a unique way, using only a single, large droplet to capture moths. While Aggregate Spidroin 1 (AgSp1) remains incomplete, AgSp2 is more than an extraordinary 62 kb of coding sequence, 20 kb longer than the longest spidroin on record. The structure of repeats from both aggregate silk proteins follows a similar pattern seen in other species, with the same strict conservation of amino acid residue number for much of the repeats' lengths. Interestingly, AgSp2 lacks the elevated number and groupings of glutamine residues seen in the other reported AgSp2 of a classic orb weaving species. The role of gene length in glue functionality remains a mystery, and thus discovering length differences across species will allow understanding and harnessing of this attribute for the next generation of bio-inspired adhesives.

Genomic Determination of Reproductive Mode in Facultatively Parthenogenetic Opiliones.

Sexual reproduction may pose myriad short-term costs to females. Despite these costs, sexual reproduction is near ubiquitous. Facultative parthenogenesis is theorized to mitigate some of the costs of sex, as individuals can participate in occasional sex to limit costs while obtaining many benefits. However, most theoretical models assume sexual reproduction is fixed following mating, with no possibility of clutches of mixed reproductive ontogeny. Therefore, we asked: if coercive males are present at high frequency in a population of facultative parthenogens, will their clutches be solely sexually produced, or will there be evidence of sexually and asexually-produced offspring? How will their offspring production compare to conspecifics in low-frequency male populations? We addressed our questions by collecting females and egg clutches of the facultatively parthenogenetic Opiliones species Leiobunum manubriatum and L. globosum. In L. manubriatum, females from populations with few males were not significantly more fecund than females from populations with higher male relative frequency, despite the potential release of the former from sexual conflict. We used 3 genotyping methods along with a custom set of DNA capture probes to reveal that offspring of L. manubriatum from these high male populations were primarily produced via asexual reproduction. This is surprising because sex ratios in these southern populations approach equality, increasing the probability for females to encounter mates and produce offspring sexually. We additionally found evidence for reproductive polymorphisms within populations. Rapid and accurate SNP genotyping data will continue to allow us to address broader evolutionary questions regarding the role of facultative reproductive modes in the maintenance of sex.

It Takes Two to Tango: Including a Female Perspective in Reproductive Biology.

Like many scientific disciplines, the field of reproductive biology is subject to biases in terminology and research foci. For example, females are often described as coy and passive players in reproductive behaviors and are termed "promiscuous" if they engage in extra-pair copulations. Males on the other hand are viewed as actively holding territories and fighting with other males. Males are termed "multiply mating" if they mate with multiple females. Similarly, textbooks often illustrate meiosis as it occurs in males but not females. This edition of Integrative and Comparative Biology (ICB) includes a series of papers that focus on reproduction from the female perspective. These papers represent a subset of the work presented in our symposium and complementary sessions on female reproductive biology. In this round table discussion, we use a question and answer format to leverage the diverse perspectives and voices involved with the symposium in an exploration of theoretical, cultural, pedagogical, and scientific issues related to the study of female biology. We hope this dialog will provide a stepping-stone toward moving reproductive science and teaching to a more inclusive and objective framework.

Nuptial gift chemistry reveals convergent evolution correlated with antagonism in mating systems of harvestmen (Arachnida, Opiliones).

Nuptial gifts are material donations given from male to female before or during copulation and are subject to sexual selection in a wide variety of taxa. The harvestman genus Leiobunum has emerged as a model system for understanding the evolution of reproductive morphology and behavior, as transitions between solicitous and antagonistic modes of courtship have occurred multiple times within the lineage and are correlated with convergence in genital morphology. We analyzed the free amino acid content of nuptial gift secretions from five species of Leiobunum using gas chromatography-mass spectrometry. Multivariate analysis of the free amino acid profiles revealed that, rather than clustering based on phylogenetic relationships, nuptial gift chemical composition was better predicted by genital morphology and behavior, suggesting that convergent evolution has acted on the chemical composition of the nuptial gift. In addition, we found that, species with solicitous courtship produce gifts consisting of a 19% larger proportion of essential amino acids as compared to those with more antagonistic courtship interactions. This work represents the first comparative study of nuptial gift chemistry within a phylogenetic framework in any animal group and as such contributes to our understanding of the evolution of reproductive diversity and the participant role of nuptial gift chemistry in mating system transitions.

Population genomics and geographical parthenogenesis in Japanese harvestmen (Opiliones, Sclerosomatidae, Leiobunum).

Naturally occurring population variation in reproductive mode presents an opportunity for researchers to test hypotheses regarding the evolution of sex. Asexual reproduction frequently assumes a geographical pattern, in which parthenogenesis-dominated populations are more broadly dispersed than their sexual conspecifics. We evaluate the geographical distribution of genomic signatures associated with parthenogenesis using nuclear and mitochondrial DNA sequence data from two Japanese harvestman sister taxa, Leiobunum manubriatum and Leiobunum globosum. Asexual reproduction is putatively facultative in these species, and female-biased localities are common in habitat margins. Past karyotypic and current cytometric work indicates L. globosum is entirely tetraploid, while L. manubriatum may be either diploid or tetraploid. We estimated species phylogeny, genetic differentiation, diversity, and mitonuclear discordance in females collected across the species range in order to identify range expansion toward marginal habitat, potential for hybrid origin, and persistence of asexual lineages. Our results point to northward expansion of a tetraploid ancestor of L. manubriatum and L. globosum, coupled with support for greater male gene flow in southern L. manubriatum localities. Specimens from localities in the Tohoku and Hokkaido regions were indistinct, particularly those of L. globosum, potentially due to little mitochondrial differentiation or haplotypic variation. Although L. manubriatum overlaps with L. globosum across its entire range, L. globosum was reconstructed as monophyletic with strong support using mtDNA, and marginal support with nuclear loci. Ultimately, we find evidence for continued sexual reproduction in both species and describe opportunities to clarify the rate and mechanism of parthenogenesis.

New records of harvestmen (Arachnida, Opiliones) from Minnesota, USA.

Harvestmen (Opiliones) are a diverse order of arachnids composed of more than 6,600 described species which together span an almost global distribution. Although these animals may occur in extremely high abundance in both pristine and disturbed habitats, much of harvestman diversity remains undescribed, undocumented, and/or in need of taxonomic attention. In the current study, we focus on the harvestman diversity of the state of Minnesota, USA, where a lack of local expertise and effort has left the species richness of the state largely undocumented. We document two genera and seven species previously unrecorded in the state.-Leiobunum aldrichi, L. calcar, L. flavum, L. politum, L. ventricosum, L. vittatum,  and Odiellus pictus.

Comparative performance of double-digest RAD sequencing across divergent arachnid lineages.

Next-generation sequencing technologies now allow researchers of non-model systems to perform genome-based studies without the requirement of a (often unavailable) closely related genomic reference. We evaluated the role of restriction endonuclease (RE) selection in double-digest restriction-site-associated DNA sequencing (ddRADseq) by generating reduced representation genome-wide data using four different RE combinations. Our expectation was that RE selections targeting longer, more complex restriction sites would recover fewer loci than RE with shorter, less complex sites. We sequenced a diverse sample of non-model arachnids, including five congeneric pairs of harvestmen (Opiliones) and four pairs of spiders (Araneae). Sample pairs consisted of either conspecifics or closely related congeneric taxa, and in total 26 sample pair analyses were tested. Sequence demultiplexing, read clustering and variant calling were performed in the pyRAD program. The 6-base pair cutter EcoRI combined with methylated site-specific 4-base pair cutter MspI produced, on average, the greatest numbers of intra-individual loci and shared loci per sample pair. As expected, the number of shared loci recovered for a sample pair covaried with the degree of genetic divergence, estimated with cytochrome oxidase I sequences, although this relationship was non-linear. Our comparative results will prove useful in guiding protocol selection for ddRADseq experiments on many arachnid taxa where reference genomes, even from closely related species, are unavailable.

Population genomic evidence for multiple Pliocene refugia in a montane-restricted harvestman (Arachnida, Opiliones, Sclerobunus robustus) from the southwestern United States.

The integration of ecological niche modelling into phylogeographic analyses has allowed for the identification and testing of potential refugia under a hypothesis-based framework, where the expected patterns of higher genetic diversity in refugial populations and evidence of range expansion of nonrefugial populations are corroborated with empirical data. In this study, we focus on a montane-restricted cryophilic harvestman, Sclerobunus robustus, distributed throughout the heterogeneous Southern Rocky Mountains and Intermontane Plateau of southwestern North America. We identified hypothetical refugia using ecological niche models (ENMs) across three time periods, corroborated these refugia with population genetic methods using double-digest RAD-seq data and conducted population-level phylogenetic and divergence dating analyses. ENMs identify two large temporally persistent regions in the mid-latitude highlands. Genetic patterns support these two hypothesized refugia with higher genetic diversity within refugial populations and evidence for range expansion in populations found outside hypothesized refugia. Phylogenetic analyses identify five to six genetically divergent, geographically cohesive clades of S. robustus. Divergence dating analyses suggest that these separate refugia date to the Pliocene and that divergence between clades pre-dates the late Pleistocene glacial cycles, while diversification within clades was likely driven by these cycles. Population genetic analyses reveal effects of both isolation by distance (IBD) and isolation by environment (IBE), with IBD more important in the continuous mountainous portion of the distribution, while IBE was stronger in the populations inhabiting the isolated sky islands of the south. Using model-based coalescent approaches, we find support for postdivergence migration between clades from separate refugia.

Male Reproductive Morphology Across Latitudinal Clines and Under Long-Term Female Sex-Ratio Bias.

SynopsisRecent work in evolutionary ecology has capitalized on the species-specific, rapidly evolving nature of male reproductive traits, but it is unclear how the strength of sexual selection mechanisms, such as female choice and intersexual conflict, changes with ecological factors-shifts in latitude, elevation, and sex-ratio. Marginal habitats, such as those found at high elevation or latitude, might be expected to exert energetic hardships on males such that they do not invest heavily in reproductive structures. Conversely, males in these habitats may adapt to limited breeding opportunities, evolving morphologies with efficacy in antagonistic mating. Sex-ratio biases elicited by facultative parthenogenesis may also signal increasing selective pressure for reproductive organs that improve coercion ability, as males must capitalize on mating opportunities when females have alternate reproductive options. We studied the reproductive organs of males of five species of a group of polygynandrous Japanese harvestmen, collected from populations that varied in their elevation, latitude, and observed male: female ratio. Morphological data were analyzed with modern comparative methods, using a phylogenomic framework built with molecular data from double-digest restriction associated DNA sequencing. Although no features with hypothesized utility in coercive mating significantly covaried with elevational change, penis length was found to decrease with increasing latitude, supporting the existence of physiological constraints in marginal habitats. We also found males of parthenogenetic species, with primarily female-biased populations, had potentially greater precopulatory clasping ability, as compared to males of non-parthenogenetic species with more even sex-ratios. Ultimately, our findings suggest long-term sex-ratio biases are one of many factors with the potential to influence evolution of primary and secondary reproductive morphology.

Biomechanical Diversity of Mating Structures among Harvestmen Species Is Consistent with a Spectrum of Precopulatory Strategies.

Diversity in reproductive structures is frequently explained by selection acting at individual to generational timescales, but interspecific differences predicted by such models (e.g., female choice or sexual conflict) are often untestable in a phylogenetic framework. An alternative approach focuses on clade- or function-specific hypotheses that predict evolutionary patterns in terms neutral to specific modes of sexual selection. Here we test a hypothesis that diversity of reproductive structures in leiobunine harvestmen (daddy longlegs) of eastern North America reflects two sexually coevolved but non-overlapping precopulatory strategies, a primitive solicitous strategy (females enticed by penis-associated nuptial gifts), and a multiply derived antagonistic strategy (penis exerts mechanical force against armature of the female pregenital opening). Predictions of sexual coevolution and fidelity to precopulatory categories were tested using 10 continuously varying functional traits from 28 species. Multivariate analyses corroborated sexual coevolution but failed to partition species by precopulatory strategy, with multiple methods placing species along a spectrum of mechanical antagonistic potential. These findings suggest that precopulatory features within species reflect different co-occurring levels of solicitation and antagonism, and that gradualistic evolutionary pathways exist between extreme strategies. The ability to quantify antagonistic potential of precopulatory structures invites comparison with ecological variables that may promote evolutionary shifts in precopulatory strategies.

Comparative analyses of reproductive structures in harvestmen (opiliones) reveal multiple transitions from courtship to precopulatory antagonism.

Explaining the rapid, species-specific diversification of reproductive structures and behaviors is a long-standing goal of evolutionary biology, with recent research tending to attribute reproductive phenotypes to the evolutionary mechanisms of female mate choice or intersexual conflict. Progress in understanding these and other possible mechanisms depends, in part, on reconstructing the direction, frequency and relative timing of phenotypic evolution of male and female structures in species-rich clades. Here we examine evolution of reproductive structures in the leiobunine harvestmen or "daddy long-legs" of eastern North America, a monophyletic group that includes species in which males court females using nuptial gifts and other species that are equipped for apparent precopulatory antagonism (i.e., males with long, hardened penes and females with sclerotized pregenital barriers). We used parsimony- and Bayesian likelihood-based analyses to reconstruct character evolution in categorical reproductive traits and found that losses of ancestral gift-bearing penile sacs are strongly associated with gains of female pregenital barriers. In most cases, both events occur on the same internal branch of the phylogeny. These coevolutionary changes occurred at least four times, resulting in clade-specific designs in the penis and pregenital barrier. The discovery of convergent origins and/or enhancements of apparent precopulatory antagonism among closely related species offers an unusual opportunity to investigate how major changes in reproductive morphology have occurred. We propose new hypotheses that attribute these enhancements to changes in ecology or life history that reduce the duration of breeding seasons, an association that is consistent with female choice, sexual conflict, and/or an alternative evolutionary mechanism.

Molecular phylogeny of the leiobunine harvestmen of eastern North America (Opiliones: Sclerosomatidae: Leiobuninae).

Phylogenetic relationships among the leiobunine harvestmen or "daddy-longlegs" of eastern North America (Leiobunum, Hadrobunus, Eumesosoma) are poorly known, and systematic knowledge of the group has been limited largely to species descriptions and proposed species groups. Here we obtained mitochondrial (NADH1, 16S and 12S rDNA) and nuclear (28S rDNA, EF-1α introns and exons) DNA sequences from representatives of each genus, virtually all Leiobunum species from the USA and Canada, four western North American outgroup species and the distantly related Phalangium opilio. We applied bayesian, maximum-likelihood and parsimony methods under various data-partition treatments to reconstruct phylogeny and to test taxonomy-based phylogenetic hypotheses. Results were largely congruent among methods and treatments and well supported by bootstrap and posterior probability values. We recovered Leiobunum as paraphyletic with respect to Eumesosoma and Hadrobunus. Most species were encompassed by five well-supported clades that broadly correspond to groups based on male reproductive morphology (Hadrobunus group, an early-season Leiobunum group, L. vittatum group, L. politum group and L. calcar group). Relationships within species groups were often ambiguous or inconsistent with morphology, suggesting the presence of gene introgression or deep coalescence and/or the need for taxonomic revision.