Machine learning approaches delimit cryptic taxa in a previously intractable species complex.

Cryptic species are not diagnosable via morphological criteria, but can be detected through analysis of DNA sequences. A number of methods have been developed for identifying species based on genetic data; however, these methods are prone to over-splitting taxa with extreme population structure, such as dispersal-limited organisms. Machine learning methodologies have the potential to overcome this challenge. Here, we apply such approaches, using a large dataset generated through hybrid target enrichment of ultraconserved elements (UCEs). Our study taxon is the Aoraki denticulata species complex, a lineage of extremely low-dispersal arachnids endemic to the South Island of Aotearoa New Zealand. This group of mite harvesters has been the subject of previous species delimitation studies using smaller datasets generated through Sanger sequencing and analytical approaches that rely on multispecies coalescent models and barcoding gap discovery. Those analyses yielded a number of putative cryptic species that seems unrealistic and extreme, based on what we know about species' geographic ranges and genetic diversity in non-cryptic mite harvesters. We find that machine learning approaches, on the other hand, identify cryptic species with geographic ranges that are similar to those seen in other morphologically diagnosable mite harvesters in Aotearoa New Zealand's South Island. We performed both unsupervised and supervised machine learning analyses, the latter with training data drawn either from animals broadly (vagile and non-vagile) or from a custom training dataset from dispersal-limited harvesters. We conclude that applying machine learning approaches to the analysis of UCE-derived genetic data is an effective method for delimiting species in complexes of low-vagility cryptic species, and that the incorporation of training data from biologically relevant analogues can be critically informative.

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.

Variation in Mating Dynamics across Five Species of Leiobunine Harvestmen (Arachnida: Opliones).

The study of mating choices often focuses on correlates of traits to the overall outcome of a mating interaction. However, mating interactions can proceed through a series of stages, with opportunities for assessment at each stage. We compared whether male or female size predicted mating interaction outcome across several stages of mating in five species of North American leiobunine harvestmen (commonly known as daddy longlegs). Leiobunine harvestmen have been previously shown to exhibit incredible morphological diversity consistent with a spectrum of male⁻female antagonism. Across all of the species, we found a general progression of female size predicting the outcome (success and timing) of early stages of interactions, and male size or male size relative to female size predicting the outcome and timing of later stages of interactions. We also found that size was not a strong predictor of outcome in the two species on the lower end of the antagonism spectrum. The variation in how female and male size predicted outcomes across species and stages of mating suggests that multiple mechanisms may operate to shape mating dynamics within and across species. Given the close relatedness of the species studied, the patterns we uncovered suggest a rapid evolution of the traits and processes predicting the outcome of mating interactions.

A dated molecular phylogeny of mite harvestmen (Arachnida: Opiliones: Cyphophthalmi) elucidates ancient diversification dynamics in the Australian Wet Tropics.

Austropurcellia, a genus of dispersal-limited arachnids endemic to isolated patches of coastal rainforest in Queensland, Australia, has a remarkable biogeographic history. The genus is a member of the family Pettalidae, which has a classical temperate Gondwanan distribution; previous work has suggested that Austropurcellia is an ancient lineage, with an origin that predates Gondwanan rifting. Subsequently, this lineage has persisted through major climatic fluctuations, such as major aridification during the Miocene and contraction and fragmentation of forest habitats during the Last Glacial Maximum (LGM). In order to understand Austropurcellia's evolutionary and biogeographic history, we generated DNA sequences from both mitochondrial and nuclear loci and combined this information with previously published datasets for the globally-distributed suborder Cyphophthalmi (i.e., all mite harvestmen). We generated phylogenetic trees using maximum likelihood and Bayesian approaches to date divergences using a relaxed molecular clock. According to our estimates, the family Pettalidae diversified in the late Jurassic, in accordance with Gondwanan vicariance. Within Pettalidae, Austropurcellia split from its sister group in the early Cretaceous and began to diversify some 15 Ma later. Therefore, its presence in Australia predates continental rifting-making it one of very few hypothesized examples of Gondwanan vicariance that have withstood rigorous testing. We found a steady rate of diversification within the genus, with no evidence for a shift in rate associated with Miocene aridification. Ages of splits between species predate the Pleistocene, consistent with a "museum" model in which forest refugia acted to preserve existing lineages rather than drive speciation within the group.

A revised dated phylogeny of scorpions: Phylogenomic support for ancient divergence of the temperate Gondwanan family Bothriuridae.

The scorpion family Bothriuridae occupies a subset of landmasses formerly constituting East and West temperate Gondwana, but its relationship to other scorpion families is in question. Whereas morphological data have strongly supported a sister group relationship of Bothriuridae and the superfamily Scorpionoidea, a recent phylogenomic analysis recovered a basal placement of bothriurids within Iurida, albeit sampling only a single exemplar. Here we reexamined the phylogenetic placement of the family Bothriuridae, sampling six bothriurid exemplars representing both East and West Gondwana, using transcriptomic data. Our results demonstrate that the sister group relationship of Bothriuridae to the clade ("Chactoidea" + Scorpionoidea) is supported by the inclusion of additional bothriurid taxa, and that this placement is insensitive to matrix completeness or partitioning by evolutionary rate. We also estimated divergence times within the order Scorpiones using multiple fossil calibrations, to infer whether the family Bothriuridae is sufficiently old to be characterized as a true Gondwanan lineage. We show that scorpions underwent ancient diversification between the Devonian and early Carboniferous. The age interval of the bothriurids sampled (a derived group that excludes exemplars from South Africa) spans the timing of breakup of temperate Gondwana.

There is no evidence that Podoctidae carry eggs of their own species: Reply to Machado and Wolff (2017).

In our recent publication (Sharma et al., 2017), we tested the hypothesis that eggs attached to the legs of male Podoctidae (Opiliones, Laniatores) constituted a case of paternal care, using molecular sequence data in tandem with multiple sequence alignments to test the prediction that sequences of the eggs and the adults that carried them would indicate conspecific identity. We discovered that the sequences of the eggs belonged to spiders, and thus rejected the paternal care hypothesis for these species. Machado and Wolff (2017) recently critiqued our work, which they regarded as a non-critical interpretation and over-reliance on molecular sequence data, and defended the traditional argument that the eggs attached to podoctids are in fact harvestman eggs. Here we show that additional molecular sequence data also refute the identity of the eggs as conspecific harvestman eggs, using molecular cloning techniques to rule out contamination. We show that individual gene trees consistently and reliably place the egg and adult sequences in disparate parts of the tree topology. Phylogenetic methods consistently place all egg sequences within the order Araneae (spiders). We submit that evidence for the paternal care hypothesis based on behavioral, morphological, and natural history approaches is either absent or insufficient for concluding that the eggs of podoctids are conspecific.

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.

A multilocus phylogeny of Podoctidae (Arachnida, Opiliones, Laniatores) and parametric shape analysis reveal the disutility of subfamilial nomenclature in armored harvestman systematics.

The taxonomy and systematics of the armored harvestmen (suborder Laniatores) are based on various sets of morphological characters pertaining to shape, armature, pedipalpal setation, and the number of articles of the walking leg tarsi. Few studies have tested the validity of these historical character systems in a comprehensive way, with reference to an independent data class, i.e., molecular sequence data. We examined as a test case the systematics of Podoctidae, a family distributed throughout the Indo-Pacific. We tested the validity of the three subfamilies of Podoctidae using a five-locus phylogeny, and examined the evolution of dorsal shape as a proxy for taxonomic utility, using parametric shape analysis. Here we show that two of the three subfamilies, Ibaloniinae and Podoctinae, are non-monophyletic, with the third subfamily, Erecananinae, recovered as non-monophyletic in a subset of analyses. Various genera were also recovered as non-monophyletic. As first steps toward revision of Podoctidae, the subfamilies Erecananinae Roewer, 1912 and Ibaloniinae Roewer, 1912 are synonymized with Podoctinae Roewer, 1912 new synonymies, thereby abolishing unsubstantiated subfamilial divisions within Podoctidae. We once again synonymize the genus Paralomanius Goodnight & Goodnight, 1948 with Lomanius Roewer, 1923 revalidated. We additionally show that eggs carried on the legs of male Podoctidae are not conspecific to the males, falsifying the hypothesis of paternal care in this group.

New species of Austropurcellia, cryptic short-range endemic mite harvestmen (Arachnida, Opiliones, Cyphophthalmi) from Australia's Wet Tropics biodiversity hotspot.

The genus Austropurcellia is a lineage of tiny leaf-litter arachnids that inhabit tropical rainforests throughout the eastern coast of Queensland, Australia. The majority of their diversity is found within the Wet Tropics rainforests of northeast Queensland, an area known for its exceptionally high levels of biodiversity and endemism. Studying the biogeographic history of limited-dispersal invertebrates in the Wet Tropics can provide insight into the role of climatic changes such as rainforest contraction in shaping rainforest biodiversity patterns. Here we describe six new species of mite harvestmen from the Wet Tropics rainforests, identified using morphological data, and discuss the biogeography of Austropurcellia with distributions of all known species. With this taxonomic contribution, the majority of the known diversity of the genus has been documented.

New species of mite harvestmen from southeast Queensland, Australia greatly extend the known distribution of the genus Austropurcellia (Arachnida, Opiliones, Cyphophthalmi).

Austropurcellia Juberthie 1988 is a genus of mite harvestmen previously known from numerous localities in the Wet Tropics of northern Queensland and from one locality in central Queensland, Australia. As a result of the current study, the genus is now also known from localities in far southeast Queensland. We describe three new species of Austropurcellia from museum lots: A. acuta sp. nov., A. barbata sp. nov., and A. superbensis sp. nov. Each new species is known from only one to two localities, and from very few specimens. In addition, we describe a pair of previously overlooked dorsal anterior cuticular structures that may be sensory in nature and are found in all Austropurcellia specimens examined, including both new and previously described species. We present a new distribution map of Austropurcellia, greatly expanding its known range to almost the entire east coast of Queensland, and discuss the biogeography of the genus.

Deep genetic divergences in Aoraki denticulata (Arachnida, Opiliones, Cyphophthalmi): a widespread 'mite harvestman' defies DNA taxonomy.

Aoraki denticulata (Arachnida, Opiliones, Cyphophthalmi, Pettalidae), a widespread 'mite harvestman' endemic to the South Island of New Zealand, is found in leaf littler habitats throughout Nelson and Marlborough, and as far south as Arthur's Pass. We investigated the phylogeography and demographic history of A. denticulata in the first genetic population-level study within Opiliones. A total of 119 individuals from 17 localities were sequenced for 785 bp of the gene cytochrome c oxidase subunit I; 102 of these individuals were from the Aoraki subspecies A. denticulata denticulata and the remaining 17 were from the subspecies A. denticulata major. An extraordinarily high degree of genetic diversity was discovered in A. denticulata denticulata, with average uncorrected p-distances between populations as high as 19.2%. AMOVA, average numbers of pairwise differences, and pairwise F(ST) values demonstrated a significant amount of genetic diversity both within and between populations of this subspecies. Phylogenetic analysis of the data set revealed many well-supported groups within A. denticulata denticulata, generally corresponding to clusters of specimens from single populations with short internal branches, but separated by long branches from individuals from other populations. No haplotypes were shared between populations of the widespread small subspecies, A. denticulata denticulata. These results indicate a subspecies within which very little genetic exchange occurs between populations, a result consistent with the idea that Cyphophthalmi are poor dispersers. The highly structured populations and deep genetic divergences observed in A. denticulata denticulata may indicate the presence of cryptic species. However, we find a highly conserved morphology across sampling localities and large genetic divergences within populations from certain localities, equivalent to those typically found between populations from different localities. Past geological events may have contributed to the deep genetic divergences observed between sampling localities; additionally, the high divergence within populations of A. denticulata denticulata suggests that the rate of COI evolution may be accelerated in this taxon. In contrast, the larger subspecies A. denticulata major shows much less differentiation between and within sampling localities, suggesting that it may disperse more easily than its smaller counterpart. The fact that the remarkable genetic divergences within populations of A. denticulata denticulata from certain localities are equivalent to divergences between localities poses a challenge to the rapidly spreading practice of DNA taxonomy.

A new model Gondwanan taxon: systematics and biogeography of the harvestman family Pettalidae (Arachnida, Opiliones, Cyphophthalmi), with a taxonomic revision of genera from Australia and New Zealand.

The phylogeny of the temperate Gondwanan harvestman family Pettalidae is investigated by means of a new morphological matrix of 45 characters, and DNA sequence data from five markers, including two nuclear ribosomal genes (18S rRNA and 28S rRNA), one nuclear protein coding gene (histone H3), and two mitochondrial genes-one protein coding (cytochrome c oxidase subunit I) and one ribosomal (16S rRNA). Phylogenetic analyses using an array of homology schemes (dynamic and static), criteria (parsimony and maximum likelihood), and sampling strategies (optimal trees versus Bayesian phylogenetics) all agree on the monophyly of Pettalidae as well as several of its subclades, each of which is restricted to a modern landmass. While most genera as traditionally defined are monophyletic, Rakaia and Neopurcellia, distributed across Queensland (Australia) and New Zealand, are not. Instead, the species from Queensland, previously described under three genera, constitute a well-supported clade, suggesting that in this case biogeography prevails over traditional taxonomy. A taxonomic emendation of the genera from Queensland and New Zealand is presented, and the new genus Aoraki is erected to include the species of the New Zealand denticulata group. A biogeographical hypothesis of the relationships of the former temperate Gondwana landmasses (with the exception of Madagascar) is presented, although ambiguity in the deep nodes of the pettalid tree renders such inference provisional. The data suggest that neither the South African fauna, the New Zealand fauna nor the Australian fauna is monophyletic but instead monophyly is found at smaller geographic scales (e.g., Western Australia, Queensland, NE South Africa).

The genus Cyphophthalmus (Arachnida, Opiliones, Cyphophthalmi) in Europe: a phylogenetic approach to Balkan Peninsula biogeography.

In this study, we present phylogenetic data to characterize the relationships among sironids centered in the Balkan region, and use these results to discuss biogeographical aspects of sironid evolution. Analysis of ca. 4.5 kb of sequence data from three nuclear and two mitochondrial genes reveals monophyly of a Balkan clade for which we resurrect the name Cyphophthalmus, considered a junior synonym of Siro for over a century. This clade diversified into several groups, and at least three of them--the duricorius group, the serbicus group, and the minutus group--are well corroborated by the data as monophyletic lineages. The members of the different groups, mostly living in troglobitic environments, have diversified in overlapping geographic regions, with evidence of an eastern origin for the group. Our data also suggest that mitochondrial and nuclear genes are all contributing towards the final resolution of the combined analysis of the data.