Australian salt lakes contain a diverse range of endemic invertebrates. The brine shrimp Parartemia is among the most speciose and salt-tolerant of these invertebrates. The morphotaxonomy of Parartemia is well established but there has only been limited molecular assessment of the phylogenetic relationships and boundaries of the morphospecies. We used multiple genetic markers (nuclear 28S and mitochondrial 16S and COI) and tree-building methods (Bayesian inference and maximum likelihood) to investigate the phylogeny of Parartemia. We also used species delimitation methods to test the validity of morphological species designations. The data set included all but 2 of the 18 described Parartemia morphospecies, collected from a total of 93 sites from across southern Australia plus some sequences from GenBank. The results identified large amounts of molecular divergence (e.g. COI P-values of up to 25.23%), some groups of closely related species (which also usually shared some morphological similarities) and some distinctive species, although the relationships among divergent lineages were generally not well resolved. The most conservative set of results from the species delimitation analyses suggests that the morphotaxonomy is largely accurate, although many morphospecies comprised divergent genetic lineages separated by COI P-values of up to 17.02%. Two putative new morphospecies, three cryptic species and one synonymy were identified. Our findings improve the knowledge of Parartemia taxonomy and will facilitate the development of future studies and conservation of this taxon.

The identification of spider species presents many challenges, since in most cases the characters used are from genital structures that are only fully developed in the adult stage, hence the identification of immatures is most often not possible. Additionally, these structures usually also present some intra-specific variability, which in some cases makes the identification of closely related species difficult. The genetic barcode technique (DNA barcodes), based on sequencing of the mitochondrial marker cytochrome c oxidase subunit I (COI), has proven a useful, complementary tool to overcome these limitations. In this work, the contribution of DNA barcoding to the taxonomy of the subfamily Amaurobioidinae is explored using the refined single linkage analysis (RESL) algorithm for the delimitation of operational taxonomic units (OTUs), in comparison with the assemble species by automatic partitioning (ASAP) algorithm, and presented in conjunction with an updated molecular phylogenetic analysis of three other markers (28S rRNA, 16S rRNA, Histone H3), in addition to COI. Of a total of 97 included species identified by morphology, 82 species were concordant with the operational taxonomic units obtained from RESL, representing an 85% correspondence between the two methods. Similar results were obtained using the ASAP algorithm. Previous observations of morphological variation within the same species are supported, and this technique provides new information on genetic structure and potentially cryptic species. Most of the discrepancies between DNA barcoding and morphological identification are explained by low geographic sampling or by divergent or geographically structured lineages. After the addition of many specimens with only COI data, the multi-marker phylogenetic analysis is consistent with previous results and the support is improved. The markers COI, closely followed by 28S, are the most phylogenetically informative. We conclude that the barcode DNA technique is a valuable source of data for the delimitation of species of Amaurobioidinae, in conjunction with morphological and geographic data, and it is also useful for the detection of cases that require a more detailed and meticulous study.

The epigean and subterranean spiders of the genus Cybaeus L. Koch, 1868 are distributed in the Holarctic, and are highly diversified in western North America and Japan. More than 100 species have been described from the Japanese Archipelago and several species assemblages have also been recognised among the Japanese Cybaeus based on their morphological similarities. However, their phylogenetic backbone remains unclear. Moreover, genus-level classification of several of the Japanese species appear to be questionable. In this study we performed extensive molecular analyses of the family Cybaeidae in East Asia, mainly the Japanese Archipelago, to clarify their fundamental phylogenetic relationships. We also conducted a divergence time estimation to provide insights into their historical biogeography and evolutionary history. Our divergence dating results indicate that the diversification of the major lineages of the East Asian cybaeid spiders might be related to the opening of the Sea of Japan c. 20 million years ago. On the basis of the morphological evaluation and obtained phylogenies, some East Asian species formerly placed in Cybaeus are transferred to the cybaeid genera Allocybaeina Bennett, 2020, Sincybaeus Wang & Zhang, 2022 and Cybaeina Chamberlin & Ivie, 1932, i.e. Allocybaeina petegarina (Yaginuma, 1972), comb. nov., Sincybaeus monticola (Kobayashi, 2006), comb. nov., Sincybaeus rarispinosus (Yaginuma, 1970), comb. nov., Sincybaeus yoshiakii (Yaginuma, 1968), comb. nov., and Cybaeina whanseunensis (Paik & Namkung, 1967), comb. nov. Our results clarify the genus-level richness of cybaeids in the Japanese Archipelago for the first time, and reveal the fine-scale phylogenetic relationships of Cybaeus species endemic to the Japanese Archipelago and adjacent regions.

ZooBank: urn:lsid:zoobank.org:pub:AF2A3C0E-7F0F-4253-85BA-D995A075F00D