Rachael A. King

Divergent Molecular Lineages and Not-So-Cryptic Species: The First Descriptions of Stygobitic Chiltoniid Amphipods (Talitroidea: Chiltoniidae) from Western Australia

ABSTRACT The Australian stygofauna comprises a unique and diverse assemblage of invertebrates, of which the amphipod crustaceans are a dominant but poorly described element. Recent exploration of the Western Australian stygofauna, in particular the Yilgarn region of central Western Australia, has shown evidence of great species diversity, with numerous individual calcrete aquifers found to contain unique assemblages of invertebrate species. A recent fine-scale biodiversity initiative, using COI barcoding, of a single calcrete aquifer (Sturt Meadows) in the Yilgarn region reported the presence of three divergent and morphologically cryptic stygobitic lineages of amphipods from Chiltoniidae, which represent undescribed taxa. This paper details the subsequent systematic analysis of these COI lineages and presents a broader phylogeny and detailed morphological analyses of the lineages. The report of cryptic species was not supported upon morphological examination and three new species from three new genera (Scutachiltonia n. gen., Stygochiltonia n. gen., and Yilgarniella n. gen.) are described from the Sturt Meadows calcrete aquifer. The three genera do not form a monophyletic group and are instead believed to have evolved from separate colonisation events from distinct ancestors rather than from speciation events within the aquifer. This work contributes to a broader research initiative, documenting the presence of a rich subterranean invertebrate fauna in the Yilgarn region.

What lies beneath: Molecular phylogenetics and ancestral state reconstruction of the ancient subterranean Australian Parabathynellidae (Syncarida, Crustacea)

The crustacean family Parabathynellidae is an ancient and significant faunal component of subterranean ecosystems. Molecular data were generated in order to examine phylogenetic relationships amongst Australian genera and assess the species diversity of this group within Australia. We also used the resultant phylogenetic framework, in combination with an ancestral state reconstruction (ASR) analysis, to explore the evolution of two key morphological characters (number of segments of the first and second antennae), previously used to define genera, and assess the oligomerization principle (i.e. serial appendage reduction over time), which is commonly invoked in crustacean systematics. The ASR approach also allowed an assessment of whether there has been convergent evolution of appendage numbers during the evolution of Australian parabathynellids. Sequence data from the mtDNA COI and nDNA 18S rRNA genes were obtained from 32 parabathynellid species (100% of described genera and ~25% of described species) from key groundwater regions across Australia. Phylogenetic analyses revealed that species of each known genus, defined by traditional morphological methods, were monophyletic, suggesting that the commonly used generic characters are robust for defining distinct evolutionary lineages. Additionally, ancestral state reconstruction analysis provided evidence for multiple cases of convergent evolution for the two morphological characters evaluated, suggesting that caution needs to be shown when using these characters for elucidating phylogenetic relationships, particularly when there are few morphological characters available for reconstructing relationships. The ancestral state analysis contradicted the conventional view of parabathynellid evolution, which assumes that more simplified taxa (i.e. those with fewer-segmented appendages and setae) are derived and more complex taxa are primitive.

Species, ESUs or populations? Delimiting and describing morphologically cryptic diversity in Australian desert spring amphipods

Abstract. Cryptic species are frequently being discovered in refugial habitats, such as desert springs and groundwater systems. Unfortunately, many of these taxa remain as unnamed entities years after their initial discovery. Recent advances in the use of molecular data and coalescent analyses allow DNA-based delimitation of species to move from single locus, tree-based methods to multilocus coalescent analyses. This study compares two DNA-based approaches to delimit species of putatively cryptic freshwater amphipods (Chiltoniidae) from desert springs in central Australia. In addition, a morphometric analysis of 11 characters was undertaken to determine whether the DNA-delimited species were morphologically distinguishable. The single locus method results in identification of lineages that are not supported as species under the multilocus coalescent analyses. We conclude that Wangiannachiltonia guzikae King, 2009, as currently circumscribed, represents six genetically distinct amphipod species, and we describe and name these species despite no clear diagnosable morphological differences. Critically, all of these newly recognised species have extremely limited distributions, which increases the biodiversity significance of their desert spring habitat.

Molecular phylogenetic, morphological and biogeographic evidence for a new genus of parabathynellid crustaceans (Syncarida:Bathynellacea) from groundwater in an ancient southern Australian landscape

Abstract. The putatively ancient subterranean crustacean family Parabathynellidae has been poorly studied, in part because of the problem of obtaining material from difficult to access subterranean habitats in which they live. Further, the systematics of the group has been complicated by their generally simplified morphology and isolated descriptions of new taxa in the absence of any phylogenetic framework. Using material from comprehensive field surveys and mitochondrial cytochrome c oxidase subunit I (COI) and nuclear 18S sequence data, plus morphology, a new genus is recognised, Arkaroolabathynella Abrams & King, gen. nov., from underground waters in the Flinders Ranges, South Australia. Arkaroolabathynella contains four genetically and morphologically distinct species, described as A. bispinosa Abrams & King, sp. nov., A. remkoi Abrams & King, sp. nov., A. robusta Abrams & King, sp. nov. and A. spriggi Abrams & King, sp. nov. Phylogenetic analysis also revealed a previously unknown diversity of parabathynellids from southern Australia, and a complex set of relationships with the eastern (New South Wales) and south-western (Western Australia) continental faunas. Additionally, this study showed that deep molecular divergences in parabathynellids are not always reflected in morphological divergence. A checklist to Australian parabathynellid genera and species is also provided.

The Australian freshwater amphipods Austrochiltonia australis and Austrochiltonia subtenuis (Amphipoda : Talitroidea : Chiltoniidae) confirmed and two new cryptic Tasmanian species revealed using a combined molecular and morphological approach

Given the complex nature of freshwater catchment divides and emerging evidence of high levels of genetic diversity, there is great potential for cryptic species to exist among Australian freshwater amphipod groups. Among the chiltoniid amphipods, two congeneric species, Austrochiltonia australis (Sayce, 1901) and A. subtenuis (Sayce, 1902), have been widely recorded across southern Australia yet are poorly known and contentiously defined. A large fragment of the mitochondrial DNA cytochrome c oxidase I (COI) gene was examined and morphological diversity among populations assessed across the reported geographic range of the two putative species. The results confirmed A. australis and A. subtenuis as morphological and molecular species. In addition, two previously undetected and cryptic species from Tasmania are recognised – sister species to A. subtenuis and A. australis. Working conclusions provide evidence towards a more comprehensive systematic revision of the Chiltoniidae and present species information relevant to conservation and management efforts of Australian river systems. A key is presented to the chiltoniid amphipods of southern Australia.

Molecular phylogenetic analyses reveal a new southern hemisphere oniscidean family (Crustacea : Isopoda) with a unique water transport system

Abstract. A significant diversity of terrestrial oniscidean isopods was recently discovered in the subterranean ‘calcrete islands’ of Western Australia, but the species and higher-level systematic status of much of the fauna are currently uncertain. Here we focus on one group of species that was initially assigned to the genus Trichorhina (Platyarthridae), based on several shared characters, and investigate the phylogenetic relationships of these species to 21 oniscidean genera, including 13 known families, using 18S rDNA sequence data. We then present phylogenetic analyses using 28S-only and combined 18S, 28S rDNA and mitochondrial cytochrome c oxidase subunit I (COI) data for a more restricted sampling of taxa, and present results for a detailed morphological study of the antennae and other cephalic structures of exemplar taxa. Bayesian and maximum likelihood analyses of the extended 18S-only, the 28S-only and multi-gene datasets provide strong evidence for a distinct well-supported monophyletic group comprising the new Western Australian and one South American taxon. This clade is unrelated to all included members of Platyarthridae, which appears to be polyphyletic, and it forms a distinct group relative to other oniscidean families. Given these findings and the results of the morphological study, a new southern hemisphere oniscidean family, Paraplatyarthridae Javidkar & King, fam. nov. is erected based on Paraplatyarthrus subterraneus Javidkar & King, gen. & sp. nov. (type genus and species), and several undescribed taxa which occur in the arid (terrestrial and subterranean) regions of Western Australia and subtropical South America. Paraplatyarthridae is distinguishable from all other oniscidian families on a combination of character states including, among others, the presence of fan-like scale setae on the dorsal body, and the ventral second antenna with leaf-like scale setae and a furrow containing elongated hair-like capillary setae that form part of a water conducting system unique within Oniscidea. This study has important implications for the higher-level classification of oniscidean crustaceans and points to the need for a more detailed molecular phylogeny that includes a comprehensive sampling of southern hemisphere taxa.

The Evolution of Epigean and Stygobitic Species of Koonunga Sayce, 1907 (Syncarida: Anaspidacea) in Southern Australia, with the Description of Three New Species.

Three new species of Koonunga were discovered in surface and subterranean waters in southern Australia, and were defined using mtDNA analyses and morphology. The new species are: Koonunga hornei Leijs & King; K. tatiaraensis Leijs & King and K. allambiensis Leijs & King. Molecular clock analyses indicate that the divergence times of the species are older than the landscape that they currently inhabit. Different scenarios explaining this apparent discrepancy are discussed in the context of the palaeography of the area. A freshwater epigean origin for Koonunga is considered the most likely hypothesis, whereby some lineages made the transition to the subterranean environment within the last few million years influenced by significant climatic cooling/drying. We discuss the possibility that one stygobitic lineage secondarily regained some of its body pigmentation as adaptation to increased photic conditions after cave collapse and forming of cenotes during the last glacial maximum.

Taxonomy of Paraplatyarthrus Javidkar and King (Isopoda: Oniscidea: Paraplatyarthridae) with description of five new species from Western Australia, and comments on Australian Trichorhina Budde-Lunde, 1908 (Platyarthridae)

The oniscidean fauna of Australia is generally poorly known but recent sampling has revealed a new family, Paraplatyarthridae, found in both terrestrial and groundwater calcretes of central Western Australia. The family was initially described based on a new genus and species, Paraplatyarthrus subterraneus Javidkar and King, 2015. Here we describe an additional five Paraplatyarthrus species from the Yilgarn region of Western Australia, based on both morphological and molecular evidence (COI divergences). Four species are subterranean: P. crebesconiscus Javidkar and King sp. nov., P. cunyuensis Javidkar and King sp. nov., P. occidentoniscus Javidkar and King sp. nov., and P. pallidus Javidkar and King sp. nov., and one is a surface species, P. nahidae Javidkar and King sp. nov. A key to their identification is provided along with information on their distribution. In addition, type material of the two described Australian species of Platyarthridae, Trichorhina australiensis Wahrberg, 1922 from Western Australia and T. tropicalis Lewis, 1998 from Queensland, are examined. Morphological reassessment of type material shows T. australiensis belongs to Paraplatyarthrus (comb. nov.) and that T. tropicalis is correctly placed in Trichorhina, confirming that the genus and family Platyarthridae occur in Australia.

Molecular evidence for mid-Pleistocene divergence of populations of three freshwater amphipod species (Talitroidea:Chiltoniidae) on Kangaroo Island, South Australia, with a new spring-associated genus and species

Abstract. Recent molecular and morphological analyses have shown that chiltoniid amphipods, once thought to be a relictual group, are a diverse and speciose family of Australian freshwater amphipods. As part of a larger examination of the family, chiltoniids from Kangaroo Island in South Australia were collected and analysed using molecular (COI and 28S) and morphological methods in order to understand species distributional patterns and relationships. Kartachiltonia moodyi gen. nov., sp. nov., a spring-associated species endemic to the island, was discovered and populations of three additional mainland species (Austrochiltonia australis, A. dalhousiensis and A. subtenuis) were examined. The island populations of A. australis, A. dalhousiensis and A. subtenuis were found to form natural groups with differing haplotype coalescence times dating from the Early to Mid-Pleistocene. Numerous cycles of regional climate change throughout the Pleistocene are likely to have driven speciation in chiltoniid amphipods in southern Australia and the presence of multiple chiltoniid species at Kangaroo Island indicates that it exists at a likely convergence of species distribution patterns. Three possible hypotheses to explain the evolution and diversity of chiltoniids in southern Australia are discussed as are evidence for potential introduction and long-distance dispersal events.

Biogeographic history of subterranean isopods from groundwater calcrete islands in Western Australia

Groundwater calcretes in arid central Western Australia contain a diverse invertebrate groundwater fauna (stygofauna). Surveys have uncovered a diverse oniscidean isopod subterranean fauna above the water table (troglofauna), including species of a recently described genus Paraplatyarthrus. The aim of this study was to investigate the biogeographic history of Paraplatyarthrus and the timing of transitions from surface to subterranean habitats. Phylogenetic relationships among the isopod troglofauna from 11 groundwater calcretes along three palaeodrainage systems were assessed using one mitochondrial gene, cytochrome c oxidase subunit 1 (COI), and two nuclear markers, lysyl‐tRNA synthetase (LysRS) and 18S rRNA (18S) genes. Phylogenetic analyses revealed multiple sister lineage relationships between troglophile and troglobite lineages and evidence for divergent mtDNA lineages within species, providing a range of nodes for dating evolutionary transitions from surface to subterranean habitats. Relaxed molecular clock analyses provided evidence that evolutionary transitions from surface to subterranean environments took place between 13.3 and 1.75 million years ago, coinciding with the onset of aridification of Australia from the late Tertiary. In cases where groundwater calcretes contained multiple species, the taxa were not closely related phylogenetically, suggesting that these calcretes were independently colonised by multiple ancestral species. The study further confirmed the role of late/post‐Miocene aridification as a key driver of the evolution of subterranean invertebrates in the calcrete islands of Western Australia, supporting the climatic relict hypothesis. Troglobites most likely evolved from the troglophile ancestors that were capable of dispersal among, and active colonisation of, calcretes.