Kathleen M. Saint

Subterranean archipelago in the Australian arid zone: mitochondrial DNA phylogeography of amphipods from central Western Australia

In 1998, a unique subterranean ecosystem was discovered in numerous isolated calcrete (carbonate) aquifers in the arid Yilgarn region of Western Australia. Previous morphological and genetic analyses of a subterranean water beetle fauna suggest that calcrete aquifers are equivalent to closed island habitats that have been isolated for millions of years. We tested this hypothesis further by phylogeographic analyses of subterranean amphipods (Crangonyctoidea: Paramelitidae and Hyalidae) using mitochondrial DNA sequence data derived from the cytochrome oxidase I gene. Phylogenetic analyses and population genetic analyses (samova) provided strong evidence for the existence of at least 16 crangonyctoid and six hyalid divergent mitochondrial lineages, each restricted in their distribution to a single calcrete aquifer, in support of the ‘subterranean island (archipelago) hypothesis’ and extending its scope to include entirely water respiring invertebrates. Sequence divergence estimates between proximate calcrete populations suggest that calcretes have been isolated at least since the Pliocene, coinciding with a major aridity phase that led to the intermittent drying of surface water. The distribution of calcretes along palaeodrainage channels and on either side of drainage divides, have had less influence on the overall phylogeographic structure of populations, with evidence that ancestral crangonyctoid and hyalid species moved between catchments multiple times prior to their isolation within calcretes. At least two potential modes of evolution may account for the diversity of subterranean amphipod populations: dispersal/vicariance of stygobitic species or colonization of calcretes by surface species and independent evolution of stygobitic characteristics.

Deep phylogeographic structuring of populations of the trapdoor spider Moggridgea tingle (Migidae) from southwestern Australia: evidence for long-term refugia within refugia

Southwestern Australia has been recognized as a biodiversity hot spot of global significance, and it is particularly well known for its considerable diversity of flowering plant species. Questions of interest are how this region became so diverse and whether its fauna show similar diverse patterns of speciation. Here, we carried out a phylogeographic study of trapdoor spiders (Migidae: Moggridgea), a presumed Gondwanan lineage found in wet forest localities across southwestern Australia. Phylogenetic, molecular clock and population genetic analyses of mitochondrial (mtDNA) COI gene and ITS rRNA (internal transcribed spacer) data revealed considerable phylogeographic structuring of Moggridgea populations, with evidence for long‐term (>3 million years) isolation of at least nine populations in different geographic locations, including upland regions of the Stirling and Porongurup Ranges. High levels of mtDNA divergence and no evidence of recent mitochondrial gene flow among valley populations of the Stirling Range suggest that individual valleys have acted as refugia for the spiders throughout the Pleistocene. Our findings support the hypothesis that climate change, particularly the aridification of Australia after the late Miocene, and the topography of the landscape, which allowed persistence of moist habitats, have been major drivers of speciation in southwestern Australia.

Subterranean archipelago: mitochondrial DNA phylogeography of stygobitic isopods (Oniscidea:Haloniscus ) from the Yilgarn region of Western Australia

The arid Yilgarn region of Western Australia contains numerous isolated calcrete aquifers, within which a diverse subterranean fauna has been discovered. Genetic and morphological studies of subterranean dytiscid beetles and amphipods have suggested that individual calcretes are equivalent to closed island habitats, which have been isolated for millions of years. Here we test this ‘subterranean island’ hypothesis further by phylogeographic analyses of subterranean oniscidean isopods (Haloniscus), using mitochondrial DNA (mtDNA) sequence data derived from the cytochrome c oxidase subunit I gene. Phylogenetic and population genetic analyses provided evidence for significant phylogeographic structuring of isopod populations, with evidence for at least 24 divergent mtDNA lineages, each restricted in their distribution to a single calcrete aquifer. The high level of divergence among calcrete populations (generally >25%) and several mtDNA lineages within calcretes (>16%) suggests that each lineage is likely to represent a distinct species. These analyses, together with comparative phylogeographic data from dytiscid beetles and amphipods, provide strong support for the ‘subterranean island’ hypothesis, applying to both air-breathing and fully aquatic arthropod groups. The finding of several epigean lineages that grouped with stygobitic Haloniscus populations, and the overall phylogeographic structure of populations, suggests that the majority of stygobitic species evolved within individual calcretes following independent colonisation by epigean ancestors.

Differential gene flow of mitochondrial and nuclear DNA markers among chromosomal races of Australian morabine grasshoppers (Vandiemenella, viatica species group)

Recent theoretical developments have led to a renewed interest in the potential role of chromosomal rearrangements in speciation. Australian morabine grasshoppers (genus Vandiemenella, viatica species group) provide an excellent study system to test this potential role of chromosomal rearrangements because they show extensive chromosomal variation and formed the basis of a classic chromosomal speciation model. There are three chromosomal races, viatica19, viatica17, and P24(XY), on Kangaroo Island, South Australia, forming five parapatric populations with four putative contact zones among them. We investigate the extent to which chromosomal variation among these populations may be associated with barriers to gene flow. Population genetic and phylogeographical analyses using 15 variable allozyme loci and the elongation factor‐1α (EF‐1α) gene indicate that the three races represent genetically distinct taxa. In contrast, analyses of the mitochondrial cytochrome c oxidase subunit I (COI) gene show the presence of three distinctive and geographically localized groups that do not correspond with the distribution of the chromosomal races. These discordant population genetic patterns are likely to result from introgressive hybridization between the chromosomal races and range expansions/contractions. Overall, these results suggest that reduction of nuclear gene flow may be associated with chromosomal variation, or underlying genetic variation linked with chromosomal variation, whereas mitochondrial gene flow appears to be independent of this variation in these morabine grasshoppers. The identification of an intact contact zone between P24(XY) and viatica17 offers considerable potential for further investigation of molecular mechanisms that maintain distinct nuclear genomes among the chromosomal races.

Opsin transcripts of predatory diving beetles: a comparison of surface and subterranean photic niches.

The regressive evolution of eyes has long intrigued biologists yet the genetic underpinnings remain opaque. A system of discrete aquifers in arid Australia provides a powerful comparative means to explore trait regression at the genomic level. Multiple surface ancestors from two tribes of diving beetles (Dytiscidae) repeatedly invaded these calcrete aquifers and convergently evolved eye-less phenotypes. We use this system to assess transcription of opsin photoreceptor genes among the transcriptomes of two surface and three subterranean dytiscid species and test whether these genes have evolved under neutral predictions. Transcripts for UV, long-wavelength and ciliary-type opsins were identified from the surface beetle transcriptomes. Two subterranean beetles showed parallel loss of all opsin transcription, as expected under ‘neutral’ regressive evolution. The third species Limbodessus palmulaoides retained transcription of a long-wavelength opsin (lwop) orthologue, albeit in an aphotic environment. Tests of selection on lwop indicated no significant differences between transcripts derived from surface and subterranean habitats, with strong evidence for purifying selection acting on L. palmulaoides lwop. Retention of sequence integrity and the lack of evidence for neutral evolution raise the question of whether we have identified a novel pleiotropic role for lwop, or an incipient phase of pseudogene development.

Re-examination of a proposed case of stasipatric speciation: phylogeography of the Australian morabine grasshoppers (Vandiemenella viatica species group)

Karyotypic differences have been used for delimiting populations or species, although whether these mutations provide strong barriers to gene flow between populations and promote speciation remains contentious. In this study, we assessed whether 11 chromosomal races of Australian morabine grasshoppers (Vandiemenella viatica species group) represent genetically distinct populations by analyses of cytological and allozyme (35 loci) data and DNA sequences of the elongation factor‐1 alpha (EF‐1α), anonymous Mvia11, and mitochondrial cytochrome c oxidase subunit I (COI) loci. While the Vandiemenella chromosomal taxa generally represent genetically distinct units, a substantial portion of the total genetic variation in our samples was not explained by the chromosomal variation. Mantel tests indicated that Vandiemenella populations were spatially structured and have maintained gene flow at a local scale within each of the taxa. The group was subdivided into 13 genetic clusters; four chromosomal taxa comprised single exclusive clusters, while others comprised more than one cluster or clusters shared with other taxa. Boundaries of these cryptic population subdivisions correspond with several biogeographical barriers, such as straits, gulfs, the Murray River, and an ancient mega‐lake, Lake Bungunnia. The viatica species group was previously proposed to have diversified without major geographical separation based on the stasipatric speciation model; however, the present study suggests the involvement of allopatric fragmentation. Given extensive nonmonophyly of chromosomal taxa and incomplete barriers to gene flow among taxa, all Vandiemenella chromosomal taxa and genetically distinct populations within chromosomal taxa, except Vandiemenella pichirichi, should be regarded as populations of one species: Vandiemenella viatica.

Cryptic grey-bellied dunnart (Sminthopsis griseoventer) discovered in South Australia: genetic, morphological and subfossil analyses show the value of collecting voucher material

The assumption that almost all mammal species are known to science has led to a recent trend away from collecting voucher specimens/tissues during field studies. Here we present a case study of a recently discovered cryptic marsupial (Sminthopsis griseoventer) in South Australia (SA) and show how such collections can contribute to rigorous biodiversity and biogeographic assessments. Morphological and genetic (allozyme and mitochondrial control region (CR) sequence data) analyses, including ancient DNA analyses of type material, were applied to 188 voucher specimens and 94 non-vouchered tissues of Sminthopsis held at the SA Museum. These data were used to confirm the presence of S. griseoventer in SA, validate means of identifying it morphologically and describe recent and pre-European distributions. Pelage differences between S. griseoventer and S. dolichura enabled their identification, but external measurements overlapped considerably. Subfossil S. griseoventer were identified from seven deposits and confirmed that in the past the species was more widespread in SA. CR divergences (>1.8%) among Western Australian and SA S. griseoventer suggested its long-term presence in SA. Discrepancies between the mitochondrial and allozyme affinities of S. aitkeni and S. griseoventer, coupled with the lack of obvious morphological differences, indicate that a taxonomic reappraisal of these species is warranted. The study strongly demonstrates an ongoing need for the routine collection of mammal voucher material in biological and environmental impact surveys.

PERMANENT GENETIC RESOURCES: Development of microsatellite markers and analysis of their inheritance in the Australian reptile tick, Bothriocroton hydrosauri.

Despite long‐term study, the mechanism explaining the parapatric distribution of two Australian reptile tick species is not understood. We describe the development of primers amplifying 10 microsatellite Bothriocroton hydrosauri loci, for the study of population structure and dispersal patterns of this tick. The numbers of alleles per locus ranged from two to seven in ticks from the study site, and the observed heterozygosity between 0.28 and 0.69. Pedigree analysis indicates that one locus is inherited in a non‐Mendelian manner in three families, which was not explained by null allele presence.

The α-Globin Gene Family of an Australian Marsupial, Macropus eugenii: The Long Evolutionary History of the θ-Globin Gene and Its Functional Status in Mammals

Comparative evolutionary analyses of gene families among divergent lineages can provide information on the order and timing of major gene duplication events and evolution of gene function. Here we investigate the evolutionary history of the α-globin gene family in mammals by isolating and characterizing α-like globin genes from an Australian marsupial, the tammar wallaby, Macropus eugenii. Sequence and phylogenetic analyses indicate that the tammar α-globin family consists of at least four genes including a single adult-expressed gene (α), two embryonic/neonatally expressed genes (ζ and ζ′), and θ-globin, each orthologous to the respective α-, ζ-, and θ-globin genes of eutherian mammals. The results suggest that the θ-globin lineage arose by duplication of an ancestral adult α-globin gene and had already evolved an unusual promoter region, atypical of all known α-globin gene promoters, prior to the divergence of the marsupial and eutherian lineages. Evolutionary analyses, using a maximum likelihood approach, indicate that θ-globin, has evolved under strong selective constraints in both marsupials and the lineage leading to human θ-globin, suggesting a long-term functional status. Overall, our results indicate that at least a four-gene cluster consisting of three α-like and one β-like globin genes linked in the order 5′–ζ–α–θ–ω–3′ existed in the common ancestor of marsupials and eutherians. However, results are inconclusive as to whether the two tammar ζ-globin genes arose by duplication prior to the radiation of the marsupial and eutherian lineages, with maintenance of exon sequences by gene conversion, or more recently within marsupials.

Development of microsatellite markers and analysis of their inheritance in the Australian reptile tick, Bothriocroton hydrosauri

Despite long‐term study, the mechanism explaining the parapatric distribution of two Australian reptile tick species is not understood. We describe the development of primers amplifying 10 microsatellite Bothriocroton hydrosauri loci, for the study of population structure and dispersal patterns of this tick. The numbers of alleles per locus ranged from two to seven in ticks from the study site, and the observed heterozygosity between 0.28 and 0.69. Pedigree analysis indicates that one locus is inherited in a non‐Mendelian manner in three families, which was not explained by null allele presence.