Abstract
Molluscs are among the most diverse animal groups. However, samples from only a small portion of mollusc populations and species are currently available frozen or in high-concentration ethanol, as presently required for most genetic approaches. Palaeogenetic techniques had previously been applied to mollusc shells, but success rates were usually low and required read numbers that would be cost-prohibitive to most lab-groups. We combined single-stranded Illumina sequence library preparation methods with hybridization-capture techniques and applied these to historical and Holocene-aged dry-preserved shell samples, generating near-complete mitogenomes with relatively high success rates and cost-efficient sequencing efficiencies.
These techniques enabled revision of the New Zealand abalone Haliotis virginea, including samples up to 60 years old, and from difficult-to-access subantarctic island marine reserves. We achieved a 100% success rate of high coverage mitogenome generation and a mean 350-fold increase in target-read efficiency over shotgun sequencing. The resulting phylogenies improved understanding of relationships between morphologically distinctive populations, and supported recognition of a new species, H. pirimoana, from the Three Kings Islands. We then applied these approaches to the global abalone fauna, generating mitogenomes for >2/3rds of the extant species, and revealing divergences as deep as the Oligocene/Eocene boundary, and strong geographic structure to the major clades. Continued work including nuclear and anatomical markers is in preparation to better resolve taxonomic positions for the deeper clades recovered.
Abalone appear to have relatively high DNA concentrations within their shells. In contrast, land snails from Holocene dunes in northern New Zealand, where temperatures and humidities are relatively high and unstable, represented more of a test. Success rates indeed dropped to 52% for such samples, which is roughly in-line with vertebrate palaeogenetic results from similar depositional settings in that region. We therefore included many extinct populations of the critically endangered land snail Maoristylus ambagiosus, together with ‘modern’ samples, within our phylogenies. Extant lineages broadly place across the combined genetic diversity of this species. Fine-scale longitudinal phylogeographic structuring was detected, but it was less strongly structured than previous studies have reported using other markers and without inclusion of extinct populations. Maintenance of protection for many extant populations, without admixture between then to prevent loss through genetic drift, is recommended to maintain genetic diversity within this species to maximise their long-term survival chances.
The methods reported in this thesis have the potential to broadly expand the mollusc natural history collection holdings available to geneticists by an order of magnitude or more. Remote sites or extinct populations may no longer be barriers to inclusion of potentially significant samples in genetic analyses. These methods also raise a suite of ethical considerations. It is now easier than ever before to sequence and name samples from distant regions and countries without any involvement of local communities and authorities. How we chose and use scientific and vernacular names, and involve and engage with indigenous communities during research, is a fraught subject. While consensus on many of these topics remains unlikely, cultural and conservation considerations relevant to the naming and management of taxa are discussed herein.