Shane Lavery

The genetic structure of Australasian green turtles (Chelonia mydas): exploring the geographical scale of genetic exchange

Ecological and genetic studies of marine turtles generally support the hypothesis of natal homing, but leave open the question of the geographical scale of genetic exchange and the capacity of turtles to shift breeding sites. Here we combine analyses of mitochondrial DNA (mtDNA) variation and recapture data to assess the geographical scale of individual breeding populations and the distribution of such populations through Australasia. We conducted multiscale assessments of mtDNA variation among 714 samples from 27 green turtle rookeries and of adult female dispersal among nesting sites in eastern Australia. Many of these rookeries are on shelves that were flooded by rising sea levels less than 10 000 years (c. 450 generations) ago. Analyses of sequence variation among the mtDNA control region revealed 25 haplotypes, and their frequency distributions indicated 17 genetically distinct breeding stocks (Management Units) consisting either of individual rookeries or groups of rookeries in general that are separated by more than 500 km. The population structure inferred from mtDNA was consistent with the scale of movements observed in long‐term mark–recapture studies of east Australian rookeries. Phylogenetic analysis of the haplotypes revealed five clades with significant partitioning of sequence diversity (Φ = 68.4) between Pacific Ocean and Southeast Asian/Indian Ocean rookeries. Isolation by distance was indicated for rookeries separated by up to 2000 km but explained only 12% of the genetic structure. The emerging general picture is one of dynamic population structure influenced by the capacity of females to relocate among proximal breeding sites, although this may be conditional on large population sizes as existed historically across this region.

Estimating the number of whales entering trade using DNA profiling and capture‐recapture analysis of market products

Surveys of commercial markets combined with molecular taxonomy (i.e. molecular monitoring) provide a means to detect products from illegal, unregulated and/or unreported (IUU) exploitation, including the sale of fisheries bycatch and wild meat (bushmeat). Capture‐recapture analyses of market products using DNA profiling have the potential to estimate the total number of individuals entering the market. However, these analyses are not directly analogous to those of living individuals because a ‘market individual’ does not die suddenly but, instead, remains available for a time in decreasing quantities, rather like the exponential decay of a radioactive isotope. Here we use mitochondrial DNA (mtDNA) sequences and microsatellite genotypes to individually identify products from North Pacific minke whales (Balaenoptera acutorostrata ssp.) purchased in 12 surveys of markets in the Republic of (South) Korea from 1999 to 2003. By applying a novel capture‐recapture model with a decay rate parameter to the 205 unique DNA profiles found among 289 products, we estimated that the total number of whales entering trade across the five‐year survey period was 827 (SE, 164; CV, 0.20) and that the average ‘half‐life’ of products from an individual whale on the market was 1.82 months (SE, 0.24; CV, 0.13). Our estimate of whales in trade (reflecting the true numbers killed) was significantly greater than the officially reported bycatch of 458 whales for this period. This unregulated exploitation has serious implications for the survival of this genetically distinct coastal population. Although our capture‐recapture model was developed for specific application to the Korean whale‐meat markets, the exponential decay function could be modified to improve the estimates of trade in other wildmeat or fisheries markets or abundance of living populations by noninvasive genotyping.

Determining the Diet of Larvae of Western Rock Lobster (Panulirus cygnus) Using High-Throughput DNA Sequencing Techniques

The Western Australian rock lobster fishery has been both a highly productive and sustainable fishery. However, a recent dramatic and unexplained decline in post-larval recruitment threatens this sustainability. Our lack of knowledge of key processes in lobster larval ecology, such as their position in the food web, limits our ability to determine what underpins this decline. The present study uses a high-throughput amplicon sequencing approach on DNA obtained from the hepatopancreas of larvae to discover significant prey items. Two short regions of the 18S rRNA gene were amplified under the presence of lobster specific PNA to prevent lobster amplification and to improve prey amplification. In the resulting sequences either little prey was recovered, indicating that the larval gut was empty, or there was a high number of reads originating from multiple zooplankton taxa. The most abundant reads included colonial Radiolaria, Thaliacea, Actinopterygii, Hydrozoa and Sagittoidea, which supports the hypothesis that the larvae feed on multiple groups of mostly transparent gelatinous zooplankton. This hypothesis has prevailed as it has been tentatively inferred from the physiology of larvae, captive feeding trials and co-occurrence in situ. However, these prey have not been observed in the larval gut as traditional microscopic techniques cannot discern between transparent and gelatinous prey items in the gut. High-throughput amplicon sequencing of gut DNA has enabled us to classify these otherwise undetectable prey. The dominance of the colonial radiolarians among the gut contents is intriguing in that this group has been historically difficult to quantify in the water column, which may explain why they have not been connected to larval diet previously. Our results indicate that a PCR based technique is a very successful approach to identify the most abundant taxa in the natural diet of lobster larvae.

www.DNA-surveillance: applied molecular taxonomy for species conservation and discovery

guished using molecular methods in addition to morphological data, but molecular methods cannot realistically be the first or only appropriate method, as has been proposed [4]. Furthermore, the genetic diversity of individuals within and among populations begs the question posed by Lipscomb et al. [3]: what are the appropriate gene sequences for objective taxonomic assessments, and the appropriate genetic/ molecular dissimilarity to define ‘taxa’? We have known for almost a decade that the biodiversity crisis is resulting in the renewed relevance of natural-history collections [7], and the wider use of morphological ‘data’ contained in such collections has been repeatedly validated [8,9]. Perhaps this DNA versus morphology debate is largely one of each camp attempting to get ‘there’ first by proving greater relevance in the competition for limited funds. However, there is, and must be, room for both approaches. References 1 Mallet, J. and Willmott, K. (2003) Taxonomy: renaissance or Tower of Babel? Trends Ecol. Evol. 18, 57–59 2 Seberg, O. et al. (2003) Shortcuts in systematics? A commentary on DNA-based taxonomy. Trends Ecol. Evol. 18, 63–65 3 Lipscomb, D. et al. (2003) The intellectual content of taxonomy: a comment on DNA in taxonomy. Trends Ecol. Evol. 18, 65–67 4 Tautz, D. et al. (2003) A plea for DNA taxonomy. Trends Ecol. Evol. 18, 70–74 5 Schaub, M. and Dunn, C.P. (2002) vPlants: a virtual herbarium of the Chicago region. First Monday 7 http://www.firstmonday.dk/issues/ issue7_5/schaub/index.html 6 Soberón, J.M. et al. (2000) The use of specimen-label databases for conservation purposes: an example using Mexican papilionid and pierid butterflies. Biodiv. Conserv. 9, 1441–1466 7 Alberch, P. (1993) Museums, collections and biodiversity inventories. Trends Ecol. Evol. 8, 372–375 8 Butler, D. (1998) Museum research comes off the list of endangered species. Nature 394, 115–119 9 Winker, K. (1999) How to bring collections data into the net. Nature 401, 524

PCR enrichment techniques to identify the diet of predators

The increasing sensitivity of PCR has meant that in the last two decades PCR has emerged as a major tool in diet studies, enabling us to refine our understanding of trophic links and to elucidate the diets of predators whose prey is as yet uncharacterized. The achievements and methods of PCR‐based diet studies have been reviewed several times, but here we review an important development in the field: the use of PCR enrichment techniques to promote the amplification of prey DNA over that of the predator. We first discuss the success of using group‐specific primers either in parallel single reactions or in multiplex reactions. We then concentrate on the more recent use of PCR enrichment techniques such as restriction enzyme digests, peptide nucleic acid clamping, DNA blocking and laser capture microdissection. We also survey the vast literature on enrichment techniques in clinical biology, to ascertain the pitfalls of enrichment techniques and what refinements have yielded some highly sensitive methods. We find that while there are several new approaches to enrichment, peptide nucleic acid clamping and DNA blocking are generally sufficient techniques for the characterization of diets of predators and highlight the most important considerations of the approach.

Changing patterns of population structure and gene flow at different spatial scales in birgus latro (The coconut crab)

The population structure of the coconut crab (Birgus latro) was studied by examining genetic variation at seven polymorphic enzyme loci. Individuals were collected from 10 locations (grouped in seven major populations) throughout the Indo-Pacific distribution of the species. Significant population differentiation was found among all seven major populations (FST = 0.078, P < 0.001) and among the six Pacific Ocean populations (FST=0.026, P < 0.01). There were no significant differences in allele frequencies among adjacent Vanuatu islands separated by up to 200 km. At any one location there were no significant changes in allele frequencies over time (up to 3 years). Estimates of gene flow varied considerably, depending on the method of calculation, but all supported the same interpretations of population subdivision. The pattern of population structure varied with the spatial scale under consideration. The Indian Ocean population (from Christmas Island) was clearly divergent from all Pacific populations, in the fashion of an island model. However, within the Pacific, the relation between genetic and geographical distance showed that the pattern of genetic variation closely resembled an isolation by distance model. Populations from adjacent Vanuatu islands exhibited panmixia.

New Zealand triplefin fishes (family Tripterygiidae): contrasting population structure and mtDNA diversity within a marine species flock

Triplefin fishes (Family Tripterygiidae) dominate the New Zealand temperate coastal fish fauna in diversity (26 endemic species, 14 genera). Most species appear to have evolved as a local radiation and mostly occupy sympatric distributions throughout New Zealand. To investigate the forces driving current gene‐flow patterns and past evolutionary histories, we searched for common patterns of population genetic subdivision within eight species sampled throughout their distributions [mitchochondrial DNA (mtDNA) control region, n = 1086]. We hypothesised that common phylogeographical and population differentiation patterns would reveal past or ongoing physical processes, with differences reflecting stochastic or species‐specific processes. Striking differences between species were apparent, with strong phylogeographical structure detected in Grahamina capito and the estuarine species G. nigripenne. G. capito fell into three distinct geographically restricted lineages. G. nigripenne largely separated into northern and southern lineages (ΦST 0.834). Strong population structuring and isolation by distance was evident in Bellapiscis medius, B. lesleyae and Forsterygion lapillum (ΦST 0.686, 0.436 and 0.115, respectively). High gene flow was apparent in G. gymnota and Ruanoho whero, and F. varium. However, for the latter species, isolation was apparent with samples collected from the offshore Three Kings Islands. Overall, a strong relationship was found between habitat depth and population structure among species, and species inhabiting shallower water habitats showed lower genetic diversity with higher levels of population subdivision. High‐latitude populations generally showed low haplotype and nucleotide diversity. These data suggest that processes resulting from intraspecific differences in habitat preference, climatic histories and/or larval ecologies have subdivided populations of shallow water triplefin species.

Using DNA barcoding and phylogenetics to identify Antarctic invertebrate larvae: Lessons from a large scale study

Ecological studies of the diversity and distribution of marine planktonic larvae are increasingly depending on molecular methods for accurate taxonomic identification. The greater coverage of reference marine species on genetic databases such as GenBank and BoLD (Barcoding of Life Data Systems; www.boldystems.org); together with the decreasing costs for DNA sequencing have made large scale larval identification studies using molecular methods more feasible. Here, we present the development and implementation of a practical molecular approach to identify over 2000 individual marine invertebrate larvae that were collected in the Ross Sea, Antarctica, during the austral summer over five years (2002-2007) as part of the LGP (Latitudinal Gradient Project). Larvae for molecular ID were morphologically identified to belong to the Phyla Mollusca, Echinodermata, Nemertea and Annelida (Class Polychaeta), but also included unidentified early developmental stages which could not be assigned a specific taxon (e.g., eggs, blastulae). The use of a 100μm mesh plankton net makes this one of the first larval identification studies to simultaneously consider both embryos and larvae. Molecular identification methods included amplification of up to three molecular loci for each specimen, a pre-identification step using BLAST with GenBank, phylogenetic reconstructions and cross-validation of assigned Molecular Operational Taxonomic Units (MOTUs). This combined approach of morphological and molecular methods assigned about 700 individuals to 53 MOTUs, which were identified to the lowest possible taxonomic level. During the course of this long-term study we identified several procedural difficulties, including issues with the collection of larvae, locus amplification, contamination, assignment and validation of MOTUs. The practical guidelines that we describe here should greatly assist other researchers to conduct reliable molecular identification studies of larvae in the future.

Population Structure and Phylogeography of the Short-Tailed Stingray, Dasyatis brevicaudata (Hutton 1875), in the Southern Hemisphere

There is accumulating evidence that the degree of vagility explains little of the extent of population subdivision found within elasmobranch species. Instead, patterns of gene flow in elasmobranchs appear more closely linked to the presence of dispersal barriers, either physical or biological. Here, we investigate the potential role of some of these isolating mechanisms in shaping the population structure of a member of the stingray family Dasyatidae (Dasyatis brevicaudata) at various scales (southern hemisphere vs. coastal New Zealand). Analyses of the mitochondrial DNA control region from 176 individuals revealed significant genetic structure between South Africa, Australia, and New Zealand populations (analysis of molecular variance [AMOVA], overall Ф(ST) = 0.67, P < 0.001), although New Zealand and Australia shared some haplotypes. Surprisingly, significant population differentiation was found among several coastal New Zealand locations (AMOVA, overall Ф(ST) = 0.05, P < 0.05). However, data did not support the genetic differentiation between individuals from an offshore breeding area and mainland individuals. Comparisons suggest that these stingrays exhibit similar levels of population differentiation as other coastal elasmobranchs, with high divergence across oceanic basins and lower differentiation along continuous coastal habitats. Differences in coastal population structuring in elasmobranch species studied to date may be attributed to species-specific preferences for coastal habitats, which may be linked to life history functions (e.g., feeding and pupping).

Stable Isotope Ratios of Carbon and Nitrogen and Mercury Concentrations in 13 Toothed Whale Species Taken from the Western Pacific Ocean off Japan

Stable isotope ratios of carbon (partial differential(13)C) and nitrogen (partial differential(15)N) and total mercury (T-Hg) concentrations were measured in red meat samples from 11 odontocete species (toothed whales, dolphins, and porpoises) sold in Japan (n = 96) and in muscle samples from stranded killer whales (n = 6) and melon-headed whales (n = 15), and the analytical data for these species were classified into three regions (northern, central, and southern Japan) depending on the locations in which they were caught or stranded. The partial differential(15)N in the samples from southern Japan tended to be lower than that in samples from the north, whereas both partial differential(13)C and T-Hg concentrations in samples from the south tended to higher than those in samples from northern Japan. Negative correlations were found between the partial differential(13)C and partial differential(15)N values and between the partial differential(15)N value and T-Hg concentrations in the combined samples all three regions (gamma= -0.238, n = 117, P < 0.01). The partial differential(13)C, partial differential(15)N, and T-Hg concentrations in the samples varied more by habitat than by species. Spatial variations in partial differential(13)C, partial differential(15)N, and T-Hg concentrations in the ocean may be the cause of these phenomena.