Population structure of pygmy (Kogia breviceps) and dwarf (Kogia sima) sperm whales in the Southern Hemisphere may reflect foraging ecology and dispersal patterns.

Little is known about the biology of pygmy (Kogia breviceps) and dwarf (K. sima) sperm whales as these animals are difficult to observe in the wild. However, both species strand frequently along the South African, Australian and New Zealand coastlines, providing samples for these otherwise inaccessible species. The use of DNA samples from tissue and DNA extracted from historical material, such as teeth and bone, allowed a first analysis of the population structure of both species in the Southern Hemisphere. A 279 base pair consensus region of the mitochondrial cytochrome b gene was sequenced for 96 K. breviceps (53 tissue and 43 teeth or bone samples) and 29 K. sima (3 tissue and 26 teeth or bone samples), and 26 and 12 unique haplotypes were identified, respectively. K. breviceps showed a higher nucleotide diversity of 0.82% compared to 0.40% in K. sima. Significant genetic differentiation was detected in the Southern Hemisphere between K. breviceps from South Africa and New Zealand (ФST = 0.042, p < 0.05). Mitochondrial control region sequences (505 bp) were available for 44 individuals (41 K. breviceps and 3 K. sima) for comparative purposes. A comprehensive global phylogenetic analysis (maternal lineage) of our sequences together with all available Kogia mtDNA sequences largely supported previously published phylogenetic findings, but highlighted some changed inferences about oceanic divergences within both species. The higher nucleotide diversity and low population differentiation observed in K. breviceps may result from its broad foraging ecology and wide distribution, which may indicate a more opportunistic feeding behaviour and tolerance towards a larger range of water temperatures than K. sima.

Genome-wide SNPs in the spiny lobster Panulirus homarus reveal a hybrid origin for its subspecies.

BACKGROUND: Evolutionary divergence and speciation often occur at a slower rate in the marine realm due to the higher potential for long-distance reproductive interaction through larval dispersal. One common evolutionary pattern in the Indo-Pacific, is divergence of populations and species at the peripheries of widely-distributed organisms. However, the evolutionary and demographic histories of such divergence are yet to be well understood. Here we address these issues by coupling genome-wide SNP data with mitochondrial DNA sequences to test the patterns of genetic divergence and possible secondary contact among geographically distant populations of the highly valuable spiny lobster Panulirus homarus species complex, distributed widely through the Indo-Pacific, from South Africa to the Marquesas Islands. RESULT: After stringent filtering, 2020 SNPs were used for population genetic and demographic analyses, revealing strong regional structure (FST = 0.148, P < 0001), superficially in accordance with previous analyses. However, detailed demographic analyses supported a much more complex evolutionary history of these populations, including a hybrid origin of a North-West Indian Ocean (NWIO) population, which has previously been discriminated morphologically, but not genetically. The best-supported demographic models suggested that the current genetic relationships among populations were due to a complex series of past divergences followed by asymmetric migration in more recent times. CONCLUSION: Overall, this study suggests that alternating periods of marine divergence and gene flow have driven the current genetic patterns observed in this lobster and may help explain the observed wider patterns of marine species diversity in the Indo-Pacific.

Genetic and particle modelling approaches to assessing population connectivity in a deep sea lobster.

The emergence of high resolution population genetic techniques, such as genotyping-by-sequencing (GBS), in combination with recent advances in particle modelling of larval dispersal in marine organisms, can deliver powerful new insights to support fisheries conservation and management. In this study, we used this combination to investigate the population connectivity of a commercial deep sea lobster species, the New Zealand scampi, Metanephrops challengeri, which ranges across a vast area of seafloor around New Zealand. This species has limited dispersal capabilities, including larvae with weak swimming abilities and short pelagic duration, while the reptant juvenile/adult stages of the lifecycle are obligate burrow dwellers with limited home ranges. Ninety-one individuals, collected from five scampi fishery management areas around New Zealand, were genotyped using GBS. Using 983 haplotypic genomic loci, three genetically distinct groups were identified: eastern, southern and western. These groups showed significant genetic differentiation with clear source-sink dynamics. The direction of gene flow inferred from the genomic data largely reflected the hydrodynamic particle modelling of ocean current flow around New Zealand. The modelled dispersal during pelagic larval phase highlights the strong connectivity among eastern sampling locations and explains the low genetic differentiation detected among these sampled areas. Our results highlight the value of using a transdisciplinary approach in the inference of connectivity among populations for informing conservation and fishery management.

Finding the adaptive needles in a population-structured haystack: A case study in a New Zealand mollusc.

Genetic adaptation to future environmental conditions is crucial to help species persist as the climate changes. Genome scans are powerful tools to understand adaptive landscapes, enabling us to correlate genetic diversity with environmental gradients while disentangling neutral from adaptive variation. However, low gene flow can lead to both local adaptation and highly structured populations, and is a major confounding factor for genome scans, resulting in an inflated number of candidate loci. Here, we compared candidate locus detection in a marine mollusc (Onithochiton neglectus), taking advantage of a natural geographical contrast in the levels of genetic structure between its populations. O. neglectus is endemic to New Zealand and distributed throughout an environmental gradient from the subtropical north to the subantarctic south. Due to a brooding developmental mode, populations tend to be locally isolated. However, adult hitchhiking on rafting kelp increases connectivity among southern populations. We applied two genome scans for outliers (Bayescan and PCAdapt) and two genotype-environment association (GEA) tests (BayeScEnv and RDA). To limit issues with false positives, we combined results using the geometric mean of q-values and performed association tests with random environmental variables. This novel approach is a compromise between stringent and relaxed approaches widely used before, and allowed us to classify candidate loci as low confidence or high confidence. Genome scans for outliers detected a large number of significant outliers in strong and moderately structured populations. No high-confidence GEA loci were detected in the context of strong population structure. However, 86 high-confidence loci were associated predominantly with latitudinally varying abiotic factors in the less structured southern populations. This suggests that the degree of connectivity driven by kelp rafting over the southern scale may be insufficient to counteract local adaptation in this species. Our study supports the expectation that genome scans may be prone to errors in highly structured populations. Nonetheless, it also empirically demonstrates that careful statistical controls enable the identification of candidate loci that invite more detailed investigations. Ultimately, genome scans are valuable tools to help guide further research aiming to determine the potential of non-model species to adapt to future environments.

Towards reproducible metabarcoding data: Lessons from an international cross-laboratory experiment.

Advances in high-throughput sequencing (HTS) are revolutionizing monitoring in marine environments by enabling rapid, accurate and holistic detection of species within complex biological samples. Research institutions worldwide increasingly employ HTS methods for biodiversity assessments. However, variance in laboratory procedures, analytical workflows and bioinformatic pipelines impede the transferability and comparability of results across research groups. An international experiment was conducted to assess the consistency of metabarcoding results derived from identical samples and primer sets using varying laboratory procedures. Homogenized biofouling samples collected from four coastal locations (Australia, Canada, New Zealand and the USA) were distributed to 12 independent laboratories. Participants were asked to follow one of two HTS library preparation workflows. While DNA extraction, primers and bioinformatic analyses were purposefully standardized to allow comparison, many other technical variables were allowed to vary among laboratories (amplification protocols, type of instrument used, etc.). Despite substantial variation observed in raw results, the primary signal in the data was consistent, with the samples grouping strongly by geographical origin for all data sets. Simple post hoc data clean-up by removing low-quality samples gave the best improvement in sample classification for nuclear 18S rRNA gene data, with an overall 92.81% correct group attribution. For mitochondrial COI gene data, the best classification result (95.58%) was achieved after correction for contamination errors. The identified critical methodological factors that introduced the greatest variability (preservation buffer, sample defrosting, template concentration, DNA polymerase, PCR enhancer) should be of great assistance in standardizing future biodiversity studies using metabarcoding.

Genetic divergence between isolated populations of the North Island New Zealand Rifleman (Acanthisitta chloris granti) implicates ancient biogeographic impacts rather than recent habitat fragmentation.

This research investigates the extent and causal mechanisms of genetic population divergence in a poorly flighted passerine, the North Island Rifleman or Titipounamu (Acanthisitta chloris granti). While this species has a historically widespread distribution, anthropogenic forest clearance has resulted in a highly fragmented current distribution. We conducted analyses of mitochondrial DNA (COI and Control Region) and 12 nuclear DNA microsatellites to test for population divergence and estimate times of divergence. diyabc and biogeobears were then used to assess likely past dispersal scenarios based on both mtDNA and nDNA. The results reveal several significantly divergent lineages across the North Island of New Zealand and indicate that some populations have been isolated for extensive periods of time (0.7-4.9 mya). Modeling indicated a dynamic history of population connectivity, with a drastic restriction in gene flow between three geographic regions, followed by a more recent re-establishment of connectivity. Our analyses indicate the dynamic influence of key geological and climatological events on the distribution of genetic diversity in this species, including support for the genetic impact of old biogeographic boundaries such as the Taupo Line and Cockayne's Line, rather than recent anthropogenic habitat fragmentation. These findings present a rare example of an avian species with a genetic history more like that of flightless taxa and so provide new general insights into vicariant processes affecting populations of passerines with limited dispersal.

Phylogeography of the dugong (Dugong dugon) based on historical samples identifies vulnerable Indian Ocean populations.

We investigated the phylogeography of the dugong (Dugong dugon) across its original range using museum material from 14 natural history museum and university collections. The mitochondrial DNA control region was successfully amplified from samples of bone or tooth powder from 162 individuals. These samples range from 1827 to 1996 and span the historical distribution range of the dugong. We were able to successfully amplify overlapping fragments of the D-loop region of the mitochondrial DNA (mtDNA) resulting in sequences of a 355 bp fragment for 162 individuals for the final analyses. This included a new sequence (189 bp) from a previously unidentified piece of skin of the extinct Steller's sea cow (Hydrodamalis gigas), as an outgroup. The resulting dugong sequences match those from previous studies of dugongs from Australia and Indonesia, but revealed several new and divergent mtDNA lineages in the Indian Ocean. One mtDNA lineage includes most specimens from the Western Indian Ocean, with another distinct lineage isolated to nearby Madagascar and Comores. There is little geographic structuring detectable among other populations in the Western Indian Ocean and all populations from that region appear to have historically contained comparatively low levels of genetic diversity. The genetic diversity of several Indian Ocean samples collected after 1950 was lower than that of the samples collected earlier from similar locations, a result coincident with the anecdotal reductions in population size. The new lineages and potential loss of diversity highlight the particular conservation importance and vulnerability of dugong populations in the Western Indian Ocean.

Sweepstakes reproductive success is absent in a New Zealand snapper (Chrysophrus auratus) population protected from fishing despite "tiny" Ne /N ratios elsewhere.

A landmark study published in 2002 estimated a very small Ne /N ratio (around 10-5 ) in a population of pink snapper (Chrysophrys auratus, Forster, 1801) in the Hauraki Gulf in New Zealand. It epitomized the tiny Ne /N ratios (<10-3 ) reported in marine species due to the hypothesized operation of sweepstakes reproductive success (SRS). Here we re-evaluate the occurrence of SRS in marine species and the potential effect of fishing on the Ne /N ratio by studying the same species in the same region, but in a population that has been protected from fishing since 1975. We combine empirical, simulation and model-based approaches to estimate Ne (and Nb ) from genotypes of 1,044 adult fish and estimate N using recapture-probabilities. The estimated Ne /N ratio was much larger (0.33, SE: 0.14) than expected. The magnitude of estimates of population-wide variance in individual lifetime reproductive success (10-18) suggested that the sweepstakes effect was negligible in the study population. After evaluating factors that could explain the contrast between studies - experimental design, life history differences, environmental effects and the influence of exploitation on the Ne /N ratio - we conclude that the low Ne of the Hauraki Gulf population is associated with demographic instability in the harvested compared to the protected population despite circumstantial evidence that the 2002 study may have underestimated Ne . This study has broad implications for the prevailing view that reproductive success in the sea is largely driven by chance, and for genetic monitoring of populations using the Ne /N ratio and Nb .

Preliminary analysis of New Zealand scampi (Metanephrops challengeri) diet using metabarcoding.

Deep sea lobsters are highly valued for seafood and provide the basis of important commercial fisheries in many parts of the world. Despite their economic significance, relatively little is known about their natural diets. Microscopic analyses of foregut content in some species have suffered from low taxonomic resolution, with many of the dietary items difficult to reliably identify as their tissue is easily digested. DNA metabarcoding has the potential to provide greater taxonomic resolution of the diet of the New Zealand scampi (Metanephrops challengeri) through the identification of gut contents, but a number of methodological concerns need to be overcome first to ensure optimum DNA metabarcoding results. In this study, a range of methodological parameters were tested to determine the optimum protocols for DNA metabarcoding, and provide a first view of M. challengeri diet. Several PCR protocols were tested, using two universal primer pairs targeting the 18S rRNA and COI genes, on DNA extracted from both frozen and ethanol preserved samples for both foregut and hindgut digesta. The selection of appropriate DNA polymerases, buffers and methods for reducing PCR inhibitors (including the use of BSA) were found to be critical. Amplification from frozen or ethanol preserved gut contents appeared similarly dependable. The COI gene was found to be more effective than 18S rRNA gene for identifying large eukaryotic taxa from the digesta; however, it was less successfully amplified. The 18S rRNA gene was more easily amplified, but identified mostly smaller marine organisms such as plankton and parasites. This preliminary analysis of the diet of M. challengeri identified a range of species (13,541 reads identified as diet), which included the ghost shark (Hydrolagus novaezealandiae), silver warehou (Seriolella punctata), tall sea pen (Funiculina quadrangularis) and the salp (Ihlea racovitzai), suggesting that they have a varied diet, with a high reliance on scavenging a diverse range of pelagic and benthic species from the seafloor.

Mechanisms of peripheral phylogeographic divergence in the indo-Pacific: lessons from the spiny lobster Panulirus homarus.

BACKGROUND: There is increasing recognition of the concordance between marine biogeographic and phylogeographic boundaries. However, it is still unclear how population-level divergence translates into species-level divergence, and what are the principal factors that first initiate that divergence, and then maintain reproductive isolation. This study examines the likely forces driving population and lineage divergences in the broadly-distributed Indo-Pacific spiny lobster Panulirus homarus, which has peripheral divergent lineages in the west and east. The study focuses particularly on the West Indian Ocean, which is emerging as a region of unexpected diversity. Mitochondrial control region (mtCR) and COI sequences as well as genotypes of 9 microsatellite loci were examined in 410 individuals from 17 locations grouped into 7 regions from South Africa in the west, and eastward across to Taiwan and the Marquesas Islands. Phylogenetic and population-level analyses were used to test the significance and timing of divergences and describe the genetic relationships among populations. RESULTS: Analyses of the mtCR revealed high levels of divergence among the seven regions (ФST = 0.594, P < 0.001). Microsatellite analyses also revealed significant divergence among regions, but at a much lower level (FST = 0.066, P < 0.001). The results reveal different patterns of mtCR v. nDNA divergence between the two distinct peripheral lineages: a subspecies in South Africa and Madagascar, and a phylogeographically diverged population in the Marquesas. The results also expose a number of other more fine-scale population divergences, particularly in the Indian Ocean. CONCLUSIONS: The divergence of peripheral lineages in the west and east of the species' range appear to have been initiated and maintained by very different processes. The pattern of mitochondrial and nuclear divergence of the western lineage, implicates processes of parapatric isolation, secondary contact and introgression, and suggests possible maintenance through adaptation and behavioural reproductive isolation. In contrast, the eastern lineage appears to have diverged through a rare colonisation event, maintained through long-term isolation, and matches expectations of the core-periphery hypothesis. The process of active peripheral speciation may be a common force in the Indo-Pacific that helps drive some of the regions' recognized biogeographic boundaries.

Evolutionary divergence of geographic subspecies within the scalloped spiny lobster Panulirus homarus (Linnaeus 1758).

Panulirus homarus is an economically important spiny lobster that is widespread through the Indo-West Pacific Region, but has an uncertain taxonomic status, with three or four geographic subspecies having been described. This study used mitochondrial (16S, COI and control region) and nuclear (18S, ITS-1) DNA sequences to examine specimens of all putative subspecies and forms from throughout their range, in order to determine their genetic validity, and understand the evolutionary history of this species. Despite the range of diversity present in the loci examined, the results were consistent across genes. P. h. rubellus from the SW Indian Ocean comprised the most divergent lineage that was reciprocally monophyletic with respect to all other P. homarus (approx. 9% divergence in COI), and has likely evolved reproductive barriers. The putative P. h. "Brown" subspecies from the Marquesas Is in the central Pacific also comprised a somewhat divergent monophyletic lineage (approx. 3% in COI), but may simply be an allopatric population. The widespread P. h. homarus was not diverged at all from the described P. h. megasculpta from the NW Indian Ocean. The degree of evolutionary divergence of populations at the extremes distribution of the species is somewhat surprising, given the long pelagic larval stage, but suggests that allopatric speciation has been an important driver in the evolution of the genus.

Phylogenetic species identification in Rattus highlights rapid radiation and morphological similarity of New Guinean species.

The genus Rattus is highly speciose, the taxonomy is complex, and individuals are often difficult to identify to the species level. Previous studies have demonstrated the usefulness of phylogenetic approaches to identification in Rattus but some species, especially among the endemics of the New Guinean region, showed poor resolution. Possible reasons for this are simple misidentification, incomplete gene lineage sorting, hybridization, and phylogenetically distinct lineages that are unrecognised taxonomically. To assess these explanations we analysed 217 samples, representing nominally 25 Rattus species, collected in New Guinea, Asia, Australia and the Pacific. To reduce misidentification problems we sequenced museum specimens from earlier morphological studies and recently collected tissues from samples with associated voucher specimens. We also reassessed vouchers from previously sequenced specimens. We inferred combined and separate phylogenies from two mitochondrial DNA regions comprising 550 base pair D-loop sequences and both long (655 base pair) and short (150 base pair) cytochrome oxidase I sequences. Our phylogenetic species identification for 17 species was consistent with morphological designations and current taxonomy thus reinforcing the usefulness of this approach. We reduced misidentifications and consequently the number of polyphyletic species in our phylogenies but the New Guinean Rattus clades still exhibited considerable complexity. Only three of our eight New Guinean species were monophyletic. We found good evidence for either incomplete mitochondrial lineage sorting or hybridization between species within two pairs, R. leucopus/R. cf. verecundus and R. steini/R. praetor. Additionally, our results showed that R. praetor, R. niobe and R. verecundus each likely encompass more than one species. Our study clearly points to the need for a revised taxonomy of the rats of New Guinea, based on broader sampling and informed by both morphology and phylogenetics. The remaining taxonomic complexity highlights the recent and rapid radiation of Rattus in the Australo-Papuan region.

A multi-locus analysis of phylogenetic relationships within cheilostome bryozoans supports multiple origins of ascophoran frontal shields.

Phylogenetic relationships within the bryozoan order Cheilostomata are currently uncertain, with many morphological hypotheses proposed but scarcely tested by independent means of molecular analysis. This research uses DNA sequence data across five loci of both mitochondrial and nuclear origin from 91 species of cheilostome Bryozoa (34 species newly sequenced). This vastly improved the taxonomic coverage and number of loci used in a molecular analysis of this order and allowed a more in-depth look into the evolutionary history of Cheilostomata. Maximum likelihood and Bayesian analyses of individual loci were carried out along with a partitioned multi-locus approach, plus a range of topology tests based on morphological hypotheses. Together, these provide a comprehensive set of phylogenetic analyses of the order Cheilostomata. From these results inferences are made about the evolutionary history of this order and proposed morphological hypotheses are discussed in light of the independent evidence gained from the molecular data. Infraorder Ascophorina was demonstrated to be non-monophyletic, and there appears to be multiple origins of the ascus and associated structures involved in lophophore extension. This was further supported by the lack of monophyly within each of the four ascophoran grades (acanthostegomorph/spinocystal, hippothoomorph/gymnocystal, umbonulomorph/umbonuloid, lepraliomorph/lepralioid) defined by frontal-shield morphology. Chorizopora, currently classified in the ascophoran grade Hippothoomorpha, is phylogenetically distinct from Hippothoidae, providing strong evidence for multiple origins of the gymnocystal frontal shield type. Further evidence is produced to support the morphological hypothesis of multiple umbonuloid origins of lepralioid frontal shields, using a step-wise set of topological hypothesis tests combined with examination of multi-locus phylogenies.

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 (Phi(ST) 0.834). Strong population structuring and isolation by distance was evident in Bellapiscis medius, B. lesleyae and Forsterygion lapillum (Phi(ST) 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.