Identification of Phyllosoma Larvae of the Lobsters Acantharctus and Biarctus (Crustacea: Scyllaridae) from the SW Indian Ocean.

The accurate assignment of cryptic larvae to species-level is a key aspect of marine ecological research and can be achieved through integrated molecular and morphological studies. A combination of two mitochondrial markers (COI and 16S) and a detailed morphological analysis was used to identify phyllosoma larvae of slipper lobster (Scyllaridae) species collected during a survey in the SW Indian Ocean. Two morphotypes were tentatively assigned to Acantharctus ornatus and Biarctus pumilus, both genera for which the larval morphology was unknown. Morphological revision of an adult specimen used to generate the putative A. ornatus sequences in GenBank revealed that it was misidentified and corresponds to B. dubius. The final phyllosoma stage of B. pumilus and subfinal and final stages of A. ornatus were described, clarifying prior misidentifications in the literature. Scyllarid biodiversity in the SW Indian Ocean is underestimated and sampling of deeper water layers is recommended to complete current knowledge of species and larval stages present in the region.

Metabarcoding analysis of marine zooplankton confirms the ecological role of a sheltered bight along an exposed continental shelf.

Zooplankton plays an essential role in marine ecosystems as the link between primary producers (phytoplankton) and higher trophic levels in food webs, and as a dynamic pool of recruits for invertebrates and fish. Zooplankton communities are diverse with a patchy distribution at different spatial scales, influenced by oceanographic processes. The continental shelf of eastern South Africa is narrow and exposed to the western-boundary Agulhas Current, with some shelter against strong directional flow provided by the broader KwaZulu-Natal Bight, a coastal offset adjacent to an estuary. We compared zooplankton species richness, diversity and relative abundance of key taxa among sheltered and exposed shelf areas using metabarcoding and community analysis, to explore the ecological role of the bight in a highly dynamic ocean region. Metabarcoding recovered higher richness and diversity at a finer resolution than could previously be achieved with traditional microscopy. Of 271 operational taxonomic units (OTUs) recovered through metabarcoding, 63% could be matched with >95% sequence similarity to reference barcodes. OTUs were dominated by malacostracan crustaceans (161 spp.), ray-finned fishes (45 spp.) and copepods (28 spp.). Species richness, diversity and the relative abundance of key taxa differed between sheltered and exposed shelf areas. Lower species richness in the bight was partly attributed to structurally homogeneous benthic habitats, and an associated reduction of meroplanktonic species originating from local benthic-pelagic exchange. High relative abundance of a ray-finned fish in the bight, as observed based on fish eggs and read counts, confirmed that the bight is an important fish spawning area. Overall, zooplankton metabarcoding outputs were congruent with findings of previous ecological research using more traditional methods of observation.

Experimental validation of taxon-specific mini-barcode primers for metabarcoding of zooplankton.

Metabarcoding to determine the species composition and diversity of marine zooplankton communities is a fast-developing field in which the standardization of methods is yet to be fully achieved. The selection of genetic markers and primer choice are particularly important because they substantially influence species detection rates and accuracy. Validation is therefore an important step in the design of metabarcoding protocols. We developed taxon-specific mini-barcode primers for the cytochrome c oxidase subunit I (COI) gene region and used an experimental approach to test species detection rates and primer accuracy of the newly designed primers for prawns, shrimps and crabs and published primers for marine lobsters and fish. Artificially assembled mock communities (with known species ratios) and unsorted coastal tow-net zooplankton samples were sequenced and the detected species were compared with those seeded in mock communities to test detection rates. Taxon-specific primers increased detection rates of target taxa compared with a universal primer set. Primer cocktails (multiple primer sets) significantly increased species detection rates compared with single primer pairs and could detect up to 100% of underrepresented target taxa in mock communities. Taxon-specific primers recovered fewer false-positive or false-negative results than the universal primer. The methods used to design taxon-specific mini-barcodes and the experimental mock community validation protocols shown here can easily be applied to studies on other groups and will allow for a level of standardization among studies undertaken in different ecosystems or geographic locations.

Genetic structure and life history are key factors in species distribution models of spiny lobsters.

AIM: We incorporated genetic structure and life history phase in species distribution models (SDMs) constructed for a widespread spiny lobster, to reveal local adaptations specific to individual subspecies and predict future range shifts under the RCP 8.5 climate change scenario. LOCATION: Indo-West Pacific. METHODS: MaxEnt was used to construct present-day SDMs for the spiny lobster Panulirus homarus and individually for the three genetically distinct subspecies of which it comprises. SDMs incorporated both sea surface and benthic (seafloor) climate layers to recreate discrete influences of these habitats during the drifting larval and benthic juvenile and adult life history phases. Principle component analysis (PCA) was used to infer environmental variables to which individual subspecies were adapted. SDM projections of present-day habitat suitability were compared with predictions for the year 2,100, under the RCP 8.5 climate change scenario. RESULTS: In the PCA, salinity best explained P. h. megasculptus habitat suitability, compared with current velocity in P. h. rubellus and sea surface temperature in P. h. homarus. Drifting and benthic life history phases were adapted to different combinations of sea surface and benthic environmental variables considered. Highly suitable habitats for benthic phases were spatially enveloped within more extensive sea surface habitats suitable for drifting larvae. SDMs predicted that present-day highly suitable habitats for P. homarus will decrease by the year 2,100. MAIN CONCLUSIONS: Incorporating genetic structure in SDMs showed that individual spiny lobster subspecies had unique adaptations, which could not be resolved in species-level models. The use of sea surface and benthic climate layers revealed the relative importance of environmental variables during drifting and benthic life history phases. SDMs that included genetic structure and life history were more informative in predictive models of climate change effects.

The design and testing of mini-barcode markers in marine lobsters.

Full-length mitochondrial cytochrome c oxidase I (COI) sequence information from lobster phyllosoma larvae can be difficult to obtain when DNA is degraded or fragmented. Primers that amplify smaller fragments are also more useful in metabarcoding studies. In this study, we developed and tested a method to design a taxon-specific mini-barcode primer set for marine lobsters. The shortest, most informative portion of the COI gene region was identified in silico, and a DNA barcode gap analysis was performed to assess its reliability as species diagnostic marker. Primers were designed, and cross-species amplification success was tested on DNA extracted from a taxonomic range of spiny-, clawed-, slipper- and blind lobsters. The mini-barcode primers successfully amplified both adult and phyllosoma COI fragments, and were able to successfully delimit all species analyzed. Previously published universal primer sets were also tested and sometimes failed to amplify COI from phyllosoma samples. The newly designed taxon-specific mini-barcode primers will increase the success rate of species identification in bulk environmental samples and add to the growing DNA metabarcoding toolkit.

Seascape genetics of the spiny lobster Panulirus homarus in the Western Indian Ocean: Understanding how oceanographic features shape the genetic structure of species with high larval dispersal potential.

This study examines the fine-scale population genetic structure and phylogeography of the spiny lobster Panulirus homarus in the Western Indian Ocean. A seascape genetics approach was used to relate the observed genetic structure based on 21 microsatellite loci to ocean circulation patterns, and to determine the influence of latitude, sea surface temperature (SST), and ocean turbidity (KD490) on population-level processes. At a geospatial level, the genetic clusters recovered corresponded to three putative subspecies, P. h. rubellus from the SW Indian Ocean, P. h. megasculptus from the NW Indian Ocean, and P. h. homarus from the tropical region in-between. Virtual passive Lagrangian particles advected using satellite-derived ocean surface currents were used to simulate larval dispersal. In the SW Indian Ocean, the dispersion of particles tracked over a 4-month period provided insight into a steep genetic gradient observed at the Delagoa Bight, which separates P. h. rubellus and P. h. homarus. South of the contact zone, particles were advected southwestwards by prevailing boundary currents or were retained in nearshore eddies close to release locations. Some particles released in southeast Madagascar dispersed across the Mozambique Channel and reached the African shelf. Dispersal was characterized by high seasonal and inter-annual variability, and a large proportion of particles were dispersed far offshore and presumably lost. In the NW Indian Ocean, particles were retained within the Arabian Sea. Larval retention and self-recruitment in the Arabian Sea could explain the recent genetic divergence between P. h. megasculptus and P. h. homarus. Geographic distance and minimum SST were significantly associated with genetic differentiation in multivariate analysis, suggesting that larval tolerance to SST plays a role in shaping the population structure of P. homarus.

A molecular phylogeny of the spiny lobster Panulirus homarus highlights a separately evolving lineage from the Southwest Indian Ocean.

Accurate species description in the marine environment is critical for estimating biodiversity and identifying genetically distinct stocks. Analysis of molecular data can potentially improve species delimitations because they are easily generated and independent, and yield consistent results with high statistical power. We used classical phylogenetic (maximum likelihood and Bayesian inference) and coalescent-based methods (divergence dating with fossil calibrations and coalescent-based species delimitation) to resolve the phylogeny of the spiny lobster Panulirus homarus subspecies complex in the Indo-West Pacific. Analyses of mitochondrial data and combined nuclear and mitochondrial data recovered Panulirus homarus homarus and Panulirus homarus rubellus as separately evolving lineages, while the nuclear data trees were unresolved. Divergence dating analysis also identified Panulirus homarus homarus and Panulirus homarus rubellus as two distinct clades which diverged from a common ancestor during the Oligocene, approximately 26 million years ago. Species delimitation using coalescent-based methods corroborated these findings. A long pelagic larval life stage and the influence of ocean currents on post-larval settlement patterns suggest that a parapatric mode of speciation drives evolution in this subspecies complex. In combination, the results indicate that Panulirus homarus rubellus from the Southwest Indian Ocean is a separately evolving lineage and possibly a separate species.