Investigating the potential roles of intra-colonial genetic variability in Pocillopora corals using genomics.

Intra-colonial genetic variability (IGV), the presence of more than one genotype in a single colony, has been increasingly studied in scleractinians, revealing its high prevalence. Several studies hypothesised that IGV brings benefits, but few have investigated its roles from a genetic perspective. Here, using genomic data (SNPs), we investigated these potential benefits in populations of the coral Pocillopora acuta from Reunion Island (southwestern Indian Ocean). As the detection of IGV depends on sequencing and bioinformatics errors, we first explored the impact of the bioinformatics pipeline on its detection. Then, SNPs and genes variable within colonies were characterised. While most of the tested bioinformatics parameters did not significantly impact the detection of IGV, filtering on genotype depth of coverage strongly improved its detection by reducing genotyping errors. Mosaicism and chimerism, the two processes leading to IGV (the first through somatic mutations, the second through fusion of distinct organisms), were found in 7% and 12% of the colonies, respectively. Both processes led to several intra-colonial allelic differences, but most were non-coding or silent. However, 7% of the differences were non-silent and found in genes involved in a high diversity of biological processes, some of which were directly linked to responses to environmental stresses. IGV, therefore, appears as a source of genetic diversity and genetic plasticity, increasing the adaptive potential of colonies. Such benefits undoubtedly play an important role in the maintenance and the evolution of scleractinian populations and appear crucial for the future of coral reefs in the context of ongoing global changes.

From genomics to integrative species delimitation? The case study of the Indo-Pacific Pocillopora corals.

With the advent of genomics, sequencing thousands of loci from hundreds of individuals now appears feasible at reasonable costs, allowing complex phylogenies to be resolved. This is particularly relevant for cnidarians, for which insufficient data is available due to the small number of currently available markers and obscures species boundaries. Difficulties in inferring gene trees and morphological incongruences further blur the study and conservation of these organisms. Yet, can genomics alone be used to delimit species? Here, focusing on the coral genus Pocillopora, whose colonies play key roles in Indo-Pacific reef ecosystems but have challenged taxonomists for decades, we explored and discussed the usefulness of multiple criteria (genetics, morphology, biogeography and symbiosis ecology) to delimit species of this genus. Phylogenetic inferences, clustering approaches and species delimitation methods based on genome-wide single-nucleotide polymorphisms (SNP) were first used to resolve Pocillopora phylogeny and propose genomic species hypotheses from 356 colonies sampled across the Indo-Pacific (western Indian Ocean, tropical southwestern Pacific and south-east Polynesia). These species hypotheses were then compared to other lines of evidence based on genetic, morphology, biogeography and symbiont associations. Out of 21 species hypotheses delimited by genomics, 13 were strongly supported by all approaches, while six could represent either undescribed species or nominal species that have been synonymised incorrectly. Altogether, our results support (1) the obsolescence of macromorphology (i.e., overall colony and branches shape) but the relevance of micromorphology (i.e., corallite structures) to refine Pocillopora species boundaries, (2) the relevance of the mtORF (coupled with other markers in some cases) as a diagnostic marker of most species, (3) the requirement of molecular identification when species identity of colonies is absolutely necessary to interpret results, as morphology can blur species identification in the field, and (4) the need for a taxonomic revision of the genus Pocillopora. These results give new insights into the usefulness of multiple criteria for resolving Pocillopora, and more widely, scleractinian species boundaries, and will ultimately contribute to the taxonomic revision of this genus and the conservation of its species.

Isolation and characterization of 21 microsatellite loci for the sea cucumber Holothuria (Halodeima) atra (Echinodermata, Holothuroidea) reveal low asexual propagation through time in Reunion Island (southwestern Indian Ocean).

BACKGROUND: Holothuria (Halodeima) atra Jaeger, 1833 is a tropical sea cucumber usually harvested for the "bêche-de-mer" trade market. It has been reported to reproduce both sexually, through gamete spawning, or asexually, through fission. To date, no study has ever investigated clonality, nor genetic connectivity, among its populations, using microsatellite markers. METHODS AND RESULTS: We isolated the first 21 microsatellite loci specific for H. atra, which were then used to investigate clonal diversity, genetic structure and diversity among 44 H. atra individuals sampled in Reunion Island (southwestern Indian Ocean), over two seasons. All 21 loci were polymorphic, with number of alleles per locus ranging from 2 to 10. No repetitive multi-locus genotype (MLG) and few clonal lineages (MLL) were found. Observed heterozygosities per locus and season ranged from 0.000 to 0.909, while expected heterozygosities ranged from 0.290 to 0.882. Four loci were at Hardy-Weinberg equilibrium for both seasons, all others presenting a deficit of heterozygotes in one or both seasons. Meanwhile, no genetic differentiation was detected between seasons, according to assignment tests and global FST. CONCLUSIONS: These results suggest low asexual propagation in this population. These loci represent useful tools to better understand reproductive strategies and population connectivity of H. atra, and thus provide relevant knowledge for efficient management.

Species and population genomic differentiation in Pocillopora corals (Cnidaria, Hexacorallia).

Correctly delimiting species and populations is a prerequisite for studies of connectivity, adaptation and conservation. Genomic data are particularly useful to test species differentiation for organisms with few informative morphological characters or low discrimination of cytoplasmic markers, as in Scleractinians. Here we applied Restriction site Associated DNA sequencing (RAD-sequencing) to the study of species differentiation and genetic structure in populations of Pocillopora spp. from Oman and French Polynesia, with the objectives to test species hypotheses, and to study the genetic structure among sampling sites within species. We focused here on coral colonies morphologically similar to P. acuta (damicornis type ß). We tested the impact of different filtering strategies on the stability of the results. The main genetic differentiation was observed between samples from Oman and French Polynesia. These samples corresponded to different previously defined primary species hypotheses (PSH), i.e., PSHs 12 and 13 in Oman, and PSH 5 in French Polynesia. In Oman, we did not observe any clear differentiation between the two putative species PSH 12 and 13, nor between sampling sites. In French Polynesia, where a single species hypothesis was studied, there was no differentiation between sites. Our analyses allowed the identification of clonal lineages in Oman and French Polynesia. The impact of clonality on genetic diversity is discussed in light of individual-based simulations.

Forensic genetic identification of sharks involved in human attacks.

Each year, 75-100 unprovoked shark attacks on humans are recorded, most of them resulting in no or minor injuries, while a few are fatal. Often, shark identification responsible for attacks relies on visual observations or bite wound characteristics, which limits species determination and preclude individual identification. Here, we provide two genetic approaches to reliably identify species and/or individuals involved in shark attacks on humans based on a non-invasive DNA sampling (i.e. DNA traces present on bite wounds on victims), depending on the knowledge of previous attack history at the site. The first approach uses barcoding techniques allowing species identification without any a priori, while the second relies on microsatellite genotyping, allowing species identification confirmation and individual identification, but requiring an a priori of the potential species involved in the attack. Both approaches were validated by investigating two shark attacks that occurred in Reunion Island (southwestern Indian Ocean). According to both methods, each incident was attributed to a bull shark (Carcharhinus leucas), in agreement with suggestions derived from bite wound characteristics. Both approaches appear thus suitable for the reliable identification of species involved in shark attacks on humans. Moreover, microsatellite genotyping reveals, in the studied cases, that two distinct individuals were responsible of the bites. Applying these genetic identification methods will resolve ambiguities on shark species involved in attacks and allow the collection of individual data to better understand and mitigate shark risk.

Seascape genomics reveals candidate molecular targets of heat stress adaptation in three coral species.

Anomalous heat waves are causing a major decline of hard corals around the world and threatening the persistence of coral reefs. There are, however, reefs that have been exposed to recurrent thermal stress over the years and whose corals appear to have been tolerant against heat. One of the mechanisms that could explain this phenomenon is local adaptation, but the underlying molecular mechanisms are poorly known. In this work, we applied a seascape genomics approach to study heat stress adaptation in three coral species of New Caledonia (southwestern Pacific) and to uncover the molecular actors potentially involved. We used remote sensing data to characterize the environmental trends across the reef system, and sampled corals living at the most contrasted sites. These samples underwent next generation sequencing to reveal single nucleotide polymorphisms (SNPs), frequencies of which were associated with heat stress gradients. As these SNPs might underpin an adaptive role, we characterized the functional roles of the genes located in their genomic region. In each of the studied species, we found heat stress-associated SNPs located in proximity of genes involved in pathways well known to contribute to the cellular responses against heat, such as protein folding, oxidative stress homeostasis, inflammatory and apoptotic pathways, and DNA damage-repair. In some cases, the same candidate molecular targets of heat stress adaptation recurred among species. Together, these results underline the relevance and the power of the seascape genomics approach for the discovery of adaptive traits that could allow corals to persist across wider thermal ranges.

Together stronger: Intracolonial genetic variability occurrence in Pocillopora corals suggests potential benefits.

We investigated the occurrence of intracolonial genetic variability (IGV) in Pocillopora corals in the southwestern Indian Ocean. Ninety-six colonies were threefold-sampled from three sites in Reunion Island. Nubbins were genotyped using 13 microsatellite loci, and their multilocus genotypes compared. Over 50% of the colonies presented at least two different genotypes among their three nubbins, and IGV was found abundant in all sites (from 36.7% to 58.1%). To define the threshold distinguishing mosaicism from chimerism, we developed a new method based on different evolution models by computing the number of different alleles for the infinite allele model (IAM) and the Bruvo's distance for the stepwise mutation model (SMM). Colonies were considered as chimeras if their nubbins differed from more than four alleles and if the pairwise Bruvo's distance was higher than 0.12. Thus 80% of the IGV colonies were mosaics and 20% chimeras (representing almost 10% of the total sampling). IGV seems widespread in scleractinians and beyond the disabilities of this phenomenon reported in several studies, it should also bring benefits. Next steps are to identify these benefits and to understand processes leading to IGV, as well as factors influencing them.

In the intimacy of the darkness: Genetic polyandry in deep-sea luminescent lanternsharks Etmopterus spinax and Etmopterus molleri (Squaliformes, Etmopteridae).

Multiple paternity seems common within elasmobranchs. Focusing on two deep-sea shark species, the velvet belly lanternshark (Etmopterus spinax) and the slendertail lanternshark (Etmopterus molleri) we inferred the paternity in 31 E. spinax litters from Norway (three to 18 embryos per litter) and six E. molleri litters from Japan (three to six embryos), using 21 and 10 specific microsatellites, respectively. At least two E. spinax litters were sired from multiple fathers each, with highly variable paternal skew (1:1 to 9:1). Conversely, no clear signal of genetic polyandry was found in E. molleri.

Isolation and characterization of 42 microsatellite loci from the prickly redfish Thelenota ananas (Echinodermata, Stichopodidae).

The prickly redfish Thelenota ananas (Jaeger, 1833) is a widely distributed tropical sea cucumber. Populations of this species have been increasingly harvested throughout its distribution area in the Indo-Pacific region, which led to significant overexploitation issues. In order to investigate the genetic structure, diversity and connectivity of its populations, 42 microsatellite loci were isolated from a T. ananas microsatellite-enriched DNA library. These loci were characterized on 24 individuals collected from the Seychelles. The number of alleles per locus ranged from 3 to 22. The observed heterozygosity ranged from 0.083 to 0.952, while the expected heterozygosity ranged from 0.162 to 0.974. No linkage disequilibrium was detected among all loci and 20 loci (48%) were at Hardy-Weinberg equilibrium. These 42 loci represent useful tools for assessing genetic diversity, structure and gene flow among T. ananas populations, providing relevant knowledge for the management and conservation of those major commercial resources.

Isolation and characterization of microsatellite loci from three widespread tropical sea cucumbers of the genus Holothuria (Echinodermata, Holothuroidea), and cross-amplification among them.

Holothuria (Microthele) fuscogilva (Cherbonnier, 1980), Holothuria sp. type "Pentard" and Holothuria (Microthele) nobilis (Selenka, 1867) are three tropical sea cucumber taxa that are heavily fished worldwide for the beche-de-mer trade market. In order to investigate the population genetic structure, diversity and connectivity of these taxa, 16, 19 and 25 microsatellite loci were isolated from H. fuscogilva, Holothuria sp. type "Pentard" and H. nobilis DNA libraries, respectively. These loci were tested on 94, 60 and eight individuals of the respective species, collected from the Seychelles. The number of alleles per locus ranged from 2 to 30. The observed heterozygosity ranged from 0.245 to 0.890 for H. fuscogilva and from 0.200 to 0.950 for Holothuria sp. type "Pentard", while the expected heterozygosity ranged from 0.231 to 0.952 and from 0.504 to 0.951, respectively. Several loci were at Hardy-Weinberg equilibrium and linkage disequilibrium was detected in only three pairs of loci. Cross-amplification was also tested and almost all loci (49 over 60) were polymorphic for at least two of the three studied taxa, showing high transferability among them. These loci represent useful tools for assessing genetic diversity and population structure of these three taxa in fishery areas, and therefore providing relevant knowledge for resource management.

Isolation and characterization of 29 and 19 microsatellite loci from two deep-sea luminous lanternsharks, Etmopterus spinax and Etmopterus molleri (Squaliformes, Etmopteridae).

Etmopterus spinax (Linnaeus, 1758) and Etmopterus molleri (Whitley, 1939) are two bioluminescent deep-sea sharks, usually caught in large numbers as bycatch by deep-water fisheries. Yet, no study has ever involved population status of these two species using genetic tools. In order to investigate population genetic structure, diversity and connectivity of these two lanternsharks, 29 and 19 microsatellite loci were isolated from E. spinax DNA library for E. spinax and E. molleri, respectively. These loci were tested on 32 E. spinax individuals from the North Sea and seven E. molleri from the East China Sea. The number of alleles per locus ranged from 2 to 13. The observed heterozygosity ranged from 0.031 to 0.839 for E. spinax and from 0.000 to 1.000 for E. molleri, while the expected heterozygosity ranged from 0.031 to 0.903 and from 0.143 to 0.821, respectively. Almost all loci (24 and 16, respectively) were at Hardy-Weinberg equilibrium for both species and no linkage disequilibrium among loci was detected. These loci represent useful tools to better understand the population structure of these two species. Besides, they could also be suitable for other lanternsharks in general, as these latter remain largely understudied, specially in terms of understanding the basic science that will serve into their conservation.

Bio Simulators: a web UI for biological simulation.

SUMMARY: A host of formal, textual languages for modeling cellular processes have recently emerged, but their simulation tools often require an installation process which can pose a barrier for use. Bio Simulators is a framework for easy online deployment of simulators, providing a uniform web-based user interface to a diverse pool of tools. The framework is demonstrated through two plugins based on the KaSim Kappa simulator, one running directly in the browser and another running in the cloud. AVAILABILITY: Web tool: bsims.azurewebsites.net. KaSim client side simulator: github.com/NicolasOury/KaSimJS. KaSim cloud simulator: github.com/mdpedersen/KaSimCloud. CONTACT: michael.d.pedersen@gmail.com or Andrew.Phillips@microsoft.com SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.