The past, the recent, and the ongoing evolutionary processes of the worldwide invasive ascidian Styela plicata.

Invasive species are one of the main threats to global biodiversity and, within marine ecosystems, tunicates feature some prominent examples. Styela plicata is an ascidian species inhabiting harbours in all temperate oceans and seas, thus being considered a thriving invasive species. However, this species' adaptive mechanisms, introduction history, and population structure have never been completely elucidated. Here, by genotyping 87 S. plicata individuals from 18 localities worldwide with 2b-RADseq, we confirm the global presence of four chromosome inversions, demonstrate population structuring on this species, detect local adaptation signals, and infer historical demographic events. We show that North Carolina individuals constitute an unrelated population, Atlanto-Mediterranean and Pacific localities form their own genetic clusters with substructuring, being the most evident the split between northern and southern Atlantic localities. The locality of South Carolina presents an intermediate genetic position between North Carolina and the other two groups pointing to a hybrid origin with recurrent gene flow. We generate and test demographic models, providing evidence of two independent introduction events to the Atlantic and Pacific, and an admixture that originated the population of South Carolina. Finally, we identify candidate loci for adaptation, with functions involved with cell processes, metabolism, development, and ion transport, among others. Overall, this study highlights the complex historical processes of S. plicata, which have led this species to its current distribution, population structure, and local adaptation footprint in oceans worldwide.

Chromosome-level genome assembly and annotation of the black sea urchin Arbacia lixula (Linnaeus, 1758).

The black sea urchin (Arbacia lixula) is a keystone species inhabiting the coastal shallow waters of the Mediterranean Sea, which is a key driver of littoral communities' structure. Here, we present the first genome assembly and annotation of this species, standing as the first Arbacioida genome, including both nuclear and mitochondrial genomes. To obtain a chromosome-level assembly, we used a combination of PacBio high fidelity (HiFi) reads and chromatin capture reads (Omni-C). In addition, we generated a high-quality nuclear annotation of both coding and non-coding genes, by using published RNA-Seq data from several individuals of A. lixula and gene models from closely related species. The nuclear genome assembly has a total span of 607.91 Mb, being consistent with its experimentally estimated genome size. The assembly contains 22 chromosome-scale scaffolds (96.52% of the total length), which coincides with its known karyotype. A total of 72,767 transcripts were predicted from the nuclear genome, 24,171 coding, and 48,596 non-coding that included lncRNA, snoRNA, and tRNAs. The circularized mitochondrial genome had 15,740 bp comprising 13 protein-coding genes, 2 rRNA, and 22 tRNA. This reference genome will enhance ongoing A. lixula studies and benefit the wider sea urchin scientific community.

Navigating spatio-temporal microbiome dynamics: Environmental factors and trace elements shape the symbiont community of an invasive marine species.

The proliferation of marine invasive species is a mounting concern. While the role of microbial communities in invasive ascidian species is recognized, the role of seasonal shifts in microbiome composition remains largely unexplored. We sampled five individuals of the invasive ascidian Styela plicata quarterly from January 2020 to October 2021 in two harbours, examining gills, tunics, and surrounding water. By analysing Amplicon Sequence Variants (ASVs) and seawater trace elements, we found that compartment (seawater, tunic, or gills) was the primary differentiating factor, followed by harbour. Clear seasonal patterns were evident in seawater bacteria, less so in gills, and absent in tunics. We identified compartment-specific bacteria, as well as seasonal indicator ASVs and ASVs correlated with trace element concentrations. Among these bacteria, we found that Endozoicomonas, Hepatoplasma and Rhodobacteraceae species had reported functions which might be necessary for overcoming seasonality and trace element shifts. This study contributes to understanding microbiome dynamics in invasive holobiont systems, and the patterns found indicate a potential role in adaptation and invasiveness.

Genome composition and GC content influence loci distribution in reduced representation genomic studies.

BACKGROUND: Genomic architecture is a key evolutionary trait for living organisms. Due to multiple complex adaptive and neutral forces which impose evolutionary pressures on genomes, there is a huge variability of genomic features. However, their variability and the extent to which genomic content determines the distribution of recovered loci in reduced representation sequencing studies is largely unexplored. RESULTS: Here, by using 80 genome assemblies, we observed that whereas plants primarily increase their genome size by expanding their intergenic regions, animals expand both intergenic and intronic regions, although the expansion patterns differ between deuterostomes and protostomes. Loci mapping in introns, exons, and intergenic categories obtained by in silico digestion using 2b-enzymes are positively correlated with the percentage of these regions in the corresponding genomes, suggesting that loci distribution mostly mirrors genomic architecture of the selected taxon. However, exonic regions showed a significant enrichment of loci in all groups regardless of the used enzyme. Moreover, when using selective adaptors to obtain a secondarily reduced loci dataset, the percentage and distribution of retained loci also varied. Adaptors with G/C terminals recovered a lower percentage of selected loci, with a further enrichment of exonic regions, while adaptors with A/T terminals retained a higher percentage of loci and slightly selected more intronic regions than expected. CONCLUSIONS: Our results highlight how genome composition, genome GC content, RAD enzyme choice and use of base-selective adaptors influence reduced genome representation techniques. This is important to acknowledge in population and conservation genomic studies, as it determines the abundance and distribution of loci.

New colonisers drive the increase of the emerging loggerhead turtle nesting in Western Mediterranean.

The loggerhead sea turtle (Caretta caretta) is sensitive to climate change and is responding by colonising the Western Mediterranean. To understand the rapid nesting increase in recent years in Spain, we sampled 45 hatchlings from 8 nests between 2016 and 2019. We sequenced a mtDNA D-loop region, genotyped 2291 SNPs using 2bRAD and collected data on clutch size, hatching success, and incubation duration. We confirmed that the colonisation has a Mediterranean and Atlantic mixed origin and we detected that these nests were laid by different females, except for two nests within the same season. Our results suggest that the recent increase in nesting is due to an increase in the number of colonising individuals rather than females born in the same area returning to breed. We hypothesize that this increase in the number of colonisers results from successful conservation efforts, feminisation of the populations of origin and earlier sexual maturation. However, the percentage of offspring females produced in Spain suggests that future returning individuals will aid to the settlement of the new population. These results allow defining the current status of this colonisation although future efforts are needed to detect remigrants to confirm the establishment of a resident population.

Going beyond a reference genome in conservation genomics.

The current biodiversity crisis demands scientifically based management. The generation of reference genomes is crucial in conservation, but is not enough to capture species diversity. By incorporating whole-genome sequencing (WGS) at the population level, Nigenda-Morales et al. provide key genomic information for the conservation of fin whale populations in the Pacific.

Multidimensional variability of the microbiome of an invasive ascidian species.

Animals, including invasive species, are complex entities consisting of a host and its associated symbionts (holobiont). The interaction between the holobiont components is crucial for the host's survival. However, our understanding of how microbiomes of invasive species change across different tissues, localities, and ontogenetic stages, is limited. In the introduced ascidian Styela plicata, we found that its microbiome is highly distinct and specialized among compartments (tunic, gill, and gut). Smaller but significant differences were also found across harbors, suggesting local adaptation, and between juveniles and adults. Furthermore, we found a correlation between the microbiome and environmental trace element concentrations, especially in adults. Functional analyses showed that adult microbiomes possess specific metabolic pathways that may enhance fitness during the introduction process. These findings highlight the importance of integrated approaches in studying the interplay between animals and microbiomes, as a first step toward understanding how it can affect the species' invasive success.

Evaluating restriction enzyme selection for reduced representation sequencing in conservation genomics.

Conservation genomic studies in non-model organisms generally rely on reduced representation sequencing techniques based on restriction enzymes to identify population structure as well as candidate loci for local adaptation. While the expectation is that the reduced representation of the genome is randomly distributed, the proportion of the genome sampled might depend on the GC content of the recognition site of the restriction enzyme used. Here, we evaluated the distribution and functional composition of loci obtained after a reduced representation approach using Genotyping-by-Sequencing (GBS). To do so, we compared experimental data from two endemic fish species (Symphodus ocellatus and Symphodus tinca, EcoT22I enzyme) and two ecosystem engineer sea urchins (Paracentrotus lividus and Arbacia lixula, ApeKI enzyme). In brief, we mapped the sequenced loci to the phylogenetically closest reference genome available (Labrus bergylta in the fish and Strongylocentrotus purpuratus in the sea urchin datasets), classified them as exonic, intronic and intergenic, and studied their function by using Gene Ontology (GO) terms. We also simulated the effect of using both enzymes in the two reference genomes. In both simulated and experimental data, we detected an enrichment towards exonic or intergenic regions depending on the restriction enzyme used and failed to detect differences between total loci and candidate loci for adaptation in the empirical dataset. Most of the functions assigned to the mapped loci were shared between the four species and involved a myriad of general functions. Our results highlight the importance of restriction enzyme selection and the need for high-quality annotated genomes in conservation genomic studies.

Genomic basis for early-life mortality in sharpsnout seabream.

Mortality at early life stages of fishes is common in nature and can be shaped by stochastic and selective processes. Selective mortality has rarely been assessed in natural conditions but can now be studied by combining genomic data with information on different life stages that realates to fitness. Here we investigate selective mortality between settlers and six-month survivors of the sharpsnout seabream by genotype-phenotype/environmental association studies in three localities along a geographic gradient. We gathered information on 105 individuals at 85,031 SNPs, obtained from individual based 2b-RAD libraries, as well as 9 phenotypic and environmental variables derived from individual otolith readings. We found common signals across localities for potential selection, such as lower survival rates for individuals hatching earlier, growing faster and experiencing higher temperatures during their planktonic phase. We identified 122 loci with parallel significant association to phenotypic and environmental variables. Importantly, one of these loci mapped to the exonic region of the il20rb, a gene involved in immune response, in the phylogenetically closest reference genome, showing parallel frequency changes in non-synonymous mutations in the three studied populations. Further temporal assessments are needed to understand how polymorphisms that are key to selective mortality are maintained.

The architecture of assisted colonisation in sea turtles: building new populations in a biodiversity crisis.

Due to changing environmental conditions, many species will have to migrate or occupy new suitable areas to avoid potential extinction in the current biodiversity crisis. Long-lived animals are especially vulnerable and ex-situ conservation actions can provide solutions through assisted colonisations. However, there is little empirical evidence on the process of founding new populations for such species or the feasibility of assisted colonisations as a viable conservation measure. Here, we combined genetics with reproductive data to study the rise of two wild populations of green turtles (Chelonia mydas) in the Cayman Islands as a possible outcome of a reintroduction program started 50 years ago. We show that both populations are highly related to the captive population but rapidly diverged due to genetic drift. Individuals from the reintroduced populations showed high levels of nest fidelity, within and across nesting seasons, indicating that philopatry may help reinforce the success of new populations. Additionally, we show that reintroduction from captive populations has not undermined the reproductive fitness of first generation individuals. Sea turtle reintroduction programs can, therefore, establish new populations but require scientific evaluation of costs and benefits and should be monitored over time to ensure viability in the long-term.

Author Correction: Population-specific signatures of intra-individual mitochondrial DNA heteroplasmy and their potential evolutionary advantages.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Individual-based population genomics reveal different drivers of adaptation in sympatric fish.

Connectivity and local adaptation are two contrasting evolutionary forces highly influencing population structure. To evaluate the impact of early-life traits and environmental conditions on genetic structuring and adaptation, we studied two sympatric fish species in the Western Mediterranean Sea: Symphodus tinca and S. ocellatus. We followed an individual-based approach and measured early-life history traits from otolith readings, gathered information on environmental variables and obtained genome-wide markers from genotyping-by-sequencing (GBS). The two species presented contrasting population structure across the same geographic gradient, with high and significant population differentiation in S. ocellatus, mostly determined by oceanographic fronts, and low differentiation and no front effect in S. tinca. Despite their different levels of genetic differentiation, we identified in both species candidate regions for local adaptation by combining outlier analysis with environmental and phenotypic association analyses. Most candidate loci were associated to temperature and productivity in S. ocellatus and to temperature and turbulence in S. tinca suggesting that different drivers may determine genomic diversity and differentiation in each species. Globally, our study highlights that individual-based approach combining genomic, environmental and phenotypic information is key to identify signals of selection and the processes mediating them.

Helping decision making for reliable and cost-effective 2b-RAD sequencing and genotyping analyses in non-model species.

High-throughput sequencing has revolutionized population and conservation genetics. RAD sequencing methods, such as 2b-RAD, can be used on species lacking a reference genome. However, transferring protocols across taxa can potentially lead to poor results. We tested two different IIB enzymes (AlfI and CspCI) on two species with different genome sizes (the loggerhead turtle Caretta caretta and the sharpsnout seabream Diplodus puntazzo) to build a set of guidelines to improve 2b-RAD protocols on non-model organisms while optimising costs. Good results were obtained even with degraded samples, showing the value of 2b-RAD in studies with poor DNA quality. However, library quality was found to be a critical parameter on the number of reads and loci obtained for genotyping. Resampling analyses with different number of reads per individual showed a trade-off between number of loci and number of reads per sample. The resulting accumulation curves can be used as a tool to calculate the number of sequences per individual needed to reach a mean depth ≥20 reads to acquire good genotyping results. Finally, we demonstrated that selective-base ligation does not affect genomic differentiation between individuals, indicating that this technique can be used in species with large genome sizes to adjust the number of loci to the study scope, to reduce sequencing costs and to maintain suitable sequencing depth for a reliable genotyping without compromising the results. Here, we provide a set of guidelines to improve 2b-RAD protocols on non-model organisms with different genome sizes, helping decision-making for a reliable and cost-effective genotyping.

Population-specific signatures of intra-individual mitochondrial DNA heteroplasmy and their potential evolutionary advantages.

Heteroplasmy is the existence of more than one mitochondrial DNA (mtDNA) variant within a cell. The evolutionary mechanisms of heteroplasmy are not fully understood, despite being a very common phenomenon. Here we combined heteroplasmy measurements using high throughput sequencing on green turtles (Chelonia mydas) with simulations to understand how heteroplasmy modulates population diversity across generations and under different demographic scenarios. We found heteroplasmy to be widespread in all individuals analysed, with consistent signal in individuals across time and tissue. Significant shifts in haplotype composition were found from mother to offspring, signalling the effect of the cellular bottleneck during oogenesis as included in the model. Our model of mtDNA inheritance indicated that heteroplasmy favoured the increase of population diversity through time and buffered against population bottlenecks, thus indicating the importance of this phenomenon in species with reduced population sizes and frequent population bottlenecks like marine turtles. Individuals with recent haplotypes showed higher levels of heteroplasmy than the individuals with ancient haplotypes, suggesting a potential advantage of maintaining established copies when new mutations arise. We recommend using heteroplasmy through high throughput sequencing in marine turtles, as well as other wildlife populations, for diversity assessment, population genetics, and mixed stock analysis.

How many came home? Evaluating ex situ conservation of green turtles in the Cayman Islands.

Ex situ management is an important conservation tool that allows the preservation of biological diversity outside natural habitats while supporting survival in the wild. Captive breeding followed by re-introduction is a possible approach for endangered species conservation and preservation of genetic variability. The Cayman Turtle Centre Ltd was established in 1968 to market green turtle (Chelonia mydas) meat and other products and replenish wild populations, thought to be locally extirpated, through captive breeding. We evaluated the effects of this re-introduction programmme using molecular markers (13 microsatellites, 800-bp D-loop and simple tandem repeat mitochondrial DNA sequences) from captive breeders (N = 257) and wild nesting females (N = 57) (sampling period: 2013-2015). We divided the captive breeders into three groups: founders (from the original stock), and then two subdivisions of F1 individuals corresponding to two different management strategies, cohort 1995 ("C1995") and multicohort F1 ("MCF1"). Loss of genetic variability and increased relatedness was observed in the captive stock over time. We found no significant differences in diversity among captive and wild groups, and similar or higher levels of haplotype variability when compared to other natural populations. Using parentage and sibship assignment, we determined that 90% of the wild individuals were related to the captive stock. Our results suggest a strong impact of the re-introduction programmme on the present recovery of the wild green turtle population nesting in the Cayman Islands. Moreover, genetic relatedness analyses of captive populations are necessary to improve future management actions to maintain genetic diversity in the long term and avoid inbreeding depression.

Author Correction: Sporadic nesting reveals long distance colonisation in the philopatric loggerhead sea turtle (Caretta caretta).

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Sporadic nesting reveals long distance colonisation in the philopatric loggerhead sea turtle (Caretta caretta).

The colonisation of new suitable habitats is crucial for species survival at evolutionary scale under changing environmental conditions. However, colonisation potential may be limited by philopatry that facilitates exploiting successful habitats across generations. We examine the mechanisms of long distance dispersal of the philopatric loggerhead sea turtle (Caretta caretta) by analysing 40 sporadic nesting events in the western Mediterranean. The analysis of a fragment of the mitochondrial DNA and 7 microsatellites of 121 samples from 18 of these nesting events revealed that these nests were colonising events associated with juveniles from distant populations feeding in nearby foraging grounds. Considering the temperature-dependent sex determination of the species, we simulated the effect of the incubation temperature and propagule pressure on a potential colonisation scenario. Our results indicated that colonisation will succeed if warm temperature conditions, already existing in some of the beaches in the area, extend to the whole western Mediterranean. We hypothesize that the sporadic nesting events in developmental foraging grounds may be a mechanism to overcome philopatry limitations thus increasing the dispersal capabilities of the species and the adaptability to changing environments. Sporadic nesting in the western Mediterranean can be viewed as potential new populations in a scenario of rising temperatures.

Population genomics of an endemic Mediterranean fish: differentiation by fine scale dispersal and adaptation.

The assessment of the genetic structuring of biodiversity is crucial for management and conservation. For species with large effective population sizes a low number of markers may fail to identify population structure. A solution of this shortcoming can be high-throughput sequencing that allows genotyping thousands of markers on a genome-wide approach while facilitating the detection of genetic structuring shaped by selection. We used Genotyping-by-Sequencing (GBS) on 176 individuals of the endemic East Atlantic peacock wrasse (Symphodus tinca), from 6 locations in the Adriatic and Ionian seas. We obtained a total of 4,155 polymorphic SNPs and we observed two strong barriers to gene flow. The first one differentiated Tremiti Islands, in the northwest, from all the other locations while the second one separated east and south-west localities. Outlier SNPs potentially under positive selection and neutral SNPs both showed similar patterns of structuring, although finer scale differentiation was unveiled with outlier loci. Our results reflect the complexity of population genetic structure and demonstrate that both habitat fragmentation and positive selection are on play. This complexity should be considered in biodiversity assessments of different taxa, including non-model yet ecologically relevant organisms.

Linking loggerhead locations: using multiple methods to determine the origin of sea turtles in feeding grounds.

Many marine megavertebrate taxa, including sea turtles, disperse widely from their hatching or birthing locations but display natal homing as adults. We used flipper tagging, satellite tracking and genetics to identify the origin of loggerhead turtles living in Amvrakikos Gulf, Greece. This location has been identified as hosting regionally important numbers of large-juvenile to adult sized turtles that display long-term residency and/or association to the area, and also presents a male biased sex ratio for adults. A total of 20 individuals were linked to nesting areas in Greece through flipper tagging and satellite telemetry, with the majority (16) associated with Zakynthos Island. One additional female was tracked from Amvrakikos Gulf to Turkey where she likely nested. Mitochondrial DNA mixed stock analyses of turtles captured in Amvrakikos Gulf (n = 95) indicated 82% of individuals originated from Greek nesting stocks, mainly from Zakynthos Island (63%), with lesser contributions from central Turkey, Cyprus and Libya. These results suggest that the male-biased sex ratio found in Amvrakikos Gulf may be driven by the fact that males breed twice as frequently on Zakynthos, resulting in their using foraging grounds of greater proximity to the breeding site. Conservation measures in localised foraging habitats for the protection of marine vertebrates, such as sea turtles, may have positive impacts on several disparate breeding stocks and the use of multiple methods to determine source populations can indicate the relative effectiveness of these measures.