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

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

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

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 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

The two sides of the Mediterranean: Population genomics of the Black Sea urchin Arbacia lixula (Linnaeus 1758) in a Warming Sea

Global environmental changes may have a profound impact on ecosystems. In this context, it is crucial to gather biological and ecological information of the main species in marine communities to predict and mitigate potential effects of shifts in their distribution, abundance, and interactions. Using genotyping by sequencing (GBS), we assessed the genetic structure of a keystone species in the Mediterranean shallow littoral ecosystems, the black sea urchin Arbacia lixula. This bioengineer species can shape their communities due to its grazing activity and it is experiencing an ongoing expansion with increasing temperatures. The population genomic analyses on 5,241 loci sequenced in 240 individuals from 11 Mediterranean sampled populations revealed that all populations were diverse and showed significant departure from equilibrium. Albeit genetic differentiation was in general shallow, a significant break separated the western and eastern Mediterranean populations, a break not detected in previous studies with less resolutive markers. Notably, no clear effect of the Almería-Oran front, an important break in the Atlanto-Mediterranean transition, could be detected among the western basin populations, where only a slight differentiation of the two northernmost populations was found. Despite the generally low levels of genetic differentiation found, we identified candidate regions for local adaptation by combining different genomic analysis with environmental data. Salinity, rather than temperature, seemed to be an important driver of genetic structure in A. lixula. Overall, from a population genomics standpoint, there is ample scope for A. lixula to continue thriving and adapting in the warming Mediterranean

East is East and West is West: Population genomics and hierarchical analyses reveal genetic structure and adaptation footprints in the keystone species Paracentrotus lividus (Echinoidea)

Aim The Atlanto‐Mediterranean edible purple sea urchin, Paracentrotus lividus, is a commercially exploited keystone species in benthic communities. Its browsing activity can deeply modify the littoral landscape, and changes in its abundance are of major conservation concern. This species is facing nowadays contrasting anthropogenic pressures linked to predator release, exploitation and sea warming. Management of this key species requires knowledge of its genetic structure, connectivity and local adaptation. Our goal was to assess the current global status of the species under a genomic perspective. Location Atlanto‐Mediterranean shores from Morocco and France to Turkey. Methods We used genotyping by sequencing (GBS) of 241 individuals belonging to 11 populations spanning the known range of distribution of the species. We obtained 3,348 loci for population genomics and outlier analyses. Results We identified significant genetic structure and a gradient matching the longitudinal position of the localities. A hierarchical analysis revealed two main clusters (Atlantic and Mediterranean) and subtler patterns of differentiation within them. Candidate markers for selection identified between and within these two main clusters were mostly different, likely indicating different selective pressures. Adaptation to maximum salinity and maximum temperature appeared as an important driver of the transition between Atlantic and Mediterranean basins. Other stressors, such as minimum temperature or range of temperature, seem to define the structuring within the Mediterranean

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 reintroduction 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 reintroduction program using molecular markers (13 microsatellites, 800bp D-loop and STR mtDNA 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 reintroduction program 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

Nesting range expansion of loggerhead turtles in the Mediterranean: phenology, spatial distribution and conservation implications.

Global warming is affecting habitat quality and availability on our planet and some species are predicted or are by now changing their distribution range. Here we show that loggerhead turtles have already started to expand their nesting range into the Western Mediterranean, which has until recently hosted only sporadic nests. We compiled information on nesting activity from beaches surrounding the Western Mediterranean and collected metadata on loggerhead turtle nests in Spain, France, Italy, and Tunisia between 2010 and 2020 to provide an exhaustive overview on the phenomenon of emerging new nest sites for loggerhead turtles. The number of recorded nests has increased drastically since 2013 from 1 to 3 nests/year to a record number of 84 registered in 2020. While this increase may partly be explained by grown awareness and reporting by citizens, there is no doubt of an upward trend in nesting activity. The nests are unevenly distributed over the study area with most nests occurring on the coasts of the warmer Tyrrhenian Sea. A hotspot analysis identified beaches in SW Italy, SE Sardinia, and NW Tunisia with statistically significant clustering of nests. Within these hotspots, three beaches in SW Italy and one in Tunisia had nests at least four out of the five last years. Nesting phenology corresponds to that of Eastern Mediterranean rookeries, and mean hatching success of naturally incubating, non-manipulated nests was 66 %, although there was variability across the region. Mean incubation durations also varied between countries indicating a diversity in inferred sex ratios, with sufficient female production to foster future colonisation of this region. Unfortunately, these beaches are already under high tourist pressure and subject to intense coastal development, imposing many threats to the females, eggs, and hatchlings. Thus, while this study reveals the unique opportunity to witness and study an ongoing new colonisation process in loggerhead turtles, it also calls for urgent proactive conservation actions to mitigate these threats and allow the turtles to establish new rookeries

Mediterranean sea turtles: current knowledge and priorities for conservation and research

The available information regarding the 2 sea turtle species breeding in the Mediterranean (loggerhead turtle Caretta caretta and green turtle Chelonia mydas) is reviewed, including biometrics and morphology, identification of breeding and foraging areas, ecology and behaviour, abundance and trends, population structure and dynamics, anthropogenic threats and conservation measures. Although a large body of knowledge has been generated, research efforts have been inconsistently allocated across geographic areas, species and topics. Significant gaps still exist, ranging from the most fundamental aspects, such as the distribution of major nesting sites and the total number of clutches laid annually in the region, to more specific topics like age at maturity, survival rates and behavioural ecology, especially for certain areas (e.g. south-eastern Mediterranean). These gaps are particularly marked for the green turtle. The recent positive trends of nest counts at some nesting sites may be the result of the cessation of past exploitation and decades of conservation measures on land, both in the form of national regulations and of continued active protection of clutches. Therefore, the current status should be considered as dependent on such ongoing conservation efforts. Mitigation of incidental catch in fisheries, the main anthropogenic threat at sea, is still in its infancy. From the analysis of the present status a comprehensive list of research and conservation priorities is proposed