Population structure within and between subspecies of the Mediterranean triplefin fish Tripterygion delaisi revealed by highly polymorphic microsatellite loci

Although F(ST) values are widely used to elucidate population relationships, in some cases, when employing highly polymorphic loci, they should be regarded with caution, particularly when subspecies are under consideration. Tripterygion delaisi presents two subspecies that were investigated here, using 10 microsatellite loci. A Bayesian approach allowed us to clearly identify both subspecies as two different evolutionary significant units. However, low F(ST) values were found between subspecies as a consequence of the large number of alleles per locus, while homoplasy could be disregarded as indicated by the standardized genetic distance G'(ST). Heterozygosity saturation was observed in highly polymorphic loci containing more than 15 alleles, and this threshold was used to define two loci pools. The less variable loci pool revealed higher genetic variance between subspecies, while the more variable pool showed higher genetic variance between populations. Furthermore, higher differentiation was also observed between populations using G'(ST) with the more variable loci. Nonetheless, a more reliable population structure within subspecies was obtained when all loci were included in the analyses. In T. d. xanthosoma, isolation by distance was detected between the eight analysed populations, and six genetically homogeneous clusters were inferred by Bayesian analyses that are in accordance with F(ST) values. The neighbourhood-size method also indicated rather small dispersal capabilities. In conclusion, in fish with limited adult and larval dispersal capabilities, continuous rocky habitat seems to allow contact between populations and prevent genetic differentiation, while large discontinuities of sand or deep-water channels seems to reduce gene flow.     

Testis-testicular gland complex of two Tripterygion species (Blennioidei, Teleostei): differences between territorial and non-territorial males

The morphological differences between the testis and testicular gland of territorial and nonterritorial males of Tripterygion tripteronotus and T. delaisi were examined and correlated with differences in reproductive behaviour. In territorial males of both species the testicular gland is much more developed than in non-territorial males. Larger cellular and nuclear sizes in the territorial males indicate that the activity of the gland cells is enhanced. These cells contain SER, numerous lipid droplets and mitochondria with lamellar cristae. Absence of 3β-HSD activity at these sites points to lack of a steroidogenic potency. In both territorial and non-territorial fish, steroid-producing Leydig cells have been demonstrated in the connective tissue betweeen the testis and the testicular gland, and around the collecting sperm duct. In addition, 3β-HSD activity has been found in the scarce interstitial Leydig cells of territorial fish. Morphometric data indicate an enhanced activity of the Leydig cells in territorial fish.     

Kinship analyses identify fish dispersal events on a temperate coastline

Connectivity is crucial for the persistence and resilience of marine species, the establishment of networks of marine protected areas and the delineation of fishery management units. In the marine environment, understanding connectivity is still a major challenge, due to the technical difficulties of tracking larvae. Recently, parentage analysis has provided a means to address this question effectively. To be effective, this method requires limited adult movement and extensive sampling of parents, which is often not possible for marine species. An alternative approach that is less sensitive to constraints in parental movement and sampling could be the reconstruction of sibships. Here, we directly measure connectivity and larval dispersal in a temperate marine ecosystem through both analytical approaches. We use data from 178 single nucleotide polymorphism markers to perform parentage and sibship reconstruction of the black-faced blenny (Tripterygion delaisi) from an open coastline in the Mediterranean Sea. Parentage analysis revealed a decrease in dispersal success in the focal area over 1 km distance and approximately 6.5% of the juveniles were identified as self-recruits. Sibship reconstruction analysis found that, in general, full siblings did not recruit together to the same location, and that the largest distance between recruitment locations was much higher (11.5 km) than found for parent–offspring pairs (1.2 km). Direct measurements of dispersal are essential to understanding connectivity patterns in different marine habitats, and show the degree of self-replenishment and sustainability of populations of marine organisms. We demonstrate that sibship reconstruction allows direct measurements of dispersal and family structure in marine species while being more easily applied in those species for which the collection of the parental population is difficult or unfeasible.     

Isolation and characterization of microsatellite loci in Tripterygion delaisi

We isolated 49 microsatellite loci from a genomic library of Tripterygion delaisi×anthosoma enriched for CA and GA repeats. Ten loci were screened in 30 individuals with high numbers of alleles per locus (averaging 15.5 ± 2.86) and observed heterozygosity (averaging 0.765 ± 0.052). No deviations from Hardy–Weinberg expectations were detected. These highly polymorphic markers will be useful in determining the spatial patterns of genetic diversity between and within subspecies of Tripterygion delaisi.     

SNP development from RNA‐seq data in a nonmodel fish: how many individuals are needed for accurate allele frequency prediction?

Single nucleotide polymorphisms (SNPs) are rapidly becoming the marker of choice in population genetics due to a variety of advantages relative to other markers, including higher genomic density, data quality, reproducibility and genotyping efficiency, as well as ease of portability between laboratories. Advances in sequencing technology and methodologies to reduce genomic representation have made the isolation of SNPs feasible for nonmodel organisms. RNA‐seq is one such technique for the discovery of SNPs and development of markers for large‐scale genotyping. Here, we report the development of 192 validated SNP markers for parentage analysis in Tripterygion delaisi (the black‐faced blenny), a small rocky‐shore fish from the Mediterranean Sea. RNA‐seq data for 15 individual samples were used for SNP discovery by applying a series of selection criteria. Genotypes were then collected from 1599 individuals from the same population with the resulting loci. Differences in heterozygosity and allele frequencies were found between the two data sets. Heterozygosity was lower, on average, in the population sample, and the mean difference between the frequencies of particular alleles in the two data sets was 0.135 ± 0.100. We used bootstrap resampling of the sequence data to predict appropriate sample sizes for SNP discovery. As cDNA library production is time‐consuming and expensive, we suggest that using seven individuals for RNA sequencing reduces the probability of discarding highly informative SNP loci, due to lack of observed polymorphism, whereas use of more than 12 samples does not considerably improve prediction of true allele frequencies.