Introduction
Four Donax species, D. semistriatus, D. trunculus, D. variegatus and D. vittatus, are common along the European coast, and live simpatrically in some areas, such as in Iberian Penisnula. D. trunculus is commercially exploited in several European countries, and constitutes an important shellfish resource due to its high economical value. However, some D. trunculus localities of the Iberian Peninsula seem to be at high long-term risk of extinction (Marie et al., 2016). In the face of the collapse of a fishery, a possible action is the assessment of similar species as an alternative resource to the exploited, allowing to increase the number of captures without increasing the pressure on the natural beds. In this context, genetic analyses on the species are essential for establishing successful stock enhancement or restocking operations.
In this study, we used information from mitochondrial and nuclear genes from three Donax species of the south Portuguese coast. Sequences for these six genes were combined and analysed to evaluate the genetic diversity and demographic patterns, and discuss the conservation implications of the obtained results. Additionally, we aim to clarify and establish the phylogenetic relationships of the genus Donax along the European coasts.
Materials and methods
Donax specimens were collected from natural beds in south Portuguese coast. Total genomic DNA was extracted from ethanol-preserved foot using a Chelex-100 (Sigma-Aldrich, USA). A total of 8 D. semistriatus specimens, 8 D. variegatus and 14 D. vittatus were amplified for three mitochondrial (16S, COI and Cytb) and three nuclear (18S, 28S and H3) markers. Sequence data were aligned with Clustal W (Thompson et al., 1994). For each gene, sequences were joined in unrooted networks using the fixed connection limit of 12-110 steps criterion implemented in TCS 1.21 (Clement et al., 2000). Number of haplotypes (h), haplotype diversity (Hd) and nucleotide diversity (π) were calculated for each gene fragment using DnaSP v5.10.01 (Librado and Rozas, 2009). In order to test for molecular signatures of demographic expansion, pairwise mismatch distribution analyses (Rogers and Harpending, 1992) as well as Tajima´s D (Tajima, 1989), Fu´s FS (Fu, 1997) and Ramos-Onsis and Rozas´s R2 tests (Ramos-Onsis and Rozas, 2002) were performed for COI gene in DnaSP v.5.10.01 (1000 replicates). Graphical outputs were used to illustrate demographic model fit. The model used for expected values was the constant population size. A final concatenated alignment, including 3 outgroups (D. trunculus, Meretrix meretrix and Venerupis philippinarum), was then analysed using Bayesian Inference (BI). Akaike Information Criterion was selected using jModelTest v.2.1.8 (Darriba et al., 2012) for each gene partition, including codon positions of coding genes. BI analysis was run using MrBayes v.3.2.6 (Ronquist et al., 2012) with sequences also partitioned according to each region, using the previously selected models. Two independent Markov chain Monte Carlo (MCMC) runs, each comprising four linked chains (one cold and three heated; as default settings), were performed 1 298 000 generations (enough to reach adequate average standard deviation (<0.01)), sampling every 1,000 generations. The first 25% trees were discarded as burn-in.
Results and discussion
Bayesian analysis produced a congruent tree topology with high support in the nodes, and showed a well-resolved phylogeny where the four species of the genus Donax from Europe form a single clade. D. trunculus is the basal clustering with a group formed by all the others, where D. variegatus is the sister clade of (D. vittatus + D. semistriatus). The results for both mitochondrial and nuclear loci showed in general high haplotype diversity in combination with a low nucleotide diversity and star-shaped networks with a predominant haplotype, indicating a recent population expansion for the examined species. Very similar networks were retrieved for mitochondrial and nuclear genes, clearly identifying the same three species as in the phylogenetic analysis. Additionally, our analyses revealed scenarios of demographic expansion in all three species (COI). The integration of nuclear markers allows a better understanding of possible causes (demography or selection) of the observed results of mitochondrial genetic variation, because demographic events are expected to affect similarly the whole genome, whereas selection will have locus-specific effect (Andolfatto, 2001). The comparison of the levels of genetic diversity between molecular markers suggest that, for the Donax species here studied, demographic expansion may be a plausible explanation.
Conclusion
The present study provides preliminary useful data for taxonomy, conservation and also resource management, as D. semistriatus D. variegatus and D. vittatus are species of interest to aquaculture for food production and here shown that could be used as a complement to the fishery of D. trunculus whose natural stocks have decreased dramatically in some areas of the Iberian Peninsula. However, further studies using a larger number of populations are necessary to provide a more accurate knowledge of the genetic structure of these species and detailed information for management of the fisheries.
This work was supported by the Ministerio de Economía y Competitividad (Spain) through project AGL2016-75288-R AEI/FEDER, UE and under the Framework of the Structured Program of R&D&I INNOVMAR (Reference NORTE-01-0145-FEDER-000035), (NORTE2020), through the European Regional Development Fund (ERDF). JFP acknowledges financial support for her pre-doctoral stay at CIIMAR awarded by INDITEX-UDC 2016.
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