Ecological niche modeling, cytogenetics and phylogeography of the genera Geomalacus and Letourneuxia (Gastropoda, Pulmonata) from the Iberian-Moroccan region

First described more that 150 years ago, the systematics of the genera Geomalacus and Letourneuxia (Arionidae, Gastropoda, Pulmonata) is still challenging. The taxonomic classification of arionid species is based on extremely labile characters such as body size or color that depends both on diet and environment, as well as age. Moreover, there is little information on the genetic diversity and population structure of the Iberian slugs that could provide extra clues to disentangle their problematic classification. The present work uses different analytical tools such as habitat suitability (Ecological Niche Modeling - ENM), cytogenetic analysis and phylogeography to establish the geographical distribution and evolutionary history of these pulmonate slugs. The potential distribution of the four Geomalacus species was modeled using ENM, which allowed the identification of new locations for G. malagensis, including a first report in Portugal. Also, it was predicted a much wider distribution for G. malagensis and G. oliveirae than previously known. Classical cytogenetic analyses were assayed with reproductive and a novel use of somatic tissues (mouth and tentacles) returning the number of chromosomes for the four Geomalacus species and L. numidica (n = 31, 2n = 62) and the respective karyotypes. G. malagensis and L. numidica present similar chromosome morphologies and karyotypic formulae, being more similar to each other than the Geomalacus among themselves. We further reconstructed the phylogeny of the genera Geomalacus and Letourneuxia using partial sequences of the mitochondrial cytochrome oxidase subunit I (COI) and the nuclear ribosomal small subunit (18S rRNA), and applied an independent evolutionary rate method, the indicator vectors correlation, to evaluate the existence of cryptic diversity within species. The five nominal species of Geomalacus and Letourneuxia comprise 14 well-supported cryptic lineages. Letourneuxia numidica was retrieved as a sister group of G. malagensis. G. oliveirae is paraphyletic with respect to G. anguiformis. According to our dating estimates, the most recent common ancestor of Geomalacus dates back to the Middle Miocene (end of the Serravallian stage). The major lineage splitting events within Geomalacus occurred during the dry periods of the Zanclean stage (5.3-3.6 million years) and some lineages were confined to more humid mountain areas of the Iberian Peninsula, which lead to a highly geographically structured mitochondrial genetic diversity. The major findings of this are the following: (1) provides updated species distribution maps for the Iberian Geomalacus expanding the known geographic distribution of the concerned species, (2) unravels the cryptic diversity within the genera Geomalacus and Letourneuxia, (3) Geomalacus oliveirae is paraphyletic with G. anguiformis and (4) Letourneuxia numidica is sister group of G. malagensis.

Phylogeography of the Atlanto-Mediterranean sea cucumber Holothuria (Holothuria) mammata: the combined effects of historical processes and current oceanographical pattern

We assessed the genetic structure of populations of the widely distributed sea cucumber Holothuria (Holothuria) mammata Grube, 1840, and investigated the effects of marine barriers to gene flow and historical processes. Several potential genetic breaks were considered, which would separate the Atlantic and Mediterranean basins, the isolated Macaronesian Islands from the other locations analysed, and the Western Mediterranean and Aegean Sea (Eastern Mediterranean). We analysed mitochondrial 16S and COI gene sequences from 177 individuals from four Atlantic locations and four Mediterranean locations. Haplotype diversity was high (H = 0.9307 for 16S and 0.9203 for COI), and the haplotypes were closely related (p = 0.0058 for 16S and 0.0071 for COI). The lowest genetic diversities were found in the Aegean Sea population. Our results showed that the COI gene was more variable and more useful for the detection of population structure than the 16S gene. The distribution of mtDNA haplotypes, the pairwise FST values and the results of exact tests and AMOVA revealed: (i) a significant genetic break between the population in the Aegean Sea and those in the other locations, as supported by both mitochondrial genes, and (ii) weak differentiation of the Canary and Azores Islands from the other populations; however, the populations from the Macaronesian Islands, Algarve and West Mediterranean could be considered to be a panmictic metapopulation. Isolation by distance was not identified in H. (H.) mammata. Historical events behind the observed findings, together with the current oceanographic patterns, were proposed and discussed as the main factors that determine the population structure and genetic signature of H. (H.) mammata

Salamandra salamandra (Amphibia: Caudata: Salamandridae) in Portugal: not all black and yellow

The fire salamander complex is quite diverse in the Iberian Peninsula where nine subspecies of Salamandra salamandra are currently recognized. Here, we analysed the geographical distribution of the subspecies S. s. gallaica and S. s. crespoi using partial sequences of the mitochondrial cytochrome b gene of 168 individuals from 12 locations in Portugal. Our results support the existence of a deep lineage divergence between the two subspecies, with non-overlapping geographical distributions except in two contact zones: one in Sesimbra on the western coast, and another in Alcoutim on the southeastern border with Spain. Moreover, S. s. crespoi displays signs of gene flow among the sampled locations whereas S. s. gallaica shows evidence of some restriction to gene flow. Present-day genetic make-up of S. s. gallaica and S. s. crespoi is a result of past historical events, fine-tuned by contemporary Iberian geoclimate. Humid mountain areas were found to harbour increased genetic diversity possibly acting as past refugia during drier interglacial periods. To analyse wider geographical patterns and lineage splitting events within S. salamandra we performed a Bayesian dating analysis completing our data set with previously published sequences. The observed divergences were associated to successive biogeographic scenarios, and to other Iberian species showing similar trends.

Phylogeographical history of the white seabream Diplodus sargus (Sparidae): Implications for insularity

Partial sequences of the mitochondrial control region and its comparison with previously published cytochrome b (cyt-b) and microsatellite data were used to investigate the influence of island isolation and connectivity on white seabream genetic structure. To achieve this, a total of 188 individuals from four island localities (Castellamare and Mallorca, Mediterranean Sea; Azores and Canary Islands, Atlantic Ocean) and five coastal localities (Banyuls, Murcia and Tunisia, Mediterranean Sea; Galicia and Faro, Atlantic Ocean) were analysed. Results showed high haplotype diversity and low to moderate nucleotide diversity in all populations (except for the Canary Islands). This pattern of genetic diversity is attributed to a recent population expansion which is corroborated by other results such as cyt-b network and demographic analyses. Low differentiation among Mediterranean/Atlantic and coastal/island groups was shown by the AMOVA and FST values, although a weak phylogeographic break was detected using cyt-b data. However, we found a clear and significant island/ distance effect with regard to the Azores islands. Significant genetic differentiation has been detected between the Azores islands and all other populations. The large geographical distance between the European continental slope and the Azores islands is a barrier to gene flow within this region and historic events such as glaciation could also explain this genetic differentiation.