Rocío Pérez-Portela

Cryptic speciation or global spread? The case of a cosmopolitan marine invertebrate with limited dispersal capabilities

The existence of globally-distributed species with low dispersal capabilities is a paradox that has been explained as a result of human-mediated transport and by hidden diversity in the form of unrecognized cryptic species. Both factors are not mutually exclusive, but relatively few studies have demonstrated the presence of both. Here we analyse the genetic patterns of the colonial ascidian Diplosoma listerianum, a species nowadays distributed globally. The study of a fragment of a mitochondrial gene in localities worldwide revealed the existence of multiple cryptic species. In addition, we found a complex geographic structure and multiple clades occurred in sympatry. One of the species showed strong population structure irrespective of geographical distances, which is coherent with stochastic dispersal linked to human transport. The present study shows the complexity of discerning the role of cryptic diversity from human-driven range shifts worldwide, as well as disentangling the effects of natural and artificial dispersal.

Phylogeny of the families Pyuridae and Styelidae (Stolidobranchiata, Ascidiacea) inferred from mitochondrial and nuclear DNA sequences

The Order Stolidobranchiata comprises the families Pyuridae, Styelidae and Molgulidae. Early molecular data was consistent with monophyly of the Stolidobranchiata and also the Molgulidae. Internal phylogeny and relationships between Styelidae and Pyuridae were inconclusive however. In order to clarify these points we used mitochondrial and nuclear sequences from 31 species of Styelidae and 25 of Pyuridae. Phylogenetic trees recovered the Pyuridae as a monophyletic clade, and their genera appeared as monophyletic with the exception of Pyura. The Styelidae, on the other hand, appeared as a paraphyletic group split into several clades. One of them was formed by solitary oviparous species, of which the Pyuridae were a sister group. A second clade included the colonial genera Botryllus, Botrylloides and Symplegma. The remaining colonial and solitary genera formed several poorly resolved clades. One of the more species genus, Polycarpa, was shown to be polyphyletic, and the species Styela plicata grouped into two genetically distant clades suggesting the existence of two cryptic species. The internal phylogeny of Styelidae has bearings on the origin of coloniality in this family. We suggest to abandon the traditional division of colonial forms into social and compound species and use instead the categories of aggregated colonies that do not have common vascular systems, and integrated colonies, that do possess such systems. Our molecular results indicate that there have been several independent acquisitions of coloniality in the Styelidae, and that viviparity may be a pre-adaptation for a colonial life-style.

Cryptic speciation and genetic structure of widely distributed brittle stars (Ophiuroidea) in Europe

Pérez‐Portela, R., Almada, V. & Turon, X. (2012). Cryptic speciation and genetic structure of widely distributed brittle stars (Ophiuroidea) in Europe. —Zoologica Scripta, 00, 000–000.

Contrasted phylogeographic patterns on mitochondrial DNA of shallow and deep brittle stars across the Atlantic-Mediterranean area

Previous studies on Ophiothrix in European waters demonstrated the existence of two distinct species, Ophiothrix fragilis and Ophiothrix sp. II. Using phylogenetic and species delimitation techniques based on two mitochondrial genes (cytochrome c oxidase I and 16S rRNA) we prove the existence of a new congeneric species (Ophiothrix sp. III), occurring in the deep Atlantic coast of the Iberian Peninsula and the Alboran Sea. We compared phylogeographic patterns of these three Ophiothrix species to test whether closely related species are differentially affected by past demographic events and current oceanographic barriers. We used 432 sequences (137 of O. fragilis, 215 of Ophiothrix sp. II, and 80 of Ophiothrix sp. III) of the 16S rRNA from 23 Atlantic-Mediterranean locations for the analyses. We observed different geographic and bathymetric distributions, and contrasted phylogeography among species. Ophiothrix fragilis appeared genetically isolated between the Atlantic and Mediterranean basins, attributed to past vicariance during Pleistocene glaciations and a secondary contact associated to demographic expansion. This contrasts with the panmixia observed in Ophiothrix sp. II across the Atlantic-Mediterranean area. Results were not conclusive for Ophiothrix sp. III due to the lack of a more complete sampling within the Mediterranean Sea.

Low genetic diversity and recent demographic expansion in the red starfish Echinaster sepositus (Retzius 1816)

Understanding the phylogeography and genetic structure of populations and the processes responsible of patterns therein is crucial for evaluating the vulnerability of marine species and developing management strategies. In this study, we explore how past climatic events and ongoing oceanographic and demographic processes have shaped the genetic structure and diversity of the Atlanto-Mediterranean red starfish Echinaster sepositus. The species is relatively abundant in some areas of the Mediterranean Sea, but some populations have dramatically decreased over recent years due to direct extraction for ornamental aquariums and souvenir industries. Analyses across most of the distribution range of the species based on the mitochondrial cytochrome c oxidase subunit I gene and eight microsatellite loci revealed very low intraspecific genetic diversity. The species showed a weak genetic structure within marine basins despite the a priori low dispersal potential of its lecithotrophic larva. Our results also revealed a very recent demographic expansion across the distribution range of the species. The genetic data presented here indicate that the species might be highly vulnerable, due to its low intraspecific genetic diversity.

Morphological and Genetic Diversity of the Wood-Boring Xylophaga (Mollusca, Bivalvia): New Species and Records from Deep-Sea Iberian Canyons

Deep-sea bivalves of the Xylophagaidae, a poorly known group, are obligate wood-borers. Deployment of wood in three submarine canyons off the Iberian coast, the Blanes and La Fonera Canyons (Mediterranean Sea) and the Avilés Canyon (Cantabric Sea, Bay of Biscay), lead to the discovery of four xylophagaid species in our samples. Xylophaga dorsalis (the dominant species), X. atlantica, X. cf. anselli and the new species X. brava, were identified on the basis of morphological data, and supported by a phylogenetic reconstruction based on the nuclear genes 18S rDNA and 28S rDNA and including several genus of Xylophagaidae. Genetic divergence between species of Xylophaga varied between genes, ranging from 0.5 to 4.0% for the 18SrDNA and from 4.1 to 16.6% for the 28SrDNA. Xylophaga brava sp. nov. appeared to be restricted to the Mediterranean and morphologically resembled the closely related X. cf. anselli from the Cantabrian Sea. However, they clearly diverged in two well-supported clades. Low levels of intraspecific variability and higher interspecific divergence between species also supported the existence of these two different species. Morphologically they differ in the number of cirri at the siphon openings, in the shape of the posterior shell and in the size of prodissoconch II. The new species is characterized by having weak, poorly mineralized mesoplax and siphons united throughout, covered by a periostracal, non-calcified tube; distinct proximal and distal siphons, the former translucent and soft, the latter muscular, with concentric rings. Xylophaga atlantica, previously known only from the western Atlantic, is reported for the first time in the Mediterranean Sea. Whether its presence in the Mediterranean indicates its natural distribution or reflects its recent introduction is unknown. Although xylophagaids have been previously reported to recruit heavily to wood deposited on the seabed, these four species colonized wood suspended 30 m above the seafloor.

Optimizing preservation protocols to extract high‐quality RNA from different tissues of echinoderms for next‐generation sequencing

Transcriptomic information provides fundamental insights into biological processes. Extraction of quality RNA is a challenging step, and preservation and extraction protocols need to be adjusted in many cases. Our objectives were to optimize preservation protocols for isolation of high‐quality RNA from diverse echinoderm tissues and to compare the utility of parameters as absorbance ratios and RIN values to assess RNA quality. Three different tissues (gonad, oesophagus and coelomocytes) were selected from the sea urchin Arbacia lixula. Solid tissues were flash‐frozen and stored at −80 °C until processed. Four preservation treatments were applied to coelomocytes: flash freezing and storage at −80 °C, RNAlater and storage at −20 °C, preservation in TRIzol reagent and storage at −80 °C and direct extraction with TRIzol from fresh cells. Extractions of total RNA were performed with a modified TRIzol protocol for all tissues. Our results showed high values of RNA quantity and quality for all tissues, showing nonsignificant differences among them. However, while flash freezing was effective for solid tissues, it was inadequate for coelomocytes because of the low quality of the RNA extractions. Coelomocytes preserved in RNAlater displayed large variability in RNA integrity and insufficient RNA amount for further isolation of mRNA. TRIzol was the most efficient system for stabilizing RNA which resulted on high RNA quality and quantity. We did not detect correlation between absorbance ratios and RNA integrity. The best strategies for assessing RNA integrity was the visualization of 18S rRNA and 28S rRNA bands in agarose gels and estimation of RIN values with Agilent Bioanalyzer chips.

Characterization of thirty two microsatellite loci for three Atlanto-Mediterranean echinoderm species

Thirty two microsatellites were optimized from 454 pyrosequencing libraries for three Atlanto-Mediterranean echinoderms: Coscinasterias tenuispina, Echinaster sepositus and Arbacia lixula. We observed different frequency of microsatellite types (di-, tri-, tetra- and penta-nucleotide) throughout the genome of the species, but no significant differences were observed in allele richness among different microsatellite repeats. No loci showed linkage disequilibrium. Heterozygosity deficit and departure from Hardy–Weinberg equilibrium were observed for some loci, in two species, probably due to high levels of inbreeding. Heterozygosity excess observed in C. tenuispina could be explained by selection against homozygotes and/or outcrossing.

Long telomeres are associated with clonality in wild populations of the fissiparous starfish Coscinasterias tenuispina

Telomeres usually shorten during an organism’s lifespan and have thus been used as an aging and health marker. When telomeres become sufficiently short, senescence is induced. The most common method of restoring telomere length is via telomerase reverse transcriptase activity, highly expressed during embryogenesis. However, although asexual reproduction from adult tissues has an important role in the life cycles of certain species, its effect on the aging and fitness of wild populations, as well as its implications for the long-term survival of populations with limited genetic variation, is largely unknown. Here we compare relative telomere length of 58 individuals from four populations of the asexually reproducing starfish Coscinasterias tenuispina. Additionally, 12 individuals were used to compare telomere lengths in regenerating and non-regenerating arms, in two different tissues (tube feet and pyloric cecum). The level of clonality was assessed by genotyping the populations based on 12 specific microsatellite loci and relative telomere length was measured via quantitative PCR. The results revealed significantly longer telomeres in Mediterranean populations than Atlantic ones as demonstrated by the Kruskal–Wallis test (K=24.17, significant value: P-value<0.001), with the former also characterized by higher levels of clonality derived from asexual reproduction. Telomeres were furthermore significantly longer in regenerating arms than in non-regenerating arms within individuals (pyloric cecum tissue: Mann–Whitney test, V=299, P-value<10−6; and tube feet tissue Students t=2.28, P-value=0.029). Our study suggests that one of the mechanisms responsible for the long-term somatic maintenance and persistence of clonal populations is telomere elongation.

Optimization of 14 microsatellite loci in a Mediterranean demosponge subjected to population decimation, Ircinia fasciculata

AbstractThe recovery potential of decimated populations of sponges will largely hinge on their populations’ size retrieval and their connectivity with conspecifics in unaffected locations. Here, we report on the development of microsatellite markers for estimation of the population connectivity and bottleneck and inbreeding signals in a Mediterranean sponge suffering from disease outbreaks, Ircinia fasciculata. From the 220,876 sequences obtained by genomic pyrosequencing, we isolated 14 polymorphic microsatellite loci and assessed the allelic variation of loci in 24 individuals from 2 populations in the Northwestern Mediterranean. The allele number per locus ranged from 3 to 11, observed heterozygosity from 0.68 to 0.73, and expected heterozygosity from 0.667 to 0.68. No significant linkage disequilibrium between pairs of loci was detected. The 14 markers developed here will be valuable tools for conservation strategies across the distributional range of this species allowing the detection of populations with large genetic diversity loss and high levels of inbreeding.