Chromosome-level genome assembly and annotation of the black sea urchin Arbacia lixula (Linnaeus, 1758).

The black sea urchin (Arbacia lixula) is a keystone species inhabiting the coastal shallow waters of the Mediterranean Sea, which is a key driver of littoral communities' structure. Here, we present the first genome assembly and annotation of this species, standing as the first Arbacioida genome, including both nuclear and mitochondrial genomes. To obtain a chromosome-level assembly, we used a combination of PacBio high fidelity (HiFi) reads and chromatin capture reads (Omni-C). In addition, we generated a high-quality nuclear annotation of both coding and non-coding genes, by using published RNA-Seq data from several individuals of A. lixula and gene models from closely related species. The nuclear genome assembly has a total span of 607.91 Mb, being consistent with its experimentally estimated genome size. The assembly contains 22 chromosome-scale scaffolds (96.52% of the total length), which coincides with its known karyotype. A total of 72,767 transcripts were predicted from the nuclear genome, 24,171 coding, and 48,596 non-coding that included lncRNA, snoRNA, and tRNAs. The circularized mitochondrial genome had 15740 bp comprising 13 protein-coding genes, 2 rRNA, and 22 tRNA. This reference genome will enhance ongoing A. lixula studies and benefit the wider sea urchin scientific community.

Genomic signatures suggesting adaptation to ocean acidification in a coral holobiont from volcanic CO2 seeps.

Ocean acidification, caused by anthropogenic CO2 emissions, is predicted to have major consequences for reef-building corals, jeopardizing the scaffolding of the most biodiverse marine habitats. However, whether corals can adapt to ocean acidification and how remains unclear. We addressed these questions by re-examining transcriptome and genome data of Acropora millepora coral holobionts from volcanic CO2 seeps with end-of-century pH levels. We show that adaptation to ocean acidification is a wholistic process involving the three main compartments of the coral holobiont. We identified 441 coral host candidate adaptive genes involved in calcification, response to acidification, and symbiosis; population genetic differentiation in dinoflagellate photosymbionts; and consistent transcriptional microbiome activity despite microbial community shifts. Coral holobionts from natural analogues to future ocean conditions harbor beneficial genetic variants with far-reaching rapid adaptation potential. In the face of climate change, these populations require immediate conservation strategies as they could become key to coral reef survival.

Exceptional population genomic homogeneity in the black brittle star Ophiocomina nigra (Ophiuroidea, Echinodermata) along the Atlantic-Mediterranean coast.

The Atlantic-Mediterranean marine transition is characterised by strong oceanographic barriers and steep environmental gradients that generally result in connectivity breaks between populations from both basins and may lead to local adaptation. Here, we performed a population genomic study of the black brittle star, Ophiocomina nigra, covering most of its distribution range along the Atlantic-Mediterranean region. Interestingly, O. nigra is extremely variable in its coloration, with individuals ranging from black to yellow-orange, and different colour morphs inhabiting different depths and habitats. In this work, we used a fragment of the mitochondrial COI gene and 2,374 genome-wide ddRADseq-derived SNPs to explore: (a) whether the different colour morphs of O. nigra represent different evolutionary units; (b) the disruptive effects of major oceanographic fronts on its population structure; and (c) genomic signals of local adaptation to divergent environments. Our results revealed exceptional population homogeneity, barely affected by oceanographic fronts, with no signals of local adaptation nor genetic differentiation between colour morphs. This remarkable panmixia likely results from a long pelagic larval duration, a large effective population size and recent demographic expansions. Our study unveils an extraordinary phenotypic plasticity in O. nigra, opening further research questions on the ecological and molecular mechanisms underpinning coloration in Ophiuroidea.

High taxonomic diversity and miniaturization in benthic communities under persistent natural CO2 disturbances.

Metabarcoding techniques have revolutionized ecological research in recent years, facilitating the differentiation of cryptic species and revealing previously hidden diversity. In the current scenario of climate change and ocean acidification, biodiversity loss is one of the main threats to marine ecosystems. Here, we explored the effects of ocean acidification on marine benthic communities using DNA metabarcoding to assess the diversity of algae and metazoans. Specifically, we examined the natural pH gradient generated by the Fuencaliente CO2 vent system, located near La Palma Island (Canary Islands). High-resolution COI metabarcoding analyses revealed high levels of taxonomic diversity in an acidified natural area for the first time. This high number of species arises from the detection of small and cryptic species that were previously undetectable by other techniques. Such species are apparently tolerant to the acidification levels expected in future oceans. Hence and following our results, future subtropical communities are expected to keep high biodiversity values under an acidification scenario, although they will tend toward overall miniaturization due to the dominance of small algal and invertebrate species, leading to changes in ecosystem functions.

Genetic homogeneity, lack of larvae recruitment, and clonality in absence of females across western Mediterranean populations of the starfish Coscinasterias tenuispina.

We here analysed the populations' genetic structure of Coscinasterias tenuispina, an Atlantic-Mediterranean fissiparous starfish, focusing on the western Mediterranean, to investigate: the distribution and prevalence of genetic variants, the relative importance of asexual reproduction, connectivity across the Atlantic-Mediterranean transition, and the potential recent colonisation of the Mediterranean Sea. Individuals from 11 Atlantic-Mediterranean populations of a previous study added to 172 new samples from five new W Mediterranean sites. Individuals were genotyped at 12 microsatellite loci and their gonads histologically analysed for sex determination. Additionally, four populations were genotyped at two-time points. Results demonstrated genetic homogeneity and low clonal richness within the W Mediterranean, due to the dominance of a superclone, but large genetic divergence with adjacent areas. The lack of new genotypes recruitment over time, and the absence of females, confirmed that W Mediterranean populations were exclusively maintained by fission and reinforced the idea of its recent colonization. The existence of different environmental conditions among basins and/or density-depend processes could explain this lack of recruitment from distant areas. The positive correlation between clonal richness and heterozygote excess suggests that most genetic diversity is retained within individuals in the form of heterozygosity in clonal populations, which might increase their resilience.

Enjoying the warming Mediterranean: Transcriptomic responses to temperature changes of a thermophilous keystone species in benthic communities.

Information about the genomic processes underlying responses to temperature changes is still limited in non-model marine invertebrates. In this sense, transcriptomic analyses can help to identify genes potentially related to thermal responses. We here investigated, via RNA-seq, whole-transcriptomic responses to increased and decreased temperatures in a thermophilous keystone sea urchin, Arbacia lixula, whose populations are increasing in the Mediterranean. This species is a key driver of benthic communities' structure due to its grazing activity. We found a strong response to experimentally induced cold temperature (7°C), with 1,181 differentially expressed transcripts relative to the control condition (13°C), compared to only 179 in the warm (22°C) treatment. A total of 84 (cold treatment) and three (warm treatment) gene ontology terms were linked to the differentially expressed transcripts. At 7°C the expression of genes encoding different heat shock proteins (HSPs) was upregulated, together with apoptotic suppressor genes (e.g., Bcl2), genes involved in the infection response and/or pathogen-recognition (e.g., echinoidin) and ATP-associated genes, while protein biosynthesis and DNA replication pathways were downregulated. At 22°C neither HSPs induction nor activation of the previously mentioned pathways were detected, with the exception of some apoptotic-related activities that were upregulated. Our results suggest a strong transcriptional response associated with low temperatures, and support the idea of low water temperature being a major limitation for A. lixula expansion across deep Mediterranean and northern Atlantic waters.

Fine-scale structure among mesophotic populations of the great star coral Montastraea cavernosa revealed by SNP genotyping.

Mesophotic reefs (30-150 m) have been proposed as potential refugia that facilitate the recovery of degraded shallow reefs following acute disturbances such as coral bleaching and disease. However, because of the technical difficulty of collecting samples, the connectivity of adjacent mesophotic reefs is relatively unknown compared with shallower counterparts. We used genotyping by sequencing to assess fine-scale genetic structure of Montastraea cavernosa at two sites at Pulley Ridge, a mesophotic coral reef ecosystem in the Gulf of Mexico, and downstream sites along the Florida Reef Tract. We found differentiation between reefs at Pulley Ridge (~68 m) and corals at downstream upper mesophotic depths in the Dry Tortugas (28-36 m) and shallow reefs in the northern Florida Keys (Key Biscayne, ~5 m). The spatial endpoints of our study were distinct, with the Dry Tortugas as a genetic intermediate. Most striking were differences in population structure among northern and southern sites at Pulley Ridge that were separated by just 12km. Unique patterns of clonality and outlier loci allele frequency support these sites as different populations and suggest that the long-distance horizontal connectivity typical of shallow-water corals may not be typical for mesophotic systems in Florida and the Gulf of Mexico. We hypothesize that this may be due to the spawning of buoyant gametes, which commits propagules to the surface, resulting in greater dispersal and lower connectivity than typically found between nearby shallow sites. Differences in population structure over small spatial scales suggest that demographic constraints and/or environmental disturbances may be more variable in space and time on mesophotic reefs compared with their shallow-water counterparts.

Correction: Digging the diversity of Iberian bait worms Marphysa (Annelida, Eunicidae).

[This corrects the article DOI: 10.1371/journal.pone.0226749.].

Digging the diversity of Iberian bait worms Marphysa (Annelida, Eunicidae).

During a visit to polychaete-rearing facilities in the vicinity of Bay of Cádiz (SW Iberian Peninsula, Atlantic Ocean), we sampled two populations of Marphysa (Annelida, Eunicidae) originally occurring at nearby intertidal soft bottoms, one being more than twice as long as the other at the same age. We analysed them using partial sequences of two mitochondrial genes, 16S rDNA and Cytochrome Oxidase I, and classical morphological observations. Our molecular results confirmed that the two populations corresponded to two different species, with PTP species delimitation values ranging from 0.973 (long-bodied species) to 0.999 (short-bodied species). Morphologically, the short-bodied species resembles the recently redescribed M. sanguinea (Montagu, 1813), but differs mainly in having some parapodia with two subacicular hooks (one bidentate and one unidentate) and three types of pectinate chaetae, Two isodont present all along the body, and one particularly large anodont asymmetric appearing only from mid-posterior parapodia. The long-bodied species resembles Marphysa aegypti Elgetany, El-Ghobashy, Ghoneim and Struck, 2018 both in size and in having very robust, unidentate subacicular hooks (single in most parapodia, two-both similar in size and form-in some posterior parapodia), but differs, among other features, in the maxillary formula, the number of acicula per parapodia and the number and shape of pectinate chaetae. Accordingly, we are here fully illustrating and formally describing the two Iberian populations as Marphysa gaditana sp. nov. (short-bodied) and Marphysa chirigota sp. nov. (long-bodied) and we are emending the description of M. aegypti based on our revision of the type material. Also, we discuss on the distribution of the species of the sanguinea-group and on the relevancy of taxonomically robust studies when dealing with species of commercial interest having the potential of being globally spread through human activities, as well as on the misunderstandings caused by the incorrect use of the "cosmopolitan species" concept.

Genetic diversity, connectivity and gene flow along the distribution of the emblematic Atlanto-Mediterranean sponge Petrosia ficiformis (Haplosclerida, Demospongiae).

BACKGROUND: Knowledge about the distribution of the genetic variation of marine species is fundamental to address species conservation and management strategies, especially in scenarios with mass mortalities. In the Mediterranean Sea, Petrosia ficiformis is one of the species most affected by temperature-related diseases. Our study aimed to assess its genetic structure, connectivity, and bottleneck signatures to understand its evolutionary history and to provide information to help design conservation strategies of sessile marine invertebrates. RESULTS: We genotyped 280 individuals from 19 locations across the entire distribution range of P. ficiformis in the Atlanto-Mediterranean region at 10 microsatellite loci. High levels of inbreeding were detected in most locations (especially in the Macaronesia and the Western Mediterranean) and bottleneck signatures were only detected in Mediterranean populations, although not coinciding entirely with those with reported die-offs. We detected strong significant population differentiation, with the Atlantic populations being the most genetically isolated, and show that six clusters explained the genetic structure along the distribution range of this sponge. Although we detected a pattern of isolation by distance in P. ficiformis when all locations were analyzed together, stratified Mantel tests revealed that other factors could be playing a more prominent role than isolation by distance. Indeed, we detected a strong effect of oceanographic barriers impeding the gene flow among certain areas, the strongest one being the Almeria-Oran front, hampering gene flow between the Atlantic Ocean and the Mediterranean Sea. Finally, migration and genetic diversity distribution analyses suggest a Mediterranean origin for the species. CONCLUSIONS: In our study Petrosia ficiformis showed extreme levels of inbreeding and population differentiation, which could all be linked to the poor swimming abilities of the larva. However, the observed moderate migration patterns are highly difficult to reconcile with such poor larval dispersal, and suggest that, although unlikely, dispersal may also be achieved in the gamete phase. Overall, because of the high genetic diversity in the Eastern Mediterranean and frequent mass mortalities in the Western Mediterranean, we suggest that conservation efforts should be carried out specifically in those areas of the Mediterranean to safeguard the genetic diversity of the species.

Spatio-temporal patterns of genetic variation in Arbacia lixula, a thermophilous sea urchin in expansion in the Mediterranean.

The genetic structure of 13 populations of the amphiatlantic sea urchin Arbacia lixula, as well as temporal genetic changes in three of these localities, were assessed using ten hypervariable microsatellite loci. This thermophilous sea urchin is an important engineer species triggering the formation of barren grounds through its grazing activity. Its abundance seems to be increasing in most parts of the Mediterranean, probably favoured by warming conditions. Significant genetic differentiation was found both spatially and temporally. The main break corresponded to the separation of western Atlantic populations from those in eastern Atlantic and the Mediterranean Sea. A less marked, but significant differentiation was also found between Macaronesia (eastern Atlantic) and the Mediterranean. In the latter area, a signal of differentiation between the transitional area (Alboran Sea) and the rest of the Mediterranean was detected. However, no genetic structure is found within the Mediterranean (excluding Alboran) across the Siculo-Tunisian Strait, resulting from either enough gene flow to homogenize distance areas or/and a recent evolutionary history marked by demographic expansion in this basin. Genetic temporal variation at the Alboran Sea is as important as spatial variation, suggesting that temporal changes in hydrological features can affect the genetic composition of the populations. A picture of genetic homogeneity in the Mediterranean emerges, implying that the potential expansion of this keystone species will not be limited by intraspecific genetic features and/or potential impact of postulated barriers to gene flow in the region.

Intraspecific genetic structure, divergence and high rates of clonality in an amphi-Atlantic starfish.

Intraspecific genetic diversity and divergence have a large influence on the adaption and evolutionary potential of species. The widely distributed starfish, Coscinasterias tenuispina, combines sexual reproduction with asexual reproduction via fission. Here we analyse the phylogeography of this starfish to reveal historical and contemporary processes driving its intraspecific genetic divergence. We further consider whether asexual reproduction is the most important method of propagation throughout the distribution range of this species. Our study included 326 individuals from 16 populations, covering most of the species' distribution range. A total of 12 nuclear microsatellite loci and sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene were analysed. COI and microsatellites were clustered in two isolated lineages: one found along the southwestern Atlantic and the other along the northeastern Atlantic and Mediterranean Sea. This suggests the existence of two different evolutionary units. Marine barriers along the European coast would be responsible for population clustering: the Almeria-Oran Front that limits the entrance of migrants from the Atlantic to the Mediterranean, and the Siculo-Tunisian strait that divides the two Mediterranean basins. The presence of identical genotypes was detected in all populations, although two monoclonal populations were found in two sites where annual mean temperatures and minimum values were the lowest. Our results based on microsatellite loci showed that intrapopulation genetic diversity was significantly affected by clonality whereas it had lower effect for the global phylogeography of the species, although still some impact on populations' genetic divergence could be observed between some populations.

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.

Natural or naturalized? Phylogeography suggests that the abundant sea urchin Arbacia lixula is a recent colonizer of the Mediterranean.

We present the global phylogeography of the black sea urchin Arbacia lixula, an amphi-Atlantic echinoid with potential to strongly impact shallow rocky ecosystems. Sequences of the mitochondrial cytochrome c oxidase gene of 604 specimens from 24 localities were obtained, covering most of the distribution area of the species, including the Mediterranean and both shores of the Atlantic. Genetic diversity measures, phylogeographic patterns, demographic parameters and population differentiation were analysed. We found high haplotype diversity but relatively low nucleotide diversity, with 176 haplotypes grouped within three haplogroups: one is shared between Eastern Atlantic (including Mediterranean) and Brazilian populations, the second is found in Eastern Atlantic and the Mediterranean and the third is exclusively from Brazil. Significant genetic differentiation was found between Brazilian, Eastern Atlantic and Mediterranean regions, but no differentiation was found among Mediterranean sub-basins or among Eastern Atlantic sub-regions. The star-shaped topology of the haplotype network and the unimodal mismatch distributions of Mediterranean and Eastern Atlantic samples suggest that these populations have suffered very recent demographic expansions. These expansions could be dated 94-205 kya in the Mediterranean, and 31-67 kya in the Eastern Atlantic. In contrast, Brazilian populations did not show any signature of population expansion. Our results indicate that all populations of A. lixula constitute a single species. The Brazilian populations probably diverged from an Eastern Atlantic stock. The present-day genetic structure of the species in Eastern Atlantic and the Mediterranean is shaped by very recent demographic processes. Our results support the view (backed by the lack of fossil record) that A. lixula is a recent thermophilous colonizer which spread throughout the Mediterranean during a warm period of the Pleistocene, probably during the last interglacial. Implications for the possible future impact of A. lixula on shallow Mediterranean ecosystems in the context of global warming trends must be considered.

Cryptic divergence and strong population structure in the colonial invertebrate Pycnoclavella communis (Ascidiacea) inferred from molecular data.

We studied sequence variation in the mitochondrial gene cytochrome c oxidase subunit I (COI) for 135 individuals from eight Mediterranean populations of the colonial ascidian Pycnoclavella communis across most of its presently known range of distribution in the Mediterranean. Three haplotypes from Atlantic locations were also included in the study. Phylogenetic, phylogeographic and population genetic analyses were used to unravel the genetic variability within and between populations. The study revealed 32 haplotypes for COI, 29 of them grouped within two Mediterranean lineages of P. communis (mean nucleotide divergence between lineages was 8.55%). Phylogenetic and network analyses suggest the possible existence of cryptic species corresponding to these two lineages. Population genetic analyses were restricted to the five populations belonging to the main genetic lineage, and for these localities we compared the information gleaned from COI sequence data and from eight microsatellite loci. A high genetic divergence between populations was substantiated using both kinds of markers (COI, global Fst=0.343; microsatellite loci, global Fst=0.362). There were high numbers of private haplotypes (COI) and alleles (microsatellites) in the populations studied. Restricted gene flow and inbreeding occur in the present range of distribution of the species. Microsatellite loci showed a strong incidence of failed amplifications, which we attribute to the marked intraspecies variability that hampered the application of these highly specific markers. Our results show important genetic variability at all levels studied, from within populations to between basins, possibly coupled to speciation processes. This variability is attributable to restricted gene flow among populations due to short-distance dispersal of the larvae.