Scientific history, biogeography, and biological traits predict presence of cryptic or overlooked species.

Genetic data show that many nominal species are composed of more than one biological species, and thus contain cryptic species in the broad sense (including overlooked species). When ignored, cryptic species generate confusion which, beyond biodiversity or vulnerability underestimation, blurs our understanding of ecological and evolutionary processes and may impact the soundness of decisions in conservation or medicine. However, very few hypotheses have been tested about factors that predispose a taxon to contain cryptic or overlooked species. To fill this gap, we surveyed the literature on free-living marine metazoans and built two data sets, one of 187,603 nominal species and another of 83 classes or phyla, to test several hypotheses, correcting for sequence data availability, taxon size and phylogenetic relatedness. We found a strong effect of scientific history: the probability of a taxon containing cryptic species was highest for the earliest described species and varied among time periods potentially consistently with an influence of prevailing scientific theories. The probability of cryptic species being present was also increased for species with large distribution ranges. They were more frequent in the north polar and south polar zones, contradicting previous predictions of more cryptic species in the tropics, and supporting the hypothesis that many cryptic species diverged recently. The number of cryptic species varied among classes, with an excess in hydrozoans and polychaetes, and a deficit in actinopterygians, for example, but precise class ranking was relatively sensitive to the statistical model used. For all models, biological traits, rather than phylum, appeared responsible for the variation among classes: there were fewer cryptic species than expected in classes with hard skeletons (perhaps because they provide good characters for taxonomy) and image-forming vision (in which selection against heterospecific mating may enhance morphological divergence), and more in classes with internal fertilisation. We estimate that among marine free-living metazoans, several thousand additional cryptic species complexes could be identified as more sequence data become available. The factors identified as important for marine animal cryptic species are likely important for other biomes and taxa and should aid many areas in biology that rely on accurate species identification.

Spineless and overlooked: DNA metabarcoding of autonomous reef monitoring structures reveals intra- and interspecific genetic diversity in Mediterranean invertebrates.

The ability to gather genetic information using DNA metabarcoding of bulk samples obtained directly from the environment is crucial to determine biodiversity baselines and understand population dynamics in the marine realm. While DNA metabarcoding is effective in evaluating biodiversity at community level, genetic patterns within species are often concealed in metabarcoding studies and overlooked for marine invertebrates. In the present study, we implement recently developed bioinformatics tools to investigate intraspecific genetic variability for invertebrate taxa in the Mediterranean Sea. Using metabarcoding samples from Autonomous Reef Monitoring Structures (ARMS) deployed in three locations, we present haplotypes and diversity estimates for 145 unique species. While overall genetic diversity was low, we identified several species with high diversity records and potential cryptic lineages. Further, we emphasize the spatial scale of genetic variability, which was observed from locations to individual sampling units (ARMS). We carried out a population genetic analysis of several important yet understudied species, which highlights the current knowledge gap concerning intraspecific genetic patterns for the target taxa in the Mediterranean basin. Our approach considerably enhances biodiversity monitoring of charismatic and understudied Mediterranean species, which can be incorporated into ARMS surveys.

Be positive: customized reference databases and new, local barcodes balance false taxonomic assignments in metabarcoding studies.

Background: In metabarcoding analyses, the taxonomic assignment is crucial to place sequencing data in biological and ecological contexts. This fundamental step depends on a reference database, which should have a good taxonomic coverage to avoid unassigned sequences. However, this goal is rarely achieved in many geographic regions and for several taxonomic groups. On the other hand, more is not necessarily better, as sequences in reference databases belonging to taxonomic groups out of the studied region/environment context might lead to false assignments. Methods: We investigated the effect of using several subsets of a cytochrome c oxidase subunit I (COI) reference database on taxonomic assignment. Published metabarcoding sequences from the Mediterranean Sea were assigned to taxa using COInr, which is a comprehensive, non-redundant and recent database of COI sequences obtained both from BOLD and NCBI, and two of its subsets: (i) all sequences except insects (COInr-WO-Insecta), which represent the overwhelming majority of COInr database, but are irrelevant for marine samples, and (ii) all sequences from taxonomic families present in the Mediterranean Sea (COInr-Med). Four different algorithms for taxonomic assignment were employed in parallel to evaluate differences in their output and data consistency. Results: The reduction of the database to more specific custom subsets increased the number of unassigned sequences. Nevertheless, since most of them were incorrectly assigned by the less specific databases, this is a positive outcome. Moreover, the taxonomic resolution (the lowest taxonomic level to which a sequence is attributed) of several sequences tended to increase when using customized databases. These findings clearly indicated the need for customized databases adapted to each study. However, the very high proportion of unassigned sequences points to the need to enrich the local database with new barcodes specifically obtained from the studied region and/or taxonomic group. Including novel local barcodes to the COI database proved to be very profitable: by adding only 116 new barcodes sequenced in our laboratory, thus increasing the reference database by only 0.04%, we were able to improve the resolution for ca. 0.6-1% of the Amplicon Sequence Variants (ASVs).

Spatial coalescent connectivity through multi-generation dispersal modelling predicts gene flow across marine phyla.

Gene flow governs the contemporary spatial structure and dynamic of populations as well as their long-term evolution. For species that disperse using atmospheric or oceanic flows, biophysical models allow predicting the migratory component of gene flow, which facilitates the interpretation of broad-scale spatial structure inferred from observed allele frequencies among populations. However, frequent mismatches between dispersal estimates and observed genetic diversity prevent an operational synthesis for eco-evolutionary projections. Here we use an extensive compilation of 58 population genetic studies of 47 phylogenetically divergent marine sedentary species over the Mediterranean basin to assess how genetic differentiation is predicted by Isolation-By-Distance, single-generation dispersal and multi-generation dispersal models. Unlike previous approaches, the latter unveil explicit parents-to-offspring links (filial connectivity) and implicit links among siblings from a common ancestor (coalescent connectivity). We find that almost 70 % of observed variance in genetic differentiation is explained by coalescent connectivity over multiple generations, significantly outperforming other models. Our results offer great promises to untangle the eco-evolutionary forces that shape sedentary population structure and to anticipate climate-driven redistributions, altogether improving spatial conservation planning.

Pan-regional marine benthic cryptobiome biodiversity patterns revealed by metabarcoding Autonomous Reef Monitoring Structures.

Autonomous Reef Monitoring Structures (ARMS) have been applied worldwide to characterize the critical yet frequently overlooked biodiversity patterns of marine benthic organisms. In order to disentangle the relevance of environmental factors in benthic patterns, here, through standardized metabarcoding protocols, we analyse sessile and mobile (<2 mm) organisms collected using ARMS deployed across six regions with different environmental conditions (3 sites × 3 replicates per region): Baltic, Western Mediterranean, Adriatic, Black and Red Seas, and the Bay of Biscay. A total of 27,473 Amplicon Sequence Variants (ASVs) were observed ranging from 1,404 in the Black Sea to 9,958 in the Red Sea. No ASVs were shared among all regions. The highest number of shared ASVs was between the Western Mediterranean and the Adriatic Sea (116) and Bay of Biscay (115). Relatively high numbers of ASVs (103), mostly associated with the genus Amphibalanus, were also shared between the lower salinity seas (Baltic and Black Seas). We found that compositional differences in spatial patterns of rocky-shore benthos are determined slightly more by dispersal limitation than environmental filtering. Dispersal limitation was similar between sessile and mobile groups, while the sessile group had a larger environmental niche breadth than the mobile group. Further, our study can provide a foundation for future evaluations of biodiversity patterns in the cryptobiome, which can contribute up to 70% of the local biodiversity.

Global invasion genetics of two parasitic copepods infecting marine bivalves.

Invasive species, and especially invasive parasites, represent excellent models to study ecological and evolutionary mechanisms in the wild. To understand these processes, it is crucial to obtain more knowledge on the native range, invasion routes and invasion history of invasive parasites. We investigated the consecutive invasions of two parasitic copepods (Mytilicola intestinalis and Mytilicola orientalis) by combining an extensive literature survey covering the reported putative native regions and the present-day invaded regions with a global phylogeography of both species. The population genetic analyses based on partial COI sequences revealed significant population differentiation for M. orientalis within the native region in Japan, while introduced populations in North America and Europe could not be distinguished from the native ones. Thus, M. orientalis' invasion history resembles the genetic structure and recent spread of its principal host, the Pacific oyster, Crassostrea gigas, while M. intestinalis lacks population genetic structure and has an overall low genetic diversity. Therefore, the native origin of M. intestinalis remains unclear. With this study, we demonstrate that even highly related and biologically similar invasive species can differ in their invasion genetics. From this, we conclude that extrapolating invasion genetics dynamics from related invasive taxa may not always be possible.

From seascape ecology to population genomics and back. Spatial and ecological differentiation among cryptic species of the red algae Lithophyllum stictiforme/L. cabiochiae, main bioconstructors of coralligenous habitats.

Ecosystem engineering species alter the physical structure of their environment and can create or modify habitats, having a massive impact on local biodiversity. Coralligenous reefs are highly diverse habitats endemic to the Mediterranean Sea built by calcareous benthic organisms among which Crustose Coralline Algae are the main engineering species. We analyzed the diversity of Lithophyllum stictiforme or L. cabiochiae in coralligenous habitats combining a multiple barcode and a population genomics approach with seascape features. Population genomics allowed disentangling pure spatial effects from environmental effects. We found that these taxa form a complex of eight highly divergent cryptic species that are easily identifiable using classic barcode markers (psbA, LSU, COI). Three factors have a significant effect on the relative abundances of these cryptic species: the location along the French Mediterranean coast, depth and Photosynthetic Active Radiation (PAR). The analysis of around 5000 SNPs for the most abundant species revealed genetic differentiation among localities in the Bay of Marseille but no differentiation between depths within locality. Thus, the effect of depth and PAR on cryptic species communities is not a consequence of restricted connectivity but rather due to differential settlement or survival among cryptic species. This differential is more likely driven by irradiance levels rather than by pressure or temperature. Both the genetic and species diversity patterns are congruent with the main patterns of currents in the Bay. Ecological differentiation among these engineering cryptic species, sensitive to ocean warming and acidification, could have important consequences on the diversity and structure of the coralligenous communities.

Lessons from photo analyses of Autonomous Reef Monitoring Structures as tools to detect (bio-)geographical, spatial, and environmental effects.

We investigated the validity of Autonomous Reef Monitoring Structures (ARMS) as monitoring tools for hard bottoms across a wide geographic and environmental range. We deployed 36 ARMS in the northeast Atlantic, northwest Mediterranean, Adriatic and Red Sea at 7-17 m depth. After 12-16 months, community composition was inferred from photographs, in six plate-faces for each ARMS. Overall, we found a highly significant effect of sea region, site (within seas), and plate-face on community composition. Plate-faces thus represent distinct micro-habitats and provide pseudo-replicates, increasing statistical power. Within each sea region taken individually, there was also a highly significant effect of site and plate-face. Because strong effects were obtained despite the fusion of taxonomic categories at high taxonomic ranks (to ensure comparability among biogeographic provinces), ARMS photo-analysis appears a promising monitoring tool for each sea region. We recommend keeping three ARMS per site and analyzing more numerous sites within a sea region to investigate environmental effects.

Species delimitation in the presence of strong incomplete lineage sorting and hybridization: Lessons from Ophioderma (Ophiuroidea: Echinodermata).

Accurate species delimitation is essential to properly assess biodiversity, but also for management and conservation purposes. Yet, it is not always trivial to accurately define species boundaries in closely related species due to incomplete lineage sorting. Additional difficulties may be caused by hybridization, now evidenced as a frequent phenomenon. The brittle star cryptic species complex Ophioderma longicauda encompasses six mitochondrial lineages, including broadcast spawners and internal brooders, yet the actual species boundaries are unknown. Here, we combined three methods to delimit species in the Ophioderma longicauda complex and to infer its divergence history: (i) unsupervised species discovery based on multilocus genotypes; (ii) divergence time estimation using the multi-species coalescent; (iii) divergence scenario testing (including gene flow) using Approximate Bayesian Computation (ABC) methods. 30 sequence markers (transcriptome-based, mitochondrial or non-coding) for 89 O. longicauda and outgroup individuals were used. First, multivariate analyses revealed six genetic clusters, which globally corresponded to the mitochondrial lineages, yet with many exceptions, suggesting ancient hybridization events and challenging traditional mitochondrial barcoding approaches. Second, multi-species coalescent-based analyses confirmed the occurrence of six species and provided divergence time estimates, but the sole use of this method failed to accurately delimit species, highlighting the power of multilocus genotype clustering to delimit recently diverged species. Finally, Approximate Bayesian Computation showed that the most likely scenario involves hybridization between brooders and broadcasters. Our study shows that despite strong incomplete lineage sorting and past hybridization, accurate species delimitation in Ophioderma was possible using a combination of complementary methods. We propose that these methods, especially multilocus genotype clustering, may be useful to resolve other complex speciation histories.

A comparative analysis of metabarcoding and morphology-based identification of benthic communities across different regional seas.

In a world of declining biodiversity, monitoring is becoming crucial. Molecular methods, such as metabarcoding, have the potential to rapidly expand our knowledge of biodiversity, supporting assessment, management, and conservation. In the marine environment, where hard substrata are more difficult to access than soft bottoms for quantitative ecological studies, Artificial Substrate Units (ASUs) allow for standardized sampling. We deployed ASUs within five regional seas (Baltic Sea, Northeast Atlantic Ocean, Mediterranean Sea, Black Sea, and Red Sea) for 12-26 months to measure the diversity and community composition of macroinvertebrates. We identified invertebrates using a traditional approach based on morphological characters, and by metabarcoding of the mitochondrial cytochrome oxidase I (COI) gene. We compared community composition and diversity metrics obtained using the two methods. Diversity was significantly correlated between data types. Metabarcoding of ASUs allowed for robust comparisons of community composition and diversity, but not all groups were successfully sequenced. All locations were significantly different in taxonomic composition as measured with both kinds of data. We recovered previously known regional biogeographical patterns in both datasets (e.g., low species diversity in the Black and Baltic Seas, affinity between the Bay of Biscay and the Mediterranean). We conclude that the two approaches provide complementary information and that metabarcoding shows great promise for marine monitoring. However, until its pitfalls are addressed, the use of metabarcoding in monitoring of rocky benthic assemblages should be used in addition to classical approaches rather than instead of them.

Understanding processes at the origin of species flocks with a focus on the marine Antarctic fauna.

Species flocks (SFs) fascinate evolutionary biologists who wonder whether such striking diversification can be driven by normal evolutionary processes. Multiple definitions of SFs have hindered the study of their origins. Previous studies identified a monophyletic taxon as a SF if it displays high speciosity in an area in which it is endemic (criterion 1), high ecological diversity among species (criterion 2), and if it dominates the habitat in terms of biomass (criterion 3); we used these criteria in our analyses. Our starting hypothesis is that normal evolutionary processes may provide a sufficient explanation for most SFs. We thus clearly separate each criterion and identify which biological (intrinsic) and environmental (extrinsic) traits are most favourable to their realization. The first part focuses on evolutionary processes. We highlight that some popular putative causes of SFs, such as key innovations or ecological speciation, are neither necessary nor sufficient to fulfill some or all of the three criteria. Initial differentiation mechanisms are diverse and difficult to identify a posteriori because a primary differentiation of one type (genetic, ecological or geographical) often promotes other types of differentiation. Furthermore, the criteria are not independent: positive feedbacks between speciosity and ecological diversity among species are expected whatever the initial cause of differentiation, and ecological diversity should enhance habitat dominance at the clade level. We then identify intrinsic and extrinsic factors that favour each criterion. Low dispersal emerges as a convincing driver of speciosity. Except for a genomic architecture favouring ecological speciation, for which assessment is difficult, high effective population sizes are the single intrinsic factor that directly enhances speciosity, ecological diversity and habitat dominance. No extrinsic factor appeared to enhance all criteria simultaneously but a combination of factors (insularity, fragmentation and environmental stability) may favour the three criteria, although the effect is indirect for habitat dominance. We then apply this analytical framework to Antarctic marine environments by analysing data from 18 speciose clades belonging to echinoderms (five unrelated clades), notothenioid fishes (five clades) and peracarid crustaceans (eight clades). Antarctic shelf environments and history appear favourable to endemicity and speciosity, but not to ecological specialization. Two main patterns are distinguished among taxa. (i) In echinoderms, many brooding, species-rich and endemic clades are reported, but without remarkable ecological diversity or habitat dominance. In these taxa, loss of the larval stage is probably a consequence of past Antarctic environmental factors, and brooding is suggested to be responsible for enhanced allopatric speciation (via dispersal limitation). (ii) In notothenioids and peracarids, many clades fulfill all three SF criteria. This could result from unusual features in fish and crustaceans: chromosome instability and key innovations (antifreeze proteins) in notothenioids, ecological opportunity in peracarids, and a genomic architecture favouring ecological speciation in both groups. Therefore, the data do not support our starting point that normal evolutionary factors or processes drive SFs because in these two groups uncommon intrinsic features or ecological opportunity provide the best explanation. The utility of the three-criterion SF concept is therefore questioned and guidelines are given for future studies.

A multispecies approach reveals hot spots and cold spots of diversity and connectivity in invertebrate species with contrasting dispersal modes.

Genetic diversity is crucial for species' maintenance and persistence, yet is often overlooked in conservation studies. Species diversity is more often reported due to practical constraints, but it is unknown if these measures of diversity are correlated. In marine invertebrates, adults are often sessile or sedentary and populations exchange genes via dispersal of gametes and larvae. Species with a larval period are expected to have more connected populations than those without larval dispersal. We assessed the relationship between measures of species and genetic diversity, and between dispersal ability and connectivity. We compiled data on genetic patterns and life history traits in nine species across five phyla. Sampling sites spanned 600 km in the northwest Mediterranean Sea and focused on a 50-km area near Marseilles, France. Comparative population genetic approaches yielded three main results. (i) Species without larvae showed higher levels of genetic structure than species with free-living larvae, but the role of larval type (lecithotrophic or planktotrophic) was negligible. (ii) A narrow area around Marseilles, subject to offshore advection, limited genetic connectivity in most species. (iii) We identified sites with significant positive contributions to overall genetic diversity across all species, corresponding with areas near low human population densities. In contrast, high levels of human activity corresponded with a negative contribution to overall genetic diversity. Genetic diversity within species was positively and significantly linearly related to local species diversity. Our study suggests that local contribution to overall genetic diversity should be taken into account for future conservation strategies.

Quantitative criteria for choosing targets and indicators for sustainable use of ecosystems.

Wide-ranging, indicator-based assessments of large, complex ecosystems are playing an increasing role in guiding environmental policy and management. An example is the EU's Marine Strategy Framework Directive, which requires Member States to take measures to reach "good environmental status" (GES) in European marine waters. However, formulation of indicator targets consistent with the Directive's high-level policy goal of sustainable use has proven challenging. We develop a specific, quantitative interpretation of the concepts of GES and sustainable use in terms of indicators and associated targets, by sharply distinguishing between current uses to satisfy current societal needs and preferences, and unknown future uses. We argue that consistent targets to safeguard future uses derive from a requirement that any environmental state indicator should recover within a defined time (e.g. 30 years) to its pressure-free range of variation when all pressures are hypothetically removed. Within these constraints, specific targets for current uses should be set. Routes to implementation of this proposal for indicators of fish-community size structure, population size of selected species, eutrophication, impacts of non-indigenous species, and genetic diversity are discussed. Important policy implications are that (a) indicator target ranges, which may be wider than natural ranges, systematically and rationally derive from our proposal; (b) because relevant state indicators tend to respond slowly, corresponding pressures should also be monitored and assessed;

Dispersal similarly shapes both population genetics and community patterns in the marine realm.

Dispersal plays a key role to connect populations and, if limited, is one of the main processes to maintain and generate regional biodiversity. According to neutral theories of molecular evolution and biodiversity, dispersal limitation of propagules and population stochasticity are integral to shaping both genetic and community structure. We conducted a parallel analysis of biological connectivity at genetic and community levels in marine groups with different dispersal traits. We compiled large data sets of population genetic structure (98 benthic macroinvertebrate and 35 planktonic species) and biogeographic data (2193 benthic macroinvertebrate and 734 planktonic species). We estimated dispersal distances from population genetic data (i.e., FST vs. geographic distance) and from ß-diversity at the community level. Dispersal distances ranked the biological groups in the same order at both genetic and community levels, as predicted by organism dispersal ability and seascape connectivity: macrozoobenthic species without dispersing larvae, followed by macrozoobenthic species with dispersing larvae and plankton (phyto- and zooplankton). This ranking order is associated with constraints to the movement of macrozoobenthos within the seabed compared with the pelagic habitat. We showed that dispersal limitation similarly determines the connectivity degree of communities and populations, supporting the predictions of neutral theories in marine biodiversity patterns.

Does natural selection explain the fine scale genetic structure at the nuclear exon Glu-5' in blue mussels from Kerguelen?

The Kerguelen archipelago, isolated in the Southern Ocean, shelters a blue mussel Mytilus metapopulation far from any influence of continental populations or any known hybrid zone. The finely carved coast leads to a highly heterogeneous habitat. We investigated the impact of the environment on the genetic structure in those Kerguelen blue mussels by relating allele frequencies to habitat descriptors. A total sample comprising up to 2248 individuals from 35 locations was characterized using two nuclear markers, mac-1 and Glu-5', and a mitochondrial marker (COI). The frequency data from 9 allozyme loci in 9 of these locations were also reanalyzed. Two other nuclear markers (EFbis and EFprem's) were monomorphic. Compared to Northern Hemisphere populations, polymorphism in Kerguelen blue mussels was lower for all markers except for the exon Glu-5'. At Glu-5', genetic differences were observed between samples from distinct regions (F CT = 0.077), as well as within two regions, including between samples separated by <500 m. No significant differentiation was observed in the AMOVA analyses at the two other markers (mac-1 and COI). Like mac-1, all allozyme loci genotyped in a previous publication, displayed lower differentiation (Jost's D) and F ST values than Glu-5'. Power simulations and confidence intervals support that Glu-5' displays significantly higher differentiation than the other loci (except a single allozyme for which confidence intervals overlap). AMOVA analyses revealed significant effects of the giant kelp Macrocystis and wave exposure on this marker. We discuss the influence of hydrological conditions on the genetic differentiation among regions. In marine organisms with high fecundity and high dispersal potential, gene flow tends to erase differentiation, but this study showed significant differentiation at very small distance. This may be explained by the particular hydrology and the carved coastline of the Kerguelen archipelago, together with spatially variable selection at Glu-5'.

Species are hypotheses: avoid connectivity assessments based on pillars of sand.

Connectivity among populations determines the dynamics and evolution of populations, and its assessment is essential in ecology in general and in conservation biology in particular. The robust basis of any ecological study is the accurate delimitation of evolutionary units, such as populations, metapopulations and species. Yet a disconnect still persists between the work of taxonomists describing species as working hypotheses and the use of species delimitation by molecular ecologists interested in describing patterns of gene flow. This problem is particularly acute in the marine environment where the inventory of biodiversity is relatively delayed, while for the past two decades, molecular studies have shown a high prevalence of cryptic species. In this study, we illustrate, based on marine case studies, how the failure to recognize boundaries of evolutionary-relevant unit leads to heavily biased estimates of connectivity. We review the conceptual framework within which species delimitation can be formalized as falsifiable hypotheses and show how connectivity studies can feed integrative taxonomic work and vice versa. Finally, we suggest strategies for spatial, temporal and phylogenetic sampling to reduce the probability of inadequately delimiting evolutionary units when engaging in connectivity studies.

Genetic data, reproduction season and reproductive strategy data support the existence of biological species in Ophioderma longicauda.

Cryptic species are numerous in the marine environment. The brittle star Ophioderma longicauda is composed of six mitochondrial lineages, encompassing brooders, which form a monophyletic group, and broadcasters, from which the brooders are derived. To clarify the species limits within O. longicauda, we compared the reproductive status of the sympatric lineages L1 and L3 (defined after sequencing a portion of the mitochondrial gene COI) during the month of May in Greece. In addition, we genotyped a nuclear marker, intron i51. Each L3 female was brooding, whereas all L1 specimens displayed full gonads, suggesting temporal pre-zygotic isolation between brooders and broadcasters. Statistical differences were found among lineages in morphology and bathymetric distribution. Finally, the intron i51 was polymorphic in L1 (60 individuals), but monomorphic in L3 (109 individuals), confirming the absence of gene flow between brooders and broadcasters. In conclusion, the broadcasting lineage L1 and the brooding lineage L3 are different biological species.

Thermotolerance and regeneration in the brittle star species complex Ophioderma longicauda: a preliminary study comparing lineages and Mediterranean basins.

Global warming is expected to change marine species distributions; it is thus critical to understand species current thermotolerance. The brittle star species complex Ophioderma longicauda comprises a broadcast spawning lineage L1 and a brooding lineage L3. We collected L1 specimens from Marseilles and Crete, and L3 specimens from Crete. We monitored survival, autotomy and arm regeneration at 17, 26 and 30°C during 14 weeks. Globally O. longicauda showed good resistance to elevated temperatures compared to other published studies on ophiuroids. The L3 sample displayed a better thermotolerance than L1 samples. Yet, more research is needed to establish whether these differences are due to lineages, geographic origin, or random effects. We provided for the first time individual regeneration trajectories, and showed that regeneration followed a growth curve and was highly influenced by temperature in both lineages. Our results highlight the importance of taking into account the presence of cryptic species when studying the potential effects of global warming.

Is the species flock concept operational? The Antarctic shelf case.

There has been a significant body of literature on species flock definition but not so much about practical means to appraise them. We here apply the five criteria of Eastman and McCune for detecting species flocks in four taxonomic components of the benthic fauna of the Antarctic shelf: teleost fishes, crinoids (feather stars), echinoids (sea urchins) and crustacean arthropods. Practical limitations led us to prioritize the three historical criteria (endemicity, monophyly, species richness) over the two ecological ones (ecological diversity and habitat dominance). We propose a new protocol which includes an iterative fine-tuning of the monophyly and endemicity criteria in order to discover unsuspected flocks. As a result nine « full ¼ species flocks (fulfilling the five criteria) are briefly described. Eight other flocks fit the three historical criteria but need to be further investigated from the ecological point of view (here called "core flocks"). The approach also shows that some candidate taxonomic components are no species flocks at all. The present study contradicts the paradigm that marine species flocks are rare. The hypothesis according to which the Antarctic shelf acts as a species flocks generator is supported, and the approach indicates paths for further ecological studies and may serve as a starting point to investigate the processes leading to flock-like patterning of biodiversity.

Differential reproductive timing in Echinocardium spp.: the first Mediterranean survey allows interoceanic and interspecific comparisons.

Echinocardium cordatum had long been considered as cosmopolitan, but molecular data revealed it is a complex of cryptic species, with two non-hybridizing species (B1 & B2) in the Mediterranean Sea living in syntopy with Echinocardium mediterraneum. Histological analyses of the gonads from a 17-month sampling period revealed a statistically significant time lag between the Maturity Indices of E. cordatum and E. mediterraneum. The main environmental stimulus may be different for the two nominal species, possibly seawater temperature for E. cordatum and chlorophyll a concentration for E. mediterraneum. Within the E. cordatum complex, spawning timing and synchrony are different according to major geographic areas (Atlantic/Pacific/Mediterranean) and/or the corresponding genetic subdivision [A/P/(B1 & B2)]. In contrast, the effects of temperature on the reproductive cycle seem rather to mirror the genetic lineages than environmental similarities of the different localities. Between the sister species (B1 & B2) no differences could be detected, maybe due to small sample sizes.