Insights into Environmental Drivers on the Reproductive Cycle of Diopatra neapolitana (Polychaeta: Onuphidae).

The reproductive cycle of Diopatra neapolitana was studied over two years in Redondela (NW Spain), considering both the monthly evolution of the percentage of individuals with oocytes and the variation of the mean oocyte size. Moreover, since three other species of Diopatra have recently been documented in regions geographically close to NW Spain, we first confirmed the identity of this species by carrying out a morphological identification of a representative number of the specimens collected. Our data showed a discontinuous reproductive season, with a period of proliferation and maturation of oocytes from January to September and asynchronous spawning events between July and October. Then, a resting period in the months of November and December was observed. We also explored the influence of some relevant environmental factors (namely, water temperature, photoperiod, salinity, primary production and upwelling index) on the observed reproductive cycle. The results suggest that water temperature is one of the most important drivers of the reproductive cycle for this species. Particularly, in Ría de Vigo, the reproductive cycle seems to be influenced by upwelling events of deep cold water that occur frequently between March and September, and that could act as a spawning-inducing switch.

A review of the empirical literature on the use of limpets Patella spp. (Mollusca: Gastropoda) as bioindicators of environmental quality.

The need to carry out monitoring programs for environmental pollution of coastal ecosystems makes it necessary to increase the number of indicator species in order to have a wide range of suitable organisms for most of the possible toxic substances and ways of exposure. With the purpose of analyzing the suitability of limpets (Patella spp.) for their use in biomonitoring programs, a literature review was performed on 88 cases found in the Web of Science online platform. The capacity of limpets to accumulate metals and hydrocarbons has been examined in several field studies. In most cases there is a clear relationship between body content of a pollutant in soft tissues of limpets and the corresponding environmental level. Likewise, different responses to pollutants have been reported in limpets, including induction of DNA damage, induction of metallothioneins, oxidative stress, decrease of Neutral Red retention or variations in heart rate. Regarding the study of changes in community structure, an uneven response to disturbances (e.g. oil spills, wastewater discharge) by species of the genus Patella has been documented. Moreover, generally limpets respond to pollutants similarly to, or even more effectively than, mussels; therefore, their inclusion as sentinel organisms in regional monitoring plans seems appropriate.

Secreted Citrate Serves as Iron Carrier for the Marine Pathogen Photobacterium damselae subsp damselae.

Photobacterium damselae subsp damselae (Pdd) is a Vibrionaceae that has a wide pathogenic potential against many marine animals and also against humans. Some strains of this bacterium acquire iron through the siderophore vibrioferrin. However, there are virulent strains that do not produce vibrioferrin, but they still give a strong positive reaction in the CAS test for siderophore production. In an in silico search on the genome sequences of this type of strains we could not find any ORF which could be related to a siderophore system. To identify genes that could encode a siderophore-mediated iron acquisition system we used a mini-Tn10 transposon random mutagenesis approach. From more than 1,400 mutants examined, we could isolate a mutant (BP53) that showed a strong CAS reaction independently of the iron levels of the medium. In this mutant the transposon was inserted into the idh gene, which encodes an isocitrate dehydrogenase that participates in the tricarboxylic acid cycle. The mutant did not show any growth impairment in rich or minimal media, but it accumulated a noticeable amount of citrate (around 7 mM) in the culture medium, irrespective of the iron levels. The parental strain accumulated citrate, but in an iron-regulated fashion, being citrate levels 5-6 times higher under iron restricted conditions. In addition, a null mutant deficient in citrate synthase showed an impairment for growth at high concentrations of iron chelators, and showed almost no reaction in the CAS test. Chemical analysis by liquid chromatography of the iron-restricted culture supernatants resulted in a CAS-positive fraction with biological activity as siderophore. HPLC purification of that fraction yielded a pure compound which was identified as citrate from its MS and NMR spectral data. Although the production of another citrate-based compound with siderophore activity cannot be ruled out, our results suggest that Pdd secretes endogenous citrate and use it for iron scavenging from the cell environment.

Hybridization between two cryptic filamentous brown seaweeds along the shore: analysing pre- and postzygotic barriers in populations of individuals with varying ploidy levels.

We aimed to study the importance of hybridization between two cryptic species of the genus Ectocarpus, a group of filamentous algae with haploid-diploid life cycles that include the principal genetic model organism for the brown algae. In haploid-diploid species, the genetic structure of the two phases of the life cycle can be analysed separately in natural populations. Such life cycles provide a unique opportunity to estimate the frequency of hybrid genotypes in diploid sporophytes and meiotic recombinant genotypes in haploid gametophytes allowing the effects of reproductive barriers preventing fertilization or preventing meiosis to be untangle. The level of hybridization between E. siliculosus and E. crouaniorum was quantified along the European coast. Clonal cultures (568 diploid, 336 haploid) isolated from field samples were genotyped using cytoplasmic and nuclear markers to estimate the frequency of hybrid genotypes in diploids and recombinant haploids. We identified admixed individuals using microsatellite loci, classical assignment methods and a newly developed Bayesian method (XPloidAssignment), which allows the analysis of populations that exhibit variations in ploidy level. Over all populations, the level of hybridization was estimated at 8.7%. Hybrids were exclusively observed in sympatric populations. More than 98% of hybrids were diploids (40% of which showed signs of aneuploidy) with a high frequency of rare alleles. The near absence of haploid recombinant hybrids demonstrates that the reproductive barriers are mostly postzygotic and suggests that abnormal chromosome segregation during meiosis following hybridization of species with different genome sizes could be a major cause of interspecific incompatibility in this system.

Species delimitation and phylogeographic analyses in the Ectocarpus subgroup siliculosi (Ectocarpales, Phaeophyceae).

The genus Ectocarpus (Ectocarpales, Phaeophyceae) contains filamentous algae widely distributed in marine and estuarine habitats of temperate regions in both hemispheres. While E. siliculosus has become a model organism for genomics and genetics of the brown macroalgae, accurate species delineation, distribution patterns and diversity for the genus Ectocarpus remain problematic. In this study, we used three independent species delimitation approaches to generate a robust species hypothesis for 729 Ectocarpus specimens collected mainly along the European and Chilean coasts. These approaches comprised phylogenetic reconstructions and two bioinformatics tools developed to objectively define species boundaries (General Mixed Yule Coalescence Method and Automatic Barcode Gap Discovery). Our analyses were based on DNA sequences of two loci: the mitochondrial cytochrome oxidase subunit 1 and the nuclear internal transcribed spacer 1 of the ribosomal DNA. Our analyses showed the presence of at least 15 cryptic species and suggest the existence of incomplete lineage sorting or introgression between five of them. These results suggested the possible existence of different levels of reproductive barriers within this species complex. We also detected differences among species in their phylogeographic patterns, range and depth distributions, which may suggest different biogeographic histories (e.g., endemic species or recent introductions).

Evolution and maintenance of haploid-diploid life cycles in natural populations: The case of the marine brown alga Ectocarpus.

The evolutionary stability of haploid-diploid life cycles is still controversial. Mathematical models indicate that niche differences between ploidy phases may be a necessary condition for the evolution and maintenance of these life cycles. Nevertheless, experimental support for this prediction remains elusive. In the present work, we explored this hypothesis in natural populations of the brown alga Ectocarpus. Consistent with the life cycle described in culture, Ectocarpus crouaniorum in NW France and E. siliculosus in SW Italy exhibited an alternation between haploid gametophytes and diploid sporophytes. Our field data invalidated, however, the long-standing view of an isomorphic alternation of generations. Gametophytes and sporophytes displayed marked differences in size and, conforming to theoretical predictions, occupied different spatiotemporal niches. Gametophytes were found almost exclusively on the alga Scytosiphon lomentaria during spring whereas sporophytes were present year-round on abiotic substrata. Paradoxically, E. siliculosus in NW France exhibited similar habitat usage despite the absence of alternation of ploidy phases. Diploid sporophytes grew both epilithically and epiphytically, and this mainly asexual population gained the same ecological advantage postulated for haploid-diploid populations. Consequently, an ecological interpretation of the niche differences between haploid and diploid individuals does not seem to satisfactorily explain the evolution of the Ectocarpus life cycle.

Population structure and range expansion: the case of the invasive gastropod Cyclope neritea in northwest Iberian Peninsula.

Biotic invasions have a reputation for unpredictable behavior. Here, we report how slight changes in human activity responsible for the introduction and range expansion of a non-native mollusk have led to detectable differences in the genetics of the invasion. Cyclope neritea is a non-predatory gastropod introduced to 2 areas of the European Atlantic: the northwest Iberian Peninsula (NWIP) and the French Atlantic coast (FAC). Shellfish seabed farming is intense in both areas but focuses on different commercial species. Using mitochondrial gene sequences, the lower genetic diversity recorded along the NWIP suggests a more homogeneous range of source populations than in the FAC. Unlike FAC, genetic diversity and haplotype composition in the NWIP correlate with the date of first occurrence of C. neritea at each site rather than with geographical location. Although this pattern evokes the genetic signature expected under a serial-founder colonization model from a single initial enclave, a comparison with samples from potential source populations suggests that the NWIP probably experienced several independent reintroductions. The jump dispersal pattern of C. neritea in the NWIP, together with the observation that populations established in the same year are genetically undifferentiated, point to human transport as the most plausible explanation for the current range expansion. Despite evidence for human-mediated dispersal, C. neritea managed to develop a seemingly non-random genetic pattern in the NWIP. It is suggested that caution must be exerted when interpreting genetic patterns in invaders.

MICROSATELLITE DEVELOPMENT IN RHODOPHYTA USING HIGH-THROUGHPUT SEQUENCE DATA(1).

Shotgun genome sequencing is rapidly emerging as the method of choice for the identification of microsatellite loci in nonmodel organisms. However, to the best of our knowledge, this approach has not been applied to marine algae so far. Herein, we report the results of using the 454 next-generation sequencing (NGS) platform to randomly sample 36.0 and 40.9 Mbp (139,786 and 139,795 reads, respectively) of the genome of two red algae from the northwest Iberian Peninsula [Grateloupia lanceola (J. Agardh) J. Agardh and a still undescribed new member of the family Cruoriaceae]. Using data mining tools, we identified 4,766 and 5,174 perfect microsatellite loci in 4,344 and 4,504 sequences/contigs from G. lanceola and the Cruoriaceae, respectively. After conservative removal of potentially problematic loci (redundant sequences, mobile elements), primer design was possible for 1,371 and 1,366 loci, respectively. A survey of the literature indicates that microsatellite density in our Rhodophyta is at the low end of the values reported for other organisms investigated with the same technology (land plants and animals). A limited number of loci were successfully tested for PCR amplification and polymorphism finding that they may be suitable for population genetic studies. This study demonstrates that random genome sequencing is a rapid, effective alternative to develop useful microsatellite loci in previously unstudied red algae.

MULTISCALE GENETIC STRUCTURE OF AN ENDANGERED SEAWEED AHNFELTIOPSIS PUSILLA (RHODOPHYTA): IMPLICATIONS FOR ITS CONSERVATION(1).

Although marine macroalgae have recently entered the lists of endangered species, conservation efforts are still limited by a lack of data, particularly for naturally rare species. One example is the turf-forming Ahnfeltiopsis pusilla (Mont.) P. C. Silva et DeCew. Albeit cataloged as vulnerable in the Northwest Iberian Peninsula (NWIP), where it occurs only at five enclaves separated by 1,200 km from the closest recorded presence of the species, nothing is known about its genetic diversity and population connectivity. We used amplified fragment length polymorphism (AFLP) and sequences of the intergenic region between the mitochondrial cytochrome oxidase subunit 2 and subunit 3 genes (cox2-3) to investigate its genetic structure at large (1,200 km), regional (<125 km), fine (<250 m), and patch (<1 m) scales. While cox2-3 variability was too low for the intraspecific study, AFLP revealed that most of the genetic diversity was due to differences between populations. Locally, genetic diversity was always low, and clones were frequent, suggesting that asexual reproduction may be common; patches of turf, however, often were composites of various genetic individuals. Genetic structure at local, regional, and large scales indicated that A. pusilla is a poor disperser, and an assignment test found no evidence of real-time dispersal between NWIP sites. Therefore, it is proposed that the five NWIP enclaves are designated independent management units (MUs). Bayesian-clustering approaches suggested that the three southernmost sites are particularly valuable for conservation since they concentrate most of the genetic heritage of A. pusilla in NWIP. Our study shows that the approaches of conservation genetics may provide useful insights for endangered seaweeds.

Genetic isolation by distance among populations of the netted dog whelk Nassarius reticulatus (L.) along the European Atlantic coastline.

Estimates of the average distances by which marine larvae disperse are generally poorly described, despite the central role that larval dispersal plays in the demographic connectivity of populations across geographic space. Here, we describe the population genetic structure and average dispersal distance of the netted dog whelk Nassarius reticulatus (L.) (Mollusca, Gastropoda, Prosobranchia), a widespread member of European intertidal communities, using DNA sequence variation in a fragment of the mitochondrial gene cytochrome c oxidase subunit I (COI). An analysis of 156 individuals from 6 locations spread across approximately 1700 km of the European Atlantic coastline revealed weak and nonsignificant population structure (overall Phi(ST) = 0.00013). However, pairwise Phi(ST) values revealed a slight but significant increase in genetic isolation with geographic distance (IBD), suggesting that populations are not panmictic across the sampled geographic range. If we assume that the isolation by distance is maintained by a stable, stepping stone model of gene flow, then the slope of the IBD is consistent with an average larval dispersal distance of approximately 70 km per generation. The spatial scale of larval dispersal in N. reticulatus is consistent with the life cycle of the species (planktotrophic veliger lasting 30-60 days before competent to settle). A mismatch analysis of the COI sequences revealed a signature of an ancient demographic expansion that began 61 500-160,000 years ago, well before the most recent Pleistocene glaciation event. The greatest levels of genetic diversity occur within the middle latitudes of the whelk's geographic range, consistent with the notion that historic populations of N. reticulatus might have expanded northward and southward from the centrally located Bay of Biscay.