Lucía Couceiro

Decreased TBT pollution and changing bioaccumulation pattern in gastropods imply butyltin desorption from sediments.

Two monitoring surveys were repeated in Galicia (NW Spain): one in 2003 concerned the rocky shore gastropod Nucella lapillus (19 populations, the reference campaign in 1996), the other dealt in 2005 with the infaunal snail Nassarius reticulatus (25 sites, the previous one in 2000). Samples were subject to a standard protocol to determine the concentrations of butyltins (BTs) in tissues. Results show that pollution in most populations has considerably decreased over the last decade: for N. lapillus the mean descent ranged from 37% (for tributyltin -TBT-) to 66% (for monobutyltin -MBT-), and TBT concentrations were on average halved in N. reticulatus. However, derivatives generally increased in this latter species, to the extent that dibutyltin -DBT- in several 2005 samples exceeded the aggregate of all three BTs in 2000. As a consequence, a major change in the bioaccumulation patterns becomes evident, particularly when computing the butyltin degradation index [BDI: (DBT+MBT)/TBT]. This shift is most marked at sites where pollution has always been lowest, and it shows significant negative correlation between both gastropods. Since sources other than antifouling paints are not important in the area, it is proposed that observations are due to BT desorption from sediments through some interplay involving the different characteristics of the chemicals and the contrasting biology of the animals.

Imposex and gender-independent butyltin accumulation in the gastropod Nassarius reticulatus from the Cantabrian coast (N Atlantic Spain).

The gastropod Nassarius reticulatus has been used as a tributyltin (TBT) biomonitor over the last two decades, and it is now endorsed by Atlantic Europe environmental agencies. However, there is one important question debated by the earliest studies still unresolved, namely whether butyltin accumulation in tissues is sex-dependent or not. Thus, a field survey was conducted along the Cantabrian coast: samples were subject to both customary imposex and modern chemical analyses. No significant bioaccumulation differences between sexes were found for any of the three butyltin species examined (i.e. TBT plus derivatives di- and mono-butyltin). In addition, both lower-than-expected absolute butyltins and minor relative TBT proportions in tissues conform with decreasing pollution in nearby areas. Imposex, though, is generally still considerable due to a lesser responsiveness of population indexes. Finally, one sample showed no sign of imposex but quantifiable butyltins; these are good news indicating that TBT is getting back down to levels around the induction threshold of this specific biological effect.

Decade-long monitoring reveals a transient distortion of baseline butyltin bioaccumulation pattern in gastropods.

Worldwide measures to restrict tributyltin (TBT) in antifouling paints have been legislated for decades, and were upgraded to a total ban on September 2008. With a view to test the response of coastal biota to changing pollution, since 1996 we have determined the concentration of TBT and derivatives di- and mono-butyltin (DBT and MBT) in NW Spain populations of two gastropods of contrasting biology, the rock-snail Nucella lapillus (n=18) and the mud-snail Nassarius reticulatus (n=24). TBT pollution in the study area has decreased consistently and considerably over time. In addition, the baseline butyltin (BT) bioaccumulation patterns showed a marked but transient distortion. These field observations are consistent with BT desorption from sediments, a natural phenomenon that is now to be expected in developing countries recently subject to the global TBT ban.

Evidence for multiple introductions of the Pacific green alga Ulva australis Areschoug (Ulvales, Chlorophyta) to the Iberian Peninsula

Abstract Earlier morpho-anatomical work showed that a non-native Ulva species is widely distributed off the northwestern Iberian Peninsula (NWIP) since the early 1990s. However, insufficient sequence data provided at the time of the earlier study led to suggestions that more genetic evidence might be required to confirm this record. Here, we present this evidence in sequence data for the nuclear-encoded ITS1 and the plastid-encoded rbcL. We confirm the occurrence of an alien distromatic Ulva in several locations along the NWIP. Additionally, its presence in the Spanish Mediterranean (>1000 km away) is reported here for the first time. Species identification yielded notably coherent results between nuclear-encoded and plastid-encoded sequences, revealing that Spanish plants are conspecific with collections of Ulva pertusa and Ulva australis from a range of Pacific locations. The probable synonymy between U. australis and U. pertusa is consistent with their morphological similarity and suggests that U. australis is the correct name for the non-native green macroalga. ITS1 sequences reveal a common source for the various sites studied in the NWIP, possibly linked to shellfish transfers into the region. However, non-native populations in southern Spain must derive from an independent introduction event. This is the first time that molecular data have been used to demonstrate multiple introductions of U. australis to European waters.

Barcoding of Cryptic Stages of Marine Brown Algae Isolated from Incubated Substratum Reveals High Diversity in Acinetosporaceae (Ectocarpales, Phaeophyceae)1

Abstract — To identify cryptic stages of marine brown macroalgae present in the “bank of microscopic forms”, we incubated natural substrata of different geographical origins and isolated emerging Phaeophyceae into clonal cultures. A total of 431 clones were subsequently identified by barcoding using 5′-COI. A proportion of 98% of the isolates belonged to the Ectocarpales. The distribution of pairwise genetic distances revealed a K2P divergence of 1.8% as species-level cut-off. Using this threshold, the samples were ascribed to 83 different species, 39 (47%) of which were identified through reference sequences or morphology. In the Ectocarpaceae, 16 lineages of Ectocarpus fulfilled the barcode criterion for different species, while three putative new species were detected. In the Chordariaceae, numerous microthalli were microstages of known macroscopic taxa. A separate cluster contained Hecatonema maculans and other microscopic species. Taxa traditionally classified in Acinetosporaceae were split in two species-rich groups containing Pylaiella and Hincksia in one and Acinetospora in the other. Feldmannia species were present in both clusters. The present study shows that the germling emergence method is suited to reveal the diversity of hidden life-history stages, albeit with a bias towards early successional species.

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.

Discovery of imposex in the gastropod Cyclope neritea now invading Galicia (north-west Spain)

A field survey to re-assess imposex and tributyltin (TBT) bioaccumulation in the gastropod Nassarius reticulatus over Galicia uncovered three populations of the invasive species Cyclope neritea , all showing some advanced imposex. One female was found to carry a mass of aborted egg capsules. The TBT pollution, now decreasing in the area, is the most likely explanation for these reproductive anomalies, and it may bear some relationship to the described population dynamics of C. neritea .

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.

Examining the bank of microscopic stages in kelps using culturing and barcoding

This paper describes a method to study the diversity of young kelp sporophytes that are recruited from the bank of microscopic stages. Small samples of rocky substratum (0.5 cm2) were collected from the low intertidal zone, which was dominated by the kelp Laminaria digitata. Samples were cultivated in the laboratory under conditions permitting gametogenesis. Sporophyte recruits in the cultures were isolated and identified at the species level using the barcoding mitochondrial marker rpl31–rns. Sixty per cent of the collected samples had at least one to a maximum of 30 kelp recruits, belonging to five different species (L. digitata, L. hyperborea, L. ochroleuca, Saccharina latissima and Sacchorhiza polyschides). As the examination of freshly collected rocky samples under a stereo microscope did not reveal any kelp sporophytes, the recruitment in these samples after culture probably occurred from the bank of microscopic forms present on the substratum. Despite the dominance of L. digitata in the field, the young sporophytes obtained after culturing were mainly S. polyschides. This study illustrates the suitability of culturing in combination with molecular identification of young sporophytes to address several key aspects of kelp ecology related to the existence of a bank of microscopic stages in the field.