Trophic niche overlap between round sardinella (Sardinella aurita) and sympatric pelagic fish species in the Western Mediterranean.

The northward expansion of round sardinella (Sardinella aurita) in the Mediterranean Sea, together with declines and fluctuations in biomass and landings of European sardine (Sardina pilchardus) and anchovy (Engraulis encrasicolus) observed in recent decades, may suggest potential inter-specific competition in the pelagic domain. The coexistence of sympatric zooplanktivorous fish species might therefore be exposed in part to trophic niche overlap and competition for food. Combining visual diet characterization under the microscope with DNA metabarcoding from stomach contents of fish collected in spring results show that predation on relatively large krill is equally important for sardinella than for the other two niche overlapping species. Furthermore, an important overlap is found in their isotopic niche, especially with anchovy, using nitrogen (δ15N) and carbon (δ13C) stable isotopes in muscle tissue. In fact, the three fish species are able to feed effectively in the whole prey size spectrum available during the sampled season, from the smallest diatoms and copepods to the larger prey (i.e., decapods and euphausiids), including fish larvae. Moreover, effective predation upon other large prey like siphonophores, which is observed only when multi-proxy analyses in stomach contents are applied, might also be relevant in the diet of sardinella. The overlapping diet composition in spring, together with the effective use of food resource by sardinella, can be of special interest in potential future scenarios with warmer water temperature leading to lower zooplankton and/or higher jellyfish availability, where sardinella may take advantage over other species due to its feeding plasticity.

A trophic latitudinal gradient revealed in anchovy and sardine from the Western Mediterranean Sea using a multi-proxy approach.

This work combines state-of-the-art methods (DNA metabarcoding) with classic approaches (visual stomach content characterization and stable isotope analyses of nitrogen (δ15N) and carbon (δ13C)) to investigate the trophic ecology of anchovy (Engraulis encrasicolus) and sardine (Sardina pilchardus) at high taxonomic and spatial resolution in the Western Mediterranean Sea. Gut contents observed are in accordance with the dietary plasticity generally described for anchovy and sardine, suggesting a diet related to the opportunistic ingestion of available prey in a certain area and/or time. Genetic tools also showed modest inter-specific differences regarding ingested species. However, inter-specific and intra-specific differences in ingested prey frequencies and prey biomass reflected a latitudinal signal that could indicate a more effective predation on large prey like krill by anchovy versus sardine, as well as a generalized higher large prey ingestion by both species southwards. In fact, both species presented lower δ15N in the northernmost area. This latitudinal gradient indicates changes in the trophic ecology of anchovy and sardine that coincide with previously described better biological conditions for fish in the southern part of the study area as well as higher landings of both species in recent years.

Tossed 'good luck' coins as vectors for anthropogenic pollution into aquatic environment.

Superstition has it that tossing coins into wells or fountains brings good luck, thereby causing a potential accumulation of microbially contaminated metal particles in the water. Here, we characterized the microbiota and the resistance profile in biofilm on such coins and their surrounding sediments. The study site was a tidal marine lake within a touristic center located in a natural reserve area. Notwithstanding the fact that coin-related biofilms were dominated by typical marine taxa, coin biofilms had specific microbial communities that were different from the communities of the surrounding sediment. Moreover, the communities were different depending on whether the coin were made mainly of steel or of copper. Sequences affiliated with putative pathogens were found on every third coin but were not found in the surrounding sediment. Antibiotic resistance genes (ARGs) were detected on most of the coins, and interestingly, sediments close to the area where coins accumulate had a higher frequency of ARGs. We suggest that the surface of the coins might offer a niche for ARGs and faecal bacteria to survive, and, thus, tossed coins are a potential source and vector for ARGs into the surrounding environment.

Isolation and characterization of fourteen polymorphic microsatellite markers in the viperine snake Natrix maura.

Nineteen polymorphic microsatellite loci were identified and developed for Natrix maura. Polymorphism was assessed for 120 individuals sampled across four sampling sites from the French Pyrenees Mountains. The number of alleles per locus ranged from 3 to 15, and expected heterozygosity per locus ranged from 0.227 to 0.863. We tested for deviation from Hardy-Weinberg equilibrium and linkage disequilibrium and assessed the presence of null alleles for all loci, resulting in a selection of 14 high-quality polymorphic markers. These markers will be extremely useful in identifying fine-scale genetic structures and providing insight into conservation management plans of this species.

Estimating belowground plant abundance with DNA metabarcoding.

Most work on plant community ecology has been performed above ground, neglecting the processes that occur in the soil. DNA metabarcoding, in which multiple species are computationally identified in bulk samples, can help to overcome the logistical limitations involved in sampling plant communities belowground. However, a major limitation of this methodology is the quantification of species' abundances based on the percentage of sequences assigned to each taxon. Using root tissues of five dominant species in a semi-arid Mediterranean shrubland (Bupleurum fruticescens, Helianthemum cinereum, Linum suffruticosum, Stipa pennata and Thymus vulgaris), we built pairwise mixtures of relative abundance (20%, 50% and 80% biomass), and implemented two methods (linear model fits and correction indices) to improve estimates of root biomass. We validated both methods with multispecies mixtures that simulate field-collected samples. For all species, we found a positive and highly significant relationship between the percentage of sequences and biomass in the mixtures (R2  = .44-.66), but the equations for each species (slope and intercept) differed among them, and two species were consistently over- and under-estimated. The correction indices greatly improved the estimates of biomass percentage for all five species in the multispecies mixtures, and reduced the overall error from 17% to 6%. Our results show that, through the use of post-sequencing quantification methods on mock communities, DNA metabarcoding can be effectively used to determine not only species' presence but also their relative abundance in field samples of root mixtures. Importantly, knowledge of these aspects will allow us to study key, yet poorly understood, belowground processes.

Enabling large-scale feather mite studies: an Illumina DNA metabarcoding pipeline.

Feather mites are among the most common and diverse ectosymbionts of birds, yet basic questions such as the nature of their relationship remain largely unanswered. One reason for feather mites being understudied is that their morphological identification is often virtually impossible when using female or young individuals. Even for adult male specimens this task is tedious and requires advanced taxonomic expertise, thus hampering large-scale studies. In addition, molecular-based methods are challenging because the low DNA amounts usually obtained from these tiny mites do not reach the levels required for high-throughput sequencing. This work aims to overcome these issues by using a DNA metabarcoding approach to accurately identify and quantify the feather mite species present in a sample. DNA metabarcoding is a widely used molecular technique that takes advantage of high-throughput sequencing methodologies to assign the taxonomic identity to all the organisms present in a complex sample (i.e., a sample made up of multiple specimens that are hard or impossible to individualise). We present a high-throughput method for feather mite identification using a fragment of the COI gene as marker and Illumina Miseq technology. We tested this method by performing two experiments plus a field test over a total of 11,861 individual mites (5360 of which were also morphologically identified). In the first experiment, we tested the probability of detecting a single feather mite in a heterogeneous pool of non-conspecific individuals. In the second experiment, we made 2 × 2 combinations of species and studied the relationship between the proportion of individuals of a given species in a sample and the proportion of sequences retrieved to test whether DNA metabarcoding can reliably quantify the relative abundance of mites in a sample. Here we also tested the efficacy of degenerate primers (i.e., a mixture of similar primers that differ in one or several bases that are designed to increase the chance of annealing) and investigated the relationship between the number of mismatches and PCR success. Finally, we applied our DNA metabarcoding pipeline to a total of 6501 unidentified and unsorted feather mite individuals sampled from 380 European passerine birds belonging to 10 bird species (field test). Our results show that this proposed pipeline is suitable for correct identification and quantitative estimation of the relative abundance of feather mite species in complex samples, especially when dealing with a moderate number (> 30) of individuals per sample.

Polymorphic microsatellite markers in Anthoxanthum (Poaceae) and cross-amplification in the Eurasian complex of the genus.

PREMISE OF THE STUDY: Nonplastid microsatellite primers were developed for the first time in the Euro-Siberian complex of Anthoxanthum (Poaceae), a genus of temperate grasses in which reticulate evolution is common. METHODS AND RESULTS: A microsatellite-enriched genomic DNA library allowed the detection of 500 fragments containing a microsatellite motif. Fifteen primer pairs were selected for an extended primer test. A preliminary analysis was conducted on the Eurasian diploid lineages of Anthoxanthum, with special emphasis on three populations of the Mediterranean A. aristatum-A. ovatum complex. Thirteen out of 15 markers tested were polymorphic in the complex, with successful cross-amplification in A. odoratum (93% polymorphic loci), A. amarum (73% polymorphic), A. alpinum (73% polymorphic), and A. maderense (60% polymorphic). CONCLUSIONS: These microsatellite markers will enable the analysis of evolution and phylogeography in diploid and polyploid lineages of this important genus.

Phylogeography of the Spanish Moon Moth Graellsia isabellae (Lepidoptera, Saturniidae).

BACKGROUND: Geographic and demographic factors as well as specialisation to a new host-plant may lead to host-associated differentiation in plant-feeding insects. We explored the phylogeography of a protected moth, Graellsia isabellae, and its two recognised host-plant species (Pinus sylvestris and P. nigra) in order to seek for any concordance useful to disentangle the evolutionary history of this iconic lepidopteran. RESULTS: DNA variation in one mitochondrial marker and nine nuclear microsatellite loci revealed a strong phylogeographic pattern across 28 populations of G. isabellae studied in Spain and France comprising six groups mostly distributed along different mountain ranges. Reanalysis of a previously published chloroplast microsatellite dataset revealed a three and two-group structure for Spanish P. sylvestris and P. nigra, respectively. Overall, the population groupings of this protected moth did not match the ones of P. sylvestris and P. nigra. CONCLUSIONS: There was no evidence of host-associated differentiation between populations using P. sylvestris and the ones inhabiting P. nigra. The two major mitochondrial clades of G. isabellae likely diverged before the Last Glacial Maximum and geographically separated the species into a "southern" (Central and Southern Iberian clusters) and a "northern" lineage (Eastern Iberian, Pyrenean and French Alpine clusters). The Eastern Iberian System, where this insect uses both host-plants, harboured the highest level of genetic diversity. Such a group independently colonised the West and East parts of the Pyrenees. Our results point to a native origin for the French populations occurring in the Alps, genetically related to the Eastern Iberian and Pyrenean sites. The Central Iberian group derived from Southern Iberian ancestors. Secondary contacts were inferred between the Southern/Central Iberian populations and Eastern Iberian cluster as well as between the two Pyrenean ones. The mito-nuclear discordance observed with regard to the Eastern Iberian cluster is congruent with a secondary contact after the evolution of mito-nuclear incompatibilities in geographically isolated areas.

Identification of the sex pheromone of a protected species, the Spanish moon moth Graellsia isabellae.

Sex attractant pheromones are highly sensitive and selective tools for detecting and monitoring populations of insects, yet there has been only one reported case of pheromones being used to monitor protected species. Here, we report the identification and synthesis of the sex pheromone of a protected European moth species, Graellsia isabellae (Lepidoptera: Saturniidae), as the single component, (4E,6E,11Z)-hexadecatrienal. In preliminary field trials, lures loaded with this compound attracted male moths from populations of this species at a number of widely separated field sites in France, Switzerland, and Spain, clearly demonstrating the utility of pheromones in sampling potentially endangered insect species.