Goncalo Silva

ISPA Instituto Universitário | ISPA

Small pelagic fishes have the ability to disperse over long distances and may present complex evolutionary histories. Here, Old World Anchovies (OWA) were used as a model system to understand genetic patterns and connectivity of fish between the Atlantic and Pacific basins. We surveyed 16 locations worldwide using mtDNA and 8 microsatellite loci for genetic parameters, and mtDNA (cyt b; 16S) and nuclear (RAG1; RAG2) regions for dating major lineage-splitting events within Engraulidae family. The OWA genetic divergences (0–0.4%) are compatible with intra-specific divergence, showing evidence of both ancient and contemporary admixture between the Pacific and Atlantic populations, enhanced by high asymmetrical migration from the Pacific to the Atlantic. The estimated divergence between Atlantic and Pacific anchovies (0.67 [0.53–0.80] Ma) matches a severe drop of sea temperature during the Günz glacial stage of the Pleistocene. Our results support an alternative evolutionary scenario for the OWA, suggesting a coastal migration along south Asia, Middle East and eastern Africa continental platforms, followed by the colonization of the Atlantic via the Cape of the Good Hope.

Natural populations of widely distributed organisms often exhibit genetic clinal variation over their geographical ranges. The European anchovy, Engraulis encrasicolus, illustrates this by displaying a two-clade mitochondrial structure clinally arranged along the eastern Atlantic. One clade has low frequencies at higher latitudes, whereas the other has an anti-tropical distribution, with frequencies decreasing towards the tropics. The distribution pattern of these clades has been explained as a consequence of secondary contact after an ancient geographical isolation. However, it is not unlikely that selection acts on mitochondria whose genes are involved in relevant oxidative phosphorylation processes. In this study, we performed selection tests on a fragment of 1044 bp of the mitochondrial cytochrome b gene using 455 individuals from 18 locations. We also tested correlations of six environmental features: temperature, salinity, apparent oxygen utilization and nutrient concentrations of phosphate, nitrate and silicate, on a compilation of mitochondrial clade frequencies from 66 sampling sites comprising 2776 specimens from previously published studies. Positive selection in a single codon was detected predominantly (99%) in the anti-tropical clade and temperature was the most relevant environmental predictor, contributing with 59% of the variance in the geographical distribution of clade frequencies. These findings strongly suggest that temperature is shaping the contemporary distribution of mitochondrial DNA clade frequencies in the European anchovy.

AimAs part of an emerging effort to understand the role played by climatic fluctuations in shaping the geographical distributions and abundances of marine organisms, we examined the genetic patterns of leading-edge populations in the European anchovy, Engraulis encrasicolus, and its American counterpart, the morphologically similar silver anchovy, Engraulis eurystole, in the North Atlantic Ocean. LocationAdults were collected from the western Atlantic, eastern Atlantic (from Norway to Ghana) and western Mediterranean. MethodsA 1045 bp fragment of the mtDNA cytochrome b gene was sequenced (n = 312) and nine microsatellite loci were genotyped (n = 462) for anchovies from 13 locations across the temperate North Atlantic. Populations were surveyed for diversity and differentiation with a range of summary statistics. Multivariate discriminant analysis of principal components was employed to detect the number of genetic clusters in the data and assign individuals to populations based on their microsatellite genotypes. Historical demographic inferences – mismatch distributions and Bayesian skyline plots – were used to observe population size changes relating to climatic oscillations. ResultsTwo mitochondrial clades were recovered, consistent with previous studies of E. encrasicolus, in which the frequency of each clade varied by latitude. Four genetic clusters corresponding loosely to large geographical regions were identified with microsatellite data. The north-western Atlantic E. eurystole was not reciprocally monophyletic for either mtDNA or microsatellite analyses and is probably conspecific with E. encrasicolus. Genetic diversity peaked in Iberian populations, but differences in genetic diversity were only statistically significant for the least diverse population, Tangier. The indications of demographic expansion were more pronounced in the southern clade and both mtDNA clades exhibited genetic diversity and expansion imprints that are likely to be older than climatic oscillations of the recent Pleistocene. Main conclusionsThe highly mobile nature of anchovies has allowed them to track their optimal thermal physiological conditions during the extreme climate shifts of the Last Glacial Maximum and avoid wholesale population reductions and genetic bottlenecks. Both north-eastern and north-western Atlantic were probably rapidly recolonized after the Last Glacial Maximum by large numbers of anchovies, such that leading-edge populations retained the genetic diversity of parent populations.

Genetic variation (mtDNA) of the European conger eel, Conger conger, was compared across five locations in the north-eastern Atlantic (Madeira, Azores, South Portugal, North Portugal and Ireland) and one location in the western Mediterranean (Mallorca). Genetic diversity of conger eel was high, and differentiation among regions was not significant. Additionally, comparisons of element:Ca ratios (Sr:Ca, Ba:Ca, Mn:Ca and Mg:Ca) in otolith cores (larval phase) and edges (3 months prior to capture) among the Azores, North Portugal, Madeira and Mallorca regions for 2 years indicated that variation among regions were greater for edges than cores. Therefore, while benthic conger may display residency at regional scales, recruitment may not necessarily be derived from local spawning and larval retention. Furthermore, data from ot-oliths suggest a separated replenishment source for western Mediterranean and NE Atlantic stocks. The combination of genetics and otolith chemistry suggests a population model for conger eel involving a broad-scale dispersal of larvae, with limited connectivity for benthic juvenile life stages at large spatial scales, although the existence of one or mul-tiple spawning grounds for the species remains uncertain.

This poster presentation is an output of SardiTemp project (FCT and POLISBOA funding; ref. LISBOA-01-0145-FEDER-032209) that results from a collaboration between several institutions. This poster presentation aims to explain the different approaches to unveil how climate change will impact marine fish, in particular small pelagic fish, using the European sardine Sardina pilchardus as a model system. The project started in July 2018 and will last until June 2021.

The Working Group on the Application of Genetics in Fisheries and Mariculture (WGAGFM) met in Olhão, Portugal, 2–5 May 2017. Nineteen participants from 11 countries discussed the four Terms of Reference (ToR) and associated matters. Specifically, there was a focused discussion concerning the proposal by the ICES Science Committee to establish Aquaculture Steering Group (ASG). It was agreed unanimously that the WGAGFM, would wish to be affiliated to the new steering group, while retaining collective interest in capture fisheries. It was further agreed that the WGAGFM would submit an application to coordinate an ICES training course broadly on the Application of Genetics and Genomics to Fisheries and Aquaculture Science. Four multiannual ToRs were considered and finalised. ToR (a) finalised consideration of infectious disease and parasite spread from seafood into wild populations. A schematic overview was designed to provide a tool for assessing infection risk based on infectious and transmission potential across specified scenarios of host-pathogen infection. The highest infection risk (value 10) corresponds to symptomatic live seafood carrying live pathogens. Moreover, a workflow to aid decision-making when analysing pathogen samples from seafood was constructed, depicting the most appropriate methods to employ, while enhancing detection, robust quantification, and assessment of viability. The mechanisms for integrating WGAGFM advice into fisheries assessment and management, ToR (b), focused on additional mechanisms for enhancing awareness of WGAGFM activities, expertise, and contributions to ICES and beyond. Such mechanisms were discussed in relation to practical applications of advances in genetics and genomics, most notably considering such issues as meta-barcoding, environmental DNA (eDNA), disease diagnosis, analysis of microbiomes, quantitative genetics of wild populations, targeting specific functional genes, and novel genetic methods to estimate population abundance. In relation to the promotion of WGAGFM activities, while interactions among potential complimentary ICES working groups is not extensive, several information requests between 2015–2017 (e.g. from the Working Group on Integrated Morphological and Molecular Taxonomy (WGIMT), Benchmark Workshop on Northern Haddock Stocks (WKHAD) and the Stock Identification Methods Working Group (SIMWG), indicates active complementarity. To promote awareness and impact of WGAGFM, two new methods of dissemination, in addition to existing ICES channels were identified: First, creation of a “Project” in “Researchgate.com” to reach the scientific community in a more targeted way (https://www.researchgate.net/project/ICES-WGAGFM-Working-Groupon-Application-of-Genetics-in-Fisheries-and-Mariculture), and design of a 2-page leaflet for targeting industry, management, national governments, EU, FAO, research councils etc. (attached herein). The challenges of elucidating the genetic basis of adaptive shifts in exploited species was considered further in relation to advances in methods and application of quantitative genetic analysis (ToR (c)). Particular emphasis was based on the range of phenotypic traits relevant to exploitation, captive propagation and environmental change, and the potential for rapid genetically-based shifts in such traits across both natural and farmed environments. An outline was produced of the scope of quantitative genetic based methods, of pedigree- and pedigree-free genomic mapping approaches, and how they can be applied in planning for promotion of evolutionary resilience, sustainable stock exploitation at MSY and in predictions for stock recovery. In particular, it was emphasised that the routine collection of appropriate tissue samples for DNA coupled with phenotypic measures on the same individuals, and associated environmental data, would enable improved monitoring of quantitative genetic change and predictions for response for shifts in harvesting practices, breeding scenarios, and ongoing influences of climate change, invasive species, and habitat deterioration. Finally, ToR (d) focused on recently developed approaches for estimating population abundance in the context of deep sea fisheries. The feasibility of the close-kin approach was assessed by consideration of a model system with sufficient background information, and representative of several deep sea species: the white anglerfish (Lophius piscatorius). Basic simulations using white anglerfish fishing data covering ICES divisions VIIIc and IX (Iberian region), indicate that, assuming a coefficient of variation (CV) of 10%, a sample size of about 17 000 individuals, 8500 adults and 8500 juveniles, would be required to obtain reliable estimates of abundance (i.e. breeding population that in this case, based on existing stock assessments, is believed to be ∼1.5 million individuals) based on the close-kin method. Using the same estimate of abundance (i.e. 1.5 million individuals), further simulations were subsequently carried out to investigate sampling requirements under distinct CV levels and, more specifically, the minimum number of parent-offspring pairs (POPs) to obtain reliable estimates of abundance (i.e. close to “real” value) based on the close-kin approach. The most important parameter was the number of POPs that need to be identified to obtain reliable estimates of abundance. A simple Excel based guide was developed to assist users in choosing the optimal sampling design, together with resource requirements. We propose that the genetic marker of choice will be Single Nucleotide Polymorphisms (SNPs) or microsatellites, ensuring that markers deployed exhibit sufficient statistical power for parentage analysis.

While studying marine organisms with complex life cycles, identifying the patterns, processes, timings and habitats associated with early-life stages becomes critical to our understanding on how populations vary over time. Here, we aimed to characterize the breeding population of a temperate cryptobenthic fish, the ringneck blenny Parablennius pilicornis, and estimate its reproductive output on the nearshore rocky reefs of the Professor Luiz Saldanha Marine Park, a marine protected area on the Portuguese west coast. Underwater visual census were performed during the breeding seasons of 2013 and 2014. A total of 197 females and 156 males were registered, from which 86 males were guarding their nests. An overall mean of 0.06 reproductive males per m2, an average of 14952.27 eggs per nest, and 892.98 eggs per m2 was recorded. Results suggest a positive correlation between the number of eggs and the estimated size of the male defending each nest. The number of adult individuals was stable between years and within the breeding season, despite some degree of variation. Due to high abundance of the ringneck blenny and its mid-trophic level in the food web, the temporal variation of this cryptobenthic species imply a high-energy turnover that will have a significant impact on the local coastal community. To our best knowledge, this is the first time that egg productivity is estimated for a temperate cryptobenthic reef fish species.

Under the hypothesis ”bigger-is-better”, it is expected that larger fish produce larger offspring. Larger initial body sized larvae were found to be able to maintain advantage to important life-history events and therefore have higher chances of survival. Here, we characterized the reproductive male population of the ringneck blenny Parablennius pilicornis and their progeny at Professor Luiz Saldanha Marine Park, Portugal, to investigate the paternal phenotypic influence on offspring morphometric traits, in two breeding seasons of 2014. Reproductive males were, on average, smaller and with larger variance in its morphological characteristics (standard length, body height, head height, body area and body perimeter) in the beginning of the reproductive season (season 1) than in the end (season 2), but sexual secondary characters (papillae diameter and papillae perimeter) tended to be larger in season 1 than in season 2. Although larvae morphometrics revealed large variance within each season and between seasons, larvae were significantly larger in season 1. Correlations between fathers’ and offspring morphometric traits suggested high variability between seasons, where standard lengths were significantly and negatively correlated in season 2 but not in season 1. Therefore, our results indicated that larger adults produced on average smaller larvae. Moreover, father’s contribution on larvae characteristics was highly variable between seasons, with larvae average standard length variation mostly explained by father’s standard length, body area and body height in season 2, while in season 1 this was not significant. These preliminary results suggested that progeny phenotypic traits are highly variable and that mother’s influence and environment may play an important role on the determination of larvae phenotype. To best of our knowledge, this is the first study on temperate cryptobenthic fishes that evaluate fathers’ contributions on progeny in the natural environment.

The European anchovy has been the focus of numerous population genetic studies, most of which exposing high levels of haplotype diversity. However, Keskin and Atar (2012) revealed rather singular results of null haplotype diversities. We therefore call for caution when considering these findings.

Ecological niche modelling (ENM) determines habitat suitability of species by relating records of occurrence to environmental variables. Here, we investigated habitat suitability of four terrestrial slugs of the genus Geomalacus from the Iberian Peninsula using ENM. The potential distribution of these species was estimated using maximum entropy modelling. For this we used published presence records, together with observations from our fieldwork, and 10 layers of environmental variables in a crossvalidation design using ‘minimum predicted area’ as a measure of success. For each species, the models predicted distributions with high accuracy, while restricting predictions to minimum areas. Precipitation, and to a lesser extent temperature, were the most important variables to predict the distributions of the four species. We then compared the predicted distributions with the currently known distributions. For G. anguiformis and G. maculosus the predicted distributions included the known distributions, but also nearby mountain areas where these species have not previously been found. For G. malagensis and G. oliveirae the models predicted much wider distributions. Subsequent dedicated fieldwork could not confirm the presence of G. oliveirae in the newly predicted areas. Conversely, G. malagensis was found at five new and distant localities, including areas in Portugal where the species has not previously been recorded.

In the marine pelagic realm, the lack of prominent barriers to dispersal is thought to facilitate the connectivity of populations and prevent their genetic differentiation. Moreover, small pelagic fishes are generally characterized by large population sizes and consequently low levels of genetic drift. The “central-marginal” theory postulates that abundance and genetic diversity tend to decrease progressively from the centre towards the limits of the distribution of a species, while genetic differentiation increases among marginal populations in response to isolation and adaption to non-optimal conditions in peripheral areas. Here, we test this hypothesis with genetic data for a small pelagic fish, the European Anchovy Engraulis encrasicolus, with samples across the northern limits of species’ distribution. The analysis of a fragment of mitochondrial cytochrome-b gene showed that anchovies exhibit lower genetic diversity and a larger proportion of shared haplotypes among populations at the leading edge of the northern distributional limit than among populations around Iberian Peninsula. Genetic diversities were significantly different between northern and southern locations, even though individual or pooled samples displayed no genetic differentiation between these regions. A single western Atlantic sample from North America showed lower genetic diversity than samples from the Iberian Peninsula and displayed no shared haplotypes with other geographical locations of the species’ distribution in the northeastern Atlantic. A sample from Tangier in the northeastern Atlantic had an exceptionally low level of genetic diversity in comparison with nearby samples. The present results show the effects of a northern post-glacial expansion, whereas southern populations show higher levels of genetic diversity, because these southern (refugial) areas likely represent regions of population persistence during glacial periods. This study supports the central-margin hypothesis for marine pelagic and large population sized organisms.

A total of 60 morphometric traits and nucleotide sequences of the entire mtDNA NADH dehydrogenase subunit 2 (ND2) gene [1047 base pair (bp)] in 23 individuals of blackmouth, Galeus melastomus, and 13 individuals of sawtail catsharks, Galeus atlanticus, caught in Southern Portugal, were examined to test the validity of these two taxa. These sharks closely resemble each other, have overlapping geographical ranges and are difficult to identify by morphological characters. Non-metric multidimensional scaling of morphometric variables indicates a clear separation between the two species, with 10 characters each contributing 2·12–2·45% of the total variability between species. Maximum likelihood, parsimony and neighbour-joining trees revealed two major mtDNA haplotype clades, corresponding to the two species, with an average corrected sequence divergence between them of 3·39 ± 0·56%. Within species divergences between haplotypes averaged 0·27 ± 0·18% in G. melastomus and 0·12 ± 0·08% in G. atlanticus. A total of 35 diagnostic nucleotide site differences and four restriction fragment length polymorphism recognition sites in the ND2 gene can be used to distinguish the two species.

Random Amplified Polymorphic DNA (RAPD-PCR) technique was used to assess the level of genetic variability and genetic relationships among 24 Portuguese isolates of pinewood nematode, Bursaphelenchus xylophilus. The isolates represent the main infested areas of Portugal. Two additional isolates of B. xylophilus representing North America and East Asia were included, and B. mucronatus was used as out-group. Twenty-eight random primers generated a total of 640 DNA fragments. The Nei and Li similarity index revealed a high genetic similarity among the Portuguese isolates (above 90%). Hierarchical cluster analysis was performed to illustrate the relatedness among the isolates. No indication for separate groups among the Portuguese isolates was obtained, and the low level of genetic diversity strongly suggests that they were dispersed recently from a single introduction. The lack of apparent relationship between the genetic and the geographic matrices of the Portuguese isolates limits the use of this technique for following recent pathways of distribution. Genetic distance of the Portuguese isolates towards an isolate from China was much lower as compared to an isolate from the USA. This confirmed previous results suggesting an East Asian origin of the Portuguese B. xylophilus.