Elena G. Gonzalez

Relative role of life-history traits and historical factors in shaping genetic population structure of sardines (Sardina pilchardus)

BackgroundMarine pelagic fishes exhibit rather complex patterns of genetic differentiation, which are the result of both historical processes and present day gene flow. Comparative multi-locus analyses based on both nuclear and mitochondrial genetic markers are probably the most efficient and informative approach to discerning the relative role of historical events and life-history traits in shaping genetic heterogeneity. The European sardine (Sardina pilchardus) is a small pelagic fish with a relatively high migratory capability that is expected to show low levels of genetic differentiation among populations. Previous genetic studies based on meristic and mitochondrial control region haplotype frequency data supported the existence of two sardine subspecies (S. p. pilchardus and S. p. sardina).ResultsWe investigated genetic structure of sardine among nine locations in the Atlantic Ocean and Mediterranean Sea using allelic size variation of eight specific microsatellite loci. Bayesian clustering and assignment tests, maximum likelihood estimates of migration rates, as well as classical genetic-variance-based methods (hierarchical AMOVA test and RSTpairwise comparisons) supported a single evolutionary unit for sardines. These analyses only detected weak but significant genetic differentiation, which followed an isolation-by-distance pattern according to Mantel test.ConclusionWe suggest that the discordant genetic structuring patterns inferred based on mitochondrial and microsatellite data might indicate that the two different classes of molecular markers may be reflecting different and complementary aspects of the evolutionary history of sardine. Mitochondrial data might be reflecting past isolation of sardine populations into two distinct groupings during Pleistocene whereas microsatellite data reveal the existence of present day gene flow among populations, and a pattern of isolation by distance.

Genetic structuring and migration patterns of Atlantic bigeye tuna, Thunnus obesus(Lowe, 1839)

BackgroundLarge pelagic fishes are generally thought to have little population genetic structuring based on their cosmopolitan distribution, large population sizes and high dispersal capacities. However, gene flow can be influenced by ecological (e.g. homing behaviour) and physical (e.g. present-day ocean currents, past changes in sea temperature and levels) factors. In this regard, Atlantic bigeye tuna shows an interesting genetic structuring pattern with two highly divergent mitochondrial clades (Clades I and II), which are assumed to have been originated during the last Pleistocene glacial maxima. We assess genetic structure patterns of Atlantic bigeye tuna at the nuclear level, and compare them with mitochondrial evidence.ResultsWe examined allele size variation of nine microsatellite loci in 380 individuals from the Gulf of Guinea, Canary, Azores, Canada, Indian Ocean, and Pacific Ocean. To investigate temporal stability of genetic structure, three Atlantic Ocean sites were re-sampled a second year. Hierarchical AMOVA tests, RSTpairwise comparisons, isolation by distance (Mantel) tests, Bayesian clustering analyses, and coalescence-based migration rate inferences supported unrestricted gene flow within the Atlantic Ocean at the nuclear level, and therefore interbreeding between individuals belonging to both mitochondrial clades. Moreover, departures from HWE in several loci were inferred for the samples of Guinea, and attributed to a Wahlund effect supporting the role of this region as a spawning and nursery area. Our microsatellite data supported a single worldwide panmictic unit for bigeye tunas. Despite the strong Agulhas Current, immigration rates seem to be higher from the Atlantic Ocean into the Indo-Pacific Ocean, but the actual number of individuals moving per generation is relatively low compared to the large population sizes inhabiting each ocean basin.ConclusionLack of congruence between mt and nuclear evidences, which is also found in other species, most likely reflects past events of isolation and secondary contact. Given the inferred relatively low number of immigrants per generation around the Cape of Good Hope, the proportions of the mitochondrial clades in the different oceans may keep stable, and it seems plausible that the presence of individuals belonging to the mt Clade I in the Atlantic Ocean may be due to extensive migrations that predated the last glaciation.

Population Proteomics of the European Hake (Merluccius merluccius)

We report the novel use of proteomics to investigate protein variation among populations of the European hake (Merluccius merluccius). The liver and brain extracts of 18 hake (N = 36) captured in the Mediterranean Sea, Cantabrian Sea, and Atlantic Ocean were examined by 2D/DIGE and mass spectrometry. Significant differences in protein expression among populations were revealed by 84 spots obtained in the gels for the liver and 145 spots for the brain. Population groups of samples were defined by multivariate analysis (PCA and hierarchical clustering). According to protein expression levels and the functions of the 55 candidate protein spots identified, which showed significant expression differences, highest population discrimination was rendered by brain proteins involved in cell signaling and metabolism/energy and by liver proteins involved in protein fate. Finally, we present a statistically robust framework to accurately classify individuals according to their population of origin. Thus, purposely identified protein isoforms were found to be competent at discriminating populations. These results suggest the possibility of identifying protein biomarkers related to environmental changes in a nonmodel species such as the hake and pave the way for more extensive research on protein variation among populations of marine fishes.

Improvement of 96-well microplate assay for estimation of cell growth and inhibition of Leishmania with Alamar Blue

The value of resazurin-based Alamar Blue redox indicator to determine multiplication of Leishmania promastigotes in 96-well microtiter plates was examined. In addition, assay was validated with amphotericin B (AmB) and allicin. The method was tested on L.donovani and L.infantum promastigotes under different culture conditions (variable air-phase, presence of phenol red, initial cell density, incubation time, use of Hepes buffer). Results showed that the gas-phase of promastigote cultures was critical. The method yielded consistent results with initial plating cell densities of 2.5 × 10⁵ promastigotes/well, up to 72 h incubation and 5% CO₂ atmosphere or reduced air availability (sealed plastic bags, film-sealed microplates). Detection of low numbers of promastigotes and earlier results could be obtained using fluorimetry instead of spectrophotometry. The addition of 20 mM Hepes improved the results. Fluorescence intensity correlated to promastigotes number in both Leishmania spp. Inhibitory concentration (IC₅₀) values for AmB and allicin using cell counting and fluorimetry were comparable. Under these conditions this one-step, low-cost redox indicator can be used in drug sensitivity assays and studies of differential proliferation rates of Leishmania isolates or strains in a 96-well format.

Genetic and demographic recovery of an isolated population of brown bear Ursus arctos L., 1758.

The brown bear Ursus arctos L., 1758 population of the Cantabrian Mountains (northwestern Spain) became isolated from other bear populations in Europe about 500 years ago and has declined due to hunting and habitat degradation. At the beginning of the 20th century, the Cantabrian population split into eastern and western subpopulations, and genetic exchange between them ceased. In the early 1990s, total population size was estimated to be < 100 bears. Subsequently, reduction in human-caused mortality has brought about an increase in numbers, mainly in the western subpopulation, likely promoting male-mediated migration and gene flow from the western nucleus to the eastern. To evaluate the possible genetic recovery of the small and genetically depauperate eastern subpopulation, in 2013 and 2014 we genotyped hair and faeces samples (116 from the eastern subpopulation and 36 from the western) for 18 microsatellite markers. Data from the annual count of females with cubs of the year (COY) during the past twenty-six years was used to analyze demographic changes. The number of females with COY fell to a minimum of seven in the western and three in eastern subpopulations in the biennium 1993–1994 and reached a respective maximum of 54 and 10 individuals in 2013–2014. We also observed increased bear dispersal and gene flow, mainly from the western to the eastern subpopulation. Of the 26 unique genotypes detected in the eastern subpopulation, 14 (54%) presented an admixture composition, and seven (27%) were determined to be migrants from the western subpopulation. Hence, the two separated and clearly structured subpopulations identified in the past currently show some degree of genetic admixture. This research shows the partial demographic recovery and a change in genetic composition due to migration process in a population of bears that has been isolated for several centuries.

Contrasting demographic history and gene flow patterns of two mangrove species on either side of the Central American Isthmus

Comparative phylogeography offers a unique opportunity to understand the interplay between past environmental events and life-history traits on diversification of unrelated but co-distributed species. Here, we examined the effects of the quaternary climate fluctuations and palaeomarine currents and present-day marine currents on the extant patterns of genetic diversity in the two most conspicuous mangrove species of the Neotropics. The black (Avicennia germinans, Avicenniaceae) and the red (Rhizophora mangle, Rhizophoraceae) mangroves have similar geographic ranges but are very distantly related and show striking differences on their life-history traits. We sampled 18 Atlantic and 26 Pacific locations for A. germinans (N = 292) and R. mangle (N = 422). We performed coalescence simulations using microsatellite diversity to test for evidence of population change associated with quaternary climate fluctuations. In addition, we examined whether patterns of genetic variation were consistent with the directions of major marine (historical and present day) currents in the region. Our demographic analysis was grounded within a phylogeographic framework provided by the sequence analysis of two chloroplasts and one flanking microsatellite region in a subsample of individuals. The two mangrove species shared similar biogeographic histories including: (1) strong genetic breaks between Atlantic and Pacific ocean basins associated with the final closure of the Central American Isthmus (CAI), (2) evidence for simultaneous population declines between the mid-Pleistocene and early Holocene, (3) asymmetric historical migration with higher gene flow from the Atlantic to the Pacific oceans following the direction of the palaeomarine current, and (4) contemporary gene flow between West Africa and South America following the major Atlantic Ocean currents. Despite the remarkable differences in life-history traits of mangrove species, which should have had a strong influence on seed dispersal capability and, thus, population connectivity, we found that vicariant events, climate fluctuations and marine currents have shaped the distribution of genetic diversity in strikingly similar ways.

Polymorphic microsatellite markers for the critically endangered Balearic shearwater, Puffinus mauretanicus.

Ten novel polymorphic microsatellite loci were isolated and characterized from the Balearic shearwater (Puffinus mauretanicus), a critically endangered seabird. The developed loci revealed a relatively low number of alleles per locus, as well as low levels of polymorphism (HO = 0.377 ± 0.241). One of the loci appeared to be W‐linked. All polymorphic loci were successfully amplified in its closely related species, the Yelkouan shearwater (Puffinus yelkouan). These microsatellite markers would be useful for assessing population structure in the Balearic shearwater and the possible hybridization process between both shearwaters species.

Comparative pattern of genetic structure in two Mediterranean killifishes Aphanius fasciatus and Aphanius iberus inferred from both mitochondrial and nuclear data

In this study, genetic variation was assessed in Aphanius fasciatus and Aphanius iberus characterized by similar ecological traits but with very different distribution ranges in the Mediterranean area. Five populations of A. iberus and five of A. fasciatus were analysed using five polymorphic microsatellite loci and partial mitochondrial control region (D-loop) sequences. Congruent results were found with both nuclear and mitochondrial molecular markers. The results showed that similar levels of genetic divergence, based on mitochondrial control region sequences, are present among populations of A. iberus and among populations of A. fasciatus despite the very different geographic distance existing among the examined populations of the two species (low geographic distance in A. iberus and high in A. fasciatus). A possible explanation could be that the populations of A. iberus were isolated for a longer time than the populations of A. fasciatus supporting the hypothesis that the split in the lineage leading to A. iberus is older than the split in the lineage leading to A. fasciatus. The possibility that the wide circum-Mediterranean distribution of A. fasciatus ensures the high connectivity of its populations, preventing, in some cases, local differentiation, however, cannot be ruled out.

Patterns of genetic variation in the endangered European mink (Mustela lutreola L., 1761).

BackgroundThe European mink (Mustela lutreola, L. 1761) is a critically endangered mustelid, which inhabits several main river drainages in Europe. Here, we assess the genetic variation of existing populations of this species, including new sampling sites and additional molecular markers (newly developed microsatellite loci specific to European mink) as compared to previous studies. Probabilistic analyses were used to examine genetic structure within and between existing populations, and to infer phylogeographic processes and past demography.ResultsAccording to both mitochondrial and nuclear microsatellite markers, Northeastern (Russia, Estonia and Belarus) and Southeastern (Romania) European populations showed the highest intraspecific diversity. In contrast, Western European (France and Spain) populations were the least polymorphic, featuring a unique mitochondrial DNA haplotype. The high differentiation values detected between Eastern and Western European populations could be the result of genetic drift in the latter due to population isolation and reduction. Genetic differences among populations were further supported by Bayesian clustering and two main groups were confirmed (Eastern vs. Western Europe) along with two contained subgroups at a more local scale (Northeastern vs. Southeastern Europe; France vs. Spain).ConclusionsGenetic data and performed analyses support a historical scenario of stable European mink populations, not affected by Quaternary climate oscillations in the Late Pleistocene, and posterior expansion events following river connections in both North- and Southeastern European populations. This suggests an eastern refuge during glacial maxima (as already proposed for boreal and continental species). In contrast, Western Europe was colonised more recently following either natural expansions or putative human introductions. Low levels of genetic diversity observed within each studied population suggest recent bottleneck events and stress the urgent need for conservation measures to counteract the demographic decline experienced by the European mink.

Island survivors: population genetic structure and demography of the critically endangered giant lizard of La Gomera, Gallotia bravoana

BackgroundThe giant lizard of La Gomera (Gallotia bravoana), is an endemic lacertid of this Canary Island that lives confined to a very restricted area of occupancy in a steep cliff, and is catalogued as Critically Endangered by IUCN. We present the first population genetic analysis of the wild population as well as of captive-born individuals (for which paternity data are available) from a recovery center. Current genetic variability, and inferred past demographic changes were determined in order to discern the relative contribution of natural versus human-mediated effects on the observed decline in population size.ResultsGenetic analyses indicate that the only known natural population of the species shows low genetic diversity and acts as a single evolutionary unit. Demographic analyses inferred a prolonged decline of the species for at least 230 generations. Depending on the assumed generation time, the onset of the decline was dated between 1200-13000 years ago. Pedigree analyses of captive individuals suggest that reproductive behavior of the giant lizard of La Gomera may include polyandry, multiple paternity and female long-term sperm retention.ConclusionsThe current low genetic diversity of G. bravoana is the result of a long-term gradual decline. Because generation time is unknown in this lizard and estimates had large credibility intervals, it is not possible to determine the relative contribution of humans in the collapse of the population. Shorter generation times would favor a stronger influence of human pressure whereas longer generation times would favor a climate-induced origin of the decline. In any case, our analyses show that the wild population has survived for a long period of time with low levels of genetic diversity and a small effective population size. Reproductive behavior may have acted as an important inbreeding avoidance mechanism allowing the species to elude extinction. Overall, our results suggest that the species retains its adaptive potential and could restore its ancient genetic diversity under favorable conditions. Therefore, management of the giant lizard of La Gomera should concentrate efforts on enhancing population growth rates through captive breeding of the species as well as on restoring the carrying capacity of its natural habitat.