Evidence of population structuring following population genetic analyses of Fasciola hepatica from Argentina.

Fasciola hepatica, the liver fluke, is a trematode parasite that causes disease of economic importance in livestock. As a zoonosis this parasite also poses a risk to human health in areas where it is endemic. Population genetic studies can reveal the mechanisms responsible for genetic structuring (non-panmixia) within parasite populations and provide valuable insights into population dynamics, which in turn enables theoretical predictions of evolutionary dynamics such as the evolution of drug resistance. Here we genotyped 320 F. hepatica collected from 14 definitive hosts from four provinces in Argentina. STRUCTURE analysis indicated three population clusters, and principal coordinate analysis confirmed this, showing population clustering across provinces. Similarly, pairwise FST values amongst all four provinces were significant, with standardised pairwise FST (F'ST) ranging from 0.0754 to 0.6327. Therefore, population genetic structure was evident across these four provinces in Argentina. However, there was no evidence of deviation from Hardy-Weinberg equilibrium, so it appears that within these sub-populations there is largely random mating. We identified 263 unique genotypes, which gave a clonal diversity of 82%. Parasites with identical genotypes, clones, accounted for 26.6% of the parasites studied and were found in 12 of the 14 hosts studied, suggesting some clonemate transmission.

Genetic diversity of Fasciola hepatica in Spain and Peru.

In the present study, molecular characterization of Fasciola flukes from Spain was performed to reveal the relation with the previously reported Peruvian F. hepatica population. The nuclear DNA markers, phosphoenolpyruvate carboxykinase (pepck) and DNA polymerase delta (pold), were used for species identification of Fasciola flukes. A total of 196 Fasciola flukes were identified as F. hepatica by pepck and pold, and 26 haplotypes were detected in mitochondrial NADH dehydrogenase subunit 1 (nad1). Only one of them was previously found in Spanish samples; which indicates the existence of high genetic diversity and population structure in F. hepatica from Spain. Three haplotypes were identical to those from Peruvian F. hepatica. The pairwise fixation index value confirmed a relatively close relationship between the Spanish and Peruvian F. hepatica samples. The Spanish samples showed clearly higher genetic variability than the Peruvian population. These results are discussed in relation with the hypothesis of the introduction of the parasite in America from Europe and recent evidence of pre-Hispanic F. hepatica from Argentina revealed by ancient DNA.

Is the "Habsburg jaw" related to inbreeding?

Background: The "Habsburg jaw" has long been associated with inbreeding due to the high prevalence of consanguineous marriages in the Habsburg dynasty. However, it is thought that mandibular prognathism (MP) is under the influence of a dominant major gene.Aim: To investigate the relationship between the "Habsburg jaw" and the pedigree-based inbreeding coefficient (F) as a relative measure of genome homozygosity.Subjects and methods: The degree of MP and maxillary deficiency (MD) of 15 members of the Habsburg dynasty was quantified through the clinical analysis of 18 dysmorphic features diagnosed from 66 portraits.Results: A statistically significant correlation (r = 0.711, p = 0.003) between MP and MD was observed among individuals. Only MP showed a statistically significant positive regression on F as evidenced from univariate analysis (b = 6.36 ± 3.34, p = 0.040) and multivariate analysis (PCA) performed from single dysmorphic features (b = 14.10 ± 6.62, p = 0.027, for the first PC).Conclusion: Both MP and MD are generally involved in the "Habsburg jaw." The results showed a greater sensitivity to inbreeding for the lower third of the face and suggest a positive association between the "Habsburg jaw" and homozygosity and therefore a basically recessive inheritance pattern.

Signatures of selection for bonamiosis resistance in European flat oyster (Ostrea edulis): New genomic tools for breeding programs and management of natural resources.

The European flat oyster (Ostrea edulis) is a highly appreciated mollusk with an important aquaculture production throughout the 20th century, in addition to playing an important role on coastal ecosystems. Overexploitation of natural beds, habitat degradation, introduction of non-native species, and epidemic outbreaks have severely affected this important resource, particularly, the protozoan parasite Bonamia ostreae, which is the main concern affecting its production and conservation. In order to identify genomic regions and markers potentially associated with bonamiosis resistance, six oyster beds distributed throughout the European Atlantic coast were sampled. Three of them have been exposed to this parasite since the early 1980s and showed some degree of innate resistance (long-term affected group, LTA), while the other three were free of B. ostreae at least until sampling date (naïve group, NV). A total of 14,065 SNPs were analyzed, including 37 markers from candidate genes and 14,028 from a medium-density SNP array. Gene diversity was similar between LTA and NV groups suggesting no genetic erosion due to long-term exposure to the parasite, and three population clusters were detected using the whole dataset. Tests for divergent selection between NV and LTA groups detected the presence of a very consistent set of 22 markers, located within a putative single genomic region, which suggests the presence of a major quantitative trait locus associated with B. ostreae resistance. Moreover, 324 outlier loci associated with factors other than bonamiosis were identified allowing fully discrimination of all the oyster beds. A practical tool which included the 84 highest discriminative markers for tracing O. edulis populations was developed and tested with empirical data. Results reported herein could assist the production of stocks with improved resistance to bonamiosis and facilitate the management of oyster beds for recovery production and ecosystem services provided by this species.

Inbreeding in the last ruling dynasty of Portugal: The house of Braganza.

OBJECTIVES: To determine whether: (1) there are high levels of inbreeding in a European royal dynasty that continues until the 20th century, and (2) whether inbreeding is negatively associated with pre-reproductive survival and longevity. METHODS: Genealogical information of all Braganza monarchs (1640-1910) was used to compute the individual's inbreeding coefficient (F) and the coefficient of kinship (θ) of the marriage which were examined in relation to two life-history traits. RESULTS: Mean F of the monarchs was 0.0530, close to that corresponding to the progeny of first cousins (F = 0.0625). A statistically significant effect of maternal inbreeding on offspring longevity (P = 0.037) and a significant effect of individual's F on survival from birth to 10 years (P = 0.023) were detected. CONCLUSIONS: Another European royal dynasty besides the Habsburgs had high levels of inbreeding, especially high during a period after the early modern age. The lineage showed evidence of inbreeding depression. Results support the hypothesis that an increase in maternal homozygosity affects the lifespan of the progeny.

Parallel evolution and adaptation to environmental factors in a marine flatfish: Implications for fisheries and aquaculture management of the turbot (Scophthalmus maximus).

Unraveling adaptive genetic variation represents, in addition to the estimate of population demographic parameters, a cornerstone for the management of aquatic natural living resources, which, in turn, represent the raw material for breeding programs. The turbot (Scophthalmus maximus) is a marine flatfish of high commercial value living on the European continental shelf. While wild populations are declining, aquaculture is flourishing in southern Europe. We evaluated the genetic structure of turbot throughout its natural distribution range (672 individuals; 20 populations) by analyzing allele frequency data from 755 single nucleotide polymorphism discovered and genotyped by double-digest RAD sequencing. The species was structured into four main regions: Baltic Sea, Atlantic Ocean, Adriatic Sea, and Black Sea, with subtle differentiation apparent at the distribution margins of the Atlantic region. Genetic diversity and effective population size estimates were highest in the Atlantic populations, the area of greatest occurrence, while turbot from other regions showed lower levels, reflecting geographical isolation and reduced abundance. Divergent selection was detected within and between the Atlantic Ocean and Baltic Sea regions, and also when comparing these two regions with the Black Sea. Evidence of parallel evolution was detected between the two low salinity regions, the Baltic and Black seas. Correlation between genetic and environmental variation indicated that temperature and salinity were probably the main environmental drivers of selection. Mining around the four genomic regions consistently inferred to be under selection identified candidate genes related to osmoregulation, growth, and resistance to diseases. The new insights are useful for the management of turbot fisheries and aquaculture by providing the baseline for evaluating the consequences of turbot releases from restocking and farming.

Influence of life history traits on the population genetic structure of parasitic helminths: a minireview.

Parasite life history traits influence the rate of gene flow between populations and the effective population size, both of which determine the levels of genetic variability and the geographic distribution of such variability. In this short review targeted to parasitologists, we summarise how life history traits influence the population genetic structure of parasitic helminths. These organisms are characterised by a wide variety of life cycles and are ecologically different from microparasites, which have been studied in more detail. In order to provide the reader a concise review that illustrates key aspects of the subject matter, we have limited ourselves to studying examples selected for their clarity and relevance.

A genome scan for candidate genes involved in the adaptation of turbot (Scophthalmus maximus).

Partitioning phenotypic variance in genotypic and environmental variance may benefit from the population genomic assignment of genes putatively involved in adaptation. We analyzed a total of 256 markers (120 microsatellites and 136 Single Nucleotide Polymorphisms - SNPs), several of them associated to Quantitative Trait Loci (QTL) for growth and resistance to pathologies, with the aim to identify potential adaptive variation in turbot Scophthalmus maximus L. The study area in the Northeastern Atlantic Ocean, from Iberian Peninsula to the Baltic Sea, involves a gradual change in temperature and an abrupt change in salinity conditions. We detected 27 candidate loci putatively under selection. At least four of the five SNPs identified as outliers are located within genes coding for ribosomal proteins or directly related with the production of cellular proteins. One of the detected outliers, previously identified as part of a QTL for growth, is a microsatellite linked to a gene coding for a growth factor receptor. A similar set of outliers was detected when natural populations were compared with a sample subjected to strong artificial selection for growth along four generations. The observed association between FST outliers and growth-related QTL supports the hypothesis of changes in growth as an adaptation to differences in temperature and salinity conditions. However, further work is needed to confirm this hypothesis.

Temporal genetic variation of Fasciola hepatica from sheep in Galicia (NW Spain).

We found low genetic differentiation between two temporal samples of Fasciola hepatica (2006 and 2008) collected from nine sheep of the same flock that shared the same pasture for at least 2 years. However, each sample, represented by four and five infrapopulations respectively, showed strong heterozygote deficits regarding Hardy-Weinberg expectations and a high degree of genetic structure at infrapopulation level. This is an unexpected result since genetic drift should increase temporal variation among years. Our findings are most likely explained by the fact that the parasite can survive many years in the definitive host. Temporal gene flow favored by high longevity probably increases levels of genetic variability of the population but could also contribute to the observed heterozygote deficits within temporal samples and infrapopulations if it favors the Wahlund effect. Despite the homogenizing effect of gene flow, the high genetic divergence observed between infrapopulations is most likely a consequence of strong genetic drift associated to the complexity of the life cycle.

A QSPR-like model for multilocus genotype networks of Fasciola hepatica in Northwest Spain.

Fasciola hepatica is a parasitic trematode that infects wild and domesticated mammals, particularly cattle and sheep, and causes significant economic losses to global livestock production. In the present study, we used codominant genetic markers to define and build, for the first time, complex genotype networks for F. hepatica isolated from cattle and sheep in NW Spain. We generated three types of random networks with a number of nodes and edges as close as possible to the observed networks, and we then calculated 14 node centrality measures for both observed and random networks. Finally, using Linear Discriminant Analysis (LDA) and these measures as inputs, we constructed a quantitative structure-property relationship (QSPR)-like model able to predict the propensity of a specific genotype of F. hepatica to infect different infrapopulations, farms and/or host species. The accuracy, sensitivity and specificity of the model were >90% for both training and cross-validation series. We also assessed the applicability domain of the model. This type of QSPR model is a potentially powerful tool for epidemiological studies and could be used to manage and prevent the spread of fasciolosis.

Development and Validation of Single Nucleotide Polymorphisms (SNPs) Markers from Two Transcriptome 454-Runs of Turbot (Scophthalmus maximus) Using High-Throughput Genotyping.

The turbot (Scophthalmus maximus) is a commercially valuable flatfish and one of the most promising aquaculture species in Europe. Two transcriptome 454-pyrosequencing runs were used in order to detect Single Nucleotide Polymorphisms (SNPs) in genes related to immune response and gonad differentiation. A total of 866 true SNPs were detected in 140 different contigs representing 262,093 bp as a whole. Only one true SNP was analyzed in each contig. One hundred and thirteen SNPs out of the 140 analyzed were feasible (genotyped), while Ш were polymorphic in a wild population. Transition/transversion ratio (1.354) was similar to that observed in other fish studies. Unbiased gene diversity (He) estimates ranged from 0.060 to 0.510 (mean = 0.351), minimum allele frequency (MAF) from 0.030 to 0.500 (mean = 0.259) and all loci were in Hardy-Weinberg equilibrium after Bonferroni correction. A large number of SNPs (49) were located in the coding region, 33 representing synonymous and 16 non-synonymous changes. Most SNP-containing genes were related to immune response and gonad differentiation processes, and could be candidates for functional changes leading to phenotypic changes. These markers will be useful for population screening to look for adaptive variation in wild and domestic turbot.

Contrasting patterns of population genetic structure of Fasciola hepatica from cattle and sheep: implications for the evolution of anthelmintic resistance.

Twelve polymorphic genetic markers, eight allozymic loci and four microsatellites, were used to characterize 20 infrapopulations of Fasciola hepatica (all flukes from 10 individual cattle and 10 sheep) from 11 farms in Northwest Spain. Results suggest different patterns of population genetic structure depending on the host species. Individuals identified as clones were much more frequent in sheep. The common presence of clones and its nonrandom occurrence among individual hosts suggests clumped transmission of liver flukes in sheep. After reducing significant repeated multilocus genotypes to one unique copy within infrapopulations, results show relatively high levels of gene diversity within infrapopulations from cattle and sheep (0.411 and 0.360 on average, respectively). However, parasites of sheep appear to show significantly more structured variation at the infrapopulation level (Standardized F(ST)=0.087 and 0.170 for parasites of cattle and sheep, respectively). Compared to the parasites from cattle, results suggest that populations from sheep show lower levels of gene flow, higher degree of aggregate transmission, higher probability of mating within clones, and lower parasitic load. These differences have implications for the evolution of anthelmintic resistance because they affect the effective population size and the degree of inbreeding. The development and rapid spread of resistance seems likely in the parasites of cattle because populations from the study area are characterized by high gene flow. However, results also suggest that the efficient selection of a new recessive advantageous mutation would be favored in parasites of sheep due to a greater potential for inbreeding.

Microsatellite marker development in the protozoan parasite Perkinsus olseni.

The analysis of an enriched partial genomic library and of public expressed sequence tag (EST) resources allowed the characterization of the first microsatellite loci in the protozoan parasite Perkinsus olseni. Clonal cultures from laboratory isolates derived from infected clams Ruditapes decussatus (from Spain), R. philippinarum (from Spain and Japan), and Austrovenus stutchburyi (from New Zealand) were used for the characterization of 12 microsatellites. Low variation was detected at most loci, with the number of alleles at polymorphic loci ranging from 2 to 7 (average 3.20 +/- 0.51) and gene diversity from 0.11 to 0.79 (average 0.40 +/- 0.07). Preliminary results show that (1) isolates of P. olseni are diploid cells, and (2) multiple infections can occur within a single host. Eight of the loci analyzed successfully cross-amplified in the congeneric species P. mediterraneus. These microsatellite markers will be useful to analyze in detail the population genetic structure of P. olseni, crucial for the efficient management of this parasitic disease.

More than meets the eye: detecting cryptic microgeographic population structure in a parasite with a complex life cycle.

Nonrandom recruitment of parasites among hosts can lead to genetic differentiation among hosts and mating dynamics that promote inbreeding. It has been hypothesized that strictly aquatic parasites with intermediate hosts will behave as panmictic populations among hosts because ample opportunity exists for random mixing of unrelated individuals during transmission to the definitive host. A previous allozyme study on the marine trematode Lecithochirium fusiforme did not support this hypothesis; in that, there was genetic differentiation among, and significant heterozygote deficiencies within, definitive hosts. We revisit this system and use microsatellites to obtain multilocus genotypes. Our goal was to determine whether cryptic subgroups and/or the presence of clones could account for the apparent deviation from 'panmixia'. We find strong evidence for cryptic subdivision (three genetic clusters) that causes the Wahlund effect and differentiation among definitive hosts. After accounting for these cryptic groups, we see panmictic genetic structure among definitive hosts that is consistent with the 'high mixing in aquatic habitats' hypothesis. We see evidence for cotransmission of clones in all three clusters, but this level of clonal structure did not have a major impact in causing deviations from Hardy-Weinberg equilibrium, and only affected genetic differentiation among hosts in one cluster. A cursory examination of the data may have led to incorrect conclusions about nonrandom transmission. However, it is obvious in this system that there is more than meets the eye in relation to the actual make-up of parasite populations. In general, the methods we employ will be useful for elucidating hidden patterns in other organisms where cryptic structure may be common (e.g. those with limited morphology or complex life histories).

Very low microsatellite polymorphism and large heterozygote deficits suggest founder effects and cryptic structure in the parasite Perkinsus olseni.

Twelve microsatellite markers were used to characterize 130 clonal cultures of Perkinsus olseni derived from 30 clams from six different geographic locations. Only two loci were polymorphic in the four populations studied from Spanish coast (mean sample size = 31.2), and a third locus was variable in only two populations. In contrast, five parasites isolated from five clams from Japan and New Zealand showed variation at nine loci. Low genetic variation (2.08 ± 0.64 alleles per locus; mean genetic diversity: 0.101 ± 0.022), and very high F(IS) values (0.857 on average) were observed in Spanish populations. A total of 39 multilocus genotypes (MLGs) were identified in the whole sample (121 clonal isolates after excluding incomplete MLGs due to missing data). A three-level hierarchical analysis of molecular variance found significant levels of genetic variation within infrapopulations (all the parasites in a single host; Φ(IS) = 0.679) and among infrapopulations within the component population (all the parasites among a host population; Φ(SC) = 0.579). Differences among the component population from different geographic locations were not significant (Φ(CT) = 0.057). These results suggest that an important fraction of F(IS) is explained by the Wahlund effect, but also strong inbreeding within infrapopulations. Another explanation for the high F(IS) within infrapopulations is the presence of haploid and diploid stages in the clam. Although fully aquatic system provides many opportunities for mixing of parasites from different clams, results are consistent with the consideration of all P. olseni in a clam as a cohesive genetic unit (i.e., deme). If the parasite was introduced into the Spanish coast with the importation of infected clams from Asia and Oceania, the low microsatellite polymorphism could be reflecting founder effects in the recent evolutionary history of P. olseni. The loss of alleles would be intensified in a scenario structured in numerous demes because of recurrent founder effects at microgeographic level.

Allozyme markers suitable for population genetic analysis of Fasciola hepatica.

Protein electrophoresis was used to study allozyme variation in Fasciola hepatica collected from three locations in Galicia (NW Spain), an area where fascioliasis is endemic. Eleven of 16 loci showed variation in at least one population and 7 loci were polymorphic in all populations studied. Five of these markers showed expected heterozygosities ranging from 0.137 to 0.569. The Nei's unbiased genetic diversity within populations ranged from 0.146 to 0.168. Genotypic frequencies were consistent with panmixia in 25 of 28 cases. Only 2 loci showed a significant deficit of heterozygotes. Genetic distances between populations were small (D(a)=0.003-0.010). These results suggest high levels of genetic variability and low population structure. This study shows that several of the markers developed are useful for study the population genetic structure of the parasite, which is essential to investigate the evolution of drug resistance that has recently emerged in populations of the study area.

Identification of the major sex-determining region of turbot (Scophthalmus maximus).

Sex determination in fish is a labile character in evolutionary terms. The sex-determining (SD) master gene can differ even between closely related fish species. This group is an interesting model for studying the evolution of the SD region and the gonadal differentiation pathway. The turbot (Scophthalmus maximus) is a flatfish of great commercial value, where a strong sexual dimorphism exists for growth rate. Following a QTL and marker association approach in five families and a natural population, we identified the main SD region of turbot at the proximal end of linkage group (LG) 5, close to the SmaUSC-E30 marker. The refined map of this region suggested that this marker would be 2.6 cM and 1.4 Mb from the putative SD gene. This region appeared mostly undifferentiated between males and females, and no relevant recombination frequency differences were detected between sexes. Comparative genomics of LG5 marker sequences against five model species showed no similarity of this chromosome to the sex chromosomes of medaka, stickleback, and fugu, but suggested a similarity to a sex-associated QTL from Oreochromis spp. The segregation analysis of the closest markers to the SD region demonstrated a ZW/ZZ model of sex determination in turbot. A small proportion of families did not fit perfectly with this model, which suggests that other minor genetic and/or environmental factors are involved in sex determination in this species.