DNA metabarcoding the diet of Podarcis lizards endemic to the Balearic Islands.

Dietary studies are essential to unravel the functioning of ecosystems and ultimately to understand biodiversity. This task, which at first may seem simple, becomes especially complex in those cases of omnivorous species with highly variable diets. In this regard, the emergence of next-generation DNA sequencing methodologies represents a powerful tool to address the problem. Here we implement a high-throughput metabarcoding strategy based on the analysis of four molecular markers aimed at sequencing both mitochondrial (animal prey) and chloroplast (diet plants) genome fragments from fecal samples of two lizard species endemic to the Balearic Archipelago (Podarcis lilfordi and P. pityusensis) obtained through non-invasive methods. The results allowed for the characterization of their diets with a high degree of taxonomic detail and have contributed a large number of new trophic records. The reported diets are based mainly on the consumption of arthropods, mollusks and plants from a diversity of taxonomic orders, as well as carrion and marine subsidies. Our analyses also reveal inter- and intra-specific differences both in terms of seasonality and geographical distribution of the sampled lizard populations. These molecular findings provide new insights into the trophic interactions of these threatened endemic lizards in their unique and isolated ecosystems.

Diet of the Insular Lizard, Podarcis lilfordi (Günther, 1874): Complementary Morphological and Molecular Approaches.

The diets of insular lizards are extremely varied, depending on the different environmental characteristics of each island population. This is particularly evident in the case of the populations of small coastal islets of the Balearic Islands, where the Balearic lizard, Podarcis lilfordi, is found. The study of trophic ecology carried out by means of traditional tools, such as morphological analysis of feces, has made it possible to detect numerous prey and nutritional elements. However, these methods are clearly insufficient, as some rare groups are not detected. It is also difficult to identify remains of marine subsidies or of foods contributed to these small islands by other predators, such as seabirds. The current study demonstrates the advantages of combining morphological diet analysis with the molecular study of individual feces samples obtained from the same populations. We obtained a greater diversity of prey groups using the combined methodologies, with each method identifying prey items that were not detected using the other method. Particularly, the study of diets at the molecular level identified plant species consumed by lizards that were, occasionally, not identified in morphological analyses. Conversely, the traditional morphological study of an equivalent number of fecal samples allowed for the identification of several prey groups that had not been detected in the molecular study. From this viewpoint, the advantages and disadvantages of each methodology are discussed.

Faecal Microbiota Divergence in Allopatric Populations of Podarcis lilfordi and P. pityusensis, Two Lizard Species Endemic to the Balearic Islands.

Gut microbial communities provide essential functions to their hosts and are known to influence both their ecology and evolution. However, our knowledge of these complex associations is still very limited in reptiles. Here we report the 16S rRNA gene faecal microbiota profiles of two lizard species endemic to the Balearic archipelago (Podarcis lilfordi and P. pityusensis), encompassing their allopatric range of distribution through a noninvasive sampling, as an alternative to previous studies that implied killing specimens of these IUCN endangered and near-threatened species, respectively. Both lizard species showed a faecal microbiome composition consistent with their omnivorous trophic ecology, with a high representation of cellulolytic bacteria taxa. We also identified species-specific core microbiota signatures and retrieved lizard species, islet ascription, and seasonality as the main factors in explaining bacterial community composition. The different Balearic Podarcis populations are characterised by harbouring a high proportion of unique bacterial taxa, thus reinforcing their view as unique and divergent evolutionary entities.

Development and validation of a new multiplex for upgrading Y-STRs population databases from 12 to 23 markers and its forensic casework application.

Y chromosomal short tandem repeats (Y-STRs) are used in forensic investigations as a useful complementary tool to autosomal markers. The ongoing development of new kits with an increasing number of markers makes it necessary to update populations typed in the Y-STR Haplotype Reference Database to reach at least 23 Y-STRs. A novel Y-STR multiplex panel was developed to offer a cost-efficient alternative to update Y-STR haplotypes from 12 to 23 loci. This panel includes the eleven markers, DYS448, DYS456, DYS458, DYS635, Y-GATA H4, DYS576, DYS481, DYS549, DYS533, DYS570 and DYS643, as well as DYS385a/b for traceability purpose. Developmental validation of this panel was conducted following the recommendations of the Scientific Working Group on DNA Analysis Methods (SWGDAM), showing high sensitivity, tolerance to common inhibitors as well as high species specificity. It was efficient for degraded DNA samples and for detection of male mixtures. When applying it for extending the current data of the Ibiza population, both the discrimination capacity and the haplotype diversity increased from 0.5952 to 0.9048 and from 0.9808 to 0.9977, respectively. Together, the study demonstrates the suitability of this panel in forensic casework.

Genomic signatures of drift and selection driven by predation and human pressure in an insular lizard.

Genomic divergence was studied in 10 small insular populations of the endangered Balearic Islands lizard (Podarcis lilfordi) using double digest restriction-site associated DNA sequencing. The objectives were to establish levels of divergence among populations, investigate the impact of population size on genetic variability and to evaluate the role of different environmental factors on local adaptation. Analyses of 72,846 SNPs supported a highly differentiated genetic structure, being the populations with the lowest population size (Porros, Foradada and Esclatasang islets) the most divergent, indicative of greater genetic drift. Outlier tests identified ~ 2% of loci as candidates for selection. Genomic divergence-Enviroment Association analyses were performed using redundancy analyses based on SNPs putatively under selection, detecting predation and human pressure as the environmental variables with the greatest explanatory power. Geographical distributions of populations and environmental factors appear to be fundamental drivers of divergence. These results support the combined role of genetic drift and divergent selection in shaping the genetic structure of these endemic island lizard populations.

Relating the outcome of HCV infection and different host SNP polymorphisms in a Majorcan population coinfected with HCV-HIV and treated with pegIFN-RBV.

Hepatitis C virus (HCV) is one of the major causes of chronic hepatitis, cirrhosis, and hepatocellular carcinoma, and the development of HCV-related disease is accelerated in individuals coinfected with human immunodeficiency-1 virus (HIV). In the present study, we correlated different host single-nucleotide polymorphisms (SNPs) in the IL28B, CTLA4, LDLr, and HFE genes and mitochondrial DNA (mtDNA) haplogroups with the outcome of HCV infection and the response to pegylated-interferon plus ribavirin (pegIFN-RBV) treatment. Our study population consisted of 63 Majorcan patients coinfected with HCV and HIV and 59 anonymous unrelated controls. Whereas the population frequency of IL28B alleles was similar to that found in a North-American cohort of European descent, the frequency of the rs12979860 C allele was lower than that determined in other cohorts from Spain. The frequencies of CTLA4 and LDLr polymorphisms were comparable to those reported in other populations. Significant differences between cases and control cohorts occurred only for the H63D mutation of the HFE gene. There were no other differences in the frequencies of other polymorphisms or mtDNA haplogroups. The IL28B rs12979860 CC genotype was shown to be associated with a rapid virological response, and the spontaneous viral clearance rate for HCV was higher in patients with the CTLA4+49 G allele. There was no relationship between SNPs in the LDLr and HFE genes and mtDNA haplogroups and the response to treatment. Our results suggest that the host genetic background plays a significant role in the pegIFN-RBV response of patients coinfected with HCV and HIV.

Relating the outcome of HCV infection and different host SNP polymorphisms in a Majorcan population coinfected with HCV-HIV and treated with pegIFN-RBV

Hepatitis C virus (HCV) is one of the major causes of chronic hepatitis, cirrhosis, and hepatocellular carcinoma, and the development of HCV-related disease is accelerated in individuals coinfected with human immunodeficiency-1 virus (HIV). In the present study, we correlated different host single-nucleotide polymorphisms (SNPs) in the IL28B, CTLA4, LDLr, and HFE genes and mitochondrial DNA (mtDNA) haplogroups with the outcome of HCV infection and the response to pegylated-interferon plus ribavirin (pegIFN-RBV) treatment. Our study population consisted of 63 Majorcan patients coinfected with HCV and HIV and 59 anonymous unrelated controls. Whereas the population frequency of IL28B alleles was similar to that found in a North-American cohort of European descent, the frequency of the rs12979860 C allele was lower than that determined in other cohorts from Spain. The frequencies of CTLA4 and LDLr polymorphisms were comparable to those reported in other populations. Significant differences between cases and control cohorts occurred only for the H63D mutation of the HFE gene. There were no other differences in the frequencies of other polymorphisms or mtDNA haplogroups. The IL28B rs12979860 CC genotype was shown to be associated with a rapid virological response, and the spontaneous viral clearance rate for HCV was higher in patients with the CTLA4+49 G allele. There was no relationship between SNPs in the LDLr and HFE genes and mtDNA haplogroups and the response to treatment. Our results suggest that the host genetic background plays a significant role in the pegIFN-RBV response of patients coinfected with HCV and HIV (AU)

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Utility and importance of accurate Hb A2 measurements for defining a strategy for ß-thalassemia screening: experience in the Balearic Islands, Spain.

The high heterogeneity in regional profiles of ß-thalassemia (ß-thal) mutations highlights the need for population-specific carrier detection strategies. Our aim was to analyze the relationship between hematological values and ß(0) and ß(+) mutations in 154 Balearic ß-thal heterozygotes, in order to establish the most optimized mutation carrier detection strategy to be used to manage the disease in our population. The Hb A2 level was the best parameter for discriminating between both types of carriers. Taking into account the cut-off point value of 4.85% (Hb A2), obtained by a receiver-operating characteristic (ROC) curve analysis, we proposed an algorithm that would use a real-time polymerase chain reaction (RT-PCR) hybridization probe assay technique to detect one of the two most common mutations in the Balearic population, namely codon 39 (C>T) and IVS-I-110 (G>A), depending on the Hb A2 value of the patient.

Variability of the mc1r gene in melanic and non-melanic Podarcis lilfordi and Podarcis pityusensis from the Balearic archipelago.

The association between polymorphism at the mc1r locus and colour variation was studied in two wall lizard species (Podarcis lilfordi and P. pityusensis) from the Balearic archipelago. Podarcis lilfordi comprises several deep mitochondrial lineages, the oldest of which originated in the Pliocene, while much shallower mitochondrial lineages are found in P. pityusensis. Here, we examined whether specific substitutions were associated with the melanic colouration found in islet populations of these species. Homologous nuclear sequences covering most of the mc1r gene were obtained from 73 individuals from melanic and non-melanic Podarcis from different populations (the entire gene was also sequenced in six selected individuals). MtDNA gene trees were also constructed and used as a framework to assess mc1r diversity. Mc1r showed greater polymorphism in P. lilfordi than in P. pityusensis. However, we observed no substitutions that were common to all melanic individuals across the two species. Only one significant association was detected in the mc1r partial sequence, but this was a synonymous A/G mutation with A alleles being more abundant in melanic populations. In addition, there were no associations between the main dominant phenotypes (green and brown, blue and yellow spots and ventral colour) and synonymous or non-synonymous substitutions in the mc1r gene. There was no statistical evidence of selection on mc1r. This study suggests no relationship between mc1r polymorphism and colour variation in Balearic Podarcis.

Generalized Linear Model (GLM) framework for the association of host variables and viral strains with liver fibrosis in HCV/HIV coinfected patients.

Chronic hepatitis C virus (HCV) infection is the main cause of advanced and end-stage liver disease world-wide, and an important factor of morbidity and mortality in Human Immunodeficiency virus-1 (HIV-1) co-infected individuals. Whereas the genetic variability of HCV has been studied extensively in monoinfected patients, comprehensive analyses of both patient and virus characteristics are still scarce in HCV/HIV co-infection. In order to find correlates for liver damage, we sought to analyze demographic, epidemiological and clinical features of HCV/HIV co-infected patients along with the genetic makeup of HCV (viral subtypes and lineage studied by nucleotide sequencing and phylogenetic analysis of the NS5B region). We used the Generalized Linear Model (GLM) methodology in order to integrate data from the virus and the infected host to find predictors for liver damage. The degree of liver disease was evaluated indirectly by means of two indexes (APRI and FIB-4) and accounting for the time since infection, to estimate fibrosis progression rates. Our analyses identified a reduced number of variables (both from the virus and the host) implicated in liver damage, which included the stage of HIV infection, levels of gamma-glutamil transferase and cholesterol, and some distinct HCV phylogenetic clades.

Effect of co-inheritance of ß-thalassemia and hemochromatosis mutations on iron overload.

Co-inheritance of mutations in the HFE gene underlying hereditary hemocromatosis (HH) may play a role in the variability of iron status in patients with ß-thalassemia (ß-thal) minor. Different studies have yielded conflicting results: some suggest iron overload might arise from the interaction of the ß-thal trait with homozygosity or even heterozygosity for HFE mutations and others that it was unrelated to the HFE genotype. Because of the high frequency of HFE mutations in the Balearic Islands, where the ß-thal trait is also moderately common, it is of interest to evaluate the effect of the co-inheritance of mutations in both genes on the severity of iron loading. A retrospective analysis of 142 individuals heterozygous for ß-thal was performed to investigate the effect of HFE mutations on iron status of these patients. No significant differences were detected between ß-thal carriers with and without HFE mutations. These results suggest that in the Balearic population the ß-thal trait does not tend to be aggravated by the co-inheritance of HFE mutations.

Relating the liver damage with hepatitis C virus polymorphism in core region and human variables in HIV-1-coinfected patients.

Hepatitis C virus (HCV) infection is the most important cause of chronic hepatitis, cirrhosis and end-stage liver disease leading to liver transplantation worldwide. Chronic infection by HCV causes liver fibrosis, which is accelerated by unknown mechanisms in patients with human immunodeficiency virus-1 (HIV-1) coinfection. Although the genetic variability of both HCV and HIV has been extensively studied in the context of monoinfections, more limited data is available regarding HCV-HIV coinfection. HCV disease progression among HIV coinfected patients may be influenced not only by demographic, epidemiological and clinical background variables, but also by genetic differences in infecting viruses. To explore this issue, we carried out a study in coinfected patients trying to associate the degree of liver damage to several demographic, clinical, and epidemiological characteristics of the patients, and also to the genetic variability of HCV between patients. For this purpose, we have applied different statistical techniques including the statistical generalized linear model (GLM) framework. The stage of fibrosis was indirectly measured by noninvasive means using the indexes Forns, APRI and FIB-4. HCV genetic variability between patients was estimated by sequencing the core region and by reconstructions of consensus maximum parsimony phylogenetic trees with 50% and 75% bootstrap majority rules. The results showed a direct correlation of the fibrosis biomarkers with the AST/ALT ratio, MoftIDU and with 3a HCV genotype clades, among others.

Nucleotide diversity of a ND5 fragment confirms that population expansion is the most suitable explanation for the mtDNA haplotype polymorphism of Drosophila subobscura.

Results from mitochondria (mt) DNA restriction site analyses (RSAs) have revealed that wild populations of Drosophila subobscura are formed by two common (I and II) and some rare, often endemic, low-frequency haplotypes. In the study reported here, we analysed nucleotide diversity in a 942-bp fragment of the mtDNA ND5 gene in 48 D. subobscura individuals captured from three populations that showed haplotypes I, II or the less common ones, as well as in one additional individual belonging to D. guanche that was taken as an outgroup. RSAs and sequencing results were compared. The two approaches yielded similar nucleotide variability parameters, suggesting a consistency in the results obtained from mtDNA dynamics in natural populations of D. subobscura. Patterns of polymorphism at ND5 are most consistent with the hypothesis of population expansion after a bottleneck that may have occurred since the last glaciation or which may occur seasonally after the summer and winter. However, we cannot rule out that selection has a role in maintaining the two major haplotypes at intermediate frequencies in worldwide populations of D. subobscura.

The genetic legacy of religious diversity and intolerance: paternal lineages of Christians, Jews, and Muslims in the Iberian Peninsula.

Most studies of European genetic diversity have focused on large-scale variation and interpretations based on events in prehistory, but migrations and invasions in historical times could also have had profound effects on the genetic landscape. The Iberian Peninsula provides a suitable region for examination of the demographic impact of such recent events, because its complex recent history has involved the long-term residence of two very different populations with distinct geographical origins and their own particular cultural and religious characteristics-North African Muslims and Sephardic Jews. To address this issue, we analyzed Y chromosome haplotypes, which provide the necessary phylogeographic resolution, in 1140 males from the Iberian Peninsula and Balearic Islands. Admixture analysis based on binary and Y-STR haplotypes indicates a high mean proportion of ancestry from North African (10.6%) and Sephardic Jewish (19.8%) sources. Despite alternative possible sources for lineages ascribed a Sephardic Jewish origin, these proportions attest to a high level of religious conversion (whether voluntary or enforced), driven by historical episodes of social and religious intolerance, that ultimately led to the integration of descendants. In agreement with the historical record, analysis of haplotype sharing and diversity within specific haplogroups suggests that the Sephardic Jewish component is the more ancient. The geographical distribution of North African ancestry in the peninsula does not reflect the initial colonization and subsequent withdrawal and is likely to result from later enforced population movement-more marked in some regions than in others-plus the effects of genetic drift.

Identifying genetic traces of historical expansions: Phoenician footprints in the Mediterranean.

The Phoenicians were the dominant traders in the Mediterranean Sea two thousand to three thousand years ago and expanded from their homeland in the Levant to establish colonies and trading posts throughout the Mediterranean, but then they disappeared from history. We wished to identify their male genetic traces in modern populations. Therefore, we chose Phoenician-influenced sites on the basis of well-documented historical records and collected new Y-chromosomal data from 1330 men from six such sites, as well as comparative data from the literature. We then developed an analytical strategy to distinguish between lineages specifically associated with the Phoenicians and those spread by geographically similar but historically distinct events, such as the Neolithic, Greek, and Jewish expansions. This involved comparing historically documented Phoenician sites with neighboring non-Phoenician sites for the identification of weak but systematic signatures shared by the Phoenician sites that could not readily be explained by chance or by other expansions. From these comparisons, we found that haplogroup J2, in general, and six Y-STR haplotypes, in particular, exhibited a Phoenician signature that contributed > 6% to the modern Phoenician-influenced populations examined. Our methodology can be applied to any historically documented expansion in which contact and noncontact sites can be identified.

Genetic linkage study for bipolar disorders on chromosomes 17 and 18 in families with a high expression of mental illness from the Balearic Islands.

Genetically, bipolar disorder is a complex genetic illness, in which both genes and environmental factors play an important role in pathogenesis. Linkage studies have reported suggestive evidence for genomic regions, especially on chromosome 18, but in most cases they have been inconclusive. A total of 12 pedigrees, from the islands of Majorca and Minorca (Balearic Archipelago), with a high expression of mental illness, have been studied. A scan of 29 polymorphic short tandem repeat markers was performed, spanning chromosomes 17 and 18 for bipolar and other affective disorder susceptibility loci. Narrow (only bipolar I disorder) and broad (bipolar plus other affective disorders) diagnosis criteria were employed. The loci D18S63, D18S452, D18S53, D18S61, D18S1161 and D17S831 showed LOD score values of less than -2. Thus, the positive linkage found by other authors on the regions 18p11.2 and 18p11.3 has not been reproduced in the families studied. The data obtained in chromosome 17 suggested two possible regions that could contain a bipolar disorder susceptibility gene: 17q11 (D17S1857, D17S798) and especially 17q24-qter (D17S949, D17S928). The maximum significant linkage was to D17S949 (17q24), following a recessive mode of inheritance. We have also found a positive LOD score value for D18S478 marker located in the region 18q12.

Mitochondrial DNA error prophylaxis: assessing the causes of errors in the GEP'02-03 proficiency testing trial.

We report the results of the Spanish and Portuguese working group (GEP) of the International Society for Forensic Genetics (ISFG) Collaborative Exercise 2002-2003 on mitochondrial DNA (mtDNA) analysis. Six different samples were submitted to the participating laboratories: four blood stains (M1-M2-M3-M4), one mixture blood sample (M5), and two hair shaft fragments (M6). Most of the labs reported consensus results for the blood stains, slightly improving the results of previous collaborative exercises. Although hair shaft analysis is still carried out by a small number of laboratories, this analysis yielded a high rate of success. On the contrary, the analysis of the mixture blood stain (M5) yielded a lower rate of success; in spite of this, the whole results on M5 typing demonstrated the suitability of mtDNA analysis in mixture samples. We have found that edition errors are among the most common mistakes reported by the different labs. In addition, we have detected contamination events as well as other minor problems, i.e. lack of standarization in nomenclature for punctual and length heteroplasmies, and indels. In the present edition of the GEP-ISFG exercise we have paid special attention to the visual phylogenetic inspection for detecting common sequencing errors.

Population dynamics of the 2 major mitochondrial DNA haplotypes in experimental populations of Drosophila subobscura.

The evolution of Drosophila subobscura mitochondrial DNA has been studied in experimental populations, founded with flies from a natural population from Calvià (Majorca, Balearic Islands, Spain). This population, like others founded in Europe, is characterized by the presence of 2 very common (>95%) mitochondrial haplotypes (named I and II) and rare and endemic haplotypes that appear at very low frequencies. Four experimental populations were established with flies having a heterogeneous nuclear genetic background, which was representative of the composition of the natural population. The populations were started with haplotypes I and II at an initial frequency of 50% each. After 33 generations, the 2 haplotypes coexisted. Random drift could be rejected as the only force responsible for the observed changes in haplotype frequencies. A slight but significant linear trend favouring a mtDNA (haploid) fitness effect has been detected, with a nonlinear deviation that could be due to a nuclear component. An analysis of chromosomal arrangements was made before the foundations of the cages and at generation 23. Our results indicated that the hypothesis that the maintenance of the frequencies of haplotypes I and II in natural populations could be due to their association with chromosomal arrangements remains controversial.