Genetic epidemiological studies of eastern Adriatic Island isolates, Croatia: objective and strategies

In this paper, the authors discuss why isolate island populations represent a particularly helpful model for genetic epidemiological studies. A thorough previous anthropological research carried out in Eastern Adriatic island isolates, Croatia, in terms of ethnohistory, geography and current demography is reviewed. The major results of the studies of population genetic structure of those populations, including model-bound and model-free approaches, the analyses of serogenetic polymorphisms and most recent studies using HLA class II, VNTR and STR DNA polymorphisms, are briefly presented. The organization of health care on the islands is analyzed and some relevant details of specific medical problems and some autochtonous diseases in these island populations is noted. The authors present in outline four illustrative examples of research opportunities which are afforded by the unique circumstances found in these isolate communities. These relate to hereditary dwarfism on Krk island, Mal de Meleda on Mljet island, extreme inbreeding on Susak island and population genetics of cancer on the islands of Brac, Hvar, Korcula, Vis and Lastovo. Finally, the authors develop objectives and strategies for a long-term genetic epidemiological research of these populations and suggest that such a programme of investigation would further our understanding of the causes of (rare) diseases which are uniquely important to these communities but also of common diseases which are important contributors to the burden of disease both in these islands and throughout the world.     

Inbreeding and cancer incidence in human isolates

This study investigates the incidence of cancer in isolate populations. Thorough anthropological research over the past 3 decades has established island populations in Middle Dalmatia, Croatia, as outstanding examples of genetic isolates. The number of cancer cases on 5 islands (Brac, Hvar, Korcula, Vis, and Lastovo) over a 20-year period (1971-1990) has been extracted from the data of the Croatian Cancer Registry. The population of coastal Dalmatia, characterized by similar environmental factors but a different population genetic structure, was used as a control population. The leading hypothesis was that, if there were genes or gene complexes (especially with recessive inheritance) responsible for genetic susceptibility to certain types of cancer, then the incidence of those cancer types should be greater in reproductively isolated island populations than in a control population because of increased manifestation of such genes or gene complexes caused by inbreeding. Furthermore, the cancer incidence should increase along with greater reproductive isolation (i.e., greater geographic distance of the islands from the mainland). After adjusting the data for sex and age, I confirmed the hypothesis: Island populations have greater total cancer incidence than the control population for both sexes. The excess incidence on the islands shows an almost linear correlation with geographic distance from the mainland. The cancer sites primarily responsible for the excess incidence are bladder cancer in males, and breast, ovarian, brain, and large bowel cancer in females, predominantly in the younger age groups.     

Ultraviolet sun radiation and incidence of age-related macular degeneration on Croatian Island Rab

This epidemiological study is completed on island Rab, located in North Adriatic Sea. For this study it is important that Adriatic Sea has one of the highest solar radiations in geographical area of Europe. The aim of this epidemiological study is to estimate correlation between incidence of Age-Related Macular Degeneration (AMD) and exposure of sunlight in two different populations on island Rab. In the first group of population of island Rab, agriculturists and fishermen (n = 1300), incidence of AMD was in 18%, but in urban population only 2.5%. It is very interesting that correlation exists between incidence of AMD and exfoliation syndrome. In the group of chronic exposure to sunlight (agriculturists and fishermen) exfoliation syndrome exists in 28%, but in urban population it has never been seen. The age in both populations was 45-65 years. We can conclude that there is a very significant correlation between chronic exposure to sunlight and appearance of AMD.     

Genetic variation at nine short tandem repeat loci among islanders of the eastern Adriatic coast of Croatia

We have analyzed the extent of genetic variation at nine autosomal short tandem repeat loci (D3S1358, VWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317, D7S820) among six populations from Croatia: five distributed in the islands of the eastern Adriatic coast and one from the mainland. The purpose is to investigate the usefulness of these loci in detecting regional genetic differentiation in the studied populations. Significant heterogeneity among the island and mainland populations is revealed in the distributions of allele frequencies; however, the absolute magnitude of the coefficient of gene differentiation is small but significant. The summary measures of genetic variation, namely, heterozygosity, number of alleles, and allele size variance, do not indicate reduced genetic variation in the island populations compared to the mainland population. In contrast to the two measures of genetic variation, allele size variance and within-locus heterozygosity, the imbalance index (beta) indicates evidence of recent expansion of population sizes in all islands and in the mainland. High mutation rates of the studied loci together with local drift effects are likely explanations for interisland genetic variation and the observed lack of reduced genetic diversity among the island populations.     

Sets of complementary phenotypic relationships across five isolated populations: Traces of expression of different gene complexes?

The aim of this study is to search for certain repeating phenotypic patterns, i.e. sets of complementary relationships across five isolated populations, which may represent the traces of expression of different genes or gene complexes. The study was conducted among isolates of five island populations of eastern Adriatic, Croatia, and the data were collected between 1979 and 1990. Selected phenotypic characteristics included measures of biological distances (e.g. anthropometrical body and head distances, physiological, dermatoglyphic and radiogrammetric bone distances), while other examined traits included sociocultural (linguistic), bio-cultural (migrational kinship) and genetic distances. The sample consisted of 6,286 examinees from 43 villages of five isolate populations. Correlations between distance matrices based on examined traits were analyzed in each of five populations using Mantel's test of matrix correspondence, and factor analysis (rotated principal component) was then performed over obtained correlation matrices. The results showed that there were several consistent and significant correlations between some analyzed traits across all of the studied isolate populations, which might indicate their regulation by the shared gene complexes or genome regions. The analyses identified three main clusters of correlations in all five isolate populations: the first one containing anthropometric measures (body and head measures and physiological properties in both sexes), the second one containing geographic distance-related traits (migrational kinship, linguistic and genetic distances), and the third one containing dermatoglyphic properties and radiogrammetric bone measures in both sexes. The higher order varimax rotation over the matrix of factor correlations revealed that the primary source of variation within all five analyzed populations was not sex-related, but rather variable-specific.     

Intact landscape promotes gene flow and low genetic structuring in the threatened Eastern Massasauga Rattlesnake

Genetic structuring of wild populations is dependent on environmental, ecological, and life‐history factors. The specific role environmental context plays in genetic structuring is important to conservation practitioners working with rare species across areas with varying degrees of fragmentation. We investigated fine‐scale genetic patterns of the federally threatened Eastern Massasauga Rattlesnake (Sistrurus catenatus) on a relatively undisturbed island in northern Michigan, USA. This species often persists in habitat islands throughout much of its distribution due to extensive habitat loss and distance‐limited dispersal. We found that the entire island population exhibited weak genetic structuring with spatially segregated variation in effective migration and genetic diversity. The low level of genetic structuring contrasts with previous studies in the southern part of the species’ range at comparable fine scales (~7 km), in which much higher levels of structuring were documented. The island population''s genetic structuring more closely resembles that of populations from Ontario, Canada, that occupy similarly intact habitats. Intrapopulation variation in effective migration and genetic diversity likely corresponds to the presence of large inland lakes acting as barriers and more human activity in the southern portion of the island. The observed genetic structuring in this intact landscape suggests that the Eastern Massasauga is capable of sufficient interpatch movements to reduce overall genetic structuring and colonize new habitats. Landscape mosaics with multiple habitat patches and localized barriers (e.g., large water bodies or roads) will promote gene flow and natural colonization for this declining species.     

Orofacial analysis on the Adriatic Islands: 1. The island of Hvar as a model for odontogenetic researches

This paper presents a preliminary orofacial analysis of a subadult population of Hvar, a Croatian island in the Adriatic. Its population represents one of the last genetic isolates in Europe and has therefore been the object of intensive crossdisciplinary research over the last 30 years. We focussed on the coefficient of endogamy on the one hand and malocclusal-related caries on the other hand, and expected differences in the latter between subgroups of the population. We analyzed 224 dental casts from children all over the island and found multiple caries in approximal surfaces in 55 percent of the children, but no significant differences between the subpopulations. Instead, significantly more caries affection was found in the boys than in the girls. The percentage of general caries affection is fairly high, even when compared to other isolated populations; it may be due to environmental influence. This would be consistent with the other results, which have putatively been caused by complex environmental influences and not solely by genetic components.     

Genetic drift and rapid evolution of viviparity in insular fire salamanders (Salamandra salamandra)

Continental islands offer an excellent opportunity to investigate adaptive processes and to time microevolutionary changes that precede macroevolutionary events. We performed a population genetic study of the fire salamander (Salamandra salamandra), a species that displays unique intraspecific diversity of reproductive strategies, to address the microevolutionary processes leading to phenotypic and genetic differentiation of island, coastal and interior populations. We used eight microsatellite markers to estimate genetic diversity, population structure and demographic parameters in viviparous insular populations and ovoviviparous coastal and interior populations. Our results show considerable genetic differentiation (F(ST) range: 0.06-0.27), and no clear signs of gene flow among populations, except between the large and admixed interior populations. We find no support for island colonization by rafting or intentional/accidental anthropogenic introductions, indicating that rising sea levels were responsible for isolation of the island populations approximately 9000 years ago. Our study provides evidence of rapid genetic differentiation between island and coastal populations, and rapid evolution of viviparity driven by climatic selective pressures on island populations, geographic isolation with genetic drift, or a combination of these factors. Studies of these viviparous island populations in early stages of divergence help us better understand the microevolutionary processes involved in rapid phenotypic shifts.     

Variation of mitochondrial DNA in Far Eastern mullet pilengas, Liza haematocheilus Temminck and Schlegel, acclimatized in the Azov-Black Sea basin

This study continues the investigation of genetic variation in the populations of native and acclimatized in the Azov-Black Sea basin pilengas from the Sea of Japan. The previous comparison based on allozyme analysis was supplemented by analysis of restriction polymorphism of a mitochondrial DNA fragment containing the cytochrome b gene and the D-loop. Five out of fifteen tested endonucleases detected polymorphic sites. In the samples of native and acclimatized pilengas, five common haplotypes were found; ten and three “unique” haplotypes were detected in the Far Eastern and the Azov populations, respectively. The differences in haplotype distributions between these populations were highly significant (P < 0.001). The mtDNA variation was lower in the Azov than in the Far Eastern population (haplotype diversity μ respectively 6.35 ± 0.27 and 9.14 ± 0.55), which is in good agreement with the decrease in the number of polymorphic loci and the mean number of alleles per locus, found earlier for allozyme markers in this population. The reasons for these differences in the acclimatized population are discussed.     

Small population size and extremely low levels of genetic diversity in island populations of the platypus, Ornithorhynchus anatinus

Genetic diversity generally underpins population resilience and persistence. Reductions in population size and absence of gene flow can lead to reductions in genetic diversity, reproductive fitness, and a limited ability to adapt to environmental change increasing the risk of extinction. Island populations are typically small and isolated, and as a result, inbreeding and reduced genetic diversity elevate their extinction risk. Two island populations of the platypus, Ornithorhynchus anatinus, exist; a naturally occurring population on King Island in Bass Strait and a recently introduced population on Kangaroo Island off the coast of South Australia. Here we assessed the genetic diversity within these two island populations and contrasted these patterns with genetic diversity estimates in areas from which the populations are likely to have been founded. On Kangaroo Island, we also modeled live capture data to determine estimates of population size. Levels of genetic diversity in King Island platypuses are perilously low, with eight of 13 microsatellite loci fixed, likely reflecting their small population size and prolonged isolation. Estimates of heterozygosity detected by microsatellites (H(E)= 0.032) are among the lowest level of genetic diversity recorded by this method in a naturally outbreeding vertebrate population. In contrast, estimates of genetic diversity on Kangaroo Island are somewhat higher. However, estimates of small population size and the limited founders combined with genetic isolation are likely to lead to further losses of genetic diversity through time for the Kangaroo Island platypus population. Implications for the future of these and similarly isolated or genetically depauperate populations are discussed.     

Variation in mitochondrial DNA in Far Eastern mullet pilengas, Liza haematocheilus Temminck and Schlegel, acclimatized in the Azov-Black Sea basin

This study continues the investigation of genetic variation in the populations of native and acclimatized in the Azov-Black Sea basin pilengas from the Sea of Japan. The previous comparison based on allozyme analysis was supplemented by analysis of restriction polymorphism of a mitochondrial DNA fragment containing the cytochrome b gene and the D-loop. Five out of fifteen endonucleases tested detected polymorphic sites. In the samples of native and acclimatized pilengas, five common haplotypes were found; ten and three "population-specific" haplotypes were detected in the Far Eastern and the Azov populations, respectively. The differences in haplotype distributions between these populations were highly significant (P < 0.001). The mtDNA variation was lower in the Azov than in the Far Eastern population (haplotype diversity mu respectively 6.35 +/- 0.27 and 9.14 +/- 0.55), which is in good agreement with the decrease in the number of polymorphic loci and the mean number of alleles per locus, found earlier for allozyme markers in this population. The reasons for these differences in the acclimatized population are discussed.     

Phylogeography of isolated freshwater three-spined stickleback Gasterosteus aculeatus populations in the Adriatic Sea basin

Analyses of mitochondrial (mt) DNA and microsatellite variation were carried out to examine the relationships between 10 freshwater populations of three-spined sticklebacks Gasterosteus aculeatus along the eastern coast of the Adriatic Sea. Partial sequences of the mtDNA control region and cytochrome b gene, in addition to 15 microsatellite loci, were used to analyse populations from four isolated river catchments. Results uncovered an Adriatic lineage that was clearly divergent from the European lineage, and confirmed that the most divergent and ancient populations are located within the Adriatic lineage as compared with other European populations. Two northern Adriatic populations formed independent clades within the European mitochondrial lineage, suggesting different colonization histories of the different Adriatic populations. Nuclear marker analyses also indicated deep divergence between Adriatic and European populations, albeit with some discordance between the mtDNA phylogeny of the northern Adriatic populations, further highlighting the strong differentiation among the Adriatic populations. The southern populations within the Adriatic lineage were further organized into distinct clades corresponding to respective river catchments and sub-clades corresponding to river tributaries, reflecting a high degree of population structuring within a small geographic region, concurrent with suggestions of existence of several microrefugia within the Balkan Peninsula. The highly divergent clades and haplotypes unique to the southern Adriatic populations further suggest, in accordance with an earlier, more limited survey, that southern Adriatic populations represent an important reservoir for ancient genetic diversity of G. aculeatus.     

Dental arch asymmetry in an isolated Adriatic community

Developmental stability reflects the ability of a genotype to develop in the same way under varying environmental conditions. Deviations from developmental stability, arising from disruptive effects of environmental and genetic stresses, can be measured in terms of fluctuating asymmetry, a particularly sensitive indicator of the ability to cope with these stresses during ontogeny. In an inbred Adriatic island population, we expected dental arch fluctuating asymmetry 1) to be higher than in an outbred sample from the same island, and 2) within this population, to increase with the level of inbreeding. Due to environmental stress, we also expected to find higher fluctuating asymmetry in the outbred island population than in an urban reference group from the same country. The material consisted of 506 dental casts of 253 children from 1) the island of Hvar, and 2) Zagreb, Croatia. Three-dimensional coordinates of 26 landmarks spanning the arches were digitized. The analysis partitioned the asymmetry of arch forms into components for directional and fluctuating bilateral asymmetry, using the appropriate Procrustes method (geometric morphometrics). The results corroborated the hypotheses. Fluctuating asymmetry was found to be higher on the island than in Zagreb in all groups and in both jaws, and increased significantly with endogamy level in the lower jaw. There was no significant directional asymmetry in the Zagreb sample and likewise none in the upper jaws of the outbred island group, but significant directional asymmetry in both jaws of the inbred population and also in the lower jaws of the outbred island group. These results suggest an environmental as well as a genetic influence on dental arch asymmetry. Although the lower jaws expressed these two stresses almost additively, the upper jaws appeared to be better buffered. The role of directional asymmetry as a potential indicator of craniofacial developmental instability clearly merits further attention.     

Biocomplexity in Populations of European Anchovy in the Adriatic Sea

The sustained exploitation of marine populations requires an understanding of a species'' adaptive seascape so that populations can track environmental changes from short- and long-term climate cycles and from human development. The analysis of the distributions of genetic markers among populations, together with correlates of life-history and environmental variability, can provide insights into the extent of adaptive variation. Here, we examined genetic variability among populations of mature European anchovies (n = 531) in the Adriatic (13 samples) and Tyrrhenian seas (2 samples) with neutral and putative non-neutral microsatellite loci. These genetic markers failed to confirm the occurrence of two anchovy species in the Adriatic Sea, as previously postulated. However, we found fine-scale population structure in the Adriatic, especially in northern areas, that was associated with four of the 13 environmental variables tested. Geographic gradients in sea temperature, salinity and dissolved oxygen appear to drive adaptive differences in spawning time and early larval development among populations. Resolving adaptive seascapes in Adriatic anchovies provides a means to understand mechanisms underpinning local adaptation and a basis for optimizing exploitation strategies for sustainable harvests.     

Genetic variability,management, and conservation implications of the critically endangered Brazilian pitviper Bothrops insularis

Information on demographic, genetic, and environmental parameters of wild and captive animal populations has proven to be crucial to conservation programs and strategies. Genetic approaches in conservation programs of Brazilian snakes remain scarce despite their importance for critically endangered species, such as Bothrops insularis, the golden lancehead, which is endemic to Ilha da Queimada Grande, coast of São Paulo State, Brazil. This study aims to (a) characterize the genetic diversity of ex situ and in situ populations of B. insularis using heterologous microsatellites; (b) investigate genetic structure among and within these populations; and (c) provide data for the conservation program of the species. Twelve informative microsatellites obtained from three species of the B. neuwiedi group were used to access genetic diversity indexes of ex situ and in situ populations. Low‐to‐medium genetic diversity parameters were found. Both populations showed low—albeit significant—values of system of mating inbreeding coefficient, whereas only the in situ population showed a significant value of pedigree inbreeding coefficient. Significant values of genetic differentiation indexes suggest a small differentiation between the two populations. Discriminant analysis of principal components (DAPC) recovered five clusters. No geographic relationship was found in the island, suggesting the occurrence of gene flow. Also, our data allowed the establishment of six preferential breeding couples, aiming to minimize inbreeding and elucidate uncertain parental relationships in the captive population. In a conservation perspective, continuous monitoring of both populations is demanded: it involves the incorporation of new individuals from the island into the captive population to avoid inbreeding and to achieve the recommended allelic similarity between the two populations. At last, we recommend that the genetic data support researches as a base to maintain a viable and healthy captive population, highly genetically similar to the in situ one, which is crucial for considering a reintroduction process into the island.     

Prevalence of metabolic syndrome in the interior of Croatia: the Baranja region

Metabolic syndrome (MS), a constellation of metabolic risk factors associated with development of cardiovascular diseases and Type 2 diabetes (T2D), has emerged as a public health problem of enormous proportions in developed and developing countries. We have reported previously its prevalence in several island populations of the Eastern Adriatic coast of Croatia. In spite of leading a relatively traditional life style pattern including adherence to a Mediterranean diet, the prevalence of MS in these populations is high and comparable to that in developed nations. However, data on prevalence of MS among the mainland Croatian populations is limited. Therefore, we conducted a study in an outbred population comprising of Croats, Hungarians and Serbs from the Baranja region of mainland Croatia. Although this is an ethnically heterogeneous population, the constituent groups exchange mates and therefore, are not reproductively isolated. The life style patterns are also similar. Overall prevalence of MS, assessed by the National Cholesterol Education Program (NCEP) criteria, is 40% (35% in males and 42% in females) with Body Mass Index (BMI) as the predictor of obesity and 42% (52% in males and 39% in females) with Waist Hip Ratio (WHR) as the predictor of obesity. It is likely that, in addition to genetic risk factors, a host of environmental factors that include dietary habits and relatively urban life style in a modernized society have influenced the levels of the constituent metabolic traits leading to an increased prevalence of MS.     

Middle Eastern and European mtDNA lineages characterize populations from eastern Crete

Throughout centuries, the geographic location of the island of Crete has been one of the leading factors shaping the composition of its population. Invasions and commercial and cultural ties at various time periods with European, Middle Eastern, and North African civilizations have created a collage of genetic and/or cultural influences from each of these regions within the island. Previous Y-chromosome diversity analyses uncovered pronounced differences in the frequency distribution of haplogroups from a mountain refugium and surrounding lowland populations of eastern Crete. In this study, the current geographic stratification of mtDNA haplotypes in eastern Crete was explored to elucidate potential sources of maternal gene flow. Our work includes a comparative characterization of two lowland collections from the Heraklion and Lasithi Prefectures in eastern Crete, as well as of an isolated mountain population from the Lasithi Plateau, all three previously examined using Y-chromosome markers. In addition to the presence of European mtDNA haplogroups in all three collections, our analyses reveal a significant contribution of Middle Eastern and Central Asian genetic signatures in the island of Crete, and particularly in the two populations from the Lasithi region at the eastern-most portion of the island. Close association between these Cretan groups and the Balkans can also be discerned, which in the case of the Lasithi Plateau corroborates previously uncovered Y-chromosome affiliations with the same geographic region.     

Large mainland populations of South Island robins retain greater genetic diversity than offshore island refuges

For conservation purposes islands are considered safe refuges for many species, particularly in regions where introduced predators form a major threat to the native fauna, but island populations are also known to possess low levels of genetic diversity. The New Zealand archipelago provides an ideal system to compare genetic diversity of large mainland populations where introduced predators are common, to that of smaller offshore islands, which serve as predator-free refuges. We assessed microsatellite variation in South Island robins (Petroica australis australis), and compared large mainland, small mainland, natural island and translocated island populations. Large mainland populations exhibited more polymorphic loci and higher number of alleles than small mainland and natural island populations. Genetic variation did not differ between natural and translocated island populations, even though one of the translocated populations was established with five individuals. Hatching failure was recorded in a subset of the populations and found to be significantly higher in translocated populations than in a large mainland population. Significant population differentiation was largely based on heterogeneity in allele frequencies (including fixation of alleles), as few unique alleles were observed. This study shows that large mainland populations retain higher levels of genetic diversity than natural and translocated island populations. It highlights the importance of protecting these mainland populations and using them as a source for new translocations. In the future, these populations may become extremely valuable for species conservation if existing island populations become adversely affected by low levels of genetic variation and do not persist.     

Reduced MHC class II diversity in island compared to mainland populations of the black-footed rock-wallaby (<Emphasis Type="Italic">Petrogale lateralis lateralis</Emphasis>)

Many animal populations that are endangered in mainland areas exist in stable island populations, which have the potential to act as an “ark” in case of mainland population declines. Previous studies have found neutral genetic variation in such species to be up to an order of magnitude lower in island compared to mainland populations. If low genetic variation is prevalent across fitness-related loci, this would reduce the effectiveness of island populations as a source of individuals to supplement declining mainland populations or re-establish extinct mainland populations. One such species, the black-footed rock-wallaby (Petrogale lateralis lateralis), exists within fragmented mainland populations and small island populations off Western Australia. We examined sequence variation in this species within a fitness-related locus under positive selection, the MHC class II DAB β1 locus. The mainland populations displayed greater levels of allelic diversity (4–7 alleles) than the island population, despite being small and isolated, and contained at least two DAB gene copies. The island population displayed low allelic diversity (2 alleles) and fewer alleles per individual in comparison to mainland populations, and probably possesses only one DAB gene copy. The patterns of DAB diversity suggested that the island population has a markedly lower level of genetic variation than the mainland populations, in concordance with results from microsatellites (genotyped in a previous study), but preserved unique alleles which were not found in mainland populations. Where possible, conservation actions should pool individuals from multiple populations, not only island populations, for translocation programs, and focus on preventing further declines in mainland populations.     

Ancestral kinship and cancer in Lastovo Island, Croatia.

The aim of this study was to compute the average kinship coefficient of cancer cases in an extreme isolate (Lastovo Island, Croatia) and to compare it to the corresponding value for the island's unaffected population. Kinship estimates were obtained by Wright's path method (phi) based on pedigree reconstruction and Lasker's coefficient of relationship by isonymy (Ri) based on surname distribution. A total of 76 cancer cases were recorded on the island between 1971 and 1995. The unaffected control population of 1144 persons was obtained from the population census in 1971. The data on five ancestral generations were collected from parish registries preserved in the local church, and included 5484 persons born on the island between 1750 and 1970. Both Wright's path method and Lasker's isonymy method indicated significantly greater kinship of female cancer cases than of the unaffected population. Cancer cases with increased values of Ri among them and Ri among their ancestors included cancers of the ovary, colon, brain, breast, and prostate and leukemias. This study confirms the role of heredity and common ancestry in the development of cancer, providing a rough insight into their relative importance for specific cancer sites.