Low levels of genetic variation within introduced Javan rusa deer (<Emphasis Type="Italic">Cervus timorensis russa</Emphasis>) in Australia

Javan rusa deer (Cervus timorensis russa) is one of six introduced wild deer species in Australia. The largest population is located in the Royal National Park (RNP), New South Wales, Australia. Although the deer are considered a pest in certain areas, they are also commercially valuable. Using molecular markers we aimed to characterise the level of genetic diversity within the RNP population and identify a possible genetic bottleneck associated with the introduction of seven founding individuals. We also aimed to identify appropriate polymorphic microsatellite loci that could assist in commercial breeding programmes and wild population management in Australia. The results indicate that Javan rusa deer from the RNP (n=37) had an average (±SE) of only 2.29±0.095 alleles/locus, which is substantially lower than that reported in source populations from New Caledonia (7.60±0.933 alleles/locus). These data combined with the presence of genetic reduction signatures (P<0.001) indicate that this population has experienced a population bottleneck possibly at the time of establishment in Australia. Despite low allelic diversity, the large suite of identified polymorphic loci (n=24) shows promise for applications in population genetics, including parentage analysis (total parentage exclusion 0.944) within Australia. These data will contribute significantly to improving commercial breeding programmes and possibly to wild population management plans.     

Genetic variability in European black grouse (<Emphasis Type="Italic">Tetrao tetrix</Emphasis>)

We studied microsatellite genetic variation in 14 different geographic populations of black grouse (Tetrao tetrix) across the European range. Populations were grouped in three different fragmentation categories: isolated, contiguous and continuous, respectively. Genetic diversity, measured as observed heterozygosity (H _O), expected heterozygosity (H _E) and allelic richness, were lower in isolated populations as compared to the other two categories that did not differ amongst one another. These results imply that lowered genetic variability in black grouse populations is negatively affected by population isolation. Our results suggest that the connectivity of small and isolated populations in Western Europe should be improved or else these face an increased risk of extinction due to genetic and demographic stochasticity.     

Non-invasive monitoring of adrenocortical activity in free-ranging fallow deer (<Emphasis Type="Italic">Dama dama</Emphasis> L.)

Measurement of faecal glucocorticoid metabolites is increasingly used as a non-invasive tool to examine disturbances in various domestic and wild animals. Because measurements of faecal glucocorticoid metabolites has previously never been reported in fallow deer, we determined 11,17-dioxoandrostanes (11,17-DOA), a group of cortisol metabolites, in the faeces of four fallow deer yearlings after an adrenocorticotropic hormone (ACTH) challenge or control saline injection by an 11-oxoaetiocholanolone enzyme immunoassay (EIA), to validate a method. A 2.9- to 4.3-fold increase in measured cortisol metabolites in challenged animals after approximately 22 h demonstrated the suitability of this group-specific EIA to monitor adrenocortical activity in respective deer species. To determine faecal cortisol metabolites in fallow deer from a Mediterranean habitat, we collected samples during a 1-year study at Veliki Brijuni Island. The study confirmed seasonal pattern of cortisol release in fallow deer. Higher 11,17-DOA concentrations (median; min–max) were determined for November (99; 50–2,035), March (112; 25–315) and May (92; 40–196 ng/g faeces). Significantly lower concentrations were measured during July (30; 10–195 ng/g faeces). This study indicates that the analysis of faecal glucocorticoid metabolites is a valuable non-invasive technique for monitoring adrenocortical activity in fallow deer. This, together with information about the seasonal pattern of glucocorticoid excretion, could help to improve fallow deer management and welfare, especially in the case of farmed and park animals.     

The genetic structure of endangered indigenous populations of the amago salmon, <Emphasis Type="Italic">Oncorhynchus</Emphasis><Emphasis Type="Italic">masou</Emphasis><Emphasis Type="Italic">ishikawae</Emphasis>, in Japan

The amago salmon, Oncorhynchus masou ishikawae, is an endemic subspecies of O. masou in Japan. Owing to the extensive stocking of hatchery fish throughout Japan, indigenous populations of O. m. ishikawae are now on the verge of extinction. We examined the genetic effects of stocking hatchery fish on wild populations in the River Koza, Japan, using microsatellite and mitochondrial DNA (mtDNA) markers. For mtDNA, haplotype mt1, which is common in wild populations, was present exclusively in isolated wild populations assumed to be unaffected by previous stocking, while it was never observed in hatchery fish. Genetic diversity was much higher in wild populations in the stocked area, which shared many mtDNA haplotypes with hatchery fish, than in isolated wild populations with haplotype mt1. Pairwise F _ST estimates based on microsatellites showed significant differentiation among the isolated populations with many microsatellite loci monomorphic. Significant deviation from Hardy–Weinberg equilibrium was observed in wild populations in the area subject to stocking, where a Bayesian-based assignment test showed a high level of introgression with hatchery fish. These results suggest that wild populations with haplotype mt1, which became isolated through anthropogenic environmental change in the 1950–1960s, represent indigenous populations of O. m. ishikawae in the River Koza. They have low genetic diversity, most likely caused by genetic bottlenecks following damming and environmental deterioration, while stocking of hatchery fish over the past 30 years apparently had a large impact on the genetic structure of wild populations in the main channel of the River Koza.     

Serum biochemical values in fallow deer (<Emphasis Type="Italic">Dama dama</Emphasis> L.) from different habitats in Croatia

Serum biochemical values were determined in blood samples collected from 48 shot fallow deer from the Brijuni islands and 45 sedated fallow deer (Dama dama L.) from hunting grounds in the continental part of Croatia. The parameters were compared with regard to age, sex and habitat. Statistically significant differences were found for serum total protein concentration, alkaline phosphatase, aspartate aminotransferase and alanine aminotransferase activity between young and adult island deer, as well as for total protein, triacylglyceride, cholesterol concentration and alkaline phosphatase activity between young and adult continental deer. In young animals, island males had higher albumin concentrations, while continental males had higher alkaline phosphatase values than females. In adult animals, island males had a higher blood urea nitrogen concentration, while continental males had higher albumin and cholesterol concentrations, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and creatine kinase activities. In this group, males exhibited lower bilirubin and triacylglyceride concentrations than females. Our results indicate that besides age, sex and sampling method, nutritional and environmental factors should be considered when evaluating serum biochemical parameters of fallow deer     

Nine microsatellite DNA primers for <Emphasis Type="Italic">Hippophae rhamnoides</Emphasis> ssp. <Emphasis Type="Italic">sinensis</Emphasis> (Elaeagnaceae)

Hippophae rhamnoides ssp. sinensis occurs mainly in the arid regions of northwest China. The wood stands of this subspecies play an important role in maintaining the local ecosystems in these regions. In addition, the genetic characteristics are essential to understand the historical range changes of this subspecies and its morphological differentiation with other subspecies. In this study, we developed nine microsatellite loci for this subspecies for the first time. We used the combining biotin capture method to enrich AG/CT/AC/GT/CG/GTG/CCA microsatellites. Twenty-six microsatellites were isolated from the enriching library and nine of them were found to be polymorphic through screening 12 distantly distributed individuals. The number of alleles per locus ranged from three to twelve and expected heterozygosity from 0.2659 to 0.4767, respectively. We further performed cross-priming tests in another subspecies and two congeneric species. These firstly isolated loci will provide a useful tool to investigate the genetic structure of this subspecies and its morphological differentiation from the other subspecies.     

Response to human presence during nocturnal line transect surveys in fallow deer (<Emphasis Type="Italic">Dama dama</Emphasis>) and wild boar (<Emphasis Type="Italic">Sus scrofa</Emphasis>)

The use of line transect methodology and portable thermal imaging for ground survey of wildlife should require a good knowledge of the behavioural response of the animals to the presence of an observer, in order to take into account the potential bias in density estimate caused by deviation from the assumption that distances are recorded at the initial position. We used ten fallow deer and eight wild boar fitted with radiocollars to investigate animals’ response during simulated nocturnal line transect surveys, carried out in a Mediterranean plain forest. The experiment consisted in radiolocating a focal animal before and after an observer walked a transect nearby (<100 m). Each transect line was followed using a Global Positioning System (GPS) navigator. We carried out a total of 64 trials on fallow deer and 57 on wild boar. Results showed that despite most of the animals moved significantly in response to the observer (mean ± standard error, wild boar—95.3 ± 10.0 m; fallow deer—149.6 ± 14.2 m), the flying patterns were different in the two species: the reaction of fallow deer turned out to be more intense and directional with respect to that showed by wild boar. Although these results sound explorative, the experiment attempted here, for the first time, is relevant for an appropriate design of nocturnal distance-sampling surveys and gives information about potential bias arising from animal’s behavioural response. We believe that these first results may foster more in-deep analyses which are now made possible with the adoption of GPS technology for animal location.     

Feed-backs between genetic structure and perturbation-driven decline in seagrass (<Emphasis Type="Italic">Posidonia oceanica</Emphasis>) meadows

We explored the relationships between perturbation-driven population decline and genetic/genotypic structure in the clonal seagrass Posidonia oceanica, subject to intensive meadow regression around four Mediterranean fish-farms, using seven specific microsatellites. Two meadows were randomly sampled (40 shoots) within 1,600 m² at each site: the “impacted” station, 5–200 m from fish cages, and the “control” station, around 1,000 m downstream further away (considered a proxy of the pre-impact genetic structure at the site). Clonal richness (R), Simpson genotypic diversity (D*) and clonal sub-range (CR) were highly variable among sites. Nevertheless, the maximum distance at which clonal dispersal was detected, indicated by CR, was higher at impacted stations than at the respective control station (paired t-test: P < 0.05, N = 4). The mean number of alleles (Â) and the presence of rare alleles ( _r) decreased at impacted stations (paired t-test: P < 0.05, and P < 0.02, respectively, N = 4). At a given perturbation level (quantified by the organic and nutrient loads), shoot mortality at the impacted stations significantly decreased with CR at control stations (R ² = 0.86, P < 0.05). Seagrass mortality also increased with  (R ² = 0.81, P < 0.10), R (R ² = 0.96, P < 0.05) and D* (R ² = 0.99, P < 0.01) at the control stations, probably because of the negative correlation between those parameters and CR. Therefore, the effects of clonal size structure on meadow resistance could play an important role on meadow survival. Large genotypes of P. oceanica meadows thus seem to resist better to fish farm-derived impacts than little ones. Clonal integration, foraging advantage or other size-related fitness traits could account for this effect.     

Isolation and characterization of polymorphic microsatellite loci in <Emphasis Type="Italic">Ardisia crenata</Emphasis> (Myrsinaceae)

Ardisia crenata, an evergreen shrub native to East Asia, has been a serious invasive plant to the southeastern USA. Here 13 polymorphic microsatellite loci were isolated and characterized from an enrichment genomic library of A. crenata. The average allele number of these microsatellites was four per locus, ranging from two to seven. The ranges of observed and expected heterozygosity were 0.000–1.000 and 0.239–0.789, respectively. These microsatellite markers will be useful for investigating population genetics and reproductive ecology of A. crenata.     

Landscape fragmentation and habitat suitability in endangered Italian hare (<Emphasis Type="Italic">Lepus corsicanus</Emphasis>) and European hare (<Emphasis Type="Italic">Lepus europaeus</Emphasis>) populations

During the last years, the population of Italian hare decreased significantly in central and south Italy. This is imputable to harvest, poaching, habitat fragmentation, and the probable competition with congeneric European hare introduced in the last decades by man for hunt. The goal of our work is to define the ecological characteristics of the two aforementioned species in order to understand how landscape facilitates or impedes movement. Spatially explicit models are used to identify a species ecological niche and to build a landscape model of suitability. To validate ecological modeling of landscape, we performed a population genetic analysis. Results suggest that the Italian hare shows an ecological requirement close to average of available resources in the considered landscape. The genetic structure of this autochthonous species validates the habitat suitability model and highlights the differences with European hare. This work analyzes for the first time the ecological relationship between those two sympatric species.     

Isolation and characterization of polymorphic microsatellite markers in <Emphasis Type="Italic">Cupressus </Emphasis><Emphasis Type="Italic">chenggiana</Emphasis> S. Y. Hu (Cupressaceae)

Cupressus chenggiana S. Y. Hu (Cupressaceae) is an endemic and endangered conifer species in southwest China. In order to study the population genetics and design the effective conservation methods, we aimed to develop microsatellite primers for this species in the present study. We developed eight new microsatellite loci for this species through biotin capture method. Polymorphism of each locus was further assessed in 18 individuals from three geographically distant populations. The number of alleles per locus ranged from 6 to 11 with an average of 8.13. The observed and expected heterozygosities ranged from 0.219 to 0.296 and from 0.374 to 0.470, with averages of 0.254 and 0.417, respectively. We further found that three of nine microsatellite loci developed previously for another congeneric species showed polymorphic banding patters. We performed primer-crossing tests of these loci in the other two congeneric species which are closely related to C. chenggiana (C. gigantea and C. duclouxiana). These microsatellite markers would be effective for analyzing genetic diversity and population genetic structure of this species and its morphological differentiation with the close relatives.     

Some minerals as well as trace and toxic elements in livers of fallow deer (<Emphasis Type="Italic">Dama dama</Emphasis>) in Slovenia

Minerals were determined in the livers of 69 fallow deer, which were shot during routine culls in Slovenian enclosures, in the winter of 2001/2002. Analysis of minerals and trace elements was done by atomic absorption spectroscopy and analysis of phosphorus was done by spectrophotometry. Mean values recorded for liver were 0.11±0.07 g/kg for calcium, 6.38±1.10 g/kg for phosphorus, 0.65±0.24 g/kg for sodium, 1.91±0.66 g/kg for potassium, 0.14±0.03 g/kg for magnesium, 31.0±21.9 mg/kg for copper, 31.3±10.8 mg/kg for zinc, 3.8±1.7 mg/kg for manganese, and 141±65 mg/kg for iron. Lead was present in high concentrations in the liver of almost all animals (1.48±0.92 mg/kg), while cadmium occurred in relatively low concentrations (0.27±0.14 mg/kg). There were significant differences in liver sodium, copper and iron concentrations between sexes and significant differences in liver phosphorus concentration between old and young animals.     

Polymorphic microsatellite loci in the Australian tree frog, <Emphasis Type="Italic">Litoria peronii</Emphasis>

Eleven novel polymorphic microsatellite loci developed from a microsatellite enriched genomic library, are presented for the Australian tree frog Litoria peronii. We screened 29 individuals from a single population and detected high levels of polymorphism for all 11 loci with the number of alleles/locus ranging from 9 to 24. Values of expected and observed heterozygosities ranged from 0.789–0.955 and 0.207–1.00, respectively. These microsatellite markers should prove useful in determining levels of genetic diversity, measuring gene flow and migration, assigning individuals to their most likely population of origin, and in the assignment of paternity.     

Development and characterization of microsatellite loci in <Emphasis Type="Italic">Ixeridium dentatum</Emphasis> (Asteraceae,Lactuceae)

We isolated and characterized seven microsatellite loci for the perennial herb Ixeridium dentatum ssp. dentatum, an apomictic triploid distributed throughout the lowland areas of East Asia. The number of alleles ranged from two to seven in 32 screened individuals of I. dentatum ssp. dentatum from Japan. The observed and expected heterozygosities were 0.000–0.950 and 0.000–0.891, respectively, calculated using genotypes of 20 individuals of I. dentatum ssp. nipponicum. One locus (msid4) deviated significantly from Hardy–Weinberg equilibrium (P = 0.0001). These microsatellites were also tested for cross-amplification in 11 other taxa of Lactuceae, including five endangered taxa. These primers should be useful genetic tools not only for Ixeridium but also for other Lactuceae taxa.     

A set of polymorphic nuclear intron markers for conservation genetics and phylogeography of <Emphasis Type="Italic">Euphorbia</Emphasis> species (<Emphasis Type="Italic">Pedilanthus</Emphasis> clade)

We developed seven nuclear intron markers for Euphorbia lomelii. New exon-primed intron-crossing (EPIC) oligonucleotides were used for initial amplification and sequencing, then locus-specific primers and restriction-fragment-length polymorphism genotyping assays were designed. Loci showed no significant deviation from Hardy–Weinberg and linkage equilibrium, and they cross-amplify in at least three congeneric species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genetic variability and structure of the remnant natural populations of <Emphasis Type="Italic">Cedrus libani</Emphasis> (Pinaceae) of Lebanon

Cedrus libani of Lebanon is a valuable natural resource and the dominant species in its natural ecosystem. Intense and diverse anthropogenic pressures over historical times raised concerns about its genetic vigor and continued survival. Our investigation of the genetic diversity included samples from all remnant natural populations. Assessment of the genetic diversity using random amplified polymorphic DNA markers revealed the persistence of considerable variation distributed within populations with low population differentiation corroborated by Bayesian and analysis of molecular variance estimates (G _ST = 0.07, Φ _ST = 0.09). Individual assignment tests were carried out to investigate measures of gene flow. Inferences concluded that this natural heritage is not currently threatened by inbreeding or by random genetic drift. Correlation studies investigated possible effects of spatial distribution and environmental conditions on genetic structure. A climatic trend corresponding to a temperature–humidity gradient correlated significantly with the level of genetic diversity, while the edaphic variation did not.     

Identification of microsatellite markers in <Emphasis Type="Italic">Hieracium pilosella</Emphasis> L.

In order to investigate the genetic diversity and structure of Hieracium pilosella L., we developed eleven pairs of primers for nuclear microsatellites through screening of three genomic DNA enriched libraries. About 127 individuals from six locations of the Trentino region (Italy) were analysed. These loci were all polymorphic and displayed 5–33 alleles per locus. These microsatellite markers constitute an efficient tool in investigating the genetic patterns of H. pilosella L. populations.     

Home range patterns of male fallow deer<Emphasis Type="Italic">Dama dama</Emphasis> in a sub-Mediterranean habitat

From 1996 to 2000 the home ranges of 14 male fallow deerDama dama (Linnaeus, 1758) were studied in the San Rossore Preserve (Italy) using radio-telemetry. Mean size of annual home ranges was 588.9 ± 278.9 ha, calculated by MCP, and 337.5 ± 178.9 ha, using Kernel method, and was larger than that reported in published literature to date. The size of the seasonal home range estimated with the MCP method was 90.6 ± 129.1 ha during spring, 73.7 ± 67.9 ha in summer, 465.0 ± 230.6 ha in fall, and 65.6 ± 60.6 ha in winter. The Kernel method gave 84.7 ± 140.2 ha in spring, 61.3 ± 64.6 ha in summer, 306.0 ± 170.5 ha in fall, and 46.5 ± 44.0 ha during winter. The seasonal analysis suggested that bucks tended to occupy the same particular area from winter to summer, which was related to rich trophic resources, even despite of anthropic disturbance. During autumn, males reached the rutting site (a lek) that was 4 km distant from the areas occupied during the other three seasons. The lekking behaviour was the main factor influencing home range size.     

Development of ten microsatellite loci for <Emphasis Type="Italic">Gentiana crassicaulis</Emphasis> (Gentianaceae)

Genetiana crassicaulis is one of famous Chinese medicinal plant. The over-collection for its root has caused its dramatic reduction. In order to devise adequate conservation and management strategies for this species, it is important to characterize its genetic diversity and understand its population structure. Here, 10 polymorphic microsatellite markers have been developed. AC/TG microsatellite was enriched by combining biotin capture method. Polymorphism of each locus was assessed in 30 individuals from six populations. The number of alleles ranged from 2 to 9 and the expected heterozygosity ranged from 0.32 to 0.78.     

Development of 10 microsatellite loci for <Emphasis Type="Italic">Rheum tanguticum</Emphasis> (Polygonaceae)

Rhubarb is an important Traditional Chinese Medicine. However, the wild resource has been declining. In order to design appropriate conservation methods for the official species across their natural distributions, it is important to characterize their genetic diversity. Here, we describe the development of 10 new microsatellite loci for AC/TG/CCA in Rheum tanguticum. The microsatellites were enriched using the combined biotin capture method. The polymorphism of each locus was further assessed in 12 individuals from four geographically distinct populations of this species. The number of alleles ranged from three to seven and the expected heterozygosity ranged from 0.53 to 0.73. All markers have been checked in the other three species in the genus and two of them together comprise the official medicinal rhubarb resource with R. tanguticum. These microsatellite markers could provide a useful tool for genetic and conservation studies of the rhubarb species.