Relative spraint density and genetic structure of otter (Lutra lutra) along the Drava River in Hungary

In this study, we used genetic-based approaches to estimate population size and structure of Eurasian otter along the Drava River in Hungary, and compared these results to traditional survey-based methods. The relative spraint density of otter was estimated based on the number of fresh (D_f) and total number (D_t) of spraints collected on standard routes over a 2-year period. Nine microsatellite loci were screened, generating 17 individual otter genotypes composed of 45 different alleles. The expected heterozygosity ranged from 0.53 to 0.89 and observed heterozygosity from 0.25 to 0.92. The mean density (D_g) estimated over six different sites was 0.17 individuals per km of shoreline. A close correlation was found between the number of genotypes and spraint counts along a standard route (fresh spraints: r_P=0.85, P<0.01; total spraints r_P=0.76, P<0.05). All genotypes found within the 50 km-long study area were closely related (D_m ranged between 0.08 and 0.21).     

An optimisation approach to increase DNA amplification success of otter faeces

Faeces have proved to be a suitable non-invasive DNA source for microsatellite analysis in wildlife research. For the success of such studies it is essential to obtain the highest possible PCR amplification success rate. These rates are still relatively low in most carnivorous species, especially in the otter (Lutra lutra). We therefore optimised the entire microsatellite genotyping process by combining our findings with results from previous studies to gain a high rate of reliable genotypes. We investigated the influence of otter faecal quality in relation to the quantity of slimy secretions and three levels of storage periods at ?20°C on amplification success. Further, we tested the cost-effective and time-saving Chelex extraction method against the profitable QIAamp^® DNA Stool Kit (Qiagen), and compared three PCR methods - a standard single-step PCR protocol, a single-locus two-step PCR procedure and a multiplex two-step PCR procedure - regarding success rate and genotyping errors. The highest amplification success rate (median: 94%; mean: 78%) was achieved using faecal samples consisting only of jelly extracted with the QIAamp^® DNA Stool Mini Kit (Qiagen) immediately after collection and amplified following the time and cost efficient multiplex two-step PCR protocol. The two-step procedure, also referred to as pre-amplification approach, turned out to be the main improvement as it increases amplification success about 11% and reduces genotyping errors about 53%, most notably allelic dropouts.     

Trophic flexibility of the otter (Lutra lutra) in southern Italy

Diet composition of otters (Lutra lutra) was investigated in 2001 by spraints analysis (N=1323) on five rivers of southern Italy, with the aim of assessing the influence of fish availability, elevation and discharge on the consumption of food resources alternative to fish. Data were expressed as per cent frequency of occurrence (%FO) and per cent volume (%V). The study confirmed the great feeding adaptability of the otter that, in rivers partially interconnected and flowing on a small area, showed a strong fish eating habit in some rivers (Sinni and Mercure-Lao) and a diet mainly constituted by alternative resources in other ones (amphibians in the rivers Cogliandrino and Frido, crustaceans in the River Battendiero). Fish consumption for the five rivers was significantly correlated with fish biomass and with mean summer discharge, while it was inversely correlated with the mean altitude of the five rivers. The lack of a clear seasonality in the consumption of food sources alternative to fish together with the correlation between fish use and fish biomass for each river indicated fish availability as the main factor affecting otter relying to non-fish preys. Otter diet seemed influenced by the characteristics of river habitats (altitude, discharge and consequently fish biomass) more than by summer drought, typical of Mediterranean regions. The %FO and the %V allowed to drawn a similar picture of otter diet. Nonetheless the %V was useful for better illustrating diet variation among the different rivers and we argue that it could be useful in habitats where the otter feeds on preys with different proportions of indigestible remains.     

Mitochondrial genetic diversity and structure of the European otter (Lutra lutra) in Britain

The European otter (Lutra lutra) is a focus for conservation efforts throughout Europe due to a population decline in recent decades and because of its importance as a biological indicator of the health of rivers and waterways. The aim of this study was to aid the conservation of this species by adding genetic information from samples originating in the United Kingdom (UK), to help build up a picture of the phylogeographic structure of the European otter throughout Europe. This was done by a comparison of 299 base pairs of the mitochondrial DNA control region. Four haplotypes were identified in the UK, one of which has not been found outside the west of the UK in the wild, and one of which was unique. Populations in the UK, and in particular the west were shown to have a higher haplotype diversity than previously found for the European otter in Europe (h = 0.7338 for the 58 UK otters sampled in this study) and an overall nucleotide diversity of π = 0.003. The western UK population was shown to have a high level of genetic distinctiveness. We discuss possible contributory population processes, the importance of the western UK population for the future conservation of the species and comment on future conservation strategies.     

Source and sink dynamics of density-dependent otter (<Emphasis Type="Italic">Lutra lutra</Emphasis>) populations in rivers of central Finland

Long-term studies were carried out in central Finland between 1985 and 2003 to examine the temporal and spatial variation in the density of otter populations. Snow tracking was used to estimate the total population and the number of litters in the study area. In total 52 otters, including 16 cubs in 11 litters, lived in the study area (1,650 km²) in 2002–2003. The otter population clearly increased during the study period. The increase in density of the otter population was sigmoid, indicating that the population had reached the local carrying capacity. The density of the population was 0.12 individuals per river ha in 1985 and 0.29 individuals per river ha in 2002. The number of cubs per litter decreased when the density of the population increased. Density-dependent offspring production, together with the auto-correlation function of growth rate, indicates intraspecific competition in otter populations. Otters in a few river systems produced most of the cubs, creating several small source populations in the entire study area. Otters in secondary (sink) habitats had a low reproduction rate. Most otters lived in river systems with large lake surfaces. The number or area of lakes within the river system correlated positively with the total number of otters, litters and cubs in the river system. The six river systems (out of 16) with the largest water area of lakes produced 81.2% of all cubs born in the study area. However, the population growth rate per river hectare or per river kilometre was equal in all kinds of river systems. Thus, among local otter populations in central Finland, a source–sink system between different habitats seems to be prevalent.     

Sex identification of Eurasian otter (<Emphasis Type="Italic">Lutra lutra</Emphasis>) non-invasive DNA samples using ZFX/ZFY sequences

We developed a new PCR/RFLP system targeted to amplify and cut a segment of the ZFX/ZFY gene in non-invasive otter (Lutra lutra) samples. This assay produced one sex-specific fragment in females (XX genotypes) and two fragments in males (XY genotypes), and is intrinsically more reliable than alternative systems (e.g., SRY genes) that are based on the amplification of a single Y-linked fragment. The new primer pair correctly identified the sex of 23 DNA extracted from sexed otter tissues. This procedure was used to sex unknown DNA extracted from otter scats. A multi-tube approach led to identify the sex of 72/91 (79%) samples, using a minimum of two PCR replicates. This procedure is currently used in non-invasive genetic monitoring of Italian otter populations.     

Genetic mapping of an ancient translocation in the genus Lens

Summary Segregation of 18 marker genes was monitored in selfed progeny of a Lens culinaris × L. ervoides hybrid; five linkage groups were mapped, one of which contained a reciprocal translocation break-point that differentiates between the parents. Four markers were found to be linked to the translocation break-point: Aco-1 and Pgm-2 on one side and Gs and Got-2 on the other. The gene pairs on both sides of the translocation are not linked in L. culinaris or in L. orientalis. The L. ervoides gene order was also found in L. odemensis but with significantly reduced map distances. Analysis of monogenic segregations in a number of Lens inter-specific crosses revealed some consistent patterns of deviations from the expected Mendelian ratios. The factors responsible for these unequal segregations, genotypic effects on recombination frequencies, negative interference, and the possible ancient origin of the translocation are discussed.     

Genetic effects of habitat contraction on Lumholtz's tree-kangaroo (Dendrolagus lumholtzi) in the Australian Wet Tropics

Lumholtz's tree-kangaroo (Dendrolaguslumholtzi) is one of two species oftree-kangaroo resident in the tropicalrainforests of north-eastern Australia. Thespecies is confined to the Wet Tropics region,with its distribution centred on the AthertonTablelands. While D. lumholtzi wasexposed to periodic large-scale climaticfluctuations during the Quaternary that haveeffectively acted as natural fragmentationevents, the species is currently under pressurefrom anthropogenic disturbance and habitatfragmentation. This study aimed to assess thelevel of genetic diversity in D.lumholtzi by examining hypervariablemicrosatellite loci and the control region ofmitochondrial DNA (mtDNA) in 21 individualsfrom a single 20 ha forest fragment, and from afurther 24 animals collected throughout theAtherton Tablelands. Results suggest that D. lumholtzi has relatively low levels ofgenetic diversity which is uniformlydistributed throughout the Atherton Tablelands;a pattern congruent with data from many othervertebrates endemic to the Australian WetTropics. It is suggested that Pleistoceneclimatic fluctuations, which resulted inlarge-scale rainforest contractions, haveimposed an ancient population bottleneck on theancestral D. lumholtzi population. Theapparent over-riding influence of thesenatural, historical effects on the geneticstructure of D. lumholtzi populations,will complicate attempts to assess the geneticimpact of current anthropogenic habitat lossand fragmentation.     

Genetic evaluation of remnant and translocated shiners,Notropis heterodon and Notropis heterolepis

Population and conservation genetics of two freshwater fish species, Notropis heterodon and Notropis heterolepis, were evaluated in north‐eastern Illinois, U.S.A., where both species have severely declined. Fishes were sampled from two remnant populations occurring in small glacial lakes (source samples) and from two man‐made ponds that had been stocked with fishes from those same lakes (sanctuary samples). The goal was to obtain information that would help inform conservation programme planning to reintroduce sanctuary fishes to areas where both species are extirpated. Microsatellite data showed that the two species were genetically quite distinct and there was no evidence of hybridization in either source or sanctuary samples. Within each species, source and sanctuary samples had moderate levels of heterozygosity and were not significantly different from each other. Many alleles observed in the source samples, however, were not detected in the sanctuary samples, indicating that translocation had resulted in reduced allelic diversity of the sanctuary samples. Sibship analysis indicated that full and half sibs occurred within source‐lake samples, thus reducing the effective population size of the reintroduced stock. Taken together, these results suggest that source‐lake stocks rather than sanctuary stocks are more appropriate for future reintroductions of both species in their native range, unless sanctuary populations can be established with hundreds of fishes. Also, fishes should be harvested from multiple locations in source lakes to avoid over‐representation of family groups.     

Genetic Differentiation of the Giant Honey Bee (Apis dorsata) in Thailand Analyzed by Mitochondrial Genes and Microsatellites

Genetic diversity and population differentiation of the giant honey bee (Apis dorsata) in Thailand were examined. Six PCR-RFLP mitotypes were generated from digestion of the COI-COII, Cytb-tRNA^ser, ATPase6-8, and lrRNA genes with Dra I and Hin fI. Low genetic diversity (h=0.074, π=0.032%) and a lack of genetic population differentiation between A. dorsata originating from geographically different regions were observed from mtDNA polymorphisms (P > 0.05). In contrast, microsatellite (A14, A24, and A88) polymorphisms revealed a relatively high level of genetic diversity in A. dorsata (H _o=0.68–0.74, average number of alleles per locus=6.0–9.0). Both A24 and A88 indicated significant population differentiation between bees from the north-to-central region (north, northeast, and central regions), peninsular Thailand, and Samui Island.     

Genetic impacts of Anacapa deer mice reintroductions following rat eradication

The Anacapa deer mouse is an endemic subspecies that inhabits Anacapa Island, part of Channel Islands National Park, California. We used mitochondrial DNA cytochrome c oxidase subunit II gene (COII) and 10 microsatellite loci to evaluate the levels of genetic differentiation and variation in ~1400 Anacapa deer mice sampled before and for 4 years after a black rat (Rattus rattus) eradication campaign that included trapping, captive holding and reintroduction of deer mice. Both mitochondrial and microsatellite analyses indicated significant differentiation between Anacapa deer mice and mainland mice, and genetic variability of mainland mice was significantly higher than Anacapa mice even prior to reintroduction. Bayesian cluster analysis and Principal Coordinates Analysis indicated that East, Middle and West Anacapa mice were genetically differentiated from each other, but translocation of mice among islands resulted in the East population becoming less distinct as a result of management. Levels of heterozygosity were similar before and after management. However, numerous private alleles in the founder populations were not observed after reintroduction and shifts in allele frequencies occurred, indicating that the reintroduced populations experienced substantial genetic drift. Surprisingly, two mitochondrial haplotypes observed in an earlier study of Anacapa deer mice were lost in the 20 years prior to the rat eradication program, leaving only a single haplotype in Anacapa deer mice. This study demonstrates how genetic monitoring can help to understand the re-establishment of endemic species after the eradication of invasive species and to evaluate the effectiveness of the management strategies employed.     

Genetic structure of Eurasian otter (Lutra lutra,Carnivora: Mustelidae) populations from the western Baltic sea region and its implications for the recolonization of north‐western Germany

We analysed 229 Eurasian otters from eastern and north‐western Germany, Denmark and southern Sweden based on sequences of the mtDNA control region and genotypes at 12 polymorphic nuclear microsatellite loci. The main focus of the study lay on the north‐west German otters from Schleswig‐Holstein, which represent a newly established and expanding population recolonizing a formerly inhabited region, thereby closing the distributional gap between the large eastern population and the long‐isolated and genetically depleted Danish population. As found before in this species, mtDNA variability was very low but we identified a hitherto unknown haplotype in Sweden. Expected heterozygosities were between 0.46 and 0.69 and thus within the range known from the literature. There were only weak signs of a founder effect in the Schleswig‐Holstein otters with respect to allelic diversity (but not heterozygosity). Nevertheless, there was a statistically significant bottleneck signal based on deviations from mutation‐drift equilibrium in Schleswig‐Holstein. All statistical approaches (amova s, factorial correspondence analysis, assignment tests and Bayesian structure analysis) unequivocally showed that north‐western Germany has so far been predominantly recolonized by otters of east German origin, but we also found evidence of admixture through immigration from Denmark. Both the Danish and the north‐west German otter population will benefit from further exchange in the wake of the ongoing range expansion.     

Detection of farm fox and hybrid genotypes among wild arctic foxes in Scandinavia

In Scandinavia, farmed arctic foxes frequently escape from farms, raising concern about hybridization with the endangered wild population. This study was performed to find a genetic marker to distinguish escaped farm foxes from wild Scandinavian foxes. Microsatellite and mitochondrial control region variation were analyzed in 41 farm foxes. The results were compared with mitochondrial and microsatellite data from the wild population in Scandinavia. The farm foxes were genetically distinct from the wild foxes (F _ST=0.254, P < 0.00001) and all farm foxes had a single control region haplotype different from those observed in the wild population. We developed a method based on Restriction Fragment Length Polymorphism (RFLP) on the mitochondrial control region to differentiate between farmed and wild arctic foxes. This test was subsequently successfully used on 25 samples from free-ranging foxes, of which four had a suspected farm origin. All four of the suspected foxes, and none of the others, carried the farm fox haplotype. Three of these were successfully genotyped for all eleven microsatellite loci. A population assignment test and a Bayesian Markov Chain Monte Carlo analysis indicated that two of these individuals were escaped farm foxes, and that the third possibly was a hybrid between a farmed and a wild arctic fox.     

Genetic identification of units for conservation in tomato frogs, genus Dyscophus

Dyscophus antongilii and D. guineti are two morphologically very similar microhylid frogs from Madagascar of uncertain taxonomy. D. antongilii is currently included in Appendix I of the Convention on the International Trade in Endangered Species (CITES) and its exportation is banned completely. In contrast, D. guineti does not receive any legal protection and it is regularly exported. Field data on ecology and behaviour are to a large extent lacking. Here we report on a genetic survey of D. antongilii and D. guineti using nuclear and mitochondrial DNA markers. Sequences of a fragment of 501 bp of the mitochondrial cytochrome b gene from one population of D. antongilii and two populations of D. guineti resulted in a single haplotype network, without haplotype sharing among the populations. However, haplotypes of D.␣guineti were only 1–4 mutational steps from those of D. antongilii, and did not form a clade. The analysis of eight microsatellites newly developed and standardized for D. antongilii revealed an excess of homozygotes and the absence of Hardy–Weinberg equilibrium. The microsatellite data clearly distinguished between D. antongilii and D. guineti, and fixed differences were observed at one locus. Although confirmation of the status of Dyscophus antongilii and D. guineti as separate species requires further data, our study supports the definition of these two taxa as different evolutionary significant units under the adaptive evolutionary conservation concept.     

A nuclear perspective on endemism in northern flying squirrels (Glaucomys sabrinus) of the Alexander Archipelago, Alaska

We used microsatellites to examine populationstructure and genetic diversity in northernflying squirrels in the Alexander Archipelagoof Southeast Alaska, with an emphasis on theendemic Prince of Wales flying squirrel(Glaucomys sabrinus griseifrons). Previouswork showed this subspecific designationcoincided with a distinct mitochondrial lineageon eleven islands (the Prince of Wales [POW]complex). To obtain a nuclear perspective onthis lineage and to further investigate geneticdiversity among insular populations, weexamined six microsatellite loci in 233 flyingsquirrels representing eight populations inSoutheast Alaska and a population from interiorAlaska (seven island and two mainlandlocalities). Island populations have lowerheterozygosity and allelic diversity thanmainland populations. Overall, populationpairs show a pattern of isolation by distance,indicating there is little long-distance geneflow across the archipelago. Analyses ofmicrosatellite allele frequencies revealsignificant differences between the POW complexpopulations and others we examined, a findingcongruent with the mitochondrial data. Thepopulation from Mitkof Island, a non-POWcomplex island, also differs significantly fromother populations in allele frequencies. Thesix POW complex populations are geneticallyvery similar, suggesting current or recent geneflow among these islands, while there seems tobe no gene flow between the POW complex andother populations in Southeast Alaska. Ourdata corroborate mitochondrial DNA resultsindicating that G. s. griseifrons isgenetically distinct and suggest a generalpattern of isolation of insular flyingsquirrels in Southeast Alaska.     

Phylogeography and subspecies assessment of vicuñas in Chile and Bolivia utilizing mtDNA and microsatellite markers: implications for vicuña conservation and management

The rearing and maintenance of wildvicuñas in semi-captivity for economicutilization is practiced mainly in Peru, butArgentina, Bolivia, and Chile are quicklydeveloping their own programs of economic use. Large scale rearing practices will likelyisolate populations and may foster selectivebreeding. In addition to these concerns, thereare also uncertainties about the distributionand validity of the currently recognizedsubspecies. To better understand the potentialimpact that economic utilization will have onthe vicuña, we describe the moleculargenetic variation among four populations andboth described subspecies. An analysis of 794~bp of mitochondrial DNA sequences (16s,cytochrome b genes, and mtDNA control region)revealed appreciable genetic diversity, low tomoderate levels of genetic differentiation, andrestricted gene flow with isolation by distanceamong populations. Analysis of microsatellitedata also indicated genetic differentiationamong populations. Past climatic and geologicevents, coupled with human history, have likelysubjected the vicuña to various episodes ofpopulation isolation and admixture. Therefore,we suggest that managers aim to ensure geneflow among adjoining populations, as observedat mtDNA and microsatellite loci, as well asmaintaining apparent restricted gene flow withisolation by distance among populationsseparated by great distances. Intensiverearing procedures like those being practicedin Peru will ultimately disrupt movements andmigration among wild populations. Furthermore,animals that are not exposed to predation byremaining inside fences, may overtime, loseportions of their behavioral repertoire thatenable them to recognize potentially dangeroussounds associated with the presence ofpredators. Therefore, we do not recommend thatanimals be placed behind fences as this couldlead to a loss of genetic and behavioraldiversity as well as halting natural ecologicalprocesses. If, however, vicuñas are placedbehind fences for commercial purposes (withlittle or no regard for the retention ofgenetic, and/or behavioral diversity as well asnatural population processes) then we suggestclose genetic monitoring of animals that are(will be) maintained in captivity.     

The origin of Indian Star tortoises (Geochelone elegans) based on nuclear and mitochondrial DNA analysis: A story of rescue and repatriation

The Indian Star tortoise (Geochelone elegans) belongs to the family Testunidae and is distributed in southwest India and Sri Lanka. In addition to facing loss of its natural habitat, the species is also illegally traded as food and as an exotic pet internationally. Here we report DNA-based analyses for identification and repatriation of these tortoises into their natural habitat. We have attempted to establish the geographical origin of these tortoises rescued from smugglers, by comparing the microsatellite and mitochondrial markers of rescued animals with animals of known provenance. Star tortoises exhibited strong genetic structure in India. The populations from western India were genetically distinct at microsatellite and mitochondrial loci from southern populations. The rescued individuals had similar multilocus genotypes and mitochondrial DNA haplotypes as the reference individuals from south India. However, the precise geographic origin of many of the rescued samples remains unresolved, because we could not assign them to southern populations and the Neighbor-Joining cluster analysis indicated that some of rescued tortoises formed distinct clusters. These data strongly suggest that the rescued group of tortoises is composed of a mix of individuals from differentiated source populations that are probably located in southern India and possibly Sri Lanka. Our study provides valuable information based on molecular markers for the assessment of genetic diversity in Indian Star tortoises.     

Genetic characterization of common carp (Cyprinus carpio) populations from Greece using mitochondrial DNA sequences

Wild common carp from two lakes and two rivers in Greece were genetically characterized with sequencing analysis of two mitochondrial DNA segments: cytochrome b (1119 bp) and D-loop (646 bp). A total of 9 variable singleton sites and 7 unique haplotypes were detected. A common haplotype was found in three out of the four populations examined, which seems to be the ancestral one and represents the European origin of common carp from Greece. This haplotype could be also justified by the introductions reported with individuals belonging to the Central European race, into many natural habitats in Greece. Limited genetic variation — in Evros and Aliakmonas populations — could be due to bottleneck effects and small effective population sizes, whereas the different haplotypes found in Lake Volvi could represent different common carp stocks. Values of sequence divergence among Greek haplotypes ranged from 0.0006 to 0.0023. The Neighbour-Joining (NJ) phylogenetic tree constructed based on the combined sequences, reveals that the populations of common carp from Greece belong to the European group of populations — which is highly divergent from the South East-Asia cluster — and to the subspecies Cyprinus carpio carpio.