Phylogenetic and phylogeographic analysis of Iberian lynx populations

The Iberian lynx (Lynx pardinus), one of the world's most endangered cat species, is vulnerable due to habitat loss, increased fragmentation of populations, and precipitous demographic reductions. An understanding of Iberian lynx evolutionary history is necessary to develop rational management plans for the species. Our objectives were to assess Iberian lynx genetic diversity at three evolutionary timescales. First we analyzed mitochondrial DNA (mtDNA) sequence variation to position the Iberian lynx relative to other species of the genus LYNX: We then assessed the pattern of mtDNA variation of isolated populations across the Iberian Peninsula. Finally we estimated levels of gene flow between two of the most important remaining lynx populations (Do?ana National Park and the Sierra Morena Mountains) and characterized the extent of microsatellite locus variation in these populations. Phylogenetic analyses of 1613 bp of mtDNA sequence variation supports the hypothesis that the Iberian lynx, Eurasian lynx, and Canadian lynx diverged within a short time period around 1.53-1.68 million years ago, and that the Iberian lynx and Eurasian lynx are sister taxa. Relative to most other felid species, genetic variation in mtDNA genes and nuclear microsatellites were reduced in Iberian lynx, suggesting that they experienced a fairly severe demographic bottleneck. In addition, the effects of more recent reductions in gene flow and population size are being manifested in local patterns of molecular genetic variation. These data, combined with recent studies modeling the viability of Iberian lynx populations, should provide greater urgency for the development and implementation of rational in situ and ex situ conservation plans.     

Looking for the Iberian lynx in central Spain: a needle in a haystack?

The Iberian lynx Lynx pardinus has suffered a dramatic reduction in its range throughout the Iberian Peninsula and at present is one of the most endangered mammals in the world. The latest studies report that, out of the 48 breeding areas that existed in 1990, only two populations are left in southern Spain. As a consequence, some of the formerly largest populations, such as Montes de Toledo (central Spain), are to all intents and purposes regarded as extinct. To determine the current distribution of Iberian lynx outside the two recognized populations, we surveyed five different areas where the species is considered extinct and collected 581 faeces for the genetic identification of the species. We identified 18 samples as belonging to Iberian lynx in four out of the five areas studied, providing clear evidence for the presence of lynx in central Spain. In some areas the species was detected repeatedly at different localities and on different dates, indicating a regular occurrence of an unknown number of individuals. The conservation implications of these results are discussed in terms of the genetic importance of the individuals found and future reintroductions of the species from an ongoing captive-breeding programme.     

Successful carnivore identification with faecal DNA across a fragmented Amazonian landscape

The use of scat surveys to obtain DNA has been well documented in temperate areas, where DNA preservation may be more effective than in tropical forests. Samples obtained in the tropics are often exposed to high humidity, warm temperatures, frequent rain and intense sunlight, all of which can rapidly degrade DNA. Despite these potential problems, we demonstrate successful mtDNA amplification and sequencing for faeces of carnivores collected in tropical conditions and quantify how sample condition and environmental variables influence the success of PCR amplification and species identification. Additionally, the feasibility of genotyping nuclear microsatellites from jaguar (Panthera onca) faeces was investigated. From October 2007 to December 2008, 93 faecal samples were collected in the southern Brazilian Amazon. A total of eight carnivore species was successfully identified from 71% of all samples obtained. Information theoretic analysis revealed that the number of PCR attempts before a successful sequence was an important negative predictor across all three responses (success of species identification, success of species identification from the first sequence and PCR amplification success), whereas the relative importance of the other three predictors (sample condition, season and distance from forest edge) varied between the three responses. Nuclear microsatellite amplification from jaguar faeces had lower success rates (15-44%) compared with those of the mtDNA marker. Our results show that DNA obtained from faecal samples works efficiently for carnivore species identification in the Amazon forest and also shows potential for nuclear DNA analysis, thus providing a valuable tool for genetic, ecological and conservation studies.     

Status survey of the critically endangered Iberian lynx <Emphasis Type="Italic">Lynx pardinus</Emphasis> in Portugal

A survey of the Iberian lynx was conducted in Portugal between January 2002 and November 2003 in order to define lynx status and distribution baselines. Intensive search for lynx scats, scat DNA analysis, and camera trapping were used in areas of potential lynx presence. Over 4,200 km were investigated during a global searching effort of 1,975 man-hours. DNA obtained from 168 scats was analyzed, producing no positive lynx amplifications. Camera trapping represented a total effort of 5,647 camera-days in three potential lynx areas, producing no positive detections. Although results cannot confirm the species’ extinction, the scenario is rather pessimistic and the Iberian lynx is probably no longer present in Portugal. Presently, considering the Portuguese lynx’s historical range, the only significantly suitable areas for the species are located in the southeastern part of the country bordering Andalusia where no recent evidence of lynx presence was recorded.     

非损伤采样提取DNA技术在猫科动物调查中的应用

<正>猫科动物广泛分布于世界各地(Johnson et al.,2006),全球现存37种猫科动物,其中25种已被国际自然保护联盟(IUCN)列为濒危和易危,86%以上的种群数量处于下降或未知状态(IUCN Red List,2011)。我国有13种猫科动物,占世界种类的35%(王应祥,2003),其中属于国家一级保护的有4种,二级保护的有8种,然而,对这些珍稀物种的生态学研究并不充分(高耀亭,     

Species-specific mitochondrial DNA markers for identification of non-invasive samples from sympatric carnivores in the Iberian Peninsula

Genetic species identification of non-invasively collected samples has become an important tool in ecological research, management and conservation and wildlife forensics. This is especially true for carnivores, due to their elusive nature, and is crucial when several ecologically and phylogenetically close species, with similar faeces, hairs, bones and/or pelts, occur in sympatry. This is the case of the Iberian Peninsula, a region with a carnivore community of 16 species—about two-thirds of the European carnivore fauna. Here we present a simple, efficient and reliable PCR-based protocol, using a novel set of species-specific primers, for the unambiguous identification to species of non-invasively collected samples or forensic materials from Iberian carnivores. For each species, from the consensus of all cytochrome b haplotypes, found here and previously reported, we designed species-specific primer pairs for short fragments, the most likely to persist in low-quantity and degraded DNA samples. The predicted specificity of each primer pair was assessed through PCR of positive DNA extracts from the carnivore species, from an exhaustive array of potential prey and from humans. The robustness of PCR amplification for non-invasively sampled DNA was tested with scat samples. The primers did not produce false positives and correctly identified all carnivore samples to the species level. In comparison with sequencing and PCR-RFLP assays, our method is, respectively, cost- and time-effective, and is especially suited for monitoring surveys targeting multiple populations/species. It also introduces an approach that works for a whole community of carnivores living sympatrically over a large geographic area.     

Ancient mtDNA haplogroups: a new insight into the genetic history of European populations

In the present work, DNA was extracted from 63 skeletal samples recovered at the Neolithic site of San Juan ante Portam Latinam (SJAPL) (Araba, Basque Country). These samples have proved useful as genetic material for the performance of population studies. To achieve this it was necessary to overcome the methodological problems arising when working with damaged DNA molecules. We succeeded in performing an amplification and restriction analysis of the polymorphisms present in the mtDNA. Ninety seven percent of the samples were classified as belonging to one of the nine mtDNA haplogroups described in Caucasians. This work shows that restriction analysis is a useful methodological tool to perform reliable population genetic analysis on archaeological remains. Tha analysis of ancient and modern haplogroup distribution can shed more light on the genetic evolution of human populations. Moreover, a more exhaustive data on prehistoric populations will allow to build stronger hypothesis on the genetic relationships among human populations.     

Gene flow in great bustard populations across the Strait of Gibraltar as elucidated from excremental PCR and mtDNA sequencing

Recent advances in molecular biology have made it possible to use the trace amounts of DNA in faeces to non-invasively sample endangered species for genetic studies. Here we use faeces as a source of DNA and mtDNA sequence data to elucidate the relationship among Spanish and Moroccan populations of great bustards. 834 bp of combined control region and cytochrome-b mtDNA fragments revealed four variable sites that defined seven closely related haplotypes in 54 individuals. Morocco was fixed for a single mtDNA haplotype that occurs at moderate frequency (28%) in Spain. We could not differentiate among the sampled Spanish populations of Cáceres and Andalucía but these combined populations were differentiated from the Moroccan population. Estimates of gene flow (Nm = 0.82)are consistent with extensive observations on the southern Iberian peninsular indicating that few individuals fly across the Strait of Gibraltar. We demonstrate that both this sea barrier and mountain barriers in Spain limit dispersal among adjacent great bustard populations to a similar extent. The Moroccan population is of high ornithological significance as it holds the only population of great bustards in Africa. This population is critically small and genetic and observational data indicate that it is unlikely to be recolonised via immigration from Spain should it be extirpated. In light of the evidence presented here it deserves the maximum level of protection. This revised version was published online in July 2006 with corrections to the Cover Date.     

Historical DNA reveals the phylogenetic position of the extinct Alpine lynx

During the last two centuries, lynx populations have undergone severe declines and extinctions in Europe. The Alpine lynx, once distributed across the whole Alpine arc, became extinct due to direct human prosecution and deprivation of its main prey in the 1930s. Similar to the Iberian lynx Lynx pardinus , its taxonomy has been subject to several controversies. Moreover, knowing the taxonomic status of the Alpine lynx will help to define conservation units of extant lynx populations in Europe. In this study, we investigated two mitochondrial DNA regions in museum specimens ( n =15) representing the autochthonous Alpine population and in samples from extant Eurasian lynx Lynx lynx populations in Europe and Asia ( n =17). Phylogenetic analysis (cytochrome b , 345 bp) placed the Alpine lynx within the Eurasian lynx lineage. Among all individuals examined, seven different haplotypes (control region, 300 bp) were observed but no unique Alpine haplotype was discovered. Haplotypes of the extinct Alpine population were identical to previously described haplotypes in Scandinavian lynx signifying a recent genetic ancestry with current European populations. Moreover, our genetic data suggest two distinct glacial refugia for the Carpathian and Balkan population. Overall this study demonstrates that historical DNA from extinct populations can help to disentangle the phylogenetic relationships and historical biogeography of taxa with only a limited number of extant populations remaining.     

A non-invasive genetic method to identify the sympatric mustelids pine marten (<Emphasis Type="Italic">Martes martes</Emphasis>) and stone marten (<Emphasis Type="Italic">Martes foina</Emphasis>): preliminary distribution survey on the northern Iberian Peninsula

The closely related mustelids European pine marten (Martes martes) and stone marten (Martes foina) sympatrically inhabit a large area of Europe. However, given our limited knowledge of their bioecological relationships, their extremely elusive behaviour and the fact that their faeces cannot be distinguished on the basis of morphology alone, it is very difficult to monitor their populations. In this study, we describe a reliable non-invasive polymerase chain reaction (PCR)–restriction fragment length polymorphism (PCR-RFLP) method for distinguishing between M. martes and M. foina based on the analysis of deoxyribonucleic acid extracted from faeces samples. The method was specifically designed to avoid possible interference from potential prey mammals and other sympatric carnivores. The procedure consists of PCR amplifying a mitochondrial D-loop region followed by digesting the resulting 276-bp-long amplicons with the restriction enzymes HaeIII and RsaI. To assess the efficiency of this technique, we conducted a preliminary field study across the potential sympatric distribution areas of both marten species in the northern Iberian Peninsula. Out of 359 faeces samples collected, we identified 80 as specimens from the stone marten and 235 from the pine marten. Unequivocal species identification was thus possible in 88% of the faeces samples collected. These findings reveal the combined use of non-invasive genetic sampling and GIS technology to be a reliable and cost-effective procedure for improving our knowledge of the spatial distributions of sympatric marten species. This protocol could also be used to identify and improve information gaps, to develop effective research and management programmes and in population and landscape genetics studies of marten species. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

短尾猴陈旧粪便中DNA的提取

分子粪便学（Molecular scatology）是一门将传统粪便分析方法与分子生物学技术相结合,以动物粪便为实验材料进行多领域研究的学科（魏辅等,2001）。虽然该方法已在野生濒危动物保护遗传学和分子生态学研究中发挥了很大作用（Kohnand Wayne,1997）,但目前大多数分子粪便学研究中使用的材料是新鲜粪便,从保存时间很长的陈旧粪便中很难提取到高质量的DNA用于PCR扩增以及序列分析,严重制约了分子粪便学的广泛应用（Wasser et al．,1997;Constable et al．,2001;Murphy et al．．2002）。     

The impact of time and field conditions on brown bear (<Emphasis Type="Italic">Ursus arctos</Emphasis>) faecal DNA amplification

To establish longevity of faecal DNA samples under varying summer field conditions, we collected 53 faeces from captive brown bears (Ursus arctos) on a restricted vegetation diet. Each faeces was divided, and one half was placed on a warm, dry field site while the other half was placed on a cool, wet field site on Moscow Mountain, Idaho, USA. Temperature, relative humidity, and dew point data were collected on each site, and faeces were sampled for DNA extraction at <1, 3, 6, 14, 30, 45, and 60 days. Faecal DNA sample viability was assessed by attempting PCR amplification of a mitochondrial DNA (mtDNA) locus (∼150 bp) and a nuclear DNA (nDNA) microsatellite locus (180–200 bp). Time in the field, temperature, and dew point impacted mtDNA and nDNA amplification success with the greatest drop in success rates occurring between 1 and 3 days. In addition, genotyping errors significantly increased over time at both field sites. Based on these results, we recommend collecting samples at frequent transect intervals and focusing sampling efforts during drier portions of the year when possible.     

Epidemiology of Ancylostoma spp. in the endangered Iberian lynx (Lynx pardinus) in the Doñana National Park, south-west Spain

The epidemiology of Ancylostoma spp. was studied in the endangered Iberian lynx (Lynx pardinus) in the Do?ana National Park, south-west Spain. Faecal samples were collected throughout a complete annual cycle (August 1997 to September 1998). The overall egg prevalence of Ancylostoma spp. was 57.8%. The pattern of abundance of Ancylostoma spp. eggs in faeces was overdispersed. Juvenile lynx demonstrated a statistically higher prevalence and abundance of Ancylostoma spp. than in adults. These levels of egg output (maximum 21195 epg), as previously reported in free ranging large felid cubs, could be close to disease involvement. The potential pathogenicity of hookworms and the influence of individual and ecological factors on hookworm transmission in the Iberian lynx from the Do?ana National Park population are discussed.     

Phylogeography of Hyalomma (Euhyalomma) lusitanicum (Acarina,Parasitiformes, Ixodidae) in Andalusia based on mitochondrial cytochrome oxidase I gene

The genetic population structure relationships of Hyalomma (Euhyalomma) lusitanicum in Andalusia (the south of the Iberian Peninsula) were examined using mtDNA sequence data from 887 bp of cytochrome oxidase subunit I (COI) gene. The sequence for the COI region was determined for 84 individuals collected in several localities of Andalusia, and 10 for other localities (i.e., five from Toledo, central Iberian Peninsula, four from Sicily (Italy) and one from Canary Island). Seventeen haplotypes were detected, including 27 polymorphic sites. The number of amino acid substitutions per site from mean diversity calculations for the entire population was 0.017. AMOVA analysis revealed a low gene flow that characterises the genetic population structure of this species in South Iberian Peninsula, with a haplotype diversity (h) value of 0.815. No geographically induced differentiation was observed, and separate evolutionary units were not detected. Our results indicate low genetic diversity across the geographical range of H. lusitanicum tick in Andalusia. Our data do not show any genetic discontinuity between the tick populations studied, including specimens from Canary Island and Sicily (Italy).

     

A new 2CTAB/PCI method improves DNA amplification success from faeces of Mediterranean (Barbary macaques) and tropical (lowland gorillas) primates

Animal genomic DNA extracts of sufficient quality to address questions about population biology or behavioural ecology can be obtained from faeces when adequate extraction procedures are used. The presence of PCR inhibitors in extract products appears generally the main factor limiting DNA amplification success. We compared DNA amplification success from faeces of a tropical primate (western lowland gorilla, Gorilla g. gorilla) and a Mediterranean primate (Barbary macaque, Macaca sylvanus) between a standardized extraction technique widely used in animals (QIAamp^® stool kit), a technique mainly used in plant species (CTAB) and a new protocol (2CTAB/PCI). Amplification success varied from 51% to 97%, the highest success being reached with the 2CTAB/PCI protocol in both species.     

Hybridization Between Canada Lynx and Bobcats: Genetic Results and Management Implications

Hybridization between taxonomically similar species is an often-overlooked mechanism limiting the recovery of threatened and endangered species. We present molecular genetic data for the first time demonstrating that Canada lynx and bobcats hybridize in the wild. We verify that two microsatellite loci Lc106 and Lc110 have non-overlapping allele ranges between Canada lynx and bobcats, and that three putative lynx from Minnesota contain DNA from both bobcats and lynx. Additionally, we use a published test for the 16S rRNA region of mitochondrial DNA (mtDNA) to determine the maternal species; all hybrids had lynx mothers. Fifteen per cent (3/20) of our putative lynx samples were hybrids, although these data are not from a representative sampling effort. Hybridization may be an under-appreciated factor limiting the distribution and recovery of lynx. The presence of hybrids is thus a new factor in the population management of both species with potential implications for hunting and trapping of bobcats.     

Quantitative analysis of prey DNA in pinniped faeces: potential to estimate diet composition?

We investigate using relative amounts of prey DNA recovered from pinniped faeces to obtain diet composition data. Faeces were obtained from captive sea lions being fed a diet containing three fish species (50%, 36% and 14% by mass). Real-time PCR was used to quantify mtDNA in undigested tissue and in the faecal samples. The percent composition of fish mtDNA extracted from tissue corresponded reasonably well to the mass of fish in the mixture. In faecal samples the absolute amount of fish mtDNA recovered varied 100-fold, but the percent composition of the samples was relatively consistent (57.5 ± 9.3%, 19.3 ± 6.6% and 23.2 ± 12.2%). These results indicate there are prey-specific biases in DNA survival during digestion. However, the biases may be less than those commonly observed in conventional diet studies.     

Extraction, amplification and characterization of wood DNA from dipterocarpaceae

A successful DNA extraction from wood yielding appropriate DNA quality for PCR amplification allows molecular genetic investigations of wood tissue. Genotypes, the origin of sampled material, and species can be identified based on an investigation of wood if suitable information on genetic variation patterns within and among species is available. Potential applications are in forensics and in the control of the timber and wood trade. We extracted DNA from wood of Dipterocarpaceae, a family that dominates rainforests and comprises many important timber species in Southeast Asia. Several different DNA isolation techniques were compared and optimized for wood samples from natural populations and from wood processing enterprises. The quality of the DNA was tested by spectrophotometry, PCR amplification, and PCR inhibitor tests. An average DNA yield of 2.2 μg was obtained per 50–100 mg of dried wood sample. Chloroplast DNA (cpDNA) regions of different length were amenable to PCR amplification from the extracted DNA. Modification of DNA isolation techniques by the addition of polyvinylpyrrolidone (PVP) addition up to 3.1% into lysis buffer reduced PCR inhibition effectively. In order to evaluate the extraction method, we analyzed leaves and wood from the same tree by PCR amplification, genotyping and sequencing of chloroplast microsatellites.     

Faeces as a source of DNA for molecular studies in a threatened population of great bustards

With recent advances in molecular biology, it is now possible to use the trace amounts of DNA in faeces to non-invasively sample endangered species for genetic studies. A highly vulnerable population of approximately 100 great bustards (Otis tarda) exists in Morocco necessitating the use of non-invasive protocols to study their genetic structure. Here we report a reliable silica-based method to extract DNA from great bustard faeces. We found that successful extraction and amplification correlated strongly with faeces freshness and composition. We could not extract amplifiable DNA from 30% of our samples as they were dry or contained insect material. However 100% of our fresh faecal samples containing no obvious insect material worked, allowing us to assess the levels of genetic variation among 25 individuals using a 542 bp control region sequence. We were able to extract DNA from four out of five other avian species, demonstrating that faeces represents a suitable source of DNA for population genetics studies in a broad range of species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Last lynxes in Portugal? Molecular approaches in a pre-extinction scenario

The Iberian lynx is the most threatened felidin the world and has suffered a declinethroughout its range. Effective monitoring ofthe species' presence is essential. Fieldwork inpreviously identified areas of lynx occurrencein Portugal has resulted in the collection of104 possible lynx scats. Recently, there hasbeen little or no evidence of lynx presence andscats could be confused with others from moreabundant carnivores such as wildcat, fox anddog. In order to confirm or not exclude thepresence of the species, identification ofscats was performed through the amplificationof lynx-specific mitochondrial DNA sequences.Two samples collected in Malcata NaturalReserve in 1997 were identified as lynx. Thisis the most recent and reliable proof of lynxpresence in Portugal^*. Given the territorialbehavior of lynx, stable resident populationswould have produced a higher proportion ofpositively identified scats. Local extinctionsmight have taken place, and this genetic datasupports a suspected national pre-extinctionscenario for the species. Genetic analysisusing a non-invasive approach has proved to bean informative part of the lynx monitoringprogram. Technical problems faced and overcomeare also presented.