Mitochondrial DNA restriction-fragment-length monomorphism in the Italian wolf (Canis lupus) population

Mitochondrial-DNA (mtDNA) restriction patterns were studied in 22 wolves (Canis lupus) sampled in central-northern Italy. A total of 60 restriction sites were detected, encompassing about 2 % of the mitochondrial genome of canids. All wolves showed the same restriction pattern. Therefore, a single mtDNA haplotype was detected in the Italian wolf population. Historical information on peninsular isolation and demographic decline suggest that low genetically effective population size and random drift may have strongly reduced the mtDNA variability of wolves in Italy over the last 100–200 years. A different mtDNA restriction pattern in feral dogs sampled from a wolf range in central Italy was detected. These findings suggest that the hybridization and introgression of female dog genomes into the Italian wolf population may be rare or absent.     

From the Apennines to the Alps: colonization genetics of the naturally expanding Italian wolf (Canis lupus) population

Wolves in Italy strongly declined in the past and were confined south of the Alps since the turn of the last century, reduced in the 1970s to approximately 100 individuals surviving in two fragmented subpopulations in the central-southern Apennines. The Italian wolves are presently expanding in the Apennines, and started to recolonize the western Alps in Italy, France and Switzerland about 16 years ago. In this study, we used a population genetic approach to elucidate some aspects of the wolf recolonization process. DNA extracted from 3068 tissue and scat samples collected in the Apennines (the source populations) and in the Alps (the colony), were genotyped at 12 microsatellite loci aiming to assess (i) the strength of the bottleneck and founder effects during the onset of colonization; (ii) the rates of gene flow between source and colony; and (iii) the minimum number of colonizers that are needed to explain the genetic variability observed in the colony. We identified a total of 435 distinct wolf genotypes, which showed that wolves in the Alps: (i) have significantly lower genetic diversity (heterozygosity, allelic richness, number of private alleles) than wolves in the Apennines; (ii) are genetically distinct using pairwise F(ST) values, population assignment test and Bayesian clustering; (iii) are not in genetic equilibrium (significant bottleneck test). Spatial autocorrelations are significant among samples separated up to c. 230 km, roughly correspondent to the apparent gap in permanent wolf presence between the Alps and north Apennines. The estimated number of first-generation migrants indicates that migration has been unidirectional and male-biased, from the Apennines to the Alps, and that wolves in southern Italy did not contribute to the Alpine population. These results suggest that: (i) the Alps were colonized by a few long-range migrating wolves originating in the north Apennine subpopulation; (ii) during the colonization process there has been a moderate bottleneck; and (iii) gene flow between sources and colonies was moderate (corresponding to 1.25-2.50 wolves per generation), despite high potential for dispersal. Bottleneck simulations showed that a total of c. 8-16 effective founders are needed to explain the genetic diversity observed in the Alps. Levels of genetic diversity in the expanding Alpine wolf population, and the permanence of genetic structuring, will depend on the future rates of gene flow among distinct wolf subpopulation fragments.     

Distribution, status and conservation of Indian gray wolf (Canis lupus pallipes) in Karnataka, India

The Indian gray wolf Canis lupus pallipes is the major large carnivore in the plains of Karnataka, India. We carried out a study on its distribution and status from November 2001 to July 2004. We estimated 555 wolves occupying about 123 330 km² of the state. In the past 40 years, wolves have disappeared from the southern plateau from an area of about 31 801 km². Their distribution is now largely restricted to the north-eastern dry plains. The wolf has also disappeared in recent years from some 'protected areas' such as Melkote Temple Wildlife Sanctuary, and their present population is largely found in 'non-protected' areas. Blackbucks are the only natural prey of wolves in Karnataka, but their density in most parts of the state is extremely low. The major prey species is domestic livestock, especially sheep. The available 'remote area' (forests or rocky terrains) in the wolf-occupied regions determined the status of the wolf. Killing of adult wolves and pups was common throughout the range of the wolf. However, such killings were made largely by local sheepherders with small sheep holdings and not by nomadic shepherds who maintained large sheep herds. The forests in the north-eastern parts of the state exist in small patches every few kilometers. Because each wolf pack ranges over large distances and is by and large a commensal species, we propose that the management of these small forest patches, considering them as components of a larger landscape, is the only effective conservation practice for the wolf. Although existing locally in low densities, because of a large ranging area of a single pack, the seemingly isolated wolf packs can become parts of a large metapopulation, providing a sustainable population.     

Evidence of genetic distinction and long-term population decline in wolves (Canis lupus) in the Italian Apennines

Historical information suggests the occurrence of an extensive human-caused contraction in the distribution range of wolves (Canis lupus) during the last few centuries in Europe. Wolves disappeared from the Alps in the 1920s, and thereafter continued to decline in peninsular Italy until the 1970s, when approximately 100 individuals survived, isolated in the central Apennines. In this study we performed a coalescent analysis of multilocus DNA markers to infer patterns and timing of historical population changes in wolves surviving in the Apennines. This population showed a unique mitochondrial DNA control-region haplotype, the absence of private alleles and lower heterozygosity at microsatellite loci, as compared to other wolf populations. Multivariate, clustering and Bayesian assignment procedures consistently assigned all the wolf genotypes sampled in Italy to a single group, supporting their genetic distinction. Bottleneck tests showed evidences of population decline in the Italian wolves, but not in other populations. Results of a Bayesian coalescent model indicate that wolves in Italy underwent a 100- to 1000-fold population contraction over the past 2000-10,000 years. The population decline was stronger and longer in peninsular Italy than elsewhere in Europe, suggesting that wolves have apparently been genetically isolated for thousands of generations south of the Alps. Ice caps covering the Alps at the Last Glacial Maximum (c. 18,000 years before present), and the wide expansion of the Po River, which cut the alluvial plains throughout the Holocene, might have provided effective geographical barriers to wolf dispersal. More recently, the admixture of Alpine and Apennine wolf populations could have been prevented by deforestation, which was already widespread in the fifteenth century in northern Italy. This study suggests that, despite the high potential rates of dispersal and gene flow, local wolf populations may not have mixed for long periods of time.     

Noninvasive molecular tracking of colonizing wolf (Canis lupus) packs in the western Italian Alps

We used noninvasive methods to obtain genetic and demographic data on the wolf packs (Canis lupus), which are now recolonizing the Alps, a century after their eradication. DNA samples, extracted from presumed wolf scats collected in the western Italian Alps (Piemonte), were genotyped to determine species and sex by sequencing parts of the mitochondrial DNA (mtDNA) control-region and ZFX/ZFY genes. Individual genotypes were identified by multilocus microsatellite analyses using a multiple tubes polymerase chain reaction (PCR). The performance of the laboratory protocols was affected by the age of samples. The quality of excremental DNA extracts was higher in samples freshly collected on snow in winter than in samples that were older or collected during summer. Preliminary mtDNA screening of all samples allowed species identification and was a good predictor of further PCR performances. Wolf, and not prey, DNA targets were preferentially amplified. Allelic dropout occurred more frequently than false alleles, but the probability of false homozygote determinations was always < 0.001. A panel of six to nine microsatellites would allow identification of individual wolf genotypes, also whether related, with a probability of identity of < 0.015. Genealogical relationships among individuals could be determined reliably if the number of candidate parents was 6-8, and most of them had been sampled and correctly genotyped. Genetic data indicate that colonizing Alpine wolves originate exclusively from the Italian source population and retain a high proportion of its genetic diversity. Spatial and temporal locations of individual genotypes, and kinship analyses, suggest that two distinct packs of closely related wolves, plus some unrelated individuals, ranged in the study areas. This is in agreement with field observations.     

Unexplained patterns of grey wolf Canis lupus natal dispersal

Natal dispersal (movement from the site of birth to the site of reproduction) is a pervasive but highly varied characteristic of life forms. Thus, understanding it in any species informs many aspects of biology, but studying it in most species is difficult. In the grey wolf Canis lupus, natal dispersal has been well studied. Maturing members of both sexes generally leave their natal packs, pair with opposite-sex dispersers from other packs, near or far, select a territory, and produce their own offspring. However, three movement patterns of some natal-dispersing wolves remain unexplained: 1) long-distance dispersal when potential mates seem nearby, 2) round-trip travels from their natal packs for varying periods and distances, also called extraterritorial movements, and often not resulting in pairing, and 3) coincidental dispersal by individual wolves from a given area in the same basic directions and over the same long distances. This perspective article documents and discusses these unexplained dispersal patterns, suggests possible explanations, and calls for additional research to understand them more clearly.     

Characterization of 12 single nucleotide polymorphisms in weathervane scallop

We describe 27 single nucleotide polymorphisms (SNPs) in a commercially important bivalve, the weathervane scallop (Patinopecten caurinus), identified using a targeted‐gene approach. We further characterize 12 of these using 5′‐nuclease and allele‐specific PCR assays. Polymorphisms were identified in both mitochondrial and nuclear genes. These are the first SNPs developed for delineating population structure in the weathervane scallop and will provide a useful complement to currently available genetic markers.     

Habitat variables associated with wolf (Canis lupus) distribution and abundance in northern Poland

Based on data collected during the National Wolf Census in 2000–01, we analysed the main habitat factors influencing the distribution and abundance of the wolf, Canis lupus, in northern Poland. The study region forms the western border of the continuous Eastern European range of wolves, although attempts at westward dispersal have been observed. Using Geographic Information System techniques, we measured nine habitat variables and three parameters related to wolf occurrence in 134 circular sample plots (radius 7 km, area 154 km² each). We compared 72 plots where wolves were recorded and 62 plots with no signs of wolf presence. Wolf plots were characterized by significantly higher forest cover, less fragmentation of forests, lower density of villages, towns, motorways, and railways than wolf‐free plots. We found a positive correlation between the sum of wolf observations in plots and forest cover. The number of domestic animals killed by wolves was higher in areas with higher indices of wolf abundance and lower forest area. In multiple regression analysis, four independent variables explained 59% of the variation in wolf distribution and abundance in northern Poland: straight‐line distance to continuous range of wolves in Eastern Europe; forest cover; forest fragmentation; and length of major motorways. We conclude that protection of wolves in Poland (since 1998) may not be an adequate conservation measure, especially because of the increasing density of highways and express motorways. Existing forest corridors should be protected and new ones should be restored to ensure long‐term conservation of wolves and allow range expansion into Western Europe.     

蒙古狼线粒体基因组序列分析及其系统发育地位

应用Long-PCR和克隆测序法得到蒙古狼(Canis lupus chanco)线粒体基因组全序列,结合GenBank中现有犬科动物线粒体基因组数据,应用最大简约法(MP)、最大似然法(ML)和Bayesian分析法对蒙古狼的系统发育地位进行了探讨。结果如下:蒙古狼线粒体基因组全长16709bp,包含13个蛋白质编码基因、22个tRNA基因、2个rRNA基因和1个非编码区。序列碱基的组成存在明显的A-T偏好性。tRNA基因中除tRNA-Ser(AGY)缺少双氢尿嘧啶(DHU)臂以外,其余均能折叠成典型的三叶草二级结构。大多数蛋白质编码基因的起始和终止密码子与犬科动物有报道相同,COXⅡ基因的起始密码子为ATA,与其他犬科动物不同。基于12S rRNA+16S rRNA+H链上的12个蛋白质编码基因的联合数据的系统发育分析发现,在已报道的狼亚种数据中,西藏狼(Canis lupus laniger)的分化时间最早,其次为阿拉伯狼(Canis lupus arabs),蒙古狼与欧亚狼(Canis lupuslupus)的系统发育地位最为接近。     

Environmental and social factors influencing wolf (Canis lupus) howling behavior

Animals communicate in a variety of ways and calls are used for a number of important behaviors. Temperature, wind, time of day, and human activities can affect animals’ use of calls, particularly over long distances. Effects of group size on the use of calls can be particularly influential in territorial social carnivores. Where gray wolves (Canis lupus) are hunted by humans, for example, howling may make it easier for hunters to locate individuals and ultimately increase mortality. We hypothesized that a suite of factors would affect wolves’ responses to simulated howling. Specifically, we predicted that howling behavior would increase with (a) group size, (b) pup age, and (c) during crepuscular time periods and howling behavior would decrease (a) where wolves were harvested and (b) when it was hot or windy. Contrary to our prediction, larger groups did not respond as quickly to simulated wolf howls as smaller groups did and minimum and maximum daily temperatures were not good predictors of wolf howling response rates. Individuals in small litters of pups may have responded more quickly to howls than those in large litters because they are eager to seek safety from and have socialization with adults returning from foraging bouts. Although harvest did not appear to affect vocal communication by wolves, group size, pup age, time of day, wind, and number of howls emitted greatly affected wolves’ behavior and responses during howling surveys. Howling responses did not change because of harvest; response rates from wolves were nearly identical with (2.2%) and without (2.3%) harvest. The year-round benefits of long-distance vocal communication may outweigh the costs of increased mortality arising from howling during harvest season.     

内蒙古达赉湖自然保护区狼冬季的生境选择

内蒙古达赉湖自然保护区狼的生境受到了自然环境变化压力和人为干扰的严重影响。为了解内蒙古达赉湖自然保护区狼的冬季生境特征,于2008年10-12月,在内蒙古达赉湖国家级自然保护区对狼的冬季生境选择进行了研究。野外共测量了59个20m×20m样方中的11个生态因子,运用聚类分析和主成分分析法,对狼的冬季生境选择因子进行了分析。结果表明,狼的生境主要特征为:隐蔽程度中等以上,水源距离100m,生境类型为高草阔地、干旱苇塘和河套,植被类型是以芦苇和柳灌丛为优势种的沼泽植被,植被高度1.0m,食物丰富度良好,居民点距离1000m,距草原道路距离1000m,牧场距离500m。影响狼冬季生境选择的主要因子为植被高度、植被类型和隐蔽级。次要因子为距围栏距离、雪深、距水源距离、生境类型、距牧场距离、食物丰富度和距居民点距离。     

Ageing and environmental factors affect PCR success in wolf (Canis lupus) excremental DNA samples

Individual genotypes determined from noninvasive DNA samples (typically extracted from shed hairs or scats) are used to estimate population size in monitoring projects of elusive species. However, polymerase chain reaction (PCR) success rates usually are lower, and genotyping errors higher than in standard population genetic surveys, due to DNA degradation or contamination in aged field samples. In this study, we evaluate the results of common garden experiments showing that DNA degradation is significant in wolf (Canis lupus) scats older than 3 days, and it is enhanced in scats in direct contact with soil. A storage test showed that samples kept frozen in 95% ethanol performed better compared to other methods. However, variance of PCR success among samples was high, independent on sample age or storage condition. The detrimental consequences of DNA degradation can be avoided by collecting scat samples as fresh as possible, and implementing efficient multitube procedures and stringent quality control of the laboratory results. Efficient multitube procedures can produce reliable data, like in this study, which showed that the consensus genotypes obtained from excremental DNA exactly matched distinct reference genotypes obtained from wolf blood samples.     

宁夏回族原发性膝骨性关节炎与瘦素受体基因多态性的相关性

为探讨宁夏回族原发性膝骨性关节炎(Osteoarthritis, OA)与瘦素受体基因(Leptin receptor, LEPR)A668G位点单核苷酸多态性(SNPs)之间的关系, 文章运用病例-对照研究, 通过聚合酶链反应-限制性片段长度多态性(PCR-RFLP)技术, 对148例兼具原发症状和影象证据的宁夏回族膝OA患者以及155名年龄、性别相匹配的对照群体进行LEPR A668G SNPs检测, 并进行测序验证, 分析LEPR基因多态性与膝OA的易感关联。研究表明, 膝OA组瘦素(Leptin, LEP)水平显著高于对照组(P<0.001), 血浆可溶性瘦素受体(sLEPR)水平较对照组明显降低(P<0.001), 膝OA组LEPR A668G位点AG/GA+GG基因型和G等位基因的分布频率和对照组存在差异(P=0.008和P=0.024)。研究结果提示, LEPR A668G位点的多态性可能与宁夏回族人群中膝OA易感性相关, 可以作为预测宁夏回族膝OA发病危险的遗传标记及早期防治的候选基因之一。     

Discovery,validation and characterization of 1039 cattle single nucleotide polymorphisms

We identified ～13 000 putative single nucleotide polymorphisms (SNPs) by comparison of repeat‐masked BAC‐end sequences from the cattle RPCI‐42 BAC library with whole‐genome shotgun contigs of cattle genome assembly Btau 1.0. Genotyping of a subset of these SNPs was performed on a panel containing 186 DNA samples from 18 cattle breeds including 43 trios. Of 1039 SNPs confirmed as polymorphic in the panel, 998 had minor allele frequency ≥0.25 among unrelated individuals of at least one breed. When Btau 4.0 became available, 974 of these validated SNPs were assigned in silico to known cattle chromosomes, while 41 SNPs were mapped to unassigned sequence scaffolds, yielding one SNP every ～3 Mbp on average. Twenty‐four SNPs identified in Btau 1.0 were not mapped to Btau 4.0. Of the 1015 SNPs mapped to Btau 4.0, 959 SNPs had nucleotide bases identical in Btau 4.0 and Btau 1.0 contigs, whereas 56 bases were changed, resulting in the loss of the in silico SNP in Btau 4.0. Because these 1039 SNPs were all directly confirmed by genotyping on the multi‐breed panel, it is likely that the original polymorphisms were correctly identified. The 1039 validated SNPs identified in this study represent a new and useful resource for genome‐wide association studies and applications in animal breeding.     

Analysis of public single nucleotide polymorphisms in commercial pig populations

More than 5500 pig single nucleotide polymorphisms (SNPs) were recently identified and deposited in the public domain. To test the usefulness of these public SNPs, 109 SNPs were analysed for polymorphism within six commercial pig populations. A functional polymerase chain reaction (PCR) assay was obtained for 103 SNPs and it was possible to validate c. 59% by PCR-restriction fragment length polymorphism. Furthermore, polymorphism was found using a relatively limited number of genomic DNA samples, indicating that these polymorphisms are segregating at a useful frequency in these populations. The high percentage of validated markers demonstrates the utility of these public pig SNPs to identify loci responsible for economically important traits in commercial pig populations.     

SNPs in ecological and conservation studies: a test in the Scandinavian wolf population

Single nucleotide polymorphisms (SNPs) have the potential to become the genetic marker of choice in studies of the ecology and conservation of natural populations because of their capacity to access variability across the genome. In this study, we provide one of the first demonstrations of SNP discovery in a wild population in order to address typical issues of importance in ecology and conservation in the recolonized Scandinavian and neighbouring Finnish wolf Canis lupus populations. Using end sequence from BAC (bacterial artificial chromosome) clones specific for dogs, we designed assays for 24 SNP loci, 20 sites of which had previously been shown to be polymorphic in domestic dogs and four sites were newly identified as polymorphic in wolves. Of the 24 assayed loci, 22 SNPs were found to be variable within the Scandinavian population and, importantly, these were able to distinguish individual wolves from one another (unbiased probability of identity of 4.33 x 10(-8)), providing equivalent results to that derived from 12 variable microsatellites genotyped in the same population. An assignment test shows differentiation between the Scandinavian and neighbouring Finnish wolf populations, although not all known immigrants are accurately identified. An exploration of the misclassification rates in the identification of relationships shows that neither 22 SNP nor 20 microsatellite loci are able to discriminate across single order relationships. Despite the remaining obstacle of SNP discovery in nonmodel organisms, the use of SNPs in ecological and conservation studies is encouraged by the advent of large scale screening methods. Furthermore, the ability to amplify extremely small fragments makes SNPs of particular use for population monitoring, where faecal and other noninvasive samples are routinely used.