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.     

Field determination of age in male great bustards (Otis tarda) in spring

During a long-term study of individually marked, free-living male great bustards captured as chicks and radio-tracked through several years in Spain, we studied the development with age of two secondary sex traits, the moustachial feathers and the neck plumage pattern. Juvenile males acquired full adult plumage between their fourth and seventh years. The main changes occurred at the neck, coinciding with the onset of sexual maturity. The grey colour typical of immature males turned to ivory white around the fourth to fifth spring, and a gradual increase was appreciated in adults in the brightness of the white colour of the upper neck and in the contrast between this and a progressively more intense chestnut brown at the neck base. Based on these changes, we proposed four neck plumage patterns that can be used to differentiate male age classes during the mating period. The development of moustachial feathers showed more interindividual variability and was not as useful as the neck plumage to estimate male age.     

Distribution, density and productivity of great bustards Otis tarda in northwestern Spain: a regional approach

This paper describes at a regional scale the distribution pattern, density, productivity and sex ratio of great bustards in northwestern Spain and explores the role played by habitat type, terrain characteristics and human disturbance on variation in its demographic parameters. Data from 136 plots covering an area of 7314 km² were obtained in two censuses carried out in the spring and summer of 1998. The density of the great bustards was 1.39 individuals/km² in the pre-breeding period, decreasing by 22% in the post-breeding period. Density was significantly higher in central plots than in peripheral plots. Mean productivity was 0.24 chicks per female and showed a high variability among plots, being significantly lower in the densest plots. The overall sex ratio was 1.35 females per male during the pre-breeding period. Productivity related positively to areas holding small fields and a high interspersion of land uses. Density and productivity were negatively affected by river density and altitude, and seasonal density variation was positively correlated with human population density.     

Genetic diversity of the great bustard in Iberia and Morocco: risks from current population fragmentation

We studied the genetic diversity of great bustards (Otis tarda) in Iberia and Morocco, the main stronghold of this globally endangered species. Samples were collected from 327 individuals covering most of the distribution range within the study area. Sequence variation in a 657 bp fragment of the mtDNA control region revealed 20 variable sites defining 22 haplotypes, two of them exclusive to Morocco. Genetic diversity showed marked regional differences (π = 0–0.53, h = 0–0.89). Multidimensional scaling analysis based on F _ST values showed a clear division between Morocco and the Iberian Peninsula, with no evidence of current gene flow between them. Our results suggest that Morocco, where few matrilines have persisted to present, was colonized from Iberia thousands of years ago. Last century reports suggest dispersal through Gibraltar, when the species was more abundant at both sides of the Strait but later population declines and the Strait’s barrier effect have favoured current genetic isolation. Within Iberia, only the most peripheral populations (Navarra, Aragón and Andalusia) differed significantly from the main ones in central Spain. The first two showed extremely low genetic diversity and are probably threatened by inbreeding depression. Diversity was higher in Andalusia, where three exclusive haplotypes were found, suggesting some degree of isolation from other populations. Andalusia and Morocco could be regarded as separate management units which hold a significant proportion of the current genetic diversity and thus deserve urgent conservation measures.     

Distribution and status of bustards in China

This article presents the distribution and status of bustards, which are listed as first-category protected animals according to the survey results during 1990–2002 in China. The Chinese populations of Otis tarda dybowskii are breeding in south-west of Heilongjiang Province, western Jilin Province, east and middle Inner Mongolia, north Ningxia Hui Autonomous Region, and Gansu Province. A few can winter in the south breeding-range. Its winter-range lies from the south to the Yellow River, as far as to Guizhou Province and Jiangxi Province. Its population number is about 200–300 or 500–800. The Chinese populations of O. t. tarda are breeding in the north and west of Xinjiang. It is unclear about its winter-range, which is presumed to be in south Asia. Recently we found individuals wintering in Chabuchaer and west Xinjiang. The population number is about 2000–3000. The habitat in breeding range includes steppe, grassland, desert grassland, and farmland. The habitat in winter range is the beach of rivers and lakes, meadows, meadow-grassland, and wheatland. The Chinese populations of Chlamydotis undulata macqueeni are breeding in the fringe of the Jungar Basin, the banks of the Ulungur River, Balikun and south Turpan Basin in Xinjiang, west Inner Mongolia, and west Gansu. NortheastMulei in eastern Jungar Basin of Xinjiang is the main breeding-range in the world. The bird uses desert and desert grassland as its habitat. Its winter-range is west Asia and south Asia. Its population number is about 2000. The Chinese populations of Tetrax tetrax are breeding in north Xinjiang, and China is located on the east border of its breeding-range. Its habitat is grassland and semi-desert, and its winter-range lies in south Asia. Its population in China is very scarce. In addition, we analyzed the causes of their endangerment and put forward protection tactics of Chinese Bustards. __________ Translated from Arid Zone Research, 2007, 24(2): 179–186 [译自: 干旱区研究]     

DNA degradation in avian faecal samples and feasibility of non-invasive genetic studies of threatened capercaillie populations

We evaluated the feasibility of using faeces as a non-invasively collected DNA source for the genetic study of an endangered bird population (capercaillie; Tetrao urogallus). We used a multitube approach, and for our panel of 11 microsatellites genotyping reliability was estimated at 98% with five repetitions. Experiments showed that free DNases in faecal material were the major cause of DNA degradation. Our results demonstrate that using avian faeces as a source of DNA, reliable microsatellite genotyping can be obtained with a reasonable number of PCR replicates.     

Evaluation of saliva as a source of human DNA for population and association studies

A simple noninvasive procedure for saliva sample collection and DNA extraction was developed. On average, the amount of human DNA (as measured by a TaqMan-based assay) was about 11.4 microg/mL saliva, which is more than can be obtained from other noninvasive samples such as cheek swabs. However, the presence of large amounts of nonhuman DNA (up to 90% of the total extracted DNA) in saliva samples does necessitate DNA quantitation methods that are specific for human DNA. We were able to reliably and accurately type different genetic markers (mDNA sequences, Y-chromosomal single-nucleotide polymorphisms, and autosomal microsatellite loci) from saliva samples stored for up to 30 days at 37 degrees C, making this method well-suited for field conditions and convenient transportation of samples back to the laboratory. Thus, saliva can be considered a reliable source of DNA for a wide variety of genetic studies.     

Distribution dynamics of a great bustard metapopulation throughout a decade: influence of conspecific attraction and recruitment

Dispersing individuals can use conspecifics as indicators of habitat quality and aggregate at traditionally occupied sites, leaving other favourable patches unoccupied. Here we test the predictions of the conspecific-based habitat selection hypothesis on a Spanish great bustard (Otis tarda) metapopulation, currently fragmented due to recent human-induced habitat changes. The number of birds had increased by 23% between 1988 and 1998, but not consistently among leks. Leks that were large in 1988 increased, while those that were small decreased, which suggests that dispersing individuals used the numbers of conspecifics as cues for breeding-site selection. Moreover, leks with high productivity increased, while those with low productivity decreased. Finally, lek distribution was markedly stable throughout the decade, with no establishment of new leks, and suitable habitat patches remained unoccupied, as predicted by the conspecific attraction hypothesis. These results were corroborated by a simulation model which incorporated natal dispersal rates between leks as obtained through radio-tracking of 15 birds that survived throughout their 4-year dispersal period. In conclusion, in spite of the apparent increase in total numbers throughout the decade, both conspecific attraction and local differences in reproductive success contributed to a more aggregated distribution, increasing the species' vulnerability to local catastrophes, and the risks of reduced genetic diversity and extinction of small leks.     

Genetic variability in mitochondrial DNA in a regional population of the great tit (Parus major)

Genetic variation of mitochondrial DNA (mtDNA) in 18 great tits (Parus major) from three neighboring localities in Sweden was investigated with eight tetranucleotide restriction endonucleases. The 18 individuals could be separated into 13 different maternal lineages. The high number of female lineages present in this regional population contrasts with a low level of sequence divergence between the different mtDNA clones, with a mean of 0.19% sequence divergence between all individuals. There was no obvious spatial structuring of mtDNA clones among the three localities. The presence of a high number of different clones with a low degree of sequence divergence could be explained by the effects of a large long-term effective population size, with the mtDNA clones having diverged about 25,000–200,000 years ago.This study was supported by the Swedish Natural Science Research Council, the Erik Philip-Sörensen Foundation, and the Nilsson-Ehle Foundation.     

Cloacal and buccal swabs are a reliable source of DNA for microsatellite genotyping of reptiles

In this study, a minimally invasive method for DNA sampling of reptiles and amphibians using cloacal and buccal swabs is described. High molecular weight DNA was isolated from the swabs, which were collected from tuatara (Sphenodon punctatus), and stored in 70% ethanol at room temperature for approximately 1 week. Amplification of mitochondrial and microsatellite DNA loci was successful from both cloacal and buccal swabs, and in all cases the genotypes matched those obtained from blood samples. These results show that cloacal and/or buccal swabbing is a useful alternative to blood sampling and toe clipping for genetic studies on reptiles. This method is rapid, inexpensive and easy to implement in field situations.     

A multi-samples, multi-extracts approach for microsatellite analysis of faecal samples in an arboreal ape

We investigated the effect of the number of faecal samples, ofextracts per sample and of PCRs per extract on the reliability ofgenotypes for a microsatellite locus in free-living orang-utans.For each individual 36 PCRs were performed using DNA extractionsfrom up to four faecal samples. We found a very largeinter-individual variation in positive PCRs (P+) (36/36 for oneindividual and 0/36 for another). As many as 30% of the cases ledto erroneous genotypes when only one P+ was obtained. It ispreferable to use at least 4 P+ per extract to reduce thisproportion to less than 1%. With 3 P+ results, erroneousgenotypes were still observed in 26% of the cases together. Theseresults indicate that it is necessary to do a minimum of 4 PCRsper extract. In order to have a chance to observe 4 P+, threeextracts should be ideally analysed for each sample. We alsorecommend that when possible two or more samples should becollected in the field to increase the chance of having extractscontaining DNA and to provide independent replicates. While werecognise the difficulty of working with faecal samples, weadvocate the use of faecal material for genetic studies ofcertain wild animal populations where the advantages of avoidingdisturbance, stress and injury are deemed of critical importance.     

Calibrating phylogenetic species formation in a threatened insect using DNA from historical specimens

Museum specimens from the late 19th and early 20th centuries were surveyed for the single nucleotide polymorphism identified previously and used to diagnose populations of the federally threatened Northeastern Beach Tiger Beetle Cicindela d. dorsalis (Coleoptera: Carabidae). Widespread polymorphism was revealed throughout the historical range of this species, suggesting a relatively recent anthropogenic character fixation event associated with the extinction and fragmentation of populations. Implications for the phylogenetic species criterion and for the reintroduction of individuals to formerly occupied sites are discussed.     

Cytoplasmic DNA variation in a potato protoclonal population

Summary Mitochondrial DNA variation was detected in potato plants (protoclones) regenerated from leaf mesophyll protoplasts. Two forms of variation were evident; (1) DNA sequence alterations within the high molecular weight mitochondrial chromosome and (2) the appearance of an additional low molecular weight mitochondrial DNA species. Variation in chloroplast DNA was not detected. The data suggests that protocloning can introduce molecular diversity into mitochondrial genomes and thereby assist in overcoming the cytoplasmic genetic uniformity prevalent in most major crops.     

Loggerhead turtle eggshells as a source of maternal nuclear genomic DNA for population genetic studies

Tagging studies on nesting beaches are commonly used to estimate nesting frequency, remigration interval and nesting population size for marine turtle rookeries. Estimates of these demographic parameters from tagging projects may be biased because of the small scale of tagging efforts relative to female nest site fidelity and the logistical difficulty of intercepting all nesting females. Therefore, alternative and supplemental means of individual identification of nesting females are required. We demonstrate that maternal nuclear microsatellite DNA can be isolated from unincubated eggshells of the loggerhead sea turtle (Caretta caretta) through comparison of DNA extracted from 59 eggs collected within 15 h of oviposition and DNA derived from skin samples from respective nesting females. Scorable microsatellite genotypes were produced in 897 of 994 (90.2%) single-locus egg amplifications attempted. Among eggs from known females, 730 of 748 (97.6%) single-locus, egg-derived genotypes matched the respective skin-derived genotypes. Allelic dropout was the most common type of error, followed by the presence of nonmaternal, presumably paternal, alleles. Genotypes derived from unincubated eggshells permit individual assignment of nests and therefore demographic parameter estimates for loggerhead turtle nesting populations, despite genotyping errors that require further optimization. Although sampling unincubated eggs is destructive, this technique is noninvasive to nesting females and is applicable in marine turtle population genetics studies when individual resolution is required but direct interception of nesting females is undesirable or logistically infeasible.     

Owl pellets as a source of DNA for genetic studies of small mammals

Owl pellets contain a good skeletal record of the small mammals consumed, and correspond to the undigested portions of prey which are regurgitated. These pellets are easy to find at the roosting site of owls. As it has been demonstrated that amplifiable DNA can be isolated from ancient bone remains, the possibility of using owl pellets as a source of DNA for small mammal genetics studies via the polymerase chain reaction has been investigated. The main uncertainties when isolating DNA from such a material are firstly the possibility that the extracted DNA would be too degraded during the digestion in the stomach of the owl, and secondly that extensive cross-contaminations could occur among the different prey consumed. The results obtained clearly demonstrate that cross-contamination does not occur, and that mitochondrial and nuclear DNA can be amplified using skulls of small mammals found in owl pellets as a source of DNA. The relative efficiency of two methods of DNA extraction is estimated and discussed. Thus, owl pellets represent a non-invasive sampling technique which provides a valuable source of DNA for studying population genetics of small mammals.     

Glycosphingolipids in feces of germ-free rats as a source for studies of developmental changes of intestinal epithelial cell surface carbohydrates

Non-acid and acid glycosphingolipids were isolated from feces of one litter of germ-free rats from day 17 to day 51. Quantitative and qualitative changes described for small intestine of conventional rats [Bouhours D, Bouhours J-F (1981) Biochem Biophys Res Commun 99:1384–89] were also found in the feces of these germ-free rats. A decrease in lactosylceramide and sialyllactosylceramide excretion and a change fromN-acetylneuraminic acid toN-glycoloylneuraminic acid, as well as an appearance of type 1 chain blood group H-active penta- and decaglycosylceramides were observed during the weaning period. Thus the dramatic changes seen in rat intestinal glycosphingolipids postnatally seem to be primarily regulated by non-microbial factors.Abbreviations G_M3 G_M3-ganglioside, II³NeuAc-LacCer or II³NeuGc-LacCer - SPG IV³NeuAc-nLcOse₄Cer - G_M1 G_M1-ganglioside, II³NeuAc-GgOse₄Cer     

Isolation of high molecular weight DNA from wholeEuglena cells

Summary A method for isolating high quality DNA from wholeEuglena cells is described. The procedure consists in: the weakening of the cell pellicle in glycerol avoiding the mechanical disruption of cells and shearing damage in DNA molecules; the decondensation ofEuglena compact chromatin directly inside the cells; the complete dissociation of cells and nucleoproteins in sarkosyl detergent; the optional digestion of proteins and RNA with DNase-free enzymes and the final purification of DNA by isopycnic banding in CsCl gradients. Degradation of DNA is prevented all along the extraction procedure by glycerol, antioxydants, EDTA and sarkosyl detergent. Using the enzymatic digestion step, DNA containing few single-stranded nicks is obtained with a yield approaching 100%. DNA with no single-stranded nick could be obtained with a 35% yield when the enzymatic digestion step was omitted. In both cases, the double-stranded DNA has an average molecular weight equal or greater than 6×10⁷. It is free of contaminants and could be easily digested with restriction enzymes. After digestion with Eco RI and size-fractionation in agarose gel this DNA has permitted specific hybridization of the rDNA sequences with a radioactive rRNA probe.Abbreviations Kbp kilobasepairs - Kb kilobases