Lisa C. Pope

Reliable microsatellite genotyping of the Eurasian badger ( Meles meles ) using faecal DNA

The potential link between badgers and bovine tuberculosis has made it vital to develop accurate techniques to census badgers. Here we investigate the potential of using genetic profiles obtained from faecal DNA as a basis for population size estimation. After trialling several methods we obtained a high amplification success rate (89%) by storing faeces in 70% ethanol and using the guanidine thiocyanate/silica method for extraction. Using 70% ethanol as a storage agent had the advantage of it being an antiseptic. In order to obtain reliable genotypes with fewer amplification reactions than the standard multiple‐tubes approach, we devised a comparative approach in which genetic profiles were compared and replication directed at similar, but not identical, genotypes. This modified method achieved a reduction in polymerase chain reactions comparable with the maximum‐likelihood model when just using reliability criteria, and was slightly better when using reliability criteria with the additional proviso that alleles must be observed twice to be considered reliable. Our comparative approach would be best suited for studies that include multiple faeces from each individual. We utilized our approach in a well‐studied population of badgers from which individuals had been sampled and reliable genotypes obtained. In a study of 53 faeces sampled from three social groups over 10 days, we found that direct enumeration could not be used to estimate population size, but that the application of mark–recapture models has the potential to provide more accurate results.

Population structure of the yellow‐footed rock‐wallaby Petrogale xanthopus (Gray, 1854) inferred from mtDNA sequences and microsatellite loci

The yellow‐footed rock‐wallaby Petrogale xanthopus is considered to be potentially vulnerable to extinction. This wallaby inhabits naturally disjunct rocky outcrops which could restrict dispersal between populations, but the extent to which that occurs is unknown. Genetic differences between populations were assessed using mitochondrial DNA (control region) sequencing and analysis of variation at four microsatellite loci among three geographically close sites in south‐west Queensland (P. x. celeris) and, for mtDNA only, samples from South Australia (P. x. xanthopus) as well. Populations from South Australia and Queensland had phylogenetically distinct mtDNA, supporting the present classification of these two groups as evolutionarily distinct entities. Within Queensland, populations separated by 70 km of unsuitable habitat differed significantly for mtDNA and at microsatellite loci. Populations separated by 10 km of apparently suitable habitat had statistically homogeneous mtDNA, but a significant difference in allele frequency at one microsatellite locus. Tests for Hardy‐Weinberg equilibrium and micro‐geographical variation at microsatellite loci did not detect any substructuring between two wallaby aggregations within a colony encircling a single rock outcrop. Although the present study was limited by small sample sizes at two of the three Queensland locations examined, the genetic results suggest that dispersal between colonies is limited, consistent with an ecological study of dispersal at one of the sites. Considering both the genetic and ecological data, we suggest that management of yellow‐footed rock‐wallabies should treat each colony as an independent unit and that conservation of the Queensland and South Australian populations as separate entities is warranted.

Phylogeography and population structure of an ecotonal marsupial, Bettongia tropica, determined using mtDNA and microsatellites.

The northern bettong, Bettongia tropica, is an endangered species of Potoroidae with a restricted distribution in the wet tropics of north Queensland, Australia. The species is only found within a thin strip of sclerophyll forest along the western margin of rainforest. This tight association with rainforest boundaries is predicted to have resulted in population isolation as rainforest contracted during the Pleistocene, though some have proposed that the northern bettong was not present in the wet tropics until the late Pleistocene. The dispersal ability of the species, and of the family, is not known. This study examined gene flow among populations within areas of continuous habitat complemented by a broader analysis of phylogeography. Individuals trapped at each of the four known regions (one region was subsampled at three different sites), were sequenced for 547 base pairs of the mitochondrial DNA (mtDNA) control region and typed for seven microsatellite loci. The mtDNA phylogeny showed congruence with a biogeographical hypothesis, a relatively deep split suggesting historical isolation in separate northern and southern refugia. The two divergent clades were both present within the Lamb Range, indicating an expansion from these refuges and subsequent admixture at one site. mtDNA allele frequencies indicated relatively limited gene flow within the Lamb Range over distances as short as nine km. Tests of population divergence using microsatellites (FST and assignment tests) strongly supported this result. A molecular signal indicative of a recent bottleneck was unexpectedly detected in one of the Lamb Range subpopulations. This lead us to examine the behaviour of the statistics used in this bottleneck test under a linear stepping‐stone model with varying migration rates. We found that it may be more difficult to detect molecular signatures for recent bottlenecks under conditions of very low migration rates than for isolated populations and, conversely, that ‘false’ bottleneck signatures may be observed at higher migration rates. The Lamb Range FST estimate clearly fell within the category of potentially ‘false’ bottleneck signals. Despite relatively limited gene flow, evidence for asymmetric dispersal suggests more complicated population dynamics than a simple linear stepping‐stone model.

Mating system of the Eurasian badger, Meles meles, in a high density population

Badgers are facultatively social, forming large groups at high density. Group‐living appears to have high reproductive costs for females, and may lead to increased levels of inbreeding. The extent of female competition for reproduction has been estimated from field data, but knowledge of male reproductive success and the extent of extra‐group paternity remains limited. Combining field data with genetic data (16 microsatellite loci), we studied the mating system of 10 badger social groups across 14 years in a high‐density population. From 923 badgers, including 425 cubs, we were able to assign maternity to 307 cubs, with both parents assigned to 199 cubs (47%) with 80% confidence, and 14% with 95% confidence. Age had a significant effect on the probability of reproduction, seemingly as a result of a deficit of individuals aged two years and greater than eight years attaining parentage. We estimate that approximately 30% of the female population successfully reproduced in any given year, with a similar proportion of the male population gaining paternity across the same area. While it was known there was a cost to female reproduction in high density populations, it appears that males suffer similar, but not greater, costs. Roughly half of assigned paternity was attributed to extra‐group males, the majority of which were from neighbouring social groups. Few successful matings occurred between individuals born in the same social group (22%). The high rate of extra‐group mating, previously unquantified, may help reduce inbreeding, potentially making philopatry a less costly strategy.

Versatile primers for the amplification of the mitochondrial DNA control region in marsupials

in vertebrates) is the most rapidly evolving portion of the mitochondrial genome and is thus well suited for detection of nucleotide polymorphism in closely related taxa and for determining intraspecific molecular population structure. Within the control region and its flanking tRNA genes there are several conserved sequence blocks, and different sets of universal primers have been designed by comparing these sequences in a wide range of taxa (e.g. Kocher et al. 1989; Palumbi 1996). However, these primers were designed from DNA sequences of placental mammals and other vertebrate species, and when used on marsupials we had limited success. In this study we describe four new primers which allow the amplification of the marsupial mtDNA control region. Using the PCR technique, we first amplified and directly sequenced the control region-flanking tRNA genes (tRNA-Pro and tRNA-Phe) in some marsupial species. The primers used were: L15774M (5 ́-ACATGAATTGGAGGACAACCNGT-3 ́), located in the cytochrome-b gene (from the alignment in Patton et al. 1996); H651B (5 ́-AAGGCYAGGACCAAACCT-3 ́), located in the 12SrRNA gene and modified from Kocher et al. (1989); and two new primers (L16517M and its reverse complement H16498M, located in the central domain of the control region). Second, we aligned the sequences obtained with the sequence of Didelphis virginiana (Janke et al. 1994), and we subsequently designed two other new primers, located in the tRNA-Pro gene and in the tRNA-Phe gene (L15999M and H605M, respectively). The sequences of these four new primers are shown in Fig. 1, together with an alignment of homologous sequences from other mammalian taxa. Finally, PCR reactions were performed with a Perkin Elmer 480 Thermocycler in 25 μl using the thermostable AmpliTaq GoldTM polymerase (Perkin Elmer/Roche); final reaction concentrations were: 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 2 mM MgCl2, 0.2 mM of each deoxynucleotide, 1 μM of primers, 0.5 U of polymerase. Five ng of extracted product was added. Negative controls were included in every experiment. PCR parameters were as follows: 9 min at 94 °C, followed by 35 cycles of 94 °C for 45 s, 50 °C for 45 s, 72 °C for 1 min 30 s; final extension of 5 min at 72 °C. The two primer pairs used in these reactions were L15999M/H16498M and L16517M/H605M. The four primers were tested by PCR on 21 species, chosen so that each of the marsupial orders were represented. All amplifications proved efficient under the conditions described above and showed a strong positive product of the approximate expected size for 20 species assayed (Fig. 2), indicating that these primers should be useful in a wide range of marsupials. In one sample (Notoryctes typhlops) the amplification was successful only for the 5 ́ portion of the control region. However, we cannot exclude the possibility that the failure to amplify the 3 ́ portion resulted from the poor quality of the DNA used. Due to the presence of tandemly repeated sequences, which are characteristic of most mammalian and vertebrate mtDNA control regions, the length of the segments amplified varied between ≈ 550 bp and 1.1 kb for the 5 ́ portion of the control region, and between 650 bp and 1.2 kb for its 3 ́ portion (representative examples in Fig. 2). To check for applicability of these primers for sequencing, five species were subsequently sequenced for both strands on an ABI373A automated sequencer, following the manufacturer’s instructions. Direct sequencing gave reliable reading (data not shown). Because of the large size of the control region in some marsupial species (> 1.5 kb), we found that amplification as two separate parts (as described above) was the most robust strategy. P R I M E R N O T E

Isolation by distance and gene flow in the Eurasian badger ( Meles meles ) at both a local and broad scale

Eurasian badgers, Meles meles, have been shown to possess limited genetic population structure within Europe; however, field studies have detected high levels of philopatry, which are expected to increase population structure. Population structure will be a consequence of both contemporary dispersal and historical processes, each of which is expected to be evident at a different scale. Therefore, to gain a greater understanding of gene flow in the badger, we examined microsatellite diversity both among and within badger populations, focusing on populations from the British Isles and western Europe. We found that while populations differed in their allelic diversity, the British Isles displayed a similar degree of diversity to the rest of western Europe. The lower genetic diversity occurring in Ireland, Norway and Scotland was more likely to have resulted from founder effects rather than contemporary population density. While there was significant population structure (FST = 0.19), divergence among populations was generally well explained by geographic distance (P < 0.0001) across the entire range studied of more than 3000 km. Transient effects from the Pleistocene appear to have been replaced by a strong pattern of genetic isolation by distance across western Europe, suggestive of colonization from a single refugium. Analysis of individuals within British populations through Mantel tests and spatial autocorrelation demonstrated that there was significant local population structure across 3–30 km, confirming that dispersal is indeed restricted. The isolation by distance observed among badger populations across western Europe is likely to be a consequence of this restricted local dispersal.

Polygynandry, extra-group paternity and multiple-paternity litters in European badger (Meles meles) social groups

The costs and benefits of natal philopatry are central to the formation and maintenance of social groups. Badger groups, thought to form passively according to the resource dispersion hypothesis (RDH), are maintained through natal philopatry and delayed dispersal; however, there is minimal evidence for the functional benefits of such grouping. We assigned parentage to 630 badger cubs from a high‐density population in Wytham Woods, Oxford, born between 1988 and 2005. Our methodological approach was different to previous studies; we used 22 microsatellite loci to assign parent pairs, which in combination with sibship inference provided a high parentage assignment rate. We assigned both parents to 331 cubs at ≥ 95% confidence, revealing a polygynandrous mating system with up to five mothers and five fathers within a social group. We estimated that only 27% of adult males and 31% of adult females bred each year, suggesting a cost to group living for both sexes. Any strong motivation or selection to disperse, however, may be reduced because just under half of the paternities were gained by extra‐group males, mainly from neighbouring groups, with males displaying a mixture of paternity strategies. We provide the strongest evidence to date for multiple‐paternity litters, and for the first time show that within‐group and extra‐group males can sire cubs in the same litter. We investigate the factors that may play a role in determining the degree of delayed dispersal and conclude that the ecological constraints hypothesis, benefits of philopatry hypothesis, and life history hypothesis may all play a part, as proposed by the broad constraints hypothesis.

Using isolation-by-distance-based approaches to assess the barrier effect of linear landscape elements on badger (Meles meles) dispersal

As the European badger (Meles meles) can be of conservation or management concern, it is important to have a good understanding of the species’ dispersal ability. In particular, knowledge of landscape elements that affect dispersal can contribute to devising effective management strategies. However, the standard approach of using Bayesian clustering methods to correlate genetic discontinuities with landscape elements cannot easily be applied to this problem, as badger populations are often characterized by a strong confounding isolation‐by‐distance (IBD) pattern. We therefore developed a two‐step method that compares the location of pairs of related badgers relative to a putative barrier and utilizes the expected spatial genetic structure characterized by IBD as a null model to test for the presence of a barrier. If a linear feature disrupts dispersal, the IBD pattern characterising pairs of individuals located on different sides of a putative barrier should differ significantly from the pattern obtained with pairs of individuals located on the same side. We used our new approach to assess the impact of rivers and roads of different sizes on badger dispersal in western England. We show that a large, wide river represented a barrier to badger dispersal and found evidence that a motorway may also restrict badger movement. Conversely, we did not find any evidence for small rivers and roads interfering with badger movement. One of the advantages of our approach is that potentially it can detect features that disrupt gene flow locally, without necessarily creating distinct identifiable genetic units.

Genetic evidence that culling increases badger movement: implications for the spread of bovine tuberculosis

The Eurasian badger (Meles meles) has been implicated in the transmission of bovine tuberculosis (TB, caused by Mycobacterium bovis) to cattle. However, evidence suggests that attempts to reduce the spread of TB among cattle in Britain by culling badgers have mixed effects. A large‐scale field experiment (the randomized badger culling trial, RBCT) showed that widespread proactive badger culling reduced the incidence of TB in cattle within culled areas but that TB incidence increased in adjoining areas. Additionally, localized reactive badger culling increased the incidence of TB in cattle. It has been suggested that culling‐induced perturbation of badger social structure may increase individual movements and elevate the risk of disease transmission between badgers and cattle. Field studies support this hypothesis, by demonstrating increases in badger group ranges and the prevalence of TB infection in badgers following culling. However, more evidence on the effect of culling on badger movements is needed in order to predict the epidemiological consequences of this control strategy. Here, analysis of the genetic signatures of badger populations in the RBCT revealed increased dispersal following culling. While standard tests provided evidence for greater dispersal after culling, a novel method indicated that this was due to medium‐ and long‐distance dispersal, in addition to previously reported increases in home‐range size. Our results also indicated that, on average, badgers infected with M. bovis moved significantly farther than did uninfected badgers. A disease control strategy that included culling would need to take account of the potentially negative epidemiological consequences of increased badger dispersal.

Age-specific breeding success in a wild mammalian population: selection, constraint, restraint and senescence

The Selection, Constraint, Restraint and Senescence Hypotheses predict how breeding success should vary with age. The Selection Hypothesis predicts between‐individual variation arising from quality differences; the other hypotheses predict within‐individual variation due to differing skills or physiological condition (Constraint), residual reproductive lifespan (Restraint), or somatic and reproductive investment (Senescence). Studies tend to focus on either the initial increase in breeding success or later decrease; however, both require consideration when unravelling the underlying evolutionary processes. Additionally, few studies present genetic fitness measures and rarely for both sexes. We therefore test these four hypotheses, which are not mutually exclusive, in a high‐density population of European badgers Meles meles. Using an 18‐year data set (including 22 microsatellite loci), we show an initial improvement in breeding success with age, followed by a later and steeper rate of reproductive senescence in male than in female badgers. Breeding success was skewed within age‐classes, indicating the influence of factors other than age‐class. This was partly attributable to selective appearance and disappearance of badgers (Selection Hypothesis). Individuals with a late age of last breeding showed a concave‐down relationship between breeding success and experience (Constraint Hypothesis). There was no evidence of abrupt terminal effects; rather, individuals showed a concave‐down relationship between breeding success and residual reproductive lifespan (Restraint Hypothesis), with an interaction with age of first breeding only in female badgers. Our results demonstrate the importance of investigating a comprehensive suite of factors in age‐specific breeding success analyses, in both sexes, to fully understand evolutionary and population dynamics.