Genetic marker investigation of the source and impact of predation on a highly endangered species

In September and October 2000, the remains of a number of apparently predated northern hairy-nosed wombats (Lasiorhinus krefftii) were discovered in Epping Forest National Park, the site of the only known population of this highly endangered species. Analysis of DNA recovered from six carcasses and a section of intestine found nearby was carried out using microsatellite and Y-specific primers. This identified seven individual wombats, the identity of three of which was inferred from a genotype database prepared from animals sampled during trapping programmes. Six victims were male and one female, suggesting that female-biased predation rates are unlikely to be the cause of the current male-biased population sex ratio. DNA isolated from four canid faeces found in the vicinity revealed three distinct canid microsatellite genotypes with very high probabilities of belonging to dingoes (Canis familiaris dingo). A wombat genotype matching that of one of the dead individuals was identified from scats of two of the dingoes. In addition, two macropod microsatellites were amplified from two dingo scats. These observations provided vital information regarding predation on northern hairy-nosed wombats, and prompted the permanent exclusion of dingoes from the park by the erection of a dingo-proof fence.     

Microsatellite variation among divergent populations of stalk-eyed flies, genus Cyrtodiopsis

Microsatellite primers are often developed in one species and used to assess neutral variability in related species. Such analyses may be confounded by ascertainment bias (i.e. a decline in amplification success and allelic variability with increasing genetic distance from the source of the microsatellites). In addition, other factors, such as the size of the microsatellite, whether it consists of perfect or interrupted tandem repeats, and whether it is autosomal or X-linked, can affect variation. To test the relative importance of these factors on microsatellite variation, we examine patterns of amplification and allelic diversity in 52 microsatellite loci amplified from five individuals in each of six populations of Cyrtodiopsis stalk-eyed flies that range from 2.2 % to 11.2% mitochondrial DNA sequence divergence from the population used for microsatellite development. We find that amplification success and most measures of allelic diversity declined with genetic distance from the source population, in some cases an order of magnitude faster than in birds or mammals. The median and range of the repeat array length did not decline with genetic distance. In addition, for loci on the X chromosome, we find evidence of lower observed heterozygosity compared with loci on autosomes. The differences in variability between X-linked and autosomal loci are not adequately explained by differences in effective population sizes of the chromosomes. We suggest, instead, that periodic selection events associated with X-chromosome meiotic drive, which is present in many of these populations, reduces X-linked variation.     

Hematologic and serum biochemical reference values for free-ranging northern hairy-nosed wombats

Hematologic and serum biochemistry values were determined for 31 adult (21 male and 10 female) and four subadult male northern hairy-nosed wombats (Lasiorhinus krefftii) from the only existing population in Epping Forest National Park, Australia. Blood samples were obtained from free-ranging northern hairy-nosed wombats during trapping for population census and health and reproductive assessment in 1999. Hematologic and biochemical values were compared between adult males and adult females, and between adult and subadult wombats. Values were also compared with those previously published for southern hairy-nosed (Lasiorhinus latifrons) and common (Vombatus ursinus) wombats. The values from this study were used to create reference intervals, and they make up the first comprehensive hematologic and biochemical study for this highly endangered species.     

Faecal progesterone metabolites and behavioural observations for the non-invasive assessment of oestrous cycles in the common wombat (Vombatus ursinus) and the southern hairy-nosed wombat (Lasiorhinus latifrons)

Wombats belong to Australia's unique marsupial species. Two of the three remaining species, the common wombat (Vombatus ursinus) and the southern hairy-nosed wombat (Lasiorhinus latifrons) are abundant. The third species, the northern hairy-nosed wombat (Lasiorhinus krefftii) has only about 115 individuals left in the wild. This study aimed to gain further insight into the basic reproductive biology of wombat species and evaluate the value of faecal progesterone metabolites and behavioural patterns as a means for non-invasive monitoring of the oestrous cycle in common and the southern hairy-nosed wombats. In an initial study, three different faecal steroid assays showed that 20alpha-OH-pregnanes were the main progesterone metabolites. These metabolites were examined in captive female common wombats (n = 5) and southern hairy-nosed wombats (n = 2). In one female common wombat 11.7 days with a follicular phase of 25.6 +/- 6.3 days and a luteal phase of 28.2 +/- 12.7 days. The data for faecal pregnanes obtained in the southern and in one male common wombat oestrous related behavioural data were obtained. Individual cycling females exhibited a significant relationship between plasma progesterone and faecal pregnanes. In the common wombat, the values for faecal pregnanes showed an oestrous cycle length of 55.1 +/- hairy-nosed wombat during the breeding season gave an oestrous cycle length of 41.1 +/- 12.8 days with a follicular phase of 27.9 +/- 12.3 days and a short luteal phase of 13.3 +/- 1.1 days. The behavioural data show that the faecal sniffing behaviour of the male, tended to increase around the time that oestrous was found. In conclusion, monitoring of 20alpha-OH-pregnanes in wombat faeces could be a useful methodology to monitor reproductive cycles in the wombat, and can possibly be applied to monitor the endangered northern hairy-nosed wombat.     

Genetic rescue of an inbred captive population of the critically endangered Puerto Rican crested toad (<Emphasis Type="Italic">Peltophryne lemur</Emphasis>) by mixing lineages

The Puerto Rican crested toad (Peltophryne lemur) is currently composed of a single wild population on the south coast of Puerto Rico and two captive populations founded by animals from the northern and southern coasts. The main factors contributing to its decline are habitat loss, inundation of breeding ponds during storms, and impacts of invasive species. Recovery efforts have been extensive, involving captive breeding and reintroductions, habitat restoration, construction of breeding ponds, and public education. To guide future conservation efforts, genetic variation and differentiation were assessed for the two captive colonies and the remaining wild population using the mitochondrial control region and six novel microsatellite loci. Only two moderately divergent mitochondrial haplotypes were found, with one fixed in each of the southern and northern lineages. Moderate genetic variation exists for microsatellite loci in all three groups. The captive southern population has not diverged substantially from the wild population at microsatellite loci (F _ST = 0.03), whereas there is little allelic overlap between the northern and southern lineages at five of six loci (F _ST > 0.3). Despite this differentiation, they are no more divergent than many populations of other amphibian species. As the northern breeding colony may not remain viable due to its small size and inbred nature, it is recommended that a third breeding colony be established in which northern and southern individuals are combined. This will preserve any northern adaptive traits that may exist, and provide animals for release in the event that the pure northern lineage becomes extirpated.     

High degree of population subdivision in a widespread amphibian

In general, amphibians are known to exhibit a higher degree of population subdivision than any other major animal taxa, but large-scale population genetic surveys of widely distributed species are still scarce, especially in the Eurasian continent. Using microsatellite markers and mitochondrial DNA sequences, we investigated the large-scale population genetic structure of the common frog (Rana temporaria)--one of the most widespread amphibians of the Palearctic region. Analyses of cytochrome b sequences revealed evidence for two distinct lineages inhabiting western and eastern parts of Europe. The separation of these lineages c. 700,000 years ago may have been induced by the onset of the Middle Pleistocene continental glaciations. Analyses of the variability of microsatellite loci within each of the clades revealed evidence for evolution of a high degree of population subdivision (FST approximately 0.23) even in northern Fennoscandia, colonized less than 10,000 years ago. The high level of substructuring is puzzling in the face of an apparently high dispersal capacity, as evidenced by the rather rapid recolonization of northern Europe. This suggests that processes other than restricted dispersal capacity need to be explored as explanations for the high degree of population subdivision in amphibians. The colonization of northern Europe has been accompanied by loss of genetic variability as evidenced by decreasing levels of intrapopulational genetic variability in microsatellite loci from south to north across Europe.     

Pouch young removal and return to oestrus in wild southern hairy-nosed wombats (Lasiorhinus latifrons)

The southern hairy-nosed wombat (Lasiorhinus latifrons) is a seasonal breeding, burrowing marsupial adapted to a semi-arid environment and the closest relative of the endangered northern hairy-nosed wombat (Lasiorhinus krefftii). Females typically give birth to one to two young every 3 years with young weaned at 360-400 days. This study examined the occurrence of polyoestry in a wild population of southern hairy-nosed wombats, and in particular the ability of this species to produce additional offspring in the same breeding season if a young was prematurely lost or removed. Pouch young were removed during the breeding seasons of 1996/1997 and 2003. No females from the 1996 (n=3)/1997 (n=3) group gave birth to a second pouch young in the same breeding season. However, two females in this group gave birth to young the following season. In contrast, all the 2003 group of females (n=6) produced a second offspring in the same breeding season after removal of pouch young (RPY). The reason for the different response to RPY between the two groups is unknown. These studies confirm that southern hairy-nosed wombats are polyoestrus in the wild and are capable of producing more than one offspring in a single breeding season. Females that failed to return to oestrus in the breeding season that pouch young were removed bred again in the following season. Rapid replacement of southern hairy-nosed wombat pouch young in the same breeding season as RPY suggests that this procedure, linked to either hand-rearing or interspecific cross-fostering, should be seriously considered as a priority conservation action to increase the population size of the critically endangered sister species, the northern hairy-nosed wombat.     

High levels of variation despite genetic fragmentation in populations of the endangered mountain pygmy-possum, Burramys parvus, in alpine Australia

In endangered mammals, levels of genetic variation are often low and this is accompanied by genetic divergence among populations. The mountain pygmy-possum (Burramys parvus) is an endangered marsupial restricted to the alpine region of Victoria and New South Wales, Australia. By scoring variation at eight microsatellite loci, we found that B. parvus populations exhibit high levels of genetic divergence and fall into three distinct groups from the northern, central and southern areas of the distribution of this species, consistent with previous assessments of mitochondrial DNA variation. F(ST) values between populations from these regions ranged from 0.19 to 0.54. Within the central area, there was further genetic fragmentation, and a linear association between genetic and geographical distance. This pattern is likely to reflect limited dispersal across barriers despite the fact that individual B. parvus can move several kilometres. Levels of genetic variation within populations were high with the exception of a southern population where there was evidence of inbreeding. From a conservation perspective, all three areas where B. parvus are found should be considered as separate gene pools; management of populations within these areas needs to take into account the low gene flow between populations, as well as threats posed by roads, resorts and other developments in the alpine region. The low genetic variability and inbreeding in the southern population is of particular concern given the high levels of variability in other B. parvus populations.     

Comparison of funding and demand for the conservation of the charismatic koala with those for the critically endangered wombat <Emphasis Type="Italic">Lasiorhinus krefftii</Emphasis>

This study contrasts the actual conservation spending and the Australian public’s demand for conservation funding for two Australian mammal species, the koala and the northern hairy-nosed wombat. It involves a survey of 204 members of the Australian public. Willingness to fund conservation action to protect the northern hairy-nosed wombat was found to be higher than that for the koala despite the koala’s immense popularity. The critically endangered status of the northern-hairy nosed wombat and the more secure conservation status of the koala is a factor likely to have influenced the comparative willingness-to-pay decisions. Actual annual conservation expenditure for both species is lower than the estimated aggregate willingness-to-pay for their conservation. Furthermore, conservation funding for the koala is much more than that for the northern hairy-nosed wombat even though the estimated public willingness-to-pay (demand) for funding koala conservation was less than for this wombat species. Reasons for this are suggested. They may also help to explain misalignment between demand for conservation funding of other species involving differences in charisma and endangerment.     

Unique and isolated: population structure has implications for management of the endangered New Zealand sea lion

Female otariids (eared seals) frequently display strong levels of philopatry, a behaviour that has the potential to influence population structure, particularly at the mitochondrial level. Conversely, male otariids often move between breeding colonies, likely facilitating nuclear gene flow between colonies. Such gender-specific movements have the potential to influence species population structure. Here we investigate the genetic population structure of the endangered New Zealand (NZ) sea lion, using nuclear (microsatellite) and mitochondrial molecular markers, with the intention to better inform conservation through identification of management units for the species. The strong levels of female philopatry in this species have potential to lead to population structure at the mitochondrial loci. In contrast, weak or no population structure is expected across nuclear loci. NZ sea lions were sampled from the main breeding areas across the species’ current distribution (three Auckland Islands sites, two Campbell Island sites, one Stewart Island site and one Otago Peninsula site). Individuals were screened for microsatellite (n?=?271; 16 loci) and mitochondrial (n?=?56; 1027 bp D-loop and 1189 bp cytb). Despite a small (c. 9880 individuals) population size, moderate levels of microsatellite variation are observed in the NZ sea lions, in contrast to low levels of mitochondrial genetic variation. Results from mitochondrial DNA analyses revealed no population structure, suggesting that the strong level of female philopatry in NZ sea lions alone is not sufficient to maintain genetic population structure. Due to the frequent male movements between breeding colonies, no population structure was detected across the nuclear loci either. The absence of genetic structure suggests that, from a genetic perspective, NZ sea lions can be considered to be a single population. Despite this, the differing impacts of threats (e.g. fisheries by-catch) to each individual breeding colony must also be taken into consideration when defining management units for this endangered species.     

Estimating the probability of identity among genotypes in natural populations: cautions and guidelines

Individual identification using DNA fingerprinting methods is emerging as a critical tool in conservation genetics and molecular ecology. Statistical methods that estimate the probability of sampling identical genotypes using theoretical equations generally assume random associations between alleles within and among loci. These calculations are probably inaccurate for many animal and plant populations due to population substructure. We evaluated the accuracy of a probability of identity (P(ID)) estimation by comparing the observed and expected P(ID), using large nuclear DNA microsatellite data sets from three endangered species: the grey wolf (Canis lupus), the brown bear (Ursus arctos), and the Australian northern hairy-nosed wombat (Lasiorinyus krefftii). The theoretical estimates of P(ID) were consistently lower than the observed P(ID), and can differ by as much as three orders of magnitude. To help researchers and managers avoid potential problems associated with this bias, we introduce an equation for P(ID) between sibs. This equation provides an estimator that can be used as a conservative upper bound for the probability of observing identical multilocus genotypes between two individuals sampled from a population. We suggest computing the actual observed P(ID) when possible and give general guidelines for the number of codominant and dominant marker loci required to achieve a reasonably low P(ID) (e.g. 0.01-0.0001).     

Detecting population expansion and decline using microsatellites

This article considers a demographic model where a population varies in size either linearly or exponentially. The genealogical history of microsatellite data sampled from this population can be described using coalescent theory. A method is presented whereby the posterior probability distribution of the genealogical and demographic parameters can be estimated using Markov chain Monte Carlo simulations. The likelihood surface for the demographic parameters is complicated and its general features are described. The method is then applied to published microsatellite data from two populations. Data from the northern hairy-nosed wombat show strong evidence of decline. Data from European humans show weak evidence of expansion.     

Weak large‐scale population genetic structure in a philopatric seabird,the European Shag Phalacrocorax aristotelis

Quantifying population genetic structure is fundamental to testing hypotheses regarding gene flow, population divergence and dynamics across large spatial scales. In species with highly mobile life‐history stages, where it is unclear whether such movements translate into effective dispersal among discrete philopatric breeding populations, this approach can be particularly effective. We used seven nuclear microsatellite loci and mitochondrial DNA (ND2) markers to quantify population genetic structure and variation across 20 populations (447 individuals) of one such species, the European Shag, spanning a large geographical range. Despite high breeding philopatry, rare cross‐sea movements and recognized subspecies, population genetic structure was weak across both microsatellites and mitochondrial markers. Furthermore, although isolation‐by‐distance was detected, microsatellite variation provided no evidence that open sea formed a complete barrier to effective dispersal. These data suggest that occasional long‐distance, cross‐sea movements translate into gene flow across a large spatial scale. Historical factors may also have shaped contemporary genetic structure: cluster analyses of microsatellite data identified three groups, comprising colonies at southern, mid‐ and northern latitudes, and similar structure was observed at mitochondrial loci. Only one private mitochondrial haplotype was found among subspecies, suggesting that this current taxonomic subdivision may not be mirrored by genetic isolation.     

Microsatellite analysis of the phylogeography, Pleistocene history and secondary contact hypotheses for the killifish, Fundulus heteroclitus

The mummichog, Fundulus heteroclitus, exhibits extensive latitudinal clinal variation in a number of physiological and biochemical traits, coupled with phylogeographical patterns at mitochondrial and nuclear DNA loci that suggest a complicated history of spatially variable selection and secondary intergradation. This species continues to serve as a model for understanding local and regional adaptation to variable environments. Resolving the influences of historical processes on the distribution of genetic variation within and among extant populations of F. heteroclitus is crucial to a better understanding of how populations evolve in the context of contemporary environments. In this study, we analysed geographical patterns of genetic variation at eight microsatellite loci among 15 populations of F. heteroclitus distributed throughout the North American range of the species from Nova Scotia to Georgia. Genetic variation in Northern populations was lower than in Southern populations and was strongly correlated with latitude throughout the species range. The most common Northern alleles at all eight loci exhibited concordant latitudinal clinal patterns, and the existence of an abrupt transition zone in allele frequencies between Northern and Southern populations was similar to that observed for mitochondrial DNA and allozyme loci. A significant pattern of isolation by distance was observed both within and between northern and southern regions. This pattern was unexpected, particularly for northern populations, given the recent colonization history of post-Pleistocene habitats, and was inconsistent with either a recent northward population expansion or a geographically restricted northern Pleistocene refugium. The data provided no evidence for recent population bottlenecks, and estimates of historical effective population sizes suggest that post-Pleistocene populations have been large throughout the species distribution. These results suggest that F. heteroclitus was broadly distributed throughout most of its current range during the last glacial event and that the abrupt transition in allele frequencies that separate Northern and Southern populations may reflect regional disequilibrium conditions associated with the post-Pleistocene colonization history of habitats in that region.     

Genetic Structure of Earthworm Populations at a Regional Scale: Inferences from Mitochondrial and Microsatellite Molecular Markers in Aporrectodea icterica (Savigny 1826)

Despite the fundamental role that soil invertebrates (e.g. earthworms) play in soil ecosystems, the magnitude of their spatial genetic variation is still largely unknown and only a few studies have investigated the population genetic structure of these organisms. Here, we investigated the genetic structure of seven populations of a common endogeic earthworm (Aporrectodea icterica) sampled in northern France to explore how historical species range changes, microevolutionary processes and human activities interact in shaping genetic variation at a regional scale. Because combining markers with distinct modes of inheritance can provide extra, complementary information on gene flow, we compared the patterns of genetic structure revealed using nuclear (7 microsatellite loci) and mitochondrial markers (COI). Both types of markers indicated low genetic polymorphism compared to other earthworm species, a result that can be attributed to ancient bottlenecks, for instance due to species isolation in southern refugia during the ice ages with subsequent expansion toward northern Europe. Historical events can also be responsible for the existence of two divergent, but randomly interbreeding mitochondrial lineages within all study populations. In addition, the comparison of observed heterozygosity among microsatellite loci and heterozygosity expected under mutation-drift equilibrium suggested a recent decrease in effective size in some populations that could be due to contemporary events such as habitat fragmentation. The absence of relationship between geographic and genetic distances estimated from microsatellite allele frequency data also suggested that dispersal is haphazard and that human activities favour passive dispersal among geographically distant populations.     

Contrasting levels of genetic differentiation among populations of wolverines (<Emphasis Type="Italic">Gulo gulo</Emphasis>) from northern Canada revealed by nuclear and mitochondrial loci

Habitat loss, fragmentation, overharvest, and other anthropogenic factors have resulted in population and distribution declines for North American wolverines (Gulo gulo). Currently, wolverines east of the Hudson Bay are endangered and possibly extinct, whereas the status of wolverines throughout the remaining Holarctic is vulnerable. Three previous studies using nuclear loci have detected little to no significant structuring among wolverines sampled across northern Canada. Based on these results it has been suggested that wolverines in northern Canada represent a single, panmictic population. However, as has been shown in numerous studies, in cases of female site fidelity, it is possible to have demographically autonomous populations even with male-biased gene flow. To better assess the genetic structure of wolverines in northern Canada, we examined nine microsatellite loci and DNA sequence variation from a 200 bp fragment of the mitochondrial (mtDNA) control region for 270 wolverines from nine collecting areas representing three regions of northern Canada. In agreement with previous studies, microsatellite analyses revealed a lack of significant population substructure (F _ST=0.0004). However, analysis of molecular variance, comparisons of pairwise F _ST values and nested-clade analysis of the mtDNA data revealed considerable genetic structuring among samples of wolverines from these three regions of northern Canada. These mitochondrial data provide evidence that wolverines in Canada are genetically structured due to female philopatry. The contrasting patterns of genetic differentiation based on nuclear and mitochondrial data highlight the importance of examining both nuclear and mitochondrial loci when attempting to elucidate patterns of genetic structure.     

Population structure and conservation genetic assessment of the endangered Pugnose Shiner,Notropis anogenus

The Pugnose Shiner is a small minnow with a fragmented distribution across the Great Lakes and Upper Mississippi River in North America. The species is listed federally as endangered in Canada, and in the United States its status varies by state, from Special Concern to Endangered (as well as Extirpated). We conducted a thorough genetic assessment of the Pugnose Shiner using both microsatellite loci and mitochondrial DNA collected for samples across the species range. Our results indicate high levels of population differentiation suggesting restricted dispersal, in some cases at very small geographical scales. We also found strong evidence of small effective population sizes and one case of a genetic bottleneck. Although significant range-wide genetic variation was observed in both microsatellite loci and mitochondrial DNA, the species is best characterized as a single evolutionarily significant unit for conservation purposes.     

Global population genetic structure and male-mediated gene flow in the green sea turtle (Chelonia mydas): analysis of microsatellite loci

We assessed the degree of population subdivision among global populations of green sea turtles, Chelonia mydas, using four microsatellite loci. Previously, a single-copy nuclear DNA study indicated significant male-mediated gene flow among populations alternately fixed for different mitochondrial DNA haplotypes and that genetic divergence between populations in the Atlantic and Pacific Oceans was more common than subdivisions among populations within ocean basins. Even so, overall levels of variation at single-copy loci were low and inferences were limited. Here, the markedly more variable microsatellite loci confirm the presence of male-mediated gene flow among populations within ocean basins. This analysis generally confirms the genetic divergence between the Atlantic and Pacific. As with the previous study, phylogenetic analyses of genetic distances based on the microsatellite loci indicate a close genetic relationship among eastern Atlantic and Indian Ocean populations. Unlike the single-copy study, however, the results here cannot be attributed to an artifact of general low variability and likely represent recent or ongoing migration between ocean basins. Sequence analyses of regions flanking the microsatellite repeat reveal considerable amounts of cryptic variation and homoplasy and significantly aid in our understanding of population connectivity. Assessment of the allele frequency distributions indicates that at least some of the loci may not be evolving by the stepwise mutation model.     

Relatedness structure detected by microsatellite analysis and attempted pedigree reconstruction in an endangered marsupial, the northern hairy-nosed wombat Lasiorhinus krefftii

The northern hairy-nosed (NHN) wombat is perhaps Australia's most endangered mammal. Being fossorial and nocturnal as well as rare, NHN wombats are difficult to observe in the wild. Hence little is known of their social biology, such as their mating and dispersal systems. A hypothesis has been advanced that adult females of the species disperse post-breeding, leaving their young to inhabit the natal burrow. Female-biased dispersal is expected to result in higher relatedness amongst males in a burrow cluster than amongst females in a burrow cluster. The usefulness of a panel of microsatellite markers in estimating the relatedness structure, and in reconstructing pedigrees for, the sole known population of NHN wombats was assessed. Microsatellite genotypes at eight or nine loci were obtained from 58 of the 85 known individuals, and used to estimate pairwise individual relatedness using Queller & Goodnight's (1989) RELATEDNESS 4.2. Our analysis gave the unexpected result that both males and females were significantly more closely related to their same-sex burrow cluster mates than random, while opposite-sex animals sharing burrows were only slightly (nonsignificantly) more related than random. This raises the possibility of dispersal patterns which lead to association of same-sex relatives. The observed relatedness structure is not expected to make likely a high incidence of inbred matings, as close relatives of the opposite sex are not significantly associated in space. Parentage analysis was attempted using genetic exclusion and LOD likelihood ratios, but proved difficult because of low genetic variation, incomplete sampling of potential parents, and paucity of ecological data such as known mother/offspring pairs and ages of individuals.     

Determination of seasonality in southern hairy-nosed wombats (Lasiorhinus latifrons) by analysis of fecal androgens

Little is known about the reproductive biology of Australia's critically endangered northern hairy-nosed wombat (Lasiorhinus krefftii), largely due to its cryptic nature and the difficulty in accessing the small remaining population of about 70 animals. Using the noninvasive technique of fecal steroid analysis, we have examined the endocrinology of the more common yet closely related southern hairy-nosed wombat (Lasiorhinus latifrons). The aims of this study were to 1) develop and validate fecal androgen analysis in this species, 2) examine and compare seasonal differences in fecal and plasma androgens in male wombats, and 3) correlate seasonal differences in androgens with changes in male accessory glands (prostate and bulbourethral gland). Fecal androgens were extracted in ether; concentrated; separated by HPLC into testosterone (T), dihydrotestosterone (DHT), and 5 alpha-androstane-3 alpha,17 beta-diol (Adiol) fractions; and quantitated by RIA. The concentrations of androgens in fecal pellets from 14 wild southern hairy-nosed wombats as determined by RIA varied over the range 6.6-25.0 ng/g dry weight for T, 4.0-24.2 ng/g dry weight for DHT, and 0-34.8 ng/g dry weight for Adiol. For each androgen, a highly significant linear correlation was observed between plasma and fecal concentrations. When individuals were grouped into either breeding season (pellets collected between August-November) or nonbreeding season (collected between February-April), significant (P < 0.05) differences between seasons were observed for both plasma and fecal T, plasma DHT, and fecal Adiol. For all androgens, the mean fecal and plasma concentrations were higher during the breeding season than the nonbreeding season. A significant (P < 0.001) correlation was observed between fecal T and prostate weight, while DHT and Adiol correlations were nonsignificant. Significant correlations were observed, however, between all three fecal androgens and bulbourethral gland weight. These studies demonstrate that fecal T is a valid indicator of reproductive status in the male southern hairy-nosed wombat, with significant correlations observed between fecal T, plasma T, and prostate and bulbourethral gland weights. These findings have important implications for the study of the reproductive endocrinology of the critically endangered northern hairy-nosed wombat.