Lyssa excreta: Defining parameters for fecal samples as a rabies virus surveillance method.

It is not possible to systematically screen the environment for rabies virus (RABV) using current approaches. We sought to determine under what conditions RABV is detectable from feces and other accessible samples from infected wildlife to broaden the number of biological samples that could be used to test for RABV. We employed a recently-developed quantitative RT-PCR assay called the "LN34 panlyssavirus real-time RT-PCR assay", which is highly sensitive and specific for all variants of RABV. We harvested and tested brain tissue, fecal, and/or mouth swab samples from 25 confirmed RABV positive bats of six species. To determine if rabies RNA lasts in feces sufficiently long post-defecation to use it as a surveillance tool, we tested fecal samples from 10 bats at the time of sample collection and after 24 hours of exposure to ambient conditions, with an additional test on six bats out to 72 hours. To assess whether we could pool fecal pellets and still detect a positive, we generated dilutions of known positives at 1:1, 1:10, 1:50, and 1:200. For six individuals for which matched brain, mouth swab, and fecal samples were tested, results were positive for 100%, 67%, and 67%, respectively. For the first time test to 24 hours, 63% of feces that were positive at time 0 were still positive after 24 hours, and 50% of samples at 72 hours were positive across all three replicates. Pooling tests revealed that fecal positives were detected at 1:10 dilution, but not at 1:50 or 1:200. Our preliminary results suggest that fecal samples hold promise for a rapid and non-invasive environmental screening system.

Dietary niche partitioning of three Sky Island Sceloporus lizards as revealed through DNA metabarcoding.

Lizard diets are highly diverse and have contributed to the diversification, biogeographical distributions, and evolution of novel traits across this global radiation. Many parts of a lizard's ecology-including habitat preferences, foraging modes, predation risks, interspecific competition, and thermal constraints, among others-interact to shape diets, and dietary niche partitioning simultaneously contributes to co-occurrence within communities. We used DNA metabarcoding of fecal samples to identify prey items in the diets of three sympatric Sceloporus lizards in the Madrean Sky Islands of Arizona, USA. We found evidence for dietary niche partitioning between interacting species concomitant with their respective ecologies. We also compared diet composition between populations to understand how conserved or plastic species' diets are between different environments. Our findings suggest that habitat generalists are also diet generalists in this system, while the same may be true for specialists. The identification of prey items to much lower taxonomic levels than previously documented further reveals hidden diversity in the diets of these species and underscores the utility of metabarcoding for understanding the full complexity of lizard diets.

Our good neighbors: Understanding ecosystem services provided by insectivorous bats in Rwanda.

Bats are prodigious consumers of agricultural and forest pests, and are, therefore, a natural asset for agricultural productivity, suppressing populations of such pests. This study provides baseline information of diet of 143 bats belonging to eight insectivorous bat species from agricultural areas of Rwanda while evaluating the effectiveness of bats as pest suppressors. Using DNA metabarcoding to analyze bat fecal pellets, 85 different insect species were detected, with 60% (n = 65), 64% (n = 11) and 78% (n = 9) found to be agricultural pests from eastern, northern and western regions, respectively. Given the high percentages of agricultural pests detected, we submit that Rwandan insectivorous bats have the capacity for biocontrol of agricultural pests. Rwandan bat populations should be protected and promoted since they may foster higher crop yields and sustainable livelihoods.

Once upon a time in Mexico: Holocene biogeography of the spotted bat (Euderma maculatum).

Holocene-era range expansions are relevant to understanding how a species might respond to the warming and drying climates of today. The harsh conditions of North American deserts have phylogenetically structured desert bat communities but differences in flight capabilities are expected to affect their ability to compete, locate, and use habitat in the face of modern climate change. A highly vagile but data-deficient bat species, the spotted bat (Euderma maculatum), is thought to have expanded its range from central Mexico to western Canada during the Holocene. With specimens spanning this latitudinal extent, we examined historical demography, and used ecological niche modeling (ENM) and phylogeography (mitochondrial DNA), to investigate historic biogeography from the rear to leading edges of the species' range. The ENM supported the notion that Mexico was largely the Pleistocene-era range, whereas haplotype pattern and Skyline plots indicated that populations expanded from the southwestern US throughout the Holocene. This era provided substantial gains in suitable climate space and likely facilitated access to roosting habitat throughout the US Intermountain West. Incongruent phylogenies among different methods prevented a precise understanding of colonization history. However, isolation at the southern-most margin of the range suggests a population was left behind in Mexico as climate space contracted and are currently of unknown status. The species appears historically suited to follow shifts in climate space but differences in flight behaviors between leading edge and core-range haplogroups suggest range expansions could be influenced by differences in habitat quality or climate (e.g., drought). Although its vagility could facilitate response to environmental change and thereby avoid extinction, anthropogenic pressures at the core range could still threaten the ability for beneficial alleles to expand into the leading edge.

Endangered Nectar-Feeding Bat Detected by Environmental DNA on Flowers.

Leptonycteris nivalis (the Mexican long-nosed bat) is an endangered nectar-feeding bat species that follows "nectar corridors" as it migrates from Mexico to the southwestern United States. Locating these nectar corridors is key to their conservation and may be possible using environmental DNA (eDNA) from these bats. Hence, we developed and tested DNA metabarcoding and qPCR eDNA assays to determine whether L. nivalis could be detected by sampling the agave flowers on which it feeds. We sampled plants with known bat visitations in the Sierra Madre Oriental in Laguna de Sanchez (LS), Nuevo León, Mexico, and in the Chisos Mountains in Big Bend National Park, TX, USA (CB). A total of 13 samples included both swabs of agave umbels and cuttings of individual flowers. DNA metabarcoding was performed as a PCR multiplex that targeted bats (SFF-COI), arthropods (ANML-COI), and plants (ITS2 and rbcL). We targeted arthropods and plants in parallel with bats because future metabarcoding studies may wish to examine all the pollinators and plants within the nectar corridor. We developed and tested the sensitivity and specificity of two qPCR assays. We found that both DNA metabarcoding and qPCR were highly successful at detecting L. nivalis (11 of 13 for DNA metabarcoding and 12 of 13 for qPCR). Swabs and flower cuttings and both qPCR assays detected the species over four replicates. We suggest that L. nivalis leaves substantial DNA behind as it forages for nectar. We also suggest that future studies examine the time since sampling to determine its effect on detection success. The DNA metabarcoding multiplex will be useful for parallel questions regarding pollination ecology, while, with further testing, the qPCR assays will be effective for large-scale sampling for the detection of migration corridors and foraging areas. This work may be relevant to other nectar-feeding bat species, which can likely be detected with similar methodologies.

Bat Use of Hollows in California's Old-Growth Redwood Forests: From DNA to Ecology.

The loss of roosting resources, either through disturbance or removal, negatively affects bats. Identifying sensitive species and determining roost requirements are critical components in conserving their habitat. Cavity-roosting bats on the North Coast of California are known to use hollows in large redwood trees. In this study, we examined the factors determining the use of basal tree hollows by different bat species at eight redwood forest sites in Del Norte, Humboldt, and Mendocino Counties, California. Bat guano was collected from 179 basal hollow roosts from 2017 to 2018, and guano mass was used as an index of roosting activity. Nine bat species and one species group were identified by analysis of DNA in guano. We made a total of 253 identifications from 83 hollows into the 10 species categories. The most prevalent species were Myotis californicus (California myotis; 28.5% of all identifications), the Myotis evotis-Myotis thysanodes group (17.4%), Corynorhinus townsendii (17.0%), and Myotis volans (15.0%). We evaluated the extent to which habitat variables at the scales of the hollow, vicinity, and site influenced the level of roost use. The correlations between guano mass and habitat variables were examined using generalized additive mixed models. At the hollow scale, guano mass increased with ceiling height above the opening. At the vicinity scale, guano mass increased with less cover of small trees. At the site scale, there was no association between guano mass and distance to foraging areas, elevation, or the number of nearby hollows. These tree hollow roost preferences can inform land managers when planning the management and conservation of redwood forests.

Vampyrum spectrum (Phyllostomidae) movement and prey revealed by radio-telemetry and DNA metabarcoding.

The spectral bat (Vampyrum spectrum), the largest bat species in the Americas, is considered Near Threatened by the International Union for Conservation of Nature and is listed as a species of special concern or endangered in several countries throughout its range. Although the species is known as carnivorous, data on basic ecology, including habitat selection and primary diet items, are limited owing to its relative rarity and difficulty in capturing the species. Leveraging advances in DNA metabarcoding and using radio-telemetry, we present novel information on the diet and movement of V. spectrum based on locations of a radio-collared individual and fecal samples collected from its communal roost (three individuals) in the Lowland Dry Forest of southern Nicaragua. Using a non-invasive approach, we explored the diet of the species with genetic markers designed to capture a range of arthropods and vertebrate targets from fecal samples. We identified 27 species of vertebrate prey which included birds, rodents, and other bat species. Our evidence suggested that V. spectrum can forage on a variety of species, from those associated with mature forests to forest edge-dwellers. Characteristics of the roost and our telemetry data underscore the importance of large trees for roosting in mature forest patches for the species. These data can inform conservation efforts for preserving both the habitat and the prey items in remnants of mature forest required by Vampyrum spectrum to survive in landscape mosaics.

RELATEDNESS AND GENETIC STRUCTURE OF BIG BROWN BAT (EPTESICUS FUSCUS) MATERNITY COLONIES IN AN URBAN-WILDLAND INTERFACE WITH PERIODIC RABIES VIRUS OUTBREAKS.

Big brown bats (Eptesicus fuscus) are the bat species in North America most frequently found to be rabid because of their high rate of human contact and thus submissions for rabies testing, of which, 4-5% are positive. The social behavior of big brown bats during the summer months may drive space use and potential viral exposure to conspecifics and mesocarnivores. We collected 88 unique genetic samples via buccal swabs from big brown bats captured at four maternity roosts surrounding a golf course during the summer of 2013. We used seven microsatellite loci to estimate genetic relatedness among individuals and genetic structure within and among colonies to infer whether females selected roosts based on kinship and used genetics and radio telemetry to determine the frequency of roost switching. We found roost switching through genetics and telemetry, and no evidence of elevated genetic relatedness within colonies or genetic structure among colonies. Social cohesion based on relatedness may not act to constrain the pathogen to a particular roost area, and thus, geographic mobility may increase viral exposure of bats in neighboring areas.

Identifying genetic relationships among tarsier populations in the islands of Bunaken National Park and mainland Sulawesi.

Eastern tarsiers (Tarsius tarsier complex) are small nocturnal primates endemic to Sulawesi Island and small adjacent islands of Indonesia. In 2004, the hybrid biogeography hypothesis predicted this species complex might contain 16 or more taxa, each corresponding to a region of endemism, based on: 1) geological evidence of the development of the archipelago, 2) biological evidence in the form of concordant distributions of monkeys and toads, and 3) the distribution of tarsier acoustic groups. Since then, 11 tarsier species have been recognized, potentially leaving more to be described. Efforts to identify these cryptic species are urgently needed so that habitat conversion, pet trade, and cultural activities will not render some species extinct before they are recognized. We gathered data to test the hypothesis of cryptic tarsier species on three volcanic islands in Bunaken National Park, North Sulawesi, namely Bunaken, Manadotua, and Mantehage, during May-August 2018. We sequenced individuals at 5 nuclear genes (ABCA1, ADORA3, AXIN1, RAG, and TTR) and made comparisons to existing genotypes at 14 mainland sites. Bayesian phylogenetic analyses revealed that island populations are genetically identical in all 5 genes, and formed a clade separated from the mainland ones. The eastern tarsiers first diverged from the western tarsiers approximately 2.5 MYA. The three island populations diverged from mainland tarsiers approximately 2,000-150,000 YA, due to either human activities or natural rafting. This study provides information for tarsier conservation, advances the understanding of biogeography of Sulawesi, and contributes to Indonesian awareness of biodiversity. Further quantitative genetics research on tarsiers, especially the island populations, will offer significant insights to establish more efficient and strategic tarsier conservation actions.

A fecal sequel: Testing the limits of a genetic assay for bat species identification.

DNA metabarcoding assays are powerful tools for delving into the DNA in wildlife feces, giving unprecedented ability to detect species, understand natural history, and identify pathogens for a range of applications in management, conservation, and research. Next-generation sequencing technology is developing rapidly, which makes it especially important that predictability and reproducibility of DNA metabarcoding assays are explored together with the post-depositional ecology of the target taxon's fecal DNA. Here, we defined the constraints of an assay called 'Species from Feces' used by government agencies, research groups, and non-governmental organizations to identify bat species from guano. We tested assay sensitivity by examining how time and humidity affect the ability to recover and successfully sequence DNA in guano, assessing whether a fecal pellet from a rare bat species could be detected in a background of feces from other bat species, and evaluating the efficacy of Species from Feces as a survey tool for bat roosts in temperate and tropical areas. We found that the assay performs well with feces over two years old in dry, cool environments, and fails by 12 months at 100% relative humidity. We also found that it reliably identifies rare DNA, has great utility for surveying roosts in temperate and tropical regions, and detects more bat species than do visual surveys. We attribute the success of Species from Feces to characteristics of the assay paired with application in taxa that are particularly well-suited for fecal DNA survival. In a time of rapid evolution of DNA metabarcoding approaches and their use with feces, this study illustrates the strengths and limitations of applied assays.

African Lineage Brucella melitensis Isolates from Omani Livestock.

Brucellosis is a common livestock disease in the Middle East and North Africa, but remains poorly described in the region both genetically and epidemiologically. Traditionally found in goats and sheep, Brucella melitensis is increasingly recognized as infecting camels. Most studies of brucellosis in camels to date have focused on serological surveys, providing only limited understanding of the molecular epidemiology of circulating strains. We genotyped B. melitensis isolates from Omani camels using whole genome SNP assays and VNTRs to provide context for regional brucellosis cases. We identified a lineage of B. melitensis circulating in camels as well as in goats, sheep, and cattle in Oman. This lineage is genetically distinct from most genotypes from the Arabian Peninsula and from isolates from much of the rest of the Middle East. We then developed diagnostic assays that rapidly identify strains from this lineage. In analyses of genotypes from throughout the region, Omani isolates were genetically most closely related to strains from brucellosis cases in humans and livestock in North Africa. Our findings suggest an African origin for B. melitensis in Oman that has likely occurred through the trade of infected livestock. Moreover, African lineages of B. melitensis appear to be undersampled and consequently are underrepresented in genetic databases for Brucella. As we begin to more fully understand global genomic diversity of B. melitensis, finding and characterizing these unique but widespread lineages is essential. We predict that increased sampling of humans and livestock in Africa will reveal little known diversity in this important zoonotic pathogen.

Evidence of Subdivisions on Evolutionary Timescales in a Large, Declining Marsupial Distributed across a Phylogeographic Barrier.

Major prehistoric forces, such as the climatic shifts of the Pleistocene, can remain visible in a species' population genetics. Inference of refuges via genetic tools is useful for conservation management as it can identify populations whose preservation may help retain a species' adaptive potential. Such investigation is needed for Australia's southern hairy-nosed wombat (Lasiorhinus latifrons), whose conservation status has recently deteriorated, and whose phylogeographic history during the Pleistocene may be atypical compared to other species. Its contemporary range spans approximately 2000 km of diverse habitat on either side of the Spencer Gulf, which was a land bridge during periods of Pleistocene aridity that may have allowed for migration circumventing the arid Eyrean barrier. We sampled from animals in nearly all known sites within the species' current distribution, mainly using non-invasive methods, and employed nuclear and mitochondrial DNA analyses to assess alternative scenarios for Pleistocene impacts on population structure. We found evidence for mildly differentiated populations at the range extremes on either side of Spencer Gulf, with secondary contact between locations neighbouring each side of the barrier. These extreme western and eastern regions, and four other regions in between, were genetically distinct in genotypic clustering analyses. Estimates indicate modest, but complex gene flow patterns among some of these regions, in some cases possibly restricted for several thousand years. Prior to this study there was little information to aid risk assessment and prioritization of conservation interventions facilitating gene flow among populations of this species. The contributions of this study to that issue are outlined.

Species From Feces: Order-Wide Identification of Chiroptera From Guano and Other Non-Invasive Genetic Samples.

Bat guano is a relatively untapped reservoir of information, having great utility as a DNA source because it is often available at roosts even when bats are not and is an easy type of sample to collect from a difficult-to-study mammalian order. Recent advances from microbial community studies in primer design, sequencing, and analysis enable fast, accurate, and cost-effective species identification. Here, we borrow from this discipline to develop an order-wide DNA mini-barcode assay (Species from Feces) based on a segment of the mitochondrial gene cytochrome c oxidase I (COI). The assay works effectively with fecal DNA and is conveniently transferable to low-cost, high-throughput Illumina MiSeq technology that also allows simultaneous pairing with other markers. Our PCR primers target a region of COI that is highly discriminatory among Chiroptera (92% species-level identification of barcoded species), and are sufficiently degenerate to allow hybridization across diverse bat taxa. We successfully validated our system with 54 bat species across both suborders. Despite abundant arthropod prey DNA in guano, our primers were highly specific to bats; no arthropod DNA was detected in thousands of feces run on Sanger and Illumina platforms. The assay is extendable to fecal pellets of unknown age as well as individual and pooled guano, to allow for individual (using singular fecal pellets) and community (using combined pellets collected from across long-term roost sites) analyses. We developed a searchable database (http://nau.edu/CEFNS/Forestry/Research/Bats/Search-Tool/) that allows users to determine the discriminatory capability of our markers for bat species of interest. Our assay has applications worldwide for examining disease impacts on vulnerable species, determining species assemblages within roosts, and assessing the presence of bat species that are vulnerable or facing extinction. The development and analytical pathways are rapid, reliable, and inexpensive, and can be applied to ecology and conservation studies of other taxa.

A simulation-based evaluation of methods for inferring linear barriers to gene flow.

Different analytical techniques used on the same data set may lead to different conclusions about the existence and strength of genetic structure. Therefore, reliable interpretation of the results from different methods depends on the efficacy and reliability of different statistical methods. In this paper, we evaluated the performance of multiple analytical methods to detect the presence of a linear barrier dividing populations. We were specifically interested in determining if simulation conditions, such as dispersal ability and genetic equilibrium, affect the power of different analytical methods for detecting barriers. We evaluated two boundary detection methods (Monmonier's algorithm and WOMBLING), two spatial Bayesian clustering methods (TESS and GENELAND), an aspatial clustering approach (STRUCTURE), and two recently developed, non-Bayesian clustering methods [PSMIX and discriminant analysis of principal components (DAPC)]. We found that clustering methods had higher success rates than boundary detection methods and also detected the barrier more quickly. All methods detected the barrier more quickly when dispersal was long distance in comparison to short-distance dispersal scenarios. Bayesian clustering methods performed best overall, both in terms of highest success rates and lowest time to barrier detection, with GENELAND showing the highest power. None of the methods suggested a continuous linear barrier when the data were generated under an isolation-by-distance (IBD) model. However, the clustering methods had higher potential for leading to incorrect barrier inferences under IBD unless strict criteria for successful barrier detection were implemented. Based on our findings and those of previous simulation studies, we discuss the utility of different methods for detecting linear barriers to gene flow.

Degree of landscape fragmentation influences genetic isolation among populations of a gliding mammal.

Forests and woodlands are under continuing pressure from urban and agricultural development. Tree-dependent mammals that rarely venture to the ground are likely to be highly sensitive to forest fragmentation. The Australian squirrel glider (Petaurus norfolcensis) provides an excellent case study to examine genetic (functional) connectivity among populations. It has an extensive range that occurs in a wide band along the east coast. However, its forest and woodland habitat has become greatly reduced in area and is severely fragmented within the southern inland part of the species' range, where it is recognised as threatened. Within central and northern coastal regions, habitat is much more intact and we thus hypothesise that genetic connectivity will be greater in this region than in the south. To test this we employed microsatellite analysis in a molecular population biology approach. Most sampling locations in the highly modified south showed signatures of genetic isolation. In contrast, a high level of genetic connectivity was inferred among most sampled populations in the more intact habitat of the coastal region, with samples collected 1400 km apart having similar genetic cluster membership. Nonetheless, some coastal populations associated with urbanisation and agriculture are genetically isolated, suggesting the historic pattern observed in the south is emerging on the coast. Our study demonstrates that massive landscape changes following European settlement have had substantial impacts on levels of connectivity among squirrel glider populations, as predicted on the basis of the species' ecology. This suggests that landscape planning and management in the south should be focused on restoring habitat connectivity where feasible, while along the coast, existing habitat connectivity must be maintained and recent losses restored. Molecular population biology approaches provide a ready means for identifying fragmentation effects on a species at multiple scales. Such studies are required to examine the generality of our findings for other tree-dependent species.

Evidence for habitat fragmentation altering within-population processes in wombats.

Habitat fragmentation and associated reduced dispersal of wildlife can lead to an accumulation of related individuals in fragments. The altered kin interactions and amplified chance of inbred matings has profound implications for mating and social systems, and ultimately population persistence. Nonetheless, within-fragment population processes are rarely studied. With this aim, we examined relatedness structure in two candidate isolated populations (Kulpara and Scrubby Peak) of southern hairy-nosed wombats (Lasiorhinus latifrons). Wombats were sampled by remote hair-trapping for genotyping at 14 microsatellite loci, enabling individual identification and estimation of space-use and associative behaviour with respect to relatedness. Genetic data indicated that Scrubby Peak was not strongly isolated, against predictions from landscape structure and history. In isolated Kulpara, inhibited female dispersal (normally the dispersing sex) was associated with high population density and altered kin relationships. First, female relatives preferentially coexisted, a radical departure from the previously reported active avoidance of female relatives in continuous habitat. This is consistent with females in altered habitat interacting with more- rather than less-related females to minimize the cost:benefit ratio of proximity to other wombats. Second, inbreeding avoidance appeared to be stronger at Kulpara than in conspecific populations with natural population structures. Although these adaptive behaviours may have contributed to persistence of the Kulpara population in the short term, they are unlikely to ensure its long-term viability in the face of ongoing isolation because they can act only to slow the rate of inbreeding and mitigate some of its negative impacts.

Female dispersal and male kinship-based association in southern hairy-nosed wombats (Lasiorhinus latifrons).

Generalizations about sociobiology require investigations of species with diverse ecological roles and phylogenetic affiliations. The southern hairy-nosed wombat (Lasiorhinus latifrons) is valuable here, in that it is a marsupial of semi-arid habitat, and one of the largest burrowing herbivores (commonly attaining 26 kg). Its sociobiology is poorly understood because the species is nocturnal, shy, and difficult to observe or capture nondisruptively. To investigate aspects of the species' sociobiology in continuous habitat, we applied high-intensity, temporally replicated, noninvasive sampling and genotyping of hairs to identify individuals and their sex. Spatial relatedness (kinship) structure was estimated, and related to patterns of burrow-use. To understand the association of kinship with burrow/warren-sharing and preferential colocation between wombats, > 100 genetically 'tagged' individuals at Brookfield Conservation Park (Murraylands, South Australia) were 'tracked' through multiple seasons between 1999 and 2001. Dispersal was female-biased, and may be performed by females after breeding. Conversely, males were philopatric. Male kin relationships were characterized by preferential burrow- and warren-sharing among closely related males, often in associations lasting for years. In contrast, females under-associated with their close female relatives and did not form matrilineal groupings with potential for favourable kin interactions. This fundamental departure from the predominant mammalian pattern raises questions about the origins and maintenance of the system, which is now known from all three species of wombat. The present study provides starting points to address those questions by adding to our knowledge of longitudinal spatiotemporal associations and habitat use of a marsupial with the unusual system of female-biased dispersal, and by outlining robust methodologies.

Does soil type drive social organization in southern hairy-nosed wombats?

Spatiotemporal distributions of key resources are hypothesized to underpin sociobiological patterns. Burrow availability and quality is of paramount importance to fossorial animals. The southern hairy-nosed wombat (Lasiorhinus latifrons) burrows in both hard and friable soils. Theoretical and empirical studies suggest that the harder substrate should promote closer geographical clustering of burrows than in softer soils. Clustered burrows are expected to be associated with larger group sizes. If sociality is driven by constraint rather than advantage, patterns of spatial and temporal distribution of animals within and among groups may show indications of avoidance or even antagonism, and 'making the best of a bad job' via positive kin associations to offset the disadvantages of high-density living. To test these ideas, we compared warren relatedness and social structure of L. latifrons on friable soils (Nullarbor Plain) and hard calcrete (Brookfield Conservation Park, BCP). Individuals were sampled by noninvasive collection of hairs for genotyping to identify individuals and to estimate their space-use and associative behaviour with respect to relatedness. Burrows in calcrete were indeed more clumped, and warren and group size larger. Differences in spatiotemporal organization and relatedness structure between sites were in the expected direction: (i) Nullarbor males associated and shared warrens less than at BCP; and (ii) Nullarbor spatial relatedness patterning data were not consistent with proposed female breeding dispersal, in contrast to those at BCP. Under Nullarbor (low density) conditions, cooperation or tolerance between males may be less advantageous, and accessing or digging burrows should be less of a constraint for juvenile females.