14-Carbon demonstrates that some illegal ivory is being taken from government stockpiles.

The 14-carbon in animal tissues records the time that the tissues are formed; since the 1960s, using the "bomb curve" for 14C, the age of animal death can be determined accurately. Using animal tissue samples of known collection and formation dates for calibration, we determine the age of ivory samples from four ivory seizures made by law enforcement agencies between 2017 and 2019. The 14C measurements from these seizures show that most ivory in the illegal wildlife trade is from animals from recent poaching activities. However, one seizure has a large fraction of ivory that is more than 30 y old, consistent with markings on the tusks indicating they were derived from a government stockpile.

Uncovering the magnitude of African pangolin poaching with extensive nanopore DNA genotyping of seized scales.

Trade in pangolins is illegal, and yet tons of their scales and products are seized at various ports. These large seizures are challenging to process and comprehensively genotype for upstream provenance tracing and species identification for prosecution. We implemented a scalable DNA barcoding pipeline in which rapid DNA extraction and MinION sequencing were used to genotype a substantial proportion of pangolin scales subsampled from 2 record shipments seized in Singapore in 2019 (37.5 t). We used reference sequences to match the scales to phylogeographical regions of origin. In total, we identified 2346 cytochrome b (cytb) barcodes of white-bellied (Phataginus tricuspis) (from 1091 scales), black-bellied (Phataginus tetradactyla) (227 scales), and giant (Smutsia gigantea) (1028 scales) pangolins. Haplotype diversity was higher for P. tricuspis scales (121 haplotypes, 66 novel) than that for P. tetradactyla (22 haplotypes, 15 novel) and S. gigantea (25 haplotypes, 21 novel) scales. Of the novel haplotypes, 74.2% were likely from western and west-central Africa, suggesting potential resurgence of poaching and newly exploited populations in these regions. Our results illustrate the utility of extensively subsampling large seizures and outline an efficient molecular approach for rapid genetic screening that should be accessible to most forensic laboratories and enforcement agencies.

Revelación de la magnitud de la caza furtiva del pangolín africano mediante el genotipo extenso de nanoporos de ADN de escamas incautadas Resumen Aunque el mercado de pangolines es ilegal, se incautan toneladas de sus escamas y productos derivados en varios puertos comerciales. Es un reto procesar estas magnas incautaciones y obtener el genotipo completo para usarlo en la trazabilidad logística ascendente e identificación de la especie y así imponer sanciones. Implementamos una canalización escalable del código de barras de ADN en el cual usamos la extracción rápida de ADN y la secuenciación MinION para obtener el genotipo de una proporción sustancial de las escamas de pangolín submuestreadas en dos cargamentos incautados en 2019 en Singapur (37.5 t). Usamos secuencias referenciales para emparejar las escamas con las regiones filogeográficas de origen. Identificamos en total 2,346 códigos de citocromo b (cytb) del pangolín de vientre blanco (Phataginus tricuspis) (de 1,091 escamas), de vientre negro (P. tetradactyla) (227 escamas) y del pangolín gigante (Smutsia gigantea) (1,028 escamas). La diversidad de haplotipos fue mayor en las escamas de P. tricuspis (121 haplotipos, 66 nuevos) que en las de P. tetradactyla (22 haplotipos, 15 nuevos) y S. gigantea (25 haplotipos, 21 nuevos). De los haplotipos nuevos, el 74.2% probablemente provenía del occidente y centro­occidente de África, lo que sugiere un resurgimiento potencial de la caza furtiva y poblaciones recién explotadas en estas regiones. Nuestros resultados demuestran la utilidad de submuestrear extensivamente las grandes incautaciones y esboza una estrategia molecular eficiente para un análisis genético rápido que debería ser accesible para la mayoría de los laboratorios forenses y las autoridades de aplicación.

Age and sex influence social interactions, but not associations, within a killer whale pod.

Social structure is a fundamental aspect of animal populations. In order to understand the function and evolution of animal societies, it is important to quantify how individual attributes, such as age and sex, shape social relationships. Detecting these influences in wild populations under natural conditions can be challenging, especially when social interactions are difficult to observe and broad-scale measures of association are used as a proxy. In this study, we use unoccupied aerial systems to observe association, synchronous surfacing, and physical contact within a pod of southern resident killer whales (Orcinus orca). We show that interactions do not occur randomly between associated individuals, and that interaction types are not interchangeable. While age and sex did not detectably influence association network structure, both interaction networks showed significant social homophily by age and sex, and centrality within the contact network was higher among females and young individuals. These results suggest killer whales exhibit interesting parallels in social bond formation and social life histories with primates and other terrestrial social mammals, and demonstrate how important patterns can be missed when using associations as a proxy for interactions in animal social network studies.

Shift of Maternal Gut Microbiota of Tibetan Antelope (Pantholops hodgsonii) During the Periparturition Period.

The maternal gut microbiota can influence and be affected by the substantial physiological changes taking place during the periparturition period. However, little information is known about the changes in the maternal gut microbiota and hormonal variations during this period in nonmodel organisms. Tibetan antelope (Pantholops hodgsonii) provide a unique system to address this issue because their summer migration cycle is synchronized with the periparturition period. Here, we used fecal microbiota as a proxy of gut microbiota. We characterized fecal microbial community of female migratory Tibetan antelope in the late pregnancy and postpartum periods using 16S rRNA gene sequencing and quantified fecal glucocorticoids (GCs) and triiodothyronine (T3) metabolite concentrations through enzyme immunoassays to identify the associations between maternal gut microbiota and physiological changes related with reproduction. We found that the fecal microbiota of Tibetan antelope was dominated by Firmicutes and Bacteroidetes. The microbial composition was significantly altered during the transition from late pregnancy to the postpartum period. Fecal T3 concentration was significantly higher in the postpartum period compared to late pregnancy, whereas GC metabolite concentration did not significantly differ between two reproductive states. We identified six genera (Anaerofustis, Bacteroides, Coprococcus_2, Ruminiclostridium_5, Ruminococcaceae_UCG-007, and Tyzzerella) that were significantly associated with reproductive states. We also found two genera (Christensenellaceae_R-7_group and Rikenellaceae_RC9_gut_group) significantly associated with GC metabolite concentration and two genera (Agathobacter and Papillibacter) significantly associated with T3 metabolite concentration, though these correlations were weak with coefficient values ranging from - 0.007 to 0.03. Our results indicate that many members of the gut microbiota are associated with the physiological changes in the transition from late pregnancy to the postpartum period, likely reflecting the metabolic and immune system dynamics during the periparturition period. This study highlights the importance of integrating microbiota, hormones and migration pattern to study the reproductive health of wildlife. By establishing a baseline of the physiological changes during the migration/periparturition period, we can have a better understanding of the impacts of increasing human activities on the Tibetan Plateau on the reproductive health of Tibetan antelope.

Dispersal limitation promotes the diversification of the mammalian gut microbiota.

The gut bacterial communities of mammals have profound effects on host fitness, but the processes that generate and maintain gut bacterial diversity remain poorly understood. We mapped compositional variation (i.e., ß-diversity) in the gut microbiotas of 136 pairs of wild mammalian species living throughout the Americas to assess how the distribution of mammals across geographic space influences the diversification of their gut bacteria. Comparing the gut microbiotas of sympatric and allopatric mammalian populations provided insights into the flow of gut bacteria within and between mammalian communities, revealing that spatial limits on bacterial dispersal promote ß-diversity between the gut microbiotas of mammalian species. Each geographic locale displayed a unique gut-microbiota composition that could not be fully explained by the diets and phylogenetic histories of the resident mammalian hosts, indicating that some gut bacteria are geographically restricted. Across the western hemisphere, the compositional overlap between the gut microbiotas of allopatric mammalian populations decayed exponentially with the geographic distance separating the hosts. The relationship between geographic distances among hosts and compositional differences among their gut microbiotas was independent of dietary and phylogenetic divergence among hosts. Within mammalian communities, we observed widespread sharing of gut bacteria between predator-prey host-species pairs, indicating horizontal transfer of gut bacteria through mammalian food chains. Collectively, these results indicate that compositional differences between the gut microbiotas of mammalian taxa are generated and maintained by limits to bacterial dispersal imposed by physical distance between hosts.

Whole-genome analysis of extraintestinal pathogenic Escherichia coli (ExPEC) MDR ST73 and ST127 isolated from endangered southern resident killer whales (Orcinus orca).

BACKGROUND: Limited studies have investigated the microbial diversity of wild marine mammals. OBJECTIVES: This study characterized Escherichia coli isolates collected from fresh faecal samples of endangered southern resident killer whales (Orcinus orca) located by detection dogs. METHODS: WGS of each strain was done to determine ST (using MLST), clonotype (C:H), antimicrobial resistance and virulence profile. Conjugation experiments were done to determine the mobility of the tet(B) tetracycline resistance gene. RESULTS: All isolates belonged to extraintestinal pathogenic E. coli (ExPEC) clonal lineages ST73 (8/9) and ST127 (1/9), often associated with human community-acquired urinary tract disease. Clonotyping using fumC and fimH alleles showed divergence in clonal lineages, with ST73 isolates belonging to the C24:H10 clade and the ST127 isolate belonging to C14:H2. The eight ST73 isolates carried multiple acquired antibiotic resistance genes, including aadA1, sul1 and tet(B), encoding aminoglycoside, sulphonamide and tetracycline resistance, respectively. Conjugative transfer of the resistance gene tet(B) was observed for three of the eight isolates. ST127 did not carry any of these acquired resistance genes. Virulence-associated genes identified included those encoding adhesins (iha, papC, sfaS), toxins (sat, vat, pic, hlyA, cnf1), siderophores (iutA, fyuA, iroN, ireA), serum survival/protectins (iss, ompT), capsule (kpsM) and pathogenicity island marker (malX). CONCLUSIONS: Orca whales can carry antibiotic-resistant potentially pathogenic strains of E. coli. Possible sources include contamination of the whale's environment and/or food. It is unknown whether these isolates cause disease in southern resident killer whales, which could contribute to the ongoing decline of this critically endangered population.

Radiocarbon dating of seized ivory confirms rapid decline in African elephant populations and provides insight into illegal trade.

Carbon-14 measurements on 231 elephant ivory specimens from 14 large ivory seizures (≥0.5 ton) made between 2002 and 2014 show that most ivory (ca 90%) was derived from animals that had died less than 3 y before ivory was confiscated. This indicates that the assumption of recent elephant death for mortality estimates of African elephants is correct: Very little "old" ivory is included in large ivory shipments from Africa. We found only one specimen of the 231 analyzed to have a lag time longer than 6 y. Patterns of trade differ by regions: East African ivory, based on genetic assignments of geographic origin, has a much higher fraction of "rapid" transit than ivory originating in the Tridom region of Cameroon-Gabon-Congo. Carbon-14 is an important tool in understanding patterns of movement of illegal wildlife products.

Comprehensive endocrine response to acute stress in the bottlenose dolphin from serum, blubber, and feces.

Several hormones are potential indicators of stress in free-ranging animals and provide information on animal health in managed-care settings. In response to stress, glucocorticoids (GC, e.g. cortisol) first appear in circulation but are later incorporated into other tissues (e.g. adipose) or excreted in feces or urine. These alternative matrices can be sampled remotely, or by less invasive means, than required for blood collection and are especially valuable in highly mobile species, like marine mammals. We characterized the timing and magnitude of several hormones in response to a stressor in bottlenose dolphins (Tursiops truncatus) and the subsequent incorporation of cortisol into blubber, and its metabolites excreted in feces. We evaluated the endocrine response to an acute stressor in bottlenose dolphins under managed care. We used a standardized stress protocol where dolphins voluntarily beached onto a padded platform and remained out of water for two hours; during the stress test blood samples were collected every 15 min and blubber biopsies were collected every hour (0, 60, and 120 min). Each subject was studied over five days: voluntary blood samples were collected on each of two days prior to the stress test; 1 and 2 h after the conclusion of the out-of-water stress test; and on the following two days after the stress test. Fecal samples were collected daily, each afternoon. The acute stressor resulted in increases in circulating ACTH, cortisol, and aldosterone during the stress test, and each returned to baseline levels within 2 h of the dolphin's return to water. Both cortisol and aldosterone concentrations were correlated with ACTH, suggesting both corticosteroids are at least partly regulated by ACTH. Thyroid hormone concentrations were generally unaffected by the acute stressor. Blubber cortisol increased during the stress test, and fecal GC excretion was elevated on the day of the stress test. We found that GCs in bottlenose dolphins can recover within hours of acute stress, and that cortisol release can be detected in alternate matrices within a few hours-within 2 h in blubber, and 3.5-5 h in fecal samples.

Modulation in Persistent Organic Pollutant Concentration and Profile by Prey Availability and Reproductive Status in Southern Resident Killer Whale Scat Samples.

Persistent organic pollutants (POPs), specifically PCBs, PBDEs, and DDTs, in the marine environment are well documented, however accumulation and mobilization patterns at the top of the food-web are poorly understood. This study broadens the understanding of POPs in the endangered Southern Resident killer whale population by addressing modulation by prey availability and reproductive status, along with endocrine disrupting effects. A total of 140 killer whale scat samples collected from 54 unique whales across a 4 year sampling period (2010-2013) were analyzed for concentrations of POPs. Toxicant measures were linked to pod, age, and birth order in genotyped individuals, prey abundance using open-source test fishery data, and pregnancy status based on hormone indices from the same sample. Toxicant concentrations were highest and had the greatest potential for toxicity when prey abundance was the lowest. In addition, these toxicants were likely from endogenous lipid stores. Bioaccumulation of POPs increased with age, with the exception of presumed nulliparous females. The exceptional pattern may be explained by females experiencing unobserved neonatal loss. Transfer of POPs through mobilization of endogenous lipid stores during lactation was highest for first-borns with diminished transfer to subsequent calves. Contrary to expectation, POP concentrations did not demonstrate an associated disruption of thyroid hormone, although this association may have been masked by impacts of prey abundance on thyroid hormone concentrations. The noninvasive method for measuring POP concentrations in killer whales through scat employed in this study may improve toxicant monitoring in the marine environment and promote conservation efforts.

Natural Variation in Stress Hormones, Comparisons Across Matrices, and Impacts Resulting from Induced Stress in the Bottlenose Dolphin.

Knowledge regarding stress hormones and how they vary in response to seasonality, gender, age, and reproductive status for any marine mammal is limited. Furthermore, stress hormones may be measured in more than one matrix (e.g., feces, blood, blubber), but the relationships between levels of a given hormone across these matrices are unknown, further complicating the interpretations of hormones measured in samples collected from wild animals. A study is underway to address these issues in a population of bottlenose dolphins trained for voluntary participation in sample collections from different matrices and across season and time of day.

Persistent Organic Pollutant Determination in Killer Whale Scat Samples: Optimization of a Gas Chromatography/Mass Spectrometry Method and Application to Field Samples.

Biologic sample collection in wild cetacean populations is challenging. Most information on toxicant levels is obtained from blubber biopsy samples; however, sample collection is invasive and strictly regulated under permit, thus limiting sample numbers. Methods are needed to monitor toxicant levels that increase temporal and repeat sampling of individuals for population health and recovery models. The objective of this study was to optimize measuring trace levels (parts per billion) of persistent organic pollutants (POPs), namely polychlorinated-biphenyls (PCBs), polybrominated-diphenyl-ethers (PBDEs), dichlorodiphenyltrichloroethanes (DDTs), and hexachlorocyclobenzene, in killer whale scat (fecal) samples. Archival scat samples, initially collected, lyophilized, and extracted with 70 % ethanol for hormone analyses, were used to analyze POP concentrations. The residual pellet was extracted and analyzed using gas chromatography coupled with mass spectrometry. Method detection limits ranged from 11 to 125 ng/g dry weight. The described method is suitable for p,p'-DDE, PCBs-138, 153, 180, and 187, and PBDEs-47 and 100; other POPs were below the limit of detection. We applied this method to 126 scat samples collected from Southern Resident killer whales. Scat samples from 22 adult whales also had known POP concentrations in blubber and demonstrated significant correlations (p < 0.01) between matrices across target analytes. Overall, the scat toxicant measures matched previously reported patterns from blubber samples of decreased levels in reproductive-age females and a decreased p,p'-DDE/∑PCB ratio in J-pod. Measuring toxicants in scat samples provides an unprecedented opportunity to noninvasively evaluate contaminant levels in wild cetacean populations; these data have the prospect to provide meaningful information for vital management decisions.

New evidence for hybrid zones of forest and savanna elephants in Central and West Africa.

The African elephant consists of forest and savanna subspecies. Both subspecies are highly endangered due to severe poaching and habitat loss, and knowledge of their population structure is vital to their conservation. Previous studies have demonstrated marked genetic and morphological differences between forest and savanna elephants, and despite extensive sampling, genetic evidence of hybridization between them has been restricted largely to a few hybrids in the Garamba region of northeastern Democratic Republic of Congo (DRC). Here, we present new genetic data on hybridization from previously unsampled areas of Africa. Novel statistical methods applied to these data identify 46 hybrid samples--many more than have been previously identified--only two of which are from the Garamba region. The remaining 44 are from three other geographically distinct locations: a major hybrid zone along the border of the DRC and Uganda, a second potential hybrid zone in Central African Republic and a smaller fraction of hybrids in the Pendjari-Arli complex of West Africa. Most of the hybrids show evidence of interbreeding over more than one generation, demonstrating that hybrids are fertile. Mitochondrial and Y chromosome data demonstrate that the hybridization is bidirectional, involving males and females from both subspecies. We hypothesize that the hybrid zones may have been facilitated by poaching and habitat modification. The localized geography and rarity of hybrid zones, their possible facilitation from human pressures, and the high divergence and genetic distinctness of forest and savanna elephants throughout their ranges, are consistent with calls for separate species classification.

Quantifying past and present connectivity illuminates a rapidly changing landscape for the African elephant.

There is widespread concern about impacts of land-use change on connectivity among animal and plant populations, but those impacts are difficult to quantify. Moreover, lack of knowledge regarding ecosystems before fragmentation may obscure appropriate conservation targets. We use occurrence and population genetic data to contrast connectivity for a long-lived mega-herbivore over historical and contemporary time frames. We test whether (i) historical gene flow is predicted by persistent landscape features rather than human settlement, (ii) contemporary connectivity is most affected by human settlement and (iii) recent gene flow estimates show the effects of both factors. We used 16 microsatellite loci to estimate historical and recent gene flow among African elephant (Loxodonta africana) populations in seven protected areas in Tanzania, East Africa. We used historical gene flow (FST and G'ST ) to test and optimize models of historical landscape resistance to movement. We inferred contemporary landscape resistance from elephant resource selection, assessed via walking surveys across ~15 400 km(2) of protected and unprotected lands. We used assignment-based recent gene flow estimates to optimize and test the contemporary resistance model, and to test a combined historical and contemporary model. We detected striking changes in connectivity. Historical connectivity among elephant populations was strongly influenced by slope but not human settlement, whereas contemporary connectivity was influenced most by human settlement. Recent gene flow was strongly influenced by slope but was also correlated with contemporary resistance. Inferences across multiple timescales can better inform conservation efforts on large and complex landscapes, while mitigating the fundamental problem of shifting baselines in conservation.

Social influences on the estrous cycle of the captive sun bear (Helarctos Malayanus).

We examined the potential influences of existing social housing arrangements on captive sun bear female reproductive cycling. Three social conditions were studied: 1.2, 1.1, and 0.2. Fecal hormone metabolites of total estrogens, progestins and glucocorticoids were compared between the three social conditions and were analyzed along with vaginal cytology data in individuals that experienced a change in social condition. Behavioral data were collected on females in each of the social conditions and summarized into agonistic, affiliative and sexual categories. Results indicated that sun bears are spontaneous ovulators, but that the presence of a male does influence hormone metabolite concentrations and cytological profiles. Male presence was also associated with a greater proportion of females cycling. In most female pairs, only one female cycled, typically the younger, subordinate female. The presence of a second female appeared to have a suppressive influence on both cycling and mating behavior. Agonistic behavior and associated stress may be a mechanism for lowering progesterone. In contrast, high estrogen levels were associated with low levels of agonistic interactions; thus, reproductive cycle monitoring could facilitate social introductions with either sex. Females in 1.2 social groupings had significantly higher GC metabolite concentrations and agonistic behavior, suggesting that 1.2 social groupings may not be advisable for captive breeding programs. Data from the North American historical captive population indicate that at most 32% of all sun bear pairs and only 18.5% of females have successfully reproduced. Implications of these social and reproductive patterns for captive management are discussed.

Evidence that ship noise increases stress in right whales.

Baleen whales (Mysticeti) communicate using low-frequency acoustic signals. These long-wavelength sounds can be detected over hundreds of kilometres, potentially allowing contact over large distances. Low-frequency noise from large ships (20-200 Hz) overlaps acoustic signals used by baleen whales, and increased levels of underwater noise have been documented in areas with high shipping traffic. Reported responses of whales to increased noise include: habitat displacement, behavioural changes and alterations in the intensity, frequency and intervals of calls. However, it has been unclear whether exposure to noise results in physiological responses that may lead to significant consequences for individuals or populations. Here, we show that reduced ship traffic in the Bay of Fundy, Canada, following the events of 11 September 2001, resulted in a 6 dB decrease in underwater noise with a significant reduction below 150 Hz. This noise reduction was associated with decreased baseline levels of stress-related faecal hormone metabolites (glucocorticoids) in North Atlantic right whales (Eubalaena glacialis). This is the first evidence that exposure to low-frequency ship noise may be associated with chronic stress in whales, and has implications for all baleen whales in heavy ship traffic areas, and for recovery of this endangered right whale population.

Elephant genotypes reveal the size and connectivity of transnational ivory traffickers.

Transnational ivory traffickers continue to smuggle large shipments of elephant ivory out of Africa, yet prosecutions and convictions remain few. We identify trafficking networks on the basis of genetic matching of tusks from the same individual or close relatives in separate shipments. Analyses are drawn from 4,320 savannah (Loxodonta africana) and forest (L. cyclotis) elephant tusks, sampled from 49 large ivory seizures totalling 111 t, shipped out of Africa between 2002 and 2019. Network analyses reveal a repeating pattern wherein tusks from the same individual or close relatives are found in separate seizures that were containerized in, and transited through, common African ports. Results suggest that individual traffickers are exporting dozens of shipments, with considerable connectivity between traffickers operating in different ports. These tools provide a framework to combine evidence from multiple investigations, strengthen prosecutions and support indictment and prosecution of transnational ivory traffickers for the totality of their crimes.

Effectiveness of scat-detection dogs in determining species presence in a tropical savanna landscape.

Most protected areas are too small to sustain populations of wide-ranging mammals; thus, identification and conservation of high-quality habitat for those animals outside parks is often a high priority, particularly for regions where extensive land conversion is occurring. This is the case in the vicinity of Emas National Park, a small protected area in the Brazilian Cerrado. Over the last 40 years the native vegetation surrounding the park has been converted to agriculture, but the region still supports virtually all of the animals native to the area. We determined the effectiveness of scat-detection dogs in detecting presence of five species of mammals threatened with extinction by habitat loss: maned wolf (Chrysocyon brachyurus), puma (Puma concolor), jaguar (Panthera onca), giant anteater (Myrmecophaga tridactyla), and giant armadillo (Priodontes maximus). The probability of scat detection varied among the five species and among survey quadrats of different size, but was consistent across team, season, and year. The probability of occurrence, determined from the presence of scat, in a randomly selected site within the study area ranged from 0.14 for jaguars, which occur primarily in the forested areas of the park, to 0.91 for maned wolves, the most widely distributed species in our study area. Most occurrences of giant armadillos in the park were in open grasslands, but in the agricultural matrix they tended to occur in riparian woodlands. At least one target species occurred in every survey quadrat, and giant armadillos, jaguars, and maned wolves were more likely to be present in quadrats located inside than outside the park. The effort required for detection of scats was highest for the two felids. We were able to detect the presence for each of five wide-ranging species inside and outside the park and to assign occurrence probabilities to specific survey sites. Thus, scat dogs provide an effective survey tool for rare species even when accurate detection likelihoods are required. We believe the way we used scat-detection dogs to determine the presence of species can be applied to the detection of other mammalian species in other ecosystems.

Inferred Paternity and Male Reproductive Success in a Killer Whale (Orcinus orca) Population.

We used data from 78 individuals at 26 microsatellite loci to infer parental and sibling relationships within a community of fish-eating ("resident") eastern North Pacific killer whales (Orcinus orca). Paternity analysis involving 15 mother/calf pairs and 8 potential fathers and whole-pedigree analysis of the entire sample produced consistent results. The variance in male reproductive success was greater than expected by chance and similar to that of other aquatic mammals. Although the number of confirmed paternities was small, reproductive success appeared to increase with male age and size. We found no evidence that males from outside this small population sired any of the sampled individuals. In contrast to previous results in a different population, many offspring were the result of matings within the same "pod" (long-term social group). Despite this pattern of breeding within social groups, we found no evidence of offspring produced by matings between close relatives, and the average internal relatedness of individuals was significantly less than expected if mating were random. The population's estimated effective size was <30 or about 1/3 of the current census size. Patterns of allele frequency variation were consistent with a population bottleneck.

Eliminating the artificial effect of sample mass on avian fecal hormone metabolite concentration.

Avian endocrinology is a productive field that could benefit from increased application of non-invasive techniques. Although assay protocols vary, most studies that measure hormone metabolites in avian feces struggle with an artificial effect of sample mass on steroid metabolite concentration. Hormone metabolite concentrations measured in small samples are consistently higher than concentrations in larger samples, and this appears to be due to multiple methodological problems. We systematically tested several causal hypotheses for the mass effect. Based on results from these tests, we modified and validated our assay protocol to effectively eliminate the mass effect. Future studies should implement the following procedures when measuring hormone metabolites from small fecal samples (particularly of birds and reptiles): (1) remove urates from the fecal sample as completely as possible; (2) lyophilize the sample prior to extraction; (3) maximize accuracy of small mass measurements; (4) increase the volume of ethanol in the extraction to 15 ml per 0.05-0.1g of dried feces; and (5) eliminate ethanol from all samples prior to radioimmunoassay by drying down extract solutions and rehydrating in buffer. By applying these precautions we successfully eliminated the mass effect from fecal samples ranging in mass from 0.001 to 0.1 g using a radioimmunoassay commonly employed for studies of fecal glucocorticoid metabolites. These corrections also resulted in a more than 3-fold increase in effect size in glucocorticoid concentrations from a controlled test of the effects of 1h motorcycle exposure on northern spotted owls. These methods have important implications not only for avian studies, but for any study measuring hormone metabolites from small fecal samples.

Non-invasive measurement of thyroid hormone in feces of a diverse array of avian and mammalian species.

We developed and validated a non-invasive thyroid hormone measure in feces of a diverse array of birds and mammals. An I(131) radiolabel ingestion study in domestic dogs coupled with High Pressure Liquid Chromatography (HPLC) analysis, showed that peak excretion in feces occurred at 24-48h post-ingestion, with I(131)-labelled thyroid hormone metabolites excreted primarily as triiodothyronine (T3) and relatively little thyroxine (T4), at all excretion times examined. The immunoreactive T3 profile across these same HPLC fractions closely corresponded with the I(131) radioactive profile. By contrast, the T4 immunoreactive profile was disproportionately high, suggesting that T4 excretion included a high percentage of T4 stores. We optimized and validated T3 and T4 extraction and assay methods in feces of wild northern spotted owls, African elephants, howler monkeys, caribou, moose, wolf, maned wolf, killer whales and Steller sea lions. We explained 99% of the variance in high and low T3 concentrations derived from species-specific sample pools, after controlling for species and the various extraction methods tested. Fecal T3 reflected nutritional deficits in two male and three female howler monkeys held in captivity for translocation from a highly degraded habitat. Results suggest that thyroid hormone can be accurately and reliably measured in feces, providing important indices for environmental physiology across a diverse array of birds and mammals.