Conservation genomics reveals possible illegal trade routes and admixture across pangolin lineages in Southeast Asia

The use of genome-wide genetic markers is an emerging approach for informing evidence-based management decisions for highly threatened species. Pangolins are the most heavily trafficked mammals across illegal wildlife trade globally, but critically endangered Sunda pangolins (Manis javanica) have not been widely studied in insular Southeast Asia. We used?>?12,000 single nucleotide polymorphic markers (SNPs) to assign pangolin seizures from illegal trade of unknown origin to possible geographic sources via genetic clustering with pangolins of known origin. Our SNPs reveal three previously unrecognized genetic lineages of Sunda pangolins, possibly from Borneo, Java and Singapore/Sumatra. The seizure assignments suggest the majority of pangolins were traded from Borneo to Java. Using mitochondrial markers did not provide the same resolution of pangolin lineages, and to explore if admixture might explain these differences, we applied sophisticated tests of introgression using?>?2000 SNPs to investigate secondary gene flow between each of the three Sunda pangolin lineages. It is possible the admixture which we discovered is due to human-mediated movements of pangolins. Our findings impact a range of conservation actions, including tracing patterns of trade, repatriation of rescue animals, and conservation breeding. In order to conserve genetic diversity, we suggest that, pending further research, each pangolin lineage should as a precaution be protected and managed as an evolutionarily distinct conservation unit.     

Monitoring the gestation period of rescued Formosan pangolin (Manis pentadactyla pentadactyla) with progesterone radioimmunoassay

Eight species of pangolin have been identified in the world. However, understanding of pangolin reproductive biology has been limited to fragmentary records. In this study, the concentration of serum progesterone in three pregnant and two nonpregnant rescued female Formosan pangolins (Manis pentadactyla pentadactyla) was monitored using a commercial progesterone radioimmunoassay kit. During gestation, the serum progesterone of pregnant pangolins A, B, and C remained at 28.5–55 ng/ml (n = 31 samples), 10.9–50.1 ng/ml (n = 34), and 12.4 and 33.5 ng/ml with a peak at 47.6 ng/ml (n = 19), respectively, whereas the serum progesterone of nonpregnant pangolins D and E remained at 1.99 ± 1.62 ng/ml (n = 80) and 2.27 ± 1.64 ng/ml (n = 27), respectively. From this study, it was found that female pangolin weighing as low as 2.14 kg was already capable of reproduction. For pregnant pangolins to give birth to viable offspring, their body weight must increase significantly, 63.89 and 134.0% in the study, from the time of inception or early pregnancy until parturition. In addition, study has found that both viable offspring were born fully developed and exceeded 80 g in weight. The period of gestation was found to be as short as 318 or longer than 372 days. Therefore, the Formosan pangolin should only be able to reproduce once a year. This study is the first insight into hormone assay for determining the gestation period of pangolin. Further investigations on the same subject are necessary to establish criteria for the recognition of reproductive status in pangolins. Zoo Biol 31:479–489, 2012. © 2011 Wiley Periodicals, Inc.     

Comparative study of gut microbiota from captive and confiscated-rescued wild pangolins

Pangolins are among the most critically endangered animals due to widespread poaching and worldwide trafficking. Captive breeding is considered to be one way to protect them and increase the sizes of their populations. However, comparative studies of captive and wild pangolins in the context of gut microbiota are rare. Here, the gut microbiome of captive and confiscated-rescued wild pangolins is compared, and the effects of different periods of captivity and captivity with and without antibiotic treatment are considered. We show that different diets and periods of captivity, as well as the application of antibiotic therapy, can alter gut community composition and abundance in pangolins. Compared to wild pangolins, captive pangolins have an increased capacity for chitin and cellulose/hemicellulose degradation, fatty acid metabolism, and short-chain fatty acid synthesis, but a reduced ability to metabolize exogenous substances. In addition to increasing the ability of the gut microbiota to metabolize nutrients in captivity, captive breeding imposes some risks for survival by resulting in a greater abundance of antibiotic resistance genes and virulence factors in captive pangolins than in wild pangolins. Our study is important for the development of guidelines for pangolin conservation, including health assessment, disease prevention, and rehabilitation of wild pangolin populations.     

EVIDENCE FOR WIDESPREAD COURTSHIP DURING COPULATION IN 131 SPECIES OF INSECTS AND SPIDERS,AND IMPLICATIONS FOR CRYPTIC FEMALE CHOICE

Male courtship behavior is generally thought to function prior to copulation, as an inducement to the female to allow the male to copulate with her; this study indicates however, that male courtship during and following copulation (“copulatory courtship”) is common in insects and spiders (81% of 131 species in 102 genera and 49 families, mostly Coleoptera, Hemiptera, Diptera, and Araneioidea). Copulatory courtship is apparently evolutionarily labile, as expected if it is under sexual selection; intrageneric variation occurred in all 17 genera in which more than one species was observed. In 81% of 94 species with copulatory courtship, the male abandoned the female soon after copulation ended; thus, copulatory courtship appears not to function generally to induce acceptance of further copulatory attempts. The most likely explanation for copulatory courtship is that it represents attempts by males to influence cryptic female choice. This suggests that an aspect of sexual selection by female choice not considered by Darwin may be more important than previously appreciated and that the common practice in evolutionary studies of measuring male reproductive success by counting numbers of copulations may sometimes be misleading because of cryptic female choice during and after copulation.     

Female sexual behavior in the absence of male-male competition in captive Japanese macaques (Macaca fuscata)

A captive group of Japanese macaques (Macaca fuscata). consisting of one adult male and three adult females, was observed for 88 hr during the 1988 mating season. We focused on the group's sexual activities because understanding how social dynamics affect reproductive behavior may enhance our ability to establish self-sustaining captive populations. The adult male exhibited distinct preferences in copulation partners, although all females were receptive and cycled during the study period. The dominant female participated in the most copulations and successfully harassed and disrupted copulations between the male and the other females. The alpha female, therefore, actively constrained the formation of mating pairs. Further, the male did not consort with his year-round female grooming partner. This female, the least dominant member of the group, engaged in the most autosexual and homosexual behavior. Birth season data show that only the dominant female bore offspring. This study emphasizes the influence of social dynamics on a group's reproductive potential and suggests an alternative means by which females can influence consort formation.     

The emergence of a commercial trade in pangolins from Gabon

Recent seizures of illegally held wildlife indicate a mounting global trade in pangolins involving all eight species. Seizures of illegally traded African pangolins are increasing as wild populations of Asian species decline. We investigated trade in pangolins and law enforcement efforts in Gabon; a country likely to have intact wild populations of three of the four species of African pangolin. We compared village sales and trade chains between 2002‐2003 and 2014. Hunters reported pangolins to be the most frequently requested species in 2014, and the value of pangolins had increased at every point along their trade chain. In Libreville, giant pangolin prices increased 211% and arboreal pangolin prices 73% whilst inflation rose only 4.6% over the same period. We documented a low rate of interception of illegally traded pangolins despite increased law enforcement. Surveys of potential export routes detected exports across forest borders, in conjunction with ivory, but not through public transport routes. We conclude that whilst there is clear potential and likelihood that a wild pangolin export trade is emerging from Gabon, traditional bushmeat trade chains may not be the primary supply route. We recommend adjusting conservation policies and actions to impede further development of illegal trade within and from Gabon .     

History and dietary husbandry of pangolins in captivity

The objective of this study was to establish a history of feeding and dietary husbandry of pangolin in captivity. Over the past 150 years, several zoos have attempted to maintain pangolins (Manis spp). Most of these zoos have not succeeded in maintaining these animals for long periods, associated largely with dietary problems. This study reviews the historic records of captive pangolins. The dietary husbandry of pangolins in Taipei Zoo is discussed in detail. Zoo Biol 0:1–8, 2007. © 2007 Wiley‐Liss, Inc.     

Courtship and copulation in Tarsius bancanus

The reproductive behavior of 6 paired, captive Bornean tarsiers was studied over an 8-month period. Seven copulations were observed. Females signalled males by visual displays and olfactory cues from vulval rubbing. Males signalled females with courtship calls heard before matings. After a courtship lasting from 1 to 2 h, copulation occurred with the male thrusting 61-190 times for 60-90 s, ending in ejaculation. The female regulated timing of mating by rejection or avoidance of the male. Multiple matings were not observed, and mating occurred once or twice a night during each night of estrus. This copulatory pattern of infrequent matings of short duration and active female solicitation and regulation of copulating timing suggests a harem or monogamous system.     

Leg impairment magnifies reproductive costs in male Mediterranean fruit flies,Ceratitis capitata

Injuries frequently accumulate with age in nature. Despite the commonality of injury and the resulting impairment, there are limited experimental data for the effects of impairment on life history trade‐offs between reproduction and survival in insects. We tested the effects of artificial injury and the resulting impairment on the reproductive costs and behavior of male medflies, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). Treatment flies were impaired by amputating tarsomere segments 2–5 from the right foreleg at either eclosion or age 22 days. The effect of impairment and age on the cost of reproduction was tested by varying the timing of female availability among the treatments. Courtship behavior and copulation rates were observed hourly from age 2–5 days to determine the effects of impairment on reproductive behavior. Female access combined with the impairment reduced the life expectancy of males more than the impairment alone, whereas the health effect of amputation was influenced by age. Conversely, the risk of death due to impairment was not influenced by the males' mating status prior to amputation. The males' copulation success was reduced due to impairment, whereas courtship behavior was not affected. Impairment does not reduce the males' impulse to mate, but decreases the females' receptivity to copulation, while also increasing the cost of each successful mating. Overall, minor impairment lowers the reproductive success of males and reduces longevity.     

Isolation and characterization of microsatellite markers in pangolins (Mammalia,Pholidota, Manis spp.)

Thirty‐four polymorphic dinucleotide microsatellite loci were developed in the Malayan pangolin Manis javanica. Of the 34 markers, 32 and 18 were also amplified, respectively, in the Chinese pangolin (Manis pentadactyla) and the African tree pangolin (Manis tricuspis). Analysis of 24 Malayan, 12 Chinese and 2 African tree pangolins showed high levels of variability (heterozygosity ranging from 0.321 to 0.708). These are the first available microsatellite markers in Pholidota and will be an invaluable tool for evolutionary and conservation genetic studies in pangolins.     

Captive reproduction in Heermann's kangaroo rat,Dipodomys heermanni

This paper summarizes 8 years of captive breeding experience with the endangered Morro Bay kangaroo rat (Dipodomys heermanni morroensis) and the non-endangered Lompoc kangaroo rat (D. h. arenae), a related form used as a surrogate. Kangaroo rats are aggressive and must be caged individually; copulation can only take place when the female is in estrus. The estrous cyle varies, but is usually 14–17 days long; gestation averages 31 days. Data on development of the young, animal health, mortality, and longevity are presented. The productivity of this project is compared with that of another long-term kangaroo rat breeding project in terms of the number of young produced per female per year. A captive breeding program can be initiated with 40–50 reproductive animals as a founder group.     

Breeding behavior in a flock of Chilean flamingos (Phoenicopterus chilensis) at Dublin zoo

The breeding behavior of a flock of Chilean flamingos was observed over a 3‐month period during the peak of breeding activity. This species’ habitat is becoming increasingly vulnerable, and concern has been raised over a decline in wild stocks numbers in the past two decades. Breeding programs in zoos have been established, but with limited success because little is known about the precise criteria and conditions required for breeding these animals. This study was undertaken to gain insight into the breeding behavior of captive Chilean flamingos. A catalogue of mating rituals, nest acquisition and defense, and egg incubation behavior was obtained. The results suggest that new additions to the colony may trigger breeding in the subsequent year and that rainfall has little effect on captive reproductive success. Individually identifiable flamingos were followed throughout the breeding season and detailed quantitative data on their nesting, copulation, and social interactions were obtained. Zoo Biol 19:227–237, 2000. © 2000 Wiley‐Liss, Inc.     

Copulatory Courtship in Drosophila: Behavior and Songs of D. birchii and D. serrata

D. birchii and D. serrata, two endemic Australian Drosophila species, have a copulatory courtship. The males of these species begin to court the female after mounting her and often go on with the courtship after the copulation is over. In the present paper we have described behavioral interactions between the male and the female and analyzed acoustic signals produced by the flies during courtship. Species differences were more pronounced in female than in male behavior. Variation within the species was obvious in the relative proportions of time the flies spent in different behaviors. Even though courtship took place nearly solely during copulation, some remains of precopulatory courtship were observed in both species. It is suggested that copulatory courtship exhibited by D. birchii and D. serrata flies is a derived rather than a primitive character.     

Captive breeding of pangolins: current status,problems and future prospects

Pangolins are unique placental mammals with eight species existing in the world, which have adapted to a highly specialized diet of ants and termites, and are of significance in the control of forest termite disaster. Besides their ecological value, pangolins are extremely important economic animals with the value as medicine and food. At present, illegal hunting and habitat destruction have drastically decreased the wild population of pangolins, pushing them to the edge of extinction. Captive breeding is an important way to protect these species, but because of pangolin’s specialized behaviors and high dependence on natural ecosystem, there still exist many technical barriers to successful captive breeding programs. In this paper, based on the literatures and our practical experience, we reviewed the status and existing problems in captive breeding of pangolins, including four aspects, the naturalistic habitat, dietary husbandry, reproduction and disease control. Some recommendations are presented for effective captive breeding and protection of pangolins.     

Encounter with heavier females changes courtship and fighting efforts of male field crickets Gryllus bimaculatus (Orthoptera: Gryllidae)

The effects of mating experience on male mating behavior are mediated by four factors: mating cost, such as resource depletion, perception of mating opportunities, self-perception of attractiveness, and female quality. For example, encountering females might increase male expectations of prospective mating opportunities, while copulation increases self-perception of attractiveness in males. To determine the relative importance of these factors, the effect of mating on the two components of reproductive effort (courtship and fighting effort) in Gryllus bimaculatus was examined. Calling activity before and after encountering females was measured, and copulation success was recorded. Subsequently, the intensity and outcome of male–male fighting behavior was recorded. Female encounter increased calling activity irrespective of copulation, thereby indicating that the perception of mating opportunities is important factor for the males. Changes in courtship effort of males were larger and fighting success was lower when they were previously paired with relatively heavier females. These results indicate that male reproductive effort is also affected by quality of previous mating partners.

     

Function of courtship feeding in the Bull-headed Shrike,Lanius bucephalus

We investigated the frequency of courtship feeding (CF) in the Bull-headed Shrike (Lanius bucephalus) during the 1981–1982 breeding seasons. Because no CF was observed just prior to, during or just after copulation, CF is not associated directly with copulation and does not function as a releaser for copulation. The absence of CF during the pair-formation period of the early breeder and the high frequency of CF behavior in the cold season and in the critical breeding stages (egg-laying and incubation stages) for females strongly suggests that the significance of CF in this species is not symbolic but substantial at least in early breeders. We assign one of the causes of this CF to the early onset of breeding.     

Black rhinoceros (Diceros bicornis) in U.S. zoos: II. behavior,breeding success,and mortality in relation to housing facilities

The captive population of black rhinoceros (Diceros bicornis) is not self‐sustaining. The reasons for suboptimal reproduction and high mortality need to be investigated. This can only be achieved by cross‐institutional analyses of environments, behavior, and performance. In this study, we collected data on 23 zoos with black rhinoceros to compare zoo environments with reproductive success, mortality, and behavior. Institutional variation was characterized by enclosure area, percentage of walls around enclosure perimeter, percentage of public access along enclosure perimeter, climate, noise level, number of years zoo has maintained black rhinoceros, frequency of chlorine use, and number of male and female black rhinoceros at a zoo simultaneously. Birth and death rates for each institution were calculated from studbook records. We found that the breeding success of a zoo since 1973 correlated positively with enclosure area, and zoos with two or more females had a lower reproductive rate than zoos with only one female. Females residing during their pre‐reproductive years at a zoo with another reproductive female gave birth for the first time on average 3 years later than sole females. Mortality since 1973 correlated positively with percentage of public access. In Part I, we developed behavior profiles of 29.31 individual black rhinoceros from keeper ratings. Scores for males on the behavior trait Fear also correlated positively to percentage of public access, and we suggest that this aspect of black rhinoceros exhibits is a stressor for this species, especially the males. We found that different aspects of captive environments are associated with male and female black rhinoceros behavior. Male scores on the behavior trait dominant were higher in smaller enclosures, and female scores for a group of behaviors suggesting agitation (chasing/stereotypy/mouthing) were positively correlated with percentage of walls in their enclosure. These two behavior traits were found in Part I to be negatively correlated with the breeding success of an individual male or female. We re‐surveyed the behavior and husbandry of 29 black rhinoceros pairs in zoos 2 years after the original data were collected. The re‐survey confirmed that compatible black rhinoceros pairs are those with assertive females and submissive males, and that enclosure area and a low percentage of concrete walls around the enclosure are positive predictors of a pair's reproductive success. We conclude that temperament traits of individuals and characteristics of their captive environments both have an impact on a pair's breeding success. Our study demonstrates that cross‐institutional comparisons of zoo facilities, when integrated with behavioral assessments of individual animals, are a valuable tool for investigating potential causes of poor reproduction and well‐being in zoo animals. Zoo Biol 18:35–52, 1999. © 1999 Wiley‐Liss, Inc.     

True deception during extra‐pair courtship feeding: cheating whiskered tern Chlidonias hybrida females perform better

Males of many bird species feed their mates during the pre‐incubation period. The food provisioned by males during these courtship feedings (CFs) represents the key source of energy for the female during egg formation. Non‐pair males may trade food for extra pair copulations (EPC) with females during extra pair courtship feeding (EPCF), while females may trade copulations for food with non‐pair males to obtain additional resources. Because EPCs can be costly to the females, they are expected to behave in ways that will deceive non‐pair males to obtain additional resources at no cost to themselves. We investigated EPCFs in whiskered terns Chlidonias hybrida breeding in food‐rich conditions, on carp ponds in southern Poland. Almost all CFs (n = 2751) took place during the female's fertile period and peaked just before clutch initiation. 10% of all CFs were performed by non‐pair males. Females tried to obtain food from the non‐pair male during 39% of EPCFs, by swindling (the female solicits a non‐social male for copulation and tries to swindle food with no cloacal contact) or by snatching (the female tries to take the gift without engaging in copulation). In the remaining 61% of EPCFs, females did not react or chased the visiting male away. The probability of a female's obtaining food during EPCF was much higher (0.69, 95% CI: 0.47–0.85) if she swindled rather than snatched (0.08, 95% CI: 0.02–0.22). Only 0.7% of EPCFs were followed by EPCs. The high availability of food in the study area allows males to perform frequent EPCFs, despite the very low probability of obtaining EPCs. This is the first time that ‘true deception’ during EPCFs has been reported in birds: swindling females obtain food from non‐pair males at no immediate detectable cost to themselves.     

Phylogeography of the heavily poached African common pangolin (Pholidota,Manis tricuspis) reveals six cryptic lineages as traceable signatures of Pleistocene diversification

Knowledge on faunal diversification in African rainforests remains scarce. We used phylogeography to assess (i) the role of Pleistocene climatic oscillations in the diversification of the African common pangolin (Manis tricuspis) and (ii) the utility of our multilocus approach for taxonomic delineation and trade tracing of this heavily poached species. We sequenced 101 individuals for two mitochondrial DNA (mtDNA), two nuclear DNA and one Y‐borne gene fragments (totalizing 2602 bp). We used a time‐calibrated, Bayesian inference phylogenetic framework and conducted character‐based, genetic and phylogenetic delineation of species hypotheses within African common pangolins. We identified six geographic lineages partitioned into western Africa, Ghana, the Dahomey Gap, western central Africa, Gabon and central Africa, all diverging during the Middle to Late Pleistocene. MtDNA (cytochrome b + control region) was the sole locus to provide diagnostic characters for each of the six lineages. Tree‐based Bayesian delimitation methods using single‐ and multilocus approaches gave high support for ‘species’ level recognition of the six African common pangolin lineages. Although the diversification of African common pangolins occurred during Pleistocene cyclical glaciations, causative correlation with traditional rainforest refugia and riverine barriers in Africa was not straightforward. We conclude on the existence of six cryptic lineages within African common pangolins, which might be of major relevance for future conservation strategies. The high discriminative power of the mtDNA markers used in this study should allow an efficient molecular tracing of the regional origin of African common pangolin seizures.     

Activity patterns and reproductive behavior of the Critically Endangered Bermuda skink (Plestiodon longirostris)

The study of rare or cryptic species in zoos can provide insights into natural history and behavior that would be difficult to obtain in the field. Such information can then be used to refine population assessment protocols and conservation management. The Bermuda skink (Plestiodon longirostris) is an endemic Critically Endangered lizard. Chester Zoo's successful conservation breeding program is working to safeguard, increase and reinforce skink populations in the wild. A key aim of this program is to develop our understanding of the behavior of this species. In this study, using 24 h video recordings, we examined the daily activity patterns, basking behavior and food preferences of four pairs of Bermuda skinks. The skinks displayed a bimodal pattern of activity and basking, which may have evolved to avoid the strength of the midday sun in exposed habitats in Bermuda. Captive Bermuda skinks appear to prefer a fruit-based diet to orthopteran prey. We also documented their reproductive behavior and compared it against two closely related species. Although there were many similarities between the courtship and mating behaviors of the three species, there was a significantly shorter period of cloacal contact in the Bermuda skink. Oophagia was also documented for the first time in this species. This knowledge has enabled the evaluation of the current ex-situ management practices of this species, filled gaps in knowledge that would be challenging to obtain in the field, and enabled the enhancement of both animal husbandry and reproductive success for the conservation breeding program.