Sequencing,annotation and comparative analysis of nine BACs of giant panda (<Emphasis Type="Italic">Ailuropoda melanoleuca</Emphasis>)

A 10-fold BAC library for giant panda was constructed and nine BACs were selected to generate finish sequences. These BACs could be used as a validation resource for the de novo assembly accuracy of the whole genome shotgun sequencing reads of giant panda newly generated by the Illumina GA sequencing technology. Complete sanger sequencing, assembly, annotation and comparative analysis were carried out on the selected BACs of a joint length 878 kb. Homologue search and de novo prediction methods were used to annotate genes and repeats. Twelve protein coding genes were predicted, seven of which could be functionally annotated. The seven genes have an average gene size of about 41 kb, an average coding size of about 1.2 kb and an average exon number of 6 per gene. Besides, seven tRNA genes were found. About 27 percent of the BAC sequence is composed of repeats. A phylogenetic tree was constructed using neighbor-join algorithm across five species, including giant panda, human, dog, cat and mouse, which reconfirms dog as the most related species to giant panda. Our results provide detailed sequence and structure information for new genes and repeats of giant panda, which will be helpful for further studies on the giant panda.     

同域分布的珍稀野生动物对放牧的行为响应策略

保护区内放牧活动对野生动物保护存在负面影响,明确不同物种对放牧干扰的行为响应对制定更有针对性的保护管理政策具有重要意义。使用红外相机研究卧龙自然保护区放牧活动对多种珍稀野生动物的影响,分析放牧激励政策实施前后大熊猫(Ailuropoda melanoleuca)及其同域分布的小熊猫(Ailurus fulgens)、川金丝猴(Rhinopithecus roxellana)、水鹿(Rusa unicolor) 4种珍稀野生动物的照片数、空间分布以及活动模式的变化,探讨这4种动物对放牧的行为响应策略。结果表明:(1)一期(2012—2013),2012年10月实施了禁马政策,同年12月实施放牧(牛羊)激励政策)家畜照片数量很少,4种野生动物照片数相对较多。二期(2014—2015)家畜的照片数显著增加(P0.01),小熊猫(P0.05)与川金丝猴(P0.01)的照片数均显著减少,大熊猫、水鹿的照片数也呈减少趋势;到三期(2016—2017),大熊猫、小熊猫及水鹿3种关注野生动物的照片数基本回升到激励政策实施前的水平,无川金丝猴照片记录。(2)一期,4种野生动物在研究区域有较广的分布;二期,大熊猫、小熊猫的空间分布范围均缩小,无川金丝猴空间分布信息,而家畜、水鹿的空间分布范围有所增加;到三期,大熊猫、小熊猫的空间分布基本恢复到放牧激励政策实施前的区域,无川金丝猴的空间分布信息。(3)放牧激励政策实施前后,大熊猫、小熊猫及川金丝猴活动模式无明显变化,但水鹿的活动更加集中于傍晚,以避开人类与家畜的活动高峰。同域分布的不同的野生动物对人类活动(如放牧)的行为响应策略不同,各保护区在制定相关保护政策时应综合考虑人类干扰对多个物种的影响,增加决策的科学性与合理性。     

大熊猫及其近缘种rDNA序列变异和系统进化关系

应用ｒＤＮＡ间隔区Ｓｏｕｔｈｅｒｎ转换技术研究大熊猫及其近缘种的分子系统关系。通过比较大熊猫、小熊猫、黑熊、马来熊、浣熊和猞猁的ｒＤＮＡ间隔区限制性内切酶图谱,用最大似然法和简约法构建它们的分子系统树。结果表明大熊猫与熊具有较近的亲缘关系,与小熊猫和浣熊的亲缘关系较远。     

Sox and Zfx genes in giant panda

By using PCR cloning techniques, the DNA sequences of the HMG box regions of sixSox genes (pSox) and the zinc finger domains of twoZfz genes (pZfx) in the giant panda were identified. The giant pandaSox genes fell into two subfamilies,SOX-S1 andSOX-S2. ThepSox andpZfx genes of the giant panda were highly homologous to the corresponding genes in mammals and revealed close substitution rates to those in the primates. Project supported by the Fok Ying Tung Education Foundation, the National Natural Science Foundation of China (Grant No. 39770392) and Wuhan Chenguang Plan.     

Impact of livestock grazing on biodiversity and giant panda habitat

Livestock grazing occurs in many protected areas for wildlife and has become a threat to wildlife worldwide. Livestock grazing within protected areas causes negative effects to rare wildlife (e.g., giant panda [Ailuropoda melanoleuca]) and their habitat. We used the 2,000-km² Wolong National Nature Reserve, Sichuan Province, southwestern China, to document the effects of livestock on the giant panda and its habitat. We monitored arrow bamboo (Bashania fangiana), wildlife sign (i.e., feces and tracks), and characteristics of plant communities in intact habitat (IH; limited livestock grazing) and disturbed habitat (DH; with grazing disturbance) to assess the effects of livestock grazing and the responses of giant pandas and sympatric species across spatial and temporal scales. Bamboo coverage and the height and basal diameter of bamboo in IH were greater than those in DH, whereas the number of herbaceous species and herbaceous coverage in IH were lower than those in DH. Wildlife signs in IH were greater than those in DH; specifically, giant panda and red panda (Ailurus fulgens) signs were greater, whereas signs of sambar (Rusa unicolor) and tufted deer (Elaphodus cephalophus) in IH were similar to those in DH. Livestock grazing reduced bamboo, which may threaten the long-term survival of the giant panda. Our results have implications for understanding and management of livestock grazing in the Wolong National Nature Reserve and elsewhere. © 2019 The Wildlife Society.     

Identifying core habitats and corridors for giant pandas by combining multiscale random forest and connectivity analysis

Habitat loss and fragmentation are widely acknowledged as the main driver of the decline of giant panda populations. The Chinese government has made great efforts to protect this charming species and has made remarkable achievements, such as population growth and habitat expansion. However, habitat fragmentation has not been reversed. Protecting giant pandas in a large spatial extent needs to identify core habitat patches and corridors connecting them. This study used an equal‐sampling multiscale random forest habitat model to predict a habitat suitability map for the giant panda. Then, we applied the resistant kernel method and factorial least‐cost path analysis to identify core habitats connected by panda dispersal and corridors among panda occurrences, respectively. Finally, we evaluated the effectiveness of current protected areas in representing core habitats and corridors. Our results showed high scale dependence of giant panda habitat selection. Giant pandas strongly respond to bamboo percentage and elevation at a relatively fine scale (1 km), whereas they respond to anthropogenic factors at a coarse scale (≥2 km). Dispersal ability has significant effects on core habitats extent and population fragmentation evaluation. Under medium and high dispersal ability scenarios (12,000 and 20,000 cost units), most giant panda habitats in the Qionglai mountain are predicted to be well connected by dispersal. The proportion of core habitats covered by protected areas varied between 38% and 43% under different dispersal ability scenarios, highlighting significant gaps in the protected area network. Similarly, only 43% of corridors that connect giant panda occurrences were protected. Our results can provide crucial information for conservation managers to develop wise strategies to safeguard the long‐term viability of the giant panda population.     

大熊猫肠道放线菌的种群组成及多样性分析

【目的】探究不同年龄、不同性别大熊猫肠道放线菌的多样性及群落结构,为寻找潜在产生活性化合物的放线菌资源提供理论依据。【方法】采用PCR-DGGE技术对大熊猫肠道放线菌进行分析,对电泳结果进行UPGMA聚类分析、主成分分析、生物多样性等多重比较。【结果】变性梯度凝胶电泳(DGGE)图谱显示,不同大熊猫肠道中放线菌的多样性及群落结构存在明显差异。随着年龄的增长,雌性大熊猫肠道中放线菌的多样性指数(H')和丰富度(S)逐渐减少,而雄性大熊猫肠道内放线菌的多样性指数(H')和丰富度(S)逐渐增多。不同个体的大熊猫肠道放线菌的群落结构存在明显差异,但相同性别之间的相似性很高。DGGE条带回收测序结果表明,获得的28条序列归属于10个放线菌属,其中链霉菌属(Streptomyces)为优势菌属,占总数的46%;北里孢菌属(Kitasatospora)、红球菌属(Rhodococcus)、棒杆菌属(Corynebacterium)、迪茨氏菌属(Dietziaceae)、大理石雕菌属(Marmoricola)、布登堡菌属(Beutenbergia)、微杆菌属(Microbacterium)、链嗜酸菌属(Streptacidiphilus)和芽生球菌属(Blastococcus)等为非链霉菌属,占总数的54%。【结论】大熊猫肠道内蕴藏着丰富的放线菌资源,其肠道菌群的结构与组成受年龄和性别的影响。     

Clonal regeneration of an arrow bamboo, <Emphasis Type="Italic">Fargesia qinlingensis</Emphasis>, following giant panda herbivory

Characteristics of giant panda herbivory sites and clonal regeneration of an arrow bamboo Fargesia qinlingensis following giant panda grazing were studied in the Qinling Mountains of China. Three types of plots were located in a pandas’ summer habitat in 2002: herbivory (naturally grazed by giant pandas), clipped (simulated panda herbivory), and control. Average area of herbivory sites was 2.92 m² and average distance from herbivory sites to the closest tree (dbh > 10 cm) was 1.0 m. Pandas avoided thin bamboo culms with basal diameters <5 mm. Average height of stumps of culms grazed by panda was 0.67 m and average density of grazed culms was 9.0 culms m⁻². Annual culm mortality rate was significantly greater in herbivory and clipped plots than in control plots while annual recruitment rate was not significantly different among the three plot types in 2003. Neither recruitment rate nor mortality rate were significantly different among the three plot types in 2004. Annual recruitment rate was significantly greater than annual mortality rate only in control plots in both 2003 and 2004, suggesting static ramet dynamics in disturbed plots (herbivory and clipped). Density of new shoots was not significantly different, but basal diameter of new shoots was significantly less in herbivory plots compared to control plots in 2002. Differences of annual mortality rate and growth of new shoots found between control plots and herbivory plots suggest that clonal regeneration of F. qinlingensis culms was negatively affected by giant panda grazing. Therefore, no evidence of a clonal integration compensatory response to panda herbivory was found in F. qinlingensis.     

Major histocompatibility complex class II variation in the giant panda (Ailuropoda melanoleuca)

Habitat destruction and human activity have greatly impacted the natural history of the giant panda (Ailuropoda melanoleuca). Although the genetic diversity of neutral markers has been examined in this endangered species, no previous work has examined adaptive molecular polymorphisms in the giant panda. Here, the major histocompatibility complex (MHC) class II DRB locus was investigated in the giant panda, using single-strand conformation polymorphism (SSCP) and sequence analysis. Comparisons of DNA samples extracted from faecal and blood samples from the same individual revealed that the two materials yielded similar quantities and qualities of DNA, as well as identical SSCP patterns and allelic sequences, demonstrating the reliability of DNA isolation from panda faeces. Analysis of faecal samples from 60 giant pandas revealed relatively low number of alleles: seven alleles. However, the alleles were quite divergent, varying from each other by a range of 7-47 nucleotide substitutions (4-25 amino acid substitutions). Construction of a neighbour-joining tree and comparisons among DRB alleles from other species revealed that both similar and highly divergent alleles survived in the bottlenecked panda populations. Despite species-specific primers used and excellent faecal DNA isolated, a lower level of heterozygosity than expected was still observed due to inbreeding. There were three types of evidence supporting the presence of balancing selection in the giant panda: (i) an obvious excess of nonsynonymous substitutions over synonymous at the antigen-binding positions; (ii) trans-species evolution of two alleles between the giant panda and other felids; and (iii) a more even distribution of alleles than expected from neutrality.     

Sox and Zfx genes in giant panda

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Phylogenetic relationships within caniform carnivores based on analyses of the mitochondrial 12S rRNA gene

The complete 12S rRNA gene of 32 carnivore species, including four feliforms and 28 caniforms, was sequenced. The sequences were aligned on the basis of their secondary structures and used in phylogenetic analyses that addressed several evolutionary relationships within the Caniformia. The analyses showed an unresolved polytomy of the basic caniform clades; pinnipeds, mustelids, procyonids, skunks,Ailurus (lesser panda), ursids, and canids. The polytomy indicates a major diversification of caniforms during a relatively short period of time. The lesser panda was distinct from other caniforms, suggesting its inclusion in a monotypic family, Ailuridae. The giant panda and the bears were joined on the same branch. The skunks are traditionally included in the family Mustelidae. The present analysis, however, showed a less close molecular relationship between the skunks and the remaining Mustelidae (sensu stricto) than between Mustelidae (sensu stricto) and Procyonidae, making Mustelidae (sensu lato) paraphyletic. The results suggest that the skunks should be included in a separate family, Mephitidae. Within the Pinnipedia, the grouping of walrus, sea lions, and fur seals was strongly supported. Analyses of a combined set of 12S rRNA and cytochromeb data were generally consistent with the findings based on each gene.     

Habitat Assessment for Giant Pandas in the Qinling Mountain Region of China

ABSTRACT Because habitat loss and fragmentation threaten giant pandas (Ailuropoda melanoleuca), habitat protection and restoration are important conservation measures for this endangered species. However, distribution and value of potential habitat to giant pandas on a regional scale are not fully known. Therefore, we identified and ranked giant panda habitat in Foping Nature Reserve, Guanyinshan Nature Reserve, and adjacent areas in the Qinling Mountains of China. We used Mahalanobis distance and 11 digital habitat layers to develop a multivariate habitat signature associated with 247 surveyed giant panda locations, which we then applied to the study region. We identified approximately 128 km²of giant panda habitat in Foping Nature Reserve (43.6% of the reserve) and 49 km²in Guanyinshan Nature Reserve (33.6% of the reserve). We defined core habitat areas by incorporating a minimum patch-size criterion (5.5 km²) based on home-range size. Percentage of core habitat area was higher in Foping Nature Reserve (41.8% of the reserve) than Guanyinshan Nature Reserve (26.3% of the reserve). Within the larger analysis region, Foping Nature Reserve contained 32.7% of all core habitat areas we identified, indicating regional importance of the reserve. We observed a negative relationship between distribution of core areas and presence of roads and small villages. Protection of giant panda habitat at lower elevations and improvement of habitat linkages among core habitat areas are important in a regional approach to giant panda conservation.     

Characterizing the spatial distribution of giant pandas (Ailuropoda melanoleuca) in fragmented forest landscapes

Aim To examine the effects of forest fragmentation on the distribution of the entire wild giant panda (Ailuropoda melanoleuca) population, and to propose a modelling approach for monitoring the spatial distribution and habitat of pandas at the landscape scale using Moderate Resolution Imaging Spectro‐radiometer (MODIS) enhanced vegetation index (EVI) time‐series data. Location Five mountain ranges in south‐western China (Qinling, Minshan, Qionglai, Xiangling and Liangshan). Methods Giant panda pseudo‐absence data were generated from data on panda occurrences obtained from the third national giant panda survey. To quantify the fragmentation of forests, 26 fragmentation metrics were derived from 16‐day composite MODIS 250‐m EVI multi‐temporal data and eight of these metrics were selected following factor analysis. The differences between panda presence and panda absence were examined by applying significance testing. A forward stepwise logistic regression was then applied to explore the relationship between panda distribution and forest fragmentation. Results Forest patch size, edge density and patch aggregation were found to have significant roles in determining the distribution of pandas. Patches of dense forest occupied by giant pandas were significantly larger, closer together and more contiguous than patches where giant pandas were not recorded. Forest fragmentation is least in the Qinling Mountains, while the Xiangling and Liangshan regions have most fragmentation. Using the selected landscape metrics, the logistic regression model predicted the distribution of giant pandas with an overall accuracy of 72.5% (κ = 0.45). However, when a knowledge‐based control for elevation and slope was applied to the regression, the overall accuracy of the model improved to 77.6% (κ = 0.55). Main conclusions Giant pandas appear sensitive to patch size and isolation effects associated with fragmentation of dense forest, implying that the design of effective conservation areas for wild giant pandas must include large and dense forest patches that are adjacent to other similar patches. The approach developed here is applicable for analysing the spatial distribution of the giant panda from multi‐temporal MODIS 250‐m EVI data and landscape metrics at the landscape scale.     

Assessing vulnerability of giant pandas to climate change in the Qinling Mountains of China

Climate change might pose an additional threat to the already vulnerable giant panda (Ailuropoda melanoleuca). Effective conservation efforts require projections of vulnerability of the giant panda in facing climate change and proactive strategies to reduce emerging climate‐related threats. We used the maximum entropy model to assess the vulnerability of giant panda to climate change in the Qinling Mountains of China. The results of modeling included the following findings: (1) the area of suitable habitat for giant pandas was projected to decrease by 281 km² from climate change by the 2050s; (2) the mean elevation of suitable habitat of giant panda was predicted to shift 30 m higher due to climate change over this period; (3) the network of nature reserves protect 61.73% of current suitable habitat for the species, and 59.23% of future suitable habitat; (4) current suitable habitat mainly located in Chenggu, Taibai, and Yangxian counties (with a total area of 987 km²) was predicted to be vulnerable. Assessing the vulnerability of giant panda provided adaptive strategies for conservation programs and national park construction. We proposed adaptation strategies to ameliorate the predicted impacts of climate change on giant panda, including establishing and adjusting reserves, establishing habitat corridors, improving adaptive capacity to climate change, and strengthening monitoring of giant panda.     

Endophytic fungi and silica content of different bamboo species in giant panda diet

The giant panda (Ailuropoda melanoleuca), one of the most threatened mammalian species in the world, has adapted to herbivorous diet consisting mainly of bamboo (Poaceae: Bambusoidea). The most acute threats to the survival of the giant panda are habitat loss and fragmentation. However, changes in habitat may influence also the quality of giant panda diet through the bamboo species composition as well as their symbiotic leaf endophytes and plant chemical properties. Here we explore species composition and frequency of endophytic fungi and silica content in different bamboo species in the range of giant panda habitat in relation to panda food preference. Silica content of the bamboos varied from 3.7 g/kg to 45.7 g/kg and did not correlate with panda preference and altitudinal gradient. Systemic and vertically in seeds transmitted fungal endophytes or bacterial endophytes were not detected in bamboo leaves. Nearly half of the identified endophytic fungi belonged to genus Arthrinium. Pandas preferred bamboo species naturally occurring in higher altitudes. Furthermore, the total amount of endophytes tended to be lower in samples collected from bamboos in higher altitudes. This draws attention to the importance of more detailed studies on the endophytic fungi-bamboo-panda trophic interactions and the effect of land use and climate change on conservation programs of giant panda.     

Genome-wide survey and analysis of microsatellites in giant panda (Ailuropoda melanoleuca), with a focus on the applications of a novel microsatellite marker system

Background

The giant panda (Ailuropoda melanoleuca) is a critically endangered species endemic to China. Microsatellites have been preferred as the most popular molecular markers and proven effective in estimating population size, paternity test, genetic diversity for the critically endangered species. The availability of the giant panda complete genome sequences provided the opportunity to carry out genome-wide scans for all types of microsatellites markers, which now opens the way for the analysis and development of microsatellites in giant panda.

Results

By screening the whole genome sequence of giant panda in silico mining, we identified microsatellites in the genome of giant panda and analyzed their frequency and distribution in different genomic regions. Based on our search criteria, a repertoire of 855,058 SSRs was detected, with mono-nucleotides being the most abundant. SSRs were found in all genomic regions and were more abundant in non-coding regions than coding regions. A total of 160 primer pairs were designed to screen for polymorphic microsatellites using the selected tetranucleotide microsatellite sequences. The 51 novel polymorphic tetranucleotide microsatellite loci were discovered based on genotyping blood DNA from 22 captive giant pandas in this study. Finally, a total of 15 markers, which showed good polymorphism, stability, and repetition in faecal samples, were used to establish the novel microsatellite marker system for giant panda. Meanwhile, a genotyping database for Chengdu captive giant pandas (n = 57) were set up using this standardized system. What’s more, a universal individual identification method was established and the genetic diversity were analysed in this study as the applications of this marker system.

Conclusion

The microsatellite abundance and diversity were characterized in giant panda genomes. A total of 154,677 tetranucleotide microsatellites were identified and 15 of them were discovered as the polymorphic and stable loci. The individual identification method and the genetic diversity analysis method in this study provided adequate material for the future study of giant panda.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1268-z) contains supplementary material, which is available to authorized users.     

Phylogenetic study of Baylisascaris schroederi isolated from Qinling subspecies of giant panda in China based on combined nuclear 5.8S and the second internal transcribed spacer (ITS-2) ribosomal DNA sequences

The nuclear ribosomal DNA (rDNA) region spanning 5.8S rDNA and the second internal transcribed spacer (ITS-2) of Baylisascaris schroederi isolated from the Qinling subspecies of giant panda in Shaanxi Province, China were amplified and sequenced. Sequence variations in the two rDNA regions within B. schroederi and among species in the family Ascarididae were examined. The lengths of B. schroederi 5.8S and ITS-2 rDNA sequences were 156 bp and 327 bp, respectively, and no nucleotide variation was found in these two rDNA regions among the 20 B. schroederi samples examined, and these ITS-2 sequences were identical to that of B. schroederi isolated from giant panda in Sichuan province, China. The inter-species differences in 5.8S and ITS-2 rDNA sequences among members of the family Ascarididae were 0-1.3% and 0-17.7%, respectively. Phylogenetic relationships among species in the Ascarididae were re-constructed by Bayesian inference (Bayes), maximum parsimony (MP), and maximum likelihood (ML) analyses, based on combined sequences of 5.8S and ITS-2 rDNA. All B. schroederi samples clustered together and sistered to B. transfuga with high posterior probabilities/bootstrap values, which further confirmed that nematodes isolated from the Qinling subspecies of giant panda in Shaanxi Province, China represent B. schroederi. Because of the large number of ambiguously aligned sequence positions (difficulty of inferring homology by positions), ITS-2 sequence alone is likely unsuitable for phylogenetic analyses at the family level, but the combined 5.8S and ITS-2 rDNA sequences provide alternative genetic markers for the identification of B. schroederi and for phylogenetic analysis of parasites in the family Ascarididae.     

cDNA cloning and expression of ghrelin in giant panda (<Emphasis Type="Italic">Ailuropoda melanoleuca</Emphasis>)

Ghrelin is an endogenous ligand for the growth hormone secretagogue receptor. It plays an important role in stimulating growth hormone secretion, food intake, body weight gain and gastric motility. cDNA sequences coding for ghrelin precursor protein (prepro-ghrelin) were isolated from the stomach of a giant panda. Two different mRNA sequences of ghrelin were obtained. The long open reading frame of ghrelin (354 bp) encodes a precursor protein of 117 amino acids with a 23 amino acid signal peptide. The short one (351 bp) encodes a precursor protein of 116 amino acids with the same 23 amino acid signal peptide. The presumed giant panda mature ghrelin proteins also had two forms. Comparative analysis showed that the first and the fourth amino acids (Gly and Phe) were completely conserved and the third amino acid (Ser) was also highly conserved in the mature ghrelin. RT-PCR analysis of giant panda ghrelin mRNA in various tissues revealed high level of expression in stomach, relative lower levels of expression in small intestine, liver and kidney, and no expression in thymus, spleen and heart.     

Nucleotide Sequences of an Important Functional Gene hnRNPA2/B1 from Ailuropoda melanoleuca and Ursus thibetanus mupinensis and Its Potential Value in Phylogenetic Study

The cDNA fragments of hnRNPA2/B1 were cloned from the giant panda and black bear using RT-PCR method, which were, respectively, 1029bp and 1026bp in length encoding 343 and 341 amino acids. Analysis indicated the cDNA cloned from the giant panda encoded variant B1 while the cDNA cloned from black bear encoded variant A2.

Analyzing the hnRNPA2B1 peptide of the giant panda and black bear, 76 glycine residues and 86 glycine residues were, respectively, found, and moreover, most glycine are concentrated in the latter halves of the hnRNPA2B1 peptides. Functional sites prediction also showed many N-myristoylation sites existed in the glycine-rich domain, which is probably related to the role of telomere maintenance.

From base bias and substitution analysis, we can conclude that the ORF of hnRNPA2/B1 biased G while hated C, and transition of the third site did not achieve the level of saturation.

Orthology analysis indicated that both the nucleotide sequence and the deduced amino acid sequence showed high identity to other 26 hnRNPA2/B1 sequences from mammals and nonmammals reported. These sequences were used to construct phylogenetic trees employing the NJ method with 1000 bootstrap, and the obtained tree demonstrated similar topology with the classical systematics, which suggested the potential value of hnRNPA2/B1 in phylogenetic analysis.

This report will be the first step to the study function of hnRNPA2/B1 in the giant panda and black bear, and will provide a scientific basis to disease surveillance, captive breeding, and conservation of the endangered species.