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Conservation genetics of Association of Zoos and Aquariums and wild Matshie's tree kangaroo (Dendrolagus matschiei) from Huon Peninsula, Papua New Guinea

ProQuest Dissertations and Theses, 2009
Dissertation
Author: Thomas Joseph Jr McGreevy
Abstract:
The Association of Zoos and Aquariums (AZA) Matschie's tree kangaroo (Dendrolagus matschiei ) population is at a critical point for assessing long-term viability; they have endured a founder effect and have low mitochondrial DNA (mtDNA) diversity. The mean kinship (MK) strategy is used to preserve their genetic diversity by minimizing the overall kinship of the population; however, the MK strategy has not been evaluated in an AZA population. Wild D. matschiei is listed as endangered and genetically unique populations may warrant management as separate conservation units. My objectives were to: (i) develop molecular markers to facilitate genetic analyses, (ii) assist AZA and wild D. matschiei management decisions, and (iii) further resolve Dendrolagus molecular systematics. To achieve my objectives I analyzed DNA extracted from AZA, captive held, and wild D. matschiei, and three additional Dendrolagus taxa using microsatellite markers and two mtDNA genes. AZA D. matschiei have similar nuclear DNA genetic diversity as captive and wild D. matschiei from Papua New Guinea, and the MK strategy has maintained their genetic diversity as well as predicted by theoretical expectations. AZA D. matschiei should continue to be managed by their studbook analyses and the MK strategy. Wild D. matschiei showed evidence of phylogeographic structure, but should be managed as one conservation unit. Preliminary genetic evidence suggests D. matschiei and Lowland tree kangaroo (Dendrolagus spadix ) are sister taxa, and supports the reclassification of Golden-mantled tree kangaroo (D. goodfellowi pulcherrimus ) and Ifola ( D. dorianus notatus ) as species. An improved understanding of Dendrolagus genetics will contribute substantially to their conservation.

TABLE OF CONTENTS ABSTRACT ii ACKNOWLEDGEMENTS ......iii PREFACE .vi TABLE OF CONTENTS ...vii LIST OF TABLES xiii LIST OF FIGURES xvi INTRODUCTION 1 Captive Matschie's Tree Kangaroo 1 2 Wild Matschie's Tree Kangaroo 2 3 Objectives 5 4 References 6 MANUSCRIPT I: Microsatellite Marker Development and Mendelian Analysis in the Matschie's Tree Kangaroo. 8 1.1 Abstract 8 1.2 Introduction 9 1.3 Methods 10 1.3.1 Microsatellite Marker Development for D. matschiei 10 1.3.2 Other Taxa Microsatellite Marker Screening 12 1.3.3 Microsatellite Analyses 12 1.3.4 Microsatellite Reliability 13 1.3.5 Concordance Between Blood and Fecal Samples 13 1.3.6 Microsatellite Analyses of Other Taxa 14 vii

1.4 Results 15 1.4.1 Microsatellite Reliability ...15 1.4.2 Concordance Between Blood and Fecal Samples 15 1.4.3 Microsatellite Analyses of Other Taxa 16 1.5 Discussion 16 1.6 References 21 MANUSCRIPT II: A Multiplex PCR Assay to Distinguish Among Three Sympatric Marsupial Taxa from Huon Peninsula, Papua New Guinea, using the Mitochondrial Control Region Gene .23 2.1 Abstract 23 2.2 Introduction .23 2.3 Methods 24 2.4 Results 26 2.5 Discussion. ..27 2.6 References 32 MANUSCRIPT III: Genetic Evaluation of the Association of Zoos and Aquariums Matschie's Tree Kangaroo (Dendrolagus matschiei) Captive Breeding Program 34 3.1 Abstract .., 34 3.2 Introduction 35 3.3 Methods 38 3.3.1 Sample Collection 39 3.3.2 DNA Extraction ..39 viii

3.3.3 Microsatellite Marker Analysis 39 3.3.4 Control Region Sequencing 40 3.3.5 D. matschiei Genetic Diversity 40 3.3.6 Change in AZA D. matschiei Genetic Diversity 41 3.3.7 Population Genetic Structure 43 3.3.8 Juvenile Mortality 43 3.4 Results 43 3.4.1 D. matschiei Genetic Diversity 43 3.4.2 Change in AZA D. matschiei Genetic Diversity 44 3.4.3 Population Genetic Structure 44 3.4.4 Juvenile Mortality .... 45 3.5 Discussion 45 3.5.1 D. matschiei Genetic Diversity 45 3.5.2 Change in AZA D. matschiei Genetic Diversity 46 3.5.3 Management Recommendations 47 3.6 Conclusion 48 3.7 Literature Cited 58 MANUSCRIPT IV: Phylogeography of Matschie's Tree Kangaroo (Dendrolagus matschiei) from Huon Peninsula, Papua New Guinea: Implications for Conservation and Management 62 4.1 Abstract 62 4.2 Introduction 63 4.3 Materials and Methods 66 IX

4.3.1 Field Sites and Sample Collection 66 4.3.2 DNA Extraction and Species Identification 68 4.3.3 Control Region Sequencing 68 4.3.4 Phylogeographic Analyses 69 4.3.5 Network Analyses 70 4.3.6 Wild D. matschiei Bottleneck and Effective Population Size 70 4.3.7 Conservation Units 70 4.4 Results 71 4.4.1 Species Identification and Haplotypes 71 4.4.2 Phylogenetic Analyses 71 4.4.3 Network Analysis 72 4.4.4 Wild D. matschiei Bottleneck and Effective Population Size 73 4.4.5 Conservation Units 73 4.5 Discussion 73 4.5.1 Species Identification and Haplotypes 73 4.5.2 Phylogeography 75 4.5.3 Distribution of AZA D. matschiei Haplotypes 76 4.5.4 Implications for Conservation and Management 77 4.6 Literature Cited 85 x

MANUSCRIPT V: Tree Kangaroo Molecular Systematics Based on Partial Cytochrome b Sequences: Are Matschie's Tree Kangaroo {Dendrolagus matschiei) and Goodfellow's Tree Kangaroo {Dendrolagus goodfellowi ssp.) Sister Taxa? 89 5.1 Abstract...... 89 5.2 Introduction 90 5.3 Methods 93 5.3.1 Sample Collection 93 5.3.2 DNA Extraction and Cytochrome b Sequencing 93 5.3.3 Phylogenetic Analyses 94 5.3.4 Evolutionary Units 96 5.3.5 Genetic Distances 96 5.4 Results 96 5.4.1 Cytochrome b Haplotypes 96 5.4.2 D. matschiei Sister Taxon 97 5.4.3 Evolutionary Units 98 5.4.4 Genetic Distances 98 5.5 Discussion 99 5.5.1 D. matschiei Sister Taxon 99 5.5.2 Evolutionary Units 100 5.5.3 Unknown Subspecies Identifications 102 5.5.4 Dendrolagus Molecular Systematics 102 5.6 Conclusion 103 XI

5.7 References I l l DICUSSION 1 Captive Matschie's Tree Kangaroo 114 2 Phylogeography.... 115 3 WildZ). matschieiManagement 116 4 D. matschiei Sister Taxon 117 5 D. matschiei Molecular Systematics. 118 6 Conclusion 118 7 References 120 BIBLIOGRAPHY .121 xn

LIST OF TABLES Table MANUSCRIPT I Page Table 1.1 Primer sequence and PCR conditions for five microsatellite markers developed for Matschie's tree kangaroo (Dendrolagus matschiei; MTK) and three microsatellite markers developed for other marsupial taxa 18 Table 1.2 Genotype and allele error rates (Hoffman and Amos 2005) for microsatellite marker analyses of DNA extracted from Matschie's tree kangaroo {Dendrolagus matschiei) fecal samples 19 Table 1.3 Genetic diversity for Goodfellow's tree kangaroo {Dendrolagus goodfellowi ssp.; n = 7), Doria's tree kangaroo {Dendrolagus dorianus ssp.; n = 6), and Grizzled tree kangaroo {Dendrolagus inustus ssp.; n = 4) based on five microsatellite markers developed in this study (GenBank accession numbers FJ937787-FJ937791) and three markers developed for other taxa: MeOOl, MeQl4 (Taylor and Cooper 1998; AF025909andAF025911),and/?/>as385 20 MANUSCRIPT II Table 2.1 Fecal samples collected by the Tree Kangaroo Conservation Program from Dendawang in 2000 (D00) to 2005 (D05), Sibidak in 2000 (S00) to 2005 (S05), and Yangorong in 2002 (Y02) to 2005 (Y05) 30 xiii

MANUSCRIPT III Table 3.1 Extant and deceased Association of Zoos and Aquariums (AZ A) Matschie's tree kangaroo {Dendrolagus matschiei) blood (B) and fecal (F) samples collected by AZ A institutions from 2001 to 2008 51 Table 3.2 Matschie's tree kangaroo {Dendrolagus matschiei) blood samples collected 16 August 1999 to 5 July 2007 by the Tree Kangaroo Conservation Program from Wasaunon, Rainforest Habitat (RFH), and Zenag (ZNG) in Papua New Guinea (PNG) 53 Table 3.3 Mean expected heterozygosity ± standard deviation values on the diagonal for Association of Zoos and Aquariums (AZA; n = 66), Rainforest Habitat (RFH; n = 16), Zenag (ZNG; n = 12), and Wasaunon (WAS; n = 22) Matschie's tree kangaroo {Dendrolagus matschiei). Below the diagonal are pairwise comparison FST values 55 Table 3.4 Average number of alleles ± standard deviation (SD) for Association of Zoos and Aquariums (AZA; n = 66), Rainforest Habitat (RFH; n = 16), Zenag (ZNG; n = 12), and Wasaunon (WAS; n = 22) Matschie's tree kangaroo {Dendrolagus matschiei) 56 MANUSCRIPT IV Table 4.1 Matschie's tree kangaroo {Dendrolagus matschiei) blood and fecal samples collected 16 August 1999 to 5 July 2007 by the Tree Kangaroo Conservation Program from Dendawang (D), Sibidak (S), Yangorong (Y), Wasaunon (W), and Rainforest Habitat (RFH) in Papua New Guinea (PNG) 79 xiv

MANUSCRIPT V Table 5.1 Matschie' s tree kangaroo {Dendrolagus matschiei), Timboyok (£>. goodfellowi buergersf), Goodfellow's tree kangaroo (D. goodfellowi ssp.), Doria's tree kangaroo (£>. dorianus ssp.), Ifola (D. d. notatus), Finch's tree kangaroo (D. inustus finschi), Grizzled tree kangaroo (D. inustus ssp.), and New Guinea pademelon (Thylogale browni) blood (B) and fecal (F) samples 105 Table 5.2 Population aggregation analysis (Davis and Nixon 1992) of cytochrome b sequence haplotypes (HAP) from tree kangaroos (Dendrolagus spp.) 109 Table 5.3 Cytochrome b mean genetic distance (d) percentage values calculated using Molecular Evolutionary Genetic Analysis version 4.1 110 xv

LIST OF FIGURES Figure Page MANUSCRIPT II Figure 2.1 One percent agarose gel image of the different sized PCR products that we produced by amplifying DNA extracted from fecal samples with our marsupial multiplex species-specific primers 29 MANUSCRIPT III Figure 3.1 Association of Zoos and Aquariums (AZA) Matschie's tree kangaroo {Dendrolagus matschiei) population size from 1970 to 2007 50 Figure 3.2 Estimated extant and deceased Association of Zoos and Aquariums Matschie's tree kangaroo {Dendrolagus matschiei; n = 66) population structure (A> 2) using STRUCTURE 2.2 ...57 MANUSCRIPT IV Figure 4.1 Geographic distribution of Matschie' s tree kangaroo {Dendrolagus matschiei) mitochondrial DNA control region sequence haplotypes (HAP) on the Huon Peninsula, Papua New Guinea 82 Figure 4.2 Neighbor-joining (NJ) phylogenetic analysis of Matschie's tree kangaroo {Dendrolagus matschiei) mitochondrial DNA control region sequences from Huon Peninsula, Papua New Guinea 83 Figure 4.3 TCS (Clement et al. 2000) network analysis of Matschie's tree kangaroo {Dendrolagus matschiei) mitochondrial DNA control region haplotypes (HAP) with a 90% connection limit 84 xvi

MANUSCRIPT V Figure 5.1 Neighbor-joining (NJ) phylogenetic analysis of cytochrome b (cyt6) sequence haplotypes (HAP) from tree kangaroos (Dendrolagus spp.) 108 XVH

INTRODUCTION 1. Captive Matschie's Tree Kangaroo The captive population of Matschie's tree kangaroo (Dendrolagus matschiei) housed in Association of Zoos and Aquariums (AZA) institutions is at a critical point for assessing long-term viability. This population was established from 19 genetically uncharacterized D. matschiei and has endured a founder effect because four individuals contributed the majority of the offspring [McGreevy et al., in press]. AZA D. matschiei have retained 90.0% of the genetic diversity from the founders [Blessington et al., 2008]. Captive populations that have lost 10% of their initial heterozygosity are susceptible to inbreeding depression and negative consequences, such as decreased juvenile survival and total fitness [Frankham et al., 2004]. AZA D. matschiei has a low reproductive rate of approximately one offspring per one and a half to two years [Dabek, 1994; Flannery et al., 1996], which reduces the population's ability to increase in size. The Tree Kangaroo Species Survival Plan® (TKSSP) is in the process of trying to import D. matschiei from international institutions to increase the size and genetic diversity of the AZA population. In 1992, the TKSSP was established to coordinate the captive management of AZA D. matschiei. The TKSSP and the majority of captive breeding programs use the mean kinship (MK) strategy to preserve genetic diversity [Ballou and Foose, 1996; Ralls and Ballou, 2004]. The objective of the MK strategy is to minimize a population's overall kinship: the probability that two alleles selected at random from a homologous locus from two different individuals or the same individual, will be identical by descent [Lacy, 1995]. Theoretical modeling, computer simulations, and 1

captive study of Drosophila melanogaster all indicate that the MK strategy preserves the highest amount of genetic diversity in a captive population [Ballou and Lacy, 1995; Montgomery et al., 1997]. The MK strategy has not been evaluated using molecular genetic techniques to determine how well it maintains the level of genetic diversity in an AZA population, however. McGreevy et al. [in press] found that AZA female-founder D. matschiei have low mitochondrial DNA (mtDNA) control region haplotype diversity compared to captive D. matschiei held in Papua New Guinea (PNG) and extant AZA D. matschiei are not representative of the high number of control region haplotypes that were found in wild D. matschiei from the Huon Peninsula of PNG. All mammals have two forms of cellular DNA, mtDNA from mitochondria and nuclear DNA (nDNA) from the nucleus. Mitochondrial DNA only partially represents the amount of genetic diversity in a mammal and is orders of magnitude smaller than the number of base pairs of nDNA. A nDNA analysis is required to more comprehensively estimate D. matschiei neutral genetic diversity and determine how much genetic diversity AZA D. matschiei has lost over one generation. 2. Wild Matschie's Tree Kangaroo D. matschiei is classified as endangered by the 2008 International Union for Conservation of Nature and Natural Resources (IUCN) Red List of Threatened Species [Leary et al., 2008] and the leading causes for their population decline have been hunting and loss of habitat [Flannery, 1995]. Wild D. matschiei reproductive rate and patterns are unknown, but if similar to those seen in captivity they could be highly sensitive to population disturbances in the wild. In 1996, the Tree Kangaroo 2

Conservation Program (TKCP) was created as a part of the AZA TKSSP to promote D. matschiei conservation in captivity and in the wild. The TKCP has worked with the local community to establish a Yupna-Urawa-Som (YUS) Conservation Area on the Huon Peninsula, PNG, to protect a portion of D. matschiei habitat from destruction and provide an area where they will not be hunted [National Geographic, 2009]. D. matschiei is endemic to the Huon Peninsula, PNG, and inhabits montane forest from 1,000 to 3,300 m on the Finisterre, Saruwaged, Cromwell, and Rawlinson Ranges [Ziegler, 1977; Betz, 2001]. D. matschiei is thought to inhabit a broader altitude range than a closely related tree kangaroo, Goodfellow's tree kangaroo {Dendrolagus goodfellowi), likely because D. matschiei is the only tree kangaroo species on the Huon Peninsula [Flannery, 1995]. Genetically unique populations of wild D. matschiei may exist at different altitudes or in isolated areas and warrant management as a separate conservation unit [Vogler and DeSalle, 1994] within the YUS Conservation Area. The sister taxa relationship of D. matschiei and D. goodfellowi ssp. has been suggested by past morphological, physiological, and behavioral evidence [Flannery et al., 1996], but this relationship has not been rigorously tested using molecular genetics. Rothschild and Dollman [1936] placed D. matschiei and D. goodfellowi into a group that only included these two species. Groves [1982] classified D. goodfellowi and Lowland tree kangaroo {Dendrolagus spadix) as subspecies of D. matschiei. Flannery et al. [1996] conducted the most extensive revision of Dendrolagus taxa, based primarily on morphological data, and classified the Goodfellow's complex as three subspecies: Goodfellow's tree kangaroo (D. g. goodfellowi), Timboyok (D. g. 3

buergersi), and Golden-mantled tree kangaroo (D. g. pulcherrimus). Flannery et al. [1996] recognized the close similarity between D. matschiei and D. goodfellowi ssp. and placed D. matschiei, D. goodfellowi ssp., and D. spadix as a monophyletic group that was unresolved. Bowyer et al. [2003] placed D. g. buergersi and D. spadix as sister taxa in a clade that included D. matschiei, but they acknowledged the need to analyze additional samples from each taxa. Dendrolagus is a unique arboreal macropodid marsupial only found on the island of New Guinea, two islands near shore of New Guinea, and northeastern Australia [Flannery, 1995]. Although they are the largest extant mammals indigenous to New Guinea, relatively little is known about New Guinea tree kangaroos compared to Australian tree kangaroos [Flannery et al., 1996; Martin, 2005]. The IUCN Red List (http://www.redlist.org) has listed the majority of New Guinea tree kangaroos as critically endangered or endangered, while the Australian tree kangaroos are considered to be near threatened and of least concern. Dendrolagus molecular systematics has not yet been fully resolved and was identified as one of the top three research priorities by a diverse international group of tree kangaroo experts at the 2005 "Ecology & Conservation of Tree-kangaroos: Current & Future Directions" workshop. Tree kangaroos have undergone an extensive radiation and diverged into at least ten species; scientists discovered two of these species in 1990 and 1995 [Flannery et al., 1996]. The 2008 IUCN Red List recently increased the number of New Guinea Dendrolagus species without explanation from eight to 12, even though an extensive molecular systematics analysis has not yet been conducted. 4

The phylogenetic species concept (PSC), a robust method for identifying evolutionary units, has numerous advantages over traditional species concepts, such as the biological species concept [Cracraft, 1989]. The PSC and a population aggregation analysis [Davis and Nixon, 1992] can be used to identify evolutionary units by finding shared characteristics that are inherited and unique to a group of organisms [Goldstein et al., 2000]. The PSC has yet to be applied to any Dendrolagus data set and could potentially help resolve tree kangaroo molecular systematics. 3. Objectives My two major objectives were to assist AZA and wild D. matschiei management decisions. My objectives in detail were to: i) develop molecular markers to estimate D. matschiei nDNA genetic diversity, ii) develop species-specific primers to facilitate the large-scale analysis of DNA extracted from wild collected fecal samples, iii) determine how well AZA D. matschiei reflect the amount of genetic diversity found in captive and wild D. matschiei from PNG, iv) evaluate the ability of the MK strategy to preserve AZA D. matschiei neutral genetic diversity, v) determine how wild D. matschiei should be managed in a proposed conservation area, and vi) identify the sister taxon to D. matschiei and further resolve Dendrolagus molecular systematics. 5

4. References Ballou JD, Foose TJ. 1996. Demographic and genetic management of captive populations. In: Kleiman DG, Allen M, Thompson K, Lumpkin S, Harris H. editors. Wild mammals in captivity. Chicago (IL): University of Chicago Press, p 263-283. Ballou JD, Lacy RC. 1995. Identifying genetically important individuals for management of genetic diversity in pedigreed populations. In: Ballou JD, Gilpin M, Foose TJ. editors. Population management for survival and recovery: analytical methods and strategies in small population conservation. New York (NY): Columbia University Press, p 76-111. Betz W. 2001. Matschie's tree kangaroo (Marsupialia: Macropodidae, Dendrolagus matschiei) in Papua New Guinea: estimates of population density and landowner accounts of food plants and natural history, [thesis]. Southampton: University of Southampton, England. Blessington J, Rodden M, Bier L. 2008. Population analysis and breeding plan: Matschie's tree kangaroo {Dendrolagus matschiei) Species Survival Plan®. Association of Zoos and Aquariums Population Management Center in Chicago, IL and Lincoln Park Zoo Lincoln, Nebraska. 19 pp. Bowyer JC, Newell GR, Metcalfe CJ, Eldridge MBD. 2003. Tree-kangaroos Dendrolagus in Australia: are D. lumholtzi and D. bennettianus sister taxa? Australian Zoologist 32:207-213. Cracraft J. 1989. Speciation and its ontology: the empirical consequences of alternative species concepts for understanding patterns and processes of differentiation. In: Otte D, Endler JA. editors. Speciation and its consequences. Sunderland (MA): Sinauer Associates, Inc. p 28-59. Dabek L. 1994. Reproductive biology and behavior of captive female Matschie's tree kangaroos, Dendrolagus matschiei. [dissertation]. Seattle: University of Washington. Davis JI, Nixon KC. 1992. Populations, genetic variation, and the delimitation of phylogenetic species. Systematic Biology 41:421—435. Flannery T. 1995. Mammals of New Guinea. New York: Cornell University Press. 568 p. Flannery T, Martin R, Szalay A. 1996. Tree kangaroos: a curious natural history. Port Melbourne Victoria: Reed Books Australia. 202 p. Frankham R, Ballou JD, Briscoe DA. 2004. Introduction to conservation genetics. Cambridge: Cambridge University Press. 617 p. 6

Goldstein PZ, DeSalle R, Amato G, Vogler AP. 2000. Conservation genetics at the species boundary. Conservation Biology 14:120-131. Groves CP. 1982. The systematics of tree kangaroos (Dendrolagus; Marsupalia, Macropodidae). Australian Mammalogy 5:157-186. Lacy RC. 1995. Clarification of genetic terms and their use in the management of captive populations. Zoo Biology 14:565-578. Leary T, Serf L, Wright D, Hamilton S, Helgen K, Singadan R, Menzies J, Allison A, James R, Dickman C, Aplin K, Flannery T, Martin R, Salas L. 2008. Dendrolagus matschiei. In: IUCN 2008. 2008 IUCN Red List of Threatened Species. . Downloaded on 14 November 2008. Martin R. 2005. Tree-kangaroos of Australia and New Guinea. Collingwood: CSIRO Publishing. 158 p. McGreevy Jr TJ, Dabek L, Gomez-Chiarri M, Husband T. in press. Genetic diversity in captive and wild Matschie's tree kangaroo {Dendrolagus matschiei) from Huon Peninsula, Papua New Guinea, based on mtDNA control region sequences. Zoo Biology, 14 pp. Montgomery ME, Ballou JD, Nurthen RK, England PR, Briscoe DA, Frankham R. 1997. Minimizing kinship in captive breeding programs. Zoo Biology 16:377-389. Ralls K, Ballou JD. 2004. Genetic status and management of California condors. Condor 106:215-228. Rothschild W, Dollman G. 1936. The Genus Dendrolagus. Transactions of the Zoological Society (London) XXL477-548. Vogler AP, DeSalle R. 1994. Diagnosing units of conservation management. Conservation Biology 8:354-363. Ziegler AC. 1977. Evolution of New Guinea's marsupial fauna in response to a forested environment. In: Stonehouse B, Gilmore D. editors. The biology of marsupials. London: The Macmillan Press Ltd. p 117-138. 7

MANUSCRIPT I: Microsatellite Marker Development and Mendelian Analysis in the Matschie's Tree Kangaroo {Dendrolagus matschiei) 1.1 Abstract We developed five microsatellite markers for endangered Matschie's Tree Kangaroo {Dendrolagus matschiei). We screened 17 additional markers developed for other marsupial taxa and identified three that were polymorphic. By analyzing 22 D. matschiei from Wasaunon on the Huon Peninsula with the set of eight markers, we estimated allelic and genetic diversity. The number of alleles ranged from two to nine and expected heterozygosity ranged from 0.440 to 0.794. We tested for null alleles and Mendelian inheritance by analyzing 19 pairs of D. matschiei parents and offspring from Association of Zoos and Aquariums institutions. Null alleles were not detected and Mendelian inheritance was followed for all eight markers. We also evaluated the reliability of using the markers to amplify DNA extracted from D. matschiei fecal samples and the ability of the markers to amplify DNA samples from Goodfellow's tree kangaroo (Dendrolagus goodfellowi ssp.), Doria's tree kangaroo (Dendrolagus dorianus ssp.), and Grizzled tree kangaroo (Dendrolagus inustus ssp.). Microsatellite markers can be used to inform management decisions to conserve D. matschiei in captivity and the wild. 8

1.2 Introduction Tree kangaroos (Dendrolagus spp.) are unique arboreal macropodid marsupials. There are eight species on the island of New Guinea and two species in Australia (Flannery 1995). Most New Guinea Dendrolagus spp. are listed as critically endangered or endangered by the 2008 International Union for Conservation of Nature and Natural Resources (IUCN) Red List of Threatened Species (http://www.redlist.org). Although they are the largest extant mammals indigenous to New Guinea, relatively little is known about tree kangaroos (Flannery et al. 1996; Martin 2005). Matschie's tree kangaroo {Dendrolagus matschiei) is endemic to the Huon Peninsula of Papua New Guinea (PNG) and is classified as endangered by the 2008 IUCN Red List (Leary et al. 2008). The leading causes for their population decline have been hunting and loss of habitat (Betz 2001; Flannery 1995). In captivity, the Association of Zoos and Aquariums (AZA) D. matschiei population has endured a founder effect and has a lower level of mitochondrial DNA (mtDNA) control region haplotype diversity than captive D. matschiei from PNG and a lower number of mtDNA haplotypes compared to wild D. matschiei (McGreevy et al. in press). A nuclear DNA analysis, however, is required to more comprehensively characterize AZA D. matschiei genetic diversity and determine how well they reflect the amount of genetic variation found in captive and wild D. matschiei from PNG. We developed five microsatellite markers for D. matschiei — the first markers developed for Dendrolagus — and identified an additional three polymorphic markers that were developed for other marsupial taxa. We estimated allelic and genetic 9

diversity by analyzing 22 D. matschiei from Wasaunon on the Huon Peninsula. The number of alleles ranged from two to nine and expected heterozygosity ranged from 0.440 to 0.794. Null alleles were not detected and Mendelian inheritance was followed for all eight markers. The eight markers produced concordant genotypes on average 80.0% ± 10.5 (standard deviation) of the time using DNA extracted from blood and fecal samples from the same animal. We also were able to polymerase chain reaction (PCR) amplify DNA samples from Goodfellow's tree kangaroo (Dendrolagus goodfellowi ssp.), Doria's tree kangaroo (Dendrolagus dorianus ssp.), and Grizzled tree kangaroo {Dendrolagus inustus ssp.) using the set of eight markers. 1.3 Methods 1.3.1 Microsatellite Marker Development for D. matschiei Total genomic DNA was extracted from blood samples from four AZA D. matschiei, studbook number 175 (AZA175), AZA 190, AZA 243, AZA 339 using DNEasy Blood and Tissue Kit, as per manufacturer's instructions (QIAGEN, Valencia, CA). DNA from the four AZA D. matschiei was pooled and a microsatellite-enriched library was constructed following Hamilton et al. (1999). Genomic DNA was digested with Hae III, Nhe I, Xmn I, and Rsa I (New England Biolabs, Ipswich, MA) and fragments were ligated to SNX linkers and then PCR amplified using a SNX primer. PCR products were hybridized to a biotinylated probe (GT)i5, purified with streptavin-coated magnetic beads (Dynal, Carlsbad, CA), washed, and PCR amplified using the SNX primer. We cloned PCR products enriched for dinucleotide repeats into a pBluescript® II SK(+) vector (Stratagene, La Jolla, CA) and PCR amplified 273 putative positive clones using primers T3 and T7. We sent 51 10

Full document contains 148 pages
Abstract: The Association of Zoos and Aquariums (AZA) Matschie's tree kangaroo (Dendrolagus matschiei ) population is at a critical point for assessing long-term viability; they have endured a founder effect and have low mitochondrial DNA (mtDNA) diversity. The mean kinship (MK) strategy is used to preserve their genetic diversity by minimizing the overall kinship of the population; however, the MK strategy has not been evaluated in an AZA population. Wild D. matschiei is listed as endangered and genetically unique populations may warrant management as separate conservation units. My objectives were to: (i) develop molecular markers to facilitate genetic analyses, (ii) assist AZA and wild D. matschiei management decisions, and (iii) further resolve Dendrolagus molecular systematics. To achieve my objectives I analyzed DNA extracted from AZA, captive held, and wild D. matschiei, and three additional Dendrolagus taxa using microsatellite markers and two mtDNA genes. AZA D. matschiei have similar nuclear DNA genetic diversity as captive and wild D. matschiei from Papua New Guinea, and the MK strategy has maintained their genetic diversity as well as predicted by theoretical expectations. AZA D. matschiei should continue to be managed by their studbook analyses and the MK strategy. Wild D. matschiei showed evidence of phylogeographic structure, but should be managed as one conservation unit. Preliminary genetic evidence suggests D. matschiei and Lowland tree kangaroo (Dendrolagus spadix ) are sister taxa, and supports the reclassification of Golden-mantled tree kangaroo (D. goodfellowi pulcherrimus ) and Ifola ( D. dorianus notatus ) as species. An improved understanding of Dendrolagus genetics will contribute substantially to their conservation.