Study on the diversity and distribution patterns of amphibians (amphibia) in karst forests of the north vietnam and conservation measures

MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY PHAM THE CUONG STUDY ON THE DIVERSITY AND DISTRIBUTION PATTERNS OF AMPHIBIANS (AMPHIBIA) IN KARST FORESTS OF THE NORTH VIETNAM AND CONSERVATION MEASURES Research field: Zoology Code: 62.42.01.03 SUMMARY OF BIOLOGY DOCTORAL THESIS HA NOI – 2018 This dissertation is submitted to the Committee of the Graduate University of Science and Technology Supervisors: 1. Dr. Nguyen Quang Truong 2. Dr. Le Duc Minh Referee 1: Ass. Prof. Dr. Le Nguyen Ngat Referee 2: Ass. Prof. Dr. Hoang Ngoc Thao Referee 3: Dr. Hoang Van Ngoc The research has been carried out at the Graduate University of Science and Technology Date of Oral Presentation:,../../2018 Copy of this dissertation is available: National Library of Viet Nam Graduate University of Science and Technology 10 INTRODUCTION 1. The necessity of the research Vietnam is recognized as one of the countries with a high level of biodiversity in the world (Conservation International 2014). In terms of amphibian diversity (Class Amphibia), the species number recorded from Vietnam has been rapidly increased in recent decades: from 82 species in 1996 to 162 species in 2005 and up to 176 species in 2009 and the current species number is ca. 230 (Nguyen & Ho 1996, Nguyen et al. 2005, 2009, Frost 2017). Karst ecosystem is considered as a natural laboratory for taxonomic, ecological, evolutionary and biogeographical research (Clements et al. 2006). Karst forests provide a variety of distinct microhabitats like “isolated islands” and are noted for their high levels of endemism (Clements et al. 2006). In Vietnam, limestone forests are extensive in the northeastern, northwestern regions and northern Truong Son Range (Sterling et al. 2006). However, limestone forest is a sensitive ecosystem to the human impacts and it is very difficult to restore. Limestone in Vietnam has been exploited for infrastructure and economic development, for example, quarrying for road construction, and cement industry. Moreover, the fauna in karst forests is threatened by decline because of forest destruction, overexploitation, tourism development and climate change, in particular, the poikilotherm like amphibians (Sterling et al. 2006). The research entitled "Study on the diversity and distribution patterns of amphibians (Amphibia) in karst ecosystem of the North Vietnam and conservation measures” aims to assess the diversity of amphibians in limestone forests of northern Vietnam with special focus on two group complexes of anuran frogs, Limnonectes and Odorrana, in Vietnam. In addition, recommendations for conservation measures are also provided. 2. Objectives - To assess the diversity of amphibians in some selected limestone forests of northern Vietnam; - To review taxonomic and phylogenetic relationships of two genus complexes, Limnonectes and Odorrana, in Vietnam; 11 - To assess conservation potential and threats to the amphibian fauna and to provide recommendations for conservation. 3. Contents Task 1: Inventory of the species diversity of amphibians in some karst forests in northern Vietnam: Hoa Binh Province in the northwestern region, Cao Bang and Ha Giang provinces in the northeastern region and Cat Ba Island, an offshore island in the Gulf of Tonkin. This research also focuses on the discovery of new species and new country records. Task 2: Evaluating distribution patterns of amphibian species across altitudinal gradient (from 0 to 1300 m), habitat types and life modes (on the tree, on the ground, and associated with water). Task 3: Evaluating biogeographic relationships of amphibian fauna amongstudy sites, between mainland and offshore islands, and between northeastern and northwestern regions to verify the evolutionary barrier of the Red River. Task 4: Evaluating taxonomy and phylogenetic relationships of two genus complexes, Odorrana and Limnonectes, based on the morphological comparison and molecular analyses. Task 5: Assessment of conservation potential based on the following criteria: species diversity, number of endemic and threatened species, possibility of population restoration. Evaluation of threats to the amphibian populations and provide recommendations for conservation measure. 4. Scientific and conservation significances Scientific significance: The research provides updated data about the diversity of amphibians in the karst forests of northern Vietnam. The research also provides novel data on taxonomy and phylogenetic relationships of Limnonectes and Odorrana from Vietnam. Conservation significance: The research provides baseline data for biodiversity conservation planning and management in northern Vietnam. 5. New findings A total 65 species of amphibans were recorded in karst forests of the North Vietnam, comprising 44 species recored from Ngoc Son-Ngo 12 Luong NR, 32 species recorded from Hang Kia-Pa Co NR, 33 species recorded from Bac Me NR, 21 species recorded from Ha Lang District, and 23 species recorded from Cat Ba NP. New species: Three new species were described as new for science, Odorrana mutschmanni from Cao Bang Province, Theloderma annae and Rhacophorus hoabinhensis from Hoa Binh Province. New records: Three species, Leptolalax minimus, Odorrana hainanensis, and O. lipuensis, were recorded for the first time from Vietnam and some other new provincial records from northern Vietnam: One species from Cao Bang Province; two species from Ha Giang Province; three species from Hai Phong city; and five species from Hoa Binh Province. Biogeographic relationships: Based on the list of amphibian species recorded from northern Vietnam, the amphibian fauna of the northeastern region was distinctly separated from that of the northwestern region (Similarity Index = 0.6087). Moreover, the amphibian fauna of the karst forests in mainland also differs from that of the offshore islands (Similarity Index = 0.4706). Distribution patterns: Most of amphibian species were recorded at elevations below 800 m (58 species), whereas 34 species were found at elevations above 800 m. In terms of habitat types, most of amphibian species were recorded in the undisturbed natural forests (58 species), while the species number recorded in the disturbed forest was 33 species and agricultural and residential areas was 16 species. The highest number of record species were found on the ground (42 species). In this study, we also provide taxonomic review of two poorly known genera of amphibians, Limnonectes (8 species) and Odorrana (25 species), based on morphological and molecular data with descriptions of two new species for science and three new country records for Vietnam. Phylogenetic relationships of these genera were also discussed with remarks on some species complexes. In addition, we evaluated the threats to the amphibian fauna and provided some recommendations for conservation in northern Vietnam. 13 Chapter 1 LITERATURE REVIEW 1.1. Overview of amphibian research in region Currently, a total 7.697 species of amphibians was recorded in the world (Frost 2017). In the neigboring countries, 432 species have been recorded from China; 152 species from Lao and 79 species from Cambodia (Frost 2017). In terms of conservation concern, Stuart et al. (2004) stated that 168 species of amphibian were extinction and at least 2,500 species are under threatened in the last 20 years (1984-2004). 1.2. Overview of amphibian research in Vietnam 1.2.1. Faunistic inventory and new discoveies Bourret (1942) reported 171 species and subspecies of amphibians in Indochina (Vietnam, Laos, Cambodia). Nguyen and Ho (1996) published a checklist of reptiles and amphibians of Vietnam with 82 recorded species of amphibians. Nguyen et al. (2005) listed 162 species of amphibians. In the recent book, Nguyen et al. (2009) recorded a total of 176 species of amphibians from Vietnam. Since 2010 more than 40 new species of amphibians have been described from Vietnam (Forst 2017). A number of studies on amphibians were conducted in the Northeast, Northwest, Central, Central Highlands, Southern and some offshore islands of Vietnam. 1.2.2. Research on biology and ecology Research on biological and ecological characters of amphibians focused on few species that were kept in captivity, the widespread species or endemic to Vietnam: Duttaphrynus melanostictus, Hoplobatrachus rugulosus, Limnonectes bannaensis, Hylarana maosonensis, Quasipaa verrucospinosa, Polypedates mutus, Paramesotriton deloustali. 1.2.3. Research on tadpoles and bioacoustics Descriptions of the tadpoles of the following species were mentioned in previous studies (Hendrix et al. 2007, Gawor et a. 2009, Wildenhues et al. 2010, Rauhaus et al. 2012, Pham et al. 2015): Rhacophorus annamensis, Hynarana nigrovittata, Rhacophorus 14 maximus, Theloderma corticale, Amolops cremnobatus. Le Thi Quy (2015) described the tadpoles of 21 amphibian species from Bach Ma National Park, Thua Thien Hue Province. In addition, the bioacoustic analysis is a useful approach to study biology and taxonomy of amphibians. Le Trung Dung (2016) described the biaocoustics of 11 amphibians species. 1.2.4. Research on phylogenetic relationships Frost et al. (2006) provided a comprehensive phylogenetic tree of amphibian species of the world. Li et al. (2008, 2009), Biju et al. (2010), Orlov et al. (2012), Kuraishi et al. (2012), Li et al. (2012), Yu et al. (2010, 2013), Nguyen et al. (2015), Poyarkov et al. (2015) studied on taxonomy and phylogenetic relationships of various groups of Rhacophoridae. 1.2.5. Research on pathology and conservation issues Rowley et al. (2013), Nguyen et al. (2013), Martel et al. (2014), Laking et al. (2017) and Nguyen et al. (2017) have identified a number of amphibian populations that infected with Batrachochytrium dendrobatidis and B. salamandrivorans. Rowley et al. (2010, 2016) assessed threats to the populations of amphibians in Southeast Asia, including Vietnam. Key impacts include habitat loss, overexploitation for food, medicinal and pet trade. 1.2.6. Research on amphibians in northern Vietnam Although numerous field excursions have been conducted in northern Vietnam, most of surveys were conducted in evergreen forests such as Tay Yen Tu, Tay Con Linh, Phia Oac-Phia Den, Hoang Lien, Muong Nhe, Sop Cop, and Copia nature reserves (Hecht et al. 2013, Bain & Nguyen 2004, Nguyen Thien Tao 2009, Orlov et al. 2001, Le Trung Dung 2016, Pham Van Anh 2016). Research in limestone karst ecosystems are still limited with descriptions of new species, namely Tylototrion ziegleri from Cao Bang and Ha Giang provinces, Liuixalus calcarius and Philautus catbaensis from Cat Ba Island (Nishikawa et al. 2013, Milto et al. 2013). 1.2.7. Research on Limnonectes and Odorrana in Vietnam Genus Limnonectes: Limnonectes is one of the most diverse groups of amphibians to date with 68 currently recognized species and 15 16 of which have been described in the last ten years (Frost 2017). According to Nguyen et al. (2009), five species of the genus Limnonectes are currently recorded from Vietnam. However, some taxonomic changes have been published in recent years. Due to their morphological similarity, species that were previously thought to be widespread are now being recognized as complexes of species with much narrower distributions Genus Odorrana: Odorrana is one of the most diverse groups of amphibians to date with 58 currently recognized species, more than 20 of which have been described in the last ten years (Frost 2017). Due to morphological similarity in the genus, species that were formally thought to be widespread are now being recognized as complexes of species with much narrower distributions. Further study on taxonomic and genetic relationships should be continued. 1.3. Natural conditions of the study area 1.3.1. Natural conditions of North Vietnam The North Vietnam bordered by China’s Guangxi Province to the northeast and Yunnan Province to the northwest, and by Laos along its western border (Sterling et al. 2006). Limestone kart forests: Most of the limestone mountain area in Vietnam is distributed in northern Viet Nam, partly in Quang Binh province. In the Gulf of Tonkin, there are thousands of limestone islands in Ha Long Bay next to each other with Cat Ba Island as the center (Sterling et al. 2006). Vegetation: The dominant forest type in northern Vietnam is evergreen, including both broad-leaved and coniferous plants, with pockets of semi-evergreen forest. Coastal mangrove formations and forests growing on limestone are also important elements of the north’s natural landscape diversity (Sterling et al. 2006). Fauna: Northern Vietnam’s animal communities are a mixture of tropical and subtropical species, many of which are unique to the northern third of the country. Within the region, the Red River may be an effective barrier to movement (or coincide with one) for some groups, such as amphibians and reptiles, resulting in different species and communities in the northeast and northwest (Sterling et al. 2006). 16 1.3.2. Natural conditions of survey sites Field surveys were conducted in the limestone forestin northern Vietnam. Northwest: Ngoc Son-Ngo Luong Nature Reserve (Hoa Binh Province): This NR is located in Tan Lac and Lac Son districts with a total area of 19.254 ha. The area is characterized by limestone mountains, with steep terrain and rugged terrain. Between the limestone mountains there are several flat valleys running in the north-west direction. The altitude ranges from 100-1.065 m (Birdlife International 2004). Hang Kia-Pa Co Nature Reserve (Hoa Binh Province): Hang Kia-Pa Co Nature Reserve is located in Mai Chau District with a total area of 5.257 ha. There are many high and pointed limestone blocks, the highest peak of which is 1.536m in the Northwest of the area, the height decreasing to the East. Most of the nature reserve stands at over 500 m (Birdlife International 2004). Northeast: Bac Me Nature Reserve (Ha Giang Province): This NR is located in Bac Me District with a total area of 9.042 ha,. Bac Me Nature Reserve is located in the depression of the Gam River to the south-east of Ha Giang Province, with steep terrain and strong splinters with many peaks above 1000 m along the Song Gam gulf (Biodiversity Report Bac Me Nature Reserve 2009). Ha Lang Forest (Cao Bang Province): Ha Lang District has a limestone karst topography that occupies most of the area, at an altitude of 100-750 m above sea level. According to the biodiversity conservation planning of Cao Bang Province, a nature reserve will be established in this district with an area of 7.343 ha (Decree No. 697/QĐ-UBND). Cat Ba National Park: Cat Ba is the largest island of the Cat Ba Archipelago with a total area of 26.241 ha. Cat Ba National Park is located in the limestone archipelago of hundreds of large and small islands at the height of 100-150 m, the highest point is Cao Vong peak 331m and the area of this protected area is 16.196 ha (www.vuonquocgiacatba.com.vn). 17 CHAPTER 2. MATERIALS AND METHODS 2.1. Field survey and study sites Fifteen field surveys with 119 days were conducted from 2014 to 2017. Survey sites: Cat Ba National Park (Hai Phong City), Ha Lang Disstrict (Cao Bang Province), Bac Me Nature Reserve (Ha Giang Province), Hang Kia-Pa Co and Ngoc Son-Ngo Luong nature reserves (Hoa Binh Province). Sampling of Limnonectes and Odorrana: Twenty field surveys with 200 days were conducted in more than twenty provinces in Vietnam from 2014 to 2017. Molecular analysis was carried out at the Institute of Ecology and Biological Resources and Vietnam National Museum of Nature, and Hanoi University of Science. 2.2. Methods 2.2.1. Sampling Excursions were carried out during the day and at night in different habitat types. Survey transects were set up along the streams, pools, small ponds or along the forest paths, under the leaf litter or on the tree branches, we thus also survey the forest near the cave entrances and cliffs. 2.2.2 Laboratory work 2.2.2.1. Morphological examination Taxonomic identification: Morphological comparisons were made with the voucher specimens which are deposited in the collections of the Institute of Ecology and Biological Resources and Vietnam National Museum of Nature. For taxonomic identification, we used the following documents: Bourret (1942), Bain et al. (2003), Ohler et al. (2011), Orlov et al. (2003, 2006, 2012), Suwannapoom et al. (2016), Taylor (1962), and othe related documents that were cited in each species account. Species names followed Nguyen et al. (2009), Frost (2017) and recently published papers. 2.2.2.2. Molecular analysis A fragment of mitochondrial gene (cytochrome b, 12S, 16S) was sequenced to investigate phylogenetic relationships of all species of the genus Limnonectes and Odorrana in Vietnam. 18 The sequences were aligned using ClustalX v2.1 (Thompson et al. 1997), selected the optimal model using Modeltest v3.7 (Posada and Crandal 1998), analyzed using Bayesian inference (BI) as implemented in Mr.Bayes v3.1.2 (Ronquist et al. 2012). We used BI and maximum-likelihood (ML) approaches to reconstruct the mitochondrial gene tree (matrilineal genealogy). The analyses used a concatenation of COI and 16S rRNA sequences. 2.2.3. Distribution patterns Habitat: Species composition were assessed in different habitat types (agricultural and residential areas, disturbed natural forests, undisturbed natural forests). Altitudinal gradient: We compared the species composition of amphibians between elevations below 800 m and above 800 m. Life modes: Species composition of amphibians was evaluated based on three life modes: on the trees, on the ground, and associated with water. 2.2.4. Conservation status Conservation status of threatened species was referred to the Vietnam Red Data Book (2007); IUCN Red List (2017); CITES appendices (2017); the Governmental Decree No. 32/2006/ND-CP; and the Governmental Decree No. 160/2013/ND-CP. The endemic species of Vietnam are species that currenly known only from Vietnam. 2.2.5. Statistic analysis Statistic analyses were performed with the softwware PAST Statistics (Hammer et al. 2001) 2.2.6. Conservation issues - Evaluation of threats to amphibians, for example habitat loss and degradation and overharvesting. - Providing recommendations for conservation with the following aspects: habitat protection and restoration, control of hunting/overharevesting, development of breeding program, and awareness campaign. 2.3. Materials A total of 524 specimens of amphibians were collected in karst forests of the North Vietnam for morphorlogical analysis. 19 A total of 148 specimens of Limnonectes and 232 specimens of Odorrana were collected in Vietnam for morphorlogical analysis. A total of 150 tissue samples, consisting of 46 tissue samples of Limnonectes, 74 tissue samples of Odorrana, and 30 tissue samples of Liuixalus, Rhacophus, and Theloderma, were use for molecular analysis. Chapter 3 RESULTS AND DISCUSSION 3.1. Species composition of amphibians 3.1.1. List of recorded species A collection of 524 specimens of amphibians was collected from different survey sites in the research areas. Species composition: A total of 65 spe