Exploration of amphibian diversity in trang an landscape complex ninh binh province

Therefore, based on that species list, a first assessment of the species richness and characteristics of the species composition of TA LC should be conducted, and new records will be des

MINISTRY OF AGRICULTURE AND RURAL DEVELOPMENT VIETNAM NATIONAL UNIVERSITY OF FORESTRY

-

STUDENT THESIS

EXPLORATION OF AMPHIBIAN DIVERSITY IN TRANG AN

LANDSCAPE COMPLEX, NINH BINH PROVINCE

Major: Natural Resources Management Code: D850101

Faculty: Forest Resources and Environmental Management

Student: Nguyen Tuan Nam Student ID: 1453090203 Class: K59A-Natural Resources Management

I would like to extend my thanks to Mr Pham Sinh Khanh – manager of Trang An Landscape Complex (TA LC) for his supporting during my survey time

Besides, I would love to thank Mr Lo Van Oanh, Mr Nguyen Huy Quang, Mr Nguyen Thanh Tung, Mr Dang Quoc Tuan for their assistance in the field

Therefore, I would like to thank Mr Thanh and his wife - who management Tran temple for supporting available transportation and shelters on the survey team

Finally, I own my gratefully to my parents for their love, support and encouragements to me throughout all my life Even though difficulties, ado, they always help me as more as possible for my dream

This research is supported by the National Foundation for Science and Technology

Development (NAFORSTED, Grant No.106.06-2017.18)

ABSTRACT

This research was conducted to provide the checklist of amphibian‟s species in Trang

An Landscape Complex, Ninh Binh Province, Vietnam Total 11 species were recorded within survey from 8th May - 28th June 2017 and 28th May - 12 June 2018 in Trang An Landscape Complex The number of amphibian species in TA LC has increased to 19

Remarkably, one new provincial species was recorded and described (Theloderma annae)

for the first time from Trang An and from Ninh Binh Province This study described 11 amphibian species in Trang An Landscape Complex The main threats to the amphibian fauna is ecotourism Therefore, the research has recommended three solutions for reducing the threats consisting of managing, keeping and protecting amphibians in Trang An Landscape Complex

TABLE OF CONTENTS

ACKNOWLEDGEMENTS i

ABSTRACT ii

TABLE OF CONTENTS iii

ABBREVIATIONS v

LIST OF FIGURES vi

LIST OF TABLES vii

CHAPTER I: INTRODUCTION 1

CHAPTER II: LITERATURE REVIEWS 3

CHAPTER III: GOAL, OBJECTIVES AND STUDY SITE 7

3.1 Goal 7

3.2 Objectives 7

3.3 Geographic location 7

3.4 Climate and hydrology 9

3.5 Topography 9

3.6 Flora and Fauna 11

3.6.1 Flora 11

3.6.2 Fauna 11

3.7 Economy – Society 11

CHAPTER IV: METHODOLOGY 12

4.1 Identify amphibian species composition in TA LC 12

4.1.1 Preparation and equipment 12

4.1.2 Line transect survey 12

4.1.3 Collecting method 14

4.1.4 Preservation of specimens 14

4.1.5 Permission and management 14

4.2 Determine distribution characteristics of the species followed by habitat types and biogeographic relationships 15

4.2.1 Distribution characteristics by habitat types in TA LC 15 4.2.2 Comparisons of the relative amphibian composition among

4.3 Determine morphological character of these species 16

CHAPTER V: RESULTS AND DISCUSSIONS 20

5.1 Amphibian diversity of TA LC 20

5.1.1 Scientific collection 20

5.1.2 The diversity of amphibian species in TA LC 20

5.1.3 New record of amphibian species in TA LC 23

5.1.4 Correlation index between regions in TA LC 24

5.2 Habitat diversity of amphibian species 25

5.2.1 Distribution of amphibian species in habitats 25

5.2.2 Comparing the amphibian list to these among karst protected areas 27

5.3 Descriptions of recorded amphibian species in TA LC 28

5.4 Conservation issues 53

5.4.1 Threats to the amphibian fauna 53

5.4.2 List of threatened species of amphibians recorded from TA LC 54

5.5 Discussions 54

CHAPTER VI: CONCLUSIONS AND RECOMMENDATIONS 56

6.1 Conclusions 56

6.2 Recommendations for protection and sustainable development of amphibian species in TA LC 56

6.2.1.Recommendations to minimize threats to amphibian species 56

6.2.2 Recommendations for Trang An Scenic Landscape Complex 57

6.2.3 Recommendations for socio-economic 57

6.3 Limitations and suggestions for further study 57

REFERENCES 58

ANNEX 63

ABBREVIATIONS

LIST OF FIGURES

Figure 2.1: Number of amphibian species in different periods 3

Figure 3.1: Map of the study site: Trang An Landscape Complex 8

Figure 3.2: Land cover in Trang An Landscape Complex 10

Figure 4.1: Map of survey locations (white dots) in TA LC 13

Figure 4.2: Morphological measures 19

Figure 5.1: Number of specimens collected in of TA LC 20

Figure 5.2: The species richness of amphibian families in TA LC 23

Figure 5.3: New recorded for amphibian species for TA LC 24

Figure 5.4: Similarity cluster analysis of the amphibian among survey sites 25

Figure 5.5: Diversity of species according to habitat 26

Figure 5.6: Comparing dice index of amphibian diversity to other region 28

Figure 5.7: (A) Dorsal and (B) ventral views of the adult male 30

Figure 5.8: Micro habitat of Fejervarya limnocharis on the ground in TA LC 30

Figure 5.9: (A) Dorsal and (B) ventral views of the one adult male Occidozyga lima (VNUF A.2017.06) 32

Figure 5.10: (A) Dorsal and (B) ventral views of the one adult male Occidozyga martensii (VNUF A.2017.55) 34

Figure 5.11: (A) Dorsal and (B) ventral views of the one adult male Sylvirana guentheri 36

Figure 5.12: Microhabitat of Sylvirana guentheri in the limestone karst forest of TA LC 36

Figure 5.13 (A) Dorsal and (B) ventral views of the one adult female Kaloula pulchra 38

Figure 5.14 Microhabitat of Kaloula pulchra in the limestone karst forest of TA LC 38

Figure 5.15 (A) Dorsal and (B) ventral views of the adult male Microhyla pulchra (VNUF A.2018.49) 40

Figure 5.16 (A) Dorsal and (B) ventral views of the adult male Rana johnsi (VNUF A.2017.21) 41

Figure 5.17: (A) Dorsal and (B) ventral views of the adult male Polypedates megacephalus (VNUF A.2017.45) in life 43

Figure 5.18: (A) Dorsal and (B) ventral views of the adult male Polypedates mutus (VNUF A.2018.22) in life 45

47

Figure 5.19: (A) Dorsal and (B) ventral views of the adult male Rhacophorus orlovi (VNUF A.2017.39) in life 47

Figure 5.20: Microhabitat of Rhacophorus orlovi in the limestone karst forest of TA LC 48

Figure 5.21 (A) Dorsal and (B) ventral views of the adult male Theloderma annae (VNUF A.2018.45) in life 51

52

Figure 5.22 Microhabitat of Theloderma annae in the limestone karst forest of TA LC 52

LIST OF TABLES

Table 4.1: Field survey sheet 14

Table 4.2: Distribution of amphibian species in each habitats 15

Table 4.3: Abbreviations of morphological characters 17

Table 5.1: List of amphibians of Trang An Landscape Complex 21

Table 5.2: Similarity index (dice index) of amphibian species among survey sites 24

Table 5.3: Distribution of amphibian species in habitats 25

Table 5.4: Comparing diversity indices of amphibian diversity to other region 27

Table 5.5: Comparing dice index of amphibian diversity to other region 27

Table 5.6: Threatened amphibians species recorded from TA LC 54

CHAPTER I: INTRODUCTION

Globally, the biodiversity plays vital roles in our planet ecosystems Deforestation, overexploitation, environmental pollutions, causes, toxicants, and climate change are the main causes of biodiversity loss (Barry et al., 2016) These factors are challenges related to extinctions of fauna over the coming century if present trends of tropical deforestation continue (Sodhi et al., 2014) Among the 1.4 million recorded (10 million estimated) extant species of organisms on the planet, amphibians comprise 7919 of these (Frost D, 2018)

The lowlands of Indochina are rich in biodiversity, but many areas are being drastically altered through rapid urbanization and ecotourism, only a few some ecosystems

to provide lowlands for amphibian species Holzer et al., 2017) Human activities were the main causes directly impact to amphibians are experiencing drastic global declines, and urbanization has been identified as one of the leading stressors (Stuart et al., 2004; Cushman, 2006) Over 95% of the primary conditions of land cover have already changed (Sloan, 2014) by human activities

Vietnam is considered as a country with high biodiversity of the world Beginning in the 1990s, because the growth in the tourism and culture heritage policy related to economic, the government saw heritage tourism as a powerful economic and diplomatic tool (Logan, 2009) Consequently, conservation activities received strong attention as the most important economic activities in Vietnam (Lask and Herold, 2004)

Up to now, there were 250 amphibian‟s species were recorded (Nguyen et al., 2009; Frost, 2018) In 1996, 82 species were recorded and the number of species is increased up to 162 species (Nguyen et al., 2005), then kept increasing to 177 species (Nguyen et al., 2009) Many researches showed their records of new species in Vietnam such as 45 amphibian‟s species in Phong Nha – Ke Bang National Park by Ziegler & Vu

(2009) Luu et al (2013) updated a list of 151 species including 50 amphibian‟s species in Phong Nha – Ke Bang National Park and Quang Binh Province According to Luu and Hoang (2015), 18 amphibian‟s species belonged to 5 families and 1 order were found in

TA LC

TA LC is located Ninh Binh Province of Northern Vietnam near the southern margin of the Red River Delta, TA LC are mixed natural property mostly within three protected area including the Hoa Lu Ancient Capital, the Trang An-Tam Coc-Bich Dong Scenic Landscape, and the Hoa Lu Special-Use Forest The property covers 6,226 hectares and surrounded by a buffer zone with 6,026 hectares, mostly rural land with paddy field (UNESCO, 2006) This area exemplifies an outstanding humid tropical tower-karst landscape in the final stages of geomorphic evolution, comprising karst cones and towers, enclosed depressions and an intricate system of freshwater-filled foot-caves (Rabet et al., 2016)

TA LC contains rich flora and fauna, some of them are endemic species Reviews of current knowledge about the biodiversity of Trang An show a lack of data for biodiversity purposes In 2015, Hoang and Luu published a list of amphibians in this area Therefore, based on that species list, a first assessment of the species richness and characteristics of the species composition of TA LC should be conducted, and new records will be described, more information about the ecology of the observed species should be collected to identify their distributional pattern Besides, threats to the biodiversity of the protected area will be pointed out and some solutions will be proposed to deal with the

current situation For those reasons, the study named “Exploration of amphibian

diversity in Trang An Landscape Complex, Ninh Binh Province” was carried out in

order to not only update a comprehensive list of amphibians in TA LC but also provide

CHAPTER II: LITERATURE REVIEWS

Vietnam (VN) belong to tropical monsoon climate and complex terrain with high biodiversity about fauna and flora It is relevant for developing wild animals, especially in

in amphibian species Study period of amphibian species in VN showing in this table:

Figure 2.1: Number of amphibian species in different periods

Before 1954, the first research about amphibian species is “Nam than duoc lieu” of Tue Tinh recorded 16 kinds of medicine from amphibian species In this period, there are some scientist did a research about the study on amphibian in Indochina According to Bourret (1936, 1941, 1942), the number of amphibians was 171 species in Indochina In this period, from 1900-1954, about 75 amphibian species were described by specimens collected in VN of Smith (in the period from1920 to 1940) and Bourret (in the period from

1930 to 1940) (Nguyen et al., 2009)

In the period from 1954 to 1975, as the Tran et al (1981) point out that there was

68 amphibian species in the North of VN

In the period from 1975 to 1986, Dao et al (1957, 1962) points out that 87 amphibians species were found in VN

250

0 50 100 150 200 250 300

Author Number of amphibian species in these period

From 1987 until now, almost research focus on the statistic, update list and find the new species to provide for science

In the period 1987- 2009: Nguyen and Ho reported 82 species of amphibians in

1996 and brought the number of amphibians to 162 species in 2005 and 177 species in

2009 According to the amphibian list in the world, up to 2018, there were 250 amphibian species in VN (Frost 2018)

The number of new species was recorded increasing in each period In the period 1976 -

1996, just 4 amphibian species has been recorded in VN However, in the period 1996 -

2005, the number of amphibian increase to 28 species and up to 66 species in the period from 2006 to 2017 In 5 years recently, 82 species have been found and described such as

Hyla annectans Fouquette and Dubois, 2014 , Hyla chinensis Fouquette and Dubois, 2014, Hyla simplex Fouquette and Dubois, 2014; Kalophrynus cryptophonus Vassilieva,

Galoyan, Gogoleva, and Poyarkov, 2014; Kalophrynus honbaensis Vassilieva, Galoyan, Gogoleva, and Poyarkov, 2014; Microhyla arboricola Tran, Le, Kretova, and Geissler, 2014; Tran, Le, Kretova, and Geissler, 2014; Microhyla darevskii Tran, Le, Kretova, and Geissler, 2014; Microhyla minuta Tran, Le, Kretova, and Geissler, 2014; Microhyla

pineticola Tran, Le, Kretova, and Geissler, 2014; Microhyla pulchella Poyarkov,

Vassilieva, Orlov, Galoyan, Tran, Le, Kretova, and Geissler, 2014; Gracilxalus ananjevae

Le, Dau, Hoang, and Cao, 2014; Gracixalus lumarius Le, Dau, Hoang, and Cao, 2014;

Kurixalus motokawa Nguyen, Matsui, and Yoshikawa, 2014; Kurxalus viridescens

Nguyen, Matsui, and Yoshikawa, 2014; Liuixalus catbaensis Nguyen, Matsui, and Yoshikawa, 2014; Limnonectes nguyenorum McLeod, Kurlbaum, and Hoang, 2015;

Leptolalax isos Rowley, Stuart, Neang, Hoang, Dau, Nguyen, and Emmett, 2015; Megophrys latidactyla Orlov, Poyarkov, and Nguyen, 2015; "Hylarana" attigua Oliver,

Indosylvirana Oliver, Prendini, Kraus, and Raxworthy, 2015; Indosylvirana milleti Oliver,

Prendini, Kraus, and Raxworthy, 2015; Odorrana fengkaiensis Wang, Lau, Yang, Chen, Liu, Pang, and Liu, 2015; Odorrana lipuensis Mo, Chen, Wu, Zhang, and Zhou, 2015; Pulchrana Oliver, Prendini, Kraus, and Raxworthy, 2015; Sylvirana guentheri Oliver, Prendini, Kraus, and Raxworthy, 2015; Liuixalus feii Yang, Rao, and Wang, 2015;

Liuixalus jinxiuensis Li, Mo, Xie, and Jiang in Qin, Mo, Jiang, Cai, Xie, Jiang, Murphy, Li,

and Wang, 2015; Nyctixalus Poyarkov, Orlov, Moiseeva, Pawangkhanant, Ruangsuwan, Vassilieva, Galoyan, Nguyen, and Gogoleva, 2015; Theloderma (Nyctixalus)

pictum Poyarkov, Orlov, Moiseeva, Pawangkhanant, Ruangsuwan, Vassilieva, Galoyan,

Nguyen, and Gogoleva, 2015; Leptolalax ardens Rowley, Tran, Le, Dau, Peloso, Nguyen, Hoang, Nguyen, and Ziegler, 2016; Leptolalax kalonensis Rowley, Tran, Le, Dau, Peloso, Nguyen, Hoang, Nguyen, and Ziegler, 2016; Leptolalax maculosus Rowley, Tran, Le, Dau, Peloso, Nguyen, Hoang, Nguyen, and Ziegler, 2016; Boulenophrys brachykolos Fei and

Ye, 2016; Boulenophrys minor Fei and Ye, 2016; Rana (Rana) johnsi Yuan, Zhou, Chen,

Poyarkov, Chen, Jang-Liaw, Chou, Matzke, Iizuka, Min, Kuzmin, Zhang, Cannatella,

Hillis, and Che, 2016; Theloderma annae Nguyen, Pham, Nguyen, Ngo, and Ziegler, 2016;

In 2017, 21 species have been found such as Limnonectes quangninhensis Pham, Le, Nguyen, Ziegler, Wu, and Nguyen, 2017; Leptolalax (Lalos) petrops Rowley, Dau, Hoang,

Le, Cutajar, and Nguyen, 2017; Leptolalax puhoatensis Rowley, Dau, and Cao, 2017;

Megophrys (Panophrys) brachykolos Mahony, Foley, Biju, and Teeling, 2017; Megophrys (Panophrys) daweimontis Mahony, Foley, Biju, and Teeling, 2017; Megophrys (Brachytarsophrys) feae Mahony, Foley, Biju, and Teeling, 2017; Megophrys (Ophryophryne) gerti Mahony, Foley, Biju, and Teeling, 2017; Megophrys (Ophryophryne) hansi Mahony, Foley, Biju, and Teeling, 2017; Megophrys (Brachytarsophrys) intermedia Mahony, Foley, Biju, and Teeling, 2017; Megophrys (Panophrys) jingdongensis Mahony, Foley, Biju, and Teeling, 2017; Gracixalus

sapaensis Matsui, Ohler, Eto, and Nguyen, 2017; Rhacophorus hoabinhensis Nguyen,

Pham, Nguyen, Ninh, and Ziegler, 2017

These studies provide an updated amphibian list for VN The number of amphibians has increased over time The results of these researchers also pointed out threats to the Vietnamese herpetofauna including overexploitation of trade and habitat loss due to the conversion of forest land to other uses

CHAPTER III: GOAL, OBJECTIVES AND STUDY SITE

3.1 Goal

This study aims to assess the amphibian diversity and provide baseline for different conservation purposes in Trang An Landscape Complex, Ninh Binh Province

3.2 Objectives

In order to achieve the goals, there are four objectives including:

Objective 1: To identify amphibian species composition and diversity indices in TA LC Objective 2: To determine distribution characteristics of the species followed by habitat

types and biogeographic relationships

Objective 3: To describe morphological characteristics of recorded amphibian species in

TA LC

Objective 4: To clarify impact factors and propose solutions for conservation management

of amphibian species in TA LC

3.3 Geographic location

- Trang An Landscape Complex inscribed as a UNESCO World Heritage Site on

23 June 2014, at the 38th session of the World Heritage Committee in Doha, Qatar

- The geographical coordinates: 20˚15‟24” N - 105˚53‟47” E

- TA LC covers an area of 6226 ha in districts of Hoa Lu, Gia Vien, Nho Quan, Tam Diep Town, and Ninh Binh City The complex is surrounded by a buffer zone of 6026

ha, mostly comprising paddy rice fields and villages (UNESCO 2017)

Trang An LC

3.4 Climate and hydrology

TA LC is located in the humid tropical region of Northern Vietnam There are four seasons, with the driest season being winter The average annual temperature from 23˚C to 26˚C Normally, the average temperature drops to 20˚C in the winter months, with the coldest months of January and February as low as 10˚C and rises to 30˚C in the hottest month of July and August The average precipitation is 1800 - 2000 mm per annum, almost occurs from May to October The humidity about 65 - 85% (UNESCO 2017)

3.5 Topography

Located in the humid tropical region, TA LC is a mountainous area that extends in

a northwest-southeast direction To the north and northwest of Trang An lie Bai Dinh karst hills, rising to approximately 185 meters a.s.l ; to the southwest and the south are Dong Tam – Son Ha and Tam Coc – Bich Dong karst ranges which are slightly lower at 160 meters a.s.l.; the highest summit is Truong Yen karst range, about 196 meters a.s.l (UNESCO 2017) Almost terrain of TA LC composed of a variety of classical karst cones and towers and a network of enclosed depressions connected by an intricate system of subterranean waterways Cover for limestone karst, some big river cover around the big mountains (Rabett 2016)

Figure 3.2: Land cover in Trang An Landscape Complex

3.6 Flora and Fauna

3.6.1 Flora

TA LC has more than 600 floral species, almost these species are Magnoliophyta

(Angiosprems) Among of them, there are 10 species which are listed in Vietnam‟s Red

book There have also been 7 species recorded in Vietnam for the first time (IUCN 2018)

Arboreal species about 16% of the total flora with 92 tree species rare wood such as

Indian mahogany Chukrasia tabularis and has high value (Burretiodendron tonkinense -

EN) (IUCN Red List, 2015) Besides, there are 310 species of medicinal plant have been

recorded in this area, for example, Stephania spp and Lonicera spp to treat arthritis and

skin diseases

3.6.2 Fauna

Information on the faunal assemblages in the property is extremely poor However, the wider scientific literature suggests that Vietnamese karst landscapes are important habitats for a diverse and unique fauna (Musser et al., 2006)

There are more than 200 animal species of animals recorded in TA LC (IUCN 2014) In addition, 18 species of amphibians belong to 6 orders were found to be the most obvious evidence of amphibian diversity found on limestone karsts (Luu et al., 2017)

According to survey data of the TA LC, said today aquatic ecosystems consist of about 30 species of zooplankton, 40 species of bottom animals including many rare species, especially striped turtles that need protection However, some of them are at rish

of extinct because of hunter and human activities

3.7 Economy – Society

There are about 14,000 residents, the majority of whom are families involved in subsistence agriculture, but much of the property is uninhabited and in a natural state In the part, agriculture were the main job to help local people survival or developed economic But, in recent years, they are going to TA LC to do works Each day, about

1000 times of boat went to beautiful landscape, help a lot of local people to make more money (average 200.000 VND/day/times)

CHAPTER IV: METHODOLOGY 4.1 Identify amphibian species composition in TA LC

4.1.1 Preparation and equipment

Before coming to field survey, we have to study, identify amphibian species,

searching information from documents and internet related to investigation and

composition of amphibian fauna in that area Besides, the map linked to the study area and

materials about amphibian identification were also prepared in advanced Therefore, there

were some special tools and chemicals for survey activities such as plastic bag, surgical

instruments, bottle for preservation, camera, headlamp, etiket, GPS, ethanol, ethyl acetate

Geographic coordinates and elevation were tracked with GPS Garmin 62st Air

temperature and humidity were measured with a digital compact thermos-hygro-barometer

(Atech, model: KW307-CSJ, accuracy 0.1% for temperature, 1% for relative humidity)

Additionally, information regarding the habitat as well as the distance to the ground and

the presence of a stream or trail were recorded

4.1.2 Line transect survey

 Site selection: Survey transects were establish with 14 days along streams,

limestone karst forest The survey followed line transects around the limestone karst forest

and search day-time and night-time We found frog species and tree frog species

 Designing transects survey before going to the field, choose where are easy to be

recognized and approached such as path, stream, pond…that have high humidity

 The length of transects from 3 to 5 kilometers up to types of habitats and terrain

 Creating transects based on topographic map, habitat, terrain and experiment of

local people

 Main collection time: 18:00h - 23:00h

Figure 4.1: Map of survey locations (white dots) in TA LC

4.1.3 Collecting method

All all specimens were caught by hand After taking photographs and detect, common species were released to their environment Individuals with potential

characteristics were collected for taxonomic identification in the laboratory of VNUF To

identify composition diversity of amphibian species in TA LC, we using Table 4.1 (below)

to note all species and took photograph on the survey time

Table 4.1: Field survey sheet

 Specimen fixing and preserving:

- Anaesthetization: Within 24 hours after collecting, the selected species were anaesthetized and euthanized in a closed vessel with a piece of cotton wool containing ethyl acetate for keep origin

- Fixing in 80% ethanol for 4-6 hours, and then transferred to 70% ethanol for permanent storage Tissue sample samples were preserved separately in 95% ethanol

providing information about the locality (e.g TA), field number and the date, month, year

of collection After that, it was also labeled with a museum number (VNUF A.)

4.1.5 Permission and management

-

- Specimen management: Collected specimens were deposited in the collection at the specimen room, T1 building, Vietnam National University of Forestry (VNUF), Hanoi, VN

- The following abiotic data was recorded for every single collected individual

4.2 Determine distribution characteristics of the species followed by habitat types and biogeographic relationships

4.2.1 Distribution characteristics by habitat types in TA LC

Each species conducted in difference habitat, it can be found on the ground, trees or river, pond, small stream We divided into 5 types of habitat: limestone karst forest; forest lands; forest lands mix limestone karst forest; pond, river, small stream and shrubs, seeding Specimens collected showing in this Table 4.2:

Table 4.2: Distribution of amphibian species in each habitats

 Using PAST Statistics software (Hammer et al 2001) for statistical analysis

and comparability of amphibian species composition of the study area with comparable regions The Sørensen index was used to compare the similarity of amphibian composition

between regions Areas with similar species composition will be grouped together This index is calculated based on the formula:

= 2M / (2M+N)

In this formula, M is the number of species which appears in both regions and N is the total number of species that occurs in only one area

Compute diversity index:

The various indicators used for statistical analysis in this study include:

Richness index (Margalef Index: d):

d = (S - 1)/logN Diversity index (Shannon index - Weiver: H '):

H ' = -∑( )

Dominant index (Simpson Index: 1 - D):

1 - D = 1- (ni/N) ²

S - Total species; N - total number of samples; ni - number of specimens of species

4.3 Determine morphological character of these species

 Morphological character

The morphological examination of the collected specimens from TA LC was conserved in the laboratory of the Vietnam National Forestry University (VNUF) Morphometric measurements were taken with an Etopoo digital caliper to the nearest 0.1 mm The terminology of the morphometric characters followed Orlov et al (2012)

Table 4.3: Abbreviations of morphological characters Abbreviation Character

Measurement

Body and Head

Fore limbs

F2L Second finger length

dermal fringe along outer finger

relative length of finger

Hind limbs

dermal fringe along outer toe

relative length of toes

Figure 4.2: Morphological measures

The ten measured traits were: SVL = snout-to-vent length; HW = head width; HH = head height; MW = mandible width; ND = nose-to-mouth distance; BN = distance between nostrils; ED = eye diameter; IW = inter-orbital width; FL = forelimb length; HLL = hindlimb length (a + b + c),(Glaw and Vences, 2007)

CHAPTER V: RESULTS AND DISCUSSIONS 5.1 Amphibian diversity of TA LC

5.1.1 Scientific collection

There were 92 specimens was collected from different survey site in the limestone karst forest of TA LC The specimens are very important evidence for taxonomic identification and further study in the future Among 92 specimens, 42 tissue samples were collected for molecular examination We did not collect specimens of common species (e.g., Asian black - spined toad, Günther's Amoy frog), however, the records were made based on direct observations or photographs All specimens were deposited in the collection of the VNUF for further taxonomic identification, exhibition and research purpose

Figure 5.1: Number of specimens collected in of TA LC

5.1.2 The diversity of amphibian species in TA LC

Comparing the results of 18 amphibian‟s species from previous studies (Hoang & Luu 2017), this study brings the confirmed species number of amphibians recorded from

TA LC to 19 The checklist of amphibian species in TA LC are shown in table 5.2.1

2 4 6 8 10

Table 5.1: List of amphibians of Trang An Landscape Complex

N

Trong Cave

Boi cave

Tam Quan

Tam Coc

Bai Dinh Temple

1 Duttaphrynus

melanostictus Asian black-spined toad Cóc nhà - - - O O

2 Fejervarya limnocharis Asian grass frog Ngoé - S - - -

6 Sylvirana guentheri Günther's Amoy frog Chẫu S S S S -

7

Sylvirana maosonensis Mao-Son frog Chàng mẫu sơn - - - - -

Hoang & Luu (2017)

8

Hyla simplex Annam tree frog Nhái bén nhỏ - - - - - Hoang & Luu

(2017)

9 Kaloula pulchra Asian narrowmouth toads Ếch ương S - - - -

10

Microhyla heymonsi Dark-sided chorus frog Nhái bầu hây môn - - - - - Hoang & Luu (2017)

5.Ranidae Rana sylvatica

Raorchetes parvulus Karin bubble-nest frog Nhái cây - - - - -

Hoang & Luu (2017)

16

Rhacophorus dennysi Blanford's whipping frog Ếch cây xanh đốm - - - - -

Hoang & Luu (2017)

Notes:

 Identification documents Nguyen et al., 2009; Hoang & Luu 2017; Laurie & Janalee 2018; Frost 2018

 Data sources: S = specimen, P = photograph, O = observation, “-” = unconfirmed record from this study

Figure 5.2: The species richness of amphibian families in TA LC

The chart showing that Rhacophoridae and Dicroglossidae are most dominant amphibian families

During the survey transect, Tran temple – Trong Cave were found the highest number of species (8 amphibian species) Besides, Tran temple – Boi Cave and Tran temple - Tam Quan have the same number of amphibians (6 families species) Finally, the number of amphibian‟s species in Tran temple - Tam Coc & Tran temple - Bai Dinh temple was only 1 species

5.1.3 New record of amphibian species in TA LC

Comparing with the previous research, this study will bring the species number of genus Theloderma to know from TA LC to one A research of Nguyen et al., (2016)

described a new amphibian species Theloderma annae with full characteristics Our research herein reports a new provincial record of Theloderma annae based on three

specimens (one male and two female‟s specimens) Therefore, we update some new characteristics for this species depend on picture and morphological of three specimens were collected in TA LC

Figure 5.3: New recorded for amphibian species for TA LC

5.1.4 Correlation index between regions in TA LC

Table 5.2: Similarity index (dice index) of amphibian species among survey sites

In this table, Trong Cave - Boi Cave are highest similarity index (djk = 0.85) with the same number of species appeared in two area Besides, similarity index between Tam Coc

- Trong cave and Tam Coc - Tam Quan (djk = 0.4) with the same number of specimens are smallest compare to others areas On the field the impacts of human quite large, the chance

mountain peaks and valleys which can contain many interesting new discoveries in the development of amphibian species Therefore, essential to continue conducting the taxonomic study in the genus because it probably database contains a high level about amphibian diversity, especially in the limestone karst forest

Figure 5.4: Similarity cluster analysis of the amphibian among survey sites 5.2 Habitat diversity of amphibian species

5.2.1 Distribution of amphibian species in habitats

Almost 11 amphibian species belong to five habitat (Table 5.4) on the field

by observation and picture

Table 5.3: Distribution of amphibian species in habitats

Habitat

5 Occidozyga martensii Cóc nước macten x x x x

Note: 1: Limestone karst forest; 2: Forest lands; 3: Forest lands mix limestone karst

forest; 4: River, stream, pond, lake; 5: Shrubs, seeding

Figure 5.5: Diversity of species according to habitat

The chart showed us the percentage of diversity species according to habitat In this pie, habitat 1, habitat 3 and habitat 5 were the highest percentage with 7 species live in that area Besides, habitat 4 dropped to 6 species, about 19% of the total Therefore, with 15 %

of the total, habitat 2 was the smallest than other habitat with just 5 species can be found

Habitat 1 22%

[CATEGORY NAME]

15%

Habitat 3 22%

Habitat 4

19%

Habitat 5 22%

Habitat 1 Habitat 2 Habitat 3 Habitat 4 Habitat 5

5.2.2 Comparing the amphibian list to these among karst protected areas

Based on the result of Nguyen et al (2003), Ziegler & Vu (2009), Nguyen et al (2009), Pham (2016), statistic used software PAST (Hammer et al 2001) showing the results (Table 5.5) the diversity indices (i.e Shannon_H, Simpson_1-D, and Margalef) are the same as above, respectively Comparing Trang An Landscape Complex with other areas where have same limestone karst forest such as Cuc Phuong National Park, Phong Nha - Ke Bang National Park and Kim Hy Nature reserve Trang An Landscape Complex and Kim Hy Nature Reserve have the same number of amphibian (20 species), but poor than Cuc Phuong National Park (35 species) and Phong Nha - Ke Bang National Park

Table 5.4: Comparing diversity indices of amphibian diversity to other region

Table 5.5: Comparing dice index of amphibian diversity to other region

Note: PN-KB: Phong Nha-Ke Bang

Based on results comparing composition of amphibian‟s species by statistic, dice index of Trang An - Cuc Phuong is highest (djk = 0.7) because of geographic location are

close and similarity of terrain, weather, climate The smallest of dice index is Phong Nha -

Ke Bang (djk = 0.56)

Figure 5.6: Comparing dice index of amphibian diversity to other region 5.3 Descriptions of recorded amphibian species in TA LC

Based on morphological, specimen identification and observation on the filed

survey, we recorded a total of 10 species Remarkably, we found a specimen of

Theloderma frog Theloderma annae in the karst forest in TA LC This is a first time,

Theloderma annae was recorded since it was described by Nguyen et al (2016) In

addition, we discovered 10 new recorded for the amphibian fauna of TA LC

DICROGLOSSIDAE

1 Fejervarya limnocharis (Gravenhorst, 1829)

Common name: Asian grass frog

Vietnamese name: Ngoé

Specimens examined (n=2) Two adult male (VNUF A.2017.74 and VNUF

A.2017.103)

Morphological characteristics of the specimens are consistent with the species

description of Gravenhorst (1829), Ziegler (2002), Orlov et al., (2007), Nguyen et al., (2009), Pham et al., (2017): medium size (SVL 44.8 - 46.1 mm); head longer than wide (HL 17.4 - 18.2 mm, HW 14.1 - 15.1 mm) snout pointed, longer than eye (SNL 6.9 - 7.6

mm, ED 4.1 - 5.1 mm), canthus rostralis obtuse; loreal region oblique; vomerine teeth present; nostrils closer to tip of snout than to the eye (NS 2.2 - 2.6 mm, EN 4.6 - 5.3 mm); internarial distance and upper eyelid broader than interorbital distance (IN 2.9 - 3.9 mm, UEW 3.2 - 3.7 mm, IOD 2.0 - 2.2 mm); tympanum distinct (TYD 2.9 - 3.9 mm) ; supratympanic fold distinct

Fore limbs length (FFL 7.9 - 9.4 mm); hand length (HAL 18.4 - 20.2 mm); relative lengths of fingers II<IV<I<III; small fingers, finger II smaller than finger I; fingers free of webbing without discs; dermal fringe along outer finger absent

Hind limbs slender (HLL 75.4 - 78.8 mm), femur length and tibia length not too much difference (FeL 19.2 - 20.5 mm, TbL 19.7 - 20.5 mm); relative lengths of toes I<II<III<V<IV; toes webbed,; inner metatarsal tubercles present, outer metatarsal tubercles small; ventral surface smooth; supratympanic fold distinct; nuptial pads present

Figure 5.7: (A) Dorsal and (B) ventral views of the adult male

Fejervarya limnocharis (VNUF A.2017.74) Photos: Nam T Nguyen

Coloration in life: Dorsal head and body greyish green with camouflage-pattern;

light vertebral stripe present or absent; lips with dark vertical bars; limbs with transverse

bars or spots; ventral surface white; black marbled throat

Figure 5.8: Micro habitat of Fejervarya limnocharis on the ground in TA LC

Photos: Vinh Q Luu

Ecological notes: Two adult males (VNUF A.2017.74 and VNUF A.2017.103) collected on the ground, at an elevation of 37 m a.s.l, temperature 27˚C and relative

16/05/2017

Distribution: In Vietnam, this species has been reported from the entire country

Elsewhere, this species is known from China, Myanmar, Laos, Cambodia, Thailand, Malaysia and Indonesia (Frost D., 2018)

Remark: In the central of dorsal view, there is no white bands from snout to anus

2 Occidozyga lima (Gravenhorst, 1829)

Common name: Pointed-tongued Floating Frog

Vietnamese name: Cóc nước sần

Specimens examined (n=5) Five specimens, two adult males (VNUF A.2017.92;

VNUF A.2017.96) and three adult females (VNUF A.2017.06, VNUF A.2017.94, VNUF A.2017.95)

Morphological characteristics of the specimens are consistent with the species

description of Ziegler (2002) Orlov (2007), Nguyen et al., (2009): body plump, small size (SVL 23.7±2.23 in males, 26.3±2.19 in females); head small, longer than head width ( HL 8.5±0.88 - 9.8±1.11, HW 6.8±0.78 - 8.4±2.16 in males-females); snout pointed, broader than eye diameter and width of upper eyelid ( SNL 2.5±0.54 - 2.6±0.35, ED 2.1±0.23 - 2.0±0.32, UEW 1.9±0.32 - 1.8±0.16, in males – females); nostrils oriented upwards, slightly raised; tympanum distinct; supratympanic fold granulated; internarial distance between nostrils longer than interorbital distance, nearly two times (IN 2.5±0.32 - 2.6±0.27, IOD 1.7±0.23 - 1.8±0.18 in males – females)

Fore limbs length (FFL 2.5±0.13 - 2.6±0.26 mm, in males - females); hand length (HAL 7.5±0.87 - 7.7±0.78 in males – females); relative lengths of fingers II<I<IV<III; small fingers and short, hands webbed; dermal fringe along outer finger absent

Hind limbs small and thin (HLL 36.3±2.41 - 38.3±2.09 mm, in males - females); the length of tibia longer than length of femur (TbL 9.3±0.53 - 9.8±0.53mm, FeL 8.9±0.81

- 9.3±0.68 mm, in males - females); relative lengths of toes I<II<III<V<IV; dermal fringe along outer toe distinct; webbing reaches toe tip

Figure 5.9: (A) Dorsal and (B) ventral views of the one adult male Occidozyga

lima (VNUF A.2017.06) Photos: Oanh V Lo

Ecological notes: Five specimens, two adult males (VNUF A.2017.92; VNUF A.2017.96) and three adult females (VNUF A.2017.06, VNUF A.2017.94, VNUF A.2017.95) almost collected in limestone karst forest on the stream or shrub, the air temperatures ranged from 22 ˚C to 26.3 ˚C and relative humidity was between 73% and 85%

Coloration in life: Skin sharply granulated through the body, with yellow dots There is a

white-yellow bar in the central dorsal view from snout to anus There are many dark marking and blotches in the body

B A