EDUCATION AND TRAINING SCIENCE AND TECHNOLOGY GRADUATED UNIVERSITY OF SCIENCE AND TECHNOLOGY --- Bui Tuan Hai THE STUDY ON TAXONOMY, DISTRIBUTION AND PHYLOGENETIC RELATIONSHIP OF EULI
Trang 1EDUCATION AND TRAINING SCIENCE AND TECHNOLOGY
GRADUATED UNIVERSITY OF SCIENCE AND TECHNOLOGY
-
Bui Tuan Hai
THE STUDY ON TAXONOMY, DISTRIBUTION AND PHYLOGENETIC RELATIONSHIP OF EULIPOTYPHLAN
SPECIES (MAMMALIA: SORICOMORPHA)
FROM VIETNAM
Curriculum: Zoology Major ID number: 9 42 01 03
SUMMARY OF BIOLOGICAL PhD THESIS
Hanoi – 2021
Trang 2Vietnam Academy of Science and Technology
Supervisor 1: Assoc Prof PhD Le Xuan Canh
Supervisor 2: Prof PhD Masaharu Motokawa
The thesis can be found at:
- Library of Graduated University of Science and Technology
- National Library of Vietnam
Trang 3INTRODUCTION
1 Rationale
The order Eulipotyphla has four families Erinaceidae, Siricidae, Talpidae, and Solenodontidae, containing a total of 530 species and 55 genera (Wilson & Mittermeier, 2018) Under IUCN (2019-2) status the species ranged from 11 Critically Threatened (CR), 41 Endangered (EN),
25 Vulnerable (VU) and 16 Near Threatened (NT)
In the last 20 years, the previous studies on Eulipotyphla in Southeast Asia (including Vietnam) focused on discovering new species and improving phylogenetic analyses of its members While the taxonomy and systematic of Eulipotyphla species has long been debated The distribution
of some species were explored by administrative units or forest areas, but there is no research assessed by zoogeography, habitats and elevation Moreover, recent studies show that knowledge on phylogenetic relationship
of Vietnam’s eulipotyphlan species/populations is still limited
Thus, this study, named “The study on taxonomy, distribution and phylogenetic relationship of eulipotyphlan species (MAMMALIA: EULIPOTYPHLA) from Vietnam” aims to assess species composition,
diversity, distribution and phylogenetic relationship of gymnures, shrews and moles from Vietnam The research was approached by combining morphological and molecular
Task 1: Explore the eulipotyphlan species diversity in Vietnam
- Provide the updated checklist of gymnures, shrews and moles
- Describe, analysis and compare the external morphology and skull
Trang 4- Provide the identification keys for eulipotyphlan species
Task 2: Discover species distribution
- Discover species distribution by zoogeography
- Discover species distribution by habitats
- Discover species distribution by elevation
Task 3: Analysis the phylogenetic relationships between populations, species and genera of Eulipotyphla
- Analysis the phylogenetic variation among the populations of wide distribution species
- Analysis the phylogenetic variation among species and between genera
- Construct the phylogenetic trees
CHAPTER 1 LITERATURE REVIEW 1.1 Overview of studies on mammal composition in the world
Scientific research on mammals is believed to officially begin with Aristotle's findings (384–322 BC) The mammal species composition of the world was studied by a lot of scientists such as Trouessart, Wilson & Reeder, Nowak, Honacki, etc To date, Burgin et al (2018) and the American Society of Zoology (2019) have listed 6495 species (96 extinct, 6399 existent) belonging to 1314 genera, 170 families, 27 orders
1.2 Overview of taxonomic classification of Eulipotyphla in the world
The “Insectivora” was used as a wastebasket taxon for a variety of small to very small, relatively unspecialised mammals that feed upon insects
by paleontologists Haeckel (1866) reclassified the order Lypotyphla including the families Soricidae, Talpidae, Erinaceidae, Tenrecidae, Chrysochloridae, Solenodontidae and Nesophontidae (extinct)
Recently, molecular and evolution studies by Nishihara et al (2009),
He et al (2010), Sato et al (2016), Brace et al (2016), Spinger et al (2017, 2018) showed a close relationship of families Soricidae, Talpidae, Erinaceidae and Solenodontidae and classified them in the Eulipotyphla order Synthesizing studies of Eulipotyphla in the world, Wilson &
Trang 5Mittermeier (2018) have listed 530 species, belonging to 55 varieties, 4 families
1.3 Overview of studies on mammals in Vietnam
1.3.1 Before 1945
The history of studying the fauna in Vietnam is considered to have started in the XVIII century with works such as "Van Dai Loai Ngu", "Phu Bien Tap Luc" by Le Quy Don (1724 - 1784)
In early XIX century, research on wild mammals from Vietnam has been offically begun with the surveys of Finlayson et al (1826) Then, the specimens of these surveys were studied and published by Dustales (1874,
1893, 1898), Germain (1887) và Gurney (1889),…
In the late XIX and early XX centuries, studies on the fauna in Vietnam continued to be carried out by European experts such as Milne - Edwards (1868 - 1874), Morice (1875), Brousmiches (1887), Heude (1894), Billet (1896), Pousargues (1898), Pavie (1904), Boutan (1906), Bonhote (1907), Osgood (1932), Blane (1932), Delacour (1934), Bourret (1942) These studies contributed many new species for Vietnamese fauna
1.3.2 The period of 1954 - 1975
During this period, the researches on mammals were widely conducted
in the Northern Vietnam A lot of domestic and international articles about mammals were published at that time especially such as Vo Quy et al (1961), Le Hien Hao (1962, 1964, 1969, 1973), Dang Huy Huynh and Vu Dinh Tuan, Dang Huy Huynh and Cao Van Sung (1965, 1973), Dao Van Tien (1966), Le Vu Khoi (1970), Van Peenen et al (1967, 1969, 1970, 1971); Duncan et al (1970, 1971)
1.3.3 The period from 1975 to present
After 1975, the studies on biological resources, including mammal fauna were promoted and had great developments with many major national projects and international cooperation The results were published in monographs, domestic and international journals Some typical publications
on this duration following: Cao Van Sung et al (1980); Dang Huy Huynh
et al (1981, 1994, 2007, 2008, 2010); Dao Van Tien (1985); Cao Van Sung
Trang 6(1989); Le Vu Khoi (2000); Kuznetsov (2006); Dang Ngoc Can et al (2008); Nguyen Xuan Dang and Le Xuan Canh (2009)
1.4 Overview of studies on Eulipotyphla in Vietnam
The first record of insectivores in Vietnam was reported by Osgood (1932) with six species Referring to recent studies, Bui Tuan Hai et al (2019) provided the latest updated checklist of Eulipotyphla in Vietnam including 37 species and sub-species, belonging to 12 genera and 3 families Regarding molecular research, Bannikova et al (2011, 2017, 2019), Zemlemerova et al (2013, 2016), Shinohara et al (2014, 2015), He et al (2014, 2018), Abramov et al (2015, 2017a, b, 2018), Li et al (2019) studied
on the genera of Crocidura, Blarinella, Chimarrogale, Chodsigoa, Episoriculus, Euroscaptor in Southeast Asia and South China, including
samples obtained from Vietnam
CHAPTER 2 SUBJECTS, SCOPE, MATERIALS AND
METHODOLOGY 2.1 Subjects, scope and materials
This study focused on taxonomy, distribution and phylogenetic relationships of insectivores (Mammalia, Eulipotyphla) in Vietnam
We examined a total of 831 specimen vouchers including 677 bodies and 778 skulls collected in Vietnam We conducted DNA analysis of a total
87 sequences from 81 specimen vouchers of 23 species Among them, we used our 60 sequences and 125 sequences data from GenBank which were published in earlier studies for reconstructing phylogenetic relationships
2.2 Duration and locations
This thesis showed my results from 2012 to 2019 with 40 field surveys, including 407 days in the field (from 2017 to 2019 (PhD course): 21 surveys and 167 days in the field) Among them, a part of the results from 2013 to
2015 were published in my master thesis named “Study on classification and phylogenetic relationship of white-toothed shrews of the genus
Crocidura (Mammalia: Soricidae) in Vietnam” which were updated and
supplemented in this study
Trang 7Laboratory studies were conducted at the Department of Nature Conservation (Vietnam National Museum of Nature), Department of vertebrate zoology (Institute of Ecology and Biological Resources, Zoological Institute of Russian Academy of Sciences in Saint-Peterburg and The Kyoto University Museum
2.2.1 General characteristics of nature in Vietnam
Vietnam is located in the tropical belt of the Northern Hemisphere, with diverse geological, topographic, climatic and hydrological features, leading
to biodiversity and abundance in general and mammal species in particular
2.2.2 Biogeographical zonation of mammals in Vietnam
On the basis of factors such as topography, geomorphology, climate, environment, adaptation and distribution of flora and fauna in Vietnam, Nguyen Xuan Dang and Le Xuan Canh (2009) and Le Vu Khoi et al (2015) have divided into 5 biogeographical distribution ranges, including Northeast, Northwest, North Central, South Central and Central Highlands, and Southern
Fig 2.1 Map of survey and
locations of studied specimens
The study was carried
out based on the specimens
collected at 66 locations in
37 provinces and cities in
Vietnam Among them, I
surveyed and collected
new specimens at 33
locations in 21 provinces
(Fig 2.1)
Trang 82.3 Methods
2.3.1 Collecting specimens
Interviewing and selecting survey areas: Interviewing experts, rangers
and villagers to collect information
Trapping: The cage traps, Sherman traps, pitfall traps and tunnel traps
were set on the trails to collect the specimens with the baits such as dried fishes, earthworms, craps, cereals or mixed instant food for cats
Field preparation of specimens: The captured individuals were
labeled, photographed and measured The external body measurements were
as follow: head and body length (HB), tail length (T), ear length (E), hind foot length (HF1, HF2), fore foot size (FF1, FF2, FFW), bristle hairs (BrH) and weight (Wt) were taken by tapeline and digital caliper Specimens were fixed and preserved in ethanol
Other information: Collect the other information about environment,
habitats, locations, elevation
2.3.2 Preparing skulls
Stripping: Using a scalpel size #11 separate the skulls from the bodies Cleaning: Boil, degrease and remove muscle on the skulls
2.3.3 Comparing classification
- Step 1: Perform the tables of skull measurements
- Step 2: Calculate descriptive statistics (Max, Min, Average, Stdev)
- Step 3: Conduct the Principal components analysis (PCA) using
PAST on skull measurements
- Step 4: Analyze, argue, compare different characteristics
- Step 5: Classify based on the results of morphological comparison, statistical analysis, and BLAST on GenBank
2.3.4 Distribution status
Geographical distribution of Eulipotyphla in Vietnam was accessed according to the actual distribution combined with information on the distribution of those species in the vicinity
Arrange distribution of Eulipotyphla from Vietnam in habitats as follows:
- Group 1: Limestone mountain (NĐV), Other mountain (NĐ)
Trang 9- Group 2: Woody forest (RCG); Bamboo forest (RTN); Shrubs and grass (CBC); Agricultural farm (ĐR); Residential area (DC)
- Group 3: Constantly affected by humans (T); Less affected by humans (I)
Arrange distribution of Eulipotyphla from Vietnam in 4 altitudinal
gradients above the sea level, including: I: : I: 0 - 600m; II: 600 - 1600m;
III: 1600 - 2600m; IV: higher than 2600m The division of 4 altitudinal
gradients was based on the temperature of the hot season on the basis of the elevational belt in the tropical monsoon climate zone and the sub-monsoonal equatorial climate zone (Vu Tu Lap, 2007)
Similarity coefficient was calculated using the formula: H = 2c / (a + b) In particular, “c” is the number of species present in both areas, habitats altitudinal zones “a” and “b” respectively in the number of species present
in each region, habitat or altitudinal zone (Sorensen’s index, Shannon và Wiener, 1963)
2.3.5 Molecular analysis
- Sampling: tissue in the liver or thigh muscles
- Studied gene locus: Cytb, 12S and Rag 1
- Primers: Cytb
SoriF TGACATGAAAAATCATCGTTG SoriR CCA TCT CTG GTT TAC AAG AC The sequence of the RAG-1 gene was amplified using the primer pair RAG1-F1851 and Rag1-R2486 (Sato et al., 2004) The 12S gene was amplified with the following primer sets: L613 (Mindell et al., 1991) and H1066 (Suzuki et al., 1997)
- DNA purification: kit DNeasy Blood and Tissue (QIA gen)
- PCR: using Ultra-Pure Taq for cytb, and Dreamtaq for Rag1 and 12S
- Gel electrophoresis: using gel agarose 1%
- Sequencing: FirstBase Sequencing INT in Malaysia
- Sequence alignment and phylogenetic analysis: using MEGA X,
Kakusan 4.0, MrBayes 3.2.7, Tracer 1.7.1, Treefinder, Figtree 1.4.4 and Ai
Trang 10CHAPTER 3 RESULTS AND DISCUSSION 3.1 Species composition of Eulipotyphla in Vietnam
3.1.1 Species composition
A total of 37 species and subspecies of insectivore mammals belonging to
13 genera in three families has been recorded from Vietnam (Table 3.1)
Table 3.1 Eulipotyphla in Vietnam
a Talpinae
10 Euroscaptor subanura* Short-tailed mole
11 Anourosorex squamipes Chinese mole shrew
12 Blarinella quadraticauda Sichuan short-tailed shrews (NT)
13 Chodsigoa caovansunga Cao Van Sung’s shrew
14 Chodsigoa hoffmanni Hoffmann’s shrew
16 Chimarrogale himalayica Himalayan water shrew
17 Chimarroalge varennei* Varenne’s water shrew
18 Episoriculus baileyi Bailey’s shrew
19 Episoriculus macrurus + Arboreal Brown-toothed Shrew
20 Episoriculus umbrinus + Hidden Brown-toothed Shrew
b Crocidurinae White-toothed shrew
Trang 11No Scientific name English name
21 Crocidura annamitensis* + Annamite Shrew
22 Crocidura attenuata Asian Grey Shrew
23 Crocidura tanakae Taiwanese Grey Shrew
24 Crocidura dracula Dracula Shrew
25 Crocidura fuliginosa Southeast Asian Shrew
27 Crocidura indochinensis + Indochinese Shrew
28 Crocidura wuchihensis Hainan Island Shrew
29 Crocidura sapaensis* Sa Pa Shrew
30 Crocidura kegoensis* Ke Go Shrew
31 Crocidura phanluongi Phan Luong’s Shrew
32 Crocidura phuquocensis* Phu Quoc Shrew
33 Crocidura rapax + Chinese White-toothed Shrew
34 Crocidura sokolovi* Sokolov’s Shrew
35 Crocidura zaitsevi* Zaitsev’s Shrew
36 Suncus etruscus Etruscan Shrew
3.1.2 Conservation status
The insectivores in Vietnam have been not listed in the Vietnam Red Data Book 2007, Decree no 160/2013/ND-CP, Decree no 06/2019/ND-CP and the appendices of CITES
As assessed by the International Union for Conservation of Nature (IUCN), in 37 insectivores species and subspecies in Vietnam with the 01 Endangered (EN), 01 near-threatened species (NT), 14 least-concern species (LC), 11 species data deficient (DD) and 9 species has not been evaluated
3.2 Description and taxonomy
3.2.1 ERINACEOMORPHA
Fairly large Gymnure, large eyes, zygomatic arch is complete, yellowish-brown feathers
3.2.1.1 Short-tailed Gymnure - Hylomys suillus
- Diagnosis: Tail short 14 – 24% of head and body length
- Dental formula: 3/3, 1/1, 4/4, 3/3 x 2 = 44
3.2.1.2 Shrew Gymnure - Neotetracus sinensis
Trang 12- Diagnosis: Tail 50 – 60% of head and body length
- Dental formula: 3/3, 1/1, 3/3, 3/3 x 2 = 40
3.2.1.3 Hai Nan Gymnura - Neohylomys hainanensis
- Diagnosis: Tail 26 – 31% length of head and body
- Dental formula: 3/3, 1/1, 4/3, 3/3 x 2 = 42
3.2.2 TALPOMORPHA
Minute eyes, tiny ears without a pinna, forefoot are greatly modified
for tunnel digging
3.2.2.1 Long-tailed Mole - Scaptonyx fusicaudus
- Diagnosis: Tail length 56% head and body
- Dental formula: 3/2 1/1 4/4 3/3 x 2 = 42
3.2.2.2 Latouche’s Mole – Mogera latouchei Thomas, 1907
- Diagnosis: Tail short, less than 14% of head and body length
- Công thức răng: 3/2, 1/1, 4/4, 3/3 x 2 = 42
3.2.2.3 Kuznetsov’s Mole – Euroscaptor kuznetsovi
- Diagnosis: P1 height = P2 height Tail long and club-shaped 10.28 –
12.6% of head and body length
- Dental formula for genus Euroscaptor: 3/3, 1/1, 4/4, 3/3 x 2 = 44 3.2.2.4 Orlov’s Mole – Euroscaptor orlovi
- Diagnosis: P1 height > P2 height Tail long, club-shaped 11 – 14%
of head and body length
3.2.2.5 Vietnamese Mole – Euroscaptor subanura
- Diagnosis: Tail very short 2 – 4,2% of head and body length
3.2.2.6 Small-toothed Mole – Euroscaptor parvidens
- Diagnosis: Caudal vertebrae 12 – 14
3.2.2.7 Ngoc Linh Mole – E p ngoclinhensis
- Diagnosis: The upper premolars P1 and P3 similar in height P1–P3
with remarkable metastyles
3.2.3 SORICOMORPHA
A Soricinae - True Brown-toothed Shrews
Very long and narrow rostrum, tiny eyes, the teeth are red-tipped or brown-tipped when unworn, characteristic odor unclear
3.2.3.1 Sichuan Short-tailed Shrew - Blarinella quadraticauda
- Diagnosis: Red-tipped teeth 5 upper unicupid teeth
Trang 13head and body length with bristle hairs covering all tail
3.2.3.4 Lowe’s Shrew - Chodsigoa parca
- Diagnosis: dark at the tip with a tuft of hair
3.2.3.5 Bailey’s Shrew - Episoriculus baileyi
- Diagnosis: The upper unicuspid row is longer than other Episoriculus species in Vietnam T/HB ~ 100%
- Dental formula for genus Episoriculus: (I 1/1, U 4/1, P1/1 M 3/3) (x2) = 30 3.2.3.6 Arboreal Brown-toothed Shrew - Episoriculus macrurus
- Diagnosis: Tail longer than head and body Semi-aboreal
3.2.3.7 Hidden Brown-toothed Shrew - Episoriculus umbrinus
- Diagnosis: I1 short, I1 talon is lower than that of U1
B Soricinae - Pseudo Brown-toothed Shrews
White teeth, larger than other species of Soricidae in Vietnam
3.2.3.8 Mole-shrew - Anourosorex squamipes
- Diagnosis: Tiny eyes There are no external ear pinna, forefoot like
shrew Very short tail
- Dental formula: (I 1/1 U 2/1 P 1/1 M 3/3) (x2) = 26
3.2.3.9 Himalayan Water Shrew - Chimarrogale himalayica
- Diagnosis: Hindfeet are lined with prominent fringes of white hairs
Posterior borders of the P1 and M1 are deeply concave and the apex of the
palatal suture is straight and extends to the level of the M2
- Dental formula for genus Chimarrogale: (I 1/1, U3/1, P1/1, M3/3) (x2) = 28 3.2.3.10 Varenne’s Water Shrew - Chimarrogale varennei
- Diagnosis: The moderately prominent protocone and hypocone of
the P1 and M1 so the posterior borders are less concave, and the palatal
suture extends anteriorly beyond the anterior border of the M2
C Crocidurinae - White-toothed Shrews
True white-toothed Suncus murinus is the biggest shrew and Suncus