MINISTRY OF EDUCATION
AND TRAINING
VIETNAM ACADEMY OF SCIENCE
AND TECHNOLOGY
INSTITUTE OF ECOLOGY AND BIOLOGICAL RESOURCES
----------
NGUYEN VAN VIET
STUDY ON CLASSIFICATION OF BATS MYOTIS GENUS AND
PIPISTRELLUS GENUS (CHIROPTERA: VESPERTILIONIDAE) IN SOME
PROVINES OF NORTHERN VIETNAM BASE ON MORPHOLOGICAL
CHARACTERISTICS, ECHOLOCATION CALL AND MOLECULAR GENETICS
Speciality: Zoology
Code: 9 42 01 03
PHD DISSERTATION SUMMARY
Hanoi - 2019
The dissertation was completed at the Institute of Ecology and Biological
Resources, Vietnam Academy of Science and Technology
Scientific Supervisors:
1. Prof. Dr. Le Vu Khoi
2. Assoc. Prof. Dr. Vu Dinh Thong
Reviewers:
1. Reviewer 1: Assoc. Prof. Dr. Le Dinh Thuy
2. Reviewer 2: Assoc. Prof. Dr. Hoang Xuan Quang
3. Reviewer 3: Assoc. Prof. Dr. Nguyen Huu Duc
The dissertation is going to be defended at the Institute of Ecology and
Biological Resources, Vietnam Academy of Science and Technology on ….
May, 2019.
Further information of this doctoral dissertation is available at:
- The library of the Institute of Ecology and Biological Resources.
- Vietnam National Library.
INTRODUCTION
Rationale of the study
The bat order (Chiroptera) contains unique mammals that have the ability to fly,
using ultrasound in their flying and catching activities. The bat order is the second
largest order of mammals, following Rodentia, the largest.
Bats play an impotant role in the natural ecosystem and argicultural ecosystem and
is one of the potential animals for some scientific majors.
Myotis genus and Pipistrells genus, of Vespertilionidae, include insectivores and
have large contribution in both plain and mountainous region, so they are useful species
for the natural ecosystem and argicultural ecosystem. Species of these two genera have
small, similar bodies, which are difficult to distinguish, so the taxonomic system of
Myotis genus and Pipistrellus genus are the most complex of the Vespertilionnidae in
general and of the bat order in particular. Some problems of the taxonomy system and
phylogeny of these two genera have been controversial and raised much arguments
among studists.
In Vietnam, studies on the taxonomy of these two genera have been rare, mostly
based on morphology. Meanwhile, studies on echolocation and molecular genetic are
still limited. In fact, up to now, there have not been any separate studies on the
taxonomy system of these two genera.
Northern Vietnam has a large area with complex and diversified terrain. These lead
to the diversification of species of all creatures in general and of bat order in particular,
including Myotis genus and Pipistrellus genus. Although many researches on bats have
been done in North Vietnam, but they mainly focus on the North-East and the NorthCentre Coast, while the North-West have rarely been researched.
Because of all the reasons above, we have chosen the subject “study on the
classification of Myotis genus and Pipistrellus genus Bats (Chiroptera: Vespertilionidae) in
some Northern Vietnam provinces, basing on their morphological characteristics,
echolocation call and molecular genetics” to identify their diversitfication in taxonomy
classification and add more data of ultrasonic call and molecular genetics so as to classify
the bats of Myotis genus and Pipistrells genus in some Northern Vietnam provinces.
Objectives of the study
1. Provide morphologic characters of the species of Myotis genus and Pipistrellus
genus in some northern Vietnam provinces.
2. Provide data about echolocation and molecular genetics of some species of
Myotis genus and Pipistrellus genus recorded in field research during the
implementation of this thesis.
3. Supplement data about the distribution area of some species of Myotis genus and
Pipistrellus genus in Vietnam.
4. Build a taxonomic key of given species of of Myotis genus and Pipistrellus genus
in field research basing on their morphologic characters.
3
Research contents of the study
1. Identify the characteristics of the species of Myotis genus and Pipistrellus genus
recorded in field research..
2. Study the characteristic echolocation and molecular genetics of some species of
Myotis genus and Pipistrellus genus recorded during the implementation of this thesis.
3. On the basis of different morphologic characteristics, build a taxonomic key of
the species of Myotis genus an Pipistrellus genus recorded in field research.
4. Update the data about the distribution area of species of Myotis genus and
Pipistrellus genus recorded in field research.
Scientific and practical signification of the thesis
1. Provide up-to-date and complete data about the species of Myotis genus and
Pipistrellus genus in some provines of northern Vietnam.
2. Provide a lot of data about the morphologic characteristics, echolocation,
molecular genetics, and distribution area of some species of of Myotis genus an
Pipistrellus genus during the implementation of the research.
3.The outcome of the thesis is the foundation for identifying the species of Myotis
genus and Pipistrellus genus, basing on a comprehensive study on their external
characteristics, echolocation and molecular genetics.
4. The outcome of the thesis is an important scientific base for future research on the
classification and ecology, as well as for managing and conserving the biological
diversity of the species of Myotis genus and Pipistrellus genus in Vietnam.
New findings and discoveries of the thesis
1. Identify a list of 14 species and subspecies of Myotis genus, 5 species of
Pipistrellus genus, in which there is one newly recorded species (M. altarium) to the bat
fauna of Vietnam and 2 subspecies (M. cf. montivagus and M. cf. laniger) different from
others descriptions before.
2. Provide some morphologic identifying characteristics in the classification of
species of Myotis genus and Pipistrellus genus, including the relative position of length
of forearm to tip of muzzle, and the character of sagittal crest and zygoma.
3. Provide data about the echolocation of 4 species of Myotis (M. chinensis, M.
pilosus, M. muricola, M. siligorensis) and 3 species of Pipistrellus (P. javanicus, P.
abramus, P. coromandra). Among them, the data of echolacation of P. coromandra, P.
abramus have been newly recorded in Vietnam.
4. Add new data about the molecular genetics of COI gene of 9 species and
subspecies of Myotis genus (M. pilosus, M. chinensis, M. annectans, M. altarium, M.
siligorensis, M. cf. laniger, M. montivagus, M. cf. montivagus, M. muricola), 2 species of
Pipistrellus genus (P. javanicus, P. tenuis) and 16S gene of P. abramus.
The structure of the study
The thesis includes 149 pages, divided to 3 chapters together with the preface,
conclusion, recommendations and references, in which there are 28 tables, 33 figures,
and 256 references. The appendix contains 3 parts: appendix 1 provides data about
4
specimen symbols and sites of specimen collection; appendix 2 offers data about the
extracting and sequencing of gene COI, 16S ; appendix 3 contains the results of
sequencing of 22 gene COI of 9 species and subspecies of Myotis genus and Pipistrellus
genus, and 1 gene 16S of 1 species of Pipistrellus genus.
5
CHAPTER 1. AN OVERVIEW OF THE STUDY
1.1. An overview of bat classification in the world
1.1.1. Classification of the bat order (Chiroptera)
1.1.1.1. Classification by morphologic characteristics
The classification of the bat order has been carried out since the mid-seventeenth
century. Before 1980, all studies on the classification of the bat order were based on their
morphologic characteristics, and some typical authors include Linnea (1758),
Blumenbatch (1780), Dobson (1785), Miller (1907), Thomas (1915).
Nowadays, the classification by morphologic characteristics has been the main
method but scientists usually combine it with the classification by echolocation and
molecular genetics to produce more precise results.
1.1.1.2. Classification by ultrasound call
From 1938 to 1944, Griffin carried out some first experiments about echolocation of
bats and concluded that bats emit the sound in the form of ultrasound and can hear
echolocation. This study is the basis for later studies about ultrasound call and the
classification by ultrasound call.
The classification by ultrasound call was done first by Fenton and Bell (1981). The
trend of these studies has been growing up in quantity, range, scale of the study.
1.1.1.3. Classification by molecular genesis
Today, the majority of findings on new species of bats are based on the combination
of analysing morphologic characteristics and molecular genesis, mainly using
technology of DNA barcoding (Hebert et al, 2003). Studies of molecular genesis have
been done mainly on some genes in cytoplasm as COI (Mytochondrial cytochrome c
oxidase subunit once), Cytochrome b etc. Besides, molecular genesis is used in
constructing a taxonomic system of taxon of upperspecies, origins of species, relation
between species and upperspecies... The results of studying of molecular genesis also
raise different viewpoints of taxonomic system of families and genara of the bat order.
1.1.2. Classification of Myotis genus and Pipistrellus genus
1.1.2.1. A brief classification of Vespertilionidae
The taxonomic system of Vespertilionidae appeared first in Gray’work (1821). Whereby,
Vespertilionidae contains 8 genera (Megadermes, Rhynolophus, Nycterus, Rhynopoma,
Thaphosores, Fespertilio, Plecotus, Barhastella). The taxonomy of this family changed in
the following works of Dobson (1875), Miller (1907), Tate(1942).
Up to now, Vespertilionidae has been divided into 3 group, 5 subfamilies
(Vespertilioninae, Myotinae, Kerivoulinae, Murininae, Miniopterinae), 45 genera and
about 350 species, distributing all over the world. In Vietnam, this family contains about
57-59 species of 20 genera.
In Vespertilionida, Myotis genus and Pipistrellus genus belong Myotinae subgenus
and Vespertilioninae subgenus respectively.
6
1.1.2.2. Classification of Myotis genus
1.1.2.2.1. Classification by morphologic characteristics
Before 1980, the classification of Myotis genus bats was completely based on
morphologic characteristics. However, each author has different viewpoints on their
classification and use of taxonomic key systems, so their results of classification are not
similar and exact.
Miller (1907) is the first one to put Myotis genus into the taxonomy system, basing
on analysing external characteristic, and the shape of skull and teeth. Penis was also
used in classifying species of Myotis genus.
By combining the analysis of morphologic characteristics and molecular genetics,
Simmons (2005) concluded that there had not been enough evidences for dividing
Myotis genus into subgenus. Accordingly, Myotis genus contains 103 species divided
into many diferent groups, and species that contain similar characteristics are put into
one group.
1.1.2.2.2. Classification by echolocation
The echolocation of the species of Myoits genus only contain FM wave, not the real
CF wave. Duty cycle is from 11 to 14 calls per second, in some species, it is from 9 to 15
calls per second; the frequency range changes a lot, from about 30 to 100 kHz, FmaxE is
about 40-55 kHz; DUR is about 3 – 5 ms. The graph of echolocation call of Myotis genus
is usually like a backslash in shape (\); some species have the inflection point at the top,
middle, or bottom of the graph, which is typical for this species. Echolocation calls
contain 2 or many harmonies; the first harmony is usually more dominant than the second
one. The exact classification of the species of Myotis genus also depends on the species,
the quantity of echolocation call analysed and the method of analysis.
1.1.2.2.3. Classification by molecular genetics
Studies of classification of species of Myotis genus by molecular genetics have been
done in many countries in the world. These studies usually combine morphologic
characteristic analysis with molecular genetics in finding new species, species with
unclear classification and studies of phylogeny and upper species.
1.1.2.3. Classification of Pipistrellus
1.1.2.3.1. Classification by morphologic characteristics
Miller (1907) is the first one to put Myotis genus into taxonomy system. The
taxonomy of this genus changed in the following studies of Kuzyakin (1944, 1950,
1965), Simpson (1945), Koopman (1984, 1985), Cobert và Hill (1992), Norwak (1994).
The classification of Pipistrellus genus is usually based on morphologic
characteristics, shape of skull, teeth and penis, and shape and relative position of
premolar.
With the combination of classification by morphologic characteristics and by
molecular genetics, Simmons (2005) demonstrated that 6 subgenus, Pipistrellus,
Vespadelus, Hypsugo, Falsistrellus, Neoromicia và Arielulus are independent genera,
except Perimyotis of Pipistrellus.
7
1.1.2.3.2. Ultrasonic calls and classification by ultrasonic calls
Ultrasonic calls of species of Pipistrellus usually contain FM waves and CF waves,
initiating at FM waves and terminating with CF waves. These species usually have over
10 calls per second; the frequency range is narrow, modulating about 35-85 kHz; FmaxE
is about 40-52 kHz; DUR is usually above 6 ms, exept for P. abramus with recorded
DUR to be 2,6 ms. The rhythm of the calls among Pipistrellus genus is usually unstable.
Up to now, studies about classification of species of Pipistrellus by echolocation call
are limited, mainly focus on describing the characters of echolocation call of one or few
species in specific area.
1.1.2.3.3. Classification by molecular genetics
Studies on molecular genetics of species of Pipistrellus genus have been done in
many places all over the world, but are more limited than those on Myoits genus. The
researches mainly focus on finding new species and relationship among species.
1.2. An overview of bat classification in Vietnam
1.2.1. A brief history of the study
A sketch of bat study in Vietnam is split into stages basing on the viewpoint of
the historic study of mammal by Huynh Dang Huy et al (2008).
1.2.1.1. Before 1945
Studies and records on bats in Vietnam in this period were rare and done by foreigners.
Many records in this period were not exact and reclassified in the following studies.
1.2.1.2. From 1945 to 1975
In this period, studies on bats were expanded, but done separately in Northern and
Southern Vietnam due to the war condition.
1.2.1.3. From 1975 to now
In this period, the bat studies were expanded and more specialized.
The period before 1994, studies on bats were usually combined in studies on
mammals.
From 1994 to now, specific studies on bat are rising more and more in quantity and
scope, which results in the increase of bat species recorded in Vietnam. Sixteen new bat
species were found in Vietnam from 1994 to 2005, among them are 2 new species and
1new genus. From 2006 to now, 22 new bat species were reported in Vietnam.
1.2.2. The context of research on ultrasonic call and molecular genetics of bat
species in Vietnam
Before 2007, studying on classification of bats in Vietnam was only based on
morphological characteristics, shape of skull, teeth and penes. Since 2007, classification
by echolocation call and molecular genetics has been rising more and more, so the
classification of bat have been more and more exact; new bat species recorded have
been rising more and more in quantity.
The studies using bat echolocation call in Vietnam have been mainly done on
species of Rhinolophidae and Hipposiderdae. The studies using bat echolocation call on
other bat families are limited.
8
Studies using molecular genetics of bat have been carried out for over a recent
decade, so the results of these studies are still limited. They mostly focus on announcing
new species or reclassifying the unclear species. The study of newly found bats by using
molecular genetics is also limited.
1.2.3. Studies on the classification of Myotis genus
Myotis adversus (Vespertilio adversus) was the first species in Myotis genus
recorded in Vietnam by Morice (1875). The following others were recorded including M.
chinensis (Menegaux, 1906); M. siligorensis alticranius và M. longipes (Osgood, 1932); M.
mystacinus và M. siligorensis (Van Peenen và cs., 1969); M. hasseltii (Topal, 1974). From
1992 to 2018, 15 species of Myotis genus have been found respectively. Especially, in
2018, basing on the analysis of COI gene of M. annatesae group species, Kruskop informed
M. cf. annatesae as a new subspecies.
Before 2008, all the studies on the classification of bats in Myotis genus were only
based on morphological characteristics, shape of skull and teeth, particularly canine and
premolar.
The classification of species in Myoits genus by echolocation call in Vietnam has
been limited with only some research outcomes of echolocation call of some species in
this genus by Borisenko et al (2008), Furey et al (2009), Kruskop (2013).
So far, the study on molecular genetics of species in this genus in Vietnam has been
limited. There were only studies of Cytochrome b gene of M. phanluongi, of COI gene of
M. muricola and M. annatesae in 2008, 2013 and 2018 respectively. In 2010, Francis and
cs concluded that it is impossible to differentiate M. laniger và M. annamiticus species by
mitochondria genetics.
1.2.4. Studies on classification of Pipistrellus genus
Thomas (1904) described a new species of Pipistrellus genus in Vietna: P. raptor.
Today, this species has been record as P. ceylonicus. From 1927 to 1928, Thomas
continued to describe 2 new species, P. tralatitius, P. coromandrus tramatus. However,
by 1992, Cobert and Hill proved these two species to be P. javanicus, P. tenuis
respectively. In 1969, Van Peenen et al reported 3 species of Pipistrellus genus in
Southern Vietnam (P. coromandra, P. javanicus (P. abramus), P. minus). In 1997, Bate et
al recorded 3 new rare species of Pipistrellus genus in Vietnam, namely P. cardonae, P.
pulvaratus, P. paterculus.
The classification of species of Pipistrellus genus by morphological
characteristics basing mainly on morphological forearm, premolar, canine, length of
penis, and shape of baculum.
Recently, the studies on echolocation call of species of Pipistrellus genus have been
limited. Furey (2009), and Kruskop (2013) recorded some characters of echolocation in
some species.
Kruskop (2013) studied the characteristic of genes in mitochondria of P. javanicus,
P. tenuis and P. coromandra.
9
CHAPTER II. LOCATION, MATERIALS AND METHODS OF RESEARCH
2.1. Location and research time
Our research were carried out from 2012 to 2017 with 15 surveys in 17 areas of 15
provinces in Northern Vietnam (Fig 2.1). Each survey lasted 3-5 days. Besides, we
collected specimens in some places including Hai Duong city (Hai Duong province),
Vietnam Academy of Science and Technology (Ha Noi capital), Hai Hau district (Nam
Dinh province). There are 18 surveys with 68 days for specimen collection in field trips
in total.
Figure 1: Diagram of location of field surveys
Note: refers to location in this research.
Numbers (1-18) refer to the ordinal number of field surveys.
Resource: Administrative Map of Vietnam
2.2. Tools, chemicals and materials of research
Dụng cụ, hóa chất: bao gồm bẫy thu cầm, lưới mờ, vợt bắt dơi, các dụng cụ và hóa
chất phục vụ cho việc làm mẫu, thu tiếng kêu siêu âm.
Tools, chemicals: involve harp trap, mist net, mobile trap, materials and chemicals
used for analysing specimens and recording echolocation calls.
Materials
+ 369 body specimens were used for studying morphological characteristics, sizes of
the body, including 30 specimens of Myotis genus and 22 specimens of Pipistrellus
genus collected in our surveys and 322 specimens collected from previous field surveys.
+ 125 skull specimens were extracted from body specimens used for studying
morphological characteristics of skull.
+ 39 specimens of breast tissues, brain tissues, spinal tissues of 14 species and
subspecies of Myotis genus, 5 species of Pipistrellus genus were collected in field
surveys to be used for analysing genetic characteristics. Two primers (primer 1,2) were
used to replicate ADN sequence of COI gene and 1 primer (primer 2) to replicate ADN
sequence of the 16S gene.
10
2.3. Methods of research
2.3.1. Methods of collecting specimens.
2.3.1.1. Collecting specimens by mist nets.
Mist nets used usually have a clathrate size of 16-20 mm, 2,5-3 m in height, 3-12
m in width. Each mist net usually contains 3-5 shelves.
2.3.1.2. Collecting specimens by harp trap
Harp traps used usually contain a metallic frame which is 1,6 x 1,8 m in height
and 1,8-2,0 m in width with 4 banks of vertical lines; the distance between banks is
5cm. The lines (thin wires of monofilament fishing lines) are fastened 2,5 cm apart, and
their consecutive vertical rows distance is from 1,5 to 2,5 cm.
Besides, sweep-net and mobile trap can be used for collecting bats in caves.
2.3.2. Methods of analysing specimens
2.3.2.1. Analyzing specimens in field surveys
Each specimen was analysed in the following steps: identifying fast, measuring the
size of body, taking photographs, and recording echolocation calls. We kept 1-3
specimens per species. Specimens were fixed by traditional methods. The specimens’
tissue with strange characters would be extracted for later molecular genetics analysis.
2.3.2.2. Analysing in laboratory
- Measure external index of all specimens collected in this study and other previous studies.
- Measure index of skull and teeth after Bates and Harrison, 1997; Scorba et al 2003;
Dietz and Von Helversen, 2004; Francis, 2008)
- Dye penes and analyse them after Bate et al, 2005.
- Identify specimens: Identify each specimen basing on references about species of
Myotis genus and Pipistrellus genus (Corbet and Hill, 1992; Bates and Harrison, 1997;
Borissenko and Kruskop, 2003; Kruskop, 2013).
- Specimens with strange morphological characters would be described in details and
identified by PCA method or analysed molecular genetics of COI gene or 16S gene. The
verification process by T-test and Geomean function were completed in Microsoft excel 2010.
2.3.3. Method of recording and analysing echolocation calls
We recorded bats’ echolocation calls by PCTage and 6700X machine (Petterson
Electronik, Sweden) in two states: stable state and moving state. All echolocation call
datas were analysed by Selena software and Batsound software.
2.3.4. Method of ADN analysing
- DNA extracting, DNA electrophoresis on agarose gel, DNA replicating by PCA,
DNA sequencing... were done at Human Immunology Department (Institute of
Genomics Research – Vietnam Academy of Science and Technology (using primer 1),
Genetic Lab – Genetic Department and Molecular – Cell Biology Lab-Center for Life
Science Research (Faculty of Biology – Vietnam University of Science, Vietnam
National University, Ha Noi (using primer 2) and Department of Molecular systematics
and Conversation genetics (using primer 3).
- All gene sequences gained were refined by Bioedit software version 7.2.5. and
then compared with correlative sequences on Genbank by Blast.
11
CHAPTER III. OUTCOMES AND DISCUSSION
3.1. Composition of bat species of Myotis and Pipistrellus in research areas
Results after analysing 378 body
specimens, 125 skull specimens, 14
species and subspecies of Myotis genus
and 5 species of Pipistrellus genus
recorded in research areas (Table 3.1).
Basing on the results of previous
research together with new records in our
studies, 21 species and subspecies of
Myotis, 6 species of Pipistrellus have been
recorded in Vietnam.
In comparison with previous researches,
this thesis adds 01 species và 02
subspecies of bats for bat ecosystem in
Northern Vietnam namely M. altarium, M.
cf. montivagus và M. cf. laniger (Table
3.1).
Ordinal
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
Scientific name
Myotis altarium*
M. annamiticus
M. annectans
M. chinensis
M. hasseltii
M. horsfieldii
M. indochinensis
M. laniger
M.cf. laniger*
M. montivagus
M.cf. montivagus*
M. muricola
M. pilosus
M. siligorensis
Pipistrellus abramus
P. coromandra
P. javanicus
P. paterculus
P. tenuis
Source
M
M, [4, 137]
M, [4, 137]
M, [4, 137]
M, [4, 137]
M, [4, 137]
M, [164]
M, [4, 137]
M
M, [4, 137]
M
M, [4, 137]
M, [4]
M, [4, 137]
M, [4, 137]
M, [4, 137]
[4, 137]
M, [4, 137]
M, [4, 137]
Note: M indicates species recorded in these studies, *
indicates species newly recorded to fauna of northern
Vietnam
3.2. Những loài dơi ghi nhận mới cho khu
3.2. Bat species recorded newly to research area and Vietnam
3.2.1. Loài Myotis altarium Thomas, 1911.
3.2.1. Myotis altarium Thomas, 1911.
We found 14 specimens with strange characters during the study. Morphologic
characteristics were described in details (pelage colour, fly membrane, naked part, shape
and size of ears, tragus, antitragus and cucar lobe of base toe). These characters are
similar to the characters of M. altarium reported before by Thomas (1911), Blood et al.
(1987), Zhang et al. (2010).
Analysing morphological characteristics, skull, teeth and molecular genetics
demonstrated that 14 specimens above belong to M. altarium (Figure 3.1 and Table 3.3).
12
I
II
Figure 3.1. Results of analysing the difference in size (I), shape (II) among M. altarium
(x), M. annectans (o), M. montivagus (□), M. indochinensis (∆) by PCA method
according to 5 indexes: FA, E, Cr, HF, HB
Table 3.3. Results of comparing between COI gene sequence of M. altarium in our study
with COI gene sequence in gene bank.
Ordinal
number
COI gene in our
study
Name of species
in gene bank
Query
Coverage (%)
Identity (%)
1
JF443976.1
M. altarium
95
99
2
KF111724.1
M. ikonnikovi
96
91
3
JF443001.1
M. ikonnikovi
95
90
4
JF442999.1
M. ikonnikovi
95
90
5
JF443028.1
M. petax
95
88
Note: Query coverage is the percentage of your sequence aligned to a sequence in gene
bank. Identity is a number that describes how similar the query sequence is to the target
sequence.
External characters, skull, teeth, structure of echolocation and distribution area of
this species in Vietnam are illustrated in Figure 3.3.
13
Figure 3.3: Shape of skull, teeth (A) and external shape (B), structure of echolocation call (C)
and distribution area (D) of M. altarium in Vietnam.
Note: Picture (B) and (C) were provided by Vu Dinh Thong.
Echolocation calls of this species have unclear second harmony with only FM
waves, no CF waves, and the frequency range modulates from 82,8 to 27,8 kHz; DUR
from 0.5-1,5 ms when flying and 0,9-1,4 ms when hands held. The structure of
echolocation call when flying are quite the same as that when hands held.
3.2.2. Myotis cf. montivagus
The study found 8 specimens with strange characters, among them, 3 specimens are
bigger in size, similar to the description of M. montivagus of Kruskop (2013), so we call
M. montivagus as a makeshift. Five remaining species are smaller in size than that of M.
montivagus; they have some characters similar to that of M. montivagus and other
characters are different from M. montivagus, so we call M. cf. montivagus as a
makeshift.
The external characters of M. cf. montivagus are relatively similar to M.
montivagus with pelage, morphologic characteristics of ears, flying membrane etc.
However, some characters are different from that of M. montivagus (Figure 3.4).
The results of comparing indexes of external characters like ears, teeth, skull and
sequence of COI gene of specimens between these two species are completely different
(Table 3.4, Figure 3.5.)
Compared with M. ater and M. muricola, M. cf. montivagus is a little bigger.
Comparing 5 specimens of M. cf. montivagus with 3 specimens of M. montivagus and
20 specimens of M. muricola by PCA on 5 indexes: FA, E, Cr, HF, HB (Figure 3.5)
showed that the graph of M. cf. montivagus is completely separate with graph of m.
montivagus and M. muricola. This proved that M. cf. montivagus species is completely
different from M. montivagus and M. muricola species in shape. The result of COI
14
sequencing of M. cf. montivagus (Table 3.5) indicated that M. cf. montivagus may be an
intermediate species of M. montivagus, M. ater và M. muricola.
Table 3.4. Some indexes of external characters, skull, teeth of M. cf. montivagus and M.
montivagus in this study and previous studies.
Mesurement
index
M. cf. montivagus
species
FA
Cr
HF
E
HB
GL
CCL
C1-C1
M3-M3
ZW
MW
IOW
BCW
BCH
C1-M3
C1-P4
ML
C1-M3
CPH
36,11-38.25
15.62-17.55
6.44-7.86
12.28-12.98
36.68-38.01
13.45-14.51
12.78-14
3.41-3.97
5.39-6.08
8.52-8.97
6.09-6.53
3.15-3.38
6.18-6.71
4.45-5.38
5.21-5.61
1.86-2.12
9.78-10.92
5.55-6.05
3.28-3.82
M. montivagus species
In this study
44.09-45.21
18.5-19.03
7.35-8.73
14.42-15.88
53.25-54.68
16.74
16.22
4.90
7.46
11.87
7.60
4.01
7.91
5.83
6.56
2.47
13.14
7.07
4.82
15
Görföl (2013)
Benda (2010)
39.2–41.5
15.62–15.76
14.07–14.10
4.39–4.51
6.50–6.79
7.93–8.01
3.74–3.88
7.15–7.19
5.73–5.76
6.09–6.17
2.89–2.94
11.48–11.85
6.35–6.47
-
11.78
15.27
14.57
4.48
6.50
3.73
7.23
5.50
6.12
11.78
6.48
3.85
Figure 3.4. Shape of skull, teeth of M. cf. montivagus (A) và M. montivagus (B).
I
II
Figure 3.5: The result of analysing the difference of size (I), shape (II) between M. cf.
montivagus (□) with M. montivagus (∆), M. muricola (o) by PCA at 5 indexes
(FA, E, HF, Cr, and HB).
Bảng 3.5. Kết quả so sánh trình tự của gen COI thuộc loài M. cf. montivagus trong
nghiên cứu này với trình tự của một số gen COI trên ngân hàng gen.
Table 3.5. The result of comparing between COI gene sequence of M. cf. montivagus in
this study and some COI gene sequence of species in gene bank.
Query
Identity
COI gene in
Name of species
coverage
Ordinal COI gene in
(%)
gene bank
in Gene bank
(%)
number
this study
1
2
3
4
5
6
7
8
9
10
11
12
T.140607.1
T.140607.4
HM541056.1
HM541053.1
HM541045.1
HM541055.1
JF443979.1
HM914900.1
HM914933.1
HM914900.1
JF443979.1
HM541055.1
HM541051.1
HM914903.1
M. cf. muricola
M. cf. muricola
M. cf. muricola
M. cf. muricola
M. cf. ater
M. cf. ater
M. cf. ater
M. cf. ater
M. ater
M. cf. muricola
M. cf. muricola
M. cf. muricola
16
96
96
96
96
96
96
96
97
97
97
97
97
100
100
100
99
99
99
99
100
99
99
99
99
3.2.3. Myotis cf. laniger subspecies
3.2.3. Myotis cf. laniger
The taxonomy system of M. laniger is relatively complex and unclear. This study found
41 specimens with some characters similar to that of M. laniger and M. annamiticus; some
other characters are strange, so we call them M. cf. laniger as a makeshift.
The external characters of M. cf. laniger are relatively similar to M. laniger’s in
terms of pelage, colour, flying membrane, shape of ears, length of forearms. However,
forearm and how flying membrane joins to foot of M. cf. laniger is approximately
similar to that of M. laniger’s. The feet of M. cf. laniger are a litte shorter than that of
M. laniger’s but thicker.
The shape of skull, and teeth of M.cf. lanige is relatively similar to M. laniger’s, but
the lambda structure of M. laniger’s skull is more convex than that of M. cf. laniger. The
zygomatics of M.cf. laniger is straight but this structure of M. laniger is concave toward
inside a bit. The upper camines are sharper and higher than the premolar ones. The
shape of these teeth are showed in figure 3.7.
Figure 3.7. Shape of skull, teeth of M. cf. laniger (A) and M. laniger (B)
So sánh hình thái ngoài của 37 mẫu thuộc loài M. cf. laniger với 12 mẫu thuộc loài
M. laniger bằng phương pháp PCA trên 5 chỉ số: FA, E, Cr, HF, HB. Kết quả phân tích
cho thấy đồ thị của loài M. cf. laniger và loài M. laniger nằm tách biệt nhau hoàn toàn
trong phân tích về kích thước (Hình 3.9). Kết quả này cùng với kết quả so sánh trình tự
gen COI của loài M. cf. laniger với gen COI trên ngân hàng gen (bảng 3.6) cho thấy loài
M. cf. laniger trong nghiên cứu này khác biệt có ý nghĩa với loài M. laniger.
17
Comparing the external characters between 37 specimens of M. cf. laniger and 12
specimens of M. laniger by PCA in 5 indexes: FA, E, Cr, HF, HB. The result showed
that graphs of M. cf. laniger and M. laniger are completely separated in size (Figure
3.9). This result together with the result of comparing between COI gene in this study
with COI genes in the gene bank (Table 3.6) indicated that M. cf. laniger in this study
are completely different from M. laniger’s.
I
II
Figure 3.9. The result of analysing the difference of size (I), shape (II) between M. cf. laniger
(□) and M. laniger (o) by PCA in 5 indexes (FA, E, HF, Cr, và HB)
Table 3.6. The result of comparing between COI gene sequence of M. cf. montivagus in
this study with some COI gene sequence in the gene bank.
Query
Name of
Identity
Ordinal
coverage
COI gene in this
COI gene in
species in Gene
(%)
number
(%)
study
Gene bank
bank
1
2
3
4
5
XN.10.10.2015.3
HM541036.1
HM541035.1
HM541025.1
HM541038.1
JF442937.1
6
HM541171.1
7
8
9
10
11
HM541036.1
HM541035.1
HM541025.1
JF442937.1
HM541020.1
12
PL.10.09.2017.21
PL.10.09.2017.3
HM541171.1
18
M. cf. laniger
M. cf. laniger
M. cf. laniger
M. cf. laniger
M. daubentonii
M. adversus
taiwanensis
M. cf. laniger
M. cf. laniger
M. cf. laniger
M. daubentonii
M. cf. laniger
M. adversus
taiwanensis
97
97
97
97
97
100
100
99
99
99
97
97
97
97
97
97
95
99
99
99
99
98
97
97
3.3. Morphological characteristics of Myoits and Pipistrellus species in the
research area
3.3.1. External characteristics
To identify species of Myotis and Pipistrellus in the research area, we measured and
analysed some indexes and external morphological characteristics including forearms,
ears, legs, feet of the specimens of 14 species of Myotis and 5 species of Pipistrellus.
3.3.1.1. Characters of forearms
Basing on the length of forearms, we can divide 14 species of Myotis into 4 groups:
Group 1 having FA of 61,12-69,66 mm (M. chinensis); Group 2 having FA of 51,31 – 57
mm (M. pilosus); Group 3 having FA of 40–49mm (M. annectans, M. montivagus, M.
altarium, M. indochinensis); Group 4 having FA of 28 – 39 mm (M. laniger, M. cf. laniger,
M. annamiticus, M. horfieldii, M. hasseltii, M. siligorensis, M. cf. montivagus, M. muricola).
Thus, the length of forearms can be used to identify species of group 1 and group 2.
The relative position of length of forearms to tip of muzzle and tip of ears can be used to
identify som species of Myotis (Table 3.9).
Table 3.9. The relative position of the length of forearms to tip of muzzle and tip of ears
of some species of Myotis in this study.
The relative position of the The relative position of
n length of forearms to tip of the length of forearm to
Species
muzzle
tip of ears
M. annamiticus
M. siligorensis
M. muricola
M. montivagus
M. laniger
M. horsfieldii
M. hasseltii
M. annectans
M. altarium
M. indochinensis
72
6
21
3
12
47
4
10
14
2
>3-5
<1-2
>1-2
>0-1
<1-3
<1-3
>1-2
<1-2
<1-2
<1-2
>2-4
<1-2
0
0
<3-7
<1-4
>0-1
<3-5
<8-10
>0-1
Note: n indicates the number of analysed specimens; symbols “>” and “<” refer to forearm
surpassing or not surpassing together with tip of muzzle and tip of ears.
Among 14 species of Myotis, M. annamiticus is the only species that has FA
surpassing tip of ears (3-5mm) and tip of muzzle (2-4 mm). This is an important
character to identify specimens of this species quickly and exactly.
Four species of group 3 can be identified by the relative position of the length of
forearms to tip of muzzle. The FA of M. montivagus is equal or surpassing tip of muzzle
a bit (0-1mm), 3 other species’ FA do not surpass tip of muzzle. However, the relative
position of length of forearm to tip of ears of 3 species M. altarium, M. annectans, M.
indochinensis are different (<8-10 mm,<3-5 mm và >0-1 mm respectively), so it is easy
to distingush them.
19
3.3.1.2. Characters of ears
The shape of ears of Myotis species has 3 types (Figure 3.10)
If we base on the shape and size of ears, it is impossible to differentiate exactly any
species of Myotis in this study. However, the relative position of length of forearms to tip
of muzzle can be used for identifying some species of Myotis (Table 3.10).
Figure 3.10. Basic types of ear shape of Myotis species
Table 3.10. Relative position of length of ears to tip of muzzle of some
Myotis species
Species name
n
Relative length of ear to tip of muzzle
(mm)
M. annamiticus
M. siligorensis
M. muricola
M. montivagus
M. laniger
M. horsfieldii
M. hasseltii
M. annectans
M. altarium
M. indochinensis
M. chinensis
M. pilosus
72
6
19
2
12
47
4
10
14
3
10
15
>0-1
>0-1
>0-1
>0-1
>3-4
>1-2
>1-2
>1-2
>7-10
<1-2
>0-1
<1-2
Note: n indicated the number of analysed specimens; symbols “>” and “<” refer to
forearm surpassing or not surpassing together with tip of muzzle and ears.
20
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