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GENETIC DIVERSITY IN BATS OF BAJAUR AGENCY,
FEDERALLY ADMINISTERED TRIBAL AREAS, PAKISTAN
MUHAMMAD IDNAN
2011-VA-608
A THESIS SUBMITTED IN THE PARTIAL FULFILLMENT OF
REQUIREMENTS FOR THE DEGREE
OF
DOCTORATE OF PHILOSOPHY
IN
WILDLIFE AND ECOLOGY
UNIVERSITY OF VETERINARY AND ANIMAL SCIENCES,
LAHORE
2020
To,
The Controller of Examination,
University of Veterinary and Animal Sciences,
Lahore.
We, the supervisory committee, certify that the contents and form of the thesis, submitted by
Muhammad Idnan, Regd. No. 2011-VA-608 has been found satisfactory and recommend that it be
processed for the evaluation by the External Examiner(s) for the award of the degree.
Chairman __________________________________
Dr. Arshad Javid
Member __________________________________
Dr. Ali Hussain
Member ___________________________________
Dr. Muhammad Tayyab
i
DEDICATION
My dissertation work is dedicated to
My Most Respected Grand Parents (Late)
Respected Father (Late)
Respected Mother
Beloved Brothers & Sister
&
My Family
ii
ACKNOWLEDGMENTS
All praises for Almighty Allah, the most merciful and the most compassionate, who enlightened me with the knowledge and enable me to complete my research work to meet another milestone of my
life.
First and foremost, I would like to express my immense gratitude and deepest appreciation to my research supervisor Dr. Arshad Javid (Associate Professor/Chairman Department of Wildlife and Ecology), University of Veterinary and Animal Sciences, Ravi Campus Pattoki, who has the attitude and substance to make a difference, and consistent encouragement. I consider it an honor to get a chance to work under his supervision and supportive attitude. Without his guidance, motivational attitude, keen interest, and persistent help this dissertation wouldn’t have been possible. It would be an injustice not to admire his patience and devotion to the knowledge that he showed during my research work. Without his constructive criticism and useful suggestions, this work would not have seen the light of day.
I’m very delighted to have my supervisory committee members as Dr. Ali Hussain (Assistant Professor, Wildlife & Ecology, University of Veterinary & Animal Sciences, Lahore) and Dr. Muhammad Tayyab (Associate Professor, Institute of Biochemistry & Biotechnology, University of Veterinary & Animal Sciences, Lahore) for their positive criticism and cooperation in every aspect of my Ph.D. Work. They were always available for any kind of help and assistance for the completion of my Ph.D. Work.
Many thanks to Dr. Waseem Shehzad, Director Institute of Biochemistry & Biotechnology, University of Veterinary & Animal Sciences, Lahore for his permission to work in this institute and his kind support, I am much obliged to Dr. Muhammad Imran, Assistant Professor of Molecular Biology, Institute of Biochemistry & Biotechnology, University of Veterinary & Animal Sciences, Lahore for his skilled pieces of bits of advice and suggestions. Moreover, I shall not forget the services of Mr. Arifullah, Ph.D. Scholar, Institute of Biochemistry & Biotechnology, University of Veterinary & Animal Sciences, Lahore for assisting me during the analysis of the samples. I’m also thankful to Mr. Salman M. Phil Scholar, Institute of Biochemistry & Biotechnology, University of Veterinary & Animal Sciences, Lahore for help and timely cooperation during lab work. I’m much thankful to Dr. Hamidullah for sampling in the difficult terrain of hilly areas of the FATA region. At least I’m very obliged to my teacher Dr. Mehboob Iqbal who courage me in the very difficult time of my Ph.D. Journey and I’m very obliged to Dr. Sajid Mansoor (Assistant Professor, Microbiology, University of Central Punjab, Lahore) who helped me to bring my research work into a paper form and to write my dissertation.
Finally, I shall like to express warm thanks to all the teachers, class fellows, and last but not least my parents.
Muhammad Idnan
CONTENTS
iii
DEDICATION (i)
ACKNOWLEDGEMENT (ii)
CONTENTS (iii)
LIST OF TABLES (iv)
LIST OF FIGURES (v)
ABSTRACT (vi)
LIS
T
OF
TA
BL
ES
SR. NO. CHAPTERS PAGE NO.
01 INTRODUCTION 01
02 REVIEW OF LITERATURE 15
03 EXPERIMENT NO.1 45
04 EXPERIMENT NO.2 55
05 EXPERIMENT NO.3 62
06 EXPERIMENT NO.4 69
07 EXPERIMENT NO.5 81
08 SUMMARY 92
TABLE NO. TITLE PAGE NO.
3.1 Morphological Parameters (mm) and mean Body mass (g) for Specimens of
Pipistrellus javanicus from Bajaur Agency, FATA, Pakistan. 51
3.2 GPS Coordinates of study area, Bajaur Agency, Pakistan 52
3.3 Genetic Identities of Pipistrellus javanicus species calculated by Kimura-2
Parameter based on cytochrome b analyses. 53
4.1 Morphological measurements (mm) of Eptesicus nasutus bats from FATA,
Pakistan 60
4.2 Phylogenetic analyses of Eptesicus nasutus from FATA, Pakistan by
Neighbor-joining method with bootstrap values on branches. 61
5.1 Morphological measurements (mm) of Pipistrellus coromondra and
Pipistrellus kuhlii lepidus from FATA, Pakistan 67
5.2 Estimates of Evolutionary Divergence between Sequences for Pipistrellus
coromondra and Pipistrellus kuhlii lepidus from FATA, Pakistan 68
iv
6.1 Morphological measurements (mm) of Kuhl‘s Pipistrelle (n=6) from Bajaur
Agency, Pakistan. 79
6.2 Estimates of Evolutionary Divergence for Sequences of Kuhl‘s Pipistrelle
from Bajaur Agency, FATA, Pakistan. 79
6.3 Estimates of interspecific and intraspecific identity matrix for Kuhl‘s
Pipistrelle from Bajaur Agency, FATA, Pakistan based on Kimura-2
parameter using 16S rRNA gene.
80
7.1 Morphological measurements (mm) of Pipistrellus bats from FATA, Pakistan 85
7.2 Estimates of Evolutionary Divergence between Sequences for Pipistrellus
species from Bajaur Agency, FATA, Pakistan 86
v
LIST OF FIGURES
FIGURE NO. TITLE PAGE NO.
3.1. Phylogenetic analysis of Pipistrellus javanicus by Neighbor-joining
Method using MEGA-X. 54
3.2. Bacular features of Pipistrellus javanicus. 54
4.1. Evolutionary analysis by Neighbor Joining method and General Time
Reversible model for Eptesicus nasutus from FATA, Pakistan. 60
5.1. Evolutionary analysis by Neighbour joining tree and General Time
Reversible method of vesper bats from FATA, Pakistan. 67
6.1. Morphological description of Kuhl‘s Pipistrelle A, B Pipistrelles kuhlii
lepidus C, D Pipistrelle kuhlii, E=Baculum of Pipistrelle kuhlii
F=Baculum of Pipistrelle kuhlii lepidus.
77
6.2. Evolutionary analysis by Neighbor Joining method and General Time
Reversible model for Kuhl‘s Pipistrelle from Bajaur Agency, Pakistan. 77
6.3. Evolutionary analysis by Maximum Likelihood method and General
Time Reversible model for Kuhl‘s Pipistrelle from Bajaur Agency,
Pakistan.
78
7.1. Evolutionary analysis by Neighbour joining tree and General Time
Reversible method of Genus Pipistrelle (Mammalia: Chiroptera) from
FATA, Pakistan.
84
7.2. Figure 2 Map of Study Area, Bajaur Agency, FATA, Pakistan 87
vi
ABSTRACT
Bats are the only mammals that are capable of true flight like birds. The species diversity in bats is
increasing day by day as more research work is being carried out to explore species diversity. In Pakistan, a
trend is provoking to investigate and carry out research work to explore chiropteran diversity. Bats are
representing one-third of mammalian fauna around the world and almost a quarter of all known mammalian
species of Pakistan. Pakistan is blessed with four seasons and different climatic regions so it is supposed as a
diverse region in the world concerning chiropteran biodiversity. In the rest of the world, this mammalian
group is extensively studied and is considered as one of the most suitable bio-indicator of environmental
health and diversity. During recent years, disturbances in the foraging habitats have seriously affected the
populations of bats and have led to migration in the areas from where the species were never reported
previously. The number of bat species in Pakistan is greater than already reported and new species records
are expected from the study area. The application of molecular genetic techniques extracts valuable
biological and behavioral information to document the population dynamics of the species. The present study
is the first initiative to explore the diversity of the bats inhabiting the Bajur Agency, FATA in Pakistan. Bat
samples were collected through mist nets and hand nets and captured specimens were identified up to species
and subspecies level based on their DNA sequences which is the most authentic technique to verify species
diversity. The main objective of this study was to find out genetic variations in chiropteran fauna inhibiting
hilly terrain of FATA region Pakistan and to establish a phylogenetic relationship among the bat species
inhabiting the study area. DNA was successfully isolated from wing tissues of representative bats‘ samples
collected from various regions of Federally Administered Tribal Areas (FATA); Pakistan described in
sampling areas. This study represents the first attempt to investigate a genetic study for bats identification
using sequencing analysis of these samples. In this study we found the bats belonging to Genus scotophillus,
(Scotophillus heathi, Scotophillus kuhlii), Genus Rhinopoma (Rhinopoma microphyllum), Genus Rousettus
(Rousettus leschenaulti), Genus myotis species (Myotis muricola, Myotis formosus), Genus Rhinolophus
(Rhinolophus hipposideros, Rhinolophus ferrumequinum) and Genus Pipistrellus (Pipistrellus kuhlii,
Pipistrellus kuhlii lepidus, Pipistrellus coromandra, Pipistrellus pipistrellus, Pipistrellus tenuis, Hypsugo
savii).
Key words: Bats identification, barcoding, chiropteran diversity, molecular identification, Pakistan.
1
CHAPTER 1
INTRODUCTION
Introduction:
Order Chiroptera: the order of the flying mammals – Bats, are widely distributed on earth except for,
a few isolated islands, tundra, and some deserts (Hutson and Mickleburgh 2001). 60 million years ago, bats
were evolved during the tertiary period. The order Chiroptera is divided into two sub-orders i.e.,
Microchiroptera and Megachiroptera. Microchiropterans (834 Species) or smaller bats are represented by 17
families i.e., Myzopodidae, Rhinolophidae, Megadermatidae, Rhinopomatdae, Hipposideridae, Furipteridae,
Craseonycteridae, Emballonuridae, Nycteridae, Mystacinidae, Phyllostomidae, Noctilionidae, Mormoopidae,
Thyropteridae, Molossidae Natilidae, and Vespertilionidae, mainly feed on the majority of insects, besides
insects, they also feed on frogs, fish, and mice and blood meals (Simmons 2005). They are having
distribution in Old World and New World regions. While the megachiropterans (186 Species) or the large
bats represented by only one family known as Pteropodidae, feed mainly on fruits, leaves, nectars, and
pollens. Around 50.2 Million years ago the Megachiropterans were separated from the Microchiropterans.
Microchiropterans have global distribution except in some isolated oceanic islands, Antarctica, and the
Arctic regions (Bastian and Schmidt 2008; Nowak 1994).
In Pakistan, there are three (3) genera and four (4) species of family pteropodidae. These four species
constituting the family pteropodidae are the short-nosed fruit bat (Cynopterus sphinx), the fulvous fruit bat
(Rousettus leschenaultii), Indian flying fox (Pteropus giganteus), and the Egyptian fruit bat (Rousettus
aegyptiacus) (Mahmood-ul-Hassan and Nameer 2006; Roberts 1977; Walker and Molur 2003a). In Pakistan,
there are about 8 families of bats, 26 genera, and 54 species have so far been discovered based on their
morphological basis (Mahmood-ul-Hassan 2009), this is equivalent to any region of the world with the same
climatic and topographic conditions and no data is yet available on barcoding of bats up till now in the
country (Horáček et al. 2000). Megachiropterans have a claw on the toe of their forelimbs to hang upside
down on a support while the microchiropterans lack this claw on their toes. They also can control and
maintain their body temperature, so they do not need to hibernate during winter. They comprise 15% of all
the bats' species in the world. Mainly the megachiropterans are restricted to the tropical regions of the Old
World regions of Asia and Africa (Neuweiler 2000).
In Pakistan, out of these 54 species, 31 species are representing 15 genera and 6 families which
belong to the Palearctic region, and the remaining species belong to the Ethiopian and Oriental region
(Mahmood-ul-Hassan 2009; Roberts 1977). It is estimated that the bats are constituting about 28% of
mammalian fauna in Pakistan but it is debatable for the exact number of bats‘ fauna within the territorial
boundary of the country (Roberts 1977; Walker and Molur 2003a; Wilson and Reeder 2005b). Bat fauna
related to the Palearctic region is existing in the North and Western mountains, Oriental bat fauna is
represented in the Indus plains while Ethiopian region diversity of bats is represented by south west through
coast belt of Makran region in the country (Mahmood-ul-Hassan 2009).
The work of systematics has started from the last 250 years, despite the majority of the species is still
unidentified. Currently, the task of species identification has been resolved by DNA barcoding, where a
specific sequence of DNA is used for species identification. Generally, the technology of DNA sequencing
has resolved the taxonomic disputes of many taxa, but some higher taxa have not yet been resolved precisely
as a species. The task of species identification by DNA barcoding is very useful to resolve the taxonomic
problems of cryptic species, extinct species, synonymous species, or matching the juvenile with adults.
However, DNA barcoding is proved as a standard for species identification. Specifically, for species
identification, the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene is used as a marker in a variety
of taxa for its efficiency in taxonomy (Waugh 2007).
Introduction
2
Globally, there are about 1100 species of bats around the world (Simmons 2005), which constitute
about 18 families and 202 genera(Wilson and Reeder 2005a). Chiroptera is divided into two sub-orders i.e.,
Microchiroptera and Megachiroptera. Microchiropterans (834 Species) or smaller bats are represented by 17
families i.e., Myzopodidae, Rhinolophidae, Megadermatidae, Rhinopomatdae, Hipposideridae, Furipteridae,
Craseonycteridae, Emballonuridae, Nycteridae, Mystacinidae, Phyllostomidae, Noctilionidae, Mormoopidae,
Thyropteridae, Molossidae Natilidae, and Vespertilionidae, mainly feed on the majority of insects, besides
insects, they also feed on frogs, fish, and mice and blood meals (Simmons 2005). Bats are the 2nd most
diverse group (order Chiroptera) of mammals, representing 21 families (Burgin et al. 2018). Chiroptera is
divided into two sub-orders i.e., Microchiroptera and Megachiroptera. Microchiropterans (834 Species) or
smaller bats are represented by 17 families i.e. Myzopodidae, Rhinolophidae, Megadermatidae,
Rhinopomatdae, Hipposideridae, Furipteridae, Craseonycteridae, Emballonuridae, Nycteridae, Mystacinidae,
Phyllostomidae, Noctilionidae, Mormoopidae, Thyropteridae, Molossidae Natilidae, and Vespertilionidae,
mainly feed on the majority of insects, besides insects, they also feed on frogs, fish, and mice and blood
meals (Simmons 2005).
They are having distribution in the Old World and New World regions. While the Megachiroptera
(186 Species) or the large bats represented by only one family known as Pteropodidae feed mainly on fruits,
leaves, nectars, and pollens. In Pakistan, there are three (3) genera and four (4) species of the family
Pteropodidae. These four species constituting the family Pteropodidae are the short-nosed fruit bat
(Cynopterus sphinx), the fulvous fruit bat (Rousettus. leschenaultii), Indian flying fox (Pteropus giganteus),
and the Egyptian fruit bat (Rousettus. aegyptiacus) (Mahmood-ul-Hassan and Nameer 2006; Roberts and
Bernhard 1977; Walker and Molur 2003b). Megachiropterans have a claw on the toe of their forelimbs to
hang upside down on a support while the Microchiroptera lack this claw on their toes. They also can control
and maintain their body temperature, so they do not need to hibernate during winter. They comprise 15% of
all the bat species in the world. Mainly the megachiropterans are restricted to the tropical regions of the Old
World regions of Asia and Africa (Neuweiler 2000).
It is estimated that the bats are constituting about 28% of mammalian fauna in Pakistan, however, the
exact number of bat fauna is a debatable topic with the territorial limits of Pakistan (Roberts 1977; Walker
and Molur 2003b; Wilson and Reeder 2005a). Bat fauna related to the Palearctic region is existing in North
and Western regions, Oriental bat fauna is represented in Indus plains while Ethiopian region diversity of
bats is represented by southwest through the coastal belt of Makran region in Pakistan, this is common to any
other region of the world – provided with the same climatological parameters, there is no data concerning the
barcoding on these bats in the country so far (Mahmood-ul-Hassan 2009).
Bats are playing both their economic and ecological roles as they are filling a wide range of
ecological niches in various ecosystems besides determining the health of an ecosystem. For example,
pteropodids from the Old World and phyllostomids from the New World play an important role in
maintaining both the economically and ecologically important plants by playing the roles of pollinators and
dispersers of seeds (Hodgkison et al. 2003). Two cactus species (cardon and organ pipe cactuses) belonging
to the Sonoran Desert are visited by Leptonycteris curasoae for pollination (Fleming and Valiente-Banuet
2002; Molina-Freaner et al. 2004). Old world bats such as i.e. Epomophorus wahlbergi, Rousettus
aegypticaus and Eidolon helvum help in pollinating The Baobab Tree, an economically important tree
species in all of Africa (Kunz et al. 2003).
The work of systematics has started in the last 250 years, despite the majority of the species are still
unidentified. Currently, the task of species identification has been resolved by DNA barcoding, where the
specific sequence of DNA is used for species identification. Generally, the technology of DNA sequencing
has resolved the taxonomic disputes of many taxa, but certain higher taxa have not been specifically
Introduction
3
classified as separate species. So, the task of species identification by DNA barcoding is very useful to
resolve the taxonomic problems of cryptic species, extinct species, synonymous species, or matching the
juvenile with adults. The mitochondrial cytochrome c oxidase subunit 1 (CO1) gene is used primarily for
species recognition as an indicator in several taxa for its efficacy in taxonomy (Waugh 2007).
Genetic analysis of species provides a piece of useful information at a level at which the wild species
are impacted by anthropogenic activities but also provides information about successful demographic
management of wild species (Sovic et al. 2016). Advancement in molecular techniques has revolutionized
the field of systematics and improved the taxonomy of some more complex chiropteran species. Many new
findings in the taxonomy of under-researched and species-rich tropical areas were highlighted in molecular
genetics (Clare et al. 2007; Francis et al. 2010), besides this, in temperate fauna where the relative species
diversity is low, molecular genetics has also resolved taxonomic uncertainties (Mayer et al. 2007; Mayer and
von Helversen 2001).
Species identification and characterization have a crucial role in the taxonomy and classification of
organisms. Modern taxonomy originated in the mid18th century has described up to 1.7 million species of
organisms (Stoeckle 2003). Besides this, to study the relationship of living beings with each other various
behavioral and morphological parameters are taken into consideration. Unsurprisingly, the larger animals are
given a priority for description and the smaller ones mostly remain unknown in sciences (Blaxter 2003).
For example, a remarkably large number of species are known to be found in fewer than 10 percent
of the vertebrates within the phylum Nematoda, most of which have not been recognized. Even among the
larger animals' species identification has also remained a taxonomic problem e.g., in the case of the African
elephant which has long been considered as a single species has become the subject of debate by a study of
mitochondrial and nuclear genomes which place it in two separate species (Comstock et al. 2002; Debruyne
2004; Roca et al. 2005a).
It is estimated that the earth‘s biota is constituting about 10 to 100 million species of eukaryotes
(Whitfield 2003). Such a large number of species is presenting a challenging task for taxonomists by
conventional identification methods. Even though, the impact of the internet and consenting for
advancements in communications, the assignment of taxonomic identification is prodigious. Besides,
variations in phenotypic characters and genotype of organisms - used for taxonomic identification can
potentially lead to identification errors, elusive species, or multiple stages of development in animal life
history can raise misperception (Hebert et al. 2003a).
Field biologists are confronted with the certainty of species diversity due to improvements of the
system for species recognition and its appropriate accessibility worldwide. Such problems of species
identification are also being faced by the people in the trade of endangered species, the fisheries sector,
identification of pest species and their control for spreading the diseases, accurate lineage identification of
extinct species, and regulation of biological materials across the world. By perceiving these issues, a concise,
simple, and accurate procedure should be employed for species identification is required to overcome these
issues for identification. As more species are being discovered day by day, the taxonomic data is becoming
more problematic. Species identification by morphological characteristics requires training and expertise
without which this process of identification is difficult. Recent advances in molecular technology have
strengthened the species identification process by using short DNA sequences, which are recognized as
species labels, in a process called DNA barcoding. The varied DNA sequences are intraspecific
differentiations that determine the order of magnitude for species identification. It is not part of the taxonomy
of DNA, nor is it a phylogenetic reconstruction tool. It simply offers a way of directly linking sample
specimens to current voucher specimens and taxonomic records. Choosing an effective portion of DNA is
essential to the effectiveness of DNA barcoding. That level of mutation must be slow enough to reduce
Introduction
4
intraspecific variance yet to illustrate interspecific variation sufficiently rapidly. To promote sequence
matching, it must be reasonably straightforward to gather and should contain as few insertions or deletions as
possible. Many advantages over nuclear DNA are provided by mitochondrial DNA (mtDNA). The DNA
mutation rate is inversely proportional to the size of the genome, according to Drake's observations.
Therefore, compared to mtDNA, nuclear DNA undergoes a comparatively slow mutation and for this reason,
it would take a much longer nucleotide sequence than is required for mtDNA to have a barcode capable of
identifying organisms. MtDNA exists in animals as a single circular double-helical molecule containing 13
protein-coding genes, 2 ribosomal genes, a control area without protein-coding, and multiple tRNAs (Waugh
2007).
That being said, ample variation to distinguish between species is shown by the nucleotides of the
gene which codes for it. Alternatively, intraspecific variation between organisms is normally <10% of that
found in this gene. In comparison, it is uncommon to insert and remove (Blaxter 2004). In the last two
decades, conservation biology and ecology have both been significantly impacted by genetics (Frankham
2005; Frankham et al. 2002; Hedrick 2001). Genetics has made valuable contributions to understanding the
impact of habitat fragmentation, genetic erosion on species extinction and endangerment, incorporating the
complexities of species adaptation to new environmental environments, contributing to the development of a
contemporary science biology file called ―Conservation Genetics‖ (Ouborg et al. 2006). Whereas several
conservation efforts measured at native scale or regional levels, they could affect the biotic consequences of
a universal phenomenon, more notably the recent global warming and climate changes and the effects on
extinction rate and population decline or imbalance are all theorized to be above the background levels
(McLaughlin et al. 2002).
The key ramifications for biodiversity on several scales of climate-induced environmental changes
are diverse, frequently dynamic, and volatile, including species interaction, species distribution spread,
population structure, and phenology (McCARTY 2001; Walther et al. 2002). From several studies, it is
observed that there is a relationship of environmental variations on population densities, growth rate, and on
varieties of species for example terrestrial birds (Sæther et al. 2005), large terrestrial herbivores (Coulson et
al. 2006), marine birds (Barbraud and Weimerskirch 2003), crabs and salmon (McCann et al. 2003), and
small mammals (Stenseth et al. 2003).
Alterations in species spatial structures could lead to habitat depletion in a population, affecting
genetic drift, resulting in a decrease in the effective population size (Ne), affecting genetic diversity and the
evolutionary degree of a species (Bijlsma et al. 2000; Spielman et al. 2004a; Spielman et al. 2004b).
Variations of species spatial arrangements are evident when it is clear from recent global warming
consequences that the spatial pattern of the species towards poles is around 6.1 km in a decade. The
"pinnacle trap syndrome" is an apparent example of a direct consequence of an average temperature rise. i.e.,
When temperatures increase, animals inhabiting mountain peaks are forced to migrate to higher elevations.
They may not have an escape route and may become regionally extinct because while the species may
survive, the limitation of the suitable habitat limits the carrying capacity and thus the size of the
population. Due to the rise in temperature per unit area, limited habitat ranges are more than accelerated by
human-induced habitat destruction, which can decrease the exchange of individuals (and subsequently gene
flow) between species. There can also be some potentially beneficial consequences of the transition in
distributional range as it can put previously separated populations into contact, raising gene transfer, which
usually increases population genetic diversity but could also hinder their local adaptation (Lenormand 2002).
In a species, the residual effect of the complex relationship between the selective pressure that acts
on the population and gene flow is the real degree of adaptation. High levels of gene transfer may either
minimize or hinder the ability to adapt to local conditions (Comins 1977) or can add important new genes for
potential adaptation or improve the ability to adapt to local conditions (Orrock 2005). Finally, outbreeding
Introduction
5
depression can also subject the populations to the possibility of decreased health (Marr et al. 2002; Sagvik et
al. 2005).
A requirement for adaptation is heritable genetic variance. Consequently, the amount of genetic
variation present is one key concern of conservation genetics. Only if the rate of adaptive adaptation at least
compares to the rate of environmental change will species persist (Bürger and Lynch 1995). The existence of
heritable variance includes all the evolutionary responses of quantitative traits (traits due to two or more
genes) to selection (Lynch and Walsh 1998). The combined effect of individual genes is represented by the
additive genetic variance effect, while the dominance effect that is not inheritable is the product of
associations between certain genes (Lynch 1995).
On the whole, small fragmented populations of bats are having low genetic diversity (Kristensen et
al. 2005; Palo et al. 2004). Such a reduction in genetic diversity has two possible intimations i.e., (1) under
fluctuating environmental conditions and in a fragmented habitat, low genetic diversity in a population poses
a long-term challenge for adaptations and growth (Lande and Shannon 1996) and (2) small but scattered
populations suffer from inbreeding depression, i.e., there is an increase in the relationship between
individuals and homozygosity, particularly autozygosity. This provides those communities with an imminent
challenge (Keller and Waller 2002).
For animals that usually outcross so they may have a genetic problem, mainly due to the presence of
recessive deleterious alleles, hence inbreeding depression is dangerous for the survival of species. When the
population becomes small, the genetic problems are expressed, which often results in the decline of extreme
fitness (Hedrick and Kalinowski 2000; Spielman et al. 2004a), and the risk of extinction is increased (Brito
and de Viveiros Grelle 2004; Brito and Grelle 2006).
There is also a general opinion that biodiversity conservation largely relies on genetic diversity
conservation. Consequently, conservation genetics appears to play a vital role in the implementation of a
short- and long-term biodiversity conservation policy. By attempting to compare genomic, socioeconomic,
and phenotypic properties of the same populations, recent experiments and simulations of conservation
genetics are starting to expand in scale and effect (Basset et al. 2001; Strand 2002).
In the genetic makeup of obstructed populations, environmental influences and their changes are
replicated. Also, minor changes in environmental factors, both by demographic and selective responses, will
influence the genetic makeup of populations (Schwartz et al. 2007). Understanding the implications of
population demographic stochasticity includes a thorough knowledge of local population size variations, the
likelihood of extinction, and prospects for colonization, as well as reproductive success, which can be
obtained from population dynamics research (Boyce et al. 2006).
Nearly two decades ago, with the introduction of molecular phylogenetic techniques, new tools
became available to distinguish groups of organisms that are morphologically identical to each other but
genetically distinct enough to be considered different organisms (Bickford et al. 2007; Pfenninger and
Schwenk 2007; Yoder et al. 2005). This analysis reveals cases of morphological features of phylogenetic
sister species that are so similar that they cannot be readily separated from each other and are thus referred to
as cryptic species. The phenotypic characters used in the taxonomic classification in such examples do not
represent the same degree of distinction as the genetic markers (Baker and Bradley 2006).
Besides, there are various cases within mammals where phenotypically related species do not form
monophyletic groups and provide evidence of morphological convergence and paraphylaxis (Funk and
Omland 2003; Ruedi and Mayer 2001). Numerous cryptic species have been described over the last few
years from across the world and through various biological groups (Pfenninger and Schwenk 2007);
Madagascar has provided a remarkable number of animal kingdom examples, including ants, beetles,
Introduction
6
amphibians, reptiles, bats, and terrestrial mammals (e.g., (Goodman et al. 2008; Monaghan et al. 2005; Olson
et al. 2004; Smith et al. 2005; Vences and Glaw 2005; Yoder et al. 2005).
Since the biological content submitted to forensic genetics laboratories is always of poor quality (trace
degradation), the analytical tool selected must be extremely sensitive and accurate. It seems that it will be
more useful to study mitochondrial DNA (mtDNA) sequences than nuclear DNA markers since the high
number of mtDNA copies found in each cell greatly enhances the specificity of the analysis. It is well known
that mtDNA can be the only source for the study of very old and extremely deteriorated specimens, and also
in the examination of samples containing very small quantities of DNA, such as hair shafts (Wilson et al.
1995).
Overall, giving priority to conservation activities in the light of scarce resources is one of the biggest
obstacles facing conservation biologists (Faith 1992). Defining these goals can include judging species on
their endangered status, financial or environmental importance, individuality, diversity, or 'charisma.'
Genetics may play a crucial role in leading to this judgment process (O'Brien 2005). The use of molecular
evidence for environmental purposes assumes greater importance. Recognizing the population dynamics of a
species can also be used to conclude both historical and present behavioral processes and thus population
history (Burland et al. 2001).
At the inter-population stage, molecular experiments have demonstrated considerable genetic variation in
bats (Burland and WILMER 2001). Even so for the majority of temperate bats, females display a pronounced
philopathy, with a clear social system impacting the population structure of the species. For example,
(Burland et al. 2001) observed genetic isolation between the Plecotus aurite maternity colonies, which were
situated in close vicinity to each other and had no physical boundaries between them (Burland and Wilmer
2001).
The vast taxonomic and functional diversity of bats makes them perfect as bioindicators (Patterson et
al. 2003). In reality, bats are one of the most varied and geographically scattered groups of live mammals.
They constitute some of the largest non-human aggregations of mammals and can be one of the most
abundant groups of mammals when counted in numbers of individuals (Kunz et al. 2003) only members of
the order Rodentia surpass bats in many species, and more than 1116 species of bats have been identified
(Simmons 2005). The recent and rapid production of next-generation benchtop sequencers such as the 454
GS Junior (Roche), Ion Torrent (Promega), and MiSeq (Illumina) have made NGS technology applicable and
feasible for people working in various fields of applied genetics (Liu et al. 2012).
The DNA barcode is a short sequence of nucleotides extracted from a suitable part of the genome of the
organism that is used to classify it at the species level. Intra-specific variation in this series is the order of
magnitude smaller than that found inter-specifically, and this provides how organisms are distinguished. It is
not part of the DNA taxonomy; neither is it a phylogenetic restoration tool. It offers a way of connecting
samples directly to current voucher specimens and taxonomic information. The identification of an effective
portion of DNA is crucial to the effectiveness of DNA barcoding. Its mutations must be gradual enough to
reduce intra-specific variation but quickly enough to illustrate inter-specific variation (Hebert et al. 2003a).
DNA was successfully isolated from wing tissues of bats‘ samples collected from various regions of
Federally Administered Tribal Areas (FATA); Pakistan described in sampling areas. This study represents
the first attempt to investigate a genetic study for bats identification using sequencing analysis of these
samples. In this study, we conducted several analyses to investigate genetic structure in the out bats
collection. Herein, we explore the species limits of another group of FATA vertebrates, the bats belonging to
Genus scotophillus, (Scotophillus heathi, Scotophillus kuhlii), Genus Rhinopoma (Rhinopoma
microphyllum), Genus Rousettus (Rousettus leschenaulti), Genus myotis species (Myotis muricola, Myotis
Introduction
7
formosus), Genus Rhinolophus (Rhinolophus hipposideros, Rhinolophus ferrumequinum) and Genus
Pipistrellus (Pipistrellus kuhlii, Pipistrellus kuhlii lepidus, Pipistrellus coromandra, Pipistrellus pipistrellus,
Pipistrellus tenuis, Hypsugo savii).
8
CHAPTER 2
REVIEW OF LITERATURE
Numerous studies have shown that cryptic species are common in a variety of organisms (Bickford et
al. 2007; Mayer and von Helversen 2001; Waugh 2007). The rate of discovery of cryptic species has
dramatically increased over the past two decades, largely due to the use of molecular data (Bickford et al.
2007). A second problem is that morphological characters under selection may produce a pattern that
contradicts the actual evolutionary history of a species. Examples of this include Anolis lizards and Myotis
bats. Morphological similarities within these two genera are a result of convergent selective pressures for
habitat type (Anolis) or foraging style (Myotis) and not common evolutionary histories (Hoofer and Bussche
2003; Losos et al. 1998; Losos and Warheitf 1997).
More than 200 years ago, the work on systematics started, but the majority of the species are still to
be recognized. Recently, advancement in DNA barcoding has resolved the problem of species identification.
In DNA barcoding, various species are identified with the help of a specific sequence of DNA. As this
technology identified many phyla still some higher genera are not identified specifically on species level.
The barcoding technique is very important to resolve many taxonomic problems of species that are
morphologically indistinguishable, superseded species, or those who have some genetic similarities with
their ancestors. However, the DNA barcoding technique is set as a scale to caliber the sequencing of many
species. Specifically, for the recognition of species, the mitochondrial cytochrome c oxidase subunit 1 (CO1)
gene is used as an identifier in a variety of taxa for its efficiency in taxonomy (Waugh 2007).
It can be inferred that the molecular data is not enough for the proper identification of a species, as it may
also suffer from a wide range of issues (Vogler and Monaghan 2007). Thus, DNA is not an ideal tool for the
detection of present biodiversity and emerging speciation because the neutral gene mutations may aggregate
at an insignificant rate than the variations that occur in the morphological traits at the time of natural
selection (Hickerson et al. 2006; May 2001; Rodriguez and Ammerman 2004). Furthermore, DNA analysis
results may also mislead to the apparent features of a certain community. Additionally, Introgression,
incomplete lineage organization, differences between the genetic as well as in the phylogenetic tree are all
some factors that can conceal the true patterns of species divergence over time (Rubinoff 2006; Rubinoff et
al. 2006).
Besides all of these hazards and perils, DNA taxonomy is still being implemented successfully to several bat
species (Order Chiroptera) (Baker and Bradley 2006; Clare et al. 2007). Numerous studies have used the
DNA sequence-based data to find out cryptic diversity within all genera and also to get a clear picture of the
phylogenetic relationships within and among different genera of bats (Hoffmann and Baker 2003; Hoffmann
and Baker 2001; Porter and Baker 2004; Ruedi and Mayer 2001).
In addition, many molecular genetic techniques have become fruitful to study population genetics, behavioral
and evolutionary biology where the use of traditional methods such as direct observation of the species‘
representative or a population is greatly is a difficult process (Burland and WILMER 2001). Moreover, most
of the bats inhabiting the temperate zone tend to move between roosting sites during the year and the sexes
frequently use different roosts. It is wonderful to know that in many species the bat females raise their
offspring in the maternal colonies and share their roost with males sometimes (Burland et al. 2001).
So, in this regard, the applications of various molecular genetic techniques show valuable biological as well
as behavioral information to the whole population dynamics of the desired species under study. Anyhow, the
Review of Literature
9
use of molecular data for conservation purposes has shown to assume greater applicability. Moreover, the
structure of the species population can be used to explore and estimate the past and present scenario of the
species from a behavioral and historical point of view (Burland et al. 2001).
Furthermore, the emergence of molecular genetic techniques based on nucleotide sequence analysis
has also provided a golden chance to describe the behavior of bats, population dynamics, and diversity in a
region (Burland et al. 2001). Bats are dispersed due to their ability to fly thus they have become capable of
covering large distances. However, many factors can surely influence the extent to which the genetic
populations are separated via specific boundaries, these include migratory and mating behavior, physical
barriers (in the way of gene flow), and historical colonization patterns (Burland and Wilmer 2001).
Moreover, the bats are considered bioindicators based on their substantial taxonomic and functional
diversity (Patterson et al. 2003). Bats are undoubtedly the most geographically diverse as well as a dispersed
group of mammals in the whole world as compared to the others. They also form some of the largest non-
human associations of mammals and lie among the most abundant groups of mammals when measured in
numbers of individuals of all mammalian species (Kunz et al. 2003; O'Shea and Bogan 2003).
For these reasons, many researchers have sought an alternative or supplementary approach to
morphological taxonomy. DNA taxonomy, the use of molecular data to describe species, has emerged over
the past two decades as an alternative to morphological species delineation (Vogler and Monaghan 2007).
Molecular markers have been used successfully to detect cryptic species (Baker and Bradley 2006)and to
provide valuable information on patterns of evolution and gene flow (Giordano et al. 2007; Hajibabaei et al.
2006; Ruedi and Mayer 2001).
Biodiversity stretches the idea of the range of variations that exist within living things. It is not long
ago that genetic diversity has also become a point of discussion when considering species conservation. It is
found that survival is directly associated with the variability of gene pool at both the species level as well as
population level. The resistance to diseases is weakened by lack of genetic variability and the prevalence of
problems related to genetics is enhanced at the same time and the fitness of a species, as a result of natural
evolutionary change can also be minimized (Frankham 2005).
The application of genetic techniques for conservation-related issues is of prime value as
conservation biology is seeking help by advances in statistical analysis and molecular biology (Avise 1996).
The fundamental element of evolutionary variations and speciation is DNA. The sequence of nucleotides is
distorted as a result of demographic evolution and selective pressures, leaving characteristic changes in
sequences to draw together in the end moreover are instructively regarded to the evolutionary history of
species or population (Page and Holmes 1998). However, it might be possible to determine the evolutionary
relationship of taxonomic units by alteration of ecological niches, habitat loss, and decline in population or
by any other selection pressure (Sherwin and Moritz 2000). Conservation genetics is working at three levels
i.e., species - level, population-level, and individual - level of studies, by their application to conserve both in
captivity and wild animals. Certainly, the basic component of concern for biologists is the conservation of
species, and up till now systematics still suffer from the lack of a practicable description of a ‗species‘ and a
variety of species concept are existing (Wayne et al. 1994). Systems of classification carry on and are based
mainly on ‗type‘ specimen that frequently relies on a small number of samples and a few morphological
characteristics (Avise 1989).
Molecular phylogenetics has progressively more significant work to contribute to systematics (Page
and Holmes 1998). This can be highlighted by the example of ‗cryptic‘ species wherever convergent
Review of Literature
10
phenotype masks the wide genotypic variations, only exposed during the divergence of genetic variability
(Warren et al. 2001). For instance, the isolation of two species of European pipistrelle bat (Pipistrellus
pipistrellus and Pipistrellus pygmaeus) with different echolocation frequencies had been confirmed through
genetic analysis (Barratt et al. 1997).
Conclusively, the debate centers on preference for the conservation of mainly genetically conflicting
taxa or the taxa with the maximum genetic variety (Diniz-Filho 2004). Policies for conservation are
frequently based on precise species identification or indeed, the recognition of evolutionarily significant units
(Fraser and Bernatchez 2001). The evolutionary history of species is complex and is associated with its
pattern of migration and confirmation of phylogeography can accompany the phylogenetic study. For
example, about 170 years ago the radiation of Galapagos island finches was not complete (Sato et al. 2001).
Phylogeography places species or population phylogenies in the perspective of their geographical
distribution and may discriminate among colonization and variations (metapopulation fragmentation
followed by divergence) (Avise et al. 1987). Co-distributed fauna and flora with a similar pattern of
phylogeography can make available helpful information on the biogeographic history of an area (Arbogast
and Kenagy 2001). Moreover, this relative phylogeographical approach can be employed to restrict areas of
larger biodiversity for targeting conservation attempts (Moritz and Faith 1998).
The assessment of genetic variability on a population level is significant for both in-situ and ex situ-
conservation (Ballou and Gilpin 1995). Genetic conservation studies at the population level focus on
intraspecific genetic variations and their association to population organization and demographic process.
The recent and past pattern of genetic drift, gene flow, dispersion, and reproductive strategies modify the
genetic variation within and between different populations (Cruzan and Templeton 2000). Studies of genetic
variations used to respond to a broad range of conservation-related queries, for example, choice of mate and
sexual selection (Tregenza and Wedell 2000); parentage analysis (Say et al. 2003); dispersal and range
expansion (Say et al. 2003); inbreeding and outbreeding (Marshall and Spalton 2000) and gene flow (Girman
et al. 2001; Roeder et al. 2001). Island colonization or Range extension by a species can result in the founder
effect while the initial colonizers signify a little percentage of genetic variability of the source populations. If
these founder populations become isolated, random genetic drift will drive to divergence. The degree of
segregation among populations that facilitate intra-specific analysis is mainly governed through gene flow
(Slatkin 1994).
If gene flow through mating occurs by a group of population (metapopulation), they will tend to
pursue the identical evolutionary genetic makeup. On the other hand, in the nonappearance of gene flow
(resulting in reproductive isolation) population will be likely to develop separately, potentially leads to
speciation events. Gene flow enhances the special effects of inbreeding by introducing new alleles to the
gene pool. On the other hand, geographic isolation limit gene flow in island species gives rise to the eminent
risk of extinction and endemism. The inbreeding effects might be sharp in bottlenecked population. The
impacts of bottleneck events will depend on a quantity of demographic behavior, but, small reproductive
output and bias in breeding success, as is characteristic of fruit bats, be likely to suffer from inbreeding
depression (Luikart and Cornuet 1998). Inbreeding depression is regarded as a decrease in fitness of traits
i.e., survivorship of organisms and reproductive success which is linked with a decline in heterozygosity and
increased the possibility of the appearance of recessive harmful genes that arise from mating among
interrelated individuals (Dudash and Fenster 2000).
Consanguinity might be taking place during inadequate mating probabilities in a bottlenecked
population or population with limited distribution. Inbreeding depression in small isolated populations has
been recognized as a matter of great concern for conservation biologists with its resulting negative effects on
birth rate, growth rate, and survivorship of individuals (Gilpin 1986). The associated loss of genetic diversity
is linked with inbreeding which might be a limiting factor for the adaptability of individuals to new
environmental conditions (Balmford et al. 1998).
Review of Literature
11
In conservation, Molecular genotyping of individuals has been extensively used for individuals‘
identification. Morin uses micro-satellites to recognize chimpanzees individually (Pan troglodytes) (Morin et
al. 2001). Ernest uses DNA to trail mountain lion (Puma concolor) and estimation of territory size (Ernest et
al. 2000). Illegally captured wild animals can also be traced to their origin by using the methodology of
genetic identification (Manel et al. 2002) and it becomes a practical means in the issues related to the illegal
trade of endangered wildlife. Gladston and Wedekind highlighted the significance of the identification of
individuals for the successful managing in captive breeding programs (Glatston 1986; Wedekind 2002).
Molecular data is not perfect, however, and suffers some significant shortcomings (Vogler and
Monaghan 2007). DNA is not ideal for detecting incipient speciation because neutral gene mutations can
accumulate at a slower rate than changes in morphological characters under selection (Hickerson et al. 2006;
Mayer et al. 2007; Rodriguez and Ammerman 2004). Also, DNA data can be just as misleading as
morphological data in identifying species. Introgression, incomplete lineage sorting, and differences between
the gene tree and species tree can all obscure true patterns of species divergence (Rodriguez and Ammerman
2004; Rubinoff 2006). Despite these potential pitfalls, DNA taxonomy has been applied very successfully to
bats (Order Chiroptera) in recent years (Baker and Bradley 2006; Clare 2011; Mayer et al. 2007). Several
studies have used DNA sequence data to discover cryptic diversity within genera (Baker and Bradley 2006;
Mayer and von Helversen 2001) and to clarify the phylogenetic relationships within and among various
genera of bats (Hoffmann and Baker 2003; Hoffmann and Baker 2001; Hoffmann et al. 2003; Porter and
Baker 2004; Ruedi and Mayer 2001).
The knowledge of the taxonomy of wild species, their demography, and the ranges at which these
species are influenced by the evolutionary activities are gained by the study of genetic species (Sovic et al.
2016). Improvement in the field of taxonomy provides appraisal to the field of systematics and classification
of more complex chiropteran species. Molecular genetics has brought out many new inventions in the
taxonomy of species of more diverse regions (Clare et al. 2007; Francis et al. 2010).
Cytochrome b is used due to identify the species which are very similar in their morphology.
Cytochrome b can also be adopted in the study of phylogeny as it‘s a reliable tool in the process of gene
sequencing, an orthodox character in phylogenetic analysis, flexibility in the masses analysis in the era of
mammals, and in this way, it can be followed to resolve problematic inquiries of systematic studies (Cardinal
and Christidis 2000; Clare et al. 2007). Cytochrome b is known as the major protein in barcoding as it makes
up the mitochondrial complex III of the oxidative phosphorylation system and is the protein that is sequenced
by the mitochondrial genome. For species identification, segment I of cytochrome b is used to discriminate
mammalian species (Dallimer et al. 2002).
One of the mtDNA regions used to create phylogenetic connections between different species and to
classify species is a fragment of the cytochrome b (Cyt b) coding gene. It has been shown that this region can
be multiplied in different animal species using a single pair of universal primers in a PCR reaction under
normal conditions (Kocher et al. 1989). Recently, Parson et al. also suggested the use of plentiful DNA
sequence data included in DNA databases to classify species of biological samples of uncertain origin
(Parson et al. 2000). This thesis presents the findings of the validation of the species identification system by
sequence analysis of the area coding Cyt b for species identification (Wilson and Reeder 1993).
Cytochrome b (Cyt b) is a gene used for most of the phylogenetic studies due to its structure and
functions of protein products (Cardinal and Christidis 2000). It is also used in the phylogenetic probe
because of its easier alignment of a protein-coding sequence, conservative role in phylogenetic analysis,
variability for population analysis, and its evolution across the period of mammalian origin hence is used in
Review of Literature
12
different systematic questions (Cardinal and Christidis 2000; Clare et al. 2007). Cyt b is one of the most
well-known proteins out of 9 to 10 proteins that make up the oxidative phosphorylation system's
mitochondrial complex III and are the only protein encoded by the mitochondrial genome. The gene portion I
of Cyt b was used to study interspecific and intraspecific differences across several mammals. Moreover, Cyt
b is advantageous as compared to those studies which are being carried out before cloning and sequencing of
vertebrate mitochondrial graded taxonomic series (Dallimer et al. 2002).
The phylogenetic utility of the Cyt-b gene has been studied at several taxonomic levels between vertebrate
taxa ((Lovejoy and De Araújo 2000; Moritz 1994). Establishing species of origin is one of the fundamental
goals of the research related to the description of biological material in the taxonomic classification of
organisms. In legal proceedings where the only material evidence is a residue of animal or plant origin, the
description of the species is of supreme importance. Defining the species of origin is also becoming
increasingly important in other areas, such as the beef industry, bat production, and environmental
conservation (Advani 1981; Forrest and Carnegie 1994).
Species identity and description have a vital role in taxonomy and type of organisms. In the mid of
18th century, about 1.7 million species of organisms have been described to date (Stoeckle 2003). In
taxonomic studies, the higher taxa are given more priorities than the lower taxa (Blaxter 2003). It has been
noted that even the higher animals are still being a puzzle in a taxonomic hierarchy. For example, African
Elephant which was considered as a single species has now become a new topic of research in the field after
studying its mitochondrial nuclear genome which exists in the other two species as well. (Comstock et al.
2002; Debruyne 2004; Roca et al. 2005b).
It is evaluated that fauna and flora of earth consist of 10 to 100 Million eukaryotic species (Whitfield
2003). A large variety of these eukaryotic species are being recognized by standards set by scientists.
Moreover, a large number of phenotypic and genotypic features of living things which are used for scientific
investigations can help to determine errors for unidentified species or different developmental stages in the
lifetime of animal which can produce the misconception for identification (Hebert et al. 2003b).
Morphological differentiation is being used as a standard referencing category to set forth as well as
elaborate various species from the period of Carolus Linnaeus up till now. With the help of this strategy,
we‘ve become able to identify a large number of species present on the Earth so far. Nearly 1.7 million
species are known to be reported by using the morphological approach (Waugh 2007). Conversely, a large
number of limitations of this identification method have been observed, among which the cryptic species is
the major hurdle which includes all the organisms possessing unique, varying, and distinguished
morphological characteristics along with contrasting evolutionary histories because of the convergent (an
independent evolution of organisms having analogous traits) evolution (Lefébure et al. 2006). According to a
large number of studies, the cryptic species are considered as a common biological group of organisms
among all the species present on this planet (Bickford et al. 2007; Mayer and von Helversen 2001; Waugh
2007).
Since the last two decades, the application of molecular techniques has contributed to discovering
cryptic species (Bickford et al. 2007). Secondly, at the time of selection, the morphological features may
represent a contradictory picture that depicts a remarkable difference in the evolutionary history of a group of
organisms. Anolis lizards and Myotis bats are common examples of it. Further, convergent selection or
convergent selective pressures give rise to resembling morphology of these two genera as it illustrates the
habitat of Anolis lizards as well as feeding and foraging behavior of Myotis bats having nonidentical
evolutionary origins and histories (Hoofer and Bussche 2003; Losos et al. 1998; Losos and Warheitf 1997).
Review of Literature
13
Field studies based on morphological features cannot easily identify or differentiate among species
(cryptic species). In fact, for the identification of >0.01% of the approximate number of species (10 to 15
million), a few taxonomists are needed, to explore species diversity based on molecular techniques while a
community comprising of 15000 taxonomic biologists are mandatory for the recognition/identification of
organisms when our studies entirely depend upon the morphological identification. However, this approach
challenges four prominent limitations for identifying different groups of organisms. Firstly, the phenotypic
and genotypic variations within the biological systems of different species may disrupt the identifications.
Secondly, this method ignores the morphologically cryptic taxa which are commonly found in several
families of organisms. Thirdly, the morphological clues are only potent for determining a specific phase or
sex of an animal, but still many characters of the individuals remain unidentified. Fourthly, although modern
species recognizing keys facilitate the scientists, the use of advanced technology requires much expertise and
knowledge to avoid any confusing research in fieldwork. However, several restrictions in the ways of
identification approaches based on the morphology of the organisms and the limited number of field workers
indicate the need for new methodologies to avail an effective reorganization of different taxa. Currently, the
microeconomic identification systems are introduced which have paved new paths to follow by providing a
precise set of informative data for the diagnosis of all the biodiversity through the molecular analysis of the
genomic sequences in detail. In general, prioritizing conservation efforts in presence of inadequate sources is
another major challenge being faced by conservation biologists (Faith 1992).
Highlighting that the main concern might involve evaluating species on their endangerment, ecological
or economic importance, distinctiveness, diversity, or ‗charisma‘. Genetics is playing a significant role in
making conservation work a successful field (O'Brien 2005). Significant work on bats identification in
Punjab (Javid et al. 2014; Javid et al. 2012; Roberts 1997), Khyber Pakhtunkhwa (Mahmood-ul-Hassan et al.
2010; Roberts 1997; Salim 2016), Baluchistan and Sindh (Roberts 1997) have been performed but no work
on barcoding of bats have been produced in Pakistan. Current projects consequently intended to investigate
genetic diversity in bats of Bajaur agency, Federally Administrated Tribal Areas (FATA), Pakistan.
STATEMENT OF PROBLEM
Cryptic speciation discovery is much more in order Chiroptera. Globally, more species are being
discovered day by day. Formally the species were recognized based on their morphological basic, but due to
advancement in molecular biology tools scientists are using various genetic markers for species identification
along with other morphological parameters of bats, hence a combination of both morphological and genetic
markers is more helpful for accurate species identification and to reveal their phylogenetic analyses, so a
complete picture of evolution and phylogeny of the species may be developed using these approaches.
Pakistan is more diverse in terms of biological species as it is the region having different climatic zones and
hence more species are present in such a diverse zone.
OBJECTIVES
The key objectives of the present study are to:
1. Morphological identification of chiropteran species from Bajaur Agency, FATA, Pakistan.
2. Molecular approaches for species identification.
3. Construction of phylogenetic analyses of different bat species from the study area and diversification
of different bat species.
References
14
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20
CHAPTER 3
Experiment No. 1
Preliminary Record of Molecular Phylogeny of Java pipistrelle (Chiroptera: Vespertilionidae) Based
on Cytochrome b Gene from Bajaur Agency, FATA, Pakistan
Muhammad Idnan1,4, Arshad Javid1, Ali Hussain1, Sajid Mansoor2, Muhammad Tayyab3, Muhammad
Imran3, Wasim Shehzad3, Arif Ullah3, Syed Mohsin Bukhari1, Hamid Ullah, Waqas Ali1 1Department of Wildlife and Ecology, University of Veterinary & Animal Sciences, Lahore, Pakistan. 2Department of Microbiology, Faculty of Life science, University of Central Punjab, Lahore, Pakistan. 3Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore,
Pakistan. 4Department of Zoology, Faculty of Sciences, University of Central Punjab, Lahore, Pakistan. 5Department of Zoology, University of Peshawar, Khyber Pakhtunkhwa, Pakistan.
Corresponding Author email; [email protected]
Abstract
An extensive study on the morphology of chiropteran taxa has been carried out but still, controversies are
existing. In order to deduce a phylogenetic relationship of Javan pipistrelle the present study was carried out
based on partial mitochondrial cytochrome b gene and is the first document on phylogenetic analysis of
Javan pipistrelle (Pipistrellus javanicus) from Bajaur Agency, Federally Administered Area (FATA),
Pakistan. The morphometric parameters of samples were measured and some samples (n = 11) were
euthanized and preserved in 70 % ethanol for molecular analyses. Other available data for cytochrome b
gene of concerned species were retrieved from GenBank and Phylogenetic analyses were conducted by the
Neighbor-joining method using MEGA X software. The Javan pipistrelle from Pakistan is making an
outgroup and showing an interspecific cladistic relation as compared to the Javan pipistrelle from Viet Nam
and Philippines. Intern the specimen reported from Viet Nam is also forming an outgroup with Philippines‘
Javan Pipistrelle. It is recommended that a detailed phylogenetic study should be employed to explore the
interspecific and intraspecific relation of chiropteran fauna from Pakistan and with reference to Asiatic bats,
based on cytochrome b analyses.
Key Words: Pipistrelle; cytochrome b; Pakistan; Distribution; Phylogenetic analysis.
Introduction: Species identification and characterization has a crucial role in taxonomy and classification of
organisms. Modern taxonomy, originated in mid-18th century has described up to 1.7 million species of
organisms (Stoeckle 2003). Besides this, to study the relationship of living beings with each other various
behavioral and morphological parameters are taken into consideration. It is very unsurprising that the larger
animals are given a priority for description and the smaller ones mostly remain unknown in sciences (Blaxter
2003). Even among the lager animals‘ species identification has also remained a taxonomic problem e.g. in
case of African elephant which has long been considered as a single species has become the subject of debate
by study of mitochondrial and nuclear genomes which place it in two separate species (Comstock et al. 2002;
Debruyne 2004; Roca et al. 2005).
Cytochrome b (cyt b) is a gene used for most of the phylogenetic studies due to its structure and
functions of protein products (Cardinal and Christidis 2000). It is also used in phylogenetic probe because of
its easier alignment of a protein coding sequence, conservative role in phylogenetic analysis, variability for
population analysis and its evolution across the period of mammalian origin (Clare et al. 2007). Genetic
analysis of species provides a useful information about the scales at which the wild species are impacted by
anthropogenic activities but also provides the information about a successful demographic management of
wild species (Sovic et al. 2016).
Globally, there are 51 species in genus Pipistrellus, (Koopman 1993), 12 from Indian subcontinent
(Bates 1997) and 8 species from Pakistan (Roberts 1997). Furthermore, these eight species are comprising
two ―species group‖ i.e. pipistrellus species-group and kuhlii species-group from south Asia (Bates and
Harrison 1998; Srinivasulu et al. 2010).
Experiment No.1
21
In Pakistan, previously the research has been conducted based on morphological parameters of bats
but no molecular studies have been conducted. So, we designed the current study based on cytochrome b
gene from Bajaur Agency, Federally Administered Tribal Areas (FATA) Pakistan to construct the
phylogenetic analyses of Javan pipistrelle and molecular species confirmation.
Materials and Methods:
Sampling: The bat sample was captured from FATA region , 32.6675° N, 69.8597° E, comprising
total area of 27,220 km² of Pakistan. The roost sites of the bat were found in cervices and holes in buildings
and in caves. The information about the roosts of the bats was also collected from the nomads. The mist nets
of different categories and different lengths (5m, 8m, 11m) were used for bats collections. The mist nets were
applied mostly before the time of the evening. The nets were applied on water bodies and the narrow ways
where the bats were more in number. The sampling was extending from June 2016 to August 2018. During
the time frame of sampling, all the potential roosting sites were searched thoroughly to collect the sample.
The study region of Bajaur Agency, FATA is 72 Km long and 32 Km broad. It is situated at a high
elevation of Kunar Valley of Pakistan and Afghanistan, separated by a continuous line of hills and on the
south are the wild mountain of Mohmand District. Towards east, beyond Panjkora River are the hills of
District Swat and to the north is a watershed and tehsil Dir. A fascinating feature in topography of the study
region is a mountain spur from the Kunar range, which, crooked eastwards, ends in the well-known peak of
Koh-i-Mor. The drainage of Bajour flows eastwards, starting from the eastern slopes of the dividing ridge,
which overlooks the Kunar and terminating in the Panjkora river, so that the district lies on a slope tilting
gradually downwards from the Kunar elevation to the Panjkora. That is why this hilly region is less explored
and least invaded by human interference, and seems to be less disturbed in sense of human settlement, hence
considered to be safe for chiropteran distribution. But war on terror has many devastating effects on human
settlements.
Sample Preservation & Measurements: The samples of bats were collected and tagged as voucher
specimen for molecular analysis (table 2). The collected samples were preserved in the 70% ethanol. The
Morphometric measurements were also observed before preservation. The comparative observational
analyses were performed with Bates and Harrison (Bates and Harrison 1998; Roberts 1997). The
morphometric data for this bat species is provided in table 1.
DNA Extraction and Sequencing: Genomic DNA was extracted from ethanol (70%) preserved
specimens (wing tissue i.e., 10 μg), which also suggests a microgram of tissue should be carried out to
reduce the specimen collection in future studies, by standard phenol-chloroform extraction method (Hoelzel
and Green 1992). Fragments of mtDNA were amplified using a set of primers described by Kocher 1989
forward primer 5-CCATCCAACATCTCAGCATGATGAAA-3 and reverse primer 3-
CCCTCAGAATGATATTTGTCCTCA-5 (Kocher et al. 1989).
Amplification was performed in a 100 μl of a solution containing 67 mM Tris (pH 8.8), 6.7 mM
MgSO4, 16.6 mM (NH4)2SO4, 10 mM 2-mercaptoethanol, each dNTP at 1 mM, each primer at 1 μl, genomic
DNA (10-1000 ng), and 2-5 units of Thermus aquaticus polymerase (Perkin-Elmer/Cetus). Denaturation for
polymerase chain reaction was carried out for 1 min at 93 °C, for the same time period hybridization at 50
°C, DNA extension was carried out at 72 °C for 2-5 min. This was repeated for 50 times. 1 µL of each DNA
sample (50ng/ µL) was separated into different tubes. The tubes were placed on ice till all samples were
prepared. The tubes were loaded into a PCR machine with the pre-set program as 94°C for 2 minutes (1
cycle); 94°C for 1minute, 60°C for 45 seconds, 72°C for 50 seconds (30cycles) and 72°C for 3 min (1 cycle).
PCR products were electrophoresed on 1.5 % agarose gel in 100 ml of TAE-I buffer. The ethanol
decontaminated PCR items were sequenced in two headings utilizing dideoxy chain end direct Sanger
sequencing on ABI 310 sequencer according to standard protocols.
Data Analyses: Sequences were aligned by ClustalW (Larkin et al. 2007), ambiguous sequences
were edited by BioEdit software (Hall 1999), sequences were submitted for accession number to National
Center for Biotechnology Information (NCBI) (MT561167, MT081430, MT081429, MT081428,
MT081427, MT081426, MT081425, MT081423, MT081422, MT081421, MT081420), the sequences of
Pipistrellus javanicus from other regions were also retrieved. We just got two sequences for cytochrome b
Experiment No.1
22
for Pipistrellus javanicus which are reported from Viet Nam and Philippines. By an extensive search we
found no other sequence for cytochrome b from NCBI for Pipistrellus javanicus. Neighbor-joining method
and General Time Reversible model with 100 Bootstrap values on MEGA X was used for the construction of
phylogenetic tree which create same type of phylogenetic trees (Kumar et al. 2018).
Results and Discussion:
Taxonomy:
Javan pipistrelle: Pipistrellus javanicus Gray, 1838
Type locality. Indonesia, Java.
Bajaur Agency, FATA, Pakistan (current study)
Synonyms:
Scotophilus javanicus: (Gray, 1838).
Pipistrellus camortae: (Miller, 1902).
Pipistrellus babu: (Thomas, 1915).
Pipistrellus peguensis: (Sinha, 1969).
Indonesian name: Sekiwen Java
Taxonomic Position: This taxon was traditionally placed to the ―javanicus‖ subgroup of the ―pipistrellus‖
species group (Corbet and Hill 1992). According to available molecular genetic data (Benda et al. 2016;
Roehrs et al. 2010), P. javanicus is a part of genetic cluster of Oriental pipistrelles. This cluster is highly
divergent from all the West Palearctic pipistrelles and may be referred to as ―javanicus‖ species group. Such
taxa as babu Thomas, 1915 and Camortae Miller, 1902 were recognized as distinct species (Das 2003;
Ellerman and Morrison-Scott 1951; Soota and Chaturvedi 1980) or as valid subspecies of P.
javanicus (Corbet and Hill 1992), DNA barcoding data definitely supports full species status for P.
babu (Francis et al. 2010).
Natural History: The habitat of Javan pipistrelle varies from primary to secondary forests, agricultural
landscapes and urban areas. Where it roosts in tree cervices, barks and holes, house ceilings, ruin buildings,
temples and signboards etc. In Ho Chi Minh city colonies of several dozen individuals were reported in
buildings (Kruskop 2013). This bat emerges early in the evening, before full darkness. Flight is moderately
speed and maneuverable, sometimes fluttering (in cluttered places) as in most pipistrelles, in Ho Chi Minh
bats were observed foraging in urban areas and city parks at about 6-15 m above ground or water (ibid.),
however they were also observed much higher in the Red river valley. Javan pipistrelle probably forages on
flies, winged ants and other small insects, though its ration was not described. There are three breeding
seasons (though probably the same females may not reproduce in two consecutive seasons) and two young
ones are born (Bates and Harrison 1997; Sanborn 1952). The morphological parameters of the species are
described in table 1.
BLASTn was performed for sequence analyses of cytochrome b. We retrieved aligned sequences for
cytochrome b of Pipistrellus javanicus from NCBI and constructed the phylogenetic tree. We just found the
cytochrome b sequences from two countries i.e. Viet Nam and Philippines. Other than these countries, there
is no record for cytochrome b reported on NCBI for Javan pipistrelle. The reported records of Javan
pipistrelle is mentioned in table 2.
Evolutionary history of the Javan pipistrelle is inferred by Neighbor-joining method based on
Bootstrap value of 100 and evolutionary distance were calculated by Kimura-2 parameter in figure 1.
Genetic identities for different species of Pipistrellus javanicus are described in Table 3. Although
systematics is very old branch of science however, majority of the species are still unidentified. Now a days
DNA barcoding is considered authentic and helps in clear cut species identification. Generally, the
technology of DNA sequencing has resolved the taxonomic disputes of many taxa, but some higher taxa
have not yet been resolved precisely as a species. The task of species identification by DNA barcoding is
very useful to resolve the taxonomic problems of cryptic species, extinct species, synonymous species or
Experiment No.1
23
matching the juvenile with adults. However, DNA barcoding is proved as a standard too for species
identification.
Advancement in molecular techniques has revolutionized the field of systematics and improved the
taxonomy of some more complex chiropteran species. Molecular genetics highlighted many new discoveries
in taxonomy of understudied and species rich tropical areas (Clare et al. 2007; Francis et al. 2010), besides
this, in temperate fauna where the relative species diversity is low, the molecular genetics has also resolved
taxonomic uncertainties (Mayer et al. 2007), from the study area of Pakistan new chiropteran species are also
being identified by using the molecular techniques. Discoveries of new species from Pakistan is suggesting a
species richness and diversity in this region.
It is estimated that the earth‘s biota is constituting about 10 to 100 million species of eukaryotes
(Whitfield 2003). Such a large number of species is presenting a challenging task for taxonomists by
conventional identification methods. Even though, the impact of internet and consenting for advancements in
communications, the assignment of taxonomic identification is prodigious. In addition, variations in
phenotypic characters and genotype of organisms, which are being employed for taxonomic identification
can primarily lead to identification errors, cryptic species or different developmental stages in the life history
of animals could increase the misperception (Hebert et al. 2003).
Field biologists are confronted with certainty of species diversity due to improvements of the system
for species recognition and its appropriate accessibility worldwide. Such problems of species identification
are also being faced by the people in trade of endangered species, fisheries sector, identification of pest
species and their control for spreading the diseases, accurate lineage identification of extinct species and
regulation of biological materials across the world. By perceiving these issues, a concise, simple and accurate
procedure should be employed for species identification is required to overcome these issues for
identification. As more species are being discovered day by day, the taxonomic data is becoming more
problematic. Species identification by morphological characteristics requires training and expertise without
which this process of identification is difficult. Recent advances in molecular technology has strengthen the
species identification process by using short DNA sequences, which are recognized as species labels, in a
process called DNA barcoding. The varied DNA sequences are intraspecific differentiations which determine
the order of magnitude for species identification.
Pipistrellus abramus was considered as a subspecies of Pipistrellus javanicus (Corbet and Hill 1980;
Corbet 1978; Gray 1838; Honacki et al. 1982), the male of Pipistrellus javanicus has a long sinous baculum,
a point of difference (Hill 1987; Thomas 1928), helped to raise categorized it to a species rank (Simmons
2005; Srinivasulu and Srinivasulu 2001).
The distributional range of this species include from Viet Nam, Phillipines, Southeast Asia to Lesser
Sunda Iseles, Thailand, Burma, North and Central India, Eastern Afghanistan, China and Northern Pakistan
(Javid et al. 2019; Roberts 1997).
Conclusion and Recommendations: Due to highly cryptic speciation and similarity in morphological
characteristics it is difficult to recognize differences in species in genus pipistrellus. So, the phylogenetic
study was designed to explore the diversity in this genus using cytochrome b as a marker along with
morphological characteristics. It is recommended that a detailed phylogenetic analysis should be carried out
of Javan pipistrelle from south Asia by cytochrome b as a marker.
Conflict of Interests: The author(s) declare no potential conflict of interests.
Acknowledgement: The author(s) is thankful to all those who have directly and indirectly helped us
complete the difficult tasks during research work. We also thank for any anonymous who helped for
constructive comments.
Data Availability Statement: Data is available on NCBI for the following accession numbers (MT561167,
MT081430, MT081429, MT081428, MT081427, MT081426, MT081425, MT081423, MT081422,
MT081421, MT081420).
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Bates P, Harrison D. 1997. Bats of the Indian subcontinent Sevenoaks. United Kingdon: Harrison Zoological
Museum.
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United Kingdom.
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Blaxter M. 2003. Molecular systematics: counting angels with DNA. Nature. 421(6919): 122.
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Corbet G, Hill J. 1980. A world list of mammalian species. British Museum of Natural History. Comstock
Publishing, London. 128: 1950-1959.
Corbet GB. 1978. The mammals of the Palaearctic region: a taxonomic review. British Museum (Natural
History). 341.
Corbet GB, Hill JE. 1992. The mammals of the Indomalayan region: a systematic review. oxford university
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Das PK. 2003. Studies on some Indian Chiroptera from West Bengal. Zoological Survey of India.
Debruyne R. 2004. Contribution of molecular phylogeny and morphometrics to the systematics of African
elephants. Journal de la Societe de biologie. 198(4): 335-342.
Ellerman JR, Morrison-Scott TCS. 1951. Checklist of Palaearctic and Indian mammals, 1758-1946. order of
the Trustees of the British Museum.
Francis CM, Borisenko AV, Ivanova NV, Eger JL, Lim BK, Guillén-Servent A, Kruskop SV, Mackie I,
Hebert PD. 2010. The role of DNA barcodes in understanding and conservation of mammal diversity
in Southeast Asia. PloS one. 5(9): e12575.
Gray J. 1838. A revision of the genera of bats (Vespertilionidae), and the description of some new genera
and species. Mag, Zool, Bot, 2: 483-505.. 1844. Mammalia. 7-36.
Hall TA editor. Nucleic acids symposium series. 1999.
Hebert PD, Cywinska A, Ball SL. 2003. Biological identifications through DNA barcodes. Proceedings of
the Royal Society of London B: Biological Sciences. 270(1512): 313-321.
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a synopsis of Pipistrellus and Eptesicus, and the description of a new genus and subgenus. Bulletin of
the British Museum (Natural History), Zoology Series. 52: 225-305.
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Molecular genetic analysis of populations: a practical approach. 159-187.
Honacki JH, Kinman KE, Koeppl JW. 1982. Mammals species of the world; a taxonomic and geographic
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Javid A, Rasheed B, Zeb J, Khan MI. 2019. Morphological Differentiation in Some Pipistrellus
sp.(Chiroptera) Captured from Bajaur Agency, Pakistan. Pakistan Journal of Zoology. 51(2): 689.
Kocher TD, Thomas WK, Meyer A, Edwards SV, Pääbo S, Villablanca FX, Wilson AC. 1989. Dynamics of
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Kruskop SV. 2013. Bats of Vietnam: Checklist and an identification manual. Tovarishchestvo nauchnykh
izdaniĭ KMK.
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Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: molecular evolutionary genetics analysis
across computing platforms. Mol. Biol. Evol. 35(6): 1547-1549.
Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM,
Wilm A, Lopez R. 2007. Clustal W and Clustal X version 2.0. bioinformatics. 23(21): 2947-2948.
Mayer F, Dietz C, Kiefer A. 2007. Molecular species identification boosts bat diversity. Front Zool. 4(1): 4.
Roberts T. 1997. The mammals of Pakistan (revised ed.) Oxford University Press. Karachi, Pakistan. 525.
Roca AL, Georgiadis N, O'Brien SJ. 2005. Cytonuclear genomic dissociation in African elephant species.
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(Chiroptera: Vespertilionidae) based on mitochondrial and nuclear sequence data. J Mammal. 91(5):
1073-1092.
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1: 312-529.
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systematic status. Rec. zool. Surv. India. 77: 83-87.
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three tree bat species at an Ohio windfarm. PeerJ. 4: e1647.
Srinivasulu C, Racey PA, Mistry S. 2010. A key to the bats (Mammalia: Chiroptera) of South Asia. Journal
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Whitfield J. 2003. DNA barcodes catalogue animals. News@ Nature. com. Published online only at: http://www. nature. com/news/2003/030512-7. html.
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Table 3.1. Morphological Parameters (mm) and mean Body mass (g) for Specimens of Pipistrellus
javanicus from Bajaur agency, FATA, Pakistan.
Body Parameters Pipistrellus javanicus (n=11)
Mean±SD
1st Phalanx on 3
rd metacarpal 11.62±0.54
1st Phalanx on 4
th metacarpal 11.54±0.88
1st phalanx on 5
th metacarpal 8.06±0.71
2nd
Phalanx on 3rd
metacarpal 9.87±0.95
3rd
metacarpal length 31.62±1.20
4th metacarpal length 31.37±1.79
5th metacarpal length 31.15±1.35
Anterior palatal width 4.35±0.21
Bacular Measurements (n=2)
Body mass 8.08±1.09
Breadth of braincase 6.68±
Calcar length 4.03±0.22
Condylo-basal length 13.1±70.41
Condylo-canine length 12.67±0.25
Cranial Measurements (n=6)
Distal branch length 0.70±0.71
Distal branch width 0.365±0.01
Ear length 8.50±1.38
Forearm length 35.13±0.53
Greatest length of skull 13.69±0.24
Head and body length 46.65±1.58
Hind foot length 5.34±0.5
Mandible length 10.21±0.45
Mandibular toothrow 5.01±0.39
Maxillary toothrow 4.80±0.14
Posterior palatal width 6.06±0.46
Postorbital constriction 3.55±0.22
Proximal branch length 1.209±0.01
Proximal branch Width 0.708±0.14
Shaft length 2.401±0.00
Tail length 30.34±2.97
Thumb with claw 4.76±0.99
Tibia length 13.46±0.88
Total baculum length 3.81±0.01
Tragus length 3.77±0.65
Wingspan 217.67±5.75
Experiment No.1
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Zygomatic breadth 8.81±0.14
Table 3.2. GPS Coordinates of study area, Bajaur Agency, Pakistan
Sr. No. Accession No. Voucher No. Locality: Area / Country GPS Coordinates
1. MT561167 WECO-PJ_001 Payshat Batkhela, Bajaur, Pakistan N 34° 52.334
E 071°31.902 2. MT081430 WECO- PJ _002
3. MT081429 WECO- PJ _003 Kariband 3 Cave Batwar Bajaur, Pakistan N 34° 55.296
E 071°30.495
4. MT081428 WECO- PJ _004 Kariband 1 Cave Batwar, Bajaur, Pakistan N 34° 55.136
E 071°30.471 5. MT081427 WECO- PJ _005
6. MT081426 WECO- PJ _006 Bajaur, Pakistan
Nawagi Darbano cave Nagibaba
N 34°39.142
E 071°21.575 7. MT081425 WECO- PJ _007
Experiment No.1
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Table 3.3 Genetic Identities of Pipistrellus javanicus species calculated by Kimura-2 Parameter based
on cytochrome b analyses.
8. MT081423 WECO- PJ _008 Chalgazy Payshat, Bajaur, Pakistan N 34°55.244
E 071°30.602 9. MT081422 WECO- PJ _009
10. MT081421 WECO- PJ _010 Lardagy Payshat, Bajaur, Pakistan N 34° 53.507
E 071°31.738 11. MT081420 WECO- PJ _011
12. KX496357 VN11-0379 Viet Nam ---
13. JX570908 FMNH 194729
DSB4612
Philippines ---
14. JX570909 FMNH 167237
LRH6080
Philippines ---
Ac. No. MT081421 MT081420 MT081429 MT081430 MT081428 MT561167 MT081425 MT081423 MT081426 MT081427 AJ504447 JX570909 JX570896 JX570908 KX496357
MT081421 ID
MT081420 1 ID
MT081429 1 1 ID
MT081430 1 1 1 ID
MT081428 1 1 1 1 ID
MT561167 1 1 1 1 1 ID
MT081425 0.833 0.833 0.833 0.833 0.833 0.833 ID
MT081423 0.833 0.833 0.833 0.833 0.833 0.833 1 ID
MT081426 0.833 0.833 0.833 0.833 0.833 0.833 1 1 ID
MT081427 0.833 0.833 0.833 0.833 0.833 0.833 1 1 1 ID
AJ504447 0.88 0.88 0.88 0.88 0.88 0.88 0.837 0.837 0.837 0.837 ID
JX570909 0.864 0.864 0.864 0.864 0.864 0.864 0.822 0.822 0.822 0.822 0.907 ID
JX570896 0.861 0.861 0.861 0.861 0.861 0.861 0.83 0.83 0.83 0.83 0.903 0.98 ID
JX570908 0.876 0.876 0.876 0.876 0.876 0.876 0.822 0.822 0.822 0.822 0.918 0.98 0.984 ID
KX496357 0.837 0.837 0.837 0.837 0.837 0.837 0.818 0.818 0.818 0.818 0.876 0.868 0.876 0.876 ID
Experiment No.1
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Figure 3.1. Phylogenetic analysis of Pipistrellus javanicus by Neighbor-joining Method using
MEGA-X.
Experiment No.1
30
Figure 3.2. Bacular features of Pipistrellus javanicus.
31
CHAPTER 4
Experiment No. 2
Range Extension and Phylogenetic Analysis of Eeptesicus nasutus (Sind Bat) (Mammalia: chiroptera)
in Bajaur Agency, FATA, Pakistan
Muhammad Idnan1,4, Arshad Javid1, Ali Hussain1, Sajid Mansoor2, Muhammad Tayyab3, Muhammad
Imran3, Wasim Shehzad3, Arif Ullah3, Waqas Ali1, Syed Mohsin Bukhari1, Hamid Ullah 1Department of Wildlife and Ecology, University of Veterinary & Animal Sciences, Lahore, Pakistan. 2Department of Microbiology, Faculty of Life science, University of Central Punjab, Lahore, Pakistan. 3Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore,
Pakistan. 4Department of Zoology, Faculty of Sciences, University of Central Punjab, Lahore, Pakistan. 5Department of Zoology, University of Peshawar, Khyber Pakhtunkhwa, Pakistan.
Corresponding Author email; [email protected]
Abstract: The lack of morphological differentiation among chiropteran species and cryptic speciation
impedes species identification. This study explores the range extension of Sind bat within the territorial
limits of Pakistan from Sindh and Baluchistan to Federally Administered Areas (FATA) of Pakistan.
Specimens were collected from Bajaur Agency, FATA during 2017. Various morphological measurements
were taken. Head and Body length was 44.3, Tail length was 43.4, Hind foot length was 8.3mm, Forearm
length was 35.7mm, and Ear length 36 while 5th Metacarpal Length, 4th Metacarpal Length and 3rd
Metacarpal Lengths were 33.2mm, 34.7mm, 35.3mm respectively (n=11). A specimen was euthanized and
preserved for genetic analyses by cytochrome b gene. Newly obtained DNA sequence was submitted to
GenBank for Accession numbers MT674673. The neighbor joining tree based on Kimura-2 parameters was
created to infer the phylogenetic analyses. Eptesicus nasutus showed a high value of genetic divergence of
94.4 % with a subspecies I from Al-Rumayliyah, Oman (Accession No. KF019043) and a minimum value of
39.9%. Approaches based on DNA barcoding reveals a high diversity of bats in study region which may be
due to low accessibility and less construction activities for habitat modification in the region by war on terror
activities. The data will enable researchers to build an improved evolutionary landscape of Eptesicus genus
from this region and subsequently to reconstruct a detailed evolutionary history of the genus. Further
research is required to assess its population status and conduct a phylogenetic analysis for Asiatic bats.
Key words: Chiroptera, cytochrome b, phylogenetic analysis, mitochondrial, eptesicus.
1. Introduction:
It is estimated that 10 to 100 million species of organisms are existing on earth out of which 1.5
million species have been described until now (Agosti 2003). Most neglected component of biodiversity is
represented by cryptic species and hence these taxa fill important ecological niches which contribute an
important role for conservation measures (Bickford et al. 2007). In recent years, the number of bat species
are increasing by newly reported species, as current estimation of recognized bat species is over 1386
(Burgin et al. 2018) which is an increase of more than 40% since 1993 (Wilson and Reeder 2005). The
increase in number of bat species is due to a surge of recent research, by the discovery of cryptic species in
order chiroptera and the use of modern approaches to explore acoustic and phylogenetic analyses (Wilson
and Reeder 2005) and molecular phylogenetics (Clare 2011; Mayer et al. 2007).
Rhyneptesicus nasutus formerly known as Eptesicus nasutus is present in Saudi Arabia, Islamic
Republic of Iran, Afghanistan, Iraq, Oman, Yemen and Pakistan (Bates 1997; Juste et al. 2013). This species
has probably never been abundant throughout its relatively restricted geographical range (Bates and Harrison
1998). It was included on List 1 of Threatened Species with the notion VU A2c ―Vulnerable with declining
population and a continuing decline of area / extent/ quality of habitat‖ (Baillie and Groombridge 1996). This
species is rare and locally distributed in Pakistan. It has been collected from near Kharan and Rajbar in
Baluchistan and from Shikarpur in Sindh (List 2004).
Modern molecular techniques also suggest that in Southeast Asia the number of bat species are
double to the currently described species. In areas of high endemism and hotspots of biodiversity, the cryptic
Experiment No.2
32
species are more prevalent as such areas are considered to have a high potential of speciation (Francis et al.
2010).
In the meantime, genetic screening methods related to a single gene or a few genes is normally
known as DNA barcoding started to establish a standard technique for species identification and the
discovery of new species to find out true number of species. Thus far, DNA barcoding is basically used to
identify species which are taxonomically poorly identified. Here, cytochrome b was used to identify species
from diverse chiropteran taxon from FATA region of Pakistan.
Synonyms:
Eptesicus nasutus (Dobson, 1877)
Vesperugo nasutus Dobson, 1877
2. Materials and Methods:
2.1. Sampling: The bat sample was captured from FATA region , 32.6675° N, 69.8597° E, comprising total
area of 27,220 km² of Pakistan. The roost sites of the bat were found in cervices and holes in buildings and in
caves. The information about the roosts of the bats was also collected from the nomads. The mist nets of
different categories and different lengths (5m, 8m, 11m) were used for bats collections. The mist nets were
applied mostly before the time of the evening. The nets were applied on water bodies and the narrow ways
where the bats were more in number. The sampling was extending from June 2016 to August 2018. During
the time frame of sampling, all the potential roosting sites were searched thoroughly to collect the sample.
2.2. Sample Preservation & Measurements: The samples of bats were collected and tagged as voucher
specimen WECO-26 for molecular analysis. The collected samples were preserved in the 70% ethanol. The
Morphometric measurements were also observed before preservation. The comparative observational
analyses were performed with Bates and Harrison (Bates and Harrison 1998; Roberts 1997). The
morphometric data for this bat species is provided in table 1.
2.3. DNA Extraction and Sequencing: Genomic DNA was extracted from ethanol (70%) preserved
specimens (wing tissue i.e., 10 μg) at Post Graduate Lab, Institute of Biochemistry and Biotechnology
(IBBt), University of Veterinary and Animal Sciences, Lahore, Pakistan, which also suggests a microgram of
tissue should be carried out to reduce the specimen collection in future studies, by standard phenol-
chloroform extraction method (Hoelzel and Green 1992). Fragments of mtDNA were amplified using a set of
primers described by Kocher 1989 forward primer 5-CCATCCAACATCTCAGCATGATGAAA-3 and
reverse primer 3CCCTCAGAATGATATTTGTCCTCA-5 (Kocher et al. 1989).
Amplification was performed in a 100 μl of a solution containing 67 mM Tris (pH 8.8), 6.7 mM
MgSO4, 16.6 mM (NH4)2SO4, 10 mM 2-mercaptoethanol, each dNTP at 1 mM, each primer at 1 μl, genomic
DNA (10-1000 ng), and 2-5 units of Thermus aquaticus polymerase (Perkin-Elmer/Cetus). Denaturation for
polymerase chain reaction was carried out for 1 min at 93 °C, for the same time period hybridization at 50
°C, DNA extension was carried out at 72 °C for 2-5 min. This was repeated for 50 times. 1 µL of each DNA
sample (50ng/ µL) was separated into different tubes. The tubes were placed on ice till all samples were
prepared. The tubes were loaded into a PCR machine with the pre-set program as 94°C for 2 minutes (1
cycle); 94°C for 1minute, 60°C for 45 seconds, 72°C for 50 seconds (30cycles) and 72°C for 3 min (1 cycle).
PCR products were electrophoresed on 1.5 % agarose gel in 100 ml of TAE-I buffer. The ethanol
decontaminated PCR items were sequenced in two headings utilizing dideoxy chain end direct Sanger
sequencing on ABI 310 sequencer according to standard protocols. Sequences were aligned by BioEdit
software and MEGA X was used for the construction of phylogenetic tree (Hall 1999; Kumar et al. 2018).
3. Results: During the study, DNA sequences of chiropteran species representing Eptesicus (formally known as
Eptesicus) genera and Vespertilionidae family were obtained. These DNA sequences have shown reliable
and clear species identifications. Recently, DNA barcoding studies of Asian bats have been carried out and
sequences of related species were available at NCBI. Closely related DNA sequences of cytochrome b were
Experiment No.2
33
retrieved from public databases in blast searches. Neighbor-joining tree based on Kimura 2-parameter
distance is shown in Figure 1.
The sequence results of query sample were run in BLASTn, the percentage identity was 95.13 % with
Eptesicus nasutus (FJ841981) and 100 % query coverage and this specimen is reported from Dehbarez,
Hormozgan, Iran. Against our query sequence, gene sequences were retrieved from GenBank, used in
subsequent phylogenetic analyses. Ambiguous sequences were trimmed. MEGA X was used for
phylogenetic analysis by Neighbor-joining method with Bootstrap values of 100 replicated. This
phylogenetic analysis revealed the Eptesicus nasutus as an out group (Figure 1). The accession number
assigned by GenBank is- MT674673. The morphometric parameters for the species under study are provided
in table 1.
4. Discussion:
Juste et al. (2013) reassigned this taxon to the genus Rhyneptesicus Bianchi, 1917 based molecular
phylogenetics (Juste et al. 2013). Four subspecies – R. n. nasutus (Southwest Pakistan, Afghanistan and
Southeast Iran), R. n. matschiei (Southwest Arabia), R. n. pellucens (Iran and Iraq), and R. n.
batinensis (Eastern Arabia including Oman and Saudi Arabia), are recognized (Benda and Reiter 2006; Juste
et al. 2013).
The available name for this taxon is Rhyneptesicus, suggested by Bianchi (1917) to distinguish
nasutus based on the lack of a diagnostic character the epiblema. This diagnostic character is also found in
other nasutus species; hence this character is not a valid diagnostic character, thereby the formal description
for and name is applicable currently. Horacek & Hanak (1986) recovered Rhyneptesicus as a genus and a
subgenus by Horacek (2000) (Horacek 1986; Horáček et al. 2000). The peculiar morphological
characteristics which differentiate this genus include a relatively short fur, narrow but pointed ears and a long
tragus. While the distinguishable dental character includes a complete molar with protocrista and a
unicuspidal 1st upper incisor.
Genetic markers like mtDNA and nuDNA describe a geographic and genetic relatedness for
discontinuous distribution of the genus Eptesicus. The taxonomic reconstruction of nasutus samples from
Iran which are close to terra typica in Pakistan validates the subspecies recognition (Juste et al. 2013).
Current molecular investigations have placed Eptesicus in tribe Nycticeini, separating it from pipistrelles
(Hoofer and Van Den Bussche 2003; Hoofer et al. 2006).
The genus Eptesicus has a worldwide distribution and high diversity, and hence represents an
entwined taxonomic puzzle among mammals. The status of this species is Least Concern as it has a wide
spread distribution and show tolerance for modified habitats. So, it is unlikely to decline fast enough to
categorize it to a threatened taxon.
The distribution record of Eptesicus nasutus is wide and patchy. It has been reported from Arabian
Peninsula to western South Asia, recorded from Oman, Saudi Arabia, Yemen, United Arab Emirates (UAE),
Qatar, Kuwait, southeastern Iran and southern Iraq. From South Asia it has been reported from Afghanistan
and Pakistan, but from the territorial boundary of Pakistan it is just reported from Baluchistan and Sind
(Bates and Harrison 1998; Molur et al. 2002; Roberts 1997; Srinivasulu et al. 2019). However, the new
record for range extension of Eptesicus nasutus is reported from Bajaur Agency, FATA, Pakistan.
The distributional record of Rhyneptesicus nasutus in Baluchistan (Seistan) is more or less
continuous while in Afghanistan (Jalalabad valley) its occurrence is 700-800 km away to the nearest record
of central Pakistan (Bates and Harrison 1998). The distributional range of the Eptesicus is variable in
different geographical area depicts its adaptability to different geographically climatic ranges. Conversely,
in the Middle East the occurrence of Rhyneptesicus nasutus has been reported in a mosaic of isolated patches
as compared to in a continuous belt (Benda et al. 2010; Benda and Vallo 2012; Harrison and Bates 1991).
The distribution in central Pakistan represents another such patch; Bates & Harrison (1997) summarized
three records from central Baluchistan (Kharan, Rajbar, junction of the Razhai and Sichk rivers; (Bates and
Harrison 1998; Roberts 1997), one from northern Sindh (near Rohri; (Blanford 1898) and current study
explores the range extension to the northern Pakistan. Where an extensive survey should be conducted to
explore more roost site and population dynamics of Eptesicus.
Experiment No.2
34
It is summarized that the effects of climate change on the range extension of Eptesicus may be
primarily determined by the weather consequences on the habitat requirements and physiological tolerances
of the species under study. Here, in the case of Eptesicus nasutus evolution in ecologically different
environment of Bajaur Agency, Pakistan may be due to variable environmental conditions as compared to its
already occurrence in other regions of the country, i.e., Baluchistan and Sind province.
Conclusion: The distributional range of chiropteran species is not thoroughly explored within the territorial
limits of Pakistan. The present record of range extension of Eptesicus nasutus is reported for the first time
from FATA region of Pakistan based cytochrome b analyses. In Pakistan, it has been reported from Sindh
and Baluchistan but a comparative analysis for habitat ecology, population genetics and a large-scale DNA
barcoding is recommended to explore the cryptic species of the genus Eptesicus.
Data availability statement: The sequence data submitted to GenBank for Eptesicus nasutus (cytochrome
b), accession numbers MT674673 is available at NCBI.
References: Agosti D. 2003. Encyclopedia of life: should species description equal gene sequence? Trends in Ecology &
Evolution. 18(6): 273.
Baillie J, Groombridge B. 1996. 1996 IUCN Red List of threatened animals. IUCN, Gland (Suiza). Species
Survival Commission.
Bates P. 1997. Bats of the Indian Subcontinent, Harrison Zoological Museum, Sevenoaks, UK. Google
Scholar.
Bates P, Harrison D. 1998. Bats of the Indian subcontinent. Biodivers Conserv. 7(10): 1383-1386.
Benda P, Al-Jumaily MM, Reiter A, Nasher AK. 2010. Noteworthy records of bats from Yemen with
description of a new species from Socotra. Hystrix, the Italian Journal of Mammalogy. 22(1).
Benda P, Reiter A. 2006. On the occurrence of Eptesicus bobrinskoi in the Middle East (Chiroptera:
Vespertilionidae). Lynx (NS). 37: 23-44.
Benda P, Vallo P. 2012. New look on the geographical variation in Rhinolophus clivosus with description of
a new horseshoe bat species from Cyrenaica, Libya. Vespertilio. 16: 69-96.
Bickford D, Lohman DJ, Sodhi NS, Ng PK, Meier R, Winker K, Ingram KK, Das I. 2007. Cryptic species as
a window on diversity and conservation. Trends in ecology & evolution. 22(3): 148-155.
Blanford WT. 1898. The Fauna of British India: Including Ceylon and Burma. Taylor & Francis.
Burgin CJ, Colella JP, Kahn PL, Upham NS. 2018. How many species of mammals are there? J Mammal.
99(1): 1-14.
Clare EL. 2011. Cryptic species? Patterns of maternal and paternal gene flow in eight Neotropical bats. PLoS
One. 6(7): e21460.
Francis CM, Borisenko AV, Ivanova NV, Eger JL, Lim BK, Guillén-Servent A, Kruskop SV, Mackie I,
Hebert PD. 2010. The role of DNA barcodes in understanding and conservation of mammal diversity
in Southeast Asia. PloS one. 5(9): e12575.
Hall T. 1999. BioEdit software, version 5.0. 9. North Carolina State University, Raleigh, NC.
Harrison D, Bates P. 1991. The mammals ofArabia. Harrison Zoological Museum, Kent.
Hoelzel A, Green A. 1992. Analysis of population-level variation by sequencing PCR-amplified DNA.
Molecular genetic analysis of populations: a practical approach. 159-187.
Hoofer SR, Van Den Bussche RA. 2003. Molecular phylogenetics of the chiropteran family
Vespertilionidae. Acta Chiropterologica. 5(suppl): 1-63.
Hoofer SR, Van Den Bussche RA, Horáček I. 2006. Generic status of the American pipistrelles
(Vespertilionidae) with description of a new genus. J Mammal. 87(5): 981-992.
Horacek I. 1986. Generic status of Pipistrellus savii and comments on classification of the genus Pipistrellus
(Chiroptera, Vespertilionidae). Myotis. 23: 9-16.
Horáček I, Hanák V, Gaisler J editors. Proceedings of the VIIIth European bat research symposium. 2000.
Juste J, Benda P, Garcia‐ Mudarra JL, Ibanez C. 2013. Phylogeny and systematics of O ld W orld serotine
bats (genus E ptesicus, V espertilionidae, C hiroptera): an integrative approach. Zool. Scr. 42(5):
441-457.
Experiment No.2
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Kocher TD, Thomas WK, Meyer A, Edwards SV, Pääbo S, Villablanca FX, Wilson AC. 1989. Dynamics of
mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers.
Proceedings of the National Academy of Sciences. 86(16): 6196-6200.
Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: molecular evolutionary genetics analysis
across computing platforms. Mol. Biol. Evol. 35(6): 1547-1549.
List IR. 2004. The IUCN red list of threatened species. Di sponí vel em:< http://www. iucn red list.
org/info/cat e go ries_cri te ria2001. html>. Aces so em. 12.
Mayer F, Dietz C, Kiefer A. 2007. Molecular species identification boosts bat diversity. Front Zool. 4(1): 4.
Molur S, Marimuthu G, Srinivasulu C, Mistry S, Hutson AM, Bates PJ, Walker S, Priya KP, Priya AB
editors. Conservation Action Management Plan (CAMP) Workshop Report, Zoo Outreach
Organisation, 320pp. 2002.
Roberts T. 1997. The mammals of Pakistan (revised ed.) Oxford University Press. Karachi, Pakistan. 525.
Srinivasulu C, Srinivasulu A, Srinivasulu B, Jones G. 2019. Integrated approaches to identifying cryptic bat
species in areas of high endemism: The case of Rhinolophus andamanensis in the Andaman Islands.
PloS one. 14(10): e0213562.
Wilson DE, Reeder DM. 2005. Mammal species of the world: a taxonomic and geographic reference. JHU
Press.
Experiment No.2
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Table 4.1. Morphological measurements (mm) of Eptesicus nasutus bats from FATA, Pakistan
Body Parameters
Species under study
Mean Sind Serotine Bat (Eptesicus nasutus)
Range (mm)
Head and Body length 44.3 40-51
Tail length 43.4 42-46
Hind foot length 8.3 -
Forearm length 35.7 35.4-36.9
5th
Metacarpal Length 33.2 31.7-34.4
4th
Metacarpal Length 34.7 33.7-35.7
3rd
Metacarpal Length 35.3 34.1-36.6
Ear length 36 -
Figure 4.1. Evolutionary analysis by Neighbor Joining method and General Time Reversible
model for Eptesicus nasutus from FATA, Pakistan.
Experiment No.2
37
Table 4. 2. Phylogenetic analyses of Eptesicus nasutus from FATA, Pakistan by Neighbor-joining
method with bootstrap values on branches.
Accession No.
EU786840 ID
EU786839 1 ID
FJ841981 1 1 ID
FJ841980 1 1 1
KF019043 0.944 0.944 0.944 0.944 ID
KF019042 0.944 0.944 0.944 0.944 1 ID
KF019057 0.928 0.928 0.928 0.928 0.934 0.934 ID
KF019056 0.928 0.928 0.928 0.928 0.934 0.934 1 ID
MF143467 0.859 0.859 0.859 0.859 0.859 0.859 0.849 0.849 ID
JN020554 0.859 0.859 0.859 0.859 0.859 0.859 0.849 0.849 1 ID
MT674673 0.431 0.431 0.431 0.431 0.418 0.418 0.428 0.428 0.399 0.399
38
CHAPTER 5
Experiment No. 3
Phylogenetic Analysis of Two Pipistrellus Species (Mammalia: Chiroptera) from Pakistan
with an Emphasis from FATA Region
Muhammad Idnan1,4, Arshad Javid1, Ali Hussain1, Sajid Mansoor2, Muhammad Tayyab3, Muhammad
Imran3, Wasim Shehzad3, Arif Ullah3, Waqas Ali1, Syed Mohsin Bukhari1, Hamid Ullah 1Department of Wildlife and Ecology, University of Veterinary & Animal Sciences, Lahore, Pakistan. 2Department of Microbiology, Faculty of Life science, University of Central Punjab, Lahore, Pakistan. 3Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore,
Pakistan. 4Department of Zoology, Faculty of Sciences, University of Central Punjab, Lahore, Pakistan. 5Department of Zoology, University of Peshawar, Khyber Pakhtunkhwa, Pakistan.
Corresponding Author email; [email protected]
ABSTRACT: Due to a high rate of cryptic speciation in chiroptera, species identification is a difficult
process, based on their morphological parameters. Bat fauna of Pakistan is poorly explored, particularly
based on molecular techniques for identification. So, the current study was designed to investigate the
genetic identification of genus Pipistrellus using 16S rRNA as a genetic marker. Bats were collected by using
mist nets from different localities of Federally Administered Areas (FATA) of Pakistan. DNA was extracted
from biopsies of wing tissues. Vesper bats i.e., Pipistrellus coromondra and Pipistrellus kuhlii lepidus were
reported for the first time by using16S rRNA as a genetic marker and their phylogenetic analyses were
carried out using Clustal X2 and MEGA-X. The overall genetic variations among Pipistrellus coromondra
and Pipistrellus kuhlii lepidus are 8% and 1% respectively. Pipistrellus kuhlii lepidus is reported as a new
record as a cryptic species of Pipistrellus kuhlii. In this study, we have provided a data about morphological
parameters and phylogenetic analyses. FATA region of Pakistan is mostly the hilly area and perhaps it has
least modified and degraded by anthropogenic activities, hence new species may be reported. Further
detailed analyses are recommended to explore the ecology, habitat management and genetic diversity in
chiropteran fauna from the study area.
Key words: Pipistrelle, Phylogeny, species identification, mitochondrial, 16s rRNA, Pakistan.
Introduction:
The genus Pipistrellus is comprising of 51 species throughout the world (Koopman 1994), 12 from
subcontinent (Bates and Harrison 1998) and 8 species from Pakistan (Roberts 1997). The distributional range
extends from Eurasia to Japan, central southern Africa, Solomon Islands, Indonesia, northern Australia,
Canada, New Guinea, USA and Mexico (Roberts 1997). Bat fauna of Pakistan is poorly explored, so an
extensive chiropteran survey is recommended to study these environment friendly creatures (Javid et al.
2014; Javid et al. 2015).
The work of systematics has started from the last 250 years, despite majority of the species is still
unidentified. Currently, the task of species identification has been resolved by DNA barcoding, where
specific sequence of DNA is used for species identification. Generally, the technology of DNA sequencing
has resolved the taxonomic disputes of many taxa, but some higher taxa have not yet been resolved precisely
as a species. The task of species identification by DNA barcoding is very useful to resolve the taxonomic
problems of cryptic species, extinct species, synonymous species or matching the juvenile with adults.
However, DNA barcoding is proved as a standard too for species identification. In most of the areas of the
world, the bat fauna is either rare or least known, consequently they have a low abundance along with their
lifestyle and hence these are the least explored taxa of mammals. Bats are also presenting minimal
morphological variations and overlapping measurements, highly cryptic species, which have been explored
by molecular analyses (Clare 2011; Dool et al. 2016; Gager et al. 2016; Miranda et al. 2011).
DNA barcoding is a fast and widely used tool for an accurate species differentiations and
identification (Clare et al. 2011; Wilson et al. 2014). Hence, the current study was designed to explore the
Experiment No.3
39
accurate species identification using 16S rRNA as a marker to explore the bat fauna of Bajaur Agency,
FATA, Pakistan.
Materials and Methods:
A total of 10 samples of morphological different bat species were captured using mist nets from
various sites in Bajaur Agency (N 34° 43' 48.7812", E 71° 28' 45.9012"), Federally Administered Tribal
Areas (FATA) of Pakistan. The samples were primarily identified on the basis of their morphology and
preserved in 70% ethanol. The morphometric measurements were also observed before preservation and
comparative observational analyses were performed (Bates and Harrison 1998; Roberts 1997).
All the lab work was performed at Institute of Biochemistry and Biotechnology (IBBt), University of
Veterinary and Animal Sciences, Lahore. DNA was extracted from ethanol (70%) preserved specimens
(wing tissue) by proteinase K digestion and standard phenol-chloroform extraction (Hoelzel and Green
1992). Universal primers for 16S rRNA Forward: 5´-AAAGACGAGAAGACCC-3´ and Reverse: 5´-
GATTGCGCTGTTATTCC-3´.
Amplification was performed in a 100 μl of a solution containing 67 mM Tris (pH 8.8), 6.7 mM
MgSO4, 16.6 mM (NH4)2SO4, 10 mM 2-mercaptoethanol, each dNTP at 1 mM, each primer at 1 μl, genomic
DNA (10-1000 ng), and 2-5 units of Thermus aquaticus polymerase (Perkin-Elmer/Cetus).
Denaturation for polymerase chain reaction was carried out for 1 min at 93 °C, for the same time
period hybridization at 50 °C, DNA extension was carried out at 72 °C for 2-5 min. This was repeated for 50
times. 1 µL of each DNA sample (50ng/ µL) was separated into different tubes. The PCR components were
made by adding the following to give a total volume of 150 µL: 113 µL sterile ddH2O, 3 µL 16S rRNA – F
primer (10pmol/ µL), 3 µL 16S rRNA – R primer (10pmol/ µL), 15 µL dNTPs (8mM), 15 µL Jefferies
Buffer (4mM MgCl2), and 1 µL Taq DNA polymerase (5 units/ µL). 20 µL of the PCR components was
added to each of the tubes containing the DNA samples. The tubes were placed on ice till all samples were
prepared. The tubes were loaded into a PCR machine with the pre-set program as 94°C for 2 minutes (1
cycle); 94°C for 1minute, 60°C for 45 seconds, 72°C for 50 seconds (30cycles) and 72°C for 3 min (1 cycle).
The electrophoresis of 5 μl PCR amplified mixture was performed in a 2% agarose gel in 100 ml of
TAE-I buffer (Tris 40mM-Acetate 20mM-EDTA 2mM) at pH 8.3 by staining with ethidium bromide.
PCR fragments were sequenced by ABI 310 sequencer. The sequences were aligned by ClustalW
method. The sequences were submitted to GenBank for accession numbers MT430902 for Pipistrellus kuhlii
lepidus and MN 719478 for Pipistrellus coromondra, available on NCBI for 16S rRNA. Phylogenetic and
molecular evolutionary analyses were conducted using MEGA version X to construct the phylogenetic trees
(Kumar et al. 2018).
Results & Discussion:
Distribution: Bats specimens were collected from different areas of Bajaur Agency (34°24′17.76″N
72°33′32.16″E), FATA, Pakistan.
Taxonomic Position: Least Pipistrelle (Temminck, 1840): Pipistrellus tenuis
Indian Pipistrelle (Gray, 1838): Pipistrellus coromondra
Common Pipistrelle (Schreber, 1774): Pipistrellus pipistrellus
Pipistrellus kuhlii lepidus (Blyth, 1845)
Morphology: Various morphological parameters like Head and Body length (HB), Tail length (TL), Hind foot length (HL),
Forearm length (FL), Wing span (WS), 5th Metacarpal Length (ML 5th), 4th Metacarpal Length (ML 4th) and
Ear length (EL) for species belonging to genus pipistrellus are mentioned in table 1.
Phylogenetic Relationship:
In Pakistan the genus pipistrellus is represented by 8 species based on their morphological
parameters. No phylogenetic survey has been conducted to explore the genetic diversity in chiroptera
taxonomy. mtDNA sequences are not available for chiropteran species belonging to Pakistan. The available
16S rRNA gene sequences for Pipistrellus kuhlii lepidus and Pipistrellus coromondra were retrieved from
NCBI website and a phylogenetic analysis conducted to build a phylogenetic tree. The DNA sequences were
Experiment No.3
40
obtained to corelate morphological parameters with genetic identification of the species which have shown
reliable and clear methods for almost all the species under study. Neighbor-joining trees based on Kimura 2-
parameter distance was used to construct the phylogenetic tree, shown in Figure 1. The morphological
parameters and their subsequent phylogenetic analysis lead to the confirmation of above-mentioned species
from Pakistan. Evolutionary divergence between Sequences for Pipistrellus coromondra and Pipistrellus
kuhlii lepidus from FATA, Pakistan is mentioned in table 2. The Pipistrellus kuhlii lepidus was considered to
be Pipistrellus kuhlii but phylogenetic analysis revealed to be P.k.lepidius. Overall, the interspecific genetic
variations among Pipistrellus coromondra and Pipistrellus kuhlii lepidus are 8% and 1% respectively.
The partial sequence of 16S rRNA confirms the species identity and this information could be used
for conservational and other ecological related studies. Another important implication of mtDNA study is to
assess the genetic diversity at inter-specific and intra-specific level. Genetic diversity is an important
component of biodiversity and it could be used to formulate conservation and management planes to
preserve the evolutionary history of a species. It is estimated that the bats are constituting about 28% of
mammalian fauna in Pakistan but it is debatable for exact number of bats‘ fauna within the territorial
boundary of the country (Roberts and Bernhard 1977; Walker and Molur 2003; Wilson and Reeder 2005).
In Pakistan there are about 8 families of bats, 26 genera and 54 species has so far been discovered
based on their morphological basis (Mahmood-ul-Hassan 2009), this is equivalent to any region of the world
with same climatic and topographic conditions and no data is yet available on barcoding of bats up till now
in the country (Horáček et al. 2000). Species identification and characterization has a crucial role in
taxonomy and classification of organisms. Modern taxonomy, originated in mid18th century has described up
to 1.7 million species of organisms (Stoeckle 2003). Besides this, to study the relationship of living beings
with each other various behavioral and morphological parameters are taken into consideration. It is very
unsurprising that the larger animals are given a priority for description and the smaller ones mostly remain
unknown in sciences (Blaxter 2003).
Genetic analysis of species provides a useful information about the scales at which the wild species
are impacted by anthropogenic activities but also provides the information about a successful demographic
management of wild species (Sovic et al. 2016). Advancement in molecular techniques has revolutionized
the field of systematics and improved the taxonomy of some more complex chiropteran species. Molecular
genetics highlighted many new discoveries in taxonomy of understudied and species rich tropical areas
(Clare et al. 2007; Francis et al. 2010), besides this, in temperate fauna where the relative species diversity is
low, the molecular genetics has also resolved taxonomic uncertainties (Mayer et al. 2007; Mayer and von
Helversen 2001).
Pipistrellus kuhlii lepidus from Pakistan making a clade with a species having accession number
HQ857597 which is also a Pipistrellus kuhlii lepidus as a sister species from Sardinia, this subspecies of
Kuhl‘s pipistrelle was not previously reported from the study area, which may be due to its morphological
non-differentiation due to cryptic species. Such a report of a new record also highlights the importance of
genetic identification as compared to the conventional methods for taxonomy. An extensive survey should be carried out in the country to explore and compare the conventional taxonomic methods with barcoding.
Pipistrellus coromandra: Indian Pipistrelle (Gray, 1838) is distributed in Afghanistan, Bangla Desh,
India (including Nicobar Isls), Sri Lanka, Pakistan, Nepal, Bhutan, Burma, Cambodia, Thailand, S China. In
Pakistan it is has been collected from Dir, Chitral and Swat districts of North Western Frontier Province.
This a small pipistrelle and is often difficult to distinguish from P. tenuis. In general P. coromandra averages
larger than P. tenuis but there is a significant overlap in all external measurements. In BLASTn results our
query sequence (MN719478) has shown a 97.45% percentage identity and 99% query coverage with
Pipistrellus coromandra (KT291766), which has been reported from India.
Since last two decades, genetics has played a major role in ecology and conservation biology
(Frankham 2005; Frankham et al. 2002; Hedrick 2001). Genetics has significant contributions to understand
the effects of habitat fragmentation, genetic erosion on extinction and endangerment of the species, the
dynamics of adaptation of species to the new environmental circumstances are added, results in the formation
of a modern scientific filed of biology called ―Conservation Genetics‖ (Ouborg et al. 2006). Whereas several
Experiment No.3
41
conservation efforts measured at native scale or regional levels, they could affect the biotic consequences of
universal phenomenon, specifically the recent climatic changes and their consequences on populations‘
extinction rate that is now believed to be on the top of the background levels (McLaughlin et al. 2002).
Conclusion: In conclusion, the taxonomic problems of cryptic species could be resolved by such a
short segment of 16S rRNA. This mitochondrial genome is an effective tool for an accurate, rapid, low cost
and easy applicable method for species identification. This could also be helpful in conservation issues and
to prevent the trade of endangered species in forensic sciences. Molecular identification of species also seeks
its importance for commercial purposes such as the mislabeling of meat and meat products.
Acknowledgement: The field work was facilitated by Dr. Hamidullah for sampling, Mr. Arifullah
and Mr. Salman assisted for lab work. We are very thankful to Dr. Muhammad Imran and Professor Dr.
Waseem Shahzad, Director Institute of Biochemistry and Biotechnology (IBBt), University of Veterinary
and Animal Sciences, Lahore for technical help and lab facilities along with technical guidelines during
difficult steps of research work. We also thank for any anonymous who helped for constructive comments.
“Data availability statement”: The sequences of this study are submitted to GenBank for accession
numbers MT430902 and HQ857597 for Pipistrellus kuhlii lepidus and MN 719478 and KT291766 for
Pipistrellus coromondra, available on NCBI for 16S rRNA.
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One. 6(7): e21460.
Clare EL, Lim BK, Engstrom MD, Eger JL, Hebert PD. 2007. DNA barcoding of Neotropical bats: species
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house bat (Scotophilus kuhlii) from Punjab, Pakistan. Mammalia. 78(1): 133-137.
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Mahmood-ul-Hassan M. 2009. The Bats of Pakistan: The least known creatures. VDM Publishing.
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Experiment No.3
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Figure 5.1. Evolutionary analysis by Neighbour joining tree and General Time Reversible
method of vesper bats from FATA, Pakistan.
Table1. 5.1. Morphological measurements (mm) of Pipistrellus coromondra and Pipistrellus kuhlii
lepidus from FATA, Pakistan
Body Parameters Species under study
Pipistrellus coromondra (n= 2) Pipistrellus kuhlii lepidus (n= 2)
Range Range
Head and Body length (HB) 43 34-49 45 40-50
Tail length (TL) 35 22-39 37 30-40
Hind foot length (HL) 7 3.4-8 6.8 5-6
Forearm length (FL) 32 25.5-34.3 33.9 30.3-37.4
Wing span (WS) 196 190-220 220 210-230
5th
Metacarpal Length (ML 5th
) 28.1 25.2-31.1 7.6 7.0-8.0
4th
Metacarpal Length (ML 4th
) 28.7 25.7-32.7 10.4 10.0-11.0
3rd
Metacarpal Length (ML 3rd
) 29.0 25.8-33.1 10.3 10.0-11.0
Ear length (EL) 11.00 7.1-14.0 12.4 12-13
Experiment No.3
44
Table 5. 2. Estimates of Evolutionary Divergence between Sequences for Pipistrellus coromondra
and Pipistrellus kuhlii lepidus from FATA, Pakistan
Ac. No MF078005 HQ857598 HQ857597 MT430902 AY495524 MN719478 KT291766 JQ039197 AY495529 KF059977
MF078005
HQ857598 0.004
HQ857597 0.000 0.004
MT430902 0.000 0.004 0.000
AY495524 0.216 0.210 0.216 0.216
MN719478 0.204 0.197 0.204 0.204 0.095
KT291766 0.196 0.190 0.196 0.196 0.100 0.012
JQ039197 0.180 0.174 0.180 0.180 0.090 0.102 0.107
AY495529 0.180 0.174 0.180 0.180 0.090 0.102 0.107 0.000
KF059977 0.207 0.201 0.207 0.207 0.086 0.020 0.024 0.104 0.104
45
CHAPTER 6
Experiment No. 4
Phylogenetic Analyses of Kuhl’s Pipistrelle from Northern Areas of Pakistan
Muhammad Idnan*1,4, Arshad Javid1, Ali Hussain1, Sajid Mansoor2, Muhammad Tayyab3, Muhammad
Imran3, Wasim Shehzad3, Arif Ullah3, Syed Mohsin Bukhari1, Hamid Ullah5, Waqas Ali1 1Department of Wildlife and Ecology, University of Veterinary & Animal Sciences, Lahore, Pakistan. 2Department of Microbiology, Faculty of Life science, University of Central Punjab, Lahore, Pakistan. 3Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore,
Pakistan. 4Department of Zoology, Faculty of Sciences, University of Central Punjab, Lahore, Pakistan. 5Department of Zoology, University of Peshawar, Khyber Pakhtunkhwa, Pakistan.
Corresponding Author email; [email protected]
Abstract: A high degree of cryptic speciation in chiropteran species hinders a clear identification. During
current study specimen representing Kuhl‘s Pipistrelle were examined for external characteristics and body
measurements from northern areas of Pakistan. We also sequenced and analyzed mitochondrial 16S rRNA
gene as a genetic marker for morphotypes of Kuhl‘s Pipistrelle. Some representative sample (n=6) for Kuhl‘
Pipistrelle were used for molecular analyses by 16S rRNA as a genetic marker. Based on molecular results
(both BLASTn and Phylogenetic Tree) we confirmed the presence of Pipistrellus kuhlii lepidus as a new
record from the study area. Neighbor-Joining and Maximum Likelihood trees produced same phylogenetic
results for Kuhl‘s pipistrelle. Pipistrellus kuhlii lepidus was an out group in both trees. Estimates of
interspecific and intraspecific identity matrix between Sequences of Pipistrellus kuhlii and Pipistrellus kuhlii
lepidus (at sub-species level) based on Kimura-2 parameter was maximum 99 % and minimum 39 % while
an evolutionary divergence was recorded as minimum 0.3 % and maximum 1 %. A detailed phylogenetic
analysis of Asiatic Kuhl‘s Pipistrelle is recommended to understand the cladistic relation of the species under
study.
Key words: Chiroptera; Kuhl‘s Pipistrelle; sub-species; species identification; 16S rRNA; Pakistan.
Introduction: During recent years, in taxonomy of bats cryptic species is a hot topic. The discoveries of new
species are the result of applications of molecular techniques. A high degree of population substructures was
not expected in a volant group of mammals (Order: Chiroptera) but the molecular tools revealed a significant
number of species and phylogenetic patterns in these animals. This is not only helpful to rearrange the
cladistic relation of chiropteran species but is also exploring the mechanisms, endured to cause such a
significant degree of cryptic speciation. Such a discoveries of cryptic species marks a question on
universality of standards to explain the sympatric conditions by range extension of allopatric species. This
demands a healthy discussion for speciation under sympatric or parapatric situations along with both
ecological and behavioral mechanisms which are affecting it (Losos and Glor 2003).
Kuhl‘s pipistrelle is a small (5-7g) vespertilionid bat, with a geographic range extending from
Mediterranean Europe to Western India (Walker and Molur 2003a). by a study of mitochondrial and nuclear
marker it is suggested that Kuhl‘s pipistrelle consists of four subpopulations i.e., the Atlantic Islands lineage,
the Eastern lineage, the Western lineage and the Middle Eastern lineage (Andriollo et al. 2015). P. kuhlii is
commonly found in anthropic environments such as agricultural and urban areas. This species is found in
diverse environments like deserts, temperate grasslands and at high altitudes (up to 2000 meters) (Aulagnier
et al. 2010). The species is classified as ‗Least Concern on the IUCN red list of threatened species (Juste and
Paunović 2016).
Kuhl‘s Pipistrelle (Kuhl, 1817) is an Afrotropical and West-Palearctic species. Apparently, its origin
is tropical, range extends from North to South Africa along the eastern coast and from the Middle East and
Turkestan, Caucasus to Uzbekistan, and Kashmir. From Europe, it is found in Islands of Canary and
Experiment No.4
46
Balearic, Atlantic coasts of Portugal and Spain all over Southern Europe. Recently Its range has expanded
northwards from Northwestern France through Switzerland, Austria, Southern Germany, Southwestern
Russia and Hungary to Northeastern Ukraine (Cel‘uch and Ševčík 2006; Sachanowicz et al. 2006) and
occasionally reported from United Kingdom (UK) (Bogdanowicz 2004).
The collection of voucher specimen is a debatable issue in current era (Russo et al. 2017) as it raises
apprehensions about redundant collections of organisms (Corthals et al. 2015), the alternates for vouchers
could be images or molecular studies (Corthals et al. 2015; Raupach et al. 2016). Species identification and
differentiation is accurately carried out by DNA barcoding techniques (Clare et al. 2011; Wilson et al. 2014).
Some studies also suggest the non-lethal methods for barcoding such as blood, fecal samples and buccal
swabs etc., (Walker et al. 2016) and from tail or wing tissues (Faure et al. 2009; Wilson et al. 2014). In case
of bats uropatagium tissue is recommended as it heals quickly and for molecular studies proved as a source
of high quality DNA (Faure et al. 2009).
The current study was designed to explore the genetic data for species confirmation from
morphological to genetic diversity by using 16S rRNA as a genetic marker and subsequently to carry out the
phylogenetic analysis of Kuhl‘s Pipistrelle from Bajaur Agency, FATA, Pakistan.
1. Materials and Methods:
1.1. Sample collection and preservation: A total of 20 specimens were captured using mist nets from
various sites of Bajaur Agency, FATA, Pakistan (N 34° 43' 48.7812", E 71° 28' 45.9012"). The
samples were identified in the field on the basis of morphology and preserved in 70% ethanol (Bates
and Harrison 1998; Roberts 1997). The preserved samples brought the Lab, at Institute of Biochemistry
and Biotechnology (IBBt), University of Veterinary and Animal Sciences, Lahore.
1.2. Morphological Identification: The samples were primarily identified on the basis of their
morphology and some samples as representatives were preserved in 70% ethanol. The morphometric
measurements were also observed before preservation and comparative observational analyses were
performed (Bates and Harrison 1998; Roberts 1997).
1.3. DNA extraction and amplification: DNA was extracted from ethanol (70%) preserved specimens
(wing tissue) by standard phenol-chloroform method (Hoelzel and Green 1992). The Purity of DNA
was checked through agarose gel electrophoresis. Total genomic DNA was amplified using 16S rRNA
universal primers set (Forward: 5´-AAAGACGAGAAGACCC-3´ and Reverse: 5´-
GATTGCGCTGTTATTCC-3´).
Amplification was performed in a 100 μl of a solution containing 67 mM Tris (pH 8.8), 6.7 mM
MgSO4, 16.6 mM (NH4)2SO4, 10 mM 2-mercaptoethanol, each dNTP at 1 mM, each primer at 1 μl,
genomic DNA (10-1000 ng), and 2-5 units of Thermus aquaticus polymerase (Perkin-Elmer/Cetus).
The PCR amplification comprised of 93 °C for 1 min, 40 cycles at 93 °C for 1 min, 50 °C for 30
seconds, 72 °C for 2 min and a final 10 min at 72 °C. The PCR products were checked through 1 %
agarose gel. PCR products were purified by the Qiagen purification kit and all the samples were Sanger
sequenced on AB3730xl sequencer Applied Bio-system, Korea.
1.4. Data analysis: The obtained DNA sequences checked on BioEdit version 7.2 and aligned using
ClustalX (Larkin et al. 2007). After trimming ambiguous bases, DNA sequences were submitted to
GenBank and accession numbers were obtained (MT430902, MT903614, MT903615, MT913567,
MT913568, MT856878). All the sequences of Pipistrellus kuhlii and Pipistrellus kuhlii lepidus were
subject to BLAST analysis to retrieved closely matched sequences. Genetic variation between and
within the species were calculated using MEGA 10 using p-distance. Neighbor-joining tree was
constructed using 100 bootstraps in MEGA 10 (Kumar et al. 2018).
2. Results:
2.1. Taxonomic Position:
Vespertilio kuhlii: (Kuhl, 1817)
Nycticeius canus: (Blyth, 1863)
Scotophilus lobatus: (Jerdon, 1867)
Vespertilio (Pipistrellus) leucotis: (Dobson, 1872)
Pipistrellus lepidus: (BLYTH, 1845)
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Pipistrellus kuhlii lepidus: (WROUGHTON, 1918)
Subspecies: Pipistrellus kuhlii lepidus: (Blyth, 1845)
2.2. Distribution: Pipistrellus kuhlii lepidus (Blyth, 1845) is reported from India (Assam, Maharashtra,
Meghalaya, and West Bengal), Afghanistan (Helmand, Kandahar, Nangarhar, Nimruz, and Paktiya
Provinces) and Pakistan [Balochistan, Punjab, Sindh and FATA (present record)].
Comments: Belongs to the kuhlii species group
2.3. Morphology: Various morphological parameters like Head and Body length (HB), Tail length (TL),
Hind foot length (HL), Forearm length (FL), Wing span (WS), 5th Metacarpal Length (ML 5th), 4th
Metacarpal Length (ML 4th) and Ear length (EL) for species belonging to genus pipistrellus are mentioned in
table 1. Based on morphological and external characteristics the specimens under study were separated into
two groups. One is of Pipistrelles kuhlii lepidus with dorsal pelage as whitish or pale sandy while the second
group is of Pipistrellus kuhlii with bright yellowish-brown coloration to dark grayish brown. Face and ears of
the former one was bright with a yellowish or orange tinge around eyes wile in later one they are light to
dark brown/black. Similarly, a color differentiation was also noted in penis. It was orange in Pipistrelles
kuhlii lepidus and pinkish brown in Pipistrelles kuhlii. A broad pale or whitish yellow wing margin was also
present in Pipistrelles kuhlii lepidus as compared to a uniformly narrow wing margin in Pipistrelles kuhlii.
The extent of the pale wing margin did not overlap in these two taxa (figure 2).
Discussion: The original description of Pipistrellus lepidus (Hutton 1845) was based only on external
characteristics, for example overall pale coloration [light yellowish-clay, pale sandy] and broad pale wing
margins. Bats from central Asia and the Caspian Sea region, considered to represent P. k. lepidus, share
similar external characteristics and/or have comparably long forearms with mean values from 35.0 to 36.2
mm (Table 3). Morphological characteristics and measurements of bats from Eastern and Central European
populations, including specimens examined in this study (Table 3), correspond to the above-mentioned bats
supporting a hypothesis that these populations represent the eastern P. k. lepidus Blyth, 1845. This
arrangement seems also consistent with genetic studies‘ results from western Russia (Kruskop et al. 2012), as
well as our genetic analysis. The genetic and morphological similarity of these populations reflects their
geographic proximity and arid habitat resemblance of central Asia and the Middle East, including eastern
Transcaucasia, which is a source area of P. k. lepidus invasion into Eastern (Strelkov et al. 1985) and Central
Europe. It seems likely that the only other Asiatic subspecies of P. kuhlii, P. k. ikhwanius (Cheesman and
Hinton 1924) is a junior synonym of P. k. lepidus, but its taxonomic status requires elucidation. Pale-
coloured representatives of P. k. ikhwanius (type locality: Hufuf, Saudi Arabia) are similar in external
characteristics (Cheesman and Hinton 1924) to P. k. lepidus, but they are visibly smaller with the forearm
length ranges (30.2–32.3 mm in three males, and 32.6–33.5 mm, mean 33.0 mm, in four females, Harrison
1964) non-overlapping with the European and Asiatic representatives of P. k. lepidus (Table 3). These Saudi
Arabian bats also seem to be smaller than P. k. kuhlii from the Balkans (Table 3). Surprisingly, we found that
P. k. lepidus was the largest representative of the genus Pipistrellus in Europe, exceeding by 1.3–2.2 mm
(males) and 0.9–1.6 mm (females) mean forearm lengths of Central European and Balkan P. nathusii (Table
3). Geographical distribution of mitochondrial haplotypes. Our results confirm the presence of two
haplogroups in both mitochondrial markers that reflect the division into western P. kuhlii/deserti and eastern
P. lepidus lineages recorded in earlier studies (Coraman et al. 2013; Mayer et al. 2007; Veith et al. 2011), and
that these lineages were allopatric. This may indicate the allopatric speciation of these sister taxa and their
recent simultaneous northward expansion from different geographic refugia in the Mediterranean (P.
kuhlii/deserti lineages) (Andriollo et al. 2015), and the Asiatic region (P. k. lepidus; areas south of the Black
Sea and the Caspian Sea) (Coraman et al. 2013). H2 haplotypes for 16S and COI markers are characteristic
for bats of the P. kuhlii/deserti lineages that in our study have been identified as P. k. kuhlii on the basis of
their morphology. This taxon is distributed in the western Balkans and reaches eastern Pannonia in the north.
These haplogroups seem to be widespread in P. kuhlii I populations across the Mediterranean basin
(Andriollo et al. 2015), and have also been recorded in Austria (Veith et al. 2011) and the Balkans
(haplotypes characteristic for this taxon based on Cytb and nd1 markers, (Coraman et al. 2013). Haplotypes
specific for P. k. lepidus, identified in our study as H1 in both markers, occurs in bats from Poland, Ukraine,
Experiment No.4
48
Slovakia, Hungary and Romania. In previous studies these and other haplotypes from mitochondrial markers
(Cytb, nd1) were identified in bats from Central and Eastern Europe (Poland, Ukraine and Russia), the
Caucasus and the Middle East (Coraman et al. 2013; Kruskop et al. 2012; Mayer et al. 2007; Veith et al.
2011). Representatives of these two lineages were recorded to be syntopic in the east of the Pannonian Basin
in Slovakia (this work), confirming that their geographic ranges had already contacted in recently invaded
parts of Central Europe (Danko 2007).
Besides, the two lineages were recorded as sympatric in southern Turkey (Coraman et al. 2013).
Therefore, a question appears about the reproductive isolation of these taxa. The issue whether they represent
separate species requires further studies, also with respect to their biology and ecology. Still, both of them
seem to be clearly separable when either molecular or morphological methods are used. Although absent
polymorphism in nuclear RAG2 marker and known moderate differentiation in mitochondrial markers in P.
k. lepidus and P. k. kuhlii may suggest evolutionally young species, it should be emphasized that DNA
barcodes fail to distinguish recently diverged species. For example, among European bats there are
morphologically very similar pairs M. m. mystacinus/M. m. bulgaricus and M. myotis/M. oxygnathus or
genetically similar but morphologically different E. serotinus and E. nilssonii (Mayer et al. 2007).
External characteristics and their usefulness in distributional study. Adult bats of P. k. kuhlii and P. k.
lepidus are readily separable on the basis of their external characteristics and measurements although some
of them overlap. Morphologically, they represent taxa that are better differentiated than some of their
congeners recognized as cryptic species, i.e. P. pipistrellus and P. pygmaeus (Dietz et al. 2009). The overall
body coloration and size (pale sandy, pale-faced larger bats vs. light or dark brown smaller bats with dark
faces), the extent of pale wing margin and its shape (> 3.5 mm and characteristically broadened vs. 0.5–1
mm and narrow), and particularly the coloration of the penis and the skin around the vagina appear to have a
diagnostic value. The distinctly pale pelage and skin coloration should be treated as a sign of the species‘
adaptation to desert and semi-desert habitats of south-western Asia, which may be defined as the native
geographic range of P. k. lepidus. Among the bat fauna of Europe where all bat species, including the
Mediterranean P. k. kuhlii, have more or less dark, brownish or greyish coloration, P. k. lepidus is unique
due to its original appearance resembling exotic bat species. Additionally, there is no other similar species
with such distinctive pale pelage as well as face and ears coloration (Dietz and Kiefer 2014). None of P. k.
kuhlii individuals from Albania and Slovakia had pelage and face coloration typical for P. k. lepidus or a
wide pale wing margin, which is similar in its extent in bats from Poland, Ukraine and Slovakia (this work),
in four females from Romania (4.2–5.6 mm, mean 5.1 mm), (Barti 2010) and in bats from Turkmenia (4.0–
7.5 mm, mean 6.0 mm), (Strelkov et al. 1978).
Apart from a few exceptions, the pale wing margin was distinctly broadened also in bats from south-
west Russia (Strelkov et al. 1985) and Azerbaijan (up to 8 mm, mean 5.0 mm), (Rahmatulina 2005). Lighter
typically colored individuals and darker adults of P. k. kuhlii may reflect animals' age differences. Similarly,
juveniles of P. k. lepidus are darker (greyish buff) and less contrasting than the adults (Rahmatulina 2005).
Clear differences in coloration of the penis and the skin around the vagina, recorded for the first time in the
present study, seem analogous but are more pronounced than these between P. pipistrellus and P. pygmaeus
(Dietz et al. 2009) with similar penis coloration in P. k. kuhlii and P. pipistrellus (pinkish brown), and in P. k.
lepidus and P. pygmaeus (bright orange and yellowish). Yellowish orange coloration of the penis was
already noticed in a juvenile male of P. k. lepidus found in December (see Fig. 1) (Sachanowicz et al. 2006),
indicating that this feature might not be limited to summer or fully adult individuals. These external
characteristics seem to be sufficient to distinguish P. k. kuhlii and P. k. lepidus, particularly in the areas of
their previously allopatric geographic ranges (eastern and southern populations of P. kuhlii s. l., (Dietz and
Kiefer 2014; Sachanowicz et al. 2006), where morphological variation is low (this work). These features may
also be used to identify bats whose morphological details and/or photographic documentation were
published. Their validity has to be tested with larger samples of bats, particularly from the limited sympatry
zone in Europe (hybrids possibility) and beyond Europe, due to uncertain taxonomic affiliation of some
populations and their largely unknown geographic variability. The distribution pattern of these two taxa in
Central Europe and the Balkans recorded in our genetic analysis corresponds to morphotype distribution. The
westernmost localities of P. k. lepidus morphotype were recorded in the south of Poland (Sachanowicz et al.
Experiment No.4
49
2006; this paper), eastern Slovakia (Danko 2007), central Romania (where at least 16 of 17 bats examined
were referred to as P. k. lepidus, Barti 2010), and as far as south-central Bulgaria (Tilova et al. 2005). The
northern and easternmost localities of P. k. kuhlii morphotype were reported from the south-east of Czech
Republic (Wawrocka et al. 2012), eastern Slovakia and Hungary (Danko 2007; P. Estók & T. Görföl pers.
comm.), as well as the north of Serbia (Paunovic & Marinkovic 1998). The contact zone of these two taxa is
narrow, ranging from the east of Slovakia to southern Hungary (Danko 2007; P. Estók & T. Görföl pers.
comm.; this work), and indicating their parapatric ranges. Because there are no real geographic barriers, the
contact zone may be expected to spread further across the Pannonian and the Carpathian regions of adjacent
countries (i.e. Poland, Czech Republic, Ukraine, Romania and Serbia), and also in the area of Bulgaria,
however its range, the extent of sympatric occurrence and the presence of possible hybrids require further
studies.
In Pakistan there are about 8 families of bats, 26 genera and 54 species has so far been discovered
based on their morphological basis (Mahmood-ul-Hassan 2009), this is equivalent to any region of the world
with same climatic and topographic conditions and no data is yet available on barcoding of bats up till now
in the country (Horáček et al. 2000). Species identification and characterization has a crucial role in
taxonomy and classification of organisms. Modern taxonomy, originated in mid18th century has described up
to 1.7 million species of organisms (Stoeckle 2003). Besides this, to study the relationship of living beings
with each other various behavioral and morphological parameters are taken into consideration. It is very
unsurprising that the larger animals are given a priority for description and the smaller ones mostly remain
unknown in sciences (Blaxter 2003).
In Pakistan Pipistrellus kuhlii has been reported from with a wide distributional range Baluchistan
(Panjgur & Chagai), Sindh (Pithoro, Jacobabad, Hyderabad, Mir Pur Khas and Sukker) and from Punjab
(Muzaffar Garh, Rajanpur and Faisalabad) (Roberts 1997; Taber et al. 1967) but no record for Pipistrellus
kuhlii lepidus is reported up till now. Several specimens of Kuhl‘s Pipistrelle were captured by mist net.
Some representative samples (n=6) were used for molecular studies. BLASTn results and phylogenetic
analysis of these specimens revealed that we have a subspecies of Kuhl‘s Pipistrelle, i.e., Pipistrellus kuhlii
lepidus. The new locality of Pipistrellus kuhlii lepidus was from Government Degree College, Nawagi N34º
41.896 E71º 20.345 at an elevation of 1031m Bajaur Agency, Pakistan. Partial sequences with the length of
340-bp of Pipistrellus kuhlii lepidus comprising the 16S rRNA gene segments sequenced and available
sequences from GenBank for different species of Pipistrellus kuhlii lepidus and Pipistrellus kuhlii were
retrieved and aligned (including gaps) using ClustalW (Larkin et al. 2007), ambiguous sequences were edited
by BioEdit software (Hall 1999), sequences were submitted for accession number to National Center for
Biotechnology Information (NCBI). The sequences for Pipistrellus kuhlii lepidus (MT430902, MT903614-
MT903615, HQ857597, HQ857598, MF078005) and for Pipistrellus kuhlii (AJ426640, KC684535,
MF078006, AJ426639, MT913567, MT913568 and MT856878) were used for molecular analysis. By using
these sequences, the phylogenetic trees were created by both Neighbor-joining and Maximum Likelihood
method, which produced same result (figure 1 & 2). The pairwise genetic distances (number of nucleotide
substitutions per site) calculated by using the Kimura two-parameter model (Kimura 1980) are shown in
Table 2. Evolutionary Divergence between Sequences of Pipistrellus kuhlii and Pipistrellus kuhlii lepidus
among all the sequences used in phylogenetic analyses range from 0.3% to 1 % (table 2).
Estimates of interspecific and intraspecific identity matrix between Sequences of Pipistrellus kuhlii and
Pipistrellus kuhlii lepidus from Bajaur Agency, Pakistan based on Kimura-2 parameter using 16S rRNA
gene are 99 % for Pipistrellus kuhlii lepidus and 39 % for Pipistrellus kuhlii (table 3). The phylogenetic tree
was constructed by Neighbor-joining (NJ) method having 100 Bootstrap replicates using MEGA-X. The
Neighbor-Joining tree is presented in figure 1 and Evolutionary analysis by Maximum Likelihood is
described in figure 2. Subspecies of Kuhl‘s Pipistrelle are forming a one group and second group at species
level by Pipistrellus kuhlii.
The partial sequence of 16S rRNA confirms the species identity and this information could be used for
conservational and other ecological related studies. Another important implication of mtDNA study is to
assess the genetic diversity at inter-specific and intra-specific level. Genetic diversity is an important
component of biodiversity and it could be used to formulate conservation and management planes to
Experiment No.4
50
preserve the evolutionary history of a species. It is estimated that the bats are constituting about 28% of
mammalian fauna in Pakistan but it is debatable for exact number of bats‘ fauna within the territorial
boundary of the country (Roberts and Bernhard 1977; Walker and Molur 2003b; Wilson and Reeder 2005)
but this study adds a new record to strengthen the chiropteran biota of Bajaur Agency, FATA within the
territorial limits of Pakistan. This also Highlights that this area is a hotspot of biodiversity which should be
further explored to record new species of bats.
Before this study, bats have been reported from Pakistan just on the basis of their morphological
parameters but the BLASTn and phylogenetic analyses confirmed the status of subspecies Pipistrellus kuhlii
lepidus as a new record. Blyth in 1845 identified the species from Kandahar, Afghanistan. Generally,
Pipistrellus lepidus is known as a subspecies of Pipistrellus kuhlii (DeBlase and AF 1980). In other records
the Pipistrellus lepidus is regarded as a synonym of the Pipistrellus kuhlii (Kuhl, 1817) (Koopman 1994).
The eastern form of the species is also viewed as a subspecies (Wilson and Reeder 2005). Between the West-
European and Middle-East populations (Iran, Syria, Israel) several genetic differences have been observed
and the restoration of Blyth‘s taxon is recommended by the introduction of Pipistrellus cf. lepidus (Blyth,
1845) (Mayer et al. 2007). Hence, in northern areas of Pakistan this is reported as a new record and further
comparison from other areas like Sindh, Baluchistan and Punjab as a comparative analysis should be carried
out to reconstruct its taxonomic position.
Genetic analysis of species provides a useful information about the scales at which the wild species are
impacted by anthropogenic activities but also provides the information about a successful demographic
management of wild species (Sovic et al. 2016). Advancement in molecular techniques has revolutionized
the field of systematics and improved the taxonomy of some more complex chiropteran species. Molecular
genetics highlighted many new discoveries in taxonomy of understudied and species rich tropical areas
(Clare et al. 2007; Francis et al. 2010), besides this, in temperate fauna where the relative species diversity is
low, the molecular genetics has also resolved taxonomic uncertainties (Mayer et al. 2007; Mayer and von
Helversen 2001).
The tree topology 100 % separates the species and subspecies of Kuhl‘s pipistrelle. Members of the
subspecies formed a separate clade from the species with their Bootstrap values while the members of
species have formed three coevolving clades with an out group, reported from Spain (figure 1 & 2). The
reasons may be unknown to us and need a further research for phylogenetic analysis. A detailed study is
recommended to explore the morphological differences at specific and subspecific level to differentiate
between Pipistrellus kuhlii lepidus and Pipistrellus kuhlii from the study area of Bajaur Agency.
After having a phylogenetic analysis of the specimens and by extensive literature survey the status of
lepidus is reported from territorial limits of Pakistan for the first time. Such a report of a new record also
highlights the importance of genetic identification as compared to the conventional methods for taxonomy.
An extensive survey should be carried out in the country to explore the genetic diversity and compare it from
the regions with different climatic conditions.
Conclusion: It is concluded that the taxonomic position of Kuhl‘s Pipistrelle is extended to subspecies level
in Pakistan after the report this subspecies Pipistrellus kuhlii lepidus. The taxonomy of this cryptic species
could be elaborated by such a short segment of 16S rRNA. We should also explore the species from other
regions with different climatic conditions and compare them with each other. This mitochondrial genome is
an effective tool for an accurate, rapid, low cost and easy applicable method for species identification and
discrimination. This could also be helpful in conservation issues and to prevent the trade of endangered
species in forensic sciences. Molecular identification of species also seeks its importance for commercial
purposes such as the mislabeling of meat and meat products.
Acknowledgement: We also thank for any anonymous who helped for constructive comments and
completion of this research work.
Conflict of Interest: The author(s) declare no conflict of interest.
Data availability statement: The sequence data submitted to GenBank for Pipistrellus kuhlii lepidus (16S
rRNA) is MT430902, MT903614, MT903615 and for Pipistrellus kuhlii is MT913567, MT913568 and
MT856878 and is available at NCBI.
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Roberts TJ, Bernhard. 1977. The mammals of Pakistan. E. Benn London.
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research: conservation, ethical implications, reduction, and alternatives. Mamm. Rev. 47(4): 237-246.
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central and eastern Europe. Acta chiropterologica. 8(2): 543-548.
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three tree bat species at an Ohio windfarm. PeerJ. 4: e1647.
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e0162342.
Walker S, Molur S. 2003a. Summary of the status of South Asian Chiroptera. ZOO'S PRINT. 18(7): 5-28.
Walker S, Molur S. 2003b. Summary of the Status of South Asian Chiroptera, Extracted from the CAMP
2002 Report, Zoo Outreach Organization, CBSG. South Asia and WILD, Coimbatore, India.
Wilson DE, Reeder DM. 2005. Mammal species of the world: a taxonomic and geographic reference. JHU
Press.
Wilson J, Sing K, Halim M, Ramli R, Hashim R, Sofian-Azirun M. 2014. Utility of DNA barcoding for rapid
and accurate assessment of bat diversity in Malaysia in the absence of formally described species. Genet Mol Res. 13(1): 920-925.
Experiment No.4
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Figure 6.1. Morphological description of Kuhl’s Pipistrelle A, B Pipistrelles kuhlii lepidus C, D
Pipistrelle kuhlii, E=Baculum of Pipistrelle kuhlii F=Baculum of Pipistrelle kuhlii lepidus.
Figure6.2. Evolutionary analysis by Neighbor Joining method and General Time Reversible model for
Kuhl’s Pipistrelle from Bajaur Agency, Pakistan.
Experiment No.4
54
Figure 6.3. Evolutionary analysis by Maximum Likelihood method and General Time Reversible
model for Kuhl’s Pipistrelle from Bajaur Agency, Pakistan.
Experiment No.4
55
Table 6. 1. Morphological measurements (mm) of Kuhl’s Pipistrelle (n=6) from Bajaur Agency,
Pakistan.
M=Male F=Female
Table 6. 2. Estimates of Evolutionary Divergence for Sequences of Kuhl’s Pipistrelle from Bajaur
Agency, FATA, Pakistan.
Ac. No. MT90361
4
MT90361
5
MT43090
2
HQ85759
7
MF07800
5
HQ85759
8
AJ42663
9
KC68453
5
MF07800
6
AJ42664
0
MT91356
7
MT91356
8
MT85687
8
MT903614
MT903615 0.006
MT430902 0.003 0.003
HQ857597 0.006 0.006 0.003
MF078005 0.006 0.006 0.003 0.000
HQ857598 0.009 0.009 0.006 0.003 0.003
AJ426639 0.035 0.035 0.032 0.028 0.028 0.032
KC684535 0.032 0.032 0.028 0.025 0.025 0.028 0.003
MF078006 0.032 0.032 0.028 0.025 0.025 0.028 0.003 0.000
AJ426640 0.035 0.035 0.031 0.028 0.028 0.031 0.035 0.032 0.032
MT913567 1.093 1.091 1.093 1.072 1.072 1.072 1.043 1.043 1.043 1.090
Morphological Parameters Species under study
Pipistrelle kuhlii Pipistrelle kuhlii lepidus
Mean ± SD Range Mean ± SD Head and Body length 45 ± 1 40-50 46.7 ± 0.12 Tail length 37.16 ± 0.28 30-40 38.2 ± 0.31
Hind foot length 6.7 ± 0.1 5-6 7.3 ± 0.11
Forearm length 34.23 ± 0.57 30.3-37.4 35.6 ± 0.76
Wing span 220 ± 0 210-230 222 ± 0.00
5th
Metacarpal Length 7.6 ± 1.08 7.0-8.0 7.9 ± 0.06
4th
Metacarpal Length 10.76 ± 0.55 10.0-11.0 11.21 ± 0.42
3rd
Metacarpal Length 10.3 ± 0 10.0-11.0 11.23 ± 0.2
Ear length 12.43 ± 0.15 12-13 13.32 ± 0.4
Body Mass 7.1F, 6.4M ---- 7.5F, 7.4M
Experiment No.4
56
MT913568 1.093 1.091 1.093 1.072 1.072 1.072 1.043 1.043 1.043 1.090 0.007
MT856878 1.090 1.088 1.090 1.069 1.069 1.069 1.054 1.054 1.054 1.100 0.003 0.003
Table 6. 3. Estimates of interspecific and intraspecific identity matrix for Kuhl’s Pipistrelle from
Bajaur Agency, FATA, Pakistan based on Kimura-2 parameter using 16S rRNA gene.
Ac. No. MT903614 MT903615 MT430902 HQ857597 MF078005 HQ857598 AJ426639 KC684535 MF078006 AJ426640 MT913567 MT913568 MT856878
MT903614 ID 0.993 0.996 0.99 0.99 0.987 0.963 0.966 0.966 0.963 0.392 0.392 0.395
MT903615 0.993 ID 0.996 0.99 0.99 0.987 0.963 0.966 0.966 0.963 0.392 0.392 0.395
MT430902 0.996 0.996 ID 0.993 0.993 0.99 0.966 0.969 0.969 0.966 0.392 0.392 0.395
HQ857597 0.99 0.99 0.993 ID 1 0.996 0.972 0.975 0.975 0.972 0.395 0.395 0.398
MF078005 0.99 0.99 0.993 1 ID 0.996 0.972 0.975 0.975 0.972 0.395 0.395 0.398
HQ857598 0.987 0.987 0.99 0.996 0.996 ID 0.969 0.972 0.972 0.969 0.395 0.395 0.398
AJ426639 0.963 0.963 0.966 0.972 0.972 0.969 ID 0.996 0.996 0.966 0.401 0.401 0.401
KC684535 0.966 0.966 0.969 0.975 0.975 0.972 0.996 ID 1 0.969 0.401 0.401 0.401
MF078006 0.966 0.966 0.969 0.975 0.975 0.972 0.996 1 ID 0.969 0.401 0.401 0.401
AJ426640 0.963 0.963 0.966 0.972 0.972 0.969 0.966 0.969 0.969 ID 0.392 0.392 0.392
MT913567 0.392 0.392 0.392 0.395 0.395 0.395 0.401 0.401 0.401 0.392 ID 0.993 0.99
MT913568 0.392 0.392 0.392 0.395 0.395 0.395 0.401 0.401 0.401 0.392 0.993 ID 0.99
Experiment No.4
57
MT856878 0.395 0.395 0.395 0.398 0.398 0.398 0.401 0.401 0.401 0.392 0.99 0.99 ID
58
CHAPTER 7
Experiment No. 5
Phylogenetic Analysis of Genus Pipistrellus (Mammalia: Chiroptera) Based on Partial
Sequences of Mitochondrial 16S rRNA Gene from Bajaur Agency, FATA, Pakistan
ABSTRACT: A high cryptic rate and minimum morphological differences in bats makes the task of species
identification a difficult process. The current study was designed to explore and investigate the genetic
identification of Genus Pipistrellus using 16S rRNA as a genetic marker. Different species belonging to
genus Pipistrelle i.e., Pipistrellus pipistrellus, Pipistrellus tenuis, Pipistrellus coromondra, Hypsugo savii,
Pipistrellus kuhlii and Pipistrellus kuhlii lepidus were reported for the first time by using16S rRNA as a
genetic marker and phylogenetic analyses carried out by MEGA-X. The overall genetic variations among
species of genus Pipistrellus are 0.78%. Pipistrellus kuhlii lepidus and Hypsugo savii are reported as a new
record of cryptic species of Pipistrellus from the territorial boundary of Bajaur Agency, FATA, Pakistan.
Hence, it is depicted from the above results that the study region is much more diverse in terms of
chiropteran diversity in Pakistan. In this study we have provided data about morphological parameters and
phylogenetic analyses. Further detailed analysis is recommended to explore biology, genetic diversity and
phylogeny of chiroptera with an emphasis of Chiropteran diversity in Pakistan.
Key words: Pipistrellus, 16s rRNA, Phylogenetic analysis, Chiropteran diversity, Pakistan.
Graphical Abstract
Introduction:
Species identification and characterization has a crucial role in taxonomy and classification of
organisms. Modern taxonomy, originated in mid-18th century has and has described up to 1.7 million species
Experiment No. 5
59
of organisms (Stoeckle 2003). Besides this, to study the relationship of living beings with each other
various behavioral and morphological parameters are taken into consideration. It is very unsurprising that the
larger animals are given a priority for description and conservation strategies while the smaller ones mostly
remain unknown in sciences (Blaxter 2003). Even among the lager animals‘ species identification has also
remained a taxonomic problem e.g., in case of African elephant which has long been considered as a single
species has become the subject of debate by study of mitochondrial and nuclear genomes which place it in
two separate species (Comstock et al. 2002; Debruyne 2004; Roca et al. 2005).
The work of systematics has started from the last 250 years, despite, the majority of the species is
still unidentified. Currently, the task of species identification has been resolved by DNA barcoding, where
specific sequence of DNA is used for species identification. Generally, the technology of DNA sequencing
has resolved the taxonomic disputes of many taxa, but some higher taxa have not yet been resolved precisely
as a species. The task of species identification by DNA barcoding is very useful to resolve the taxonomic
problems of cryptic species, extinct species, synonymous species or matching the juvenile with adults.
However, DNA barcoding is proved as a standard tool for species identification.
In most of the areas of the world, the bat fauna is either rare or least known, consequently they have a
low abundance along with their lifestyle and hence these are the least explored taxa of mammals. Bats are
also presenting minimal morphological variations and overlapping measurements, highly cryptic species,
which have been explored by molecular analyses (Clare 2011; Dool et al. 2016; Gager et al. 2016; Miranda
et al. 2011).
Genetic analysis of species provides a useful information about the level at which the wild species
are impacted by anthropogenic activities but also provides the information about a successful demographic
management of wild species (Sovic et al. 2016). DNA barcoding is a fast and widely used tool for an
accurate species differentiations and identification (Clare et al. 2011; Wilson et al. 2014). The genus
Pipistrellus is comprising of 51 species throughout the world (Koopman 1994), 12 from subcontinent (Bates
and Harrison 1998) and 8 species from Pakistan (Roberts 1997). The distributional range extends from
Eurasia to Japan, central southern Africa, Solomon Islands, Indonesia, northern Australia, Canada, New
Guinea, USA and Mexico (Roberts 1997). Bat fauna of Pakistan is poorly explored, so an extensive
chiropteran survey is recommended to study these environment friendly creatures (Javid et al. 2014; Javid et
al. 2015).
Hence, the current study was designed to explore the accurate species identification using 16S rRNA
as a marker to explore the bat fauna of Bajaur Agency, FATA, Pakistan.
Materials and Methods:
A total of 200 samples of morphological different bat species were captured (2016-18) using mist
nets from various sites in Bajaur Agency (N 34° 43' 48.7812", E 71° 28' 45.9012"), Federally Administered
Tribal Areas (FATA) of Pakistan. The samples were primarily identified on the basis of their morphology
and preserved in 70% ethanol. The morphometric measurements were also observed before preservation and
comparative observational analyses were performed (Bates and Harrison 1998; Roberts 1997).
DNA was extracted from ethanol (70%) preserved specimens (wing tissue) by proteinase K digestion
and standard phenol-chloroform extraction (Hoelzel and Green 1992), at Institute of Biochemistry and
Biotechnology (IBBt), University of Veterinary and Animal Sciences, (UVAS), Lahore, Pakistan. Universal
primers for 16S rRNA Forward: 5´-AAAGACGAGAAGACCC-3´ and Reverse: 5´-
GATTGCGCTGTTATTCC-3´. The PCR fragments were sequenced by ABI 310 sequencer. The sequences
were aligned by ClustalW method. The sequences were submitted to GenBank for accession numbers
[(MT949662, MT949663 for Hypsugo savii), (MT430902, MT903614, MT903615 for Pipistrellus kuhlii
lepidus, MT913567, MT913568, MT856878 for Pipistrellus kuhlii), (MT539133 for Pipistrellus
pipistrellus), (MT645245, MW342585, MW342586 for Pipistrellus tenuis), (MN719478, MW342602,
MW342603, MW342604, MW342605 for Pipistrellus coromondra)], available on NCBI for 16S rRNA.
Experiment No.5
60
Phylogenetic and molecular evolutionary analyses were conducted using MEGA version X to construct the
phylogenetic trees (Kumar et al. 2018).
Results:
Distribution: Bats specimens were collected from different areas of Bajaur Agency (34°24′17.76″N
72°33′32.16″E), FATA, Pakistan.
Taxonomic Position: Least Pipistrelle (Temminck, 1840): Pipistrellus tenuis
Indian Pipistrelle (Gray, 1838): Pipistrellus coromondra
Common Pipistrelle (Schreber, 1774): Pipistrellus pipistrellus
Pipistrellus kuhlii lepidus (Blyth, 1845)
Morphology: Various morphological parameters like Head and Body length (HB), Tail length (TL), Hind foot length (HL),
Forearm length (FL), Wing span (WS), 5th Metacarpal Length (ML 5th), 4th Metacarpal Length (ML 4th) and
Ear length (EL) for species belonging to genus pipistrellus are mentioned in table 1.
Phylogenetic Relationship:
In Pakistan the genus pipistrellus is represented by 8 species based on their morphological
parameters. No phylogenetic survey has been conducted to explore the genetic diversity in chiroptera
taxonomy. mtDNA sequences are not available for chiropteran species belonging to Pakistan. Currently,
eight species belonging to genus pipistrellus have been reported from Pakistan (Bates and Harrison 1998;
Srinivasulu et al. 2010), but during current study from Bajaur Agency, FATA Pakistan six species have been
reported which highlight the importance of study area as a hotspot for chiropteran diversity.
Experiment No.5
61
The species identified during current study are Hypsugo savii, Pipistrellus kuhlii lepidus, Pipistrellus
kuhlii, Pipistrellus pipistrellus, Pipistrellus tenuis for Pipistrellus coromondra (figure 2). Two species i.e.,
Pipistrellus kuhlii lepidus and Hypsugo savii have not reported from the current study region, hence these
results are describing the range extension or discovery of these species from the study area. Due to small
size, the Japanese pipistrelle is often confused with its other congeners‘ subgroups (Pipistrellus ceylonicus
and Pipistrellus. coromandra). The DNA sequences were obtained to corelate morphological parameters
with genetic identification of the species which have shown reliable and clear methods for almost all the
species under study. Neighbor-joining trees based on Kimura 2-parameter distance was used to construct the
phylogenetic tree, shown in Figure 1. The morphological parameters and their subsequent phylogenetic
analysis led to the confirmation of above-mentioned species from Pakistan. The Pipistrellus kuhlii lepidus
was considered to be Pipistrellus kuhlii but phylogenetic analysis revealed to be P.k. lepidius. Overall, the
interspecific genetic variations among different species of genus Pipistrellus are mentioned in table 2.
Experiment No.5
62
Figure 1. Evolutionary analysis by Neighbour joining tree and General Time Reversible
method of Genus Pipistrelle (Mammalia:Chiroptera) from FATA, Pakistan.
Table1. Morphological measurements (mm) of Pipistrellus bats from FATA, Pakistan
Body Parameters Species under study
Pipistrellus
tenuis (n= 40)
Pipistrellus
pipistrellus (n= 53)
Pipistrellus
coromondra (n= 38)
Pipistrellus kuhlii
lepidus (n= 23)
Hypsugo savii
(n=46)
Head and Body length
(HB) mm
38.2 (33-45) 44.0 (40.0-48.0) 43 (34-49) 45(40-50) 51.0 (47.0–60.0)
Tail length (TL) mm 27.9 (20-35) 32.9 (29.0-35.0) 35 (22-39) 37(30-40) 34.0 (30.0–35.0)
Hind foot length (HL)
mm
5.4 (3-7.0) 6.1 (6.0-7.0) 7 (3.4-8) 6.8(5-6) 7.2 (6.4–8.0)
Forearm length (FL)
mm
27.9 (25-30.2) 31.0 (30.0-31.6) 32 (25.5-34.3) 33.9(30.3-37.4) 30.0 (32.1–38.0)
Wing span (WS) mm 225.5 (180-240) 190 (180-240) 196 (190-220) 220(210-230) 236.5(226-251)
5th
Metacarpal Length
(ML 5th
) mm
24.8 (23.5-28.5) 28.9 (28.4-29.8) 28.1 (25.2-31.1) 7.6(7.0-8.0) 31.4 (29.1–31.3)
4th
Metacarpal Length
(ML 4th
) mm
25.9 (23.7-29.2) 29.6 (28.7-30.8) 28.7 (25.7-32.7) 10.4(10.0-11.0) 32.2 (30.2–34.0)
3rd
Metacarpal Length
(ML 3rd
) mm
25.8 (23.9-29.7) 29.9 (29.5-31.0) 29.0 (25.8-33.1) 10.3(10.0-11.0) 32.3 (30.4–33.2)
Ear length (EL) mm 9.6 (5.0-11.0) 11.1 (10.5-12.0) 11.00 (7.1-14.0) 12.4(12-13) 11.6 (10.0–14.0)
Experiment No.5
63
Ac.
No. MT9
4966
2
MT9
4966
3
MT5
3913
3
MW
3426
03
MW
3426
05
MN7
1947
8
MW
3426
04
MW
3426
02
MT9
0361
4
MT9
0361
5
MT4
3090
2
MT9
1356
7
MT9
1356
8
MT8
5687
8
MT6
4524
5
MW
3425
86
MW
3425
85
MT9
49662
MT9
49663
0.007
MT5
39133
0.193 0.193
MW3
42603
0.231 0.237 0.228
MW3
42605
0.240 0.240 0.231 0.014
MN7
19478
0.227 0.227 0.218 0.007 0.007
Experiment No.5
64
Table 2. Estimates of Evolutionary Divergence between Sequences for Pipistrellus species from
Bajaur Agency, FATA, Pakistan
MW3
42604
0.236 0.236 0.227 0.014 0.022 0.007
MW3
42602
0.242 0.242 0.233 0.022 0.018 0.011 0.018
MT9
03614
0.193 0.193 0.033 0.217 0.216 0.207 0.216 0.217
MT9
03615
0.193 0.193 0.033 0.211 0.211 0.202 0.211 0.217 0.007
MT4
30902
0.188 0.188 0.029 0.211 0.211 0.202 0.211 0.217 0.004 0.004
MT9
13567
1.160 1.184 1.290 1.252 1.357 1.299 1.270 1.343 1.366 1.352 1.331
MT9
13568
1.184 1.210 1.321 1.222 1.321 1.266 1.240 1.308 1.405 1.389 1.366 0.007
MT8
56878
1.185 1.211 1.322 1.224 1.323 1.268 1.241 1.310 1.406 1.391 1.368 0.003 0.003
MT6
45245
1.307 1.307 1.743 1.421 1.577 1.490 1.438 1.552 2.030 1.967 1.904 0.117 0.117 0.116
MW3
42586
1.320 1.320 1.757 1.434 1.590 1.503 1.451 1.565 2.043 1.980 1.917 0.117 0.117 0.116 0.007
MW3
42585
1.333 1.333 1.715 1.409 1.557 1.475 1.426 1.534 1.972 1.917 1.860 0.129 0.129 0.128 0.010 0.017
Experiment No.5
65
Figure 2 Map of Study Area, Bajaur Agency, FATA, Pakistan
Experiment No.5
66
Discussion:
Although systematics is very old branch of science however, majority of the species are still
unidentified. Now a days DNA barcoding is considered authentic and helps in clear cut species
identification. Generally, the technology of DNA sequencing has resolved the taxonomic disputes of many
taxa, but some higher taxa have not yet been resolved precisely as a species. The task of species
identification by DNA barcoding is very useful to resolve the taxonomic problems of cryptic species, extinct
species, synonymous species or matching the juvenile with adults. However, DNA barcoding is proved as a
standard too for species identification (Avise 1989; Francis et al. 2010).
The partial sequence of 16S rRNA confirms the species identity and this information could be used
for conservational and other ecological related studies. Another important implication of mtDNA study is to
assess the genetic diversity at inter-specific and intra-specific level. Genetic diversity is an important
component of biodiversity and it could be used to formulate conservation and management planes to
preserve the evolutionary history of a species. It is estimated that the bats are constituting about 28% of
mammalian fauna in Pakistan but it is debatable for exact number of bats‘ fauna within the territorial
boundary of the country (Roberts and Bernhard 1977; Walker and Molur 2003; Wilson and Reeder 2005).
In Pakistan there are about 8 families of bats, 26 genera and 54 species has so far been discovered
based on their morphological basis (Mahmood-ul-Hassan 2009), this is equivalent to any region of the world
with same climatic and topographic conditions and no data is yet available on barcoding of bats up till now
in the country (Horáček et al. 2000). Species identification and characterization has a crucial role in
taxonomy and classification of organisms. Modern taxonomy, originated in mid18th century has described up
to 1.7 million species of organisms (Stoeckle 2003). Besides this, to study the relationship of living beings
with each other various behavioural and morphological parameters are taken into consideration. It is very
unsurprising that the larger animals are given a priority for description and the smaller ones mostly remain
unknown in sciences (Blaxter 2003).
Genetic analysis of species provides a useful information about the scales at which the wild species
are impacted by anthropogenic activities but also provides the information about a successful demographic
management of wild species (Sovic et al. 2016). Advancement in molecular techniques has revolutionized
the field of systematics and improved the taxonomy of some more complex chiropteran species. Molecular
genetics highlighted many new discoveries in taxonomy of understudied and species rich tropical areas
(Clare et al. 2007a; Francis et al. 2010), besides this, in temperate fauna where the relative species diversity
is low, the molecular genetics has also resolved taxonomic uncertainties (Mayer et al. 2007; Mayer and von
Helversen 2001).
Pipistrellus kuhlii lepidus as a sister species from Sardinia, this subspecies of Kuhl‘s pipistrelle was
not previously reported from the study area, which may be due to its morphological non-differentiation due
to cryptic speciation. Such a report of a new record also highlights the importance of genetic identification as
compared to the conventional methods for taxonomy. An extensive survey should be carried out in the
country to explore and compare the conventional taxonomic methods with barcoding. Advancement in
molecular techniques has revolutionized the field of systematics and improved the taxonomy of some more
complex chiropteran species. Molecular genetics highlighted many new discoveries in taxonomy of
understudied and species rich tropical areas (Clare et al. 2007b; Francis et al. 2010), besides this, in
temperate fauna where the relative species diversity is low, the molecular genetics has also resolved
taxonomic uncertainties (Mayer et al. 2007), from the study area of Pakistan new chiropteran species are also
being identified by using the molecular techniques. Discoveries of new species from Pakistan is suggesting a
species richness and diversity in this region.
It is estimated that the earth‘s biota is constituting about 10 to 100 million species of eukaryotes
(Whitfield 2003). Such a large number of species is presenting a challenging task for taxonomists by
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conventional identification methods. Even though, the impact of internet and consenting for advancements in
communications, the assignment of taxonomic identification is prodigious. In addition, variations in
phenotypic characters and genotype of organisms, which are being employed for taxonomic identification
can primarily lead to identification errors, cryptic species or different developmental stages in the life history
of animals could increase the misperception (Hebert et al. 2003).
Field biologists are confronted with certainty of species diversity due to improvements of the system
for species recognition and its appropriate accessibility worldwide. Such problems of species identification
are also being faced by the people in trade of endangered species, fisheries sector, identification of pest
species and their control for spreading the diseases, accurate lineage identification of extinct species and
regulation of biological materials across the world. By perceiving these issues, a concise, simple and accurate
procedure should be employed for species identification is required to overcome these issues for
identification. As more species are being discovered day by day, the taxonomic data is becoming more
problematic. Species identification by morphological characteristics requires training and expertise without
which this process of identification is difficult. Recent advances in molecular technology have strengthen the
species identification process by using short DNA sequences, which are recognized as species labels, in a
process called DNA barcoding. The varied DNA sequences are intraspecific differentiations which determine
the order of magnitude for species identification.
The status of Pipistrellus tenuis is Least Concern by IUCN 2018 survey. The distributional range of
this species includes Laos, Isles, S China, Afghanistan to the Moluccas; Cocos Keeling, Vietnam, and
Christmas Isle (Indian Ocean). In Pakistan this species has been found at Chakri Gambat, Sukkur, Malakand,
Karachi, Malir, Chitral, Multan, Chaklala (HINTON 1926; Roberts 1997; Siddiqi 1961; Sinha 1980;
WALTON 1974). It is the smallest pipistrelle in subcontinent with average forearm length 27.7 mm.
However, on the basis of forearm length, the differentiation of this species is difficult from smaller
individuals of Pipistrellus coromandra. So, the phylogenetic analysis reveals the more accurate way for
species identification. This species is found throughout Punjab and Sindh and seems to avoid desert areas
like Cholistan. It is found throughout the Indus plains from Karachi to the north where it is common in the
older towns (Roberts 1997).
Pipistrellus coromandra: Indian Pipistrelle (Gray, 1838) is distributed in Afghanistan, Bangla Desh,
India (including Nicobar Isls), Sri Lanka, Pakistan, Nepal, Bhutan, Burma, Cambodia, Thailand, S China. In
Pakistan it is has been collected from Dir, Chitral and Swat districts of North Western Frontier Province.
This a small pipistrelle and is often difficult to distinguish from P. tenuis. In general P. coromandra averages
larger than P. tenuis but there is a significant overlap in all external measurements. In BLASTn results our
query sequence (MN719478) has shown a 97.45% percentage identity and 99% query coverage with
Pipistrellus coromandra (KT291766), which has been reported from India.
Pipistrellus pipistrellus is a ―Least Concern‖ species by IUCN 2018. The distribution range includes
British Isles, Kazakhstan, Taiwan, S Denmark, Israel and Lebanon to Afghanistan, Burma, Kashmir,
Pakistan, W Europe to the Volga and Caucasus, Morocco; Greece, Turkey, Sinkiang, perhaps Korea and
Japan.
Since last two decades, genetics has played a major role in ecology and conservation biology
(Frankham 2005; Frankham et al. 2002; Hedrick 2001). Genetics has significant contributions to understand
the effects of habitat fragmentation, genetic erosion on extinction and endangerment of the species, the
dynamics of adaptation of species to the new environmental circumstances are added, results in the formation
of a modern scientific filed of biology called ―Conservation Genetics‖ (Ouborg et al. 2006). Whereas several
conservation efforts measured at native scale or regional levels, they could affect the biotic consequences of
universal phenomenon, specifically the recent climatic changes and their consequences on populations‘
extinction rate that is now believed to be on the top of the background levels (McLaughlin et al. 2002).
Conclusion: In conclusion, the taxonomic problems of cryptic species could be resolved by such a
short segment of 16S rRNA. This mitochondrial genome is an effective tool for an accurate, rapid, low cost
and easy applicable method for species identification. Over all eight species have been reported from
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Pakistan, and six have been reported from study area of Bajaur Agency, FATA, Pakistan, this highlights the
rich bat fauna diversity in this region. This could also be helpful in conservation issues and to prevent the
trade of endangered species in forensic sciences. Molecular identification of species also seeks its importance
for commercial purposes such as the mislabeling of meat and meat products.
Authors contributions: Muhammad Idnan performed all the lab work, Arshad Javid supervised the research
work, Ali Hussain, Sajid Mansoor and Muhammad Tayyab helped with data analysis, critical review and
manuscript writing, the fieldwork was facilitated by Hamidullah, and Syed Mohsin Bukhari for sampling and
handling of bats, Muhammad Imran and Waqas Ali facilitated for lab work and technical assistance.
Conflict of Interest: The author(s) declare(s) no conflict of interest for this study.
Data Availability Statement: The sequence data for the following Pipistrelle species is available on NCBI
[(MT949662, MT949663 for Hypsugo savii), (MT430902, MT903614, MT903615 for Pipistrellus kuhlii
lepidus, MT913567, MT913568, MT856878 for Pipistrellus kuhlii), (MT539133 for Pipistrellus
pipistrellus), (MT645245, MW342585, MW342586 for Pipistrellus tenuis), (MN719478, MW342602,
MW342603, MW342604, MW342605 for Pipistrellus coromondra)].
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CHAPTER 6
SUMMARY
Bats are representing the one third of mammalian fauna around the world and almost a quarter of all
known mammalian species of Pakistan however, they are amongst the least known and hence least studied
taxa in our country. Pakistan is blessed with wifferent seasons and climatic conditions and is considered as a
diverse region in world with respect to biodiversity. In rest of the world, this mammalian group is
extensively studied and is considered as one of the most suitable bio-indicator as the bats are the only
mammals capable of true flight and can cross the barriers other mammals can‘t. During recent years,
disturbances in the foraging habitats have seriously affected the populations of bats and have led to migrate
in the areas from where the species were never reported previously. The number of bat species in Pakistan is
greater than already reported and new species records are expected from the study area.
The application of molecular genetic techniques extracts valuable biological and behavioral
information to document population dynamics of the species. The present study is the first initiative to
explore diversity of the bats inhabiting Bajur agency, FATA in Pakistan. Bat samples were collected through
mist nets and hand nets and captured specimens were identified up to species and subspecies level on the
basis of their DNA sequences which is the most authentic technique to verify species diversity. The main
objective of this study was to find out genetic variations in chiropteran fauna inhibiting hilly terrain of FATA
region Pakistan and to establish phylogenetic relationship among the bat species inhabiting the study area.
DNA was successfully isolated from wing tissues of representative bats‘ samples collected from
various regions of Federally Administered Tribal Areas (FATA); Pakistan described in sampling areas. This
study represents the first attempt to investigate genetic study for bats identification using sequencing analysis
of these samples. In this study we found the bats belonging to Genus scotophillus, (Scotophillus heathi,
Scotophillus kuhlii), Genus Rhinopoma (Rhinopoma microphyllum), Genus Rousettus (Rousettus
leschenaulti), Genus myotis species (Myotis muricola, Myotis formosus), Genus Rhinolophus (Rhinolophus
hipposideros, Rhinolophus ferrumequinum) and Genus Pipistrellus (Pipistrellus kuhlii, Pipistrellus kuhlii
lepidus, Pipistrellus coromandra, Pipistrellus pipistrellus, Pipistrellus tenuis, Hypsugo savii).