GENETIC DIVERSITY IN BATS OF BAJAUR AGENCY, FEDERALLY ...

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





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


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


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).


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


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).


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).

mailto:[email protected]

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

https://en.wikipedia.org/wiki/Kunar_River

https://en.wikipedia.org/wiki/Panjkora

https://en.wikipedia.org/wiki/Panjkora

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.



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).

References:

Bates P, Harrison D. 1998. Bats of the Indian subcontinent. Biodivers Conserv. 7(10): 1383-1386.

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Bates P, Harrison D. 1997. Bats of the Indian subcontinent Sevenoaks. United Kingdon: Harrison Zoological

Museum.

Bates PJ. 1997. Bats of the Indian Subcontinent: Harrison Zoological Museum publication. Sevenoaks, Kent,

United Kingdom.

Benda P, Reiter A, Uhrin M, Varadínová Z. 2016. A new species of pipistrelle bat (Chiroptera:

Vespertilionidae) from southern Arabia. Acta Chiropterologica. 18(2): 301-323.

Blaxter M. 2003. Molecular systematics: counting angels with DNA. Nature. 421(6919): 122.

Cardinal B, Christidis L. 2000. Mitochondrial DNA and morphology reveal three geographically distinct

lineages of the large bentwing bat (Miniopterus schreibersii) in Australia. Aust. J. Zool. 48(1): 1-19.



Comstock KE, Georgiadis N, Pecon‐ Slattery J, Roca AL, Ostrander EA, O'Brien SJ, Wasser SK. 2002.

Patterns of molecular genetic variation among African elephant populations. Mol. Ecol. 11(12):

2489-2498.

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

press Oxford.

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.




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.

Hill JE. 1987. The baculum in the Vespertilioninae (Chiroptera: Vespertilionidae) with a systematic review,

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.

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.

Honacki JH, Kinman KE, Koeppl JW. 1982. Mammals species of the world; a taxonomic and geographic

reference.

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.




Koopman KF. 1993. Order chiroptera. Mammal species of the world: a taxonomic and geographic reference.

137-241.

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.



Roca AL, Georgiadis N, O'Brien SJ. 2005. Cytonuclear genomic dissociation in African elephant species.

Nat. Genet. 37(1): 96-100.

Roehrs ZP, Lack JB, Van Den Bussche RA. 2010. Tribal phylogenetic relationships within Vespertilioninae

(Chiroptera: Vespertilionidae) based on mitochondrial and nuclear sequence data. J Mammal. 91(5):

1073-1092.

Sanborn CC. 1952. Philippine zoological expedition, 1946-1947; Mammals. Fieldiana (Zoology). 33: 87-

158.

Simmons NB. 2005. Order chiroptera. Mammal species of the world: a taxonomic and geographic reference.

1: 312-529.

Soota T, Chaturvedi Y. 1980. New locality record of Pipistrellus camortae Miller from Car Nicobar and it‘s

systematic status. Rec. zool. Surv. India. 77: 83-87.



Srinivasulu C, Racey PA, Mistry S. 2010. A key to the bats (Mammalia: Chiroptera) of South Asia. Journal

of Threatened Taxa. 2(7): 1001-1076.

Srinivasulu C, Srinivasulu B. 2001. Bats of the Indian subcontinent–An update. Curr. Sci. 80(11): 1378-

1380.


Thomas O editor. Proceedings of the Zoological Society of London. 1928.

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

27

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

28

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

29

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


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,



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


https://www.iucnredlist.org/species/7935/22117147#taxonomy

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).



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



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.


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.


One. 6(7): e21460.




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.



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.


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

35






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.


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.


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

36

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


3rd


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


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,



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




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.


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´.



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



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


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


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.

Reference:


Blaxter M. 2003. Molecular systematics: counting angels with DNA. Nature. 421(6919): 122-124.


One. 6(7): e21460.



Clare EL, Lim BK, Fenton MB, Hebert PD. 2011. Neotropical bats: estimating species diversity with DNA

barcodes. PloS one. 6(7): e22648.

Dool SE, Puechmaille SJ, Foley NM, Allegrini B, Bastian A, Mutumi GL, Maluleke TG, Odendaal LJ,

Teeling EC, Jacobs DS. 2016. Nuclear introns outperform mitochondrial DNA in inter-specific

phylogenetic reconstruction: lessons from horseshoe bats (Rhinolophidae: Chiroptera). Molecular

phylogenetics and evolution. 97: 196-212.




Frankham R. 2005. Genetics and extinction. Biol. Conserv. 126(2): 131-140.

Frankham R, Briscoe DA, Ballou JD. 2002. Introduction to conservation genetics. Cambridge university

press.

Gager Y, Tarland E, Lieckfeldt D, Ménage M, Botero-Castro F, Rossiter SJ, Kraus RH, Ludwig A,

Dechmann DK. 2016. The value of molecular vs. morphometric and acoustic information for species

identification using sympatric molossid bats. PLoS One. 11(3): e0150780.

Hedrick PW. 2001. Conservation genetics: where are we now? Trends in Ecology & Evolution. 16(11): 629-

636.




Javid A, Mahmood-ul-Hassan M, Hussain SM, Iqbal KJ. 2014. Recent record of the Asiatic lesser yellow

house bat (Scotophilus kuhlii) from Punjab, Pakistan. Mammalia. 78(1): 133-137.

Javid A, Shahbaz M, Mahmood-ul-Hassan M, Hussain SM. 2015. The Blasius‘ horseshoe bat Rhinolophus

blasii (Chiroptera, Rhinolophidae) still extends to Pakistan. Mammalia. 79(2): 249-251.

Koopman K. 1994. Chiroptera: Systematics. Handbook of Zoology. Volume VIII. Mammalia. Part 60. In:

Berlin-New York: Walter de Gruyter.



Experiment No.3

42





McLaughlin JF, Hellmann JJ, Boggs CL, Ehrlich PR. 2002. Climate change hastens population extinctions.


Miranda J, Bernardi I, Passos F. 2011. Chave ilustrada para a determinação dos morcegos da Região Sul do

Brasil. João MD Miranda, Curitiba.

Ouborg N, Vergeer P, Mix C. 2006. The rough edges of the conservation genetics paradigm for plants. J.

Ecol. 94(6): 1233-1248.






Walker S, Molur S. 2003. Summary of the Status of South Asian Chiroptera, Extracted from the CAMP 2002

Report, Zoo Outreach Organization, CBSG. South Asia and WILD, Coimbatore, India.


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.3

43

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,



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´).


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)

Experiment No.4

47

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.







with each other various behavioral and morphological parameters are taken into consideration. It is very



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



Experiment No.4

50



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




(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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51

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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.

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Figure 6.3. Evolutionary analysis by Maximum Likelihood method and General Time Reversible

model for Kuhl’s Pipistrelle from Bajaur Agency, Pakistan.

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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

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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




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


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.


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





boundary of the country (Roberts and Bernhard 1977; Walker and Molur 2003; Wilson and Reeder 2005).







with each other various behavioural and morphological parameters are taken into consideration. It is very



Genetic analysis of species provides a useful information about the scales at which the wild species





(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.



Experiment No.5

67

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


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

Experiment No.5

68

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).

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