UNIVERSITY INSTITUTE OF BIOTECHNOLOGY

DEPARTMENT OF BIOSCIENCES
Master of Science (Zoology)

SYNOPSIS
(DISSERTATION)

“IN SILICO PHYLOGENTIC STUDIES

ON BUTTERFLIES OF FAMILY PIERIDAE”

Dissertation Guide:
Dr Arvinder Pal Singh
Professor in Zoology
Submitted By:
Ashish George
UID- 22MSZ10103

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INDEX TO CONTENTS

S. No. Item Page No.

1 Topic 3
2 Abstract 3
3 Introduction 4-5
4 Taxonomical Position Of Family 5
5 Objective Of Study 5-6
6 Significance Of Study 6
7 Literature Review 6-8
8 Morphology Of Butterflies 8
9 Methodology 8-10
10 Worldwide Stats/ Pictures 9-12
11 Timeline Of Study 12-13
12 Discussion 13
13 Acknowledgment 14
14 Reference 14-16

TOPIC

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 “IN SILICO PHYLOGENTIC STUDIES ON BUTTERFLIES OF FAMILY PIERIDAE”

ABSTRACT –

In silico phylogenetic studies refers to computer-based simulations or analyses conducted in a

virtual environment. This study utilizes in silico tools and techniques to unravel the phylogenetic
relationship of butterflies belonging to the Pieridae family. This research will analyze genetic
sequences and molecular data of Pierid butterflies. The study delves into the intricate patterns of
divergence that have occurred during the evolutionary process, leading to the development of
distinct traits and behaviors in response to environmental factors and interactions with co-species
members.

To construct the phylogenetic tree, genetic sequences will be gathered from reputable sources
such as BOLD (The Barcode Of Life Data System), NCBI (National Center Of Biotechnology
Information) and GenBank. Sequence alignment will be facilitated using software tools like
CLUSTAL W and MUSCLE, while the comprehensive functionality of MEGA 11 is employed
for aligning sequences, identifying similarities and dissimilarities, and ultimately constructing
the phylogenetic tree.

The findings and insights derived from these in silico analyses will contribute to a deeper
understanding of the evolutionary dynamics within the Pieridae family, shedding light on the
intricate relationships and adaptations that have shaped the diverse butterfly species in this
taxonomic group.

KEYWORDS – in silico, Phylogenetics, Pieridae family, NCBI, GenBank, CLUSTAL W,

MUSCLE, MEGA 11, Butterfly.

INTRODUCTION

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In the modern world of science, the term in silico (Pedro Miramontes,1987) is generally used for
the experimentations based on computer simulation. It is a Latin phrase that means in silicon ,
referring to silicon in computer chips. It was coined in 1987 as a parallel to Latin phrases such
as in vivo, in vitro, and in situ, which are commonly used in biology. Computer-based
technology, modeling, and computational methodologies typically involve a variety of analyses,
including but not limited to cell analysis, protein structure analysis, drug analysis, metabolic
pathways, and DNA sequencing. Originally, the term in silico was specifically associated with
computer simulations replicating natural or laboratory processes across the natural sciences
(Sieburg, H.B. et.al.1990).

Examining both genes and proteins requires the use of DNA sequencing. Sequencing is the
method of identifying the precise sequence of nucleotides in DNA or RNA molecules. This
process enables the comprehension of genetic patterns and the specific functions of individual
genes.
The significance of sequencing lies in its ability to furnish the genetic data essential for giving
the evolutionary connections among various species and for constructing a phylogenetic tree.
These sequences serve as the foundational data for conducting phylogenetic analyses (Sabir, J. S.
M. et.al. 2019).

The majority of computational phylogenetic investigations concerning Pieridae butterflies

depend on DNA sequences extracted from mitochondrial and nuclear genes (Sabir, J. S. M. et.al.
2019). These sequences supply the molecular information essential for conducting phylogenetic
analyses. In contemporary studies, there is a growing trend towards utilizing genomic data,
including whole-genome sequencing, which offer a more comprehensive perspective on genetic
relationships. However, prior to sequencing, it is imperative to align the sequences, a task
typically accomplished through performing BLAST (Basic Local Alignment Search Tools) for
sequence alignment.

The study of phylogenetics (Haeckel,1866), also known as phylogenetic study, centers on

understanding the evolutionary connections among various species, organisms, or genes. It
elucidates the evolutionary history and branching patterns of life through the creation of
phylogenetic trees. These trees facilitate the interpretation of relationships between species or
genes, indicating their closeness. To construct a phylogenetic tree, the initial step involves

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gathering data, which may encompass genetic sequencing (DNA or RNA sequencing),
morphological characteristics, and other relevant information (Jeffrey Rizzo et.al.2007)

This paper will aimed to provide an descriptive information about the in silico phylogenetic
study on the butterflies of family Pieridae of order Lepidoptera following the class insecta of
phylum Arthropoda.

Phylum Arthropoda (Johann L.C.G. 1843) is a major phylum with a vast and diverse group of
invertebrate animals mainly featured by jointed appendages (Lankester et.al.1904). This phylum
involves all kinds of insects, crustaceans making it most wide spread phylum of kingdom
Animalia (Linn.,1866).

Lepidoptera, which can be considered the most extensive group of plant-feeding insects, play
diverse ecological roles as both pollinators and prey. Their influence extends to humans and
various other species, showcasing significant ecological impact. Furthermore, Lepidoptera serve
as valuable model organisms for research in genetics, physiology, development, ecology, and
evolutionary biology (Mitter et.al.2017).

TAXONOMIC POSITION OF FAMILY

Kingdom: Animalia

Phylum: Arthropoda

Class: Insecta

Order: Lepidoptera

Suborder: Rhopalocera

Superfamily: Papilionoidea

Family: Pieridae

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 OBJECTIVES OF STUDY

In silico phylogenetic studies will be performed using computational methods and bioinformatics
tools to –

 Construct the evolutionary relationship between different members of species, different species,
family, order.
 To align DNA, RNA or protein sequences, which are important for finding similarities and
differences between species.
 To depict the common ancestry through phylogenetic tree.
 To identify the taxonomic classification of different species.
 To estimate the divergence timing between different lineages.

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SIGNIFICANCE OF STUDY

Approach to find out the evolution of current day species of butterflies.

Approach to find out the evolutionary relationship between different species of butterflies.

Give information about the divergence occurred during evolution among species of butterflies.

Give information about the different traits and behavior in the species.

LITERATURE REVIEW

The expression in silico (Pedro Miramontes,1987) was first used to characterize biological
experiments carried out entirely in a computer in 1989, in the workshop "Cellular Automata:
Theory and Applications" in Los Alamos, New Mexico, by mathematician Pedro Miramontes
(Sieburg, H.B. et.al.1990). The first referenced paper where in silico appears was written by a
French team in 1991. In 1990, the first referenced book chapter where in silico appears was
written by Hans B. Sieburg (Sieburg, H.B. et.al.1990). In silico phylogenetic studies play a
crucial role in unraveling the evolutionary history of these butterflies. It also help to know recent
advancements in computational phylogenetics within this family.

The Oxford English Dictionary stated that the common word “butterflies” being originated from
Ancient English language. The term “butterfly” originated from word “buttorfleoge” i.e; pale
yellow color (Phil Cousineau et.al.2012). The order Lepidoptera (Linn.1746) that includes
butterflies and moths. The term “Lepidoptera was first been used by Carl Linnaeus in 1746 and
derived from Greek word ‘lepidos’ meaning scale and ‘pteron’ meaning wings (Emmet A.
et.al.1991). In order Lepidoptera, mainly there are two suborders – and mainly focused suborder
is Rhopalocera (Butterflies). The term is derived again from Greek word ‘rhopalon’ meaning
club and ‘keras’ meaning horn, named after the shape of antennae of butterflies (Hulme
et.al.1903). The Pieridae family (William John,1820) is a large family of butterflies consisting
about 1,100 species approximately. The family was discovered by William John Swainson in
1820. Coliadinae and Pierinae are the sub families both are being focused as well as for
comparing their phylogenetic situation (Braby et.al.2006).
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The Barcode of Life Data System, widely recognized as BOLD, is a web-based platform
dedicated exclusively to the field of DNA barcoding. Established in 2005 at the Centre for
Biodiversity Genomics in Canada, BOLD serves as a cloud-based system for the storage and
analysis of DNA data (Stoeckle et.al.2006). The software Molecular Evolutionary Genetics
Analysis (MEGA) (Masatoshi Nei,1993) is widely used for performing statistical analysis of
molecular evolution and creating phylogenetic trees. The initiative to develop this software
originated under the guidance of Masatoshi Nei in his laboratory at the Pennsylvania State
University, where he collaborated with his graduate student Sudhir Kumar and postdoctoral
fellow Koichiro Tamura. The software encompasses tasks such as sequence alignment,
evolutionary analysis, phylogenetic analysis, tree visualization, and statistical analysis (Sohpal
et,al. 2010). (MEGA 1.0 version in 1993 – MEGA 11 version in 2022 February).

There are generally two recognized categories for constructing phylogenetic trees: distance-
based methods and character-based methods. Within distance-based methods (Fitch-
Margoliash,1967), the widely used approaches include the Unweighted Pair Group Method using
Arithmetic Averages (UPGMA) (Sokal et.al.1973), it always make rooted tree and Neighbor
Joining (Ernst Finkenzimmerer,1980) it makes unrooted tree, when identify the neighboring
species. On the other hand, character-based methods (Woose, 1977), like Maximum Parsimony
(James S. Farris,1970), it requires simple or fewest number of evolutionary changes and
Maximum Likelihood (Felsenstein,1981), it is specified and also includes associated parameters.
They offer alternative approaches for phylogenetic tree construction (Jeffrey Rizzo et.al.2007).

MORPHOLOGY OF PIERIDAE BUTTERFLIES

The family Pieridae, commonly known as the white and sulphur butterflies, is characterized by
certain morphological features that aid in their identification.

Coloration: Pieridae butterflies often exhibit distinctive coloration, with white and yellow being
predominant. Some species may have orange or black markings on the wings (Giraldo
et.al.2008).

Wing Shape: Pieridae butterflies typically have relatively pointed and angular wings (Stavenga,
D. G., et. al.2004).

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Vein Patterns: The venation pattern on the wings, including the arrangement and presence of
specific veins, can be characteristic of the Pieridae family (Patil et.al.2017).

Antennae: The antennae of Pieridae butterflies are often club-shaped, with a bulbous tip. The
length and structure of the antennae can provide additional characteristics for identification
(Ehrlich et.al.1957).

METHODOLOGY

This research will focuses primarily on in silico methodologies, that relying on computational
analysis or simulation, eliminating the extra need for physical materials. The required sequences
will be obtained from the "Barcode of Life Database" (BOLD System) as it offers user-friendly
access for collecting and analyzing specific sequences. The BOLD System facilitates easy
repository management and analysis of the available sequences (Stoeckle et.al.2006).

Other software or public data can also be used for collecting the sequence such as NCBI (1988),
GEN BANK (1982) etc.

Information of butterflies are been considered based on “Butterflies Of India”. It provide

extensive and detailed information about butterfly species in India. This could include taxonomy,
distribution, behavior, and lifecycle details, making it a valuable source for researchers as well as
for students.

Sequence must be in “FASTA FORMAT”. The FASTA format serves as a text-based

representation for nucleotide or protein sequences and is widely utilized in the field of
bioinformatics.

For construction and analysis of phylogenetic study “MEGA 11” (Masatoshi Nei,1993) will be
used. It provide all in one software. The software encompasses tasks such as sequence alignment,
evolutionary analysis, phylogenetic analysis and statistical analysis (Sohpal et,al. 2010).

For construction different methods can be used viz. Neighbor joining, UPGMA, Maximum
parsimony, Maximum likelihood etc.

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For BLAST or to align “CLUSTAL W (Thompson et.al.1994), MUSCLE” (Edger et.al.1795)
etc. software can be used. CLUSTAL W align the sequences slowly, and is have less accuracy in
comparison to MUSCLE.

WORLDWIDE STATS

During British India approximately 1439 species of butterfly recorded. After 1947, the rise of
several new nations, it led to a reduction of the area forming part of India proper, and the number
of species has been noted to be an estimated 1339 species.

The family Pieridae constitutes an extensive butterfly family comprising around 76 genera and
approximately 1,100 species, predominantly found in tropical regions of Africa and Asia.

INDIA STATS

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 GLOBAL STATS

BARCODE OF LIFE DATA SYSTEM (Stoeckle et.al.2006)

BUTTERFLIES OF INDIA

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MEGA 11 SOFTWARE (Sohpal et,al. 2010)

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 PUBLIC DATA PORTAL – RECORD LIST

TIMELINE OF STUDY –

The study will be conducted at Chandigarh University, Gharuan, district Mohali, under the
Department of Bioscience in semester 3rd and 4th, session 2022-2024.

AUGUST  Selection of appropriate topic

SEPTEMBER  Initiation of literature survey

OCTOBER – NOVEMBER  Continuation of literature review for

updation

DECEMBER – JANUARY  Continuation of literature review for

updation

 Preparation of synopsis and

presentation

FEBRUARY – MARCH  Continuation of literature review

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  Collection of DNA sequences of
butterfly

APRIL  Continuation of literature review

 Sequencing, Alignment and other

procedure to construct the
phylogenetic tree in MEGA 11.

MAY  Completion of phylogenetic tree

construction.

 Submission of Dissertation

 Prepare for Publication of Research

Paper

DISCUSSION –
The term "in silico" (Pedro Miramontes,1987) originally referred specifically to computer
simulations modeling natural or laboratory processes, including the creation of phylogenetic
trees. In the context of phylogenetic studies conducted in silico, researchers employ
computational analysis of genetic sequences or molecular data to discern the evolutionary
relationships among different butterfly species. The study typically involves summarizing key
findings and insights derived from these analyses (Sieburg, H.B. et.al.1990; Sabir, J. S. M. et.al.
2019; Jeffrey Rizzo et.al.2007)

During the course of evolution within a species lineage, various divergences occur (Niklas
Wahlberg et.al.2013) leading to distinct traits and behaviors exhibited by species in response to
their environment and interactions with co-species members. In order to construct a phylogenetic
tree, genetic sequences are essential. Researchers often visit platforms such as NCBI and
GenBank to obtain the required sequences. Additionally, software tools like CUSTLE W and
MUSCLE are utilized for sequence alignment. However, for a comprehensive approach
encompassing sequence alignment, similarity and dissimilarity analysis, and overall tree

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construction, MEGA 11, which integrates these functionalities, can be preferred (Sohpal et,al.
2010; Edger et.al.1795; Thompson et.al.1994).

ACKNOWLEDGMENT –
I would like to express my deepest appreciation to Dr. Arvinder Pal Singh, for his unwavering
guidance, valuable insights, and continuous support. His expertise, encouragement, and
constructive feedback have been instrumental in shaping the direction of my study.

I want to thank my friends and peers for their camaraderie, intellectual discussions, and moral
support. Your diverse perspectives and shared experiences have enriched my academic journey.

I would like to acknowledge other mentors too who have offered guidance and advice beyond
the formal requirements of my dissertation. Your wisdom and encouragement have been
invaluable.

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