Perbandingan Filogeografi Dari Ikan Air Tawar Di Jawa Dan Bali Menggunakan Barcode Dna

COMPARATIVE PHYLOGEOGRAPHY OF JAVA AND BALI
FRESHWATER FISHES USING DNA BARCODES

ARIEF ADITYA HUTAMA

GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2016

STATEMENT LETTER

I hereby declare that thesis entitled Comparative Phylogeography of Java
and Bali Freshwater Fishes Using DNA Barcodes is based on original results of
my own research supervised under advisory committee and has never been
submitted in any form at any institution before. All information from other
authors cited here are mentioned in the text and listed in the reference at the end
part of the thesis.

Bogor, January 2016

Arief Aditya Hutama
Student ID G352130201

SUMMARY

ARIEF ADITYA HUTAMA. Comparative Phylogeography of Java and Bali
Freshwater Fishes Using DNA Barcodes. Supervised by BAMBANG
SURYOBROTO, NICOLAS HUBERT, ACHMAD FARAJALLAH.
The information about the origin of the ichthyodiversity in Java is still
poorly understood and spatio-temporal dynamic also still unknown. Therefore,
biogeographic studies aiming at exploring the spatio-temporal dynamics of
settlement of freshwater fishes in Java are certainly needed. Fishes are the most
diverse group of vertebrates, and because of their large phenotypic diversity and
profound changes during their ontogenetic development, fish identification is
difficult.
DNA barcoding is a new approach to species identification based on a
short fragment of DNA from a standardized region of the mitochondrial genome.
In this study, we aim at using DNA barcoding to document and characterize the
intra-specific diversity of several widespread species of freshwater fishes in Java
and Bali. The objective is to explore the population structure of each species,

identify common patterns and propose potential mechanisms to explain observed
pattern. Due the high diversity of habitat in the study area, this research was
conducted in a total of fifty one locations in Java and Bali islands in order to cover
altitudinal and ecological gradients. Three species were selected as model:
Channa gachua (Perciformes, Channidae), Glyptothorax platypogon(
Siluriformes, Sisoridae), and Barbodes binotatus (Cypriniformes, Cyprinidae).
C. gachua, G. platypogon and B. binotatus display a high population
genetic structure shown by a high genetic differentiation of the populations and
the presence of several genetic clusters. High genetic variations among groups but
low genetic variability within population according to nucleotidic diversity is
likely to be the consequence of an important fragmentation of the populations as a
high number of closely related haplotypes were observed within populations and
genetic clusters. The age estimates of each genealogy suggest each species
originated during the Pliocene at 3.05 Ma, 3 Ma and 2.6 Ma for C. gachua,
G.platypogon and B. barbodes, respectively, the colonization of Java and Bali
islands happened mainly during the Pleistocene. The lack of significance of the
mantel tests showed that the differentiation of the populations was not the result
of geographic distance but a consequence of habitat fragmentation. A genetic
differentiation between the West and East populations was observed for all three
species and a dynamic of secondary contact was observed in Central Java.

Key words : Phylogeography, population structure, Channa gachua, Glyptothorax
platypogon, Barbodes binotatus

RINGKASAN

ARIEF ADITYA HUTAMA. Perbandingan Filogeografi dari Ikan Air Tawar di
Jawa dan Bali Menggunakan Barcode DNA. Dibimbing oleh BAMBANG
SURYOBROTO, NICOLAS HUBERT, ACHMAD FARAJALLAH.
Informasi mengenai asal – usul keragaman diversitas ikan serta dinamika
keadaan geologi terkait waktu di pulau Jawa dan Bali masih kurang dipahami.
Oleh karena itu, dibutuhkan studi tentang biogeografi yang bertujuan untuk
mempelajari dinamika geologi terkait waktu pada kolonisasi ikan air tawar di
pulau Jawa dan Bali Ikan merupakan grup vertebratra yang paling beragam,
memiliki diversitas fenotipik yang tinggi dan adanya perubahan selama
perkembangan ontogenetik, identifikasi ikan merupakan hal yang sulit.
DNA barcoding merupakan pendekatan baru untuk mengidentifikasi
spesies didasarkan pada fragmen pendek DNA yang berasal dari bagian
mitokondria yang telah dibakukan. Penelitian ini bertujuan menggunakan DNA
barcoding untuk mendokumentasi dan mengkarakterisasi keragaman intraspesifik
dari beberapa spesies ikan air tawar di pulau Jawa dan Bali yang memiliki

wilayah persebaran luas. Sasaran utama dari penelitian ini adalah untuk
mengetahui struktur populasi dari setiap spesies, mengidentifikasi pola dan
mengajukan mekanisme potensial untuk menjelaskan pola yang teridentifikasi.
Pengambilan sampel untuk penelitian dilaksanakan di 51 lokasi di pulau Jawa dan
Bali dengan tujuan untuk mewakili diversitas habitat di pulau Jawa dan Bali yang
tinggi. Tiga spesies dipilih menjadi model untuk penelitian ini : Channa gachua
(Perciformes, Channidae), Glyptothorax platypogon ( Siluriformes, Sisoridae),
and Barbodes binotatus (Cypriniformes, Cyprinidae).
C. gachua, G. platypogon and B. binotatus menunjukan tingginya struktur
genetika populasi. Hal ini dapat dilihat dari tingginya diferensiasi populasi genetik
dan keberadaan dari beberapa kluster genetic. Tingginya variasi genetik antar
grup namun rendah di dalam satu populasi yang sama menurut diversitas
nukleotida diperkirakan karena adanya fragmentasi populasi yang dapat dilihat
dari tingginya nilai diversitas haplotipe namun jarak genetiknya kecil pada satu
populasi yang sama. Perkiraan waktu secara genealogy memperkirakan ketiga
spesies tersebut telah ada semenjak masa Pliocene yakni 3,05 juta tahun yang lalu
untuk C. gachua, 3juta tahun yang lalu intuk G.platypogon dan 2,6 juta tahun
yang lalu untuk B. barbodes dan berkolonisasi di pulau Jawa dan Bali pada masa
Pleistocene. Hasil yang tidak signifikan dari mantel test menunjukkan bahwa
diferensiasi populasi bukan disebabkan karena jarak geografi, namun dikarenakan

adanya fragmentasi habitat. Adanya diferensiasi genetic antara populasi di Jawa
Barat dan populasi di Jawa Timur terdapat pada ketiga spesies tersebut serta
diperkirakan adanya secondary contact yang terjadi di daerah Jawa Tengah.

Key words : Filogeografi, struktur populasi, Channa gachua, Glyptothorax
platypogon, Barbodes binotatus

Copyright © 2016 Bogor Agricultural University

All Rights Reserved

It is prohibited to cite all or a part of this thesis without referring to and mentioning
the source. Citation is permitted for the purposes of education, research, scientific
paper, report, or critism writing only; and it does not defame the name and honour of
Bogor Agricultural University.
It is prohibited to republish and reproduce all or a part of this thesis without
permission from Bogor Agricultural University

COMPARATIVE PHYLOGEOGRAPHY OF JAVA AND BALI
FRESHWATER FISHES USING DNA BARCODES

ARIEF ADITYA HUTAMA

Thesis
Submitted in partial fulfillment of the requirements for a
Master Degree
in Animal Bioscience Major
Graduate School of Bogor Agricultural University

GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2016

Examiners : Dr. Nurlisa Alias Butet

ENDORSEMENT PAGE

Title
Name

NIM
Major

: Comparative Phylogeography of Java and Bali Freshwater Fishes
Using DNA Barcodes
: Arief Aditya Hutama
: G352130201
: Animal Bioscience

Endorsed by,
Supervisor Committee

Dr. Bambang Suryobroto
Chair

Dr. Nicolas Hubert
Committee

Dr. Ir. Achmad Farajallah, M.Si
Committee

Acknowledged by,

Coordinator of Major
Animal Bioscience

p.p. Dean of Postgraduate School
Secretary of Master Program

Dr.Ir.R R Dyah Perwitasari, M.Sc.

Prof. Dr.Ir. Nahrowi, M.Sc.

Examination date :

Graduation Date :

x

PREFACE

This research is a part of the general research program Inventory of
Indonesian freshwater fishes through DNA Barcoding jointly developed by the
Institut de Recherche pour le Développement (IRD, France) and the Indonesian
Institute of Sciences (LIPI, Indonesia). This research program has been started in
2012 with the aim to characterize the diversity of Indonesian freshwater fishes
through DNA barcoding. This international collaboration is ruled by a
Memorandum of Understanding signed on 29th of October 2012 by the Chairman
of LIPI (Prof. Dr. Likman Hakim) and the president of IRD (Prof. Dr. Michel
Laurent). The research developed in the present thesis had been conducted from
December 2012 to November 2015 in the context of this joint research program
and as such, this research and dissemination of the results are bound by the
obligations defined by the Memorandum of understanding between IRD and LIPI.
I want to send my gratitude to my supervisor Dr. Bambang Suryobroto,
Dr. Nicolas Hubert and Dr. Achmad Farajallah for all the support. I wish thank to
all of the IRD and LIPI staff that gave me the opportunity to develop this
research: Dr. Renny Hadiaty, Sopian Sauri, Sumanta and Hadi Darhuddin. A
gratitude to Mr. M Syamsul Arifin Zein, M.Si for allowing and helping me while I
was working in Genetic Laboratory, Research Center for Biology LIPI, Cibinong.
Thanks to all of the Animal Bioscience 2013, especially Esa and Maulana. Special
thanks to my parents, my brother, and my sister for their love and support. I thank

Allah SWT for all the blessings in my life.

Bogor, January 2016

Arief Aditya Hutama

xi

xii

CONTENTS

SUMMARY ........................................................................................................ iv
ENDORSEMENT PAGE ................................................................................... ix
PREFACE ............................................................................................................ x
CONTENTS ....................................................................................................... xii
LIST OF TABLES ............................................................................................ xiii
LIST OF FIGURES .......................................................................................... xiii
LIST OF APPENDIX ....................................................................................... xiii
INTRODUCTION ............................................................................................... 1

MATERIAL AND METHOD ............................................................................. 2
Geological Framework .................................................................................... 2
Sample Collection ........................................................................................... 3
Genetic Analyses ............................................................................................. 4
Data Analyses .................................................................................................. 5
Phylogenetic Analyses of Gene genealogies ............................................... 5
Population Structure Analyses ..................................................................... 5
RESULT .............................................................................................................. 6
Phylogenetic Analyses..................................................................................... 6
Population structure ....................................................................................... 11
DISCUSSION .................................................................................................... 13
CONCLUSION .................................................................................................. 15
REFERENCES .................................................................................................. 16
APPENDIX ........................................................................................................ 18
CURRICULUM VITAE .................................................................................... 35

xiii

LIST OF TABLES

1 Amova statistics and fixation indexes................................................................ 11
2 Genetic diversity ................................................................................................ 13
LIST OF FIGURES

1 Late Miocene and Early Pliocene reconstructions of land and sea in the IndoAustralian Archipelago (Lohmann et al 2011) .................................................... 2
2 Pleistocene map of South East Asia illustrating depth contours of 120m below
present sea levels (Voris 2000) ............................................................................ 3
3 Sampling locations in Java and Bali islands. One point may represent several
sampling sites. ...................................................................................................... 4
4 The three hierarchical levels of population structure and fixation index ........... 6
5 Maximum Likehood tree of haplotypes for Channa gachua (5A), Glyptothorax
platypogon (5B) and Barbodes binotatus (5C). ................................................... 9
6 Neighbour-joining tree of the population of C. gachua (6A), G. platypogon (6B)
and B. binotatus (6C). SAMOVA result of C. gachua (6D), G. platypogon (6E)
and B. binotatus (6F) with CT(green), ST( red) and SC(blue), number of
group as y-axis and variance number as x-axis.................................................. 10
7 Relationship between geographic distance and corrected pairwise Fst (Fst(1Fst)) in C. gachua (7A), G. platypogon (7B) and B. binotatus (7C) ................. 12
8 Postulated distribution of land and sea in South East Asia from Hall (1998) ... 14

LIST OF APPENDIX

1 Barbodes binotatus site sampling locations….........……………………….….18
2 Channa gachua site sampling locations…...………..........………………..….23
3 Glyptothorax platypogon site sampling locations………….........………….…28
4 Maximum Likelihood tree best model for Channa gachua..............................33
5 Maximum Likelihood tree best model for Glyptothorax platypogon...............34
6 Maximum Likelihood tree best model for Barbodes binotatus........................34

INTRODUCTION

Phylogeography is a field of research concerned with the principles and
processes governing the geographic distributions of genetic diversity, either
within or among closely related species. Phylogeography deals with the historical
origin of the spatial distribution of genes. Analysing and interpreting the
distribution of genetic diversity usually requires extensive input from population
genetics, phylogeny, paleontology and geology. Thus, phylogeography is an
integrative field of research that lies at an important crossroad among diverse
microevolutionary and macroevolutionary disciplines (Avise 2001).
Phylogeographic studies of freshwater fishes are still scarce in Indonesia. Thus,
the present study may be expected to produce valuable insights into the
biogeography of Indonesian aquatic biotas.
Among the 25 world‟s terrestrial hotspots, Indonesian hotspots
(Sundaland, Wallacea) are currently the world‟s most threaten by human activities
(Lamoureux et al 2006, Hoffman et al 2010). This is particularly evident in Java
island where habitat destruction for palm oil plantation, species introduction
through the ornamental fish market and pollution have deeply affected freshwater
ecosystems. A previous study of extinction rates in West Java determined that
92.5 % and 76.5% of the fish species were extirpated, in the Ciliwung and
Cisadane rivers, respectively (Hadiaty 2011). The origin of the ichthyodiversity
in Java still poorly understood and spatio-temporal dynamics are also unknown.
Therefore, biogeographic studies aiming at exploring the spatio-temporal
dynamics of settlement of freshwater fishes in Java are certainly needed.
Given the diversity of ecological habitats in Java, cryptic diversity (two or
more biological species previously assigned to the same taxon due to their
morphological similarity) may be expected. The close morphological similarity
among closely related and cryptic species makes them difficult to distinguish
based on morphological characters and this issue currently limits the usefulness of
a traditional taxonomy approach based on morphological characters. For instance,
two new species from Java have been recently described based on the
combination of morphological and molecular characters (Keith et al. 2014a,
2014b). Fishes are the most diverse group of vertebrates, and because of their
large phenotypic diversity and profound changes during their ontogenetic
development, fish identification is difficult.
DNA barcoding is a new approach to species identification based on a
short fragment of DNA from a standardized region of the mitochondrial genome.
To that end, DNA Barcoding uses a 648-bp segment derived from the 5‟ end of
the mitochondrial cytochrome c oxidase subunit I (COI) gene as a target for
species identification (Hebert et al, 2003). DNA barcoding provides a rapid and
effective way in characterizing the complexity of biodiversity. It helps delineate
species generally well by a particular set of sequences that allow unambiguous
identifications and provides accurate characterization across all life stages and
species (Hubert et al. 2008). The use of DNA barcoding to reveal
phylogeographic pattern proved to be useful, as previous research successfully
revealed population genetic structure using COI sequences (Yu et al. 2014;

2

Koizumi et al. 2012). The substitution rate in COI is high enough to allow the
discrimination of not only closely related species, but also phylogeographic
groups within a single species.
In this study, we aim at evaluating the utility of DNA barcoding to
document and characterize the intra-specific diversity of several widespread
species of freshwater fishes in Java and Bali. The objective is to explore the
factors and mechanisms that contributed to population structure. To that end, the
genetic diversity and population genetic structure in three widespread species has
been used to estimate the impact of the geological on phylogeographic patterns
and population structures.

MATERIAL AND METHOD

Geological Framework

Java and bali islands are part of Sundaland, which during the low sea
levels had half the coastline and twice the land area as today. Sundaland has
diverse and highly endemic biotas, although, it only consists of 4% of the planet‟s
land area. At the beginning of the Cenozoic approximately 65 Mya, Sundaland
was a continental promontory at the southern end of Eurasia (Hall 1996).
Northward subduction of the Australian plate resumed beneath Indonesia, causing
widespread volcanism at the active margin and producing chains of islands (arcs)
similar to those of the West Pacific today. Sundaland was surrounded by
subduction zones until the Miocene and was probably mainly close to sea level,
with elevated areas in western Borneo and the Thai-Malay Peninsula (Lohmann et
al 2011).

Figure 1 Late Miocene and Early Pliocene reconstructions of land and sea in the
Indo-Australian Archipelago (Lohmann et al 2011)

3

In the Early Miocene approximately 23 Mya, the Australian plate made
contact with the submerged Sundaland margin near Sulawesi, and the Sunda
region began to rotate, initially keeping pace with Australia‟s northward
movement. There was a gradual increase in the area of shallow seas and reduction
in land area on Sundaland until the Pliocene approximately 5 Mya). During the
Quaternary, Australia continued to move north, and subduction-related
deformation led to the gradual emergence of Sumatra and Java as major land areas
during the Pliocene (Figure 1).

Figure 2 Pleistocene map of South East Asia illustrating depth contours of 120m
below present sea levels (Voris 2000)
Sea level change driven by polar ice volume changes modified land area
during the Quaternary. It greatly affect mass configuration in Sundaland. The
major East Sunda River System existed when sea levels were below present day
levels (Figure 2). This huge river system ran east across what is now the Java Sea
to enter the sea near Bali. This system included virtually all the modern day river
of the north coast of Java, south coast of Borneo and the northern portion of the
east coast of Sumatra (Lohmann et al. 2011).

Sample Collection

Due the high diversity of habitat and complex geological and
paleocological history in the study area, this research was conducted in a total of
fifty one locations across Java and Bali with the aim to explore phylogeographic
patterns in South Sundaland. Three widely distributed species were selected as
model: Channa gachua known as Bogo as local name (Perciformes, Channidae),
Glyptothorax platypogon known as kehkel as local name (Siluriformes,
Sisoridae), and Barbodes binotatus known as beunteur as local name
(Cypriniformes, Cyprinidae).
Specimens were captured using electrofishing, castnets and gillnets. The
minimum number of individual per site was set to five specimens per species, in

4

order to cover as much genetic diversity as possible and get enough statistical
power to explore population genetic structure. All the specimens were
photographed, individually labeled and voucher specimens were preserved in a
96% ethanol solution. Voucher specimens were deposited at the Muzeum
Zoologicum Bogoriense (MZB) in the Research Centre for Biology (LIPI). DNA
barcodes, photographs and collecting data were deposited on the Barcode of Life
Datasystem (BOLD) in the projects Barcoding Indonesian Fishes - part VI.
Phylogeography of Javanese Fish (BIFG) and Barcoding Indonesian Fishes - part
VIb. Widespread primary freshwater fishes of Java and Bali (BIFGA) in the
container Barcoding Indonesian Fishes of the Barcoding Fish (FishBOL)
campaign. DNA barcodes were also deposited in GenBank, accessions number are
available through the specimen records in BIFG and BIFGA projects

Figure 3 Sampling locations in Java and Bali islands. One point may represent
several sampling sites.

Genetic Analyses

A total of 109 specimens of Channa gachua (Perciformes, Channidae),
123 specimens of Glyptothorax platypogon (Siluriformes, Sisoridae) and 122
specimens of Barbodes binotatus (Cypriniformes, Cyprinidae) were sequenced.
Genomic DNA was extracted using the QIAGEN DNeasy 96 Blood and Tissue
Kit according to manufacturer specifications and further used with no dilution for
amplification and sequencing. The 5‟ end of the cytochrome oxydase I gene
(COI) will amplified (651-bp) using primer cocktails C_FishF1t1/C_FishR1t1
including a M13 tails(Ivanova et al. 2007). PCR amplifications were done on a
Veriti 96-well Fast (ABI-AppliedBiosystems) thermocycler with a final volume of
10.0μl containing 5.0μl Buffer 2X, 3.3μl ultrapure water, 1.0μl each primer
(10μM), 0.2μl enzyme Phire® Hot Start II DNA polymerase (5U) and 0.5μl of
DNA template (~50 ng). Amplifications were conducted as follow: initial
denaturation at 98°C for 5 min followed by 30 cycles. denaturation at 98°C for 5s,
annealing at 56°C for 20s and extension at 72°C for 30s, followed by a final
extension step at 72°C for 5 min. The PCR products were purified with ExoSapIT® (USB Corporation, Cleveland, OH, USA) and sequenced in both directions.
Sequencing reactions were performed using the “BigDye® Terminator v3.1 Cycle

5

Sequencing Ready Reaction” and sequencing was performed on the automatic
sequencer ABI 3130 DNA Analyzer (Applied Biosystems).

Data Analyses

Phylogenetic Analyses of Gene genealogies
Haplotype phylogenies were constructed using the maximum likelihood
(ML) algorithm as implemented in phyml (http://atgc.lirmm.fr/phyml) following
the algorithm developed by Guindon and Gascuel (2003). The Akaike information
criterion (AIC) identified the optimal model, among the 88 available, as
implemented in modeltest 3.7 (Posada & Crandall 1998) and was further used for
ML tree searches. Number of clades in ML haplotypes phylogenies were resolved
based on Barcode Index Number (BIN) algorithm, a clustering algorithm
delineating operational taxonomic unit detected through departure of genetic
distances among molecular lineages (Ratnasingham & Hebert 2013). Estimation
of the age of each species genealogies was inferred using the maximum pairwise
divergence among haplotypes in the haplotype trees and the canonical fish
substitution rate of 1% of divergence per million years (Bermingham et al. 1997).

Population Structure Analyses
Population structure was explored through pairwise ST as implemented in
Arlequin version 3.5 (Excoffier et al.2005) and significance of ST estimates was
tested though permutation procedures including 1000 random replicates.
Population structure was further explored through a Neighbor-Joining (NJ) tree
constructed using the ape R package (Paradis et al. 2004) based on population
pairwise ST estimates. A spatial analysis of molecular variance was conducted
using the SAMOVA algorithm to explore the spatial partition of genetic diversity
through an increasing number of genetically distinct groups of populations (Figure
3). The optimal number of groups (k) was determined in parallel based on the NJ
population tree. Subsequently, the hierarchical analysis of molecular variance
(AMOVA) was extracted from SAMOVA results for predefined sets of
populations to infer the relative contribution of several levels of spatial
partitioning of the variance including among groups (CT), among populations
within groups (SC) and within populations (ST). In this hierarchical framework,
ST means genetic differentiation between individual from the same population,
SC means genetic differentiation between individual from different population in
different area and CT means genetic differentiation between individual from
different population and area (Figure 4). Genetic diversity within populations was
estimated through several parameters including haplotype diversity, nucleotidic
diversity, number of haplotypes, and polymorphic sites using the software DNAsp
(Rozas et al. 2003).
A test of isolation by distance was performed using the R stats package.
ST between pairwise comparisons were used to test for a potential isolation by

6

distance (IBD) pattern in the data by plotting (1 – ) against the logarithm of
geographical distance (Rousset 1997; Slatkin 1993). Isolation by Distance is a
term for determining the distribution of gene frequencies over a geographic region
according to the geographic distance among populations. Significance of the
correlation between genetic and geographic distances was tested with the Mantel
test (Legendre & Legendre 1998) as implemented in the ade4 package in R (Dray
& Dufour 2007).

Group

ST
SC

Group

Population

Population

CT
Population

Population

Population

Population

Figure 4 The three hierarchical levels of population structure and fixation index

RESULT

Phylogenetic Analyses
The AIC pointed to the TIM3+G model as the most likely substitution
model in C. gachua. This model includes 4 base frequencies and 6 substitution
categories as well as a gamma distribution accounting for differences in rates of
substitution among sites. The TIM3+G most likely tree displayed a score of -lnL=
2924.20. The estimated nucleotide frequencies were A = 0.2326, C = 0.3090, G =
0.1763 and T = 0.2822, and the substitution rates were [A–C] = 2,1514, [A–G] =
24.132, [A–T] = 1.000, [C–G] = 2.1514, [C–T] = 12.6982, [G–T] = 1.0000. The
BIN algorithm detected nine major clades in the tree of C. gachua (Figure 5A).
The AIC pointed to TIM1+I+G as the most likely substitution model for
G. platypogon (Figure 2B). This substitution model includes 4 base frequencies
and 6 substitution categories, the proportion of invariable sites (I) and the gamma
distribution parameter. The likelihood score of G. platypogon ML tree was lnL=2372.05. The estimated nucleotide frequencies were A = 0.2775, C = 0.2792,
G = 0.1653 and T=0.2780, and the substitution model incorporated the

7

substitution rates [A–C] = 1.000, [A–G] = 12.1212, [A–T] = 0.1306, [C–G] =
0.1306, [C–T] = 4.6442, [G–T] = 1.0000. BIN algorithm detected seven major
clades in G. platypogon tree (Figure 5B).
The AIC pointed the TPM3uf+I+G as the most likely substitution model
for B. binotatus. This model includes 4 base frequencies and 6 substitution
categories, the proportion of invariable sites and the gamma distribution
parameter. The resulting ML score was -lnL=3233.88. The estimated nucleotide
frequencies were A = 0.3023, C = 0.2763, G = 0.1404 and T=0.2809 and the
substitution model incorporated the substitution rates [A–C] = 0.6322, [A–G] =
10.5781, [A–T] = 1.000, [C–G] = 0.6322, [C–T] = 10.5781, [G–T] = 1.0000.
There were 3 major lineages (Figure 5C).

5A

8

5B

9

5C

Figure 5 Maximum Likehood tree of haplotypes and its distribution in Java and
Bali island for Channa gachua (5A), Glyptothorax platypogon (5B) and
Barbodes binotatus (5C).

10
Cluster III

Cluster II

6A

Cluster I

Cluster VI

6C

Cluster IV

Cluster II

Cluster V

6B

Cluster IV

Cluster
V

Clus
ter I

Cluster III
Cluster I
Cluster III

Cluster II

6E

6F

6D
Figure 6 Neighbour-joining tree of the population of C. gachua (6A), G. platypogon (6B) and B. binotatus (6C). SAMOVA result of
C. gachua (6D), G. platypogon (6E) and B. binotatus (6F) with  CT(green), ST( red) and SC(blue), number of group as
y-axis and variance number as x-axis.

11
We further estimated the absolute age of the maximum divergence times
for each species using the canonical 1% per million year substitution rate. In C.
gachua, the maximum age estimate was 3.05 Ma (with a maximum intraspecific
pairwise divergence of 6,1 %). In G. platypogon the maximum divergence was
estimated to happen at 3 Ma (with a maximum intraspecific divergence of 6,0 %).
In B. binotatus, the age estimate of the maximum divergence was 2.60% (with a
maximum intraspecific divergence of 5.2 %).
Population structure
The NJ population trees provided information about the population
structure of each species. From the topology of the trees, several clusters were
delineated within each species including five clusters in C. gachua (6A), six
clusters in G. platypogon (6B) and three clusters in B. binotatus (6C). Below the
NJ tree, the relationships between the fixation index among groups (CT), among
populations within groups (SC) and within populations (ST) according to the
number of population clusters indicate that among groups and among populations
within group fixation index are very similar for C. gachua (6D), G. platypogon
(6E) and B. binotatus (6F).
Table 1 Amova statistics and fixation indexes
Number of Group
SC
ST
CT
Variation among groups (%)
Variation among populations within
groups (%)
Variation within population (%)
p-value ofSC
p-value ofST
p-value ofCT

C. gachua G. platypogon B. binotatus
5
6
3
0.83863
0.59904
0.69865
0.9525
0.90405
0.90928
0.70564
0.7607
0.69896
70.56
76.07
69.9
24.69
14.34
21.03
4.75
0.000
0.000
0.000

9.6
0.000
0.000
0.000

9.07
0.000
0.000
0.000

Amova statistics for C. gachua (Table 1) showed high levels of structure
within population (ST), among population group (SC) and among group (CT).
This structure is supported by significant p-values at all levels (Table 1). Most of
the variance in C. gachua, however, is explained by variations among groups
(70,56%). Amova statistic of G. platypogon showed high levels of structure
within population (ST), among population group (SC) and among group (CT)
as evidenced by significant p-values at all levels (Table 1). Most of the variance in
G. platypogon, however, is explained by variations among groups (76,07%).
Amova statistics of B. binotatus showed high levels of structure within population
(ST), among population group (SC) and among group (CT) as supported by
significant p-values at all levels. Similarly, most of the variance in B. binotatus is
explained by variations among groups (69.90%).

12

7A
R-squared = 0.032

7B
R-squared = 0.0422

P-value = 0.6396

P-value = 0.9319

R-squared = 0.0036

7C

P-value = 0.5733

Figure 7 Relationship between geographic distance and corrected pairwise Fst
(Fst(1 Fst)) in C. gachua (7A), G. platypogon (7B) and B. binotatus
(7C)
The Figure 7 showed that the relationship between geographic distance
and corrected pairwise st was weak for all three species. This result is confirmed
by the low R-squared (R2=0.032 for C. gachua, R2= 0.0422 for G. platypogon and
R2=0.0036 for B. binotatus). The mantel test (P-value) confirmed this trend as the
correlation between geographic and genetic distances was not significant for all
three species (Fig. 4). The genetic diversity of the three species provided
contrasted results depending on the kind of genetic diversity examined (Table 2).
The numbers of haplotype and haplotypic diversity were high for all the clusters
of populations, excepting in G. platypogon V and VI.

13
Table 2 Genetic diversity of C. gachua, G. platypogon and B. binotatus
Population

Sampl
e Size
(n)

Variable
sites

Number of
Haplotypes

C. gachua( Cluster I)
C. gachua (Cluster II)
C. gachua (Cluster III)
C. gachua (Cluster IV)
C. gachua (Cluster V)
G. platypogon (ClusterI)
G. platypogon (Cluster II)
G. platypogon (Cluster III)
G. platypogon (Cluster IV)
G. platypogon (Cluster V)
G. platypogon (Cluster VI)
B. binatotus (ClusterI)
B. binatotus (Cluster II)
B. binatotus (Cluster III)
Overall (C. gachua)
Overall (G. platypogon)
Overall (B. binatotus)

30
13
11
36
14
40
37
16
8
10
12
51
52
19
104
123
122

22
10
1
22
25
46
9
3
48
6
33
7
23
41
87
66
53

14
7
2
10
4
8
6
4
6
2
3
6
14
12
37
22
28

Haplotype
diversity
(Hd)

0.921
0.871
0.509
0.806
0.582
0.496
0.740
0.575
0.928
0.200
0.318
0.695
0.884
0.929
0.956
0.892
0.915

Nucleotide
Diversity
(µ)

0.0103
0.0057
0.0008
0.0094
0.0113
0.0093
0.0062
0.0012
0.0371
0.0020
0.0092
0.0023
0.0066
0.0228
0.0318
0.0282
0.0125

DISCUSSION

The ML trees of the three species had a different topology suggesting
different patterns for each species. In C. gachua tree, the most basal BINs were
observed in East Java and Bali (BIN:ACQ0292 and BIN:ACQ0290). This result
suggests that C. gachua expanded to the West, started from East Java and Bali and
resulting in the younger lineages in West and Central Java (BIN:ACQ6941,
BIN:ACQ3951, BIN:ACQ6941, BIN ACQ6940, BIN ACQ6939, ACQ3952 and
ACQ3950). For G. platypogon, three basal lineages were observed in West Java
(BIN:ACP5850, BIN:AAY1028, BIN:ACP5898). Then, G. platypogon expanded
to the east resulting in four recently derived lineages (BIN:ACP6225, BIN:
ACQ6223, BIN:ACP6117, BIN:ACQ6224). In the case of B. binotatus, three
different lineages were observed and two of them were restricted to small areas in
West Java (BIN:ACP6290, BIN:6025), while the third one was wide-spread in
Java (BIN:ACP5712). Within the wide-spread lineage (BIN:ACP5712), two
subclades were observed, one in West Java and one in East Java and both
subclades display very low genetic distances. C. gachua most basal lineage is
observed in Bali and east Java. This suggests that the colonization of Central and
West Java were more recent than in East Java and Bali. For G. platypogon and B.
binotatus, the oldest lineages are observed in the West, thus it suggested that the
colonization of Central and East Java is more recent than in East Java and Bali

14
Overall, genetic differentiation within populations, among population
within group and within group was high for the three species. This result was
supported by the result of the SAMOVA with the values of all tests being
significant (P