Research Article Characterisation of Asian Snakehead ...downloads.hindawi.com/journals/tswj/2013/917506.pdfResearch Article Characterisation of Asian Snakehead Murrel Channa striata

Hindawi Publishing CorporationThe Scientific World JournalVolume 2013 Article ID 917506 16 pageshttpdxdoiorg1011552013917506

Research ArticleCharacterisation of Asian Snakehead MurrelChanna striata (Channidae) in Malaysia An Insight intoMolecular Data and Morphological Approach

Li Min Song1 Kaviarasu Munian12 Zulkafli Abd Rashid3 and Subha Bhassu14

1 Genomics and Evolutionary Biology Laboratory Department of Genetics amp Molecular Biology Institute of Biological SciencesFaculty of Science University of Malaya 50603 Kuala Lumpur Malaysia

2 Zoology Branch Forest Biodiversity Division Forest Research Institute Malaysia (FRIM) 52109 Kepong Selangor Malaysia3 Department of Fisheries Malaysia Freshwater Fisheries Research Division FRI Glami Lemi Jelebu71650 Titi Negeri Sembilan Malaysia

4Centre for Biotechnology in Agriculture Research Division of Genetics amp Molecular Biology Institute of Biological SciencesFaculty of Science University of Malaya 50603 Kuala Lumpur Malaysia

Correspondence should be addressed to Subha Bhassu subhabhassugmailcom

Received 19 September 2013 Accepted 23 October 2013

Academic Editors A C Manna and A Walley

Copyright copy 2013 Li Min Song et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Conservation is imperative for the Asian snakeheads Channa striata as the species has been overfished due to its high marketdemand Using maternal markers (mitochondrial cytochrome c oxidase subunit 1 gene (COI)) we discovered that evolutionaryforces that drove population divergence did not show any match between the genetic and morphological divergence patternHowever there is evidence of incomplete divergence patterns between the Borneo population and the populations from PeninsularMalaysia This supports the claim of historical coalescence of C striata during Pleistocene glaciations Ecological heterogeneitycaused high phenotypic variance and was not correlated with genetic variance among the populations Spatial conservationassessments are required to manage different stock units Results on DNA barcoding show no evidence of cryptic species in Cstriata in Malaysia The newly obtained sequences add to the database of freshwater fish DNA barcodes and in future will provideinformation relevant to identification of species

1 Introduction

The freshwater snakehead Channa striata (Bloch 1793)from the family Channidae has a wide range of habitatsranging from rivers swamps ponds canals lakes and landof rice fields Their natural populations are extensively dis-tributed across southern Asia southern China Indochinaand Sunda Islands [1ndash3] This carnivore snakehead murrellocally known as ldquoHaruanrdquo is able to tolerate to adverseenvironments due to its hardiness and air-breathing capabil-ities assisted with a suprabranchial chamber an air-breathingorgan [4] which is unique to Channidae but exclusive inother freshwater fish families [5ndash7]The localmarket demandon C striata is greatly expanding due to its commercialvalue agreeable flavor local food [3] and its postoperative

medicinal application to enhance wound healing and reducepostoperative pain and discomfort [8] A population struc-ture of C striata is urgently needed in handling this speciesin aquaculture and to provide spatial conservation value onthis species

To date the exact taxonomic status at the level of speciesof the genus of Channa remains unclear as C striata wasplaced as one of the ldquospecies complexesrdquo under this men-tioned genus [9]This had raised the interest to investigate thephylogenetic relationship of the snakehead particularly theorigin of the Asian snakehead and its evolution towards for-mation of different species of genusChanna Previous studiesshowed that the occurrence of the Asian snakehead in south-ern Asia particularly in Southeast Asia was mainly due tophylogeographical influences [10] The population structure

2 The Scientific World Journal

of C striata surveyed in Perak Malaysia was adduced toa historical Pleistocene event [11] strongly suggesting thecoalescence of freshwater taxa The Pleistocene event mayexplain the most historical coalescence of a species wherebymultiple episodes of glaciations and deglaciations occurredwith the accompanied of lowering and rising in sea waterlevel happened approximately 2 million years ago HistoricalPleistocene glaciations with themaximum lowering sea waterlevel greatly affected the land mass configuration and thusthe biodiversity where PeninsularMalaysia island of BorneoSouthern Thailand Southern Indo china Sumatera andJava were interconnected [12] The land bridge through theformation of Sunda River permitted the migration and geneflow of fauna between Peninsular Malaysia and the island ofBorneo as the sea-water level rose that allowed submersionof this river system during the last Pleistocene Epoch [113] This phenomenon is shown by the genetic divergencepattern of Hampala macrolepidota [14] Tor tambroides [15]and Barbonymus schwanenfeldii [16] with the witness ofnonsignificant genetic differentiation and sharing of the samehaplotype between Peninsular Malaysia and the island ofBorneo

Environmental factors were recognized as an additivedeterminant on the phenotype of an organism Human activ-ities such as construction of dwellings that often involve forestdestruction leading to sedimentation in the river coupledwith biotic environmental factors such as food competitionas a whole will influence the trait development for survivalfitness The physical changes as a response to pollutedaquatic habitats were shown in previous studies in relation todeformity of fish morphology [17] deformation [18 19] andfin erosion [20 21] The study of Roberts and Khaironizam[22] revealed that the mouth polymorphism ofNeolissochilussoroides due to feeding behavior had occurred at sympatricpopulation This in turn led us to further investigate thepattern of phenotypic variation among allopatric populationsof C striata across Malaysia

In this study we adopted a mitochondrial cytochrome119888 oxidase subunit 1 (COI) gene as a genetic marker toassess the genotypic variationThis genersquos comparatively slowmutation rate in relation to microsatellite markers and itsmaternal inheritancemakes it possible to reveal any historicalpopulation dispersal pattern of a species A population fromSarawak was included as a representative of the islandof Borneo to test the hypothesis of Pleistocene event ongenotypic divergence pattern ofC striataThemorphologicaldivergence pattern will be assessed through Truss NetworkMeasurement [23] Understanding the correlations betweenthe genotypic and phenotypic divergence pattern would beinformative to conservationists in proposing conservationassessments The aim of this study is to characterize the pat-tern of population divergence of C striata in order to providea spatial conservation planning in different localities andinvestigate the possible hidden cryptic taxa of C striata usinga molecular species identification approach-DNA bar codingthat was introduced byHebert et al [24]Thismethod for tax-onomic identification avoids potential confusion surround-ing accurate classification of discriminating morphologicalcharacters by focusing directly on the molecular level [25]

and has been used as a universal method that is applicableacross all eukaryote taxonomic groups [23 26ndash32] In relationto this study mitochondrial (mtDNA) gene cytochrome 119888oxidase (COI) is the marker of choice [24] Focusing on thisstandardized short sequence of DNA aims to benefit the tax-onomist by assisting in the classification of known species aswell as by identifying cryptic diversity potentially leading tothe discovery of new undescribed taxa [26 33]This approachappears to have been successful in differentiating amongspecies within a range of animal groups [28 34 35] includingfreshwater andmarine fishes [36 37] In this study where thelevel of interspecies divergence will be taken into accountadditional freshwater fishes of 26 fish species were chosenand where possible multiple individuals of each species werecollected to examine diversity in the COI region at theinterspecific and intraspecific level Our suspected cryptictaxa ofC striatawas sampled extensively across its geograph-ical range in Malaysia in order to represent the extent ofphylogeographical divergence that is likely to be present forthis taxon across the region This widely distributed taxon isthought to comprise a species complex [9] and has recentlybeen found to occur in genetically divergent forms in SE Asia[10] and therefore is of particular taxonomic interest as it mayshow higher than average intraspecific divergence

2 Materials and Methods

A total of 220 specimens of C striata representing eightlocations of Kedah Pulau Pinang Selangor Negeri SembilanJohor Pahang Terengganu and Sarawak were collected andused in this study Samples from Peninsular Malaysia werecaught by simple fishing methods such as nets and samplesfrom Sarawak were purchased from local market These fishsamples were collected by Freshwater Fisheries ResearchDivision of Jelebu (Department of Fisheries Malaysia Fresh-water Fisheries Research Division FRI Glami Lemi JelebuTiti Negeri Sembilan Malaysia) Collection was made from8 November 2006 to 27 August 2007 The samples fromSarawak were purchased by the author on 26 March 2011In the case of DNA bar coding approach an additional 83fishes were sampled at Pahang (Kuala Mai and Lubuk Paku)Selangor (Kepong) and Perak (Temenggor) representingmembers of 26 previously described species from 23 genera7 families and 3 orders (see Figure 1 for sampling locationand details) Multiple individuals per species were collectedwhere possible to provide some indication of the levelof intraspecific variation present in the DNA bar codingfragment In particular 43 C striata individuals with four toten individuals as representatives from each eight sites acrossa wide geographical range in Malaysia were considered inDNA bar coding to provide an estimate of the likely extent ofintraspecific divergence present in this species and to investi-gate possible cryptic taxa that might occur across Malaysia(see Table 1 for selected fishes used in DNA bar coding)These 43 C striata samples were also genetically surveyedfor population study in the case of examining amount ofgene flow and genetic differentiation All additional 83 fisheswere caught by electrofishing and all the collected samples

The Scientific World Journal 3

Malaysia

N

W E

S100 km

State Site Latitude LongitudeA Perak Temenggor 551 10136B Selangor Kepong 3 230603 101634806C Pahang Lubuk Paku 35101406 1027738548D Pahang Kuala Mai 37736179 10236643411 Kedah Kubur Panjang 611427 100549282 Pulau Pinang Permatang Kuang 540665 100367833 Selangor Tanjung Karang 344285 10118344 Negeri Sembilan Sikamat 275925 101949015 Johor Batu Pahat 18586 102937776 Pahang Pahang River 369172 102895167 Terengganu Terengganu River 53795 103088848 Sarawak Miri River 46134368 1143129719

AmdashD sampling site of additional selected 26 freshwater fish species

Malaysia

N

W E

S100 km

1mdash8 sampling site of Channa striata

Figure 1 Sampling locations of 220 specimens of Channa striata and additional 83 freshwater fishes constituting selected 26 freshwater fishspecies used in this study Exact latitude and longitude for each sampling sites are being recordedNumbers 1ndash8 and alphabets AndashD correspondto locations listed in Table 1

in this study were positively identified to species level at thetime of collection The GPS location of collection sites wasrecorded Muscle tissues of each sample collected were storedindividually in absolute ethanol prior to genetic analysisand voucher specimens were preserved in absolute ethanoland lodged at the Repository Store located at FreshwaterFisheries Research Division of Jelebu Ten vouchers samplesofC striata from Sarawakwere kept frozen in aminus80∘C freezerlocated at laboratory of University of Malaya (see Figure 1 forC striata samples used in both molecular and morphometricof population study)

For samples brought back to the laboratory 38 morpho-metric measurements were taken to the nearest centimeterbased on Truss Network Measurement (see Figures 2(a)and 2(b) for all the linear measurements) [22] A box-plotanalysis was done using SPSS software version 160 (SPSSInc Chicago IL) to exclude the extreme measurementswhich indicate outlier Prior to multivariate discriminantanalysis transformation of size-independent shape variableswas carried out using SPSS software version 160 (SPSS IncChicago IL USA) with the formula of 119872adj = 119872(119871 119904119871119900)

119887

[38] where119872adj is the transformed morphometric measure-ment to be used in subsequent multivariate analysis 119872 is

the original morphometric measurement 119871119904is the overall

mean of standard length obtained from all samples for eachvariable119871

119900is the standard length of the fish and 119887 is the slope

of regression of log119872 over log 119871119900 Parameter 119887 is calculated

to check the effect of size on the measurement and trans-formed119872adj variables were used in the followingmultivariatediscriminant analysis when the transformed variable did notshow significant correlation with standard length

Multivariate discriminant analysis was then carried outusing software STATISTICAversion 8 (StataCorp 2003 StataStatistical Software Release 8 College Station TX StataCorpLP) based on transformed morphometric measurementsThis analysis highlighted the two traits which had the greatestcontribution in discriminating populations and the patternof variation will be illustrated in two-dimensional graphSpecifically there are unobserved patterns of variation lyingbehind each observedmeasure andmultivariate discriminantanalysis aims to group those underlying variation whichare correlated to each other under the same root Thepattern of variation underlined by each root can be seen intwo-dimensional graph in which those populations locatedat positive region of each root acquired unique extrememeasurements of the two highly correlated traits that could


Table 1 List of 27 selected freshwater fishes species used in this study for DNA barcoding Numbers and letters under location refer toFigure 1

Order Family Genus Species Number of individuals Local name Location

Cypriniformes Cyprinidae

Amblyrhynchicthys Amblyrhynchicthys truncatus 5 Tempuling (C)Barbichthys Barbichthys laevis 4 Bentulu (C)Barbonymus Barbonymus schwanenfeldii 5 Lampam Sungai (C)Chirrinus Cirrhinus caudimaculatus 2 Selimang batu (D)

Cyclocheilichthy Cyclocheilichthys apogon 3 Temperas (D)Devario Devario regina 3 Danio (A)Hampala Hampala macrolepidota 3 Sebarau (C A)

Hypsibarbus Hypsibarbus wetmorei 5 Kerai Kunyit (C)Luciosoma Luciosoma setigerum 2 Nyenyuar (D)Osteochilus Osteochilus vittatus 4 Terbul (C)Poropuntius Poropuntius smedleyi 5 Tengas daun (B)Probarbus Probarbus jullieni 1 Temoleh (C)

Puntioplites Puntioplites bulu 5 Tengalan (D)Puntioplites proctozystron 4 Kerengkek (C)

Puntius Puntius banksi 3 Tengas (B)

Rasbora Rasbora sumatrana 3 Seluang (C D)Rasbora paviana 2 Seluang (A B)

Thynnichthys Thynnichthys thynnoides 2 Lomah (D)

PerciformesAnabantidae Anabas Anabas testudineus 1 Puyu (D)

Channidae Channa Channa striata 43 Haruan (1 2 3 4 5 6 7 8)Channa lucius 1 Bujuk (B)

Siluriformes

Bagridae Hemibagrus Hemibagrus wyckii 2 Baung kelulang (D)Hemibagrus nemurus 3 Baung (A)

Pangasiidae Pangasius Pangasius nasutus 4 Patin buah (D)Pseudolais Pseudolais micronemus 5 Patin juara (D)

Siluridae Belodontichthys Belodontichthys dinema 2 Gerahak (D)Sisoridae Bagarius Bagarius yarrelli 4 Kenderap (D)

3 Order 7 families 23 genus 27 species 126 fishes 27 species 12 sites

discriminate them from other population located at negativeregion of the same root These two discriminant traits will bereferred to the ldquoList of StandardizedCoefficients of CanonicalVariablesrdquo which will highlight the two highly correlatedtraits that had the greatest contribution of the variation foreach root These discriminant traits are those traits with thetwo top coefficient values in thementioned list assigned to theldquopositiverdquo and ldquonegativerdquo sign of the value The comparativesmall or comparative large measurement of the trait inrelation to others will then be referred to the ldquonegativerdquo andldquopositiverdquo sign of the coefficient value respectively [38]

Initially a multivariate discriminant function whichgroup the populations based on similarity on certain charac-ters was performed with all the populations from PeninsularMalaysia Secondly in order to have a balance sample sizefor data analysis populationswith overlapping centroidmeanare considered as single group and subsets of each grouptogether with Sarawak population were included in furthermultivariate discriminant analysis

A total of 43 C striata specimens were used in thisanalysis Between four to ten samples representing each site

(sites 1ndash8 refer to Figure 1) were used in genetic analysisOnce morphometric measurement was taken approximately2 cm times 1 cm of tissue samples were dissected and preservedin absolute ethanol prior to genetic analysis and stored atminus80∘C freezer for long-term storage Genomic DNA wasextracted from each tissue sample using AxyPrep GenomicDNA Extraction Kit according to the animal tissues spinprotocol provided by the company (AxyPrep GenomicDNA Extraction Kit website httpwwwaxygenbiocomcollectionsvendorspage=2ampq=Axygen) Polymerase chainreactions (PCRs) were then performed to obtain sim700 bpCOI amplicons with the primer pair FishF1 51015840TCAACC-AACCACAAAGACATTGGCAC31015840 and FishR1 51015840TAG-ACTTCTGGGTGGCCAAAGAATCA31015840 [39] PCR was per-formed with no modifications to standard PCR reactionSuccessful amplification was determined through 1 agarosegel electrophoresis with the products visualized by stainingwith ethidium bromide After agarose gel electrophoresisPCRproductswere abscised from the gel and purificationwasperformed using AxyPrepGel DNAExtraction Kit accordingto the provided protocol (AxyPrep Gel DNA Extraction


a1

a2a3 a4

a5

a6a7

a8a10 a9a11a12

b1b4

c2c3

d1

d2

e1e2 f1

f2

g1g2 g3

g4

tl

sl

ed

b2b3

c1

cfl

(a)

h1

h2

h3

SnW

SnL

(b)

Figure 2 Schematic diagram of 38 morphometric measurements of Channa striata (total length (tl) standard length (sl) caudal fin length(cfl) eyes diameter (ed) snout width (SnW) and snout length (SnL) and measurement of each body part (a1ndasha12 b1ndashb4 c1ndashc3 d1-d2 e1-e2f1-f2 g1ndashg4 and h1ndashh3)) based on the Truss Network Measurement [23]

Kit) After purification the products were sequenced in bothdirections using the Big Dye version 31 chemistry followingthe manufacturerrsquos instructions (Applied Biosystem) Aftersequencing reactions the sequencing products were purifiedby ethanol precipitation and analyzed on an ABI 3730capillary sequencer (Applied Biosystems)

Prior to data analysis nucleotide sequences weretrimmed to a length of 582 bps to remove any areas wherebase calling was uncertain and then all sequence readswere checked against chromatograph data using ChromasSoftware (Technelysium Pty Ltd) and Mega Softwareversion 41 [40]The final alignment began at position 6552 ofthe complete mitochondrial genome ofOncorhynchus mykiss(GenBank accession numberNC 0017171) All the sequenceswere submitted to the GenBank database with accessionnumbers HM156340ndashHM156344 HM156350ndashHM156372HM156377ndashHM156390 HM156392 JF781170ndashJF781242 andJN695688 to JN695697 and to the Barcode of Life Database(BOLD httpwwwbarcodinglifecom or [41])

To ensure that the desired COI region had been obtainedDNA sequences were aligned using the Basic Local Align-ment Search Tool (BLAST) [41] to check for homology toother vertebrate COI sequences In some cases nontargetfragments such as nuclear mtDNA pseudo genes (numts)may be amplified during bar coding [42 43] To ensurethat this was not a problem for the current data set allsequences were screened for the presence of stop codonsand insertion-deletion mutations using the software Mega

version 41 [42] The software DnaSP v5 [44] was used tosummarize haplotype information For DNA bar codinganalysis within the group mean genetic distances werecalculated within species within genera and where samplingwas appropriate within one family (Cyprinid) using theKimura 2 Parameter (K2P) distance model [45] in thesoftware Mega version 41 Interspecific K2P differentiationwas also calculated for all possible species through pairwisecomparisons using Mega software Phylogenetic trees wereconstructed to illustrate the patterns of divergence amongspecies and within species where multiple haplotypes hadbeen detected Where multiple individuals possessed identi-cal haplotypes only one representative was included in thephylogenetic analyses Two species Amia calva (genBankaccession number EU5244351) and Atractosteus tristoechus(genBank accession number FN5455921) were included asout-group taxa A neighbour-joining (NJ) tree (Saitou andNei 1987) was constructed inMega version 41 using the K2Pdistance model with 1000 bootstrap replicates Secondly aBayesian tree was estimated usingMrBayes software [46] Forthis analysis parameters not part of the default settings werea 4by4 nuclear model an nst of 6 and an invariable site withgamma model with analyses run for 200000 generations ofsimulations under a Markov Chain Monte Carlo (MCMC)model The first 25 of samples were discarded as burn-in and trees were sampled every 100 generations For thepopulation study at a genetic level a haplotype networkconnected by all the haplotypes by mutational step was


Table 2 Pairwise population distance represented by 119865st value with bold and ldquolowastrdquo indicates significant 119865st (119875 lt 005)

Site (1) (2) (3) (4) (5) (6) (7) (8)(1)(2) 000000(3) 100000lowast 100000lowast

(4) 095455lowast 095455lowast 045205(5) 100000lowast 100000lowast 100000 089992lowast

(6) 100000lowast 100000lowast 100000lowast 093478lowast 100000lowast

(7) 100000lowast 100000lowast 100000lowast 081250lowast 100000lowast 100000lowast

(8) 095271lowast 095271lowast minus012150 040239 092243lowast 092817lowast 077941lowast

Number of site (1) Johor (2) Negeri Sembilan (3) Kedah (4) Pulau Pinang (5) Terengganu (6) Pahang (7) Selangor and (8) Sarawak

performed using software TCS version 121 [47]The 119865st valuewhich determines the population differentiation was thencalculated using software Arlequin version 311 [48] Pop-ulation comparison was performed by computing pairwisedifferenceswith number of permutation of 100 and significantlevel at 119875 lt 005

3 Results

A total of 126 samples obtained from27 freshwater fish specieswere characterized for a 582-base-pair length of mtDNACOI sequence All fragments aligned well with other COIbarcodes in the GenBank database and showed no evidenceof the presence of indels or in-frame stop codons that mayhave indicated that the regions were numts Therefore itwas assumed that the nucleotide sequences generated heretruly represented the mtDNA COI barcode region and weresuitable for subsequent analysis

For all 43C striata sequences the aligned COI sequencesconsist of nine nucleotide variable sites contributing 155of intra-species nucleotide variation All the variable sitesare parsimony informative Overall there are seven uniquehaplotypes (hap) (Figures 3(a) and 3(b)) identified from 43C striata specimens used in this study with high haplotypediversity value (ℎ = 08018) being recorded The haplotypedistribution in Peninsular Malaysia is parallel to the partitiondivision in Mohsin and Ambak (Figure 3(c)) [1 49] with nooverlapping of haplotype between the three regions of thenorth central and south division of Peninsular MalaysiaHowever there are three unique haplotypes (hap4 hap5 andhap6) identified with no repetition in the three locationswithin the central division of Peninsular Malaysia Hap2 isthe most common haplotype and interestingly this haplotypesequence overlapped West (Kedah and Pulau Pinang) andEastMalaysia (Sarawak) sequencesThe population structureofC striatawas obtained by using the119865st value (Table 2) fromthe underlying population differentiation with the bench-mark of 119875 lt 005 as the borderline of significant differenceIn this study there are no significant differences (119875 lt 005)between populations which are in the north coast (Kedahand Pulau Pinang) and population in the south coast (NegeriSembilan and Johor) However the degree of populationdifferences is not correlated to the geographical distance due

to the absence of significant difference between EastMalaysia(Sarawak) and the north coast of West Malaysia (Kedah andPulau Pinang) which are separated by South China Sea

For the DNA bar coding results among the 27 fishspecies surveyed 46 unique haplotypes were detected withno haplotypes shared among different species The K2Pdivergences are presented in Table 3 and summarized withinand among taxonomic levels in Table 4 The divergencebetween individuals within each species was low with K2Pdistances ranging from 000 to 090 whereas averagecongeneric distance ranged from 170 to 1010 Despite therelatively intensive intraspecific sampling of C striata thisspecies did not show the highest level of intraspecific diver-gence which was instead observed among the two Rasborapaviana collected from sites A and B (090)The maximumdivergence among C striata individuals was 060 less thanthe mean divergence between C striata and the congenericC lucius (170) indicating that the divergence betweenthese species was comparable to the divergence observed atthe same taxonomic level among other congeneric groupsOn average there was almost 38-fold more variation amongcongeneric species (mean divergence of 603) than amongcon-specific individuals (mean divergence of 016) with theminimum congeneric divergence (170) more than twicethe maximum intraspecific divergence (090) This patternof divergence is reflected in the phylogenetic tree (Figure 4)where all haplotypes from the same species are clusteredtogether with a high statistical support under both NJ andBayesian inference methods The mean genetic distanceamong the 18 species sampled within the Cyprinid familywas 157 almost 3-fold greater than the mean divergenceobserved among genera

Discriminant analysis based on 30 successfullytransformed morphometric measurements had successfullydiscriminate locations within Peninsular Malaysia as wellas between the West and the East of Malaysia For thediscriminant analysis within Peninsular Malaysia fourdistinct groups had been discovered with nonoverlappingcentroid mean with each other as shown in the two-dimensional graph of discriminant function (Figure 5)There are group of multiple populations consists of SelangorPahang and Terengganu group of two populations of PulauPinang and Johor group of single population of NegeriSembilan and another group of single population of Kedah


Haplotype 1 (H 1)Haplotype 2 (H 2)Haplotype 3 (H 3)Haplotype 4 (H 4)

Haplotype 5 (H 5)Haplotype 6 (H 6)Haplotype 7 (H 7)

Malaysia

100 km

Malaysia

100 km

N

W E

S

(a)

H 1

H 4

H 2

H 3 H 6

H 5

H 7

(b)

Taiping

0 100

(km)

Kelang

Northwest

Northeastand

central division

division

South ChinaSea

KualaRompin

Southerndivision

N

(c)

Figure 3 (a) Haplotype distribution of seven haplotypes generated from eight populations of Channa striata across Malaysia (b) Haplotypenetwork connecting all generated haplotypes the size of each haplotype is proportional to the number of each individual of the correspondinghaplotype each line represents onemutational step (c)Threemain divisions of PeninsularMalaysia Adapted fromMohsin andAmbak [1 49]cited in [16]

These resulted groups are further assigned as groups A B Cand D respectively for further discriminant analysis whichinclude Sarawak as group E as a representative populationfrom East Malaysia The eigenvalues are the greatest on root

1 and root 2 of the discriminant function among populationsin Peninsular Malaysia and hence contributing 5854 and1849 of the total variance respectively in discriminationof morphometric character Refer to Table 5 for details


Table3Pairw

isegenetic

K2Pdistancesa

mon

gtaxaIntraspecificv

aluessho

wnin

bold

andmarkedby

asterix

where

multip

ledistancesw

erep

resent

betweenintraspecific

haplotypes

the

largestvalue

isshow

n[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27]

[1]B

schw

anenfeldiilowast

010

[2]A

truncatus

1130lowast

010

[3]B

laevis

1480

1450lowast

010

[4]Ovittatus

1740

1840

1650lowast

010

[5]Hw

etmorei

1110

1180

1150

1570lowast

000

[6]Pproctozystron

1040

930

1370

1840

1070lowast

000

[7]Hm

acrolep

idota

1850

1880

1780

2030

1510

1650lowast

050

[8]Lsetigerum

2030

1950

2040

2200

1880

1870

2130lowast

020

[9]B

yarrelli

2380

2310

2240

2660

2260

2470

2700

2750lowast

000

[10]R

sumatrana

1990

1850

1790

2120

1880

1930

2190

2140

2300lowast

000

[11]Pmicronem

us2350

2400

2380

2590

2440

2380

2470

2410

1800

2350lowast

000

[12]Pjullieni

1430

1480

1360

1440

1190

1250

1630

2160

2210

2130

2450lowast

NA

[13]Pna

sutus

2410

2230

2340

2500

2400

2350

2400

2390

1800

2180

800

2530lowast

030

[14]Tthynnoides

1430

1490

1290

1540

1420

1270

1740

1880

2310

1960

2310

1490

2130lowast

000

[15]Hw

yckii

2510

2140

2290

2660

2380

2400

2440

2440

1930

2430

1720

2520

1770

2410lowast

000

[16]C

caud

imaculatus

1360

1590

1250

1630

1510

1370

2070

2020

2380

1720

2230

1680

2250

1240

2280lowast

000

[17]C

apogon

1270

1130

1290

1820

1160

1040

1770

1830

2250

1840

2180

1360

2310

1380

2420

1320lowast

050

[18]A

testu

dineus

2470

2620

2520

2770

2570

2670

2870

2500

2990

2540

2580

2520

2460

2420

2620

2250

2610lowast

NA

[19]B

dinema

2360

2560

2460

2580

2350

2510

2560

2530

2100

2330

2220

2660

2140

2580

2410

2260

2470

2630lowast

000

[20]Pbu

lu1110

990

1460

1910

1220

630

1720

1860

2420

1900

2280

1450

2200

1200

2260

1380

1030

2490

2640lowast

050

[21]C

striata

2450

2730

2290

2530

2610

2640

2780

2550

2440

2610

2340

2650

2370

2200

2250

2520

2580

2530

2340

2550lowast

060

[22]R

pavian

a1970

1850

1790

2160

1820

1900

2000

1870

2420

1430

2240

1980

2090

1810

2400

1650

1760

2620

2460

1800

2540lowast

090

[23]Hnem

urus

2360

2360

2220

2470

2230

2400

2600

2540

2020

2510

1540

2260

1790

2380

1680

2350

2240

3040

2410

2440

2400

2540lowast

000

[24]Dregina

2750

2450

2640

2900

2640

2590

2850

3120

3030

2880

3020

2760

3220

2820

3330

2950

2790

3400

3620

2780

3270

2820

3140lowast

000

[25]C

luciu

s2210

2100

2180

2230

2070

2220

2360

2110

2460

2380

2420

2430

2390

2170

2310

2100

2260

2530

2310

2200

2530

2220

2420

3110lowast

NA

[26]Pbanksi

1660

1580

1570

1790

1480

1430

1770

1760

2560

1960

2280

1720

2060

1450

2420

1410

1460

2670

2350

1490

2400

1820

2380

3060

2340lowast

000

[27]Psm

edley

i1190

1280

1290

1570

1040

1220

1600

1830

2430

1820

2470

1470

2470

1490

2450

1290

1090

2560

2500

1390

2750

1850

2480

2940

2170

1700lowast

000


Table 4 Comparison of divergence distance (K2P percent) calculated using K2P model

Comparison of K2Pdistances within groups

Number of groupsincluded incomparison

Minimum K2Ppercentage ()

Maximum K2Ppercentage ()

Mean K2P percentage()

Within species 24 species 000 090 016Within genera (amongspecies) 4 generalowast 170 1010 603

Within family (amonggenera) 2 familiesdagger 450 1570 1010lowastChanna Hemibagrus Puntioplites RasboradaggerCyprinidae Pangasiidae

The greatest contribution of character to the root 1 was madeby measurement of c2 and e1 which are located at posteriorpart of the head whereas the greatest contribution to root 2was contributed by measurement of c3 and d2 which are alsolocated at posterior part of the head Specifically the group ofSelangor Pahang andTerengganuwas characterized by com-paratively small c2 measurement and comparatively large e1measurement in comparison to other locations the group ofPulau Pinang Johor and Negeri Sembilan was characterizedby comparatively small c3 measurement and comparativelysmall d2 measurement in comparison to other locationsThis data suggested that C striata population in PeninsularMalaysia was significantly discriminated from each otherby distinct size of the head When compared to Sarawakpopulation using discriminant analysis the discriminantfunction graph shown in Figure 6 root 1 had the greatestvariance (7442) to populations of Peninsular Malaysia andthe Sarawak population (group E) formed a distinct groupand is well discriminated by root 1 The greatest contributionof measurement to root 1 was made by b1 measurement ande2 measurement and hence West Malaysia populations wereuniquely characterized by a comparatively large b1 measure-ment and comparatively large e2 measurement which are themeasurements of the size of the mouth and posterior partof the head respectively in contrast to population from EastMalaysia (refer to Table 6 for details)

Briefly the population molecular divergence pattern dis-covered in this research study did not show a match tomorphological divergence pattern Populations from EastMalaysia (Sarawak) and West Malaysia (Kedah and PulauPinang) are genetically alike with the evidence of sharingof the same haplotype (hap2) and nonsignificant 119865st valuewhereas morphologically the island of Borneo in thisstudy case was discriminated from Peninsular Malaysia aswitnessed by the second stage of multivariate discriminantanalysis

4 Discussions

In our study in order to make any judgment of unidentifiedcryptic taxa in C striata validation of COI barcode indiscriminating freshwater fish species inMalaysia is requiredAmong all 27 freshwater fish species surveyed for DNA barcoding divergence among these 27 species surveyed here

was relatively high suggesting that the COI marker wasable to differentiate between the Malaysian freshwater fishspecies examined in this study In every case where multiplehaplotypes were sampled from a single species all haplotypeswere closely related with no haplotypes common amongmore than one species and strong statistical support that eachindividual species sampled was monophyletic Previouslybar coding studies have reported the same incidences ofshared haplotypes among species (see [37]) These cases areprobably most often associated with sister taxa where thetime since speciation has not been sufficient for lineagesorting and divergence to result in monophyly althoughintrogressive hybridization among more divergent taxa hasalso been observed [26 37] The finding here that there wereno haplotypes common to multiple species is encouragingfrom a bar coding perspective however greater taxonomicsampling of more closely related taxa may yet uncover sharedhaplotypes and introgressive hybridization among Malaysiarsquosmany freshwater fish species Ideally the appropriate thresh-old level for determining interspecific versus intraspecificshould be high enough to separate samples that are likely tobelong to different species but not that representing differenthaplotypes of the same species Hebert et al [26] proposedthat the standard threshold level for designating two taxa asdifferent species should be 10X the mean intraspecific varia-tion for the group under study In the current study very lowmean divergence was observed among individual haplotypesbelonging to the same species (mean intraspecific divergenceof 016 maximum divergence 090) and in contrastmean overall congeneric divergence was 603 Overallthe minimum congeneric distance (170) was almost 11-fold higher than the average variation among con-specificindividuals (016) thus clearly dividing the haplotypes intointraspecific and interspecific groups under Hebert et alrsquos[26] 10X benchmark This ratio of congeneric to averagecon-specific variation parallels the findings of other DNAbarcode studies on animals (13X-fold higher in Neotropicalbats [34] 18X-fold higher in birds [26] and 27X-fold higherin Canadian freshwater fish [37] In the current studytaxonomic sampling was relatively sparse and it is likely thatas more Malaysian freshwater species are surveyed for eachgenus within families the average level of congeneric geneticdistance may decrease Nonetheless the data presented hereimplies that this value is likely to remain higher than the 10Xthreshold It is also likely that within species higher mtDNA


A tristoechusA calva

A testudineus (1)D regina (3)

P banksi (3)B schwanenfeldii (4)B schwanenfeldii (1)

A truncatus (1)A truncatus (4)

P proctozystron (4)P bulu (2)P bulu (3)C apogon (1)C apogon (2)P jullieni (1)

H pierrei (5)P smedleyi (5)

H macrolepidota (1)H macrolepidota (1)H macrolepidota (1)

T thynnoides (2)C caudimaculatus (2)B laevis (3)B laevis (1)

O vittatus (3)O vittatus (1)L setigerum (1)L setigerum (1)

R sumatrana (3)R paviana (1)R paviana (1)

B yarrelli (4)H wyckii (2)

B dinema (2)H nemurus (3)

P micronemus (5)P nasutus (1)P nasutus (1)P nasutus (1)P nasutus (1)

C lucius (1)C striata (10)C striata (4)C striata (5)C striata (1)C striata (15)C striata (3)C striata (5)

lowast099

99840099

lowast099

99840099

lowast099

99840099

lowast100

99840099

lowast100

99840099

lowast100

99840099

lowast100

99840099

lowast100

99840099

lowast097

99840099

lowast099

99840099

lowast099

99840099

01

Figure 4 Phylogenetic tree of mtDNA COI barcodes of 27 fish species with the number of individuals of each haplotype listed in bracketsafter the species name Posterior probability and bootstrap among different haplotypes of same species are shown by lowast and 1015840 respectively

COI divergence may be present among individuals sampledacross the entire species in its natural geographical range Inthis case the 10X method may be employed to investigate ifthe level of divergence present may represent the presenceof cryptic speciation or was simply a result of regionalphylogeographical structuring [27 50] This study employedextensive geographical sampling of one species of freshwaterfish in Malaysia C striata and for this taxon intraspecific

divergence was 060 less than the interspecific divergenceobserved among C striata and Channa lucius (170)Although when considered independently from divergencesamong the entire dataset the pattern of divergence withinthe Channa does not conform to the 10X threshold forspecies delineation proposed by Hebert et al [26] and thismay be a result of sparse taxonomic sampling as greatersampling effort that includedmoreChanna species is likely to


Table 5 List of standardised coefficients of canonical variables based on all Channa striata populations surveyed in Peninsular MalaysiaMeasurements in ldquoboldrdquo and ldquoitalicrdquo indicate the greatest contribution of characters to each root The variables are corresponding to TrussNetwork Measurement illustrated in Figure 2

Variable Standardised coefficients for canonical variablesRoot 1 Root 2 Root 3 Root 4 Root 5 Root 6

c2 minus141184 0499825 0398591 0054463 0119042 minus0238883f2 047170 0408845 minus0615390 minus0168215 minus0230770 minus0211542b2 028101 minus0278591 minus0284479 0174256 minus042253 0458817a10 041539 0096533 minus0367550 minus0432404 0259111 minus0268992c3 066377 minus0858751 0014077 0472265 minus0250660 minus0080690a5 036065 0314906 minus0225384 minus0008893 0267024 0198322e2 037393 0331761 minus0266962 0681100 minus0058005 minus0360663b4 minus019458 0132053 0252415 minus0497241 minus0523356 0359599SnL minus040295 minus0046967 0003316 minus0148526 0532470 minus0133889b3 minus047636 0368229 minus0728886 0429959 0008980 minus0328389a8 008207 minus0149248 0373287 minus0265477 0015430 minus0064601b1 minus043399 minus0523266 minus0084456 minus0507840 minus0286558 minus0003799SnW 004462 0228839 0184555 0652408 minus0226785 minus0053505e1 067757 minus0152426 0587134 0740656 0442494 0845807d1 minus046682 0329635 0231088 minus0655794 minus0133296 minus0085119g2 043378 minus0040481 0309560 04676630 minus0394074 0556584g4 minus040523 minus0077577 0026801 minus0205765 0396488 0091170h2 minus048672 0340375 minus0166280 0050386 0055264 minus0023362a3 027658 0063105 0280636 0288647 0163754 0117652a6 minus025723 0396127 0321013 0032397 minus0299612 0211321d2 007330 minus0585637 0007170 minus0075163 minus0137986 minus0503075c1 017793 0309891 minus0142353 minus0043706 0811599 minus0243137a4 025777 0062941 minus0042803 minus0302267 0331285 minus0248307g3 minus025389 minus0259770 minus0346391 0242604 minus0036979 0115660a12 003025 0253880 0294079 minus0172015 minus0278936 0015935g1 minus020226 0291106 minus0012690 0010235 minus0403767 minus0146930f1 014296 minus0046102 0320983 minus0248611 0212844 minus0372136h3 minus014463 minus0158514 minus0212149 minus0032284 minus0166438 0353353a9 008448 0017800 minus0010266 minus0270678 0233016 0126766tl 007500 minus0141272 minus0182243 0040925 0012208 0065811Eigenvalue 1489929 4705745 3882054 1482290 0334059 0148039Cum Prop 058540 (5854) 0770291 (1849) 0922818 (1525) 0981058 (582) 0994183 (131) 1000000 (058)

increase estimates of average congeneric divergence amongthis group Furthermore this study reveals that althoughhighly divergent populations of C striata have been found inother parts of its natural geographical distribution [10] thereis no evidence of cryptic diversity in this species in MalaysiaWhile generating these 126 barcodes in revealing unidentifiedcryptic taxa the successfully identified barcodes will then aidin future study to act as a reference so that in the future whenan unknown taxon is sampled it can be identified bymatchingits sequence against a DNA barcode database (ie BOLD)This is complementary to the aim of the DNA bar codingapproach which is to assign a unique DNA barcode or set ofDNA barcodes to a particular species in such a way that thesame barcode will never be common among two species [24]Previous studies on the same specimens from similar localityin PeninsularMalaysia reveal the possibility of cryptic taxa of

C striata in Negeri Sembilan through themolecular dispersalpatterns of C striata using rapidly evolving microsatellitemarker [51]

In the population study gene flow is predicted to occurin adjacent populations such as between Kedah and PulauPinang and between Negeri Sembilan and Johor whichrecorded a nonsignificant 119865st value (119875 lt 005) This couldbe explained by the possibility of migration of fish acrossadjacent drainage systems due to flooding [52] This thenfollows the one-dimensional stepping stone models [52] thatallow migration to adjacent population This is typical of themigration pattern of Acrossocheilus paradoxus found in themodel in Taiwan [52]

Physical separation of an inhabited geographical rangeallows a particular organism to acquire different geneticstructures It appears that freshwater species is a casualty of


Selangor Johor N Sembilan Pahang

Terengganu P Pinang Kedah

0 2 4 6 8 10Root 1

0

2

4

6

8

10

Root

2

Group C

Group B Group A

Group D

minus10 minus8

minus8

minus6

minus6

minus4

minus4

minus2

minus2

Root 1 versus root 2

Figure 5 Multivariate discriminant analysis based on 7 locationsplotted on root 1 and root 2 axes Grouping of Groups AndashD wasbased on the overlapping pattern of each population centroid meanThe two discriminant traits that contribute most to the varianceunderlined by each root are referred to as the coefficient values listedin Table 6

physical barriers such as mountains and dam constructioneffectively disconnecting them from the drainage basin Aninteresting finding in this study was the sharing of hap2between West Malaysia (Kedah and Pulau Pinang) and EastMalaysia (Sarawak) and the nonsignificant population pair-wise difference among these two isolated locations indicatingthe presence of gene flow and migration of fish among themainland of Peninsular Malaysia and the island of Borneoduring the historical event of Pleistocene Epoch This epochencountered with the multiple scenarios of glaciations anddeglaciations with the resulted rising and lowering sea-water level and hence periodically changes the exposedland mass The historical event during Pleistocene withthe accompanied fluctuation in sea level could explain theincomplete divergence of C striata between West and Eastof Malaysia Although physical barriers could eventuallyseparate the allopatric populations in central region in Penin-sular Malaysia the historical event of Pleistocene as a wholewill explain the historical genetic divergence pattern of Cstriata in Malaysia Our finding with overlapping haplotypedistribution between theWest and the EastMalaysia indicatesthat the fluctuating sea-water level during Pleistocene playeda predominant role in permitting genetic exchange betweenthemainland of PeninsularMalaysia and the island of Borneothrough the formation of Sunda River which allowmigrationof C striata and thus permitting gene flow

The Pleistocene glaciations [12] period happened approx-imately 250000 years ago The mainland of Peninsular

A B C D E

0 10 20 30Root 1

0

5

10

15

20

Root

2

minus5

minus10

minus15

minus40 minus30 minus20 minus10

lowastA = Selangor Pahang TerengganuB = Johor Pulau Pinang C = Negeri Sembilan D = Kedah E = Sarawak


Figure 6 Multivariate discriminant analysis based on 8 locationsplotted on root 1 and root 2 axes The two discriminant traits thatcontribute most to the variance underlined by each root are referredto as the coefficient values listed in Table 6

Malaysia and the island of Borneo together with southernThailand southern Indo-China and Sumatra was once partof the submerged Sunda Shelf [1] Because of the effectof maximum sea-water lowering during Pleistocene themainland of Peninsular Malaysia and the island of Borneowas once connected through the formation of Sunda Riverwhich is rich in fish diversity due to water draining out fromthe eastern coast of Peninsular Malaysia and western coastof Borneo and Sumatra [1 13] This historical land bridgeallowed themigration of fish species between these regions asin this caseC striata until the end of the Pleistocene erawhenthis great river system was submerged due to rising in sea-water level as a result of the deglaciations period [1 13] Recentincrease in sea-water level followed by the formation of SouthChina Sea is suspected to have isolated the island of Borneofrom the mainland of Peninsular Malaysia It is possiblethat once the hap7 had colonized the east and the south ofPeninsular Malaysia and could have mutated spatially Theoverlapping haplotype pattern between Negeri Sembilan andJohor suggested that gene flow was most likely to happenbetween them laterally which further suggested the lateraldispersal behavior ofC striata It is suspected that hap1 hap4and hap5were formed after separation of PeninsularMalaysiafrom the island of Borneo due to these haplotypes not beingany haplotype leading towards formation of those Sarawakrsquoshaplotypes (hap2 and hap7) Hence we predict that completepopulation divergence will occur between West and EastMalaysia after a long period of separation by SouthChina SeaIndeed in our study it is believed that there is no colonizationof C striata on the island of Borneo and it is expected that


Table 6 List of standardised coefficients of canonical variables based on all Channa striata populations surveyed across entire MalaysiaMeasurements in ldquoboldrdquo and ldquoitalicrdquo indicate the greatest contribution of characters to each root The variables are corresponding to TrussNetwork Measurement illustrated in Figure 2

Variable Standardised coefficients for canonical variablesRoot 1 Root 2 Root 3 Root 4

e2 21872 391106 minus020063 021126c2 04818 minus084864 minus170885 008811a5 minus08042 minus237991 135470 minus082680b1 22417 minus037858 006038 minus061243a10 00702 013883 minus014192 004250f2 minus10685 minus079491 075069 minus053315a4 minus00686 minus299113 023824 minus109478g4 minus20462 minus056517 minus082163 minus107446c3 10960 383485 minus140422 020379d1 minus21132 minus506293 115619 083281SnL 17179 077327 minus108747 041713a3 minus20655 090394 minus030638 106254b4 minus16337 072442 009383 144342g2 01400 189031 minus014720 051732a6 minus20150 096549 042037 minus075643h2 14260 minus270404 060591 013971e1 12073 065500 018580 041229g3 13471 136426 minus057506 minus092315a9 minus01035 minus083578 018981 035164a12 02179 minus071198 024452 091279a8 minus05912 189025 minus170222 092930c1 09959 006944 127592 minus077418d2 13882 minus048643 069451 minus086227SnW minus03964 minus058803 077795 minus050801a1 minus14941 minus067406 028236 minus067413tl 09918 minus012765 minus075142 035329a7 minus07121 048801 072751 040213Eigenvalue 2229919 5048606 1921761 694981Cum Prop 07442 (7442) 091267 (1685) 097681 (641) 100000 (232)

the population in Sarawak is the migrated population fromthe mainland of Peninsular Malaysia This is supported byprevious study ofAdamson et al [10]which hypothesized thatthe evolution of majority of the Channa species including Cstriata happened in South East Asia specifically the northpart of Indochina The biogeographically structure betweenIndia and South East Asia coupled with historical climaticchanges caused fish flow through southeastern route fromIndia Geographically the population on the east coast ofPeninsular Malaysia which is closer to the island of Borneohas a greater tendency to migrate than the west coast dueto the draining of water from the east of mainland towardsSunda River However the population group of Kedah inthe northwest of Peninsular Malaysia may have migratedcloser to the northern region of Peninsular Malaysia andthen eventually flowed through the river streams drainingout towards Sunda River during Pleistocene glaciations This

hypothesis is supported by previous study of Esa et al [15]on Tor tambroides which shows a possibility of a migrationroute between northern PeninsularMalaysia and Borneo dueto the same haplotype between populations from BatangAi Sarawak and Perak northern Peninsular Malaysia Inshort historical Pleistocene glaciations and deglaciationsaccompanied with changing in land mass configuration willdescribe the historical coalescence of a freshwater speciessuch as in previous studies on Hampala macrolepidota [17]Tor tambroides [15] and Barbonymus schwanenfeldii [16]

The morphology divergence pattern of C striata wasnot correlated to genotypic divergence pattern due to themorphology of certain terrestrial and marine animal havingan additive effect of genetic components and environmentalfactors A simple explanation would be that there is noconnection between variation at the single gene investigatedand variation in body size and shape


5 Conclusion

In summary the current dataset did not include samplersquossex details due to the evidence of the absence of cytologicaldistinguishable sex chromosome [53] The morphologicaldivergence pattern of C striata is more appropriate to besignificantly affected by environmental heterogeneity thatinfluence the development of certain morphology charactersand such development changes might be a response toadaptive survival in certain harsh environmental conditionsThese developmental changes need not be correlated with theunderlying genetic changes due to morphology as it is theadditive effect of the genetic component and environmentalconditions that induce changes in certain observable charac-ter for survival fitness Although C striata is able to survivein harsh environments conservation planning on this speciesis required due to its highly commercialize value which hasled overfishing to further reduce its effective population sizeIn this study we provided information on spatial pattern ofconservation planning on this species Although our studydid not reveal any high diversity population in one of thesurveyed locations however conservation of this species atits natural variation level is required as C striata forms adiverse group of taxa across Malaysia with seven haplotypesdistributed across Malaysia and the majority of populationsshow significant differences among each other Our studyalso revealed that there are two groups with populationswithin each group which are genetically alike and henceexchangeable for stockmanagement purpose there are groupof Negeri Sembilan and Johor and another group of KedahPulau Pinang and Sarawak The current data compiled didnot reveal any commercial characters such as larger bodysize which will produce more flesh but the morphologicaldivergence pattern of C striata indicates that environmentalfactors had a significant impact on the evolution of certaintraits Our molecular divergence pattern is not congruentto the pattern of morphology variation due to environ-mental conditions playing a significant role in populationmorphology divergence or simply the genetic variation acrossthe whole genome would have to be investigated if wewanted to explore the connections between genotype andphenotype The divergence patterns of both genotypic andphenotypic data reveals that spatial conservation assessmenton C striata is needed to conserve this species Spatialconservative management could be applied to maintain thegenetic diversity and to improve aquaculture quality

Acknowledgments

The authors are grateful to the Department of FisheriesMalaysia (Division of Jelebu Negeri Sembilan) for the provi-sion of tissue samples This work is supported by IPPP grantfrom the University of Malaya (Grant no PV0712011A) Theauthors would like to thank Ellie Adamson and acknowledgethe Australian Endeavour Foundation for the support of a2010 Endeavour Postdoctoral Research Fellowship for assist-ing them in the data analysis The authors also thank twoanonymous reviewers for constructive comments on a previ-ous draft of the paperThe authors would also thank Emeritus

Professor K T Joseph for editing the paperMohd Rashidi formorphological measurements and Dr Hasmahzaiti Omarfor her assistance in the morphometric analysis

References

[1] A K M Mohsin and M A Ambak Freshwater Fishes ofPenisular Malaysia Penerbitan Universiti Pertanian MalaysiaKuala Lumpur Malaysia 1983

[2] P G Lee and P K L Ng ldquoThe systematics and ecology ofsnakeheads (Pisces Channidae) in Peninsular Malaysia andSingaporerdquo Hydrobiologia vol 285 no 1-3 pp 59ndash74 1994

[3] M K Hossain G A Latifa and M M Rahman ldquoObservationson induced breeding of snakehead murrel Channa striatus(Bloch 1793)rdquo International Journal of Sustainable Crop Produc-tion vol 3 pp 65ndash68 2008

[4] S Chandra and T K Banerjee ldquoHistopathological analysisof the respiratory organs of Channa striata subjected to airexposurerdquo Veterinarski Arhiv vol 74 no 1 pp 37ndash52 2004

[5] J S D Munshi ldquoOn the accessory respiratory organs ofOphicephalus punctatus and O striatus (Bloch)rdquo Journal of theLinnean Society of London vol 179 pp 616ndash626 1962

[6] A Ishimatsu and Y Itazawa ldquoVentilation of the air-breathingorgan in the snakehead Channa argusrdquo Japanese Journal ofIchthyology vol 28 pp 176ndash282 1981

[7] A Ishimatsu and Y Itazawa ldquoAnatomy and physiology of thecardiorespiratory system in air-breathing fish Channa argusrdquo inAdvances in Fish Research B R Singh Ed vol I pp 361ndash408Narendra Publishing House Delhi India 1993

[8] A M Mat Jais Y M Dambisya and T-L Lee ldquoAntinociceptiveactivity ofChanna striatus (haruan) extracts in micerdquo Journal ofEthnopharmacology vol 57 no 2 pp 125ndash130 1997

[9] W R Courtenay and J D Williams Snakeheads (Pisces Chan-nidae) ABiological Synopsis andRiskAssessment USGeologicalSurvey US Geological Survey Circular Denver Colo USA2004

[10] E A S Adamson D A Hurwood and P B Mather ldquoAreappraisal of the evolution of Asian snakehead fishes (PiscesChannidae) usingmolecular data frommultiple genes and fossilcalibrationrdquoMolecular Phylogenetics and Evolution vol 56 no2 pp 707ndash717 2010

[11] J A F Jamaluddin T M Pau and M N Siti-Azizah ldquoGeneticstructure of the snakehead murrel Channa striata (channidae)based on the cytochrome c oxidase subunit i gene influence ofhistorical and geomorphological factorsrdquo Genetics and Molecu-lar Biology vol 34 no 1 pp 152ndash160 2011

[12] H K Voris ldquoMaps of Pleistocene sea levels in SoutheastAsia shorelines river systems and time durationsrdquo Journal ofBiogeography vol 27 no 5 pp 1153ndash1167 2000

[13] J J Dodson F Colombani and P K Ng ldquoPhylogeographicstructure in mitochondrial DNA of a South-east Asian fresh-water fish Hemibagrus nemurus (Siluroidei Bagridae) andPleistocene sea-level changes on the Sunda shelfrdquo MolecularEcology vol 4 no 3 pp 331ndash346 1995

[14] J R J Ryan and Y B Esa ldquoPhylogenetic analysis of Hampalafishes (subfamily cyprininae) in Malaysia inferred from partialmitochondrial cytochrome b DNA sequencesrdquo Zoological Sci-ence vol 23 no 10 pp 893ndash901 2006

[15] Y B Esa S S Siraj S K Daud K A A Rahim J R R Japningand S G Tan ldquoMitochondrial DNA diversity of Tor tambroides


Valenciennes (Cyprinidae) from five natural populations inMalaysiardquo Zoological Studies vol 47 no 3 pp 360ndash367 2008

[16] K R Kamarudin and Y Esa ldquoPhylogeny and phylogeographyof Barbonymus schwanenfeldii (Cyprinidae) from Malaysiainferred using partial cytochrome b mtDNA generdquo Journal ofTropical Biology and Conservation vol 5 pp 1ndash13 2009

[17] P L Sun W E Hawkins R M Overstreet and N J Brown-Peterson ldquoMorphological deformities as biomarkers in fishfrom contaminated rivers in Taiwanrdquo International Journal ofEnvironmental Research and Public Health vol 6 no 8 pp2307ndash2331 2009

[18] H W Ferguson Systemic Pathology of Fish Iowa State Univer-sity Ames Iowa USA 1989

[19] E Lindesjoo and J Thulin ldquoA skeletal deformity of northernpike (Esox lucius) related to pulp mill effluentsrdquo CanadianJournal of Fisheries and Aquatic Sciences vol 49 no 1 pp 166ndash172 1992

[20] C J Sindermann ldquoPollution-associated diseases and abnormal-ities of fish and shellfish a reviewrdquo Fishery Bulletin vol 764 pp717ndash749 1979

[21] R J Reash and T M Berra ldquoIncidence of fin erosion andanomalous fishes in a polluted stream and a nearby cleanstreamrdquo Water Air and Soil Pollution vol 47 no 1-2 pp 47ndash63 1989

[22] T R Roberts and M Z Khaironizam ldquoTrophic polymorphismin the Malaysia fish Neolissochilus soroides and other old worldbarbs (Teleostei Cyprinidae)rdquo Natural History Bulletin SiamSociety vol 56 no 1 pp 25ndash53 2008

[23] S Bhassu and Z A Rashid ldquoMolecular and morphologicalApproaches in Discrimination of endangered Probarbus jullieniof Malaysia and Thailand stocksrdquo Russian Journal of Geneticsvol 45 no 9 pp 1092ndash1097 2009

[24] P D N Hebert A Cywinska S L Ball and J R DeWaardldquoBiological identifications through DNA barcodesrdquo Proceedingsof the Royal Society B vol 270 no 1512 pp 313ndash321 2003

[25] R Eisler Eislerrsquos Encyclopedia of Environmentally HazardousPriority Chemicals Elsevier Science 1st edition 2007

[26] P D N Hebert M Y Stoeckle T S Zemlak and C M FrancisldquoIdentification of birds through DNA barcodesrdquo PLoS Biologyvol 2 no 10 2004

[27] P D N Hebert E H Penton J M Burns D H Janzen andWHallwachs ldquoTen species in one DNA barcoding reveals crypticspecies in the neotropical skipper butterflyAstraptes fulgeratorrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 101 no 41 pp 14812ndash14817 2004

[28] R D H Barrett and P D N Hebert ldquoIdentifying spidersthrough DNA barcodesrdquo Canadian Journal of Zoology vol 83no 3 pp 481ndash491 2005

[29] W J Kress K J Wurdack E A Zimmer L A Weigt and DH Janzen ldquoUse of DNA barcodes to identify flowering plantsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 102 no 23 pp 8369ndash8374 2005

[30] G W Saunders ldquoApplying DNA barcoding to red macroalgaea preliminary appraisal holds promise for future applicationsrdquoPhilosophical Transactions of the Royal Society B vol 360 no1462 pp 1879ndash1888 2005

[31] S M Scicluna B Tawari and C G Clark ldquoDNA barcoding ofBlastocystisrdquo Protist vol 157 no 1 pp 77ndash85 2006

[32] R C Summerbell C A Levesque K A Seifert et alldquoMicrocoding the second step in DNA barcodingrdquo Philosoph-ical Transactions of the Royal Society B vol 360 no 1462 pp1897ndash1903 2005

[33] J Huang Q Xu Z J Sun G L Tang and Z Y Su ldquoIdentifyingearthworms through DNA barcodesrdquo Pedobiologia vol 51 no4 pp 301ndash309 2007

[34] E L Clare B K Lim M D Engstrom J L Eger and P DN Hebert ldquoDNA barcoding of Neotropical bats species iden-tification and discovery within Guyana barcodingrdquo MolecularEcology Notes vol 7 no 2 pp 184ndash190 2007

[35] S U Pauls R J Blahnik X Zhou C T Wardwell andR W Holzenthal ldquoDNA barcode data confirm new speciesand reveal cryptic diversity in Chilean Smicridea (Smicridea)(TrichopteraHydropsychidae)rdquo Journal of the North AmericanBenthological Society vol 29 no 3 pp 1058ndash1074 2010

[36] J V Ward and J A Stanford ldquoThe serial discontinuity conceptof river ecosystemsrdquo in Dynamics of Lotic Ecosystems T DFontaine and S M Bartell Eds pp 29ndash42 Science Publica-tions Ann Arbor Mich USA 1983

[37] N Hubert R Hanner E Holm et al ldquoIdentifying Canadianfreshwater fishes throughDNA barcodesrdquo PLoS ONE vol 3 no6 Article ID e2490 2008

[38] N G Elliott K Haskard and J A Koslow ldquoMorphometricanalysis of orange roughy (Hoplostethus atlanticus) off thecontinental slope of Southern Australiardquo Journal of Fish Biologyvol 46 no 2 pp 202ndash220 1995

[39] R D Ward T S Zemlak B H Innes P R Last and P D NHebert ldquoDNA barcoding Australiarsquos fish speciesrdquo PhilosophicalTransactions of the Royal Society B vol 360 no 1462 pp 1847ndash1857 2005

[40] K Tamura J Dudley M Nei and S Kumar ldquoMEGA4 Molec-ular Evolutionary Genetics Analysis (MEGA) software version40rdquo Molecular Biology and Evolution vol 24 no 8 pp 1596ndash1599 2007

[41] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997

[42] K S Shafer T Hanekamp K H White and P E ThorsnessldquoMechanisms of mitochondrial DNA escape to the nucleus inthe yeast Saccharomyces cerevisiaerdquo Current Genetics vol 36no 4 pp 183ndash194 1999

[43] D Bensasson D-X Zhang D L Hartl and G M HewittldquoMitochondrial pseudogenes evolutionrsquos misplaced witnessesrdquoTrends in Ecology and Evolution vol 16 no 6 pp 314ndash321 2001

[44] P Librado and J Rozas ldquoDnaSP v5 a software for comprehen-sive analysis of DNA polymorphism datardquo Bioinformatics vol25 no 11 pp 1451ndash1452 2009

[45] M Kimura ldquoA simple method for estimating evolutionary ratesof base substitutions through comparative studies of nucleotidesequencesrdquo Journal ofMolecular Evolution vol 16 no 2 pp 111ndash120 1980

[46] J P Huelsenbeck and F Ronquist ldquoMRBAYES Bayesian infer-ence of phylogenetic treesrdquo Bioinformatics vol 17 no 8 pp754ndash755 2001

[47] M Clement D Posada and K A Crandall ldquoTCS a computerprogram to estimate gene genealogiesrdquo Molecular Ecology vol9 no 10 pp 1657ndash1659 2000

[48] L Excoffier G Laval and S Schneider ldquoArlequin ver 30an integrated software package for population genetics dataanalysisrdquo Evolutionary Bioinformatics Online vol 1 pp 47ndash502005

[49] A K M Mohsin and M A Ambak Freshwater Fish in Penin-sular Malaysia Dewan Bahasa dan Pustaka Kuala LumpurMalaysia 1991


[50] D Bickford D J Lohman N S Sodhi et al ldquoCryptic species asa window on diversity and conservationrdquo Trends in Ecology andEvolution vol 22 no 3 pp 148ndash155 2007

[51] Y Salah and S Bhassu ldquoNovel microsatellites from partialgenomic library and population structure of wild Channastriata (Bloch 1793)rdquoMolecular Biology Report 2010

[52] J P Wang K C Hsu and T Y Chiang ldquoMitochondrial DNAphylogeography of Acrossocheilus paradoxus (Cyprinidae) inTaiwanrdquoMolecular Ecology vol 9 no 10 pp 1483ndash1494 2000

[53] W Supiwong P Jearranaiprepame and A Tanomtong ldquoA newreport of karyotype in the chevron snakehead fish Channa stri-ata (Channidae Pisces) from Northeast Thailandrdquo Cytologiavol 74 no 3 pp 317ndash322 2009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


of C striata surveyed in Perak Malaysia was adduced toa historical Pleistocene event [11] strongly suggesting thecoalescence of freshwater taxa The Pleistocene event mayexplain the most historical coalescence of a species wherebymultiple episodes of glaciations and deglaciations occurredwith the accompanied of lowering and rising in sea waterlevel happened approximately 2 million years ago HistoricalPleistocene glaciations with themaximum lowering sea waterlevel greatly affected the land mass configuration and thusthe biodiversity where PeninsularMalaysia island of BorneoSouthern Thailand Southern Indo china Sumatera andJava were interconnected [12] The land bridge through theformation of Sunda River permitted the migration and geneflow of fauna between Peninsular Malaysia and the island ofBorneo as the sea-water level rose that allowed submersionof this river system during the last Pleistocene Epoch [113] This phenomenon is shown by the genetic divergencepattern of Hampala macrolepidota [14] Tor tambroides [15]and Barbonymus schwanenfeldii [16] with the witness ofnonsignificant genetic differentiation and sharing of the samehaplotype between Peninsular Malaysia and the island ofBorneo

Environmental factors were recognized as an additivedeterminant on the phenotype of an organism Human activ-ities such as construction of dwellings that often involve forestdestruction leading to sedimentation in the river coupledwith biotic environmental factors such as food competitionas a whole will influence the trait development for survivalfitness The physical changes as a response to pollutedaquatic habitats were shown in previous studies in relation todeformity of fish morphology [17] deformation [18 19] andfin erosion [20 21] The study of Roberts and Khaironizam[22] revealed that the mouth polymorphism ofNeolissochilussoroides due to feeding behavior had occurred at sympatricpopulation This in turn led us to further investigate thepattern of phenotypic variation among allopatric populationsof C striata across Malaysia

In this study we adopted a mitochondrial cytochrome119888 oxidase subunit 1 (COI) gene as a genetic marker toassess the genotypic variationThis genersquos comparatively slowmutation rate in relation to microsatellite markers and itsmaternal inheritancemakes it possible to reveal any historicalpopulation dispersal pattern of a species A population fromSarawak was included as a representative of the islandof Borneo to test the hypothesis of Pleistocene event ongenotypic divergence pattern ofC striataThemorphologicaldivergence pattern will be assessed through Truss NetworkMeasurement [23] Understanding the correlations betweenthe genotypic and phenotypic divergence pattern would beinformative to conservationists in proposing conservationassessments The aim of this study is to characterize the pat-tern of population divergence of C striata in order to providea spatial conservation planning in different localities andinvestigate the possible hidden cryptic taxa of C striata usinga molecular species identification approach-DNA bar codingthat was introduced byHebert et al [24]Thismethod for tax-onomic identification avoids potential confusion surround-ing accurate classification of discriminating morphologicalcharacters by focusing directly on the molecular level [25]

and has been used as a universal method that is applicableacross all eukaryote taxonomic groups [23 26ndash32] In relationto this study mitochondrial (mtDNA) gene cytochrome 119888oxidase (COI) is the marker of choice [24] Focusing on thisstandardized short sequence of DNA aims to benefit the tax-onomist by assisting in the classification of known species aswell as by identifying cryptic diversity potentially leading tothe discovery of new undescribed taxa [26 33]This approachappears to have been successful in differentiating amongspecies within a range of animal groups [28 34 35] includingfreshwater andmarine fishes [36 37] In this study where thelevel of interspecies divergence will be taken into accountadditional freshwater fishes of 26 fish species were chosenand where possible multiple individuals of each species werecollected to examine diversity in the COI region at theinterspecific and intraspecific level Our suspected cryptictaxa ofC striatawas sampled extensively across its geograph-ical range in Malaysia in order to represent the extent ofphylogeographical divergence that is likely to be present forthis taxon across the region This widely distributed taxon isthought to comprise a species complex [9] and has recentlybeen found to occur in genetically divergent forms in SE Asia[10] and therefore is of particular taxonomic interest as it mayshow higher than average intraspecific divergence

2 Materials and Methods

A total of 220 specimens of C striata representing eightlocations of Kedah Pulau Pinang Selangor Negeri SembilanJohor Pahang Terengganu and Sarawak were collected andused in this study Samples from Peninsular Malaysia werecaught by simple fishing methods such as nets and samplesfrom Sarawak were purchased from local market These fishsamples were collected by Freshwater Fisheries ResearchDivision of Jelebu (Department of Fisheries Malaysia Fresh-water Fisheries Research Division FRI Glami Lemi JelebuTiti Negeri Sembilan Malaysia) Collection was made from8 November 2006 to 27 August 2007 The samples fromSarawak were purchased by the author on 26 March 2011In the case of DNA bar coding approach an additional 83fishes were sampled at Pahang (Kuala Mai and Lubuk Paku)Selangor (Kepong) and Perak (Temenggor) representingmembers of 26 previously described species from 23 genera7 families and 3 orders (see Figure 1 for sampling locationand details) Multiple individuals per species were collectedwhere possible to provide some indication of the levelof intraspecific variation present in the DNA bar codingfragment In particular 43 C striata individuals with four toten individuals as representatives from each eight sites acrossa wide geographical range in Malaysia were considered inDNA bar coding to provide an estimate of the likely extent ofintraspecific divergence present in this species and to investi-gate possible cryptic taxa that might occur across Malaysia(see Table 1 for selected fishes used in DNA bar coding)These 43 C striata samples were also genetically surveyedfor population study in the case of examining amount ofgene flow and genetic differentiation All additional 83 fisheswere caught by electrofishing and all the collected samples


Malaysia

N

W E

S100 km



Malaysia

N

W E

S100 km





119887





















Siluriformes










a1

a2a3 a4

a5

a6a7

a8a10 a9a11a12

b1b4

c2c3

d1

d2

e1e2 f1

f2

g1g2 g3

g4

tl

sl

ed

b2b3

c1

cfl

(a)

h1

h2

h3

SnW

SnL

(b)















3 Results









Malaysia

100 km

Malaysia

100 km

N

W E

S

(a)

H 1

H 4

H 2

H 3 H 6

H 5

H 7

(b)

Taiping

0 100

(km)

Kelang

Northwest

Northeastand

central division

division

South ChinaSea

KualaRompin

Southerndivision

N

(c)





Table3Pairw

isegenetic

K2Pdistancesa

mon

gtaxaIntraspecificv

aluessho

wnin

bold

andmarkedby

asterix

where

multip

ledistancesw

erep

resent


haplotypes

the

largestvalue

isshow

n[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27]

[1]B

schw

anenfeldiilowast

010

[2]A

truncatus

1130lowast

010

[3]B

laevis

1480

1450lowast

010

[4]Ovittatus

1740

1840

1650lowast

010

[5]Hw

etmorei

1110

1180

1150

1570lowast

000

[6]Pproctozystron

1040

930

1370

1840

1070lowast

000

[7]Hm

acrolep

idota

1850

1880

1780

2030

1510

1650lowast

050

[8]Lsetigerum

2030

1950

2040

2200

1880

1870

2130lowast

020

[9]B

yarrelli

2380

2310

2240

2660

2260

2470

2700

2750lowast

000

[10]R

sumatrana

1990

1850

1790

2120

1880

1930

2190

2140

2300lowast

000

[11]Pmicronem

us2350

2400

2380

2590

2440

2380

2470

2410

1800

2350lowast

000

[12]Pjullieni

1430

1480

1360

1440

1190

1250

1630

2160

2210

2130

2450lowast

NA

[13]Pna

sutus

2410

2230

2340

2500

2400

2350

2400

2390

1800

2180

800

2530lowast

030

[14]Tthynnoides

1430

1490

1290

1540

1420

1270

1740

1880

2310

1960

2310

1490

2130lowast

000

[15]Hw

yckii

2510

2140

2290

2660

2380

2400

2440

2440

1930

2430

1720

2520

1770

2410lowast

000

[16]C

caud

imaculatus

1360

1590

1250

1630

1510

1370

2070

2020

2380

1720

2230

1680

2250

1240

2280lowast

000

[17]C

apogon

1270

1130

1290

1820

1160

1040

1770

1830

2250

1840

2180

1360

2310

1380

2420

1320lowast

050

[18]A

testu

dineus

2470

2620

2520

2770

2570

2670

2870

2500

2990

2540

2580

2520

2460

2420

2620

2250

2610lowast

NA

[19]B

dinema

2360

2560

2460

2580

2350

2510

2560

2530

2100

2330

2220

2660

2140

2580

2410

2260

2470

2630lowast

000

[20]Pbu

lu1110

990

1460

1910

1220

630

1720

1860

2420

1900

2280

1450

2200

1200

2260

1380

1030

2490

2640lowast

050

[21]C

striata

2450

2730

2290

2530

2610

2640

2780

2550

2440

2610

2340

2650

2370

2200

2250

2520

2580

2530

2340

2550lowast

060

[22]R

pavian

a1970

1850

1790

2160

1820

1900

2000

1870

2420

1430

2240

1980

2090

1810

2400

1650

1760

2620

2460

1800

2540lowast

090

[23]Hnem

urus

2360

2360

2220

2470

2230

2400

2600

2540

2020

2510

1540

2260

1790

2380

1680

2350

2240

3040

2410

2440

2400

2540lowast

000

[24]Dregina

2750

2450

2640

2900

2640

2590

2850

3120

3030

2880

3020

2760

3220

2820

3330

2950

2790

3400

3620

2780

3270

2820

3140lowast

000

[25]C

luciu

s2210

2100

2180

2230

2070

2220

2360

2110

2460

2380

2420

2430

2390

2170

2310

2100

2260

2530

2310

2200

2530

2220

2420

3110lowast

NA

[26]Pbanksi

1660

1580

1570

1790

1480

1430

1770

1760

2560

1960

2280

1720

2060

1450

2420

1410

1460

2670

2350

1490

2400

1820

2380

3060

2340lowast

000

[27]Psm

edley

i1190

1280

1290

1570

1040

1220

1600

1830

2430

1820

2470

1470

2470

1490

2450

1290

1090

2560

2500

1390

2750

1850

2480

2940

2170

1700lowast

000












4 Discussions


















lowast099

99840099

lowast099

99840099

lowast099

99840099

lowast100

99840099

lowast100

99840099

lowast100

99840099

lowast100

99840099

lowast100

99840099

lowast097

99840099

lowast099

99840099

lowast099

99840099

01















0 2 4 6 8 10Root 1

0

2

4

6

8

10

Root

2

Group C

Group B Group A

Group D

minus10 minus8

minus8

minus6

minus6

minus4

minus4

minus2

minus2





A B C D E

0 10 20 30Root 1

0

5

10

15

20

Root

2

minus5

minus10

minus15














5 Conclusion


Acknowledgments



References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Malaysia

N

W E

S100 km



Malaysia

N

W E

S100 km





119887





















Siluriformes










a1

a2a3 a4

a5

a6a7

a8a10 a9a11a12

b1b4

c2c3

d1

d2

e1e2 f1

f2

g1g2 g3

g4

tl

sl

ed

b2b3

c1

cfl

(a)

h1

h2

h3

SnW

SnL

(b)















3 Results









Malaysia

100 km

Malaysia

100 km

N

W E

S

(a)

H 1

H 4

H 2

H 3 H 6

H 5

H 7

(b)

Taiping

0 100

(km)

Kelang

Northwest

Northeastand

central division

division

South ChinaSea

KualaRompin

Southerndivision

N

(c)





Table3Pairw

isegenetic

K2Pdistancesa

mon

gtaxaIntraspecificv

aluessho

wnin

bold

andmarkedby

asterix

where

multip

ledistancesw

erep

resent


haplotypes

the

largestvalue

isshow

n[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27]

[1]B

schw

anenfeldiilowast

010

[2]A

truncatus

1130lowast

010

[3]B

laevis

1480

1450lowast

010

[4]Ovittatus

1740

1840

1650lowast

010

[5]Hw

etmorei

1110

1180

1150

1570lowast

000

[6]Pproctozystron

1040

930

1370

1840

1070lowast

000

[7]Hm

acrolep

idota

1850

1880

1780

2030

1510

1650lowast

050

[8]Lsetigerum

2030

1950

2040

2200

1880

1870

2130lowast

020

[9]B

yarrelli

2380

2310

2240

2660

2260

2470

2700

2750lowast

000

[10]R

sumatrana

1990

1850

1790

2120

1880

1930

2190

2140

2300lowast

000

[11]Pmicronem

us2350

2400

2380

2590

2440

2380

2470

2410

1800

2350lowast

000

[12]Pjullieni

1430

1480

1360

1440

1190

1250

1630

2160

2210

2130

2450lowast

NA

[13]Pna

sutus

2410

2230

2340

2500

2400

2350

2400

2390

1800

2180

800

2530lowast

030

[14]Tthynnoides

1430

1490

1290

1540

1420

1270

1740

1880

2310

1960

2310

1490

2130lowast

000

[15]Hw

yckii

2510

2140

2290

2660

2380

2400

2440

2440

1930

2430

1720

2520

1770

2410lowast

000

[16]C

caud

imaculatus

1360

1590

1250

1630

1510

1370

2070

2020

2380

1720

2230

1680

2250

1240

2280lowast

000

[17]C

apogon

1270

1130

1290

1820

1160

1040

1770

1830

2250

1840

2180

1360

2310

1380

2420

1320lowast

050

[18]A

testu

dineus

2470

2620

2520

2770

2570

2670

2870

2500

2990

2540

2580

2520

2460

2420

2620

2250

2610lowast

NA

[19]B

dinema

2360

2560

2460

2580

2350

2510

2560

2530

2100

2330

2220

2660

2140

2580

2410

2260

2470

2630lowast

000

[20]Pbu

lu1110

990

1460

1910

1220

630

1720

1860

2420

1900

2280

1450

2200

1200

2260

1380

1030

2490

2640lowast

050

[21]C

striata

2450

2730

2290

2530

2610

2640

2780

2550

2440

2610

2340

2650

2370

2200

2250

2520

2580

2530

2340

2550lowast

060

[22]R

pavian

a1970

1850

1790

2160

1820

1900

2000

1870

2420

1430

2240

1980

2090

1810

2400

1650

1760

2620

2460

1800

2540lowast

090

[23]Hnem

urus

2360

2360

2220

2470

2230

2400

2600

2540

2020

2510

1540

2260

1790

2380

1680

2350

2240

3040

2410

2440

2400

2540lowast

000

[24]Dregina

2750

2450

2640

2900

2640

2590

2850

3120

3030

2880

3020

2760

3220

2820

3330

2950

2790

3400

3620

2780

3270

2820

3140lowast

000

[25]C

luciu

s2210

2100

2180

2230

2070

2220

2360

2110

2460

2380

2420

2430

2390

2170

2310

2100

2260

2530

2310

2200

2530

2220

2420

3110lowast

NA

[26]Pbanksi

1660

1580

1570

1790

1480

1430

1770

1760

2560

1960

2280

1720

2060

1450

2420

1410

1460

2670

2350

1490

2400

1820

2380

3060

2340lowast

000

[27]Psm

edley

i1190

1280

1290

1570

1040

1220

1600

1830

2430

1820

2470

1470

2470

1490

2450

1290

1090

2560

2500

1390

2750

1850

2480

2940

2170

1700lowast

000












4 Discussions


















lowast099

99840099

lowast099

99840099

lowast099

99840099

lowast100

99840099

lowast100

99840099

lowast100

99840099

lowast100

99840099

lowast100

99840099

lowast097

99840099

lowast099

99840099

lowast099

99840099

01















0 2 4 6 8 10Root 1

0

2

4

6

8

10

Root

2

Group C

Group B Group A

Group D

minus10 minus8

minus8

minus6

minus6

minus4

minus4

minus2

minus2





A B C D E

0 10 20 30Root 1

0

5

10

15

20

Root

2

minus5

minus10

minus15














5 Conclusion


Acknowledgments



References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














Siluriformes










a1

a2a3 a4

a5

a6a7

a8a10 a9a11a12

b1b4

c2c3

d1

d2

e1e2 f1

f2

g1g2 g3

g4

tl

sl

ed

b2b3

c1

cfl

(a)

h1

h2

h3

SnW

SnL

(b)















3 Results









Malaysia

100 km

Malaysia

100 km

N

W E

S

(a)

H 1

H 4

H 2

H 3 H 6

H 5

H 7

(b)

Taiping

0 100

(km)

Kelang

Northwest

Northeastand

central division

division

South ChinaSea

KualaRompin

Southerndivision

N

(c)





Table3Pairw

isegenetic

K2Pdistancesa

mon

gtaxaIntraspecificv

aluessho

wnin

bold

andmarkedby

asterix

where

multip

ledistancesw

erep

resent


haplotypes

the

largestvalue

isshow

n[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27]

[1]B

schw

anenfeldiilowast

010

[2]A

truncatus

1130lowast

010

[3]B

laevis

1480

1450lowast

010

[4]Ovittatus

1740

1840

1650lowast

010

[5]Hw

etmorei

1110

1180

1150

1570lowast

000

[6]Pproctozystron

1040

930

1370

1840

1070lowast

000

[7]Hm

acrolep

idota

1850

1880

1780

2030

1510

1650lowast

050

[8]Lsetigerum

2030

1950

2040

2200

1880

1870

2130lowast

020

[9]B

yarrelli

2380

2310

2240

2660

2260

2470

2700

2750lowast

000

[10]R

sumatrana

1990

1850

1790

2120

1880

1930

2190

2140

2300lowast

000

[11]Pmicronem

us2350

2400

2380

2590

2440

2380

2470

2410

1800

2350lowast

000

[12]Pjullieni

1430

1480

1360

1440

1190

1250

1630

2160

2210

2130

2450lowast

NA

[13]Pna

sutus

2410

2230

2340

2500

2400

2350

2400

2390

1800

2180

800

2530lowast

030

[14]Tthynnoides

1430

1490

1290

1540

1420

1270

1740

1880

2310

1960

2310

1490

2130lowast

000

[15]Hw

yckii

2510

2140

2290

2660

2380

2400

2440

2440

1930

2430

1720

2520

1770

2410lowast

000

[16]C

caud

imaculatus

1360

1590

1250

1630

1510

1370

2070

2020

2380

1720

2230

1680

2250

1240

2280lowast

000

[17]C

apogon

1270

1130

1290

1820

1160

1040

1770

1830

2250

1840

2180

1360

2310

1380

2420

1320lowast

050

[18]A

testu

dineus

2470

2620

2520

2770

2570

2670

2870

2500

2990

2540

2580

2520

2460

2420

2620

2250

2610lowast

NA

[19]B

dinema

2360

2560

2460

2580

2350

2510

2560

2530

2100

2330

2220

2660

2140

2580

2410

2260

2470

2630lowast

000

[20]Pbu

lu1110

990

1460

1910

1220

630

1720

1860

2420

1900

2280

1450

2200

1200

2260

1380

1030

2490

2640lowast

050

[21]C

striata

2450

2730

2290

2530

2610

2640

2780

2550

2440

2610

2340

2650

2370

2200

2250

2520

2580

2530

2340

2550lowast

060

[22]R

pavian

a1970

1850

1790

2160

1820

1900

2000

1870

2420

1430

2240

1980

2090

1810

2400

1650

1760

2620

2460

1800

2540lowast

090

[23]Hnem

urus

2360

2360

2220

2470

2230

2400

2600

2540

2020

2510

1540

2260

1790

2380

1680

2350

2240

3040

2410

2440

2400

2540lowast

000

[24]Dregina

2750

2450

2640

2900

2640

2590

2850

3120

3030

2880

3020

2760

3220

2820

3330

2950

2790

3400

3620

2780

3270

2820

3140lowast

000

[25]C

luciu

s2210

2100

2180

2230

2070

2220

2360

2110

2460

2380

2420

2430

2390

2170

2310

2100

2260

2530

2310

2200

2530

2220

2420

3110lowast

NA

[26]Pbanksi

1660

1580

1570

1790

1480

1430

1770

1760

2560

1960

2280

1720

2060

1450

2420

1410

1460

2670

2350

1490

2400

1820

2380

3060

2340lowast

000

[27]Psm

edley

i1190

1280

1290

1570

1040

1220

1600

1830

2430

1820

2470

1470

2470

1490

2450

1290

1090

2560

2500

1390

2750

1850

2480

2940

2170

1700lowast

000












4 Discussions


















lowast099

99840099

lowast099

99840099

lowast099

99840099

lowast100

99840099

lowast100

99840099

lowast100

99840099

lowast100

99840099

lowast100

99840099

lowast097

99840099

lowast099

99840099

lowast099

99840099

01















0 2 4 6 8 10Root 1

0

2

4

6

8

10

Root

2

Group C

Group B Group A

Group D

minus10 minus8

minus8

minus6

minus6

minus4

minus4

minus2

minus2





A B C D E

0 10 20 30Root 1

0

5

10

15

20

Root

2

minus5

minus10

minus15














5 Conclusion


Acknowledgments



References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


a1

a2a3 a4

a5

a6a7

a8a10 a9a11a12

b1b4

c2c3

d1

d2

e1e2 f1

f2

g1g2 g3

g4

tl

sl

ed

b2b3

c1

cfl

(a)

h1

h2

h3

SnW

SnL

(b)















3 Results









Malaysia

100 km

Malaysia

100 km

N

W E

S

(a)

H 1

H 4

H 2

H 3 H 6

H 5

H 7

(b)

Taiping

0 100

(km)

Kelang

Northwest

Northeastand

central division

division

South ChinaSea

KualaRompin

Southerndivision

N

(c)





Table3Pairw

isegenetic

K2Pdistancesa

mon

gtaxaIntraspecificv

aluessho

wnin

bold

andmarkedby

asterix

where

multip

ledistancesw

erep

resent


haplotypes

the

largestvalue

isshow

n[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27]

[1]B

schw

anenfeldiilowast

010

[2]A

truncatus

1130lowast

010

[3]B

laevis

1480

1450lowast

010

[4]Ovittatus

1740

1840

1650lowast

010

[5]Hw

etmorei

1110

1180

1150

1570lowast

000

[6]Pproctozystron

1040

930

1370

1840

1070lowast

000

[7]Hm

acrolep

idota

1850

1880

1780

2030

1510

1650lowast

050

[8]Lsetigerum

2030

1950

2040

2200

1880

1870

2130lowast

020

[9]B

yarrelli

2380

2310

2240

2660

2260

2470

2700

2750lowast

000

[10]R

sumatrana

1990

1850

1790

2120

1880

1930

2190

2140

2300lowast

000

[11]Pmicronem

us2350

2400

2380

2590

2440

2380

2470

2410

1800

2350lowast

000

[12]Pjullieni

1430

1480

1360

1440

1190

1250

1630

2160

2210

2130

2450lowast

NA

[13]Pna

sutus

2410

2230

2340

2500

2400

2350

2400

2390

1800

2180

800

2530lowast

030

[14]Tthynnoides

1430

1490

1290

1540

1420

1270

1740

1880

2310

1960

2310

1490

2130lowast

000

[15]Hw

yckii

2510

2140

2290

2660

2380

2400

2440

2440

1930

2430

1720

2520

1770

2410lowast

000

[16]C

caud

imaculatus

1360

1590

1250

1630

1510

1370

2070

2020

2380

1720

2230

1680

2250

1240

2280lowast

000

[17]C

apogon

1270

1130

1290

1820

1160

1040

1770

1830

2250

1840

2180

1360

2310

1380

2420

1320lowast

050

[18]A

testu

dineus

2470

2620

2520

2770

2570

2670

2870

2500

2990

2540

2580

2520

2460

2420

2620

2250

2610lowast

NA

[19]B

dinema

2360

2560

2460

2580

2350

2510

2560

2530

2100

2330

2220

2660

2140

2580

2410

2260

2470

2630lowast

000

[20]Pbu

lu1110

990

1460

1910

1220

630

1720

1860

2420

1900

2280

1450

2200

1200

2260

1380

1030

2490

2640lowast

050

[21]C

striata

2450

2730

2290

2530

2610

2640

2780

2550

2440

2610

2340

2650

2370

2200

2250

2520

2580

2530

2340

2550lowast

060

[22]R

pavian

a1970

1850

1790

2160

1820

1900

2000

1870

2420

1430

2240

1980

2090

1810

2400

1650

1760

2620

2460

1800

2540lowast

090

[23]Hnem

urus

2360

2360

2220

2470

2230

2400

2600

2540

2020

2510

1540

2260

1790

2380

1680

2350

2240

3040

2410

2440

2400

2540lowast

000

[24]Dregina

2750

2450

2640

2900

2640

2590

2850

3120

3030

2880

3020

2760

3220

2820

3330

2950

2790

3400

3620

2780

3270

2820

3140lowast

000

[25]C

luciu

s2210

2100

2180

2230

2070

2220

2360

2110

2460

2380

2420

2430

2390

2170

2310

2100

2260

2530

2310

2200

2530

2220

2420

3110lowast

NA

[26]Pbanksi

1660

1580

1570

1790

1480

1430

1770

1760

2560

1960

2280

1720

2060

1450

2420

1410

1460

2670

2350

1490

2400

1820

2380

3060

2340lowast

000

[27]Psm

edley

i1190

1280

1290

1570

1040

1220

1600

1830

2430

1820

2470

1470

2470

1490

2450

1290

1090

2560

2500

1390

2750

1850

2480

2940

2170

1700lowast

000












4 Discussions


















lowast099

99840099

lowast099

99840099

lowast099

99840099

lowast100

99840099

lowast100

99840099

lowast100

99840099

lowast100

99840099

lowast100

99840099

lowast097

99840099

lowast099

99840099

lowast099

99840099

01















0 2 4 6 8 10Root 1

0

2

4

6

8

10

Root

2

Group C

Group B Group A

Group D

minus10 minus8

minus8

minus6

minus6

minus4

minus4

minus2

minus2





A B C D E

0 10 20 30Root 1

0

5

10

15

20

Root

2

minus5

minus10

minus15














5 Conclusion


Acknowledgments



References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










3 Results









Malaysia

100 km

Malaysia

100 km

N

W E

S

(a)

H 1

H 4

H 2

H 3 H 6

H 5

H 7

(b)

Taiping

0 100

(km)

Kelang

Northwest

Northeastand

central division

division

South ChinaSea

KualaRompin

Southerndivision

N

(c)





Table3Pairw

isegenetic

K2Pdistancesa

mon

gtaxaIntraspecificv

aluessho

wnin

bold

andmarkedby

asterix

where

multip

ledistancesw

erep

resent


haplotypes

the

largestvalue

isshow

n[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27]

[1]B

schw

anenfeldiilowast

010

[2]A

truncatus

1130lowast

010

[3]B

laevis

1480

1450lowast

010

[4]Ovittatus

1740

1840

1650lowast

010

[5]Hw

etmorei

1110

1180

1150

1570lowast

000

[6]Pproctozystron

1040

930

1370

1840

1070lowast

000

[7]Hm

acrolep

idota

1850

1880

1780

2030

1510

1650lowast

050

[8]Lsetigerum

2030

1950

2040

2200

1880

1870

2130lowast

020

[9]B

yarrelli

2380

2310

2240

2660

2260

2470

2700

2750lowast

000

[10]R

sumatrana

1990

1850

1790

2120

1880

1930

2190

2140

2300lowast

000

[11]Pmicronem

us2350

2400

2380

2590

2440

2380

2470

2410

1800

2350lowast

000

[12]Pjullieni

1430

1480

1360

1440

1190

1250

1630

2160

2210

2130

2450lowast

NA

[13]Pna

sutus

2410

2230

2340

2500

2400

2350

2400

2390

1800

2180

800

2530lowast

030

[14]Tthynnoides

1430

1490

1290

1540

1420

1270

1740

1880

2310

1960

2310

1490

2130lowast

000

[15]Hw

yckii

2510

2140

2290

2660

2380

2400

2440

2440

1930

2430

1720

2520

1770

2410lowast

000

[16]C

caud

imaculatus

1360

1590

1250

1630

1510

1370

2070

2020

2380

1720

2230

1680

2250

1240

2280lowast

000

[17]C

apogon

1270

1130

1290

1820

1160

1040

1770

1830

2250

1840

2180

1360

2310

1380

2420

1320lowast

050

[18]A

testu

dineus

2470

2620

2520

2770

2570

2670

2870

2500

2990

2540

2580

2520

2460

2420

2620

2250

2610lowast

NA

[19]B

dinema

2360

2560

2460

2580

2350

2510

2560

2530

2100

2330

2220

2660

2140

2580

2410

2260

2470

2630lowast

000

[20]Pbu

lu1110

990

1460

1910

1220

630

1720

1860

2420

1900

2280

1450

2200

1200

2260

1380

1030

2490

2640lowast

050

[21]C

striata

2450

2730

2290

2530

2610

2640

2780

2550

2440

2610

2340

2650

2370

2200

2250

2520

2580

2530

2340

2550lowast

060

[22]R

pavian

a1970

1850

1790

2160

1820

1900

2000

1870

2420

1430

2240

1980

2090

1810

2400

1650

1760

2620

2460

1800

2540lowast

090

[23]Hnem

urus

2360

2360

2220

2470

2230

2400

2600

2540

2020

2510

1540

2260

1790

2380

1680

2350

2240

3040

2410

2440

2400

2540lowast

000

[24]Dregina

2750

2450

2640

2900

2640

2590

2850

3120

3030

2880

3020

2760

3220

2820

3330

2950

2790

3400

3620

2780

3270

2820

3140lowast

000

[25]C

luciu

s2210

2100

2180

2230

2070

2220

2360

2110

2460

2380

2420

2430

2390

2170

2310

2100

2260

2530

2310

2200

2530

2220

2420

3110lowast

NA

[26]Pbanksi

1660

1580

1570

1790

1480

1430

1770

1760

2560

1960

2280

1720

2060

1450

2420

1410

1460

2670

2350

1490

2400

1820

2380

3060

2340lowast

000

[27]Psm

edley

i1190

1280

1290

1570

1040

1220

1600

1830

2430

1820

2470

1470

2470

1490

2450

1290

1090

2560

2500

1390

2750

1850

2480

2940

2170

1700lowast

000












4 Discussions


















lowast099

99840099

lowast099

99840099

lowast099

99840099

lowast100

99840099

lowast100

99840099

lowast100

99840099

lowast100

99840099

lowast100

99840099

lowast097

99840099

lowast099

99840099

lowast099

99840099

01















0 2 4 6 8 10Root 1

0

2

4

6

8

10

Root

2

Group C

Group B Group A

Group D

minus10 minus8

minus8

minus6

minus6

minus4

minus4

minus2

minus2





A B C D E

0 10 20 30Root 1

0

5

10

15

20

Root

2

minus5

minus10

minus15














5 Conclusion


Acknowledgments



References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Malaysia

100 km

Malaysia

100 km

N

W E

S

(a)

H 1

H 4

H 2

H 3 H 6

H 5

H 7

(b)

Taiping

0 100

(km)

Kelang

Northwest

Northeastand

central division

division

South ChinaSea

KualaRompin

Southerndivision

N

(c)





Table3Pairw

isegenetic

K2Pdistancesa

mon

gtaxaIntraspecificv

aluessho

wnin

bold

andmarkedby

asterix

where

multip

ledistancesw

erep

resent


haplotypes

the

largestvalue

isshow

n[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27]

[1]B

schw

anenfeldiilowast

010

[2]A

truncatus

1130lowast

010

[3]B

laevis

1480

1450lowast

010

[4]Ovittatus

1740

1840

1650lowast

010

[5]Hw

etmorei

1110

1180

1150

1570lowast

000

[6]Pproctozystron

1040

930

1370

1840

1070lowast

000

[7]Hm

acrolep

idota

1850

1880

1780

2030

1510

1650lowast

050

[8]Lsetigerum

2030

1950

2040

2200

1880

1870

2130lowast

020

[9]B

yarrelli

2380

2310

2240

2660

2260

2470

2700

2750lowast

000

[10]R

sumatrana

1990

1850

1790

2120

1880

1930

2190

2140

2300lowast

000

[11]Pmicronem

us2350

2400

2380

2590

2440

2380

2470

2410

1800

2350lowast

000

[12]Pjullieni

1430

1480

1360

1440

1190

1250

1630

2160

2210

2130

2450lowast

NA

[13]Pna

sutus

2410

2230

2340

2500

2400

2350

2400

2390

1800

2180

800

2530lowast

030

[14]Tthynnoides

1430

1490

1290

1540

1420

1270

1740

1880

2310

1960

2310

1490

2130lowast

000

[15]Hw

yckii

2510

2140

2290

2660

2380

2400

2440

2440

1930

2430

1720

2520

1770

2410lowast

000

[16]C

caud

imaculatus

1360

1590

1250

1630

1510

1370

2070

2020

2380

1720

2230

1680

2250

1240

2280lowast

000

[17]C

apogon

1270

1130

1290

1820

1160

1040

1770

1830

2250

1840

2180

1360

2310

1380

2420

1320lowast

050

[18]A

testu

dineus

2470

2620

2520

2770

2570

2670

2870

2500

2990

2540

2580

2520

2460

2420

2620

2250

2610lowast

NA

[19]B

dinema

2360

2560

2460

2580

2350

2510

2560

2530

2100

2330

2220

2660

2140

2580

2410

2260

2470

2630lowast

000

[20]Pbu

lu1110

990

1460

1910

1220

630

1720

1860

2420

1900

2280

1450

2200

1200

2260

1380

1030

2490

2640lowast

050

[21]C

striata

2450

2730

2290

2530

2610

2640

2780

2550

2440

2610

2340

2650

2370

2200

2250

2520

2580

2530

2340

2550lowast

060

[22]R

pavian

a1970

1850

1790

2160

1820

1900

2000

1870

2420

1430

2240

1980

2090

1810

2400

1650

1760

2620

2460

1800

2540lowast

090

[23]Hnem

urus

2360

2360

2220

2470

2230

2400

2600

2540

2020

2510

1540

2260

1790

2380

1680

2350

2240

3040

2410

2440

2400

2540lowast

000

[24]Dregina

2750

2450

2640

2900

2640

2590

2850

3120

3030

2880

3020

2760

3220

2820

3330

2950

2790

3400

3620

2780

3270

2820

3140lowast

000

[25]C

luciu

s2210

2100

2180

2230

2070

2220

2360

2110

2460

2380

2420

2430

2390

2170

2310

2100

2260

2530

2310

2200

2530

2220

2420

3110lowast

NA

[26]Pbanksi

1660

1580

1570

1790

1480

1430

1770

1760

2560

1960

2280

1720

2060

1450

2420

1410

1460

2670

2350

1490

2400

1820

2380

3060

2340lowast

000

[27]Psm

edley

i1190

1280

1290

1570

1040

1220

1600

1830

2430

1820

2470

1470

2470

1490

2450

1290

1090

2560

2500

1390

2750

1850

2480

2940

2170

1700lowast

000












4 Discussions


















lowast099

99840099

lowast099

99840099

lowast099

99840099

lowast100

99840099

lowast100

99840099

lowast100

99840099

lowast100

99840099

lowast100

99840099

lowast097

99840099

lowast099

99840099

lowast099

99840099

01















0 2 4 6 8 10Root 1

0

2

4

6

8

10

Root

2

Group C

Group B Group A

Group D

minus10 minus8

minus8

minus6

minus6

minus4

minus4

minus2

minus2





A B C D E

0 10 20 30Root 1

0

5

10

15

20

Root

2

minus5

minus10

minus15














5 Conclusion


Acknowledgments



References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table3Pairw

isegenetic

K2Pdistancesa

mon

gtaxaIntraspecificv

aluessho

wnin

bold

andmarkedby

asterix

where

multip

ledistancesw

erep

resent


haplotypes

the

largestvalue

isshow

n[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27]

[1]B

schw

anenfeldiilowast

010

[2]A

truncatus

1130lowast

010

[3]B

laevis

1480

1450lowast

010

[4]Ovittatus

1740

1840

1650lowast

010

[5]Hw

etmorei

1110

1180

1150

1570lowast

000

[6]Pproctozystron

1040

930

1370

1840

1070lowast

000

[7]Hm

acrolep

idota

1850

1880

1780

2030

1510

1650lowast

050

[8]Lsetigerum

2030

1950

2040

2200

1880

1870

2130lowast

020

[9]B

yarrelli

2380

2310

2240

2660

2260

2470

2700

2750lowast

000

[10]R

sumatrana

1990

1850

1790

2120

1880

1930

2190

2140

2300lowast

000

[11]Pmicronem

us2350

2400

2380

2590

2440

2380

2470

2410

1800

2350lowast

000

[12]Pjullieni

1430

1480

1360

1440

1190

1250

1630

2160

2210

2130

2450lowast

NA

[13]Pna

sutus

2410

2230

2340

2500

2400

2350

2400

2390

1800

2180

800

2530lowast

030

[14]Tthynnoides

1430

1490

1290

1540

1420

1270

1740

1880

2310

1960

2310

1490

2130lowast

000

[15]Hw

yckii

2510

2140

2290

2660

2380

2400

2440

2440

1930

2430

1720

2520

1770

2410lowast

000

[16]C

caud

imaculatus

1360

1590

1250

1630

1510

1370

2070

2020

2380

1720

2230

1680

2250

1240

2280lowast

000

[17]C

apogon

1270

1130

1290

1820

1160

1040

1770

1830

2250

1840

2180

1360

2310

1380

2420

1320lowast

050

[18]A

testu

dineus

2470

2620

2520

2770

2570

2670

2870

2500

2990

2540

2580

2520

2460

2420

2620

2250

2610lowast

NA

[19]B

dinema

2360

2560

2460

2580

2350

2510

2560

2530

2100

2330

2220

2660

2140

2580

2410

2260

2470

2630lowast

000

[20]Pbu

lu1110

990

1460

1910

1220

630

1720

1860

2420

1900

2280

1450

2200

1200

2260

1380

1030

2490

2640lowast

050

[21]C

striata

2450

2730

2290

2530

2610

2640

2780

2550

2440

2610

2340

2650

2370

2200

2250

2520

2580

2530

2340

2550lowast

060

[22]R

pavian

a1970

1850

1790

2160

1820

1900

2000

1870

2420

1430

2240

1980

2090

1810

2400

1650

1760

2620

2460

1800

2540lowast

090

[23]Hnem

urus

2360

2360

2220

2470

2230

2400

2600

2540

2020

2510

1540

2260

1790

2380

1680

2350

2240

3040

2410

2440

2400

2540lowast

000

[24]Dregina

2750

2450

2640

2900

2640

2590

2850

3120

3030

2880

3020

2760

3220

2820

3330

2950

2790

3400

3620

2780

3270

2820

3140lowast

000

[25]C

luciu

s2210

2100

2180

2230

2070

2220

2360

2110

2460

2380

2420

2430

2390

2170

2310

2100

2260

2530

2310

2200

2530

2220

2420

3110lowast

NA

[26]Pbanksi

1660

1580

1570

1790

1480

1430

1770

1760

2560

1960

2280

1720

2060

1450

2420

1410

1460

2670

2350

1490

2400

1820

2380

3060

2340lowast

000

[27]Psm

edley

i1190

1280

1290

1570

1040

1220

1600

1830

2430

1820

2470

1470

2470

1490

2450

1290

1090

2560

2500

1390

2750

1850

2480

2940

2170

1700lowast

000












4 Discussions


















lowast099

99840099

lowast099

99840099

lowast099

99840099

lowast100

99840099

lowast100

99840099

lowast100

99840099

lowast100

99840099

lowast100

99840099

lowast097

99840099

lowast099

99840099

lowast099

99840099

01















0 2 4 6 8 10Root 1

0

2

4

6

8

10

Root

2

Group C

Group B Group A

Group D

minus10 minus8

minus8

minus6

minus6

minus4

minus4

minus2

minus2





A B C D E

0 10 20 30Root 1

0

5

10

15

20

Root

2

minus5

minus10

minus15














5 Conclusion


Acknowledgments



References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












4 Discussions


















lowast099

99840099

lowast099

99840099

lowast099

99840099

lowast100

99840099

lowast100

99840099

lowast100

99840099

lowast100

99840099

lowast100

99840099

lowast097

99840099

lowast099

99840099

lowast099

99840099

01















0 2 4 6 8 10Root 1

0

2

4

6

8

10

Root

2

Group C

Group B Group A

Group D

minus10 minus8

minus8

minus6

minus6

minus4

minus4

minus2

minus2





A B C D E

0 10 20 30Root 1

0

5

10

15

20

Root

2

minus5

minus10

minus15














5 Conclusion


Acknowledgments



References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















lowast099

99840099

lowast099

99840099

lowast099

99840099

lowast100

99840099

lowast100

99840099

lowast100

99840099

lowast100

99840099

lowast100

99840099

lowast097

99840099

lowast099

99840099

lowast099

99840099

01















0 2 4 6 8 10Root 1

0

2

4

6

8

10

Root

2

Group C

Group B Group A

Group D

minus10 minus8

minus8

minus6

minus6

minus4

minus4

minus2

minus2





A B C D E

0 10 20 30Root 1

0

5

10

15

20

Root

2

minus5

minus10

minus15














5 Conclusion


Acknowledgments



References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












0 2 4 6 8 10Root 1

0

2

4

6

8

10

Root

2

Group C

Group B Group A

Group D

minus10 minus8

minus8

minus6

minus6

minus4

minus4

minus2

minus2





A B C D E

0 10 20 30Root 1

0

5

10

15

20

Root

2

minus5

minus10

minus15














5 Conclusion


Acknowledgments



References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




0 2 4 6 8 10Root 1

0

2

4

6

8

10

Root

2

Group C

Group B Group A

Group D

minus10 minus8

minus8

minus6

minus6

minus4

minus4

minus2

minus2





A B C D E

0 10 20 30Root 1

0

5

10

15

20

Root

2

minus5

minus10

minus15














5 Conclusion


Acknowledgments



References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









5 Conclusion


Acknowledgments



References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


5 Conclusion


Acknowledgments



References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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