A MORPHOLIGICAL AND MOLECULAR PHYLOGENETIC … morpholigical and molecular phylogenetic analysis...

A MORPHOLIGICAL AND MOLECULAR PHYLOGENETIC ANALYSIS OF MALAYSIAN

Kerivoula (Chiroptera: Vespertilionidae)

Noor Haliza binti Hassan @ Ahmad

QL 737 C595 N818 2009

Master of Science 2009

P. KHIDMAT MAKLUMAT AKADEMIK UNIMAI

11111 IIIIINIIIIIIIIN 1000212172 A Morphological and Molecular Phylogenetic Analysis of Malaysian Kerivoula (Chiroptera:

Vespertilionidae)

NOOR HALIZA BINTI HASAN

A thesis submitted in fulfillment of the requirement for the degree of

Master of Science

Faculty of Resource Science and Technology UNIVERSITI MALAYSIA SARAWAK

2009

DECLARATION

I hereby declare that no portion of the work referred to this thesis has been submitted in support

of an application for another degree or qualification to this or any other university or institute of

higher learning.

(Noor Haliza binti Hasan @ Ahmad)

JUNE 2009

1

Acknowledgements

Bismillahhirrahmannirrahim. Alhamdulillah and grace the Allah Almighty to have given me

strength and determination, for have given me the health and faith to finally complete my work

for my master's degree. My deep appreciation goes to my supervisor, Prof. Dr. Mohd Tajuddin

Abdullah (MTA) and co-supervisor, Assoc. Prof. Dr. Edmund Sim for their guidance, advice,

constructive comments, concern and support throughout this study. This study would not have

been possible without various administrative and financial support from UNIMAS. I would like

to thank UNIMAS for granting me the Zamalah UNIMAS 2007/09 scholarship. This study was

also funded by three grants; the Ministry of Science, Technology and Innovation IRPA grant

number 09-02-09-1022-AE001 lead by MTA with co-researchers, Yuzine Esa and Awg Ahmad

Sallehin Awg Husaini; grant FRGS 06(08)6602007(25) and UNIMAS Small Grant Scheme

awarded to MTA. I would also like to thank Sarawak Forestry Corporation and Sarawak

Forestry Department for granting permission with license number 07775 under the State Wild

Life Protection Rules 1998; for research permit number NPW. 907.4.2(II)-54 and permit to enter

park number 45/2007. My thanks also go to the Institute of Biological Diversity (IBD) Krau

Pahang, Department of Wildlife and National Park (DWNP) Kuala Lumpur and Musuem of

Sabah, Kota Kinabalu, Sabah.

I wish to thank members of the Department of Zoology; Mr Besar Ketol, Mr. Isa Sait, Mr. Huzal

Irwan Husin, Mr. Wahab Mami and Mr. Mohd Jalani Mortada for their hard work and

assistances throughout this study. To Molecular Ecology Laboratory (MEL) seniors; Andy Kho

Han Guan, Jayaraj Vijaya Kumaran, Fong Pooi Har and Siti Nurlydia Sazali, I wish to thank

them for being the steady mentors for me since 2005 until the very end of this study. My thanks

It

also go especially for Mr. Faisal Anwarali Khan for sharing his samples, ideas and thoughts

throughout these years, and Ms. Ratnawati Hazali, for being a steady supporter and a big sister

whom I can always turn to for advice. I thank my MEL colleagues; Hung Tze Mau, Roberta

Chaya Tawie Tingga, Eileen Lit, Mohd Ridwan Abd Rahman, Anang Setiawan Achmadi, Sigit

Wiantoro, Muhd Ikhwan Idris, Mohd Fizl Sidq Mohd Ram i, Eric Pui Yong Meng and Nur

Salmizar Azmi for their wonderful friendship, for the great times, shared knowledge and support.

Never forgotten, to my best friends, Shamsyah Hamid, Rohanie Bohan, Ahmad Farizzulkhairi

Ahmad Sobri, Khairul Anwar Othman and to my good friends, Nur Hafizah Azizan, Ida Nivina

Pathe, Aminah Imat and Pang Sing Tyan; thanks for all the wonderful memories, friendship,

support and encouragement.

I would like to thank my beloved parents, Hasan @ Ahmad bin Anis and Jama'yah binti Kedri

for their never ending encouragement, support and prayers. To my siblings, Muhammad Najib

bin Hasan, Muhammad Fakhri bin Hasan, Muhammad Al-Hadi Akmal bin Hasan, Muhammad

Fakhrul Aiman bin Hasan and Muhammad Fakhrul Izzat bin Hasan; always have faith and

always be thankful. And to Omu, thank you for always being there and always being supportive.

111

Abstract

(Morphometric and phylogenetic analyses were done on six species of Kerivoula from Malaysia

(Morphological studies could only be done on five out of the six Kerivoula species available for

this research, namely K. papillosa, K. lenis, K. pellucida, K. hardwickii and K. minutaJ No

sample of K intermedia was available for morphological analysis. (Thirty-one

characters of the

external body, skull and dentition were taken from 47 adult individuals of Kerivoula(Three

separate analyses were done on the morphological data; (1) clustering analysis, (2) principal

component analysis (PCA) and (3) discriminant function analysis (DFA) were applied to the

data)(The findings from all the three analyses supported the groupings of the Kerivoula samples

into six different groups; namely, K minuta, K. hardwickii, K. pellucida, K. lenis and K.

papillos) Cryptic samples of K papillosa were further separated into two types; K. papillosa

type small (K papillosa type S) and K papillosa type large (K papillosa type L). Phylogenetic

analysis was done on six available Kerivoula samples utilising three mitochondrial genes; 409

basepair (bp) of cytochrome b (cyt b), 478 bp of cytochrome oxidase I (COI) and 1044 bp of

NADH dehydrogenase subunit 2 gene (ND2). This was followed by another analysis ustilising

one nuclear gene - 1054 bp of recombinant activating gene subunit 2 (Rag2). The

reconstructions of phylogenetic trees depicting the relationship of Kerivoula were retrieved using

all four inferring methods through three analyses namely, neighbor joining (NJ), maximum

parsimony (MP) and maximum likelihood (ML). All analyses consistently resulted in seven

groups, namely K minuta, K pellucida, K. hardwickii, K. lenis and K. papillosa, with K.

papillosa further separated into two different subgroups which were congruent with those of

morphological analyses, and also the addition of K intermedia samples. The same samples were

used in both analyses and out of 17 samples identified as K papillosa, five were classified as K.

iv

papillosa type L (forearm length of 44.5 mm to 49.0 mm); with one sample from the Madai Cave

in Sabah and the other four from the Niah National Park of Sarawak. Another 12 samples were

classified as K papillosa type S (forearm length of 40.0 mm to 44.5 mm) with their distribution

scattered around Sarawak and Peninsular Malaysia. The separation of these two types of K

papillosa were also supported by a notable genetic distance of >10% which was comparable to

those of biologically different species. This suggested the presence of cryptic species within the

K papillosa groups. It was also noted that K hardwickii samples were separated into the Eastern

and the Western Borneo samples with 100% support of bootstrap for the cyt b and the ND2

genes analyses. The existence of a potential phylogroup was suggested with a genetic distance of

4.6% to 6.0% in the cyt b as >5% was the value proposed by Baker and Bradley (2006) for such

definition. Separation of K minuta samples into two subgroups was also observed using at least

three analyses and the existence of a subspecies was suggested. It was concluded that the

analysis using ND2 gene gave the best tree in depicting the phylogenetic relationship of

Kerivoula. The comparison of the K papillosa type S and the K papillosa type L identified in

this study to the type specimen would justify the taxonomic revision of the cryptic species within

the genus. Population studies of K papillosa, K hardwickii and K minuta were suggested to

further verify the findings of the present study. Further analysis onto both forms of K papillosa

together with the other nine Kerivoula species occurring in Malaysia would provide better

insights into the phylogenetic relationship of genus Kerivoula. (The findings of this study were

expected to aid in the taxonomy and future management and conservation plans for this genus

Keywords: Kerivoula, Malaysia, morphology, molecular phylogenetic, cryptic species

V

Ana/isis morfologi dan filogenetik moleku/ Kerivoula (Chiroptera: Vespertilionidae) darf

Malaysia

Abstrak

Analisis ke alas morfomelrik dan hubungan ftlogenetik telah dijalankan ke alas enam spesies

Kerivoula yang lerdapat di Malaysia. Kajian morfologi hanya dijalankan ke alas lima daripada

enam spesies Kerivoula, iaitu K. papillosa, K. lenis, K. pellucida, K. hardwickii dan K. minula

herikutan liada sampel K. intermedia yang dimiliki semasa kajian dijalankan. Tiga puluh salu

ciri ukuran luar hadan, iengkorak dan gigi telah diambil ke alas seliap 47 individu Kerivoula

yang dewusa dan tiga analisis yang berbeza telah di jalankan ke alas semua data ukuran yang

telah diamhil; (1) "clustering analysis ", (2) analisa "principal component " dan (3) analisa

"discrimirnrnt . /unction". Ketiga-tiga analisis ini telah menyokong pembahagian . sampel

Kerivoula yang ada kepada enam spesies, iailu K. minula, K. hardwickii, K. pellucida, K. lenis

dan K. papillosa. Sampel-sampel K. papillosa yang kriptik kemudiannya leluh dihahagi kepuda

duu jenis, iailu K. papillosa jenis kecil (K. pa illosa jenis S- "small ") dun K. papillosu jenis

bestir (K. papillosa jenis L- "large'). Analisis filogenetik pula telah dijalankan ke alas enam

spesies Kerivoula den gun menggunakan liga gen mitokondria, iuitu 409 pasungan hes dari

jujukun gen sitokrom h (cyt J, 478 pasangan hes dari jujukran gen . sitokrom ok. sidu I(C'C)1) dan

1044 pasangan bes dari jujukan gen NADH "dehydrogenuse " subunit 2 (ND2). Ini diikuli

unulisis yang menggunukun 1054 pasangan bes dari jujukan gen nuklear ukiivusi rekomhinun

subunit 2 (Rag2). Pembinaan semula pokok yang menggambarkan hubungan. filogenetik uniuk

Kerivoula mcný, >gunakan keempal-empal gen terse but lelah dijalankan melalui tiKu unulisis iuilu

"neighbor-joining" (NJ), "maximum parsimony" (MP) dan "maximum likelihood" (Ml, ).

vi

Penambahan sampel dari spesies K intermedia untuk analisis filogenetik ini telah

membahagikan sampel-sampel yang ada kepada tujuh kumpulan, iaitu K. intermedia, K. minuta,

K pellucida, K. hardwickii dan K lenis, dengan K. papillosa yang kemudiannya dibahagikan

lagi kepada dua jenis, sebagaimana yang didapati dari hasil kajian morfologi. Dari 17 sampel

yang dikenalpasti sebagai K. papillosa, lima daripadanya diklasifikasikan sebagai K. papillosa

jenis L (ukuran lengan 44.5-49.0 mm), dengan satu sampel didapati dari Gua Madai di Sabah

dan empat sampel yang lain didapati dari Taman Negara Niah, Sarawak. Dua belas sampel

yang seterusnya diklasifikasikan sebagai K. papillosa jenis S (ukuran lengan 40.0-44.5 mm) dan

didapati dari pelbagai lokasi di sekitar Sarawak danjuga Semenanjung Malaysia. Pembahagian

K. papillosa kepada dua jenis ini juga disokong oleh peratusan jarak genetik >10%, yang mana

bersamaan dengan peratusan di antara spesies yang berbeza dari segi biologi. Ini mungkin

menunjukkan bahawa terdapat kewujudan spesies kriptik di dalam kumpulan K papillosa.

Kajian ini juga telah mendapati bahawa kumpulan K. hardwickii telah dibahagikan kepada

kumpulan Borneo Barat dan Borneo Timur. Ini disokong oleh nilai 'bootstrap' sebanyak 100%

di dalam analisis untuk gen `cyt b' dan juga gen ND2. Perbezaan peratusan jarak genetik di

antara kedua-dua kumpulan ini adalah sebanyak 4.6-6.0%, dan peratusan jarak genetik

sehanyak >5% telah dicadangkan oleh Baker dan Bradley (2006) untuk kemungkinan wujudnya

phylogroup' yang belum dikenalpasti di dalam kumpulan berkenaan. Pembahagian kumpulan

K minuta kepada dua subkumpulan juga telah didapati daripada liga analisis genetik dari

kajian ini. Dengan ini juga dicadangkan bahawa terdapat kewujudan subspesies bagi K. minuta.

Dengan ini, disimpulkan bahawa analisis yang menggunakan gen ND2 memberi gambaran yang

terbaik tentang hubungan filogenetik di antara spesies dalam genus Kerivoula untuk kajian ini.

Perhandingan di antara K. papillosa jenis L dan K. papillosa jenis S terhadap -type specimen "

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K. papillosa mungkin akan dapat memberi penjelasan ke Was status sampel ini di dalam genus

Kerivoula. Kajian lanjutan ke atas populasi genetik untuk spesies K. papillosa, K. hardwickii

dan K. minuta adalah dicadangkan bagi memberi gambaran yang lebih jelas terhadap hasil

yang didapati dari kajian ini. Kajian selanjutnya ke atas kedua-dua jenis sampel K papillosa

bersama sembilan lagi spesies Kerivoula yang terdapat di Malaysia mungkin akan memberi

gambaran yang lebih lengkap ke Was hubungan filogenetik di antara spesies di dalam genus

Kerivoula di Malaysia. Hasil kajian ini diharapkan dapat membantu didalam pengurusan dan

pelan-pelan pemeliharaan genus ini di masa hadapan.

Kata kunci: Kerivoula, Malaysia, morfologi, filogenetik molekul, spesies kriptik

Vlll

Pusai Ktuuwii, . v. as,;. tuat ruc. adenilc UNIVERSITI MALAYSIA SARAWAIC

Table of Contents

Declaration

Acknowledgement

Abstract

Abstrak

Table of content

List of Figures

List of Tables

Abbreviations

CHAPTER ONE General Introduction

1.1 Chiroptera (Bats)

1.1.1 Classifications of Chiroptera

(i) Megachiroptera

(ii) Microchiroptera

1.1.2 Taxonomic Studies of Chiroptera

1.1.3 New Classifications of Bats

1.2 Study Taxa: The Wooly Bats (Kerivoula sp. )

1.2.1 New Species in Kerivoula

1.2.2 Distribution and Conservation Status

1

11

iv

V1

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xviii

xxiii

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1

3

4

5

7

9

10

12

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1.3 Morphology Analysis

1.3.1 Cluster Analysis

1.3.2 Principal Component Analysis

1.3.3 Discriminant Function Analysis

1.3.4 Other Statistical Analysis

1.4 Molecular Approaches

1.4.1 Mitochondrial DNA

(i) Cytochrome b

(ii) Cytochrome Oxidase I

(iii) NADH Dehydrogenase gene subunit 2

1.4.2 Nuclear Gene

(i) Recombinant Activating Gene 2

1.4.3 Inferring Methods

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16

18

19

21

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24

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25

26

26

27

1.5 Rationale 28

1.6 Aims of Study

1.6.1 Morphological Analysis

1.6.2 Molecular Phylogenetic Analysis

1.6.3 Morphological and Phylogenetic Analysis

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32

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32

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1.7 Outline of thesis

CHAPTER TWO Material and Methodologies

2.1 Specimen Collections

2.1.1 Collecting Methods and Sites of Study

2.1.2 Specimen Identification

2.1.3 Specimen Preservation

2.1.4 Museum Voucher Samples

2.2 Morphological Study Materials

2.2.1 Skull Extraction and Measurements

2.3 Morphological Analysis

2.3.1 Cluster Analysis

2.3.2 Basic Statistical Test

2.3.3 Principal Component Analysis (PCA)

2.3.4 Discriminant Function Analysis (DFA)

2.4 Molecular Study

2.4.1 Sources of DNA

2.4.2 Laboratory Materials

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34

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35

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41

41

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43

44

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2.5 Molecular Methodologies

2.5.1 DNA Extraction

2.5.2 DNA Visualization

2.5.3 Amplification using Polymerase Chain Reaction

(a) Mitochondrial Gene

(b) Nuclear Gene

2.5.4 Purification and Sequencing

2.6 Molecular Analysis

2.6.1 Distance-based Method

2.6.2 Character-based Methods

CHAPTER THREE Morphology Analysis Results

3.1 Morphological Data and Cluster Analysis

3.2 Statistical Tests

3.2.1 Multiple Regression

3.2.2 Normality Test and Data Transformation

3.2.3 Homogeneity of Variance Test

3.2.4 Multicolinearity Test

3.3 Principal Component Analysis

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3.4 Discriminant Function Analysis

CHAPTER FOUR Molecular Analysis Results

4.1 Mitochondrial Gene Analysis

4.1.1 Cytochrome b

4.1.2 NADH Dehydrogenase gene subunit 2

4.1.3 Cytochrome Oxidase I

4.2 Nuclear Gene Analysis

4.2.1 Recombinant Activating Gene subunit 2

CHAPTER FIVE Discussion

5.1 Morphological Analysis of Kerivoula

5.2 Phylogenetic Relationship of Kerivoula

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69

81

90

99

107

5.2.1 Mitochondrial Gene Analysis 114

5.2.2 Nuclear Gene Analysis in Comparison with the mtDNA Gene 118

Analysis

X111

5.3 Comparison of Morphology and Molecular Results 119

CHAPTER SIX General Conclusions and Recommendations 123

REFERENCES 126

List of Publications 143

APPENDIX A Samples used for morphometrics analysis. 144

APPENDIX B Multiple regression for testing sex dimorphism for each 152

species.

APPENDIX Cl P-values of Kolmogorov-Smirnov goodness-of-fit test for 153

all characters in K papillosa type S.

APPENDIX C2 P-values of Kolmogorov-Smirnov goodness-of-fit test for 154

all characters in K papillosa type L.


all characters in K. pellucida.

xiv


all characters in K hardwickii.


all characters in K minuta.

APPENDIX D Box's M test results for standardised characters. 158

APPENDIX E Variables in the analysis and not in the analysis for 159

normally distributed character and all characters.

APPENDIX F1 Scree plot describing the eigenvalue for each component 165

in the analysis.

APPENDIX F2 Total variance explained by each factor. Extracted factor 166

used in analysis are being bold.

APPENDIX F3 Communalities tables for the extracted component in the 167

analysis.

APPENDIX F4 KMO and Bartlett's test. 168

xv

APPENDIX F5 Component score coeffiecient matrix for all 31 variables 168

used in the analysis.

APPENDIX F6 Descriptive statistics for the variables used in the analysis. 169

APPENDIX G1 Classification results for all characters in analysis. 170

APPENDIX G2 Statistical test used at each step of the stepwise analysis 172

(Wilk's lambda) for all characters.

APPENDIX G3 Wilk's Lambda test of functions and eigenvalues for all 173

functions.

APPENDIX H The aligned sequences of the 409 bp of partial cyt b 174

sequences for each species used in Chapter Four.

APPENDIX I The aligned sequences of the complete ND2 sequences for 180

each species used in Chapter Four.

APPENDIX J The aligned sequences of the partial COI sequences for 191


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APPENDIX K The aligned sequences of the partial Rag2 sequences for 196


APPENDIX L Comparison of skulls of K papillosa type L, K papillosa 204

type S, K lenis, K pellucida and K hardwickii.

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List of Figures

Figure 2.1 Sampling sites for Kerivoula samples used in present study, namely as

the following; 1-Kubah National Park, Swk; 2-Batang Ai National Park,

Swk; 3-Bau, Swk; 4-Bintulu, Swk; 4-Niah National Park, Swk; 5-

Loagan Bunut National Park, Swk; 6- Lambir Hills National Park, Swk;

7-Similajau National Park, Swk; 8-Gua Madai, Sbh; 9-Taman Negara

Pahang, PM; 10-Kelantan, PM; 11-Perak, PM; 12-Johor, PM and 13-

Terengganu, PM. *Swk=Sarawak; Sbh=Sabah and PM=Peninsular

Malaysia.

Figure 2.2 Thirty-one characters used for measurements and analysis in Kerivoula.

Drawing is not to scale.

Figure 3.1 Figure above showed the dendogram for cluster analysis result using

UPGMA analysis and average Euclidean distance method. K papillosa

S1=Sarawak (SWK) locality; K papillosa S2=Peninsular Malaysia

(PM) locality; K pellucida 1=SWK locality; K pellucida 2=PM

locality; K minuta 1=SWK locality; K minuta 2=PM locality;

FA=forearm length; mm=millimeter.

Figure 3.2 Regression factor plot for species grouping for Factor 1 versus Factor 2.

XVlil

Figure 3.3 Regression factor plot for species grouping for Factor 1 versus Factor 3.

Figure 3.4 Canonical variate analysis for all characters using pooled covariance

matrix.

Figure 3.5 Canonical variate analysis for all characters using separate covariance

matrix.

Figure 4.1 Plot of transition and transversion against divergence using Kimura

(1980) distance method onto the third codon position shows no

saturation occurrence in cyt b gene region.

Figure 4.2 Phylogenetic relationships of six species of Kerivoula under study based

on 409 bp partial cyt b mtDNA gene sequences. The phylogeny is a

single tree recovered using NJ analysis. Values on the branches

represent NJ bootstrap estimates, based on 1000 replicates. Only

bootstrap values >50% are shown.

Figure 4.3 Unweighted and rooted MP tree based on nucleotide data set of 409 bp

partial cyt b mtDNA gene (tree length=440; CI=0.4295; RI=0.7794).

Values on the branches represent MP bootstrap estimates, based on

1000 replicates. Only bootstrap values >50% are shown.

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Figure 4.4 Rooted ML tree (-Ln likelihood= 2652.61175) generated based on

nucleotide data set of 409 bp partial cyt b mtDNA gene. Values on the

branches represent ML bootstrap estimates, based on 100 replicates.

Only bootstrap values >50% are shown.



saturation occurrence of the ND2 gene region.


on 1044 bp complete ND2 mtDNA gene sequences. The phylogeny is a





complete ND2 mtDNA gene (tree length=1256; CI=0.5645;

RI=0.7760). Values on the branches represent MP bootstrap estimates,

based on 1000 replicates. Only bootstrap values >50% are shown.

xx


nucleotide data set of 409 bp partial cyt b mtDNA gene. Values on the





saturation occurrence of COI gene region.


on 478 bp partial COI mtDNA gene sequences. The phylogeny is a





partial COI mtDNA gene (tree length=339; CI=0.6077; RI=0.8222).




nucleotide data set of 478 bp partial COI mtDNA gene. Values on the


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(1980) distance method shows no saturation occurrence in the Rag2

gene region.


on 1054 bp partial Rag2 nuclear gene sequences. The phylogeny is a





partial Rag2 nuclear gene (tree length=118; CI=0.7542; RI=0.8564).




nucleotide data set of 1054 bp partial Rag2 nuclear gene. Values on the



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A MORPHOLIGICAL AND MOLECULAR PHYLOGENETIC … morpholigical and molecular phylogenetic analysis...

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