Phylogeography of tropical eel (Anguilla spp) in Indonesia Waters
PHYLOGEOGRAPHY OF TROPICAL EELS
(
Anguilla spp
) IN INDONESIAN WATERS
MELTA RINI FAHMI
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
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STATEMENT OF DISSERTATION
AND INFORMATION SOURCE
Hereby I express that the dissertation entitled: “
Phylogeography of
Tropical Eel (
Anguilla spp
) in Indonesian Water
” is the original result of
my research and has never been submitted to obtain a Doctorate in similar
mayor at other universities. All of data and information that I had provided
in this dissertation are based on evidence and available references.
Bogor, March 2013
Melta Rini Fahmi
NRP G362080061
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SUMMARY
MELTA RINI FAHMI. Phylogeography of Tropical Eel (Anguilla spp) in Indonesia Waters. Supervised by DEDY DURYADI SOLOHIN, PATRCIK BERREBI, KADARWAN SOEWARDI and LAURENT POUYAUD
The freshwater eels (Anguilla spp: Anguillidae) are popular as a commercial important food, because of good nutritional value. These fish are also a well known for their unique catadromous life histories. These species breed far from offshore after migrate thousands kilometers from their growth habitats in freshwater and estuarine to their spawning area in oceanic waters. Most of the investigations concerning eels are concentrated on temperate species, in the north hemisphere mainly because of the economic importance of these species. Nowadays, the population of temperate eel, dramatically decrease is caused by habitat damage, illegal fishing and climatic changes in the ocean. As a consequence, tropical eels become important eel nowadays in the market, as well as the research on tropical eels become a new challenge. One of primary problems in tropical eels is that they have overlapping range of most morphological character, so species identification on this genus are no more sufficient. Then molecular approaches have been proposed for eel identification.
This study has four main objectives: (1) to establish quick methods of identification of tropical eels; (2) to understand exact distribution and dispersal of tropical eel in Indonesia water; (3) to reveal the phylogenetic relationship among population in Indonesia and (4) to uncover population genetic structure of
Anguilla bicolor between two Ocean. All of information obtained in this study
revealed the conservation management of tropical eel in Indonesia water.
The semi-multiplex method was proposed in this study has demonstrated the efficiency for in identifying seven species and sub-species of tropical eels with only one step PCR. By using this method, one could reduce the number of necessary sequences while the results are very sure for each species determination (we easily identified 1112 specimens). All of species were obtained in this study showed overlapping morphology and distribution. Especially for small specimens (mainly glass eels), the molecular method appears as indispensable. This method has proven to be most simple, quicker, lower cost (no acrilamide migration), specific/sensitive, and highly reliable way.
The geographic distribution of tropical eels of the genus Anguilla in Indonesian waters was establish by identified genetically of all sample that obtain during this study. The genetically identification applied the semi-multiplex PCR method that recently developed in this study. We recognized four species and subspecies with wide distribution: A. b. bicolor, A. b. pacifica, A. marmorata and
A. interioris, two species with limited distribution, close to endemism: A.
celebesensis and A. borneensis and one subspecies A. nebulosa nebulosa that
is only spread in river flowing into Indian Ocean.
The phylogenetic relationship and genetic diversity was cosntructed and calculated based on mitochondria DNA cytochrome b gene sequence of Anguilla
that covering all of the geograpical distribution in Indonesia waters. The genetic structure of populations that responsible for a given genetic architecture and evolutionary factors is an important objective of population genetics. In this study we present genetic diversity and phylogenetic relationship among and within species in genus Anguilla to understanding the evolutionary process of Anguillid that inhabit in Indonesia waters.Seven species of Indonesian tropical eel showed a higher haplotype and nucleotide diversity at the cyt b locus with haplotype and nucleotide diversity (π) were 0,98 and 4,57% respectively. Phylogenetic tree showed A. borneensis a likely most basal species in Indonesia water.
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The economic important shortfinned eel, Anguilla bicolor, has a relatively wide geographic distribution compared to the 19 species and subspecies of genus Anguilla, it is distributed longitudinally from the eastern coasts of Africa through the seas around Indonesia to New Guinea in Pacific Ocean. The genotypes of seven microsatellite DNA were analysed for 180 specimens collected from 10 representative location where two subspecies have been found. Analysis with seven microsetallite loci showed Expected (He) and observed (Ho) heterozigosities of each locus range from 0,594 to 0,921 and from 0,250 to 1,000 respectively. All off locus showed high polymorphic. Based on FST value and clustering test, there is no structure and fragmentation of A. bicolor in Indonesia water.
A critical first step in species conservation is to gain a clear understanding of its taxonomy. Considering Indonesian waters that are inhabited by several sympatric species of tropical eels, almost all morphological character in this genus are overlapping. So a rapid and efficient identification technique is needed. Semi multiplex PCR that has been developed in the present study has successfully distinguished seven species that inhabit the waters of Indonesia through one-step PCR. After established the identification species subspecies methods then we constructed distribution and dispersal pattern, phylogenetic relationship among species that inhabit Indonesian water and population genetic structure of species that have widespread distribution. All of information obtained in this study was needed for conservation management of anguillid in Indonesian water.
Key words: Tropical eel, Anguilla spp, semi-multiplex PCR, Indonesia water and population genetic
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a. Citation only permitted for sake of education, research, scientific problem.
b. Citation doesn’t inflict the name and honor of Bogor Agricultural University.
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Dissertation’s title : Phylogeogphy of Tropical Eels (Anguilla spp) In Indonesian Waters
Name : Melta Rini Fahmi Student ID : G 362080061 Departement : Biology
Aproved by the Supervisor Commite
Dr. Dedy Duryadi Solihin Head
Prof. Dr. Patrick Berrebi Prof. Dr.Kadarwan Soewardi, M.Sc
Member Member
Dr.Laurent Pouyoud M.Sc Member
Acknowledged by:
Head of Biology Programme of Dean of Graduate School Graduate School Bogor Agricultural University
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TABLE OF CONTENTS
Page
TABLE OF CONTENTS ... i
LIST OF TABLES ... iii
LIST OF FIGURES ... iv
LIST OF APPENDIX ... v
I. GENERAL INTRODUCTION Background ... 1
Questions to Solve ... 7
Framework ... 7
II. A NOVEL SEMI-MULTIPLEX PCR ASSAY FOR IDENTIFICATION OF TROPICAL EEL GENUS Anguilla IN INDONESIAN WATER Abstract ... 10
Introduction ... 10
Material and Methods ... 12
Result ... 16
Discussion ... 18
III. DISTRIBUTION OF TROPICAL EEL GENUS Anguilla IN INDONESIAN WATER BASED ON SEMI-MULTIPLEX PCR Abstract ... 20
Introduction ... 20
Material and Methods ... 23
Result ... 25
Discussion ... 28
IV. A NEW MOLECULAR PHYLOGENY AND GENETIC DIVERSITY OF FRESHWATER EEL GENUS Anguilla IN INDONESIAN WATER BASED ON MITOCHONDRIAL GENES Abstract ... 35
Introduction ... 35
Material and Methods ... 38
Result ... 41
Discussion ... 52
V. POPULATION GENETIC STRUCTURE OF TROPICAL EEL Anguilla bicolor IN INDONESIAN WATER Abstract ... 55
Introduction ... 55
Material and Methods ... 58
i
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Result ... 62
Discussion ... 65
VI. GENERAL DISCUSSION ... 67
REFERENCES ... 71
APPENDIX ... 76
ii
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LIST OF TABLES
Page
1. Position of nine species-specific primers for semi-multiplex PCR on cytochrome b and 16s rRNA genes respectively. Two positions for forward primers (FCYT-EEL and F16S-EEL) and seven positions for
reverse primers ... 15
2. Species-specific primer sequences for semi- multiplex PCR, and PCR product lengths expected for the seven Anguilla species and subspecies ... 16
3. Identification eel by morphology (only 796 eels were classified into the four groupa) and semi-multiplex-PCR (1112 eels were identified among 1115 eels) ... 18
4. List of sampling locations of tropical eels in Indonesia ... 25
5. List specimens, sampling location and date in this study ... 39
6. Matrix nucleotide substitution based on HKY+G+I methods ... 42
7. Genetic diversity and neutral test of species/sub species of tropical eel genus Anguilla from Indonesia waters ... 43
8. Nucleotide diagnostic of each species and clade on the species ... 47
9. Matrix genetic distance between all of species-subspecies on genus Anguilla. Grey highlights one is species-subspecies during this study ... 48
10. Genetic distance within species and sub species ... 48
11. Intraspecific genetic differentiation measured within A. bicolor species ... 51
12. Mutation on nucleotide sequence (above) and amino acid (below) between A. b. bicolor and A. b. pacifica ... 51
13. Intraspecific genetic differentiation measured within A. marmorata species ... 52
14. List of specimen used for microsatellite analyses in the present study .... 59
15. List locus that using during this study ... 61
16. Genetic variability of population parameters at 7 microsatellite loci on tropical eel in Indonesia water ... 63
17. Genetic divergence (Fst) (above) and genetic distance (below) among population during present study ... 64
iii
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LIST OF FIGURES
Page
18. World production of eels (FAO, 2009) ... 3 19. Research framework ... 9 20. Measurement morphology character of specimen ... 15 21. Identification species and subspecies of Anguilla by semi-multiplex PCR
samples ... 18 22. Map of the stations around Indonesia Sea where samples have been
collected for this study ... 25 23. Distribution of the seven species and subspecies of freshwater eels
which were found around Indonesia during this study ... 28 24. The sampling location of genus Anguilla in the origin region Indonesia
water ... 41 25. Neighbor Joining (NJ) phylogentic tree based on 1042 bp of
mitochondrial DNA cyt b gene fragmen under Kimura 2-parameter model, with Serrivomer sector, Synaphobranchus kaupi and Conger myriaster as outgroup (up to 75%) ... 46 26. Neighbor Joining (NJ) phylogentic tree based on 1042 bp of
mitochondrial DNA cyt b gene fragmen under Kimura 2-parameter model, with Synaphobranchus kaupi as outgroup (up to 75%) ... 47 27. Neighbor Joining (NJ) phylogentic tree of all sequence in this study
based on 1042 bp of mitochondrial DNA cyt b gene fragmen under
Kimura 2-parameter model (up 75%) ... 51 28. Dendogram of genetic distance of Indonesian tropical eel based on cyt b
sequence ... 56
iv
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v
LIST OF APPENDIX
Page
1. Sampling Location during this Study ... 76 2. Number haplotype for the cytochrome b gene among all specimen on
present study ... 77 3. Sequencing Alignment for Cytochrome b gene among all haplotype of
Anopicall eel Anguilla spp ... 79 4. Frequency allele for each locus, each localities for all specimen ... 83 5. Fis for each locus, each localities for all specimen ... 86
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LIST OF TABLES
Page
1. Position of nine species-specific primers for semi-multiplex PCR on cytochrome b and 16s rRNA genes respectively. Two positions for forward primers (FCYT-EEL and F16S-EEL) and seven positions fo reverse primers ... 2. Species-specific primer sequences for semi- multiplex PCR, and PCR
r
... 15
... 16 into the
18 .... 25 . 39
... 61
64 product lengths expected for the seven Anguilla species and
subspecies ... 3. Identification eel by morphology (only 796 eels were classified
four groupa) and semi-multiplex-PCR (1112 eels were identified among 1115 eels) ... 4. List of sampling locations of tropical eels in Indonesia ... 5. List specimens, sampling location and date in this study ... 6. Matrix nucleotide substitution based on HKY+G+I methods ... 42 7. Genetic diversity and neutral test of species/sub species of tropical eel
genus Anguilla from Indonesia waters ... 43 8. Nucleotide diagnostic of each species and clade on the species ... 47 9. Matrix genetic distance between all of species-subspecies on genus
Anguilla. Grey highlights one is species-subspecies during this study ... 48 10. Genetic distance within species and sub species ... 48 11. Intraspecific genetic differentiation measured within A. bicolor species ... 51 12. Mutation on nucleotide sequence (above) and amino acid (below)
between A. b. bicolor and A. b. pacifica ... 51 13. Intraspecific genetic differentiation measured within A. marmorata
species ... 52 14. List of specimen used for microsatellite analyses in the present study .... 59 15. List locus that using during this study ...
16. Genetic variability of population parameters at 7 microsatellite loci on
tropical eel in Indonesia water ... 63 17. Genetic divergence (Fst) (above) and genetic distance (below) among
population during present study ...
iii
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LIST OF FIGURES
Page
1. World production of eels (FAO, 2009) ... 3 2. Research framework ... 9 3. Measurement morphology character of specimen ... 15 4. Identification species and subspecies of Anguilla by semi-multiplex PCR
25
1
del,
... 51 t b samples ... 18 5. Map of the stations around Indonesia Sea where samples have been
collected for this study ... 6. Distribution of the seven species and subspecies of freshwater eels
which were found around Indonesia during this study ... 28 7. The sampling location of genus Anguilla in the origin region Indonesia
water ... 4 8. Neighbor Joining (NJ) phylogentic tree based on 1042 bp of
mitochondrial DNA cyt b gene fragmen under Kimura 2-parameter model, with Serrivomer sector, Synaphobranchus kaupi and Conger myriaster as outgroup (up to 75%) ... 46 9. Neighbor Joining (NJ) phylogentic tree based on 1042 bp of
mitochondrial DNA cyt b gene fragmen under Kimura 2-parameter mo
with Synaphobranchus kaupi as outgroup (up to 75%) ... 47 10. Neighbor Joining (NJ) phylogentic tree of all sequence in this study
based on 1042 bp of mitochondrial DNA cyt b gene fragmen under Kimura 2-parameter model (up 75%) ... 11. Dendogram of genetic distance of Indonesian tropical eel based on cy
sequence ... 56
iii
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I. GENERAL INTRODUCTION
Background
The freshwater eels (Anguilla spp: Anguillidae) are a well known for their catadromous life histories. They migrate between freshwater and marine environment. These species are breeding far offshore after a migration of thousands kilometers from their growth habitats in freshwater and estuarine to their spawning area in oceanic waters (Ege 1939; Tesch 1977). Most of the temperate species spawn within a narrow tropical area. Furthermore, the eels larvae, known as leptocephali, passively swim to their growth habitat along subtropical currents (Tesch 1977; Tsukamoto 1992).
The anguillid leptocephalus is one of the most distinctive larvae of Anguilliform fishes and has an olive leaf-like shape, with a depth of about one fifth of total length (TL), no melanophores, transparent body, relatively few myomeres and high water content contribute to the buoyancy, which would be advantageous for passive transport by ocean current (Tesch 1977; Mochioka 2003). Lepthocephalus will be metamorphose, their morphological and physiological change before into glass eel, which occurs before entering freshwater. Eel larvae undergo marked changes the somatic structure from the leaf-like shape of the lepthocephali to the adult-like shape of the glass eel. Glass eel is a developmental stage from the end of metamorphosis to the beginning of pigmentation, following this stage the young eels are called “elver” (Tesch 1977; Mochioka 2003). The next development stage is the yellow eel or sexually immature adult stage. Those inhabit in estuarine until freshwater environment, which tend to live longer and attain much larger sizes (Tesch 1977). Further freshwater eels will continue to the sexually mature stage are called silver eel. In this stage, they begin downstream migration from the growth habitat, freshwater to the open oceanic, after transformation from yellow-phase to silver-phase eels. The silver eel spent their entire growth phase in the marine environment and apparently they never entered freshwater (Tesch 1977; Aoyama et al. 2003a).
Ege (1939) divided the Anguilla species into four groups, based upon color, body proportions, dentition, and meristic characters: First group, variegated
species with broad undivided maxillary and mandibular bands of teeth;
A. celebesensis Kaup 1856; A. interioris Whitley 1938; and A. megastoma Kaup
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1856; second group, variegated species with a toothless longitudinal groove in the maxillary and mandibular bands of teeth; A. nebulosa (A. n. labiata Peters 1852; A. n. nebulosa McClelland 1844); A. marmorata Quoy and Gaimard 1824;
A. reinhardti Steindachner 1867; and A. ancestralis Ege 1939; third group,
species without variegated markings and with a long dorsal fin; A. anguilla
Linnaeus 1758; A. rostrata Lesueur 1817; A. mossambica Peters 1852;
A. borneensis Popta 1924; A. japonica Temminck and Schlegel 1846; and
A. dieffenbachii Gray 1842; and fourth group, species without variegated
markings and with a short dorsal fin; A. bicolor (A. b. bicolor McClelland 1844;
A. b. pacifica Schmidt 1928); A. australis (A. a. australis Richardson 1841;
A. a. schmidti Phillips 1925); and A. obscura Gunther 1871. Castle and
Williamson (1974) postulated that A. ancestralis is an invalid species, based on similarities with juvenile A. celebesensis. Currently, after scientist discovered a new species A. luzonensis in Philippines water at 2009, accepted number of species in the genus Anguilla is 16 species (Watanabe et al. 2009).
At the population level, most of the freshwater eel showed randomly mating panmictic structure on the whole species or sub-species distribution (Avice et al. 1986). Panmixia is realized on the unique species or subspecies spawning area where all individuals are potential partners. There are no mating restrictions, neither genetics or behavioral and all recombination is possible (Avise et al. 1986).
Apart from their unique life history, eels are also popular as a commercial important food, because of good nutritional value, with protein and fat content of 65% and 28% respectively (Hainsbroek 2007). Among the many popular eel dishes consumed around the world, kabayaki (marinated grilled eel) is a national dish in Japan, while smoked eel is favored in Europe and North America, and eel larvae are eaten as appetizers in Spain (Ringuet 2002).
The international market for cultured eels exceeds 200,000 ton in year 2000. Based on FISHSTAT (FAO 2009) data, total production of eels rose from 17,750 ton in 1950 year to 284,274 ton in 2007 year. In Japan, the Japanese eel
(Anguilla japonica) has long been esteemed as an important food fish, as much
as 130,000 tons of eels are consumed per year followed by China, Korea, America and some European countries, like Denmark, France, Italy, Belgium and Germany. However most of this production is based on catching wild of adults and rearing of wild-caught juvenile “glass eels”. The catching activity of glass eel
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since the mid-1990s has been increased rapidly (Figure 1). The impact of exploitation of glass eel populations is unknown, although the yield of yellow and silver eel has declined. Ecologists consider that the decline of eel production is caused by habitat damage, illegal eel fisheries, climatic changes in the ocean, and parasites (especially Anguillicola crassus in European eel). According to Ringuet (2001), the overall production of A. anguilla and A. japonica has declined, landings of European eels, Japanese eels and American eels dropped to 43.5%, 64% and 8.3%, respectively, over a period of 17 years (1984 to 2000). As a consequence, tropical eels became most important nowadays in the market, as well as the research on tropical eel which becomes a new challenge.
Figure 1 World production of eels (FAO 2009), graphic adapted from FAO 2009
On the other hand, the knowledge on tropical eel species occupying southern or tropical zones is still limited. Two thirds of the recognized 18 Anguilla
species and subspecies are found in the tropical Pacific, while only 6 in temperate regions of both the Pacific and Atlantic Oceans and seven occupy the western Pacific around Indonesia (Ege 1939; Castle & Williamson 1974; Arai et al. 1999). The main differences between tropical and temperate eels are the length of larval phase, the distribution, the spawning season and the population structure. The temperate eel generally have a longer and farther migration distances (Cheng and Tzeng 1996; Arai et al. 1999, 2001). The larvae of temperate eel enter estuaries is in spring, while that of tropical eels are found in estuaries throughout the year with different abundance (Arai et al. 1999).
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Most studies of population genetic structure of eels have focused just on temperate species in the northern hemisphere and few have examined tropical species. Some studies on population genetic structure of tropical eel have been conducted such as; A. marmorata by Ishikawa et al. (2004), Tseng (2012) and Minegishi et al. (2008); A. bicolor by Minegishi et al. (2012) and A. reinhardtii by Shen and Tzeng (2007, 2012).
To uncover the existence of tropical eel, several studies have been conducted through the collaboration between the Japanese institution Ocean Research Institute and research institutes of Indonesia (LIPI) to investigate the distribution of tropical eels , using the research vessels KM Baruna Jaya VII and RV Haruko Maru in the period 1998-2003. Both research vessels collected lepothocephali around the Indonesian sea: A. borneensis caught in the Celebes Sea, A. b. bicolor caught in the Mentawai Islands and A. celebesensis in the Tomini bay (Arai et al. 1999; Wouthuyzen et al. 2009; Aoyama et al. 2007; Setiawan et al. 2001; Miller 2003).
Beside looking for the spawning areas of these eels, this expedition confirmed also some observations quite interesting for taxonomy. According to Watanabe et al. (2004a), the tropical eels show geographic distribution and morphological characters heavily overlapping. The comprehensive identification of eels proposed by Ege (1939), divided genus Anguilla into 15 species, three of which were subdivided into two subspecies by using morphological character. But when the geographic distribution of each species is plotted on a map, several species have overlapping geographic range. They have also overlapping range of morphological character. While Ege's taxonomy has long been accepted since its publication (because most of the first freshwater eel studied came from temperate region and are not geographically overlapping), some doubts were expressed (Watanabe et al. 2004a).
That means the identification of eels depends on the location of collection. Watanabe et al. (2004a) deduced that if only morphological characters were used for identification, the freshwater eels could be classified into only four groups. This can be also a problem for the freshwater eels that have been transported around the world in recent years for aquaculture.
Since morphological studies are no more sufficient for freshwater eels species identification, the molecular genetics approaches become a new challenge to develop technically and theoretically nowadays. Molecular genetics
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have been used to evaluate certain taxa, for identification, evolution purposes and phylogenies reconstructions (Freeland 2005). The application of molecular genetics to confirm identification of freshwater eel have been conducted in several research (Aoyama et al. 1999, 2001; Watanabe et al. 2004b, 2008; Sezaki et al. 2005; Itoi et al. 2005; Gagnaire et al. 2007 and Trautner et al. 2006), most of them apply RFLP, RAPD and Real Time-PCR methods with 16S rRNA and Cyt b gene as marker.
Multiplex PCR is a variant of PCR permitting simultaneous amplification of several targets in one reaction by using more than one pair of primers. For taxa identification purpose, the multiplex PCR produce amplicons on varying sizes that are specific to different DNA sequences (Rompler 2006). Multiplex PCR assay that using several species-specific primers enable to identify several species in a simple, quick, low cost, sensitive, and highly reliable method (Catanese et al. 2010)
The molecular genetics techniques also have been used for new hypotheses of phylogeny and evolution of the genus Anguilla (Tagliavini et al.
1996; Aoyama and Tsukamoto 1997; Lin et al. 2001; Bastrop et al. 2000; Aoyama et al. 2001; Inoue et al. 2001 and Minegishi et al. 2005). Molecular phylogenetic studies upon all species of genus Anguilla have been conducted by
Lin et al. (2001) who examined mitochondrial 12SrRNA and cytochrome bgenes,
and Aoyama et al. (2001) examined 16SrRNA and cytochrome b genes. Both studies presented almost the same topology defining species groups and their geographic distribution. However these contributions diverged on the position of basal species. Aoyama et al. (2001) concluded that A.borneensis was most likely the basal species of the genus while Lin et al. (2001) suggested that the ancestor species are A. marmorata and A.nebulosa.In recent year, Minegishi et al (2005) produced a new phylogenetic analysis based on the complete mitochondrial DNA (mtDNA) sequence of all species of genus Anguilla. This phylogenetic analysis showed a better statistical support that mean more sure analysis than previous studied. Minegishi et al. (2005) suggest A. mossambica to be most basal species using Baysian analysis, but based on MP (maximum parsimony) analysis, similarly with Aoyama et al. (2001), Minegishi et al. (2005) A. borneensis appears as basal species. Moreover, because the tropical and Indo-Pacific zones have a highest species diversity, these authors said that the Indonesia waters are the
“origin of the eels” and “Indonesia is homeland of eels”.
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A. bicolor and A. marmorata are tropical eels that have been widely analyzed due to their exceptional large distribution (nearly 20,000 km east-west in Indopacific oceans). This wide distribution is mostly in sympatry from the eastern coasts of Africa through the seas around Indonesia to New Guinea in the Pacific Ocean. They are strongly suspected to have several spawning areas. According to Minegishi et al. (2008), A. marmorata is not taxonomically divided into subspecies because of its morphological stability, but molecular studies have demonstrated its structure into four differentiated populations: North Pacific, South Pacific, Indian Ocean, and Mariana (Minegishi et al. 2008; Gagnaire et al.
2009). The shortfined eel, A. bicolor, of high abundance, is considered to be structured into two subspecies A. b. bicolor in Indian Ocean especially at the west of Indonesia and A. b. pacifica in Pacific Ocean (Ege 1939; Minegishi et al. 2012). However, the population structure and evolutionary history of A. bicolor
needs to be investigated in detail especially in Indonesia. These fish have high economic value, and are believed to be the best candidate eels to replace Japanese eel (A. japonica) and European eel (A. anguilla) which have been decline for food, including fish farm growth.
Indonesia is an archipelagic country that has a long coastline of 91,000 km and 71,480 islands. The western part of Indonesia is connected with Indian Ocean and the eastern was one with the Pacific Ocean, making Indonesia an important biogeographic crossroad. Until now, there are seven recognized species that occupy Indonesia waters: A. bicolor (two subspecies: A. b. bicolor
and A. b. pacifica), A. marmorata, A. celebesensis, A. borneensis, A. interioris, A.
obscura and A. nebulosa (subspecies: A. n. nebulosa) (Ege 1939; Castle &
Williamson 1974; Tsukamoto & Aoyama 1998 and Sugeha et al. 2008). But information about distribution, evolution, phylogenetic relationship and structure population still are limited and without details.
Questions to solve
This study was intended to solve some questions:
a. The distribution of seven eel species living in Indonesia is not really known. This project aimed first at establishing quick methods of identification of tropical eels. This is partly due to the difficulty to determine the species just using morphological character. From here we begin to get a clear description of the distribution of the species that live in Indonesian water. while it is quite
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b. The genetic relationships among Anguillide in Indonesian water can be used generate a new phylogenetic tree which can help to understand the evolution of this genus in tropical areas especially in Indonesian water.
c. Indonesia is an important biogeographic crossroad, at the contact between Indian and Pacific ichthyofaunas. Concerning the two widespread eel species (A. marmorata and A. bicolor), Indonesia harbors a part of the Indian and a part of the Pacific populations or subspecies, but nearly nothing is known on the exact distribution of each lineage in Indonesian rivers. Biogeography of both species and populations genetics of A. bicolor will be
used to understand the population structure and to know the gene flow
pattern among Anguillidae populations in the Indonesian waters.
Framework
The decreasing of temperate eel populations in the subtropical zone have encouraged biologists to help conservation and aquaculture of this group. As a result, research on tropical eel are become a new challenge especially in Indonesian waters. Many scientists consider Indonesia as ”homeland and origin
of eels”, however, knowledge about distribution and biological traits of eel in
Indonesian waters is still limited. Understanding biological aspects and population is an important point in the development of sustainable aquaculture.
In sustainable aquaculture, phylogeography and genetic population studies are two first crucial steps to manage the fisheries resources. Such studies should provide valuable information about their status, population fragmentation and dispersal pattern. To analyze phylogeography of eels, we should begin by their distribution map and phylogenetic relationships. Therefore samples collection and identification become an important point. Recently many biologists agree that identification of eels is more appropriate using molecular biology approach. Semi-multiplex PCR is one method that provides genetic information to identification of target species, by using the species-specific primers. Semi-multiplex PCR assays can identify several species in a simple, quick, low cost, sensitive and highly reliable way on one step PCR. Phylogenetic relationships can be analyzed based on the information resulting from mtDNA sequencing. The PCR and DNA sequencing constitute easy and fast methods, furthermore,
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computer software provide convenient analytical methods to infer phylogenetic relationships and evolution and some statistical calculation.
The analysis of polymorphic microsatellites will provide inter and intra populations information structure. This population analysis should be conducted on widespread species.
As a summary, the outputs of this research are (i) a quick identification method of ells species living in Indonesian waters; (ii) distribution of eels species, furthermore (iii) the information on systematics, phylogenetics and populations genetics structure, which will provide data for conservation and aquaculture strategy. The framework of this study is summarized in the Fig. 2.
Phylogeography tropical eels genus Anguilla in Indonesian waters
Distribution and species composition of Anguilla spp in Indonesian water
Phylogenetic tree tropical eel in Indonesian waters
Population genetic structure of tropical eel: Anguilla bicolor in Indonesian waters
CYT b and 16SrRNA genes to construct phylogenetic
relationships among populations and species Morphometric and
Semi-Multiplex PCR for identification
Polymorphic microsatellites to determine the population genetic structure of
A. bicolor
Quick identification methods, dispersal and distribution, systematic and phylogenetic and population genetic structure of tropical eels in
Indonesian water
Population connectivity and conservation-aquaculture strategy of tropical eel in
Indonesian waters
Figure 2. Research framework
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II. A Novel Semi-Multiplex PCR Assay for Identification of
Tropical Eel Genus
Anguilla
in Indonesian Water
Abstract
A
o
ne step semi-multiplex PCR is proposed for distinguishing seven species andsubspecies of tropical eels including Anguilla bicolor bicolor, A. bicolor pacifica, A. marmorata, A. interioris, A. celebesensis, A. borneensis, and A. nebulosa nebulosa in Indonesian waters. Seven pairs of species-specific primers, including two forward and seven reverse primer sequences, were designed after the alignment of complete mitochondrial cytochrome b (1140 bp) and 16S rRNA (1120 bp) genes. All species-specific primer pairs are included in one PCR, but only one pair of them can amplify a specific fragment from the template DNA that is analyzed. The semi-multiplex PCR amplified a fragment of 230 bp for A. b. bicolor, 372 bp for A. n. nebulosa, 450 bp for A. borneensis, 620 bp for A. marmorata, 670 bp for A. b. pacifica, 720 bp for A. celebesensis, and 795 bp for A. interioris, which are then separated by DNA agarose gel electrophoresis.
KEY WORDS: semi-multiplex PCR, Anguilla, tropical eels, molecular identification, species-subspecies specific primer
Introduction
Order Anguilliformes contains 400 genera and 800 species, most of them lives in the oceans and only genus Anguilla migrates to the freshwater for growth Nelson (2006). Genus Anguilla is composed of freshwater eel having a catadromous life history characterized by spawning in ocean waters and by a migration of the larvae back to the parents growing habitats in freshwater or estuarine areas. The three temperate species spawn in remote tropical waters after a long adult migration. Their larvae, called leptocephali are passively returning to their growth habitat with the influence of subtropical currents, and perform long migration distance (Tesch 1977; Tsukamoto 1992). Oppositely, the tropical eels have shorter migration distance for both adults and leptocephali than those of the temperate species (Arai et al. 1999; Wouthuyzen et al. 2009 and Aoyama 2009)
A first comprehensive identification of the genus Anguilla was proposed by Kaup (1856), who recognized 45 species. In 1870, Gunther reduced this number to 23 species. Lastly, a revision of the genus was done by Ege (1939) who
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divided the genus Anguilla into 16 species, three of which were subdivided into two subspecies. Based on morphological characters, the systematic organization of Ege (1939) have long been widely accepted by many biologists. However, Watanabe et al. (2004a) recently found that the morphological character described by Ege (1939) were not sufficient to classify all species of this genus without including the information on the geographic distribution of the specimens, which he used as a taxonomic character. Watanabe et al. (2004a) considered that the Ege’s (1939) key for species identification is partly invalid because many morphological characters used are overlapping in most species. Species recognition becomes especially important for tropical eels because nowadays they are commercially transported around the world for food (frozen) and aquaculture (a live) purposes. Ege's (1939) key is considered as insufficient especially in tropical areas, where geographic and morphological characters of eels are heavily overlapping and where scientific data are still scarce.
Indonesia is a wide equatorial characterized by archipelagos composed of around 71,480 islands and coastline of around 91,000. Biogeographically, Indonesia is at the crossroad of the Indian and Pacific Oceans, the western part of Indonesia is connected with Indian Ocean and the eastern is connected with the Pacific Ocean. Two thirds of the recognized 18 Anguilla species and subspecies are found in the tropical Pacific and seven species and subspecies of tropical eels range around Indonesia (Ege 1939; Castle & Williamson 1974, Arai
et al. 1999). Those are A. bicolor (two subspecies: A. b. bicolor and A. b.
pacifica), A. marmorata, A. celebesensis, A. borneensis, A. interioris, A. obscura
and A. nebulosa (subspecies: A. n. nebulosa). As a result, the position of
Indonesia appears to be center of origin the diversity of eel and is strategic in the knowledge of their evolution.
Several methods for species identification have been used on fish like conventional morphology and electrophoresis, immunoassay, liquid chromatography or molecular genetics assay (O’Reilly and Wright, 1995). Concerning eels, after demonstration that morphological characters were not sufficient to classify all species, molecular genetics have been recommended (Watanabe et al. 2004a). Most of the genetic approaches to species identification are based on the amplification of a partial sequence of mitochondria (mtDNA). The mtDNA, of maternal inheritance, shows no recombination, so that its sequences are more conservative. The gene 16S rRNA, relatively well
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conserved, is considered as a good marker for genera and species identification (Aoyama et al. 2000, 2001; Watanabe et al. 2004b, 2005; Gagnaire et al. 2007). However, it is not polymorphic enough between subspecies (Watanabe 2003). Moreover, it is important to develop the markers that could distinguish subspecies. One of the other genes used is cytochrome b (Cytb). This functional gene is positioned between tRNAGlu and tRNAThr genes. Many investigations on
Cytb focus on inheritance and evolution (Freenlad 2005). Several studies have used Cytb as a marker for identification of subspecies (Jain et al. 2008; Hyde et al. 2005).
Recently, several simple PCR techniques have been used to distinguish A.
japonica and A. anguilla (Sezaki et al. 2005), A. interioris and A. celebesensis
(Aoyama et al. 2000) and to distinguish A. anguilla and A. rostrata (Trautner 2006). Application of Real-Time PCR technique allowed identification of A.
japonica leptocephali (Watanabe et al. 2004b), using single nucleotide
polymorphism (SNP) (Itoi et al. 2005), Random Amplified Polymorphic DNA (RAPD) (Kim et al. 2009; Lehmann 2000), Restriction Fragment Length Polymorphism (RFLP) (Lin et al. 2001), most of these researches dealing with temperate area.
Rapid molecular identification of tropical eel began with Gagnaire et al. (2007), who developed semi-multiplex PCR and RFLP to identify four eel species in Indian Ocean. Multiplex PCR is a variant of PCR enabling simultaneous amplification of several targets in one reaction by using more than one pair of primers. The multiplex PCR produces amplicons on varying sizes that are specific to different DNA sequences (Rompler 2006). By using the species-specific primers in multiplex-PCR assays, the identification of several species in a simple, quick, low cost, sensitive, and highly reliable amplification is possible (Catanese et al. 2010).
In the present study we developed a method derived from multiplex PCR assay. In this method, the PCR is based on two “universal” forward Anguilla
primers (and so, called semi-multiplex) and seven specific reverse primers, one for each eel species or subspecies know from Indonesian waters. The method is based on the complete sequence of cytochrome b and 16S rRNA extracted from the specimens that was collected around Indonesia waters.
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Material and Methods
Specimens
The 1115 specimens examined in this study were collected around the Indonesian waters, covering all the geographic distribution of Anguilla species that were expected to occur in the country (see Appendix 1). Around 800 specimens are silver eels while the others are glass eels. Samples collection was conducted in rivers estuaries along the coasts of Indian Ocean, Pacific Ocean and around Arafura and Celebes Seas. The specimens were collected from 2008 to 2012. Species assignation was preliminary performed by using available morphological keys (Watanabe et al. 2004a and Reveilac et al. 2007). The first morphological characters, which are measurements in this study, a quantitative one as follows: the total length (LT), the dorsal fin length (LD), the anal fin length (LA). These measurements were used to calculate the distance between the origin of the dorsal and anal fins (DA) using the formulation
DA=100(LD – LA)LT-1 (Reveillac et al. 2007) (Fig.3). This character determined whether an individual was short-fin (FS) or long fin (FL). The accuracy of the
measurement is 0.01 mm by using the digital caliper. The second measurement of morphological character was qualitative parameters that are presence or absence of marbling and breadth of maxillary bands. According to Watanabe et al. (2004a) genus Anguilla can be divided into four groups based on three characters: presence or absence of marbling, wide and narrow maxillary band of teeth and origin of the dorsal fin; group 1, groups long dorsal fin with marbling skin and broad maxillary bands of teeth; group 2, groups long dorsal fin with marbling skin and narrow maxillary bands of teeth; group 3, groups long dorsal fin and no marbling and group 4, groups short dorsal fin without marbling skin. Tissues from anal fin, which were immediately stored in 95% ethanol, were used for genetic analysis.
Design of PCR primer
Nine semi-multiplex PCR primers were designed from sequence alignment performed on the cytochrome b (cyt b) and the 16S rRNA genes. The Cyt b and 16S rRNA sequence dataset include sequence from GenBank (ref. AP007236, AP007237, AP007238, AP007239, AP007241, AP007242, AP007246) and 100 sequences obtained during the present study. Nine position of original sets for semi-multiplex PCR primers are shown in Table 1. Each
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Haplotype_69 C A . . T A . . A C . T . . A . A T . . T G . . T . C . . . T . . . G . T . A . . . . T C T . . . T . . C . A T T C T T . . A . . . A . . A . . . T . . . C . . A . C . C Haplotype_70 C A . . T A . . A C . T . . A . A T . . T G . . T . C . . . T . . . G . T . A . . . . T C T . . . T . . C . G T T C T T . . A . . . A . . A . . . T . . . C . . A . C . C Haplotype_71 C A . . T A . . A C . T . . A . A T . . T G . . T . C . . . T . . . G . T . A . . . . T C T . . . T . . C . A T T C T T . . A . . . A . . A . . . T . . . C . . A . C . C Haplotype_72 C A . . T A . . A C . T . . A . A T . . T G . . T . C . . . T . . . G . T . A . . . . T C T . . . T . . C . A T T C T T . . A . . . A . . A . . . T . . . C . . A . C . C Haplotype_73 C A . . T A . . A C . T . . A . A T . . T G . . T . C . . . T . . . G . T . A . . . . T C T . . . T . . C . A T T C T T . . A . . . T . . . A . . A . . . T . . . C . . A . C . C Haplotype_74 C A . . T A . . A C . T . . A . A T . . T G . . T . C . . . T . . T . . . G . T . A . . . . T C T . . . T . . C . A T T C T T . . A . . . A . . A . . . T . . . C . . A . C . C Haplotype_75 C A . . . A . . A C . T . . A . A T . . T G . . T . C . . . T . . . G . T . A . . . . T C T . . . T . . C . A T T C T T . . A . . . A . . A . . . T . . . C . . A . C . C Haplotype_76 C A . . T A . . A C . T . . A . A T . . T G . . T . C . . . T . T . . . G . T . A . . . . T C T . . . T . . C . A T T C T T . . A . . . A . . A . . . T . . . C . . A . C . C Haplotype_77 C A . . T A . . A C . T . . A . A T . . T G . . T . C . . . T . . . T . A . . . . T C T . . . T . T C . A T T C T T . . A . . . A . . A . . . T . . . C . . A . C . C Haplotype_78 C . . . T A . . A C . T . . A . G T . . T . . . T . C . . . T . . . T . . G . T . A T T . . T C T . . . T C . A T T C T . . . A . . G . . . A . . A . . . T . . . C . . A . C . C Haplotype_79 C . . . T A . . A C . T . . A . G T . . T . . . T . C . . . T . . . T . . G . C . A T . . . T C T . . . T C . A T T C T . . . A . . G . . . A . . A . . . T . . . C . . A . C . C Haplotype_80 C . . . T A . . A C . T . . A . G T . . T . . . T . C . . . T . . . T . . G . T . A T . . . T C T . . . T C . A T T C T . . . A . . G . . . A . . A . . . T . . . . G . C . . A . C . C Haplotype_81 C . . . T A . . A C . T . . A . G T . . T . . . T . C . . . T . . . T . . G . T . A T . . . T C T . . . T C . A T T C T . . . A . . G . . . A . . A . . . T . . . C . . A . C . C Haplotype_82 C . . . T A . . A C . T . . A . G T . . T . . . T . C . . . T . . . T . . G . T . A . . . . T C T . . . T C . A T T C T . . . A . . G . . . A . . A . . . T . . . C . . A . C . C Haplotype_83 C . . . T A . . A C . T . . A . G T . . T . . . T . C . . . T . . . C . . T . . G . T . A T . . . T C T . . . T C . A T T C T . . . A . . G . . . A . . A . . . T . . . C . . A . C . C Haplotype_84 C . . . T A . . A C . T . . A . G T . . T . . . T . C . . . T . . . T . . G . T . A T . . . T C T . . . T C . A T T C T . . . A . . G . . . A . . A . . . T . . . C . . A . C . C Haplotype_85 C . . . T A . . A C . T . . A . G T . T T . . . T . C . . . T . . . T . . G . T . A T . . . T C T . . . T C . A T T C T . . . A . . G . . . A . . A . . . T . . . C . . A . C . C Haplotype_86 C . . . T A . . A C . T . . A . G T . . T . . . T . C . . . T . . . T . . G . T . A T . . . T C T . . . T C . A T T C T . . . A . . G . . . A . . A . . . T . . . C . . A . C . C Haplotype_87 C . . . T A . . A C . T . . A . G T . . T . . . T . C . . . T . . . T . . G . T . A T . . . T C T . . . T C . A T T C T . . . A . . G . . . A . . A . . . T . . -Haplotype_88 C . . . T A . . A C . T . . A . G T . . T . . . T . C . . . T . . . G . T . A T . . . T C T . . . T C . A T T C T . . . A . . G . . . A . . A . . . T . . . C . . A . C . C Haplotype_89 C . . . T A . . A C . T . . A . A T . . T . . . T . C . . . T . . . T . . G . T . A T . . . T C T . . . T C . A T T C T . . . A . . G . . . A . . A . . . T . . . C . . A . C . C Haplotype_90 C . . . T A . . A C . T . . A . G T . . T . . . T . C . . . T . . . T . . . . T . A T . . . T C T . . . T C . A T T C T . . . A . . G . . . A . . A . . . T . . . C . . A T C . C Haplotype_91 C . . . T A . . A C . T . . A . G T . . T . . . T . C . . . T . . . T . . G . T . A T . . . T C T . . . T C . A T T C T . . . A . . G . . . A . . A . . . T . . . C . . A . . . C Haplotype_92 C . . . T A . . A C . T . . A . G T . . T . . . T . C . . . T . . . . G . T . T G . T . A T . . . T C T . . . T C . A T T C T . . . A . . G . . . A . . A . . . T . . . C . . A . C . C Haplotype_93 C . . . T A . . A C . T . . A . G T . . T . . . T . C . . . T . . . . G . T . T G . T . A T . . . T C T . . . T C . A T T C T . . . A . . G . . . A . . A . . . T . . . C . . A -Haplotype_94 C . . . T A . . A C . T . . A . G T . . T . . . T . C . . . T . . . . G . T . T G . T . A T . . . T C T . . . T C . A T T C T . . . A . . G . . . A . . A . . . T . . . C . . A . C . C Haplotype_95 C . . . T A . . A C . T . . A . G T . . T . . . T . C . G . . . T . . . . G . T . T G . T . A T . . . T C T . . . . T T C . A T T C T . . . A . . G . . . A . . A . . . T . . . C . . A . C . C Haplotype_96 C . . . T A . . A C . T . . A . G T . . T . . . T . C . . . T . . . T . T G . T . A T . . . T C T . . . T C . A T T C T . . . A . . G . . . A . . A . . . T . . . C . . A . C . C Haplotype_97 C A . . T A . . A C . T . . A . G T . . T . . . T . C . . . T . T G . T . A T . . . T C T . . C . . T C . A T T C T . . . A . . . C . . . A . . A . . . T . . . C . . A . C . C Haplotype_98 C A . . T A . . A C . T . . A . G T . . T . . . T . C . . . T . T G . T . A T . . . T C T . . C . . T C . A T T C T . . . A . . . A . . A . . . T . . . C . . A . C . C Haplotype_99 C A . G T A . . A C . T . . G . G T . . T . . . T . C . . . T . T G . T . A T . . . T C T . . C . . T C . A T T C T . . . A . . . A . . A . . . T . . . C . . A . C . C Haplotype_100 C A . G T A . . A C . T . . A . G T . . T . . . T . C . . . T . T G . T . A T . . . T C T . . C . . T C . A T T C T . . . A . . . A . . A . . . T . . . C . . A T C . C Haplotype_101 C A . G T A . . A C . T . . A . G T . . T . . . T . C . . . T . T . . T . A T . . . T C T . . C . . T C . A T T C T . . . A . . . A . . A . . . T . . . C . . A . C . C Haplotype_102 C A . G T A . . A C T T . . A . G T . . T . . . T . C . . . T . T G . T . A T . . . T C T . . C . . T C . A T T C T . . . A . . . A . . A . . . T . . . C . . A . C . C Haplotype_103 C A . G T A . . A C . T . . A . G T . . T . . . T . C . . . T . T G . T . A T . . . T C T . . C . . T C . A T T C T . . . A . . . A . . A . . . T . . . C . -Haplotype_104 C A . G T A . . A C . T . . A . G T . . T . . . T . C . . . T . T G . T . A T . . . T C T . . C . . T C . A T T C T . . . A . . . A . T A . . . T . . . C . . A . C . C Haplotype_105 C A . G T A . . A C . T . . A . G T . . T . . . T . C . . . T . T . . T . A T . . . T C T . . C . . T C . A T T C T . . . A . . . A . . A . . . T . . . C . . A . C . C Haplotype_106 C A . G T A . . A C . T . . A . G T . . T . . . T . C . . . T . T . . T . A T . . . T C T . . C . . T C . A T T C T . . . A . . . A . . A . . . T . . . C . . A . C . C Haplotype_107 C A . G T A . . A C . T . . A T G T . . T . . . T . C . . . T . T . . T . A T . . . T C T . . C . . T C . A T T C T . . . A . . . A . . A . . . T . . . C . . A . C . C Haplotype_108 C A . G T A . . A C . T . . A . G T . . T . . . T . C . . . T T . T . . T . A T . . . T C T . . C . . T C . A T T C T . . . A . . . A . . A . . . T . . . C . . A . C . C Haplotype_109 C A . G T A . . A C . T . . A . G T . . T . . . T . C . . . T T . T G . T . A T . . . T C T . . C . . T C . A T T C T . . . A . . . A . . A . . . T . . . C . . A . C . C Haplotype_110 C A . G T A . . A C . T . . A . G T . . T . . . T . C . . . T T . T G . T . A T . . . T C T . . C . . T C . A T T C T . . . A . . . A . . A . . . T . . . C . . A . C . C Haplotype_111 C A . G T A . . A C . T . . A . G T . . T . . . T . C . . . T T . T G . T . A T . . . T C T . . C . . T C . A T T C T . . . A . . . A . . A . . . T . . . C . . A . C . C Haplotype_112 C A . G T A . . A C . T . . A . G T . . T . . . T . C . . . T . T G . T . A T . . . T C T . . C . . T C . A T T C T . . . A . G . . . A . . A . . . T . . . C . . A . C . C Haplotype_113 C A . G T A . . A C . T . . A . A T . . T . . . T . C . . . T . T G . T . A T . . . T C T . . C . . T C . A T T C T . . . A . . . A . . A . . . T . . . C . . A . C . C Haplotype_114 C A . G T A . . A C . T . . A . G T . . T . . . T . C . . . T . T G . T . A T . . . T C T . . C . . T C . A T T C T . . . A . . . A . . A . . . T . . . C . . A . C . C Haplotype_115 C A . G T A . . A C . . . . A . G T . . T . . . T . C . . . T . T G . T . A T . . . T C T . . C . . T C . A T T C T . . . A . . . A . . A . . . T . . . C . . A . C . C Haplotype_116 C A . G T A . . A C . T . . A . G T . . T . . . T . C . . . T . T G . T . A T . . . T C T . . C . . T C . A T T C T . . . A . . . A . . A . . . T . . . C . . A . C . C Haplotype_117 C A . G T A . . A C . T . . A . G T . . T . . . T . C . . . T . . . T . T G . T . A T . . . T C T . . C . . T C . A T T C T . . . A . . . A . . A . . . T . . . C . . A . C . C Haplotype_118 C A . G T A . . A C . T . T A . G T . . T . . . T . C . . . T . T G . T . A T . . . T C T . . C . . T C . A T T C T . . . A . . . A . . A . . . T . . . C . . A . C . C Haplotype_119 C A . G T A . . A C . T . . A . G T . . T . . . T . C . . . T . T G . T . A T . . . T C T . . C . . T C . A T T C T . . . A . . . A . . A . . . T . . . C . . A . C T C Haplotype_120 C A . G T A . . A C . T . . A . G T G . T . . . T . C . . . T . T G . T . A T . . . T C T . . C . . T C . A T T C T . . . A . . . A . . A . . . T . . . C . . A . C . C Haplotype_121 C A . G T A . . A C . T . . A . G T . . T . . . T . C . . . T . T G . T . A T . . . T C T . . C . . T C . A T T C T . . . A . . . A . . A . . . T . . . C . . A . C . C Haplotype_122 C A . G T A . . A C . T . . A . G T . . T . . . T . C . . . T . T G . T . A T . . . T C T . . C . . T C . A T T C T . . . A . . . A . . A . . . T . . . C . . A . C . C Haplotype_123 C A . G T A . . A C . T . . A . G T . . T . . . T . C . . . T . T G . T . A T . . . T C T . . C . . T C . A T T C T . . . A . . . A . . A . . . T . . . C . . A . C . C Haplotype_124 . A . . T A T . A C . . . A T . . T . . G . . C . . . . C . . T T . . T T . . . C C . C . . . C . . . C . . . C . C . . C T . G . . . C . . T . . . G . C . . . C . . . C . . A . . . C Haplotype_125 . A . . T A T . A C . . . A T . . T . . G . . C . . . . C . . T T . . T T . . . C C . C . . . C . . . C . . . C . C . . C T . G . . . C . . T . . . C . . . C . . A . . . C Haplotype_126 . A . . T A T . A C . . . A T . . T . . G . . C . . . . C . . T T . . T T . . . C C . C . . . C . . . C . . . C . C . . C T . G . . . C . . T . . . C . . . C . . A . . . C Haplotype_127 . A G . T A T . A C . . . A T . . T . . G . . C . . . . C . . T T . . T T . . . C C . C . . . C . . . C . . . C . C . . C T . G . . . C . . T . . . T . . . C . . A . . . C Haplotype_128 . A G . T A T . A C . . . A T . . T . . G . . C . . . . C . . T T . . T T . . . C C . C . . . C . . . C . . . C . C . . C T . G . . . C . . T . . . G T . . . C G . A . . . C Haplotype_129 . A G . T A T . A C . . . A T . . . G . . C . . . . C . . T T . . . T . . . C C . C . . . C . . . C . . . C . C . . C T . G . . . C . . T . . . C T . . . G . . . . C . . . C Haplotype_130 . A G . T A T . A C . . . A T . . . G . . C . . . . C . . T T . . . T . . . C C . C . . . C . . . C . . . C . C . . C T . G . . T . . . C . . T . . . C T . . . C . . A . . . C Haplotype_131 . A G . T A T . A C . . G . . . A T . . . G . . C . . . . C . . T T . . . T . . . C C . C . . . C . . . C . . . C . C . . C T . G . . T . . . C . . T . . . C T . . . C . . A . . . C Haplotype_132 . A G . T A T . A C . . . A T . . . G . . C . . . . C . . T T . . . T . . . C C . C . . . C . . . C . . . C . C . . C T . G . . T . . . C . . T . . . C T . . . C . . A . . . C Haplotype_133 . A G . T A T . A C . . . A T . . . G . . C . . . . C . . T T . . . T . . . C C . C . . . C . . . C . . . C . C . . C T . G . . T . . . C . . T . . . C T . . . C . . A . . . C Haplotype_134 C A . . . A C . . . A . . T . . . C . . C . . G . C T . . . T . . . T . A . . C . C T . T . . C C C . . C . . . C C . . . C C T . . A . . . T . . . A . C . C
720 840 960 1042
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Appendix 4. Frequency allele for each locus, each localities for all specimen
LOCUS Ace Pad Ben PelR Pang Bal Obi Poig Dong Beng
AjTR12
(Nspeciemen) 22 24 22 24 21 23 7 4 18 11
140 0.0000 0.0000 0.0227 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 142 0.0000 0.0000 0.0000 0.0000 0.0238 0.0435 0.0000 0.0000 0.0000 0.0000 146 0.0000 0.0208 0.0227 0.0417 0.0476 0.0435 0.1429 0.0000 0.0278 0.0455 148 0.0000 0.0000 0.0227 0.0208 0.0000 0.0000 0.0000 0.0000 0.0278 0.0455 150 0.0682 0.0417 0.0227 0.1458 0.0476 0.1087 0.1429 0.0000 0.0000 0.0000 152 0.1818 0.0833 0.0227 0.0417 0.0238 0.0870 0.0000 0.0000 0.0000 0.0909 154 0.2727 0.2292 0.2273 0.0417 0.2857 0.2391 0.0000 0.0000 0.0833 0.1364 156 0.1136 0.1250 0.1591 0.1042 0.1429 0.0217 0.0714 0.1250 0.3333 0.2273 158 0.1591 0.0625 0.2045 0.1667 0.1429 0.0652 0.3571 0.5000 0.3333 0.1818 160 0.0909 0.0417 0.0455 0.1667 0.0952 0.0217 0.0714 0.0000 0.1111 0.0455 162 0.0682 0.1458 0.0682 0.1458 0.0476 0.2174 0.0714 0.0000 0.0556 0.0000 164 0.0000 0.0625 0.0227 0.0625 0.0476 0.0435 0.0000 0.0000 0.0278 0.1364 166 0.0000 0.0000 0.0909 0.0208 0.0000 0.0217 0.0000 0.0000 0.0000 0.0000 168 0.0000 0.0417 0.0000 0.0000 0.0000 0.0217 0.0000 0.0000 0.0000 0.0909 170 0.0455 0.0417 0.0000 0.0208 0.0000 0.0217 0.0000 0.3750 0.0000 0.0000 172 0.0000 0.0000 0.0227 0.0208 0.0476 0.0000 0.0000 0.0000 0.0000 0.0000 174 0.0000 0.0000 0.0000 0.0000 0.0238 0.0435 0.1429 0.0000 0.0000 0.0000 176 0.0000 0.0208 0.0227 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 178 0.0000 0.0208 0.0000 0.0000 0.0238 0.0000 0.0000 0.0000 0.0000 0.0000 180 0.0000 0.0000 0.0227 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 182 0.0000 0.0417 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 186 0.0000 0.0208 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 AjTR45
(Nspeciemen) 24 24 22 23 20 22 7 4 17 11
135 0.0625 0.0625 0.0227 0.0000 0.1000 0.0909 0.0000 0.0000 0.0294 0.0000 137 0.1875 0.2708 0.2955 0.2174 0.3500 0.2500 0.0714 0.1250 0.0882 0.0909 139 0.1458 0.0417 0.0000 0.1739 0.1250 0.0455 0.0714 0.0000 0.1176 0.0455 141 0.1458 0.1042 0.1136 0.1304 0.0250 0.0455 0.0000 0.0000 0.0294 0.0455 143 0.0833 0.0625 0.0909 0.0870 0.0500 0.1591 0.0000 0.2500 0.0588 0.0000 145 0.0625 0.1042 0.0455 0.0435 0.0500 0.0909 0.1429 0.0000 0.0882 0.0455 147 0.2708 0.1458 0.1364 0.1522 0.1500 0.2500 0.0714 0.3750 0.1471 0.3636 149 0.0417 0.1667 0.1136 0.1522 0.0500 0.0682 0.5000 0.1250 0.3235 0.3182 151 0.0000 0.0208 0.1364 0.0435 0.0500 0.0000 0.1429 0.1250 0.1176 0.0909 153 0.0000 0.0208 0.0227 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 155 0.0000 0.0000 0.0227 0.0000 0.0500 0.0000 0.0000 0.0000 0.0000 0.0000 Aro63
(Nspeciemen) 24 24 21 22 20 23 6 4 15 10
160 0.0000 0.0000 0.0000 0.0455 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 162 0.0000 0.0000 0.0000 0.0227 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 166 0.0000 0.0000 0.0000 0.0000 0.0250 0.0000 0.0000 0.0000 0.0000 0.0000 168 0.0208 0.0000 0.0000 0.0227 0.0500 0.0000 0.0000 0.0000 0.0000 0.0000 170 0.0208 0.0000 0.0000 0.0227 0.0000 0.0217 0.0000 0.0000 0.0000 0.0000 172 0.0000 0.0208 0.0000 0.0227 0.0250 0.0000 0.0000 0.0000 0.0000 0.0000 174 0.0000 0.0208 0.0000 0.0000 0.0500 0.0000 0.0000 0.0000 0.0000 0.0000 176 0.0000 0.0417 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 178 0.0000 0.0000 0.0000 0.0682 0.0000 0.0435 0.0000 0.0000 0.0000 0.0000 180 0.0000 0.0208 0.0000 0.0455 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 182 0.0417 0.0000 0.0000 0.0227 0.0000 0.0435 0.1667 0.0000 0.0667 0.2500 184 0.0000 0.0417 0.0000 0.0227 0.0000 0.0000 0.0833 0.0000 0.1333 0.0000 186 0.0625 0.0833 0.0476 0.0000 0.0000 0.0000 0.0000 0.0000 0.0667 0.0000 188 0.0417 0.0625 0.1429 0.0227 0.1250 0.0435 0.0000 0.2500 0.0667 0.1000 190 0.0833 0.1458 0.1429 0.1364 0.0000 0.0870 0.1667 0.0000 0.0000 0.0500 192 0.0625 0.0625 0.0000 0.0682 0.0750 0.0652 0.0000 0.1250 0.0000 0.2000 194 0.2083 0.0000 0.0952 0.0455 0.0500 0.1304 0.0833 0.0000 0.1333 0.0000 196 0.0625 0.0417 0.0000 0.0909 0.1250 0.0217 0.3333 0.1250 0.0667 0.1000 198 0.1458 0.0833 0.0238 0.1136 0.0250 0.0870 0.0833 0.2500 0.1000 0.0000 200 0.0625 0.0625 0.0238 0.0455 0.0000 0.1087 0.0000 0.0000 0.0000 0.0000 202 0.0417 0.0000 0.0238 0.0227 0.1500 0.0000 0.0000 0.1250 0.0000 0.1500 204 0.0208 0.0833 0.1190 0.0227 0.1250 0.0000 0.0000 0.1250 0.1000 0.0500 206 0.0833 0.0000 0.0238 0.0455 0.0000 0.0652 0.0000 0.0000 0.0000 0.0500 208 0.0000 0.0833 0.0714 0.0000 0.0000 0.0217 0.0833 0.0000 0.0000 0.0500 210 0.0000 0.0208 0.0000 0.0000 0.0000 0.1304 0.0000 0.0000 0.0000 0.0000 212 0.0208 0.1042 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0667 0.0000 214 0.0000 0.0208 0.0952 0.0227 0.0250 0.0000 0.0000 0.0000 0.0667 0.0000 216 0.0000 0.0000 0.0238 0.0000 0.0250 0.0000 0.0000 0.0000 0.0667 0.0000 218 0.0000 0.0000 0.0000 0.0000 0.0500 0.0870 0.0000 0.0000 0.0000 0.0000 220 0.0000 0.0000 0.0714 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 222 0.0208 0.0000 0.0714 0.0455 0.0250 0.0435 0.0000 0.0000 0.0000 0.0000 224 0.0000 0.0000 0.0000 0.0000 0.0250 0.0000 0.0000 0.0000 0.0667 0.0000 226 0.0000 0.0000 0.0000 0.0227 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 230 0.0000 0.0000 0.0238 0.0000 0.0250 0.0000 0.0000 0.0000 0.0000 0.0000 AjTR37
(Nspeciemen) 23 24 22 24 21 24 7 4 18 11
186 0.0000 0.0000 0.0227 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 188 0.0435 0.0208 0.0000 0.0208 0.0952 0.0417 0.0000 0.1250 0.0278 0.0000 190 0.0217 0.0208 0.0000 0.0208 0.0476 0.0417 0.0000 0.0000 0.0278 0.0000 192 0.0870 0.0417 0.0227 0.0833 0.0476 0.0625 0.0714 0.1250 0.0556 0.0909 194 0.0000 0.0000 0.0909 0.1250 0.0000 0.1042 0.0000 0.0000 0.0556 0.0909 196 0.1087 0.0833 0.0909 0.0833 0.1190 0.0625 0.0000 0.0000 0.0278 0.1364 198 0.1087 0.1458 0.1136 0.1458 0.1190 0.1250 0.0000 0.2500 0.0278 0.0455 200 0.2609 0.1458 0.2500 0.2083 0.1905 0.1667 0.2143 0.0000 0.1389 0.0000 202 0.0435 0.1250 0.1136 0.0833 0.0238 0.0625 0.2857 0.2500 0.1111 0.0000 204 0.0435 0.0625 0.0682 0.0833 0.0476 0.0625 0.0000 0.0000 0.0833 0.1364 206 0.0870 0.1667 0.0682 0.0208 0.0952 0.0625 0.1429 0.0000 0.0556 0.0909 208 0.0435 0.0208 0.0682 0.0417 0.0476 0.0208 0.1429 0.1250 0.1111 0.1818
(3)
210 0.0000 0.1042 0.0227 0.0625 0.0714 0.0833 0.0000 0.1250 0.0833 0.0000 212 0.0435 0.0208 0.0000 0.0000 0.0238 0.0417 0.0000 0.0000 0.0278 0.0455 214 0.0435 0.0208 0.0000 0.0208 0.0238 0.0208 0.0714 0.0000 0.0556 0.0000 216 0.0435 0.0000 0.0227 0.0000 0.0238 0.0208 0.0000 0.0000 0.0000 0.0000 218 0.0217 0.0000 0.0227 0.0000 0.0238 0.0208 0.0000 0.0000 0.0000 0.0000 220 0.0000 0.0000 0.0227 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0909 222 0.0000 0.0208 0.0000 0.0000 0.0000 0.0000 0.0714 0.0000 0.0000 0.0455 224 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0278 0.0000 230 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0278 0.0455 232 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0278 0.0000 234 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0278 0.0000 Aro54
(Nspeciemen) 22 24 21 24 20 20 4 3 16 11 136 0.0000 0.0208 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 142 0.0000 0.0208 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 146 0.0000 0.0208 0.0000 0.0000 0.0250 0.0000 0.0000 0.0000 0.0000 0.0000 148 0.0227 0.0000 0.0238 0.0208 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 150 0.0227 0.0208 0.0000 0.0208 0.0000 0.0500 0.0000 0.0000 0.0313 0.0000 152 0.1136 0.0208 0.0000 0.0000 0.0000 0.0750 0.0000 0.0000 0.0938 0.0909 154 0.1364 0.0417 0.0952 0.0208 0.0750 0.0250 0.0000 0.0000 0.0625 0.0455 156 0.1591 0.1042 0.0000 0.0208 0.0750 0.1500 0.2500 0.0000 0.0938 0.0455 158 0.0909 0.1667 0.2143 0.0208 0.1000 0.2250 0.0000 0.1667 0.0625 0.0455 160 0.0000 0.0000 0.0238 0.4583 0.0500 0.3000 0.1250 0.1667 0.0000 0.0000 162 0.0682 0.1250 0.2857 0.0208 0.2750 0.0250 0.0000 0.1667 0.0625 0.0455 164 0.1364 0.1667 0.1429 0.3125 0.0250 0.1000 0.2500 0.1667 0.1563 0.1818 166 0.0455 0.1042 0.0952 0.0417 0.1750 0.0500 0.0000 0.3333 0.1563 0.0909 168 0.0455 0.0000 0.0238 0.0000 0.0000 0.0000 0.0000 0.0000 0.1250 0.1364 170 0.0000 0.0833 0.0000 0.0208 0.0000 0.0000 0.1250 0.0000 0.0625 0.1818 172 0.0227 0.0208 0.0238 0.0000 0.0000 0.0000 0.2500 0.0000 0.0625 0.0455 174 0.0455 0.0000 0.0000 0.0208 0.0750 0.0000 0.0000 0.0000 0.0000 0.0455 176 0.0227 0.0417 0.0238 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 178 0.0000 0.0000 0.0238 0.0208 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 180 0.0227 0.0000 0.0000 0.0000 0.0250 0.0000 0.0000 0.0000 0.0000 0.0455 182 0.0000 0.0208 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 184 0.0000 0.0208 0.0238 0.0000 0.0500 0.0000 0.0000 0.0000 0.0000 0.0000 186 0.0000 0.0000 0.0000 0.0000 0.0250 0.0000 0.0000 0.0000 0.0000 0.0000 188 0.0227 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 194 0.0227 0.0000 0.0000 0.0000 0.0250 0.0000 0.0000 0.0000 0.0000 0.0000 200 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0313 0.0000 Aro95
(Nspeciemen) 24 24 22 23 21 24 7 4 16 11 86 0.0000 0.0208 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 90 0.0000 0.0208 0.0000 0.0217 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 92 0.0000 0.0208 0.0000 0.0000 0.0000 0.0000 0.0000 0.2500 0.0000 0.0000 94 0.0208 0.0417 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 96 0.0417 0.0833 0.0000 0.0217 0.0238 0.0000 0.0000 0.1250 0.0313 0.0455 98 0.0208 0.0208 0.0000 0.0435 0.0000 0.0625 0.0000 0.0000 0.0000 0.0000 100 0.1042 0.1667 0.0227 0.1304 0.1429 0.2292 0.0000 0.0000 0.0625 0.0000 102 0.0625 0.1875 0.2045 0.2174 0.0952 0.1458 0.0000 0.0000 0.2188 0.2273 104 0.0208 0.0625 0.1364 0.0435 0.1190 0.0833 0.2143 0.2500 0.0625 0.0909 106 0.2500 0.1667 0.0455 0.2174 0.1667 0.1875 0.3571 0.1250 0.1250 0.2727 108 0.1042 0.0417 0.0455 0.0435 0.0952 0.0833 0.3571 0.0000 0.1250 0.0000 110 0.0417 0.0417 0.0909 0.0217 0.0476 0.0417 0.0000 0.0000 0.1250 0.0909 112 0.0625 0.0417 0.0000 0.0435 0.0714 0.0625 0.0000 0.1250 0.0625 0.0000 114 0.0417 0.0208 0.0909 0.0217 0.1190 0.0208 0.0714 0.0000 0.0313 0.0000 116 0.0000 0.0208 0.0227 0.0435 0.0238 0.0417 0.0000 0.0000 0.0625 0.0000 118 0.1042 0.0208 0.0455 0.0652 0.0238 0.0208 0.0000 0.1250 0.0000 0.1364 120 0.0000 0.0000 0.0455 0.0652 0.0476 0.0208 0.0000 0.0000 0.0938 0.0455 122 0.0625 0.0208 0.1136 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 124 0.0208 0.0000 0.0000 0.0000 0.0238 0.0000 0.0000 0.0000 0.0000 0.0000 126 0.0208 0.0000 0.0455 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 128 0.0000 0.0000 0.0455 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 130 0.0208 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0909 134 0.0000 0.0000 0.0227 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 136 0.0000 0.0000 0.0227 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 AjTR04
(Nspeciemen) 23 24 22 24 21 24 7 4 18 11 177 0.0000 0.0417 0.0000 0.0000 0.0238 0.0000 0.0000 0.0000 0.0000 0.0000 179 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0455 181 0.0000 0.0000 0.0000 0.0208 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 183 0.0000 0.0208 0.0455 0.0625 0.0952 0.0417 0.0000 0.0000 0.0556 0.0000 185 0.0000 0.0208 0.0227 0.0000 0.0238 0.0000 0.0714 0.0000 0.0278 0.0455 187 0.0870 0.0833 0.1818 0.0833 0.0476 0.1250 0.0000 0.2500 0.0278 0.0909 189 0.0652 0.0833 0.1591 0.1042 0.1905 0.1042 0.0714 0.1250 0.0833 0.0455 191 0.1087 0.0833 0.1136 0.0417 0.0238 0.1250 0.0000 0.0000 0.0278 0.0000 193 0.1522 0.0000 0.0682 0.1250 0.1190 0.0625 0.1429 0.0000 0.0556 0.0909 195 0.1304 0.0417 0.0682 0.1042 0.1429 0.1042 0.0000 0.0000 0.0278 0.0000 197 0.0000 0.0417 0.0455 0.1042 0.0238 0.1667 0.0000 0.0000 0.0278 0.1818 199 0.1739 0.1458 0.0909 0.0625 0.0476 0.0833 0.1429 0.0000 0.0556 0.0909 201 0.0870 0.0000 0.0455 0.0625 0.0476 0.0208 0.0000 0.0000 0.1667 0.1818 203 0.0000 0.0417 0.0000 0.0208 0.0476 0.0000 0.3571 0.1250 0.0278 0.0909 205 0.0435 0.0833 0.0000 0.0208 0.0476 0.0417 0.0000 0.0000 0.0556 0.0000 207 0.0217 0.0417 0.0000 0.0625 0.0238 0.0625 0.0000 0.0000 0.0000 0.0000 209 0.0217 0.1042 0.0227 0.0625 0.0238 0.0208 0.0714 0.0000 0.0556 0.0000 211 0.0000 0.0000 0.0227 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 213 0.0000 0.0000 0.0455 0.0208 0.0000 0.0000 0.0000 0.2500 0.0000 0.0000 215 0.0000 0.0417 0.0227 0.0208 0.0000 0.0000 0.0714 0.0000 0.0833 0.0000 217 0.0435 0.1042 0.0000 0.0000 0.0000 0.0208 0.0000 0.0000 0.0556 0.0000 219 0.0000 0.0000 0.0000 0.0000 0.0238 0.0000 0.0000 0.0000 0.0000 0.0000 221 0.0435 0.0000 0.0455 0.0000 0.0000 0.0000 0.0000 0.0000 0.0556 0.0000 225 0.0217 0.0000 0.0000 0.0208 0.0000 0.0208 0.0000 0.0000 0.0000 0.0000
(4)
227 0.0000 0.0208 0.0000 0.0000 0.0238 0.0000 0.0000 0.1250 0.0000 0.0455 229 0.0000 0.0000 0.0000 0.0000 0.0238 0.0000 0.0000 0.1250 0.0000 0.0000 231 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0556 0.0000 233 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0714 0.0000 0.0000 0.0000 241 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0556 0.0000 245 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0455 247 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0455
(5)
Appendix 5. F
is
for each locus, each localities for all specimen
LOCUS Ace Pad Bal PelR Obi Poig Dong Pang Beng Ben
AjTR12
(Nspeciemen) 22 24 23 24 7 4 18 21 11 22
142 0 0 0,023 0 0 0 0 0 0 0
146 0 0 0,023 1 0,091 0 0 0,026 0 0
150 0,050 0,022 0,1 0,184 0,091 0 0 0,026 0 0
152 0,706 0,471 0,073 0,022 0 0 0 0 1 0
154 0,106 0,428 0,421 0,022 0 0 0,653 0,143 0,643 0,012
156 0,105 0,122 0 0,349 0 0 0,521 0,626 0,268 0,167
158 0,173 0,045 0,048 0,179 0,143 1 0,277 0,245 0,429 0,045
160 0,468 0,022 0 0,418 0 0 1 0,081 0 0,024
162 0,656 0,184 0,254 0,514 0 0 1 0,026 0 0,656
164 0 0,045 0,023 0,657 0 0 0 0,026 0,643 0
166 0 0 0 0 0 0 0 0 0 0,077
168 0 0,022 0 0 0 0 0 0 1 0
170 0,024 0,022 0 0 0 0,571 0 0 0 0
172 0 0 0 0 0 0 0 0,026 0 0
174 0 0 0,023 0 0,091 0 0 0 0 0
182 0 1 0 0 0 0 0 0 0 0
Tous W&C 0,259 0,174 0,114 0,263 0 0,667 0,505 0,077 0,505 0,021
AjTR45
(Nspeciemen) 22 24 23 24 7 4 18 21 11 22
135 0,045 0,045 0,077 0 0 0 0 0,086 0 0
137 0,211 0,353 0,313 0,257 0 0 0,067 0,295 0,053 0,258
139 0,184 0,022 0,024 0,189 0 0 0,103 0,337 0 0
141 0,184 0,095 0,024 0,128 0 0 0 0 0 0,105
143 0,07 0,045 0,508 0,073 0 1 0,032 0,027 0 0,077
145 0,045 0,095 0,077 0,023 0,091 0 0,652 0,027 0 0,024
147 0,281 0,184 0,068 0,158 0 0,5 0,143 0,24 0,259 0,135
149 0,022 0,179 0,05 0,158 0,217 0 0,089 0,027 0 0,105
151 0 0 0 0,023 0,091 0 0,458 0,027 1 0,25
155 0 0 0 0 0 0 0 0,027 0 0
Tous W&C 0,07 0,113 0,024 0,158 0,048 0,143 0,103 0,016 0,191 0,047
Aro63
(Nspeciemen) 22 24 23 24 7 4 18 21 11 22
160 0 0 0 0,024 0 0 0 0 0 0
168 0 0 0 0 0 0 0 1 0 0
174 0 0 0 0 0 0 0 1 0 0
176 0 0,022 0 0 0 0 0 0 0 0
178 0 0 1 0,656 0 0 0 0 0 0
180 0 0 0 1 0 0 0 0 0 0
182 0,022 0 1 0 1 0 1 0 0,25 0
184 0 1 0 0 0 0 1 0 0 0
186 0,045 1 0 0 0 0 1 0 0 1
188 0,022 0,045 1 0 0 1 1 0,782 0,059 0,626
190 0,07 0,514 0,47 1 0,111 0 0 0 0 0,626
192 0,657 0,657 0,656 0,656 0 0 0 0,655 0,419 0
194 0,511 0 0,63 0,024 0 0 1 1 0 0,467
196 0,045 0,022 0 0,468 1 0 1 0,337 0,059 0
198 0,184 0,07 1 0,105 0 1 0,65 0 0 0
200 0,045 0,045 0,784 1 0 0 0 0 0 0
202 0,022 0 0 0 0 0 0 0,24 0,64 0
204 0 0,471 0 0 0 0 0,65 0,782 0 0,111
206 0,471 0 0,048 0,024 0 0 0 0 0 0
208 0 0,471 0 0 0 0 0 0 0 0,053
210 0 0 0,63 0 0 0 0 0 0 0
212 0 0,785 0 0 0 0 1 0 0 0
214 0 0 0 0 0 0 1 0 0 0,081
216 0 0 0 0 0 0 1 0 0 0
218 0 0 1 0 0 0 0 0,027 0 0
220 0 0 0 0 0 0 0 0 0 0,655
222 0 0 1 0,024 0 0 0 0 0 0,655
224 0 0 0 0 0 0 1 0 0 0
Tous W&C 0,186 0,385 0,679 0,339 0,455 0,5 0,931 0,471 0,222 0,338
AjTR37
(Nspeciemen) 22 24 23 24 7 4 18 21 11 22
188 0,023 0 0,022 0 0 0 0 0,081 0 0
190 0 0 0,022 0 0 0 0 0,026 0 0
192 0,073 0,022 0,045 0,07 0 0 0,03 0,026 0,053 0
194 0 0 0,095 0,258 0 0 0,03 0 0,053 0,077
196 0,1 0,07 0,045 0,07 0 0 0 0,341 0,111 0,077
198 0,1 0,184 0,258 0,15 0 0,2 0 0,111 0 0,105
200 0,343 0,514 0,179 0,011 0,625 0 0,329 0,098 0 0,313
202 0,023 0,258 0,045 0,07 0,333 0,2 0,46 0 0 0,105
204 0,023 0,657 0,045 0,07 0 0 0,063 0,026 0,111 0,05
206 0,47 0,418 0,045 0 0,091 0 0,03 0,081 0,053 0,05
(6)
210 0 0,095 0,07 0,045 0 0 0,063 0,053 0 0
212 0,023 0 0,022 0 0 0 0 0 0 0
214 0,023 0 0 0 0 0 0,03 0 0 0
216 0,023 0 0 0 0 0 0 0 0 0
220 0 0 0 0 0 0 0 0 0,053 0
Tous W&C 0,082 0,223 0,029 0,015 0,027 0,091 0,125 0,019 0,024 0,116
Aro54
(Nspeciemen) 22 24 23 24 7 4 18 21 11 22
150 0 0 0,027 0 0 0 0 0 0 0
152 0,105 0 0,056 0 0 0 0,071 0 1 0
154 0,135 1 0 0 0 0 0,034 0,056 0 0,467
156 0,167 0,349 0,24 0 0,2 0 0,071 0,655 0 0
158 0,077 0,121 0,587 0 0 0 0,034 0,086 0 0,034
160 0 0 0,309 0,154 0 0 0 0,027 0 0
162 0,05 0,632 0 0 0 0 0,034 0,395 0 0,322
164 0,135 0,418 0,086 0,241 1 0 0,318 0 0,176 0,245
166 0,024 0,349 0,027 0,022 0 1 0,318 0,159 0,053 0,081
168 0,024 0 0 0 0 0 0,111 0 0,111 0
170 0 0,471 0 0 0 0 0,034 0 0,176 0
172 0 0 0 0 0,2 0 0,034 0 0 0
174 0,024 0 0 0 0 0 0 0,056 0 0
176 0 0,022 0 0 0 0 0 0 0 0
184 0 0 0 0 0 0 0 0,027 0 0
Tous W&C 0,086 0,32 0,227 0,132 0,182 0,333 0,056 0,152 0,02 0,163
Aro95
(Nspeciemen) 22 24 23 24 7 4 18 21 11 22
92 0 0 0 0 0 0,2 0 0 0 0
94 0 0,022 0 0 0 0 0 0 0 0
96 0,022 0,07 0 0 0 0 0 0 0 0
98 0 0 0,045 0,023 0 0 0 0 0 0
100 0,095 0,179 0,04 0,63 0 0 0,034 0,626 0 0
102 0,045 0,064 0,514 0,254 0 0 0,477 0,467 0,268 0,596
104 0 0,657 0,07 0,023 0,625 0,2 0,034 0,341 1 0,135
106 0,09 0,121 0,211 0,254 0,727 0 0,455 0,167 0,574 0,024
108 0,095 0,022 0,471 0,023 0,727 0 0,455 0,467 0 0,024
110 0,022 0,022 0,022 0 0 0 1 1 0,053 0,077
112 0,045 0,022 0,045 0,023 0 0 0,034 0,655 0 0
114 0,022 0 0 0 0 0 0 0,111 0 0,468
116 0 0 0,022 0,023 0 0 1 0 0 0
118 0,095 0 0 0,048 0 0 0 0 0,643 0,024
120 0 0 0 0,656 0 0 0,071 0,026 0 1
122 0,045 0 0 0 0 0 0 0 0 0,344
126 0 0 0 0 0 0 0 0 0 0,024
128 0 0 0 0 0 0 0 0 0 1
130 0 0 0 0 0 0 0 0 1 0
Tous W&C 0,061 0,036 0,057 0,22 0,636 0,091 0,388 0,329 0,487 0,261
AjTR04
(Nspeciemen) 22 24 23 24 7 4 18 21 11 22
177 0 1 0 0 0 0 0 0 0 0
183 0 0 0,022 0,045 0 0 1 0,081 0 0,024
187 1 0,07 0,258 0,07 0 1 0 0,026 0,053 0,706
189 0,048 1 0,349 0,349 0 0 0,653 0,403 0 0,837
191 0,784 1 0,258 1 0 0 0 0 0 0,105
193 0,179 0 0,657 0,632 1 0 0,03 0,341 1 0,656
195 0,63 1 0,349 0,349 0 0 0 0,626 0 0,656
197 0 0,022 0,418 0,095 0 0 0 0 0,429 0,024
199 0,413 0,514 0,07 0,045 1 0 0,03 0,026 1 1
201 1 0 0 0,657 0 0 1 0,026 0,429 1
203 0 0,022 0 0 0,727 0 0 0,026 0,053 0
205 0,023 0,471 1 0 0 0 0,03 0,026 0 0
207 0 0,022 0,657 0,657 0 0 0 0 0 0
209 0 0,785 0 0,045 0 0 0,03 0 0 0
213 0 0 0 0 0 1 0 0 0 1
215 0 1 0 0 0 0 0,063 0 0 0
217 0,023 0,785 0 0 0 0 0,03 0 0 0
221 1 0 0 0 0 0 0,03 0 0 0,024
231 0 0 0 0 0 0 0,03 0 0 0
241 0 0 0 0 0 0 1 0 0 0