Jean -Christophe Avarre

DR Dr Odang Utut Carman

Pr. DR M Widyastuti

Pr Zairin Junior

Dr

Komar Sumantadina

D R Jean

DR Paul Marc Toutain

DR Etienne Legendre

DR Remi Baras

DR Emmanuel Dugue

DR. Dominico Paradis

DR Carrusso

Francois Jean

Madame Monique Agnese

M Saltel

Ibu Christophe Coche

Etny

Sumanta Bapak

Thank

Pelangia 1 Mbutaensis Allen

Rhadinocentrus 1 Ornatus Regan

B Pseudomugilidae

Arguni Bay

M Angfa Allen

Bird's Neck

M Arfakensis Allen

Bird's Head

M Arguni Kadarusman

Jasu Ck

M Batanta Allen

M Irianjaya Allen

Bomberai

M Kokasensis

Kokas Ck

M Longispina Nugraha

Klahfot R Bird's Head

M Manibuii Nugraha

Tisbo R Bird's Head

M Misoolensis Allen

Wai Tama

Misool Is

M Multiradiata

Doktor Ck

Salawati

M Batanta

Allen & Renyaan

Warmon Ck

Batanta Is

Bird

Irianjaya Allen

Kf491340

M Salawati Kadarusman

Kf491390

M Sembrae Nugraha

Sembra R Bird's Head

M Sikuensis Nugraha

Siku Ck

M Sneideri

Allen & Hadiaty

Small Ck In Kumawa

M Susii Nugraha

Susi Ck

M Synergos

Allen & Unmack

Wai Bin Ck

Pulau Batanta

L Sewiki

Gebiasi Ck

Wermura Ck

Aifuf Ck* Birds Head No samples

Keywords: Aquaculture, Biodiversité génétique, Microsatellites, Les poissons arc-en-ciel, Melanotaeia boesemani, Nouvelles espèces Melanotaenia, Phylogénie, Papouasie occidentale

genetic, conservation and evolution. Many biodiversity in New Guinea Island, particularly West Papua makes many misinterpretations about flora and fauna, particularly fresh water fish in West Papua area.

9. Dr. Kadarusman, thank you for sharing and knowledge about Papua and rainbow fish, also the exploration and expedition therefore there are many fish revealed there. 10. All academic staffs in IPB and Université Montpellier II France.

11. Franch embassy and Campus France -Mle. Apreita Putri, M. Farid Saadoun and M. Florian Libourel, thank you for all financial and administration supports.

12. CROUS Montpellier having helped all my life aspects during my studying in Montpellier. 13

GENERAL INTRODUCTION

The ornamental fish hobby has continuously grown over the last decades.

Some endemic fish from Indonesia have been very successfully sold abroad, like arowana (genus Scleropages) from Borneo island, botia (Chromobotia macracanthus)

from Borneo and Sumatera islands, Tiger barb (Puntigrus tetrazona) from Sumatera island, and rainbowfish (genus Melanotaenia) from Papua. Talking about rainbowfish (Melanotaeniidae), at the moment there are only 3 species cultivated in Indonesia, namely Glossolepis incisus [START_REF] Weber | Eine zoogeographische Prophezeiung[END_REF], Iriatherina werneri Meinken 1974, and Melanotaenia boesemani [START_REF] Allen | [END_REF]. Gerald Allen, an ichtiologist from Australia, was the first to perform deep researches on rainbowfish.

In The first species of rainbowfish was scientifically described in 1843 from a collection of freshwater fishes collected in King River (Northen Australia) and was named Atherina nigrans. In 1862, Thomas Gill proposed to place this species into a new genus called Melanotaenia; Melanotaenia nigrans then became the first member (i.e. nominal species) of this genus. The subfamily Melanotaeniidae was subsequently created by Gill in 1894.More recently, Munro (1964) claimed that Melanotaeniidae must represent an independant family. Between 1843 and 1964, there were about 30 species described as belonging to this family. Over the last four decades, many expeditions were conducted in New Guinea, and led to the discovery of an impressive level of rainbowfish diversity. Today, this family includes 102 species in 7 genera following [START_REF] Nugraha | Eight new species of Rainbowfishes (Melanotaeniidae) from the Birds Head Region, West Papua, Indonesia[END_REF] and Allen et al., (2015a, b):

Cairnsichthys (1 species), Chilatherina (11 species), Glossolepis (9 species),

Iriatherina (1 species), Melanotaenia (78 species), Pelangia (1 species) and

Rhadinocentrus (1 species) [START_REF] Eschmeyer | Catalog of fishes: genera, species, references[END_REF]Allen et al., 2014a.b). The greatest diversity of Melanotaeniidae is found on the island of New Guinea with 87 endemic species, while there are 13 species in Australia, and only 2 species spread between the two biogeographic regions [START_REF] Allen Gr Hadiaty | Melanotaenia sneideri, a new species of rainbowfish (Melanotaeniidae), from West Papua Province, Indonesia[END_REF].

Melanotaeniidae spread out throughout the island of New Guinea, as well as the large islands around the western part of the region, including Raja Ampat, Aru and Yapen islands. This fish can also be found throughout Northern Australia, along the east to the south coast of Murray Darling drainage, and in the Western part of Australia [START_REF] Allen | Field guide to the freshwater fishes of New Guinea[END_REF]. Melanotaeniidae belong to the most popular freshwater fish from the island of New Guinea and northern Australia [START_REF] Eschmeyer | Catalog of fishes: genera, species, references[END_REF]. They represent the most speciose group of pure freshwater fishes within the Australia-New Guinea region. They typically possess a compressed body covered by relatively large scales, two separate dorsal fins (the first with 3-7 spines and the second with a single spine and 6-22 segmented rays), a long-based anal fin, and no lateral line (Allen et al., 2008). The family is characterized by relatively small (usually less than 10 cm) and often brightly colored fish. Sexual dimorphism is often apparent and males tend to be deeper-bodied and more vividly colored than females [START_REF] Allen | Field guide to the freshwater fishes of New Guinea[END_REF]. Generally, they are locally abundant and occupy the full spectrum of freshwater habitats including arid-zone waterholes, swamps, rainforest streams, karst rocks and oxbow mountainous lakes and streams in the mountains.

The Boesman's rainbowfish Melanotaenia boesemani [START_REF] Allen | Descriptions of five new rainbowfish (Melanotaeniidae) from New Guinea[END_REF] is certainly the most popular species of rainbowfish both in Indonesia and abroad (Figure 1). This species is very different from most other rainbowfish because it harbors different colors between the anterior and posterior. The color of anterior (front) is bright blue and the color of posterior (back) is bright yellow. This species was first described by [START_REF] Allen | Descriptions of five new rainbowfish (Melanotaeniidae) from New Guinea[END_REF], from specimens caught by Marinus Boeseman in 1954-1955, which were kept in the museum Rijksmuseum van Natuurlijke Histoire of Leiden (Netherlands). This species is native from two distinct lakes in West Papua (i.e. Ayamaru and Uter), distant by around 30 km and separated by limestone mountains [START_REF] Kadarusman | Rainbowfishes from West Papua (Melanotaeniidae): Evolution and Systematics[END_REF]. Because of its popularity, the species has been greatly exploited in its natural habitat and is currently in the verge of extinction. Since 2004, this species is included in the CITES list as an endangered species (IUCN, 2013), and theoretically only fishery products are now allowed to be exported. However, Allen reported in 2007 that around 60,000 males of M. boesemani were caught monthly and exported for trading [START_REF] Allen | Fishes of Papua[END_REF]. Although Melanotaenia boesemani has been domesticated by Indonesian farmers since 1983, the number of farms that exploit this species in Indonesia is still very low. Besides, farmers claim that there is a decline in the production, in terms of quantity and quality, such as a higher proportion of females than males per spawning, a loss of body coloration, lower growth rate and fecundity, or frequent morphological abnormalities. They interpret this as a consequence of a loss of genetic variability and inbreeding.

In this context, my work aimed at gathering new genetic information that would be useful for the aquaculture and conservation of the Melanotaeniidae family.

Specifically, the objectives of the research were: 1) to develop new microsatellite DNA markers from the endangered M. boesemani, 2) to evaluate the genetic diversity of wild populations of Melanotaenia and refine their taxonomy, 3) to describe the geographic origins of M. boesemani reared by ornamental fish farmers in Indonesia, and to evaluate the genetic diversity and inbreeding pressure resulting from this domestication. My PhD work was a continuation of the PhD realized by Dr.

Kadarusman in the frame of the Lengguru program. Among other activities, this scientific program consists in describing and characterizing the genetic structure and diversity of Rainbowfishes in West Papua, through scientific expeditions jointly conducted by the Institut de Recherche pour le Développement (France), the Academy of Fisheries of Sorong (West Papua, Indonesia), the Aquaculture Research and Development Center for Ornamental Fish (Java, Indonesia), and the Indonesian Institute of Sciences (LIPI).

The present manuscript is divided into five sections: the first one consists in a bibliographic review; the next three ones present the results relating to 1) the development and succesfull validation of 12 polymorphic microsatellite DNA markers from Melanotaenia boesemani; 2) the use of these markers for analyzing the genetic diversity of wild populations of Melanotaenia from West Papua; 3) their use to investigate the genetic diversity and origin of reared strains of M. boesemani in Indonesia. Finally, a general discussion followed by perspectives will constitute the last section of this manuscript.

II. LITERATURE REVIEW

RAINBOWFISH DISTRIBUTION AND DIVERSITY

Rainbowfishes are very famous for ornamental trade since the 1930s [START_REF] Tappin | Rainbowfish Fishes, their care & keeping in captivity[END_REF], because of their vivid coloration that is reminiscent of a rainbow. They are endemic from New Guinea and Australia. The first rainbowfish (Melanotaenia nigrans) was scientifically described in 1843 from the collection of freshwater fish obtained in the northern region of Australia from the King River, near Victoria Settlement -Northern Territory-Australia. In 1964, Ian Munro determined that Rainbowfishes belonged to their own family. Gerald Allen, an ichthyologist from Western Australian Museum, gave a very important contribution to the systematics of Rainbowfishes inhabiting Australia and New Guinea with the description of dozen of new species and and several new genera. There are many endemic species of rainbowfish in various systems of river and lake waters with close phylogenetic and biogeography relationships both in Australia and New Guinea [START_REF] Zhu | Sequence evolution and phylogenetic signal in control-region and cytochrome b sequences of rainbow fishes (Melanotaeniidae)[END_REF][START_REF] Mcguigan | Phylogenetic relationships and historical biogeography of melanotaeniid fishes in Australia and New Guinea[END_REF][START_REF] Unmack | Biogeography of Australian freshwater fishes[END_REF]. Taxonomy of Melanotaeniidae has therefore a long story. There were as many as 30 species that had been discovered between 1843 and 1964 but several expeditions in New Guinea over the past four decades led to the description of more than 40 new additional species [START_REF] Kadarusman | Description of Melanotaenia fasinensis, a new species of rainbowfish (Melanotaeniidae) from West Papua, Indonesia with comments on the rediscovery of M. ajamaruensis and the endangered status of M. parva[END_REF]. Melanotaeniidae is believed to be part of the Atheriniforme order, which has evolved from the ancestors of atherinoids at sea (Allen, 1980).

There are currently 2 families of rainbowfish that have been described until now, and which are distributed on the islands of Australia and New Guinea (the region of Indonesia and Papua New Guinea) (Table 1): the family Melanotaeniidae, which includes 7 genera and 102 species, and the family Pseudomugilidae, composed of 18 species spread into 3 genera (Table 1). Melanotaenia is the largest genus of the Melanotaeniidae family [START_REF] Ivantsoff | A redfinned blue-eye, a new spesies and genus of the family Pseudomugilidae from central western Queensland[END_REF]. There are thirty-two species that are endemic from the Vogelkop Province [START_REF] Nugraha | Eight new species of Rainbowfishes (Melanotaeniidae) from the Birds Head Region, West Papua, Indonesia[END_REF]. The Vogelkop province belongs to the territory of West Papua, Indonesia and biogeographically should include for Melanotaeniids the western side of Lengguru Range (i.e. all watersheds flowing to Arguni Bay), the Bomberai and the Birds Head Peninsulas, the Birds Head Isthmus joining both peninsulas and the four major Raja Ampat islands (i.e. Misool, Batanta, Salawati, Waigeo) (Kadarusman et al., 2012a). [START_REF] Rubripinnis Allen & Renyaan | [END_REF]11. boesemani Allen & Cross, 198012. catherinae (de Beaufort, 1910) 13. caerulea Allen, 1996 New Guinea has a mountainous cordillera which runs along the central mountain which maximum height is reached at the peak Jaya (5030 meters), the highest point of Papua-New Guinea.

Monophyletic groups of strictly freshwater fishes have special significance in continental biogeography because they require freshwater for dispersal, and thus their distributions are correlated with the evolution of topography and watershed [START_REF] Lundberg | So many fishes, so little time: An overview of recent ichthyological discovery in continental waters[END_REF]. In general, the fish fauna of New Guinea is closely linked to that of northern Australia. The most diverse taxa are Eleotrididae and Gobiidae of about 115 species, followed by Melanotaeniidae of about 102 species. Around fifty species distributed in southern New Guinea are also found in northern Australia and are limited to these two regions [START_REF] Lundberg | So many fishes, so little time: An overview of recent ichthyological discovery in continental waters[END_REF]. Allen (1980) proposed a generic revision of Melanotaeniidae based on osteological characters and recognized Melanotaenia, Glossolepis and Chilatherina as three closely related genera, sharing a rigid fin spine at beginning of first dorsal, anal and pelvic fins (versus soft for the other members of the family). Allen (1998) described Pelangia and stated that this new genus was closely related to Glossolepis, particularly with regards to dentition, and morphology of the premaxillary, pelvic girdle, and pectoral fin.

THE MELANOTAENIIDAE FAMILY

Melanotaenia [START_REF] Gill | Notice of a collection of the fishes of California presented to the Smithsonian Institution by Mr. Samuel Hubbard[END_REF] is the most diverse genus with around 80 species and sub-species (Allen et al., 2008;Allen et al., 2015a, b;Kadarusman et al., 2012;[START_REF] Nugraha | Eight new species of Rainbowfishes (Melanotaeniidae) from the Birds Head Region, West Papua, Indonesia[END_REF]. The genus consists of 15 taxa in Australia and 89 taxa in New Guinea. The species Melanotaenia macculochi (Ogilby 1915) and M. splendida [START_REF] Peters | Mittheilung über Fische (Protopterus, Auliscops, Labrax, Labracoglossa, Nematocentris, Serranus, Scorpis, Opisthognathus, Scombresox, Acharnes, Anguilla, Gymnomuraena, Chilorhinus, Ophichthys, Helmichthys)[END_REF] are shared in both areas of Australia and New Guinea [START_REF] Rubripinnis Allen & Renyaan | [END_REF]. (M. arguni, M. fasinensis, M. salawati, M. urisa, M. veoliae, and M. wanoma) were consequently described [START_REF] Kadarusman | Description of Melanotaenia fasinensis, a new species of rainbowfish (Melanotaeniidae) from West Papua, Indonesia with comments on the rediscovery of M. ajamaruensis and the endangered status of M. parva[END_REF]2011;2012b).

Based on the PhD thesis of [START_REF] Kadarusman | Rainbowfishes from West Papua (Melanotaeniidae): Evolution and Systematics[END_REF], the biogeography of Melanotaeniidae distribution consisted of 4 groups (Figure 2):

1. Western New Guinea or Vogelkop Province including the four Raja Ampat islands, the Birds Head and Bomberai Peninsulas, and the western side of the Birds Head Isthmus.

2. The Central part of the Birds Head Isthmus.

3. Northern part of New Guinea (Northern New Guinea). The region covers an area of Great Northern part and the North Eastern Province [START_REF] Allen | Field guide to the freshwater fishes of New Guinea[END_REF].

This region includes the islands of Yapen, all streams in the northern part of Weyland Plateau (Wapoga, Siriwo etc) and the north New Guinea (Memberamo, Sepik, Markham etc).

4. Southern New Guinea and Australia. This section covers the Arafuru sea to Triton Lake, Cenderawasih Bay (from Munuari to Nabire) (Figure 2). These results gave a new view that there is a great diversity of Melanotaeniidae in the area of New Guinea and Australia, particularly West Papua which is the center of this family. 32 species belonging to the genus Melanotaenia have been described, [START_REF] Nugraha | Eight new species of Rainbowfishes (Melanotaeniidae) from the Birds Head Region, West Papua, Indonesia[END_REF]. Based on species composition, Bird's head Papua is believed to be the center of rainbowfish diversity [START_REF] Allen | Rainbowfishes in Nature and the Aquarium[END_REF]. Kadarusman et al. (2012a) demonstrated that species of Melanotaenia from the area of Bird's head are monophyletic compared to other clades. The result of the research done by Kadarusman et al. (2012a) showed an unpredictable diversity level, and it also showed that the diversity of Melanotaenia was still underestimated. (Figure 3).

Species extinction is generally caused by the introduction of alien species, excessive exploitation of species and illegal logging causing river habitat degradation and lake habitat siltation or dessication, like in the case of Kurumoi and Ayamaru lakes [START_REF] Allen | Fishes of Papua[END_REF][START_REF] Kadarusman | Description of Melanotaenia fasinensis, a new species of rainbowfish (Melanotaeniidae) from West Papua, Indonesia with comments on the rediscovery of M. ajamaruensis and the endangered status of M. parva[END_REF][START_REF] Tappin | Rainbowfish Fishes, their care & keeping in captivity[END_REF]. Melanotaenia boesemani is incontestably the most famous rainbowfish in the aquarium hobby. According to [START_REF] Kadarusman | Rainbowfishes from West Papua (Melanotaeniidae): Evolution and Systematics[END_REF], M. boesemani lives in 2 distinct locations, i.e. Ayamaru and Uter lakes. Until now, the exact origin of the strains reared in Indonesia has not been found out.

3. THREATS ON THE MELANOTAENIIDAE

The West Papua Province (115,363 km2; 760,855 people), which encompasses the Vogelkop Peninsula and the adjacent islands of the Raja Ampat, is one of the least economically developed regions in the world. Human activities concentrate mostly in the vicinity of Sorong, Manokwari, Teminabuan and Bintuni.

Papua, which includes the province of West Papua and Papua Province, is house of 50% of the Indonesian biodiversity and sinificantly contributes to Indonesia's status as one of the biologicaly richest countries in the world with Brazil [START_REF] Supriatna | The Irian Jaya biodiversity conservation priority-setting workshop[END_REF].

Currently, Papua is home of 400 species of freshwater fish [START_REF] Allen | Field guide to the freshwater fishes of New Guinea[END_REF]. Until now, there have been many unspoiled areas because the access is difficult. The habitat physical topology is very steep as a result of complex geological events. This is probably the reason why new species can still be found every year [START_REF] Allen | Field guide to the freshwater fishes of New Guinea[END_REF][START_REF] Novotny | An altitudinal comparison of caterpillar (Lepidoptera) assemblages on Ficus trees in Papua New Guinea[END_REF].

In spite of this, the province of West Papua Indonesia is now facing new challenges. Flora and fauna in this region face a threat of extinction due to human activities such as logging and burning of forests to agricultural areas, transmigration, poaching, mining, etc. All human activities have been increasingly disturbing and destroying the habitat of flora and fauna, particularly freshwater fish. Moreover, since the 1950s, more than thirty species of freshwater fish have been introduced into the waters of New Guinea essentially for human consumption [START_REF] Allen | Fishes of Papua[END_REF]. Those species are principally the tilapias Oreochromis mossambicus and O. niloticus, the walking catfish Clarias batrachus, the common carp Cyprinus carpio, the snakehead Channa striata, and also other species such as Tor putitora and Anabas testudineus.

The information about the biology and ecology of these fish species in their natural habitat is not much documented. An in-depth study is therefore needed, such as reproductive condition in their natural habitat, water quality, frequency of spawning and habitat preferences. Ecological research on rainbowfish families is very crucial for helping efficiently their conservation.

4. HISTORY OF RAINBOWFISH AS WORLD AQUARIUM PEARLS AND AQUACULTURE

Rainbowfish from New Guinea have been imported to Australia since around the mid-1950s. They have been continuously cultivated, and their farmers had no idea that these fish will be popular for the international aquarium hobby. Some specimens were introduced, including Melanotaenia affinis, M. goldiei, M. rubrostriata, M. papuae and M. sexlineata, which were developed by hobbyists from Australia in early 1959. In 1982, the Australian and Papua New Guinea rainbowfish were promoted through a book by Gerald Allen and Norbert Cross [START_REF] Allen | Rainbowfish of Australia and Papua New Guinea[END_REF], which resulted in the increase in popularity and curiosity about the rainbowfish species found in New Guinea and Australia. During this period, there was no significant limitation, and there was an important number of different species brought from New Guinea to Australia by private collectors, who then distributed them to hobbyists (Smith, 2007;[START_REF] Tappin | Rainbowfish Fishes, their care & keeping in captivity[END_REF].

The excessive trade of rainbowfish from New Guinea to Australia attracted the attention of the "Advisory Committee on Live Fish (ACOLF)". At the end of 1983, ACOLF prohibited the import of fish into Australia for all freshwater fish species from New Guinea. In the mid-1980s, aquarium fish collectors began collecting species from New Guinea, cultivating and distributing them to the international aquarium hobbyists (Smith et al., 2007). This trend has continued over the last two decades and has grown side by side with the increasing discovery of new species. Ichtyologists and rainbowfish hobbyists have continued to look for these fish to the inland, to collect different shapes and colors, to keep them survive in the aquarium by fulfilling all the physiological needs of the fish outside their natural habitat. However, when these fish are kept in aquarium, they do not produce bright colors as those living in their natural habitat. This resulted in the widespread sales of these fish under 5 cm in size, in which the colors have not come out well [START_REF] Tappin | Rainbowfish Fishes, their care & keeping in captivity[END_REF].

Rainbowfish from Australia were domesticated for the first time as decoration in the aquarium, as reported by Albert Gale in early 1915, in his book entitled "Aquarian nature Studies and Economic Fish farming made known the hobby of keeping Australian freshwater fishes". At the beginning of the 1920s and 1930s, ornamental fish aquarium hobbyists were set up in major cities in Europe such as Germany. The delivery used ship, and there was no change of oxygen. When they got in the country of destination, the fish were in poor condition, but there were some survivors and they could be successfully cultivated and saved by the hobbyists.

Thus this became the community of hobbyists in European countries.

During this period, these fish were known as sunfish. In January 1934, the magazine "National Geographic" published an article written by Walter H Chute, and then the director of the Shed Aquarium in Chicago USA called these as "Tropical Fish Immigrants Reveal New Nature Wonders". This raised the reference of Australian rainbowfish. The earliest record was found in the Aquarium German magazine "Wochenschrift für Aquarien und Terrarienkunde" in September 1931 by Erich Henzelmann, who wrote an article about 'Regenbogenfisch' Melanotaenia nigrans. The earliest reference to the name of a rainbowfish that had been found in Australia was in a report entitled "The Aquarium and Terrarium Society of Queensland". On March 6, 1932, Amandus Rudel reported the results of his expedition and mentioned all the fish with the name "rainbowfish", while the name "Sunfish" were designated only for Rhadinocentrus ornatus which he described as "Moreton Island Sunfish" [START_REF] Tappin | Rainbowfish Fishes, their care & keeping in captivity[END_REF].

In 1927 Amandous Rudel, a founder of Aquarium and Terrarium Society of Queensland, introduced Australian rainbowfish on the international community, by sending Melanotaenia duboulayi [START_REF]eachamensis Allen & Cross[END_REF] to Germany. These fish were then raised in the aquarium of Berlin and then the fish were promoted to America.

Amandous Rudel also introduced another rainbowfish for the international aquarium hobbyists. In 1934, he sent 12 specimens of Melanotaenia maccullochi, which were gathered near the river Cairns, northern Australia, and then sent to Fritz Mayer in Hamburg, Germany. Four specimens arrived and lived safely, and they developed into two pairs. These fish have become one of the most popular aquarium fish originating from Australia. This news was written in a German aquarium magazine [START_REF] Allen | Descriptions of five new rainbowfish (Melanotaeniidae) from New Guinea[END_REF] on the description of M. boesemani. Overfishing of M. boesemani males then started to affect Ayamaru Lake, the natural habitat of M. boesemani [START_REF] Allen | Fishes of Papua[END_REF]. Rainbowfish cultivation in Indonesia is carried out indoor and outdoor, involves hatcheries and grow-out ponds (Fig 5 ab), with semi-natural water bodies (Fig 5 c,d,e) and indoor aquaria installations (fig 5f). According to the quarantine authorities in Jakarta, producers export nearly 2,500 live M. boesemani per week, at a price of around 1 US$ per individual. Though there are no official numbers, this is unlikely to account for the totality of M. boesemani that are exported worldwide from Indonesia.

MAJOR MOLECULAR MARKERS USED IN FISH GENETIC POPULATION STUDIES

A genetic marker is a measurable character of Mendelian inheritance [START_REF] Liu | DNA marker technologies and their applications in aquaculture genetics[END_REF] and is considered ideal when polymorphic (variable between individuals), discriminatory (different related individuals), multiallelic (has many alleles on the same locus), codominant (heterozygotes are visible), not epistatic (independent of the expression of other markers), neutral (whatever the allele present at the locus, the fitness of the individual is the same), reproducible from one experiment to the next, to handle large scale and economical. The distribution of markers over the whole genome is also a criterion to remember. The main sources of molecular markers are from either a sequence polymorphism (eg: substitution, insertion, deletion) or a polymorphism number of repetitions units (microsatellites and minisatellites). There are two major types of genetic markers that are widely used to characterize fish populations, namely mitochondrial markers and microsatellite markers.

MITOCHONDRIAL DNA MARKERS

Mitochondrial DNA is a popular marker for study of evolution like phylogenetic inference, identification of species, phylogeography, and analysis of population structure. Characteristics of mitochondrial DNA are small genome with simple structure and organization, ubiquitous presence, high copy number, thereby easy to isolate, effective haploidy in DNA sequences, maternal inheritance, lack of recombination in introns or other non coding sequence, mosaic molecule with aster and slower evolving DNA regions allowing to design conserved primers and to address phylogenetic questions at various taxonomic levels (Zhang & Hewiit 1996;Harrison, 1989).

For identification of species and evolution studies, maternal inheritance has important consequences, because only a fraction of the population (half if the sex ratio is 1:1) pass on their mtDNA to offspring and the effective population size for mtDNA is smaller than that for nuclear genes. In stochastic processes, it will be particularly important to determine frequencies of mtDNA genotypes. Genetic affinities defined by mtDNA genotypes reflect matriarchal phylogeny. MtDNA is suited for studying population patterns including founder populations/founder events.

Because females transmit many copies of the mtDNA molecule to each offspring, a new mtDNA variant arises as mutation in a single molecule within a single cell lineage (Harrison, 1989).

MtDNA analysis has been used in three ways in this context; (1) to measure genetic variation within populations, especially ones thought to have declined recently, (2) to identify evolutionarily divergent sets of populations, including the resolution of evolutionarily significant units, and (3) to assess conservation value of populations or areas from an evolutionary or phylogenetic perspective.

Mitochondrial DNA variation is more sensitive in population phenomena such as bottlenecks and hybridizations. Sex-specific differences in gene flow could also be revealed by contrasting nuclear with mitochondrial DNA. And also mtDNA is intensively studied and sequences in some parts of the molecule are highly conserved across species (Okumus & Ϛiftei, 2003).

A prerequisite for managing biodiversity is the identification of populations with independent evolutionary histories. Such groupings are variously categorized as species. MtDNA phylogenies can provide unique insights into population history [START_REF] Avise | Intraspecific phylogeography: The mitochondrial DNA bridge between population genetics and systematics Ann[END_REF] and can suggest hypotheses about the boundaries of genetically divergent groups (e.g. cryptic species). Several studies about cryptic species and DNA barcoding have been succesfully implemented in the identification of previously described species. DNA barcoding studies have focused on the identification of pre-defined species (Hebert et al. 2003). Applying mtDNA analyses on rainbowfish, Kadarusman et al. (2012) succesfully described the cryptic diversity in Indo-Australian Rainbowfishes. As much as 14 species of rainbowfish have been discovered in populations exhibiting private barcode clusters diverging from their nearest neighbor by K2P distances similar to those observed among valid species characterized by diagnostic morphological characters. DNA barcoding was not only effective for the identification of species, but it proved to be effective for the discovery of provisional cryptic diversity awaiting further screening through integrative approaches as previously predicted by other species like butterfly [START_REF] Hebert | Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator[END_REF], diptera (Smith et al. 2007) and reef fishes [START_REF] Hubert | Cryptic diversity in Indo-Pacific coral reef fishes revealed by DNA-barcoding provides new support to the centre-ofoverlap hypothesis[END_REF] .

In spite of many advantages, mtDNA marker shows several drawbacks for genetic studies, such as non-neutrality, non-constant molecular clock, length and sequence heteroplasmy and even mitochondrial bottleneck [START_REF] Stewart | Keeping mtDNA in Shape between Generations[END_REF].

MICROSATELLITE MOLECULAR MARKERS (SSR / SIMPLE SEQUENCE REPEAT)

The selection of microsatellite molecular genetic markers was found in the 1980s. They represent a unique type of tandemly repeated genomic sequences, which are abundantly distributed across genomes and demonstrate high levels of allele polymorphism. They are also called simple sequence repeats (SSRs). They are codominant markers with relatively small size, around 20 repetitions of base maximum in length, and can be easily amplified by polymerase chain reaction. They are ubiquitous in prokaryotes and eukaryotes, present even in the smallest bacterial genomes. The majority of microsatellites (30-67%) found are dinucleotides. In the genomes of vertebrates, (AC)n is the most common dinucleotide motif. It is 2.3-fold more frequent than (AT)n, the second most general type of dinucleotides [START_REF] Tóth | Microsatellites in Different Eukaryotic Genomes: Survey and Analysis[END_REF]. In all vertebrates, (G+C)-rich motives (e.g., CCG, CAG) are the most common among trinucleotides. Microsatellites can be found anywhere in the genome, both in protein coding and noncoding DNA [START_REF] Tóth | Microsatellites in Different Eukaryotic Genomes: Survey and Analysis[END_REF]. In eukaryotic organisms, SSRs have been shown to be in excess in noncoding regions compared to a random distribution pattern [START_REF] Metzgar | Evidence for the Adaptive Evolution of Mutation Rates[END_REF].

Applications of microsatellites include: genetic mapping, individual DNA identification and parentage assignment, phylogeny, population and conservation genetics, molecular epidemiology and pathology, quantitative trait loci mapping, marker-assisted selection. Also, the use of microsatellite markers may benefit the genetic dissection of complex and quantitative traits in order to map, identify and eventually clone and characterize the candidate genes controlling economically important traits. In aquaculture, SSRs represent the markers of choice for genetic monitoring of farmed stocks in view of breeding programs through the analysis of genetic variablity and pedigree structure to design beneficial crosses, select genetically improved stocks, minimize inbreeding and increase selection response [START_REF] Davis | The impact of genetic markers on selection[END_REF][START_REF] Knibb | Genetic improvement of marine fish -which method for industry?[END_REF]. Microsatellite-based techniques are applied in genome scans and quantitative trait loci (QTL) mapping in natural populations to search for the genetic basis of adaptive selection and biodiversity in an increasing number of species [START_REF] Rogers | FAST-TRACK: Integrating QTL mapping and genome scans towards the characterization of candidate loci under parallel selection in the lake whitefish (Coregonus clupeaformis)[END_REF].

Microsatellites are highly abundant in various eukaryotic genomes including all aquaculture species studied to date. In most of the vertebrate genomes, microsatellites make up a few percent of the genomes in terms of the involved base pairs, depending on the compactness of the genomes [START_REF] Zhan | Isolation and characterization of novel microsatellite markers for yellow perch (Perca Flavescens)[END_REF]. In fish, one microsatellite was found every 1.87 kb of DNA. For comparison, in the human genome, one microsatellite was found every 6 kb of DNA (Beckmann & Weber 1992). It is reasonable to predict that in most aquaculture fish species, one microsatellite should exist every 10 kb or less of the genomic sequences, on average (Wright 1993;[START_REF] Duran | Molecular Genetic Markers: Discovery, Applications, Data stoarage and visualization[END_REF]. Their high polymorphism, together with their PCR-based analysis, have made them one of the most popular genetic markers [START_REF] Duran | Molecular Genetic Markers: Discovery, Applications, Data stoarage and visualization[END_REF][START_REF] Boris | Genetic diversity of six populations of red hybrid tilapia, using Microsatellite genetic Markers[END_REF]. Some microsatellite loci have very high numbers of alleles per locus (>20), making them very useful for applications such as parent-offspring identification in mixed populations, while others have lower numbers of alleles and may be more suited for population genetics and phylogeny [START_REF] Tabbaa | Analysis of genetic fat tailed sheep in southern region of Jordan[END_REF]. Primers developed for one species will often cross-amplify microsatellite loci in closely related species [START_REF] Boris | Genetic diversity of six populations of red hybrid tilapia, using Microsatellite genetic Markers[END_REF]. Genotyping of microsatellite markers is usually straightforward [START_REF] Castoe | Rapid identification of thousands of copperhead snake (Agkistrodon contortrix) microsatellite loci from modest amounts of 454 shotgun genome sequence[END_REF].

Microsatellite markers are co-dominant markers, which means they can distinguish heterozygous and homozygous alleles. They are usually distributed throughout the genome, and they are independent markers [START_REF] Chambers | Microsatellite: consensus and controversy[END_REF], neutral (any allele present at this locus has the same selective value on all individuals), and can be used from one experiment to another experiment, at an affordable cost [START_REF] Db | Microsatellite evolution and application[END_REF]. In the case of high allelic diversity with repetitions of mono, di, tri, tetra, penta and hexa nucleotides, the theory is based on the concept of neutrality, and the variability can differentiate individuals, populations and different species. This characteristic is very important for population genetics studies, and other genetic studies such as inbreeding pressure analysis of heredity or genetic mapping [START_REF] Vartia | Multiplexing with three-primer PCR for rapid and economical microsatellite validation[END_REF][START_REF] Bruford | Microsatellites and their application to population genetic studies[END_REF][START_REF] Brockmann | Sperm competition in horseshoe crabs ( Limulus polyphemus )[END_REF][START_REF] Knapik | A microsatellite genetic linkage map for zebrafish ( Danio rerio )[END_REF][START_REF] Goldstein | The use of microsatellite variation to infer population structure and demographic history in a natural model system[END_REF]Primmer et al., 2000).

Microsatellites consist of four models: 1) Perfect microsatellites consist of a single continuously repeated motif without being distracted by other repeated motifs or patterns, like ctctctctctctctctctct. 2) Imperfect microsatellites are microsatellites in which one or more repetitions carries base pairs that are not in accordance with the structure of repetition, like ctctctctctgtctct. 3) Interrupted microsatellites are microsatellites with the insertion of a small number of base pairs that do not fit the structure of repetition, like ctctctctctgggctctctct. 4) Compound microsatellites consist of two or more adjacent microsatellites with different repetition types, for example ctctctctctctgatgatgatgatgatgat [START_REF] Db | Microsatellite evolution and application[END_REF][START_REF] Jarne | Microsatellites, from molecules to populations and back[END_REF].

Microsatellites are very abundant in eukaryotic genomes because of their process of mutations. Mutations in microsatellite loci usually involve a change in one repetition, but sometimes mutations invole multiple repetition units. There are two processes of mutation that affect the formation of microsatellite loci, namely: 1)

Unequal crossing over (UCO), occurring during the process of meiosis, and 2)

Slipped-strand mispairing (SSM), and occurring due to mistakes/errors during DNA replication [START_REF] Db | Microsatellite evolution and application[END_REF].

The microsatellite mutation models as addressed by Estoup and Cournet (1998) in [START_REF] Db | Microsatellite evolution and application[END_REF] [START_REF] Db | Microsatellite evolution and application[END_REF][START_REF] Anmarkrud | Microsatellite evolution: Mutations, sequence variation, and homoplasy in the hypervariable avian microsatellite locus HrU10[END_REF].

The key feature of SSRs as molecular markers is their hypermutability and, hence, their hypervariability in species and populations. SSR grow very fast with a high mutation rate of 10 -2 -10 -6 per locus per gamete per generation, when compared with point mutations in the gene encoding regions [START_REF] Anmarkrud | Microsatellite evolution: Mutations, sequence variation, and homoplasy in the hypervariable avian microsatellite locus HrU10[END_REF]. This high level of mutation leads to a high polymorphism. The abundance of microsatellites in the genome occurs because replication slippage are not the same in sister chromatid exchange, nucleotide substitutions and duplication events as well as crossovers in the process of meiosis. Many factors might be important for the mutational processes in microsatellites, such as allele size, motif size, gender, and G/C content. Mutation pattern also depends on the specific location in the genome, e.g functional potential of the product when transcribed, as well as the effectiveness of various repair enzymes. In addition, microsatellite mutation rate is also influenced by the pattern of stabilization and potential secondary structure [START_REF] Anmarkrud | Microsatellite evolution: Mutations, sequence variation, and homoplasy in the hypervariable avian microsatellite locus HrU10[END_REF].

A very high degree of polymorphism is the most striking character of microsatellites, but not all of microsatellites are polymorphic at the same point and in the same time. Microsatellite mutations may occur during replication of chromosomes, either as part of mitosis or meiosis, and can also occur during DNA synthesis. Therefore, the frequency of microsatellite mutations increases rapidly when the cells are under stress and when the cells undergo active repair due to damage. Each mutation that occurs during meiosis or mitosis in early cleavage embryo will develop well, and if it is not selected during the development process, it will form a new microsatellite allele. In contrast, mutations that occur in the cell, as somatic mutations, will not affect other cells. There are many factors affecting the level of microsatellite mutation that makes the locus and microsatellite motif abundant [START_REF] Jarne | Microsatellites, from molecules to populations and back[END_REF]. Besides being multiallelic and codominant at a single locus, SSRs offer several advantages over other molecular markers. For instance genetic variability can be easily detected by PCR using primers designed around the SSR repeats and by analyzing length fragments, and they are transferrable across closely related species.

Among the mechanism of this mutation, an error in the replication plays a major role in producing new alleles in the SSR loci; therefore, it increases the diversity of SSR. Replication error is the most important mechanism in the formation of SSR. During DNA replication, longer stretches of repeated units pose more of a problem to DNA polymerase than do shorter stretches making longer alleles more prone to slipped-strand mispairing. Interaction in replication errors and crossovers is an important factor to the diversity of repetition motifs and the number of SSR repetitions, such as (CA) n, (CT) n, and (CA) n (TA).

Replication error in chromosomes in each species is different depending on the rate of recombination of these species and SSR motif models in the patterns of evolution that occurs in these loci [START_REF] Li | Genetic effects on microsatellite diversity in wild emmer wheat (Triticum dicoccoides) at the yehudiyya microsite[END_REF]. The imperfect SSR pattern makes the pattern more complex than that from the perfect SSR (Orti 'et al., 1997).

Consequently, a genome can evolve faster than other genomes, such as in the study of [START_REF] Li | Genetic effects on microsatellite diversity in wild emmer wheat (Triticum dicoccoides) at the yehudiyya microsite[END_REF] in Triticum dicoccoides. SSR sequences are more abundant and longer in vertebrates than in invertebrates, and among the vertebrates, the SSR motifs are longer in cold-blooded species such as reptiles, amphibians and fish (Chambers and McAvoy 2000).

Transcribed microsatellites located in 5' untranslated regions (UTRs) could form specific and unusual DNA structures. In that case, the length of the repeat region could affect the translation level from the target mRNA. Microsatellites situated in the 3'-UTR could affect gene expression through their influence on the stability of transcribed products. O'Brien (1991) devided molecular markers into type I markers associated with genes of known functions and type II markers associated with anonymous genomic sequences. Microsatellites usually represent type II markers, since they are commonly located in noncoding intergenic regions.

Type 1 markers are more difficult do develop (Liu et al., 1996). While non-gene sequences are free to mutate, causing higher levels of polymorphism, sequences within protein-coding regions generally show lower levels of polymorphism because of functional selection pressure. In addition, type II markers can be rapidly developed from SSRs isolated previously from closly related species [START_REF] Bruford | Microsatellites and their application to conservation genetics[END_REF]. Cross-species amplification of SSRs provides a possibility to superimpose the genetic information or a genetic linkage map from one species to another and therefore to use SSR markers for population genetics, parentage analysis and other applications without having to invest in the isolation of polymorphic microsatellites [START_REF] Cairney | Characterization of microsatellite and minisatellite loci in Atlantic salmon (Salmo salar L.) and cross-species amplification in other salmonids[END_REF]. Previous research reported highly conserved flanking regions in fish [START_REF] Rico | 470 million years of conservation of microsatellite loci among fish species[END_REF] allowing cross amplification from species that diverged as long ago as 470 million years.

As previously mentioned, the various types and patterns of mutations in microsatellites cause the great diversity of patterns and lengths. This also generates what is called microsatellite homoplasy. Size homoplasy occurs when alleles show similar length in microsatellite sequences, but they are different in heredity. Thus, homoplasic microsatellites are divided into two categories: 1) microsatellite alleles that are identical in length, but not in sequence (distinguished by sequencing), 2) identical allele in both length and sequence with different evolutionary history (only detected through mutation and documented through a known pedigree) [START_REF] Anmarkrud | Microsatellite evolution: Mutations, sequence variation, and homoplasy in the hypervariable avian microsatellite locus HrU10[END_REF].

ISOLATION OF MICROSATELLITE MARKERS IN NON MODEL ORGANISMS

Microsatellite markers are one of the most informative and versatile DNAbased markers used in fish genetic research, but their development has traditionally been a difficult and costly process. This requires a lot of energy and a lot of work, as well as huge costs, and it takes a long time to find and design appropriate primer at non models organisms. [START_REF] Telles | Isolation and characterization of microsatellite markers in the armored catfish Hypostomus gymnorhynchus (Loricariidae)[END_REF][START_REF] Vartia | Multiplexing with three-primer PCR for rapid and economical microsatellite validation[END_REF]. Emergence of next generation sequencing (NGS) technologies has enabled the efficient identification of large numbers of microsatellites at a fraction of the cost and effort of traditional approaches. The major advantage of NGS methods is their ability to produce large amounts of sequence data from which to isolate and develop numerous genome-wide microsatellite loci. The two major methods for SSR isolation are direct shotgun sequencing of microsatellite-enriched libraries [START_REF] Malausa | High-throughput microsatellite isolation through 454 GS-FLX Titanium pyrosequencing of enriched DNA libraries[END_REF][START_REF] Guichoux | Current trends in microsatellite genotyping[END_REF]. NGS are extremely high-throughput technologies that produce millions of sequences at once at a fraction of the cost of traditional Sanger methods [START_REF] Shendure | Next-generation DNA sequencing[END_REF][START_REF] Ekblom | Applications of next generation sequencing in molecular ecology of non-model organisms[END_REF]. A direct sequencing approach has a high risk and may not detect SSR [START_REF] Parchman | Transcriptome sequencing in an ecologically important tree species: assembly, annotation, and marker discovery[END_REF]. In this case, SSR enrichment must be considered. The technique of enriched libraries has proven very efficient and effective to isolate SSR in non model species [START_REF] Malausa | High-throughput microsatellite isolation through 454 GS-FLX Titanium pyrosequencing of enriched DNA libraries[END_REF][START_REF] Zalappa | Using next-generation sequencing approaches to isolate simple sequence repeat (SSR) loci in the plant science[END_REF] (Figure 6).

Over the available NGS platforms, 454 pyrosequencing has shown a higher success in SSR isolation, probably due to the higher length of the reads it produces, which increase the probability of properly design SSRs primers under low to medium sequencing coverage [START_REF] Zalappa | Using next-generation sequencing approaches to isolate simple sequence repeat (SSR) loci in the plant science[END_REF]. The 454 technology was the first next- Validation and microsatellite genotyping can be achieved through the use of capillary electrophoresis, in presence of fluorescent primers to detect the amplicons.

These markers are then amplified in a sole PCR reaction or combined to multiplex PCRs that contain fluorescent markers with different wavelengthes [START_REF] Guichoux | Current trends in microsatellite genotyping[END_REF], like in Figure 7. sample that is heterozygous at the same locus (two major peaks are observed).

Homozygote allele

Heterozigote allele

THE APPLICATION OF MICROSATELLITES IN AQUACULTURE

In the field of aquaculture, microsatellites represent workhorse markers, which are useful for the characterization of genetic stocks, broodstock selection, constructing of dense linkage maps, mapping economically important quantitative traits, identifying genes responsible for these traits and application to marker-assisted breeding programmes (Chistiakov et al., 2006). In fish, one microsatellite was found every 1.87 kb of DNA. For comparison, in the human genome, one microsatellite was found every 6 kb of DNA (Beckmann and Weber 1992). It is reasonable to predict that in most aquaculture fish species, one microsatellite should exist every 10 kb or less of the genomic sequences, on average [START_REF] Duran | Molecular Genetic Markers: Discovery, Applications, Data stoarage and visualization[END_REF].

Polymorphism can be studied from microsatellite markers which give important information to be considered in the management of fish stock (Alam and Islam 2005), population and biodiversity conservation analyses (Romana-Eguia et al., 2004). [START_REF] Boris | Genetic diversity of six populations of red hybrid tilapia, using Microsatellite genetic Markers[END_REF] have applied microsatellites to study the various genetics of hybrid red tilapia fish population, and [START_REF] Norris | Microsatellite genetic variation between and within farmed and wild Atlantic salmon (Salmo salar) populations[END_REF] have applied such markers for Atlantic salmon (Salmo salar).

Molecular markers can be a useful tool in identifying the stock population and monitoring the potential of parent changes, as DNA fingerprint [START_REF] Fjalestad | Prospects for genetic technology in salmon breeding programees[END_REF][START_REF] Ahmad | Diversity and distribution of fishes of Gaji River, Bauchi state Nigeria[END_REF]. The main molecular markers that have been used widely in monitoring the variation of genetics in hatchery were derived from allozymes and microsatellite; however, the use of microsatellite markers is more efficient [START_REF] Sekino | Genetic Diversity Within and Between Hatchery Strains of Japanese Flounder Paralichthys olivaceus Assessed by Means of Microsatellite and Mitochondrial DNA Sequencing Analysis[END_REF][START_REF] Ramos-Paredes | Allozyme genetic analysis in hatchery strains and wild blue shrimp, Penaeus (Litopenaeus) stylirostris (Stimpson), from the Gulf of California[END_REF][START_REF] Samy-Yehya | Phylogenetc differentiation of wild and cultured Nile Tilapia (Oreochromis niloticus) populations based on phenotype and genotype analysis[END_REF]. The research on genetic variation in hatchery stock (such as in salmon) has shown the loss of genetic variation as the different results including the number of effective parental specimens, the selection of domestication or mating design [START_REF] Samaradivakara | Morphological variation of four Tilapia population in selected Reservoirs in Sri Lanka[END_REF]. [START_REF] Sekino | Genetic Diversity Within and Between Hatchery Strains of Japanese Flounder Paralichthys olivaceus Assessed by Means of Microsatellite and Mitochondrial DNA Sequencing Analysis[END_REF] evaluated the genetic difference among the cultivation hatchery population and wild fish population on Paralichthys olivaceus fish with microsatellite and mtDNA sequencing. They identified a smaller number haplotypes with mtDNA in the hatchery strains than in the wild populations. [START_REF] Desvignes | Genetic variability in reared stocks of common carp (Cyprinus carpio L.) based on allozymes and microsatellites[END_REF] studied the genetic diversity of golden fish (Cyprinus carpio) strains from France and Czechoslovakia using allozyme and microsatellite markers. They detected different strains of both countries with microsatellite markers. [START_REF] Bartfai | Genetic analysis of two common carp broodstocks by RAPD and microsatellite markas[END_REF] analyzed the whole parents from two Hungarian golden fish stockbreedings (80 and 196 fish) by using RAPD test and microsatellite analysis. The result was that the analysis of microsatellite was more informative than RAPD analysis. Microsatellite is usually the marker chosen to analyze the stock farming [START_REF] Duran | Molecular Genetic Markers: Discovery, Applications, Data stoarage and visualization[END_REF][START_REF] Boris | Genetic diversity of six populations of red hybrid tilapia, using Microsatellite genetic Markers[END_REF].

The monitoring of genetic changes on stock farming with microsatellite has succeeded greatly in detecting the impact of genetic changes [START_REF] Zhan | Isolation and characterization of novel microsatellite markers for yellow perch (Perca Flavescens)[END_REF]. The higher the detection sensitivity from the microsatellite towards the phenomenon like genetic drift and founder effect makes this marker ideal to monitor the consequence of the founder effect and propagation in farming if compared to mtDNA and allozyme markers [START_REF] Duran | Molecular Genetic Markers: Discovery, Applications, Data stoarage and visualization[END_REF]. Microsatellite marker has been used to minimize the inbreeding on rainbow trout [START_REF] Fishback | Optimization of semiautomated microsatellite multiplex PCR systems for rainbow trout (Oncorhynchus mykiss)[END_REF]. The analysis of F1 generation from Greek gilthead sea bream broodstock revealed the decrease of 15% in the number of alleles and the increase of 1.5% homozygosity [START_REF] Magoulas | Application of molecular markas to aquaculture and broodstock management with special emphasis on microsatellite DNA[END_REF].

Thus, SSR marker has important application in monitoring the pressure of inbreeding.

Microsatellite markers were also used to find chromosome areas that are responsible for the inbreeding pressure. Research on this is very likely to do in the future on cultivation species, in which the genitors and their offspring can be managed and tracked in close and controlled system. Moreover, this can be used to map some chromosome inheritance on a progeny and its parents [START_REF] Morelli | Isolation and characterization of eight microsatellite loci in Leporinus macrocephalus (Characiformes: Anostomidae) and cross-species amplification[END_REF].

Management of mixed fisheries (multiple species, different stocks) can be very tricky. To address this, genetic data have increasingly been used to investigate stock structure and stock contribution to mixed-stock fisheries. When fisheries are composed of only two different stocks, estimation of relative contribution is simple and straightforward, like for the Atlantic salmon case at West Greendland, where salmon from Europe and North America were differentiated with molecular microsatellite and minisatellite DNA markers (McConnell et al., 1995).

Microsatellites were found to give much higher degree of accuracy and precision than those available from allozymes (Avis, 1994).

Analysis of breeders and their progeny in programs of selective breeding uses both information about their prospective parents and relatives to increase the accuracy in parent selection and its reaction selection. In breeding, usually the number of parents used is limited and some of them fail to spawn. In this case, SSRs and other genetic markers become useful to estimate the relative success of the potential genitors. The progeny can be determined by using minisatellite or microsatellite markers after spawning [START_REF] Moran | Genetic assessment of the reproductive success of Atlantic salmon precocious parr by means of VNTR loci[END_REF][START_REF] Thompson | Comparison, using minisatellite DNA profiling, of secondary male contribution in the fertilisation of wild and ranched Atlantic salmon (Salmo salar) ova[END_REF].

One of the biggest obstacles in applying the program of selective and effective fish breeding is new born offsprings that are too small to be marked physically. It is thus mandatory to keep the progeny separated from the rest of the cheptel until they become big enough to be marked. It is very expensive to do this and it is time consuming [START_REF] Doyle | The use of DNA fingerprinting for high intensity, within-family selection in fish breeding[END_REF]. This problem can be overcome by applying DNA microsatellite markers. Of the farming result, there are a lot of tillers from fish cultivation stock that can be kept without using separated container during the initial stage. These markers have been widely used to identify relatives or progenies, and these can be used to distinguish fish in aquaculture mixed with other species [START_REF] Herbinger | DNA fingerprint-based analysis of paternal and maternal effects on offspring growth and survival in communally reared rainbow trout[END_REF][START_REF] Fjalestad | Prospects for genetic technology in salmon breeding programees[END_REF][START_REF] Duran | Molecular Genetic Markers: Discovery, Applications, Data stoarage and visualization[END_REF][START_REF] Boris | Genetic diversity of six populations of red hybrid tilapia, using Microsatellite genetic Markers[END_REF][START_REF] Tabbaa | Analysis of genetic fat tailed sheep in southern region of Jordan[END_REF].

A cultivation program can start from a stockbreeding which origin is not known. The method termed "walk back" consists in tracing lineages backward [START_REF] Doyle | The use of DNA fingerprinting for high intensity, within-family selection in fish breeding[END_REF]. The conventional way implies physical markers (tagging), and the marking of the individuals is performed when they are big enough.

However, microsatellite analysis based on biopsy can be used without waiting the individual to be big enough. Some empirical studies with micosatellite loci have allowed to reconstruct the fish population genealogy and their family that are mixed when they hatch; this resulted in 99% right assignations of male and female parents and their offsprings (Norris et al., 2000;[START_REF] Morelli | Isolation and characterization of eight microsatellite loci in Leporinus macrocephalus (Characiformes: Anostomidae) and cross-species amplification[END_REF][START_REF] Zhan | Isolation and characterization of novel microsatellite markers for yellow perch (Perca Flavescens)[END_REF][START_REF] Olivatti | Heterologous amplification and characterization of microsatellite markers in the Neotropical fish Leporinus friderici[END_REF]. Doyle et al., (1994) used these DNA markers to distinguish the group of cod fish family (Gadus morhua). One of the microsatellite marker challenges in parent management is to assess the contribution of the potential genitors in mass spawning because it often happens that many breeders fail to spawn. The use of

III. DEVELOPMENT OF NEW MICROSATELLITE MARKERS FROM M. BOESEMANI USING NEXT GENERATION SEQUENCING TECHNOLOGY

INTRODUCTION

Rainbowfishes of the family Melanotaeniidae are commonly distributed throughout New Guinea and Australia below elevations of 1500 m in most freshwater habitats including streans, lakes and swamps [START_REF] Allen | Field guide to the freshwater fishes of New Guinea[END_REF].

Rainbowfishes are very popular ornamental fish because of their vivid colouration, placid nature and ability to thrive in captivity [START_REF] Kadarusman | Description of Melanotaenia fasinensis, a new species of rainbowfish (Melanotaeniidae) from West Papua, Indonesia with comments on the rediscovery of M. ajamaruensis and the endangered status of M. parva[END_REF] 2000) and showed that Glossolepis genus was polyphyletic, which challenged the validity of its taxonomic status. More recently, the use of a new morphological approach and of mitochondrial and nuclear molecular markers resulted in the description of many new species (Kadarusman et al., 2012a;[START_REF] Kadarusman | Description of Melanotaenia fasinensis, a new species of rainbowfish (Melanotaeniidae) from West Papua, Indonesia with comments on the rediscovery of M. ajamaruensis and the endangered status of M. parva[END_REF][START_REF] Kadarusman | Description of Melanotaenia salawati, a new species of rainbowfish (Melanotaeniidae) from Salawati Island, West Papua, Indonesia[END_REF] and showed that the diversity of rainbowfishes in West Papua was largely underestimated (Kadarusman et al., 2012b). Specifically, it revealed that the distribution of Melanotaeniidae consists of 4 monophyletic groups endemic to discrete geographic regions: Western New Guinea, Central Lengguru, Northern New Guinea and Southern New Guinea plus Australia (Kadarusman et al., 2012b). To date, the family consists of 105 species spread in seven genera, which are broadly distributed throughout the region. West Papua is the center of rainbowfish diversity with Melanotaenia genus [START_REF] Allen | Rainbowfishes in Nature and the Aquarium[END_REF]Allen et al., 2008, Kadarusman et al., 2010[START_REF] Mcguigan | Phylogenetic relationships and historical biogeography of melanotaeniid fishes in Australia and New Guinea[END_REF]. At the moment, 24 Considering the high economic value of rainbowfish and the continous pressure from human towards their habitat, research on their populations in their natural habitat is very important and may lead to the discovery of new candidates for ornamental fish aquaculture. Furthermore, this can be used for conservation and endangered species rescue, as well as for cultivation comodity. In order to address these questions, new DNA microsatellite markers were developed and validated on several species of Melanotaenia that live in West Papua region, Indonesia.

We made the choice to use microsatellite markers to study the genetic diversity of the Melanotaeniidae family because the emergence of NGS has enabled to quickly and cost-effectively identify simple sequence repeats (SSR) in unstudied genomes and because they can usually be applied to closely related species [START_REF] Rico | Combining next generation sequencing and online data base for microsatellite development in non model organisms[END_REF]. Microsatellites are markers consisting of mono-, di-, tri-or tetranucleotides tandemly repeated. Besides being generally highly polymorphic, microsatellites are generally neutral, codominant and usually abundant in the genomes of eukaryotic organisms. These characteristics are very important for population genetics studies whose theories are usually based on concepts of neutrality, independence and variability to differentiate populations and individuals.

MATERIAL AND METHODS

Extraction of genomic DNA

In all cases, DNA was extracted from a small fragment (10 mg) of anal fin clip with the NucleoSpin® 96 Tissue kit (Macherey-Nagel). Tissue samples, which consisted of 1-cm 2 caudal fin preserved in absolute ethanol and stored at -20°C, were first incubated overnight at 37°C in the lysis buffer. DNA was extracted the following day according to the manufacturer's instructions, and eluted in 70 µl of the provided elution buffer. When the number of samples exceeded 24, extractions were performed with the same kit, but in 96-well plates, with a Janus automated Workstation (Perkin Elmer), in the technical facilities of the labex "Centre Méditerranéen de l'Environnement et de la Biodiversité" (Montpellier).

Isolation of microsatellite markers

Seven individuals of M boesemani were used for the isolation of microsatellites markers. DNA was extracted as described above. The concentration of DNA was checked with a Nanodrop ND8000 (Thermo Scientific), and an equimolar mix of the 7 DNAs was prepared. Isolation of microsatellites was performed at Genoscreeen (Lille, France), according to the method previously described by [START_REF] Malausa | High-throughput microsatellite isolation through 454 GS-FLX Titanium pyrosequencing of enriched DNA libraries[END_REF]. The mix of genomic DNA was subjected to sonication, ligation to standard adapters and purification on a Nucleofast PCR plate (Macherey-Nagel). DNA was denatured and then hybridized for 20 minute at 56°C to eight biotin-labeled oligonucleotides, the sequences of which are microsatellite motifs [(TG)10, (TC)10, (AAC)8, (AGG)8, (ACG)8, (AAG)8, (ACAT)6 and

(ATCT)6]. the microsatellite-enriched DNA was then amplified using the primers corresponding to the adapters (5'-GTTTAAGGCCTAGCTAGCAGAATC-3' and 5'-GATTCTGCTAGCTAGGCCTT-3') enriched libraries were subsequently prepared and sequenced using the 454 GS FLX Titanium, following the manufacturer's protocol. Sequences were trimmed and analyzed at Genoscreen, using the QDD pipeline [START_REF] Meglecz | QDD : a user-friendly program to select microsatellite markas and design primers from large sequencing projects[END_REF], which enables to search for simple sequence repeats (SSR) motifs and design primers in the corresponding flanking regions.

Amplification and size analysis of microsatellite markers

From all the microsatellite loci provided by Genoscreen, sixty one were primarily tested. The corresponding primer pairs were synthesized by Eurofins (Germany) and resuspended in water at a concentration of 100 µM. Table 2, presents the main characteristics of these primers.

Test of the Mendelian inheritance pattern of the selected microsatellite markers

Five pairs of M. boesemani purchased from a retailer (Botanic, Montpellier)

were constituted. They were reared in distinct 60L aquariums and allowed to mate.

During the two following months, aquaria were frequently checked for the presence of offspring. When available, progeny of each parental pair was collected. It consisted of 23 to 43 hatchlings, depending on the spawn size (Table 3). Juveniles were individually transferred into 1.5 ml tubes containing absolute ethanol for further DNA extractions. Parents were anaesthetized with 0.1 mL/L Eugenol and a ~1-cm 2 piece of anal fin was collected and stored in absolute ethanol. Fish were then allowed to recover from anesthetic and were released back into their tank. Crossing 5 1 1 43

Microsatellite data analysis

Allele sizing and genotyping were achieved with the Peak Scanner v1.0 and GeneMapper® v5.0 software (Applied Biosystems). Allelic diversity (Na) and average observed (Ho) heterozygoties were calculated using the GENETIX 4.05 software [START_REF] Belkhir | GENETIX 4.05, logiciel sous Windows TM pour la génétique des populations[END_REF]. The GENETIX software is a complete program on the indices in population genetics to assess the diversity and genetic divergence. It was used to study the genetic variability in terms of heterozygosity and allelic richness rates. The markers under selection were detected from estimates of differentiation between and within samples (Fst, [START_REF] Weir | Estimating F-Statistics for the Analysis of Population Structure[END_REF] and by simulating the expected Fst under Model islands and under a mutation model (IAM) with 1,000 simulations / permutation. A significance test was applied using an exact test based on the default settings. The estimator Fis [START_REF] Weir | Estimating F-Statistics for the Analysis of Population Structure[END_REF] was also calculated for each locus using this software. Microsatellite data were checked from Hardy-Weinberg equilibrium by estimating the significance of the multilocus inbreeding coefficient (Fis) from sets of 1000 random allelic permutations of the original dataset as implemented by GENETIX 4.05 software. The P value of Fis was defined as the probability to obtaining absolute values higher than or equal to the observed one under the null hypothesis (i.e. the individuals can be considered as the sample from a panmictic population at Hardy-Weinberg equilibrium). In order to assess the possible presence of genotyping errors, the presence of null alleles, stuttering or large allele dropout was tested with the Micro-Checker v. 2.2.3 software (CI 95%, bootstrap simulation x1000) [START_REF] Van Oosterhout | Micro-Checker: software for identifying and correcting genotyping errors in microsatellite data[END_REF]. A factorial component analysis (FCA) was also conducted to assess the level of differentiation between crossing families. This analysis involves projecting the genetic distances between individuals on a set of axes that maximize the differences between populations. It was carried out using the GENETIX 4.05 software [START_REF] Belkhir | GENETIX 4.05, logiciel sous Windows TM pour la génétique des populations[END_REF].

RESULTS

Selection and test of candidate microsatellite markers on several

Melanotaenia species.

Sequencing of the SSR-enriched libraries with the 454 GS-FLX Titanium sequencer resulted in a total of 88,432,250 bases and 14,922 microsatellite locus sequences from the genome of M. boesemani. Moreover, there were as much as 1,012 loci for which primers could be designed. From this list, we selected 61 loci according to the following criteria: the repetition sequence motif should contain 1, 2, 3, 4, 5 or 6 nucleotides ; the microsatellite motif should be pure (i.e. contain perfect reptitions); there should be at least 8 repetitions; G-C content should be comprised between 30 and 70%; product size of amplification should be comprised between 60 and 300 bp; primers should be separated from the recurring motif by at least 20 nucleotides; annealing temperatures should range between 50 and 64 o C (Table 1). From the agarose gel observations, these loci also showed polymorphism.

These 51 primer pairs were subsequently tested on 11 rainbowfish species ( 5specimens / species) distributed in Western New Guinea and Central Lengguru phylogenetic groups. Eventually, twelve of them successfully amplified all the tested specimens under the above-mentioned PCR conditions. The corresponding loci were renamed (Table 4) and their sequence submitted to GenBank (http://www.ncbi.nlm.nih.gov/nuccore/?term=Microsatellite+Melanotaenia+boesema ni).

The 12 loci were subsequently analyzed by capillary electrophoresis. In order to reduce the costs of the genotyping experiments, they were tested in a 4-plex format as displayed in Table 3. Four PCR products were mixed together as described in the M&M section prior to capillary electrophoresis. Results obtained on a large panel of samples revealed that mixing 4 PCR products did not alter the electrophoresis quality nor the allele sizing with Peak Scanner and Gene Mapper (Figure 8).

Experimantal validation of the 12 new microsatellite markers

Microsatellites represent codominant single-locus DNA markers. For each SSR, a progeny inherits one allele from the male parent and another allele from the female parent. We therefore tested this simple inheritance pattern on 5 mate-pairs of M. boesemani and their progeny, in order to make sure that the new microsatellite markers did not give raise to null alleles. Results indicated that all genotypes observed in the offspring complied with those expected from the parental ones.

Besides, there were no heterozygous genotypes different from the predicted ones, and genotype frequencies were similar to those expected (Table 5). The expected (He)

and observed (Ho) heterozygosity values were not significantly different (Table 6). 9). Consequently, it could be concluded that there was no null allele and that all microsatellite loci followed a Mendelian inheritance pattern.

DISCUSSION

The use of next generation sequencing enabled very quick development of microsatellite markers in a species for which no or limited genomic resources were available. The 12 new markers described here proved efficient and polymorphic on 12 species belonging to the Melanotaenia genus, among which 4 species (M. boesemani, M. catherinae, M. misoolensis and M. parva) are classified in the red list of IUCN. The possible presence of null alleles in the 12 microsatellite markers was checked in silico and results suggested that M. boesemani species was not affected. However, this needed to be experimentally validated in order to definitely exclude this possibility. The cross-breeding experiments presented here showed that the 12 microsatellite markers follow a Mendelian inheritance, and ruled out the existence of null alleles due to primer misamplifications, as well as genotyping errors due to stuttering or large allele dropout. Twelve microsatellite markers had already been described for the Australian species Melanotaenia australis (Castelnau, 1875) [START_REF] Young | Isolation and characterization of 12 novel DNA microsatellite in the Western rainbowfish, Melanotaenia australis[END_REF].

Tested on 32 individuals, these loci showed a moderate polymorphism, with a number of alleles 10 for all but one of them. Two of them showed a significant deviation from Hardy-Weinberg equilibrium, suggesting the possibility of null alleles.

These results indicate that the 12 new microsatellite markers seem suitable to evaluate the genetic diversity among wild populations of rainbowfish from West Papua. Considering their effectiveness in cross-species amplification, we can reasonably expect to apply them on a larger number of species belonging to the genus Melanotaenia. They can also be applied to monitor the genetic changes in the aquaculture strains of M. boesemani, (for example genetic M. boesemani in Indonesian farmers is different with M. boesemani from Uter lake) as is already currently done for other important fish groups [START_REF] Sekino | Genetic Diversity Within and Between Hatchery Strains of Japanese Flounder Paralichthys olivaceus Assessed by Means of Microsatellite and Mitochondrial DNA Sequencing Analysis[END_REF][START_REF] Samy-Yehya | Phylogenetc differentiation of wild and cultured Nile Tilapia (Oreochromis niloticus) populations based on phenotype and genotype analysis[END_REF]. Alternatively, they will also be useful in assessing the reproductive success of M. boesemani [START_REF] Moran | Genetic assessment of the reproductive success of Atlantic salmon precocious parr by means of VNTR loci[END_REF][START_REF] Thompson | Comparison, using minisatellite DNA profiling, of secondary male contribution in the fertilisation of wild and ranched Atlantic salmon (Salmo salar) ova[END_REF], since

Indonesian farmers claim a decline in the spawning rates and in the number of offspring per spawning (personal communication).

IV. GENETIC DIVERSITY OF WILD POPULATIONS OF MELANOTAENIA FROM WEST PAPUA

INTRODUCTION

Rainbowfishes of the family Melanotaeniidae are the most speciose group of pure freshwater fishes within the Australia-New Guinea region [START_REF] Eschmeyer | Catalog of fishes: genera, species, references[END_REF]. They typically possess a compressed body covered with relatively large scales, two separate dorsal fins (the first with 3-7 spines and the second with a single spine and 6-22 segmented rays), a long-based anal fin, and no lateral line (Allen et al. 2008). The family is characterized by relatively small (usually less than 10 cm) and often brightly colored fish. Rainbowfishes are very popular ornamental fish with their peaceful disposition and their ease of breeding. Sexual dimorphism is often apparent and males tend to be deeper-bodied and more vividly colored than females [START_REF] Allen | Field guide to the freshwater fishes of New Guinea[END_REF]. Generally, they are locally abundant and occupy the full spectrum of freshwater habitats including arid-zone waterholes, swamps, rainforest streams and oxbow or mountainous lakes. They are generalist omnivores and show little ecological or morphological specialization like many other freshwater species of the Australian region [START_REF] Roberts | An ichthyological survey of the Fly River in Papua New Guinea with descriptions of new species[END_REF]McDowall, 1981).

Melanotaeniidae occur throughout northern Australia, along the eastern costal strip until the southern Murray-Darling drainages, and also in the western part of the country. They are also found throughout the island of New Guinea below 1600 m elevation, as well as on most surrounding large islands of the western half of the mainland, including the Raja Ampat and Aru Archipelagos and Yapen Island [START_REF] Allen | Field guide to the freshwater fishes of New Guinea[END_REF].

According to [START_REF] Eschmeyer | Catalog of fishes: genera, species, references[END_REF] [START_REF] Allen Gr Hadiaty | Melanotaenia sneideri, a new species of rainbowfish (Melanotaeniidae), from West Papua Province, Indonesia[END_REF].

The tectonic history of the New Guinea region shows that the Bird's Head

Peninsula is a section of the Australian craton that became separated from the main continental mass at Early Cretaceous (Pigram and Davies, 1987). At the same period, the main section of New Guinea was expanded by the collision of the northward-moving Indo-Australian plate with the westward-moving Pacific tectonic plate, and a series of smaller plates [START_REF] Hamilton | Plate Tectonic and the Island Arcs[END_REF][START_REF] Charlton | Tertiary evolution of the Eastern Indonesia Collision Complex[END_REF][START_REF] Hill | Mesozoic-Cenozoic evolution of Australia's New Guinea margin in a west Pacific context[END_REF]. During the Late Miocene (c. 10-12 MYA), a northeast-southwest compression coupled with several subduction processes resulted in the uplift of the rugged Lengguru fold-and-thrust belt which unified the Bird's Head to the greater New Guinea [START_REF] Bailly | Deformation zone 'jumps' in a young convergent setting; the Lengguru fold-and-thrust belt[END_REF]. This geologic episode was concomitantly marked by the uplift of the current Central Highlands that extend east-west through New Guinea [START_REF] Hall | Cenozoic geological and plate tectonic evolution of SE Asia and the SW Pacific: computer-based reconstruction, model and animations[END_REF]. These intense tectonic and orogenic events resulted in the isolation of three major aquatic biotic provinces: northern New Guinea, the combined southern New Guinea/Australia, and the Bird's Head region including the Lengguru Range [START_REF] Allen | Field guide to the freshwater fishes of New Guinea[END_REF][START_REF] Abell | Freshwater ecoregions of the world: a new map of biogeographic units for freshwater biodiversity conservation[END_REF]. ammeri Allen et al., 2008[START_REF] Allen | [END_REF], M. arfakensis Allen, 1990, M.

arguni Kadarusman et al., 2012, M. batanta Allen & Renyaan, 1996, M. boesemani [START_REF] Allen | [END_REF], M. catherinae (de Beaufort, 1910), M.

ericrobertsi Allen et al., 2014, M. fasinensis Kadarusman et al., 2010, M. flavipinnis Allen et al., 2014, M. fredericki (Fowler, 1939), M. irianjaya Allen, 1985, M. kokasensis Allen et al., 2008, M. laticlavia Allen et al., 2014, M. misoolensis Allen, 1982, M. multiradiata Allen et al., 2014[START_REF] Allen | [END_REF], M. salawati Kadarusman et al., 2011, M. sneideri Allen & Hadiaty, 2013, M. synergos Allen & Unmack, 2008, M. urisa Kadarusman et al., 2012, M. veoliae Kadarusman et al., 2012, M. wanoma Kadarusman et al., 2012. Considering their increasing economic value and the ever-growing human pressures on their habitats, more research on the genetic variability of these fishes is needed to i) reveal cryptic diversity and occurrence of additional new Melanotaenia species and ii) identify candidate species for aquaculture and for conservation pur poses. To address these questions, a large number of populations from the Indo-Australian region were first genotyped with the newly developed DNA microsatellite markers. Then, a special focus was made on the Specific Bird's head region of West Papua. Whenever precise taxa could not be determined, a combination of molecular and morphologic approaches were used to assess whether morphological similar specimens from distinct locations were conspecifics or whether sufficient genetic variation and reproductive isolation existed to consider these specimens as distinct species. This led to the identification of eight new species from the Bird's Head region.

automated Workstation (Perkin Elmer). It was eluted in 70 µl of the provided elution buffer, and its concentration measured using a Nanodrop ND8000 spectrophotometer (Thermo Scientific). Microsatellite amplifications were performed with fluorescently labeled primers (Eurofins) and each reaction contained 5 µl of 2x Master mix (Roche), 0.1 µM of forward primer, 0.4 µM of reverse primer and 0.5 µl of template DNA.

Cycling conditions were as follow: initial denaturation at 95°C for 4 min followed by 30 cycles of 95°C for 20 s, 56°C for 20 s and 72 °C for 30 s, and a final elongation step of Allelic diversity (Na), average expected (He) and average observed (Ho) heterozygoties were calculated using the GENETIX 4.05 software [START_REF] Belkhir | GENETIX 4.05, logiciel sous Windows TM pour la génétique des populations[END_REF].

Locus-by-locus and multilocus Hardy-Weinberg equilibrium was tested for each species by estimating the significance of the inbreeding coefficient (Fis) from sets of 1000 random allelic permutations of the original dataset, as implemented by GENETIX 4.05 software. The P value of Fis was defined as the probability to obtain absolute values higher than or equal to the observed one under the null hypothesis. In order to assess the possible presence of genotyping errors, the presence of null alleles, stuttering or large allele dropout was also tested for all the analyzed loci across all the species with the Micro-Checker v. 2.2.3 software (CI 95%, bootstrap simulation x1000) [START_REF] Van Oosterhout | Micro-Checker: software for identifying and correcting genotyping errors in microsatellite data[END_REF]. Genetic relationships between multilocus genotypes were assessed using factorial correspondence analysis (FCA) in GENETIX 4.05 software.

Genetic structure among species was also investigated by calculating pairwise Fst values with 1000 random allelic permutations of the original dataset as implemented by GENETIX 4.05 software. Significance level of P value for Fst was defined as above for Fis.

Partial 650-bp sequences from the 5' region of the cytochrome oxydase I gene (COI) were obtained from GenBank for 20 valid species and 8 undetermined taxa (according to Kadarusman et al., 2012a) for which DNA was available (Table 9). Phylogenetic analysis of the COI data set was inferred with neighbor joining (NJ) and maximum likelihood (ML) methods in MEGA5 [START_REF] Tamura | MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance and Maximum Parsimony Methods[END_REF]. The Kimura 2-parameter (K2P) substitution model was used for the NJ analysis with 1000 bootstrap replicates to infer evolutionary distances. We also performed ML analyses with 500 bootstrap replicates using the Kimura 2parameter model with a discrete Gamma distribution (K2P+G), which was the most appropriate nucleotide substitution model for our dataset based on the Bayesian Information Criterion (BIC) in the ML model selection feature of MEGA5.

Specimen deposition and new species description

Specimens of the new species were deposited at the Museum Zoologicum Bogoriense (MZB, Indonesia). The methods for counting and measuring were derived from [START_REF] Allen | [END_REF] with some modifications and additions [START_REF] Kadarusman | Description of Melanotaenia fasinensis, a new species of rainbowfish (Melanotaeniidae) from West Papua, Indonesia with comments on the rediscovery of M. ajamaruensis and the endangered status of M. parva[END_REF]. Measurements were performed with digital dial callipers under lightening monocular lens (x2) and counts were made under lightening binocular lenses (x4). Measurements were made on the left side and were expressed to the nearest 0.1 mm. All proportions were expressed as percentage of the standard length. Counts and measurements were performed by Dr. Kadarusman, as this part of my thesis is the continuation of the work initiated by this latter in the frame of his PhD.

The comparative material included the 20 valid species described from the Bird's Head region with their COI barcode. This material consisted of type specimens and additional specimens caught at the type localities during the three expeditions (2007-2008-2010). These additional specimens were deposited at MZB, RMNH and MNHN. M. catherinae In order to assess the genetic relationships between the predicted new species and all other closely related nominal species (according to the COI phylogeny), several factorial correspondence analyses (FCA) were computed using the genotypes obtained with the 12 microsatellite markers. The results were as follow:

M. batanta ------ -0.087 ------ ------ ------ -0.026 ------ ------ ------ -0.132 ------ ------ - 0
0.094 ------ ------ -0.078 ------ 0.107 ------ ------ ------ -0.108 ------ -0.115 -0.006 M. ericrobertsi ------ ------ ------ ------ ------ ------ ------ ------ ------ ------ ------ ------ ------

M. veoliae

------ ------ ------ -0.007 ------ ------ -0.206 ------ 0.051 ------ ------ ------ -0.048

M. wanoma

(1) The three new species M. klasioensis, M. longispina and M. susii were genetically differentiated from each other as well as from M. arfakensis and all the remaining species that constitute the "Central Ayamaru Plateau" cluster (Fig. 11). All pairwise Fst values showed highly significant genetic divergence (p < 0.001) among these species (Table 13). Pairwise Fst values between the 3 new species (i.e. M. klasioensis, M. longispina, M. susii) were comprised between 0.254 and 0.418. While the new species M. klasioensis showed a low genetic distance with M. fasinensis on the COI (Fig. 17), the microsatellite data indicated that these two species constitute distinct genetic entities (Fig. 11) characterised by a significant Fst value of 0.633 (Table 13).

The same results were observed between M. susii and M. ericrobertsi and between M. longispina and all other species belonging to the "Central Ayamaru Plateau" cluster. (2) The FCA presented in figure 19 shows that the new species M. sembra is genetically differentiated from its sister species M. multiradiata (cluster "Southern Ayamaru Plateau"; Fig. 12). This new species was also genetically differentiated from M. arfakensis and from all nominal species (i.e. M. ajamaruensis, M. boesemani, M. ericrobertsi, M. fasinensis) belonging to the cluster "Central Ayamaru Plateau". All pairwise Fst values computed between these species were highly significant (p < 0.001) and are given in table 14.

Figure 12. Factorial correspondence analysis of genotypes (200 individuals; 12 loci) from all valid species belonging to cluster "Southern Ayamaru Plateau", to cluster "Central Ayamaru Plateau" and to M. arfakensis. Projection on axis 1 and axis 3 segregating M. sembra sp. nov, with other species.

(3) The FCA in figure 13 shows that the new species M. manibuii is genetically separated from M. arfakensis, M. multiradiata, M. fredericki and from all the species distributed in the Raja Ampat Islands (i.e. M. batanta, M. misoolensis, M. salawati). In addition, the FCA presented in figure 14 indicates that M.

manibuii is genetically separated from all nominal species composing the cluster "Central Ayamaru Plateau". (5) The three new species M. naramasae, M. rumberponensis and M. sikuensis are genetically differentiated from each other, as well as from the remaining species M. angfa and M. parva that compose the "Bird's Neck" cluster (Figure 16). All pairwise Fst values showed highly significant genetic divergence (p < 0.001) among these species (Table 15). Pairwise Fst values between the 3 new species were comprised between 0.356 and 0.714.

Interestingly, M. angfa and M. parva, the two species that shared the same COI haplotype, displayed a significant pairwise Fst value of 0.460. The locus Mb_tri1 revealed diagnostic for these two species, with a fixed allele (104)

for M. angfa and two alleles (110 (75%) and 113 (25%)) for M. parva.

Figure 16. Factorial correspondence analysis of genotypes (164 individuals; 12 loci) from all the species belonging to cluster "Bird's Neck". Projection on axis 1 and axis 2 segregating the three new species M. naramasae, M. rumberponensis, and M. sikuensis with other species composing this cluster. The ML tree obtained from partial COI sequences obtained from 81 individuals belonging to 20 valid species and the 8 potential new species (Table 9) is presented in figure 24. The same topology was obtained for the K2P-NJ tree; thus, only bootstrap values were displayed together with those obtained for the ML tree.

Three major clades are highlighted on the ML tree (Figure 17). These results are congruent with the topology of the ML tree based on cytochrome b provided in Allen et al., (2014 b) 17).

The new species M. manibuii has a basal position in the first clade (Figure 17) and displays an important phylogenetic distance with all the species composing this clade (average K2P distance: 0.053 ± 0.008). M. manibuii and M. Bootstrap values obtained for ML on 500 replicates and for NJ (within brackets).

The new species M. sembrae forms together with M. multiradiata the "Southern Ayamaru Plateau" cluster within the first clade (bootstrap: ML 98%, NJ 99%). The two species are characterized by low genetic divergence of less than 1% (K2P distance: 0.007 ± 0.003).

The new species M. klasioensis, M. susii and M. longispina belong to the cluster "Central Ayamaru Plateau" together with M. ajamaruensis, M. boesemani, M. ericrobertsi and M. fasinensis (bootstrap: ML 88%, NJ 86%). These species are characterized by low phylogenetic distances (average K2P distance: 0.015 ± 0.003) and are all distributed in the fragmented river systems of the karstic Ayamaru Plateau. This cluster displays higher phylogenetic distances with M.

arfakensis (K2P distance: 0.030 ± 0.005) as well as with the "Southern Ayamaru

Plateau" cluster (K2P distance: 0.029 ± 0.005).

Finally, the 3 new species M. naramasae, M. sikuensis and M.

rumberponensis are genetically closely related with M. angfa and M. parva. They belong to the cluster "Bird's Neck" and are characterised by phylogenetic distances of less than 1% (K2P distance: 0.005 ± 0.002). This cluster, however, displays a higher phylogenetic distances with the remaining species composing the "Southern Bird's Head" clade (K2P distance: 0.046 ± 0.007). Interestingly, the COI failed to differentiate M. angfa from M. parva, which display the same barcode sequence. This absence of genetic differentiation among these species was also revealed by Allen et al., (2014 b) for the cytochrome b gene and by [START_REF] Unmack | Phylogeny and biogeography of rainbowfishes (Melanotaeniidae) from Australia and New Guinea[END_REF] on one nuclear gene (S7) and seven mitochondrial markers (ND1, ND2, ND4L, ND4, ATPase6/8, cyt b, and partial COIII).

Melanotaenia manibuii

Only from the Tisbo River.

Melanotaenia naramasae

Type locality of Melanotaenia naramasa sp.nov.

Naramasa river, Bird's Neck, west Papua-Indonesia

Melanotaenia rumberponensis

Only from the Rumberpon Island.

Melanotaenia sikuensis

Only from the Siku Creek.

DISCUSSION

The only five species (i.e. M. ammeri, M. fredericki, M. irianjaya, M. manibuii, M. naramasae) presenting significant multilocus Hardy-Weinberg deviations consisted of a mix of individuals sampled at various locations along the same river drainage, because of an insufficient number of specimens that could be caught. These results suggest the possible occurrence of genetically differentiated sub populations patchily distributed along the same river and must be taken into consideration in future sampling strategies or population monitoring of Melanotaenia species. The majority of Hardy-Weinberg disequilibria reported for the different species were probably due to Wahlund effect, as reported by [START_REF] Johnson | The Wahlund effect and the geographical scale of variation in the intertidal limpet Siphonaria sp[END_REF]. The Wahlund effect refers to the reduction of heterozygosity in a population caused by a structuration into subpopulations. Namely, if two or more subpopulations have different allele frequencies, then the overall heterozygosity is reduced, even if the subpopulations themselves are under Hardy-Weinberg equilibrium.

The mitochondrial DNA phylogeny tree combined with the microsatellite genotyping revealed previously undetected diversity among West Papua rainbowfishes. However, the use of the 12 new microsatellite markers proved useful and allowed to confirm these taxa as new species. It revealed an unexpected species diversity of West Papuan Rainbowfishes. Prior to this work, 24 valid species had been described from the region. The discovery of 8 new species in the area emphasized a cryptic diversity that was largely underestimated.

Seven of these 8 newly discovered species are confined to a single river system localized in fragmented karstic environments such as in the limestone karst formations from Ayamaru Plateau or in the northern extension of Lengguru Range.

Melanotaenia manibuii, which was discovered on the southern foothills of Arfak mountains, is the only species distributed outside a karstic formation.

Due to intense tectonic movements of uplift or subsidence and important erosion activity that happened during more than 10 million years, karst systems are characterized by endorheic drainage basins consisting of isolated lakes or fragmented rivers with aerial course which length varies from only several hundred meters to a few kilometers. These unique geological formations have therefore promoted intense diversification and numerous speciation events in Rainbowfishes from Western Papua [START_REF] Kadarusman | Description of Melanotaenia fasinensis, a new species of rainbowfish (Melanotaeniidae) from West Papua, Indonesia with comments on the rediscovery of M. ajamaruensis and the endangered status of M. parva[END_REF](Kadarusman et al., , 2012b;;[START_REF] Allen Gr Hadiaty | Melanotaenia sneideri, a new species of rainbowfish (Melanotaeniidae), from West Papua Province, Indonesia[END_REF]Allen et al., 2014a, b). The total area covered during expedition The Boeseman´s Rainbowfish Melanotaenia boesemani [START_REF] Allen | [END_REF] is one of the most popular rainbowfish species in the aquarium hobby. When fully matured, males display a very distinct pattern of coloration compared to other species with a half-and-half coloration marked by a brilliant blue anteriorly and by a bright yellow to orange-red posteriorly (Figure 19) [START_REF] Kadarusman | Rainbowfishes from West Papua (Melanotaeniidae): Evolution and Systematics[END_REF][START_REF] Tappin | Rainbowfish Fishes, their care & keeping in captivity[END_REF]. History in Leiden (Netherlands) (Allen, 1980). According to several studies [START_REF] Allen | Rainbowfishes in Nature and the Aquarium[END_REF]Allen, 1982;[START_REF] Kadarusman | Rainbowfishes from West Papua (Melanotaeniidae): Evolution and Systematics[END_REF], the species is only known from Ayamaru Lakes and tributaries and also from Uter-Aitinyo Lake (or Uter Lake).

The two locations are distant by 30 km and are separated by rugged karsts (Fig. 20). boesemani (Allen, 1980), a great interest arose for the potential commercial value of this species. It was introduced to the aquarium hobby in 1983 and has steadily increased in popularity since then [START_REF] Polhemus | Freshwater Biogeography of Papua[END_REF]. In the mid 1980s, more than 60,000 males were caught monthly from Ayamaru for exportation purposes (Smith et al., 2007). This over-exploitation has therefore quickly conducted this species to the verge of extinction in its natural habitat [START_REF] Allen | Rainbowfishes in Nature and the Aquarium[END_REF]. As a consequence, it has been placed on the red list of endangered species since 2004, and only aquaculture products are now supposed to be exported (IUCN, 2015). Farm Warso, Tambun Utara, Bekasi 2012 30

DNA extraction and microsatellite amplification

Individual samples were genotyped with the 12 nuclear microsatellite markers developed in chapter III. DNA was extracted from a small fragment ( 10

Genetic diversity analysis

Allelic numbers (Na), average observed (Ho) and expected (He) heterozygosities were calculated using the GENETIX 4.05 software [START_REF] Belkhir | GENETIX 4.05, logiciel sous Windows TM pour la génétique des populations[END_REF]. Data were analyzed for possible departures from Hardy-Weinberg equilibrium by estimating the significance of the multilocus inbreeding coefficient (Fis) from sets of 1000 random allelic permutations of the original datasets. The genetic structure among the investigated populations was also evaluated by calculating pairwise Fst values with 1000 random allelic permutations. The significance level of P value for Fis and Fst was defined as the probability to obtaining absolute values higher than or equal to the observed one under the null hypothesis. The geographic origin of the 6 reared strains was assessed through a factorial analysis of correspondences (Genetix 4.05), by comparing their genotype frequencies to those of the wild populations of M. boesemani from Ayamaru and Uter lakes.

RESULTS

Genetic variability and heterozygosity of M. boesemani in aquaculture settings and in natural populations

A total of 151 alleles were detected in the 183 farmed and 77 wild specimens across the 12 microsatellite loci. The total number of alleles per locus for all populations varied from 5 to 22 and all loci were polymorphic in each strain and wild population (Table 17). Generally, the domesticated populations showed a lower genetic variability (total number of alleles comprised between 62 and 86) compared to Ayamaru wild population (107 alleles), but comparable with that observed in the wild population of Uter (82 alleles) (Table 17). Considering only the wild populations, there were 23 private alleles for Uter Lake population and 48 private alleles for Ayamaru Lake. When all samples were included, there were 17 private alleles for Uter Lake, 16 private alleles for Ayamaru Lake and 21

private alleles for the domesticated populations (i.e. only present in one or more strains and absent in wild populations). There were 6 alleles shared between Uter Lake and one or more strains, and 32 alleles shared between Ayamaru lake and at least one strain. There were 51 alleles shared between both wild populations and one or more strains, and 8 alleles shared between Ayamaru and Uter lakes but absent from any strains (Table 20). Likewise, the heterozygosities calculated for the captive populations (Hobs comprised between 0.53 and 0.64) were lower than that calculated for the Ayamaru wild population (0.68) but similar to that of Uter population (0.56) (Table 18). Moreover, the Ho were never significantly different from the He. The multilocus Fis values for all populations (wild and captive) were comprised between -0.010 and 0.061 and none of them was significant except that obtained for the Sukri strain (Fis = 0.061, P < 0.05) (Table 18). These results strongly suggest that, with the exception of Sukri farm, there is no deficit in heterozygotes in any population. Finally, the pairwise Fst values calculated between each population were all significant (P < 0.05), except between Warso and Yahya and between Warso and Didi farms, indicating that most of the populations are genetically differentiated from each other (Table 19).

DISCUSSION

Ayamaru and Uter lakes are located in Indonesia in the western part of New Guinea island. These two lakes are not connected and are separated by rugged karsts, with a distance of 30km. Though they are considered to belong to the same species, the fish from these two lakes display significant morphological differences: those from Ayamaru lake harbor a bright blue anterior and bright yellow anterior coloration, whereas those from Uter show a bright blue anterior and a reddish posterior coloration (Figure 19). Nevertheless, previous analyses based on both the mitochondrial cytochrome oxydase I gene and morphomeristic traits failed to separate these two populations, which are slightly overlapping [START_REF] Kadarusman | Rainbowfishes from West Papua (Melanotaeniidae): Evolution and Systematics[END_REF]. Here, the use of 12 microsatellite markers brought clear evidence that they are genetically differentiated, and emphasizes that population mixtures should be strictly avoided during any upcoming broodstock constitution.

From the samples investigated here, many private alleles were evidenced between the two wild populations of M. boesemani (23 for Uter lake and 48 for Ayamaru lake). Among the 12 markers, Mb_di1, Mb_di3, Mb_di4 and Mb_tetra2 accounted for more than 50% of the private alleles found between the two populations (42 over 71).

Among the investigated farms, Gusi, Hasan and Didi started to exploit M.

boesemani in 1983 and 1986, respectively, i.e. quickly after the description of M.

boesemani species by [START_REF] Allen | Descriptions of five new rainbowfish (Melanotaeniidae) from New Guinea[END_REF] and the publication of Rainbowfishes of Australia and Papua New Guinea (Allen, 1982). This book greatly contributed to increase the popularity of rainbowfishes, especially the newly discovered New Guinea species, among which M. boesemani [START_REF] Tappin | Rainbowfish Fishes, their care & keeping in captivity[END_REF]. In spite of the establishment of these farms, exploitation of this species from its natural environment in Ayamaru has persisted until now. The geography of Uter Lake, surrounded by mountains and characterized by steep and rocky shores, is less favorable to fishing activities than that of Ayamaru, more easily accessible. This is probably the main reason why all investigated domesticated populations originated from Ayamaru.

The core population of Ayamaru sampled 30 years ago for the constitution of the cultivated strains was genetically different from that of the wild population sampled in 2007. This was especially evidenced by the 21 private alleles observed in the strains and absent in the wild population of Ayamaru Lake. The presence of these private alleles may suggest a loss of variability that the wild population of Ayamaru has undergone over the last 30 years. However, the fact that heterozygosity and allele number values calculated from the Ayamaru population are comparable with those of other rainbowfish species [START_REF] Nugraha | Development of twelve novel polymorphic microsatellite DNA markas for the Boeseman's rainbowfish (Melanotaenia boesemani) and test for their cross-utility in 21 rainbowfish species from West Papua (Indonesia)[END_REF], is not in favor of a loss of genetic diversity. Most likely, these private alleles may indicate that the genetic diversity of the Ayamaru specimens investigated here are probably not representative of the whole genetic diversity of M. boesemani in this area (i.e. Ayamaru Lake and tributaries). The wild fish sampled in 2007 were indeed collected in Tiwit River, a tributary of Ayamaru Lake, and could represent a distinct genetic entity from the whole population. In this case, the species could constitute a metapopulation composed of several population subdivisions according to their geographic distribution in the mosaic of habitats characterizing Ayamaru Lake. It was recently shown on a wide range of marine fishes that overfishing could result in a significant reduction of genetic diversity [START_REF] Pinsky | Meta-analysis reveals lower genetic diversity in overfished populations[END_REF]. Another recent meta-population study using many marine fish species demonstrated that the renewal of a fish population depends much less on the amount of available genitors than on environmental factors [START_REF] Cury | Resolving Hjort's Dilemma How Is Recruitment Related to Spawning Stock Biomass in Marins Fish?[END_REF]. If the data presented here is not sufficient to tackle these questions, it nonetheless highlights the urgent need to perform a complete and exhaustive sampling campaign for assessing the exact genetic structure and diversity of this endangered species, in order to efficiently contribute to its conservation. This implies to collect fish in many more locations of Ayamaru Lake and its tributaries.

Indeed, in addition to overfishing, Ayamaru Lake also experiences important environmental threats (deforestation, urbanization) and episodic droughts. Based on the sequence of a mitochondrial DNA fragment of the native Australian freshwater fish Rhadinocentrus ornatus, Mather et al. showed that habitat degradation caused by urbanization entailed a significant reduction in genetic diversity [START_REF] Mather | Urban development explains reduced genetic diversity in a narrow range endemic freshwater fish[END_REF].

Finally, the genetic variability assessed within the 6 farmed populations was comparable to that of the natural population of Uter Lake. Besides, no deficit in heterozygotes was evidenced, suggesting that there was no major inbreeding in these reared populations. Therefore, the problems experienced by the farmers (i.e. decline of production, higher proportion of females per spawning, loss of coloration, lower growth rate and fecundity, morphological abnormalities) are obviously not due to inbreeding depression and are probably caused by other factors such as unsuitable management and/or poor water quality. It is noteworthy that all investigated farms are located in industrial and densely populated periurban areas. Considering that Jakarta and its suburbs have no waste water treatment system, the water used for rearing these fish is probably of very poor quality, and may contain many chemical and hormonal pollutants, which have both been proven to alter major fish traits such as reproduction, growth. It has indeed been shown that exposure to low concentrations of endocrine disruptive chemicals (among which estrogens) can impede gonadal function, reduce fertilization success, decrease fecundity, alter mating behavior and reverse sex of various aquatic species [START_REF] Martinović | Environmental estrogens suppress hormones, behavior, and reproductive fitness in male fathead minnows[END_REF][START_REF] Sumpter | Endocrine Disrupters in the Aquatic Environment: An Overview[END_REF][START_REF] Vos | Health Effects of Endocrine-Disrupting Chemicals on Wildlife, with Special Reference to the European Situation[END_REF].

It can even cause the collapse of fish populations at trace concentrations [START_REF] Kidd | Collapse of a fish population after exposure to a synthetic estrogen[END_REF].

Regarding the coloration pattern, it is known that rainbowfish are able to change color according to the turbidity level of their environment [START_REF] Kelley | Changes in the visual environment affect colour signal brightness and shoaling behaviour in fresh water fish[END_REF][START_REF] Rodgers | Colour change and assortment in the western rainbowfish[END_REF]. For instance, an increased brightness of red colors in environments rich in organic matter (i.e. more turbid) may help enhance conspicuousness, and then allow individuals to maintain communication in altered visual environments. Thus, the alteration of fish color brightness observed in reared animals may be the consequence of an adaptation to the aquaculture setting conditions, where ponds are very shallow and mimic habitats with full-spectrum lighting. However, a better understanding of the visual system of Melanotaeniidae is required to better predict how changes in the aquatic light environment affect the physiology and ecology of these fishes, and in turn, to allow farmers to adapt their rearing conditions in order to maintain bright body colors. boesemani conducted this species to the verge of extinction [START_REF] Allen | Fishes of Papua[END_REF]. The results presented here (i.e. no significant loss of heterozygotes in the wild populations and no inbreeding depression in the reared strains) indicate that, in spite of the threats that Ayamaru is facing, it is still possible to prevent the extinction of M. boesemani. This, however, implies to increase its aquaculture production in order to quickly alleviate the overfishing pressure. This, in turn, involves a better management of the quality of waters used for rearing M.

boesemani, which is a general concern in Indonesia.

VI. GENERAL DISCUSSION AND PERSPECTIVES

Indonesia is one of the countries that hold the highest biodiversity in the world [START_REF] Fretes | Opportunities and challenges for doing conservation in Papua in Marshall AJ and Beehler BM[END_REF]. New Guinea Island is located in the northern quadrant of the Australian tectonic plate. Papua has more than a thousand fringing islands, from tiny to quite large ones. The temperature gradient due to altitude elevation in mountainous Papua has contributed distinct biotas to inhabit adjacent patches of land, separated only by elevation. Together with the geology of this island, it certainly explains much of the species richness of Papua.

Polhemus and Allen (2007) defined 29 areas of freshwater endemism within New Guinea and closely adjacent islands that need further assessment for conservation strategies. These hypotheses of regional endemism were further refined for western New Guinea, especially for freshwater fish and and more widely for aquatic organisms for the whole New Guinea. These authors also Tropical Wilderness Area" remaining in Asia. For illustration, all the species of melanotaeniids from Western New Guinea are endemic from the area. Because many rainbowfish species have restricted distributions and are confined to specific habitats, such as isolated lacustrine environments or small parts of a single river system, they are highly vulnerable to environmental disturbance and overharvesting. For instance, Ayamaru Lake is affected by the development of residential areas and ecotourism. As a result, forest clearance has increased channel obstructions by sediments and led to partial drying of the lake.

Meanwhile, simultaneous over-catching of the endemic M. boesemani conducted this species to the verge of extinction [START_REF] Allen | Fishes of Papua[END_REF]. The results presented in this manuscript (i.e. no significant loss of heterozygotes in the wild populations and no inbreeding depression in the reared strains) indicate that, in spite of the threats that Ayamaru Lake is facing, it is still possible to prevent the extinction of M. boesemani.

Our results also revealed an unexpected species diversity of West Papuan

Rainbowfishes. Prior to this work, 24 nominal species were known from West Papua. The discovery of 8 new species in the area emphasized a cryptic diversity largely underestimated. Seven of these 8 newly discovered species were confined to a single river system localized in fragmented karstic environment. This, again, highlights the high vulnerability of these populations, and illustrates the need to protect West Papua. For the past half century, it has been widely recognized that the rate of species extinction was increasing and that many other species were in imminent danger of extinction. The major factors related to these extinctions and declines were over-harvesting from hunting, fishing, trapping, and other killing; loss, degradation, and fragmentation of habitat; and introduction of non-native species such as pathogens, parasites, predators, and competitors [START_REF] Diamond | Overview of recent extinctions[END_REF].

This is typically what West Papua is facing today.

Considering the many endemic species in Papua, there is an urgent need to conduct floral and faunal surveys throughout Papua to identify areas of special conservation value. Many freshwater fish, particularly the rainbowfish (Melanotaeniidae), appear to be restricted to an isolated lake or small part of single river system. These species are highly vulnerable to environmental disturbances such as clear-cut logging, mining and building construction. For environment and biodiversity preservation, the government must increase its conservation policies. This implies that local populations be aware of the uniqueness of their plants and animals, and of the need to protect them. This implementation is of course difficult, especially in a context of economic development. However, this is part of our responsibilities to make citizens understand that biodiversity is important to people because it supports their livelihoods and welfare either directly through the use of biological resources, or indirectly through its contribution towards the maintenance of ecosystem function.

Land with high biodiversity like Papua is able to provide services like watershed The primary aim of conservation is to save the species in their natural environment. If the importance of conserving the wild ancestors and related species both in situ and ex situ is widely recognized, domestication is however often seen as the first (and even the only) way of conservation. The purpose of reorganization of wild populations into domestic and cultivated forms varies according to the interests of people: conserve genetic resources and/or use them in breeding programs. A problem resulting from domestication is the possibility of significant reduction of genetic variability, as has been observed in most species with advanced level of domestication [START_REF] Arnaiz | Evolution of polaskia Chichipe (Cactaceae) under domestication in the Thehuacán valley, Central Mexico: Reproductive biology[END_REF]. In Indonesia, the aquaculture of ornamental fish has become an important industry. However, farming of the endangered Melanotaenia boesemani has remained anecdotic, which is reflected by the low number of farms breeding this species. As a consequence, aquaculture cannot fulfill the high demand for this fish. The farmers claimed a decrease of both quantity and quality of their production, and attributed it to loss of genetic variability and possible inbreeding. From the results reported here, it is now clear that this decline in production is not due to genetic factors and is probably caused by other problems such as poor water quality. It is now crucial to increase the aquaculture production of this species in order to quickly alleviate the overfishing pressure. This, of course, involves a better management of the quality of waters used for rearing M. boesemani, which is a general concern in Indonesia.

Finally, priority strategies for conservation of genetic diversity need to consider not only the level of diversity in an area but also the current threat to the condition of a particular region. Results obtained in the present study suggest that in situ conservation of the Melanotaenia complex should focus on metapopulations which have a higher level of genetic variation than populations from other groups within this species complex. For ex situ conservation, sampling more populations rather than intense sampling of a few populations is a preferable strategy. Ex situ conservation can be achieved through genetic management like captive breeding programs. It also has the advantage to provide people with economical value and high quality nutrients. All the species characterized in this manuscript deserve to be immediately domesticated and cultivated in order to preserve them. They also represent possible new candidates in aquaculture commodity that may have high economy on the international market. Study of the population, distribution and genetic variation of Melanotaenia species should provide useful information in the active domestication of these species and allow prioritizing areas for future collection and conservation. Genetic diversity data are scarce for West Papua Melanotaenia species, and the genetic markers developed here should prove valuable for both in situ and ex situ conservation strategies.

Ikan pelangi (Melanotaeniidae) didistribusikan antara New Guinea dan Australia. Mereka sangat dicari oleh hobiis akuarium karena karena warna mereka yang luar biasa bagus. Sejumlah besar spesies, berada di daftar merah spesies terancam punah. Spesies Melanotaenia boesemani, adalah salah satu spesies yang paling populer pada famili Melanotaenia dan sudah terancam punah. Budidaya spesies ini muncul sebagai solusi yang menjanjikan untuk membatasi penangkapan di habitat alam. Namun jumlah peternakan penangjaran M. boesemani sangat rendah. Ini mungkin karena masalah yang dihadapi oleh pembudidaya, yaitu proporsi yang lebih tinggi pada betina, hilangnya warna, tingkat pertumbuhan dan kesuburan rendah, serta sering terjadinya cacat morfologi. Dalam konteks disertasi ini, adalah untuk menghasilkan data genetik baru dalam meningkatkan budidaya dan konservasi famili Melanotaenia. Secara khusus, tujuan adalah: 1) untuk mengembangkan penanda mikrosatelit baru dari DNA M. boesemani, 2) menilai keragaman genetik populasi liar dari Melanotaenia dan memperbaiki taksonomi mereka, 3) untuk menentukan asal geografis dari strain M. boesemani yang telah dibudidayakan di Indonesia, dan untuk menilai tekanan inbreeding yang dihasilkan dari perkawinan sedarah pada domestikasi ini. Untuk sekuensing, sebanyak 12 penanda mikrosatelit dikembangkan dan divalidasi pada spesies M. boesemani. Lokus yang sesuai semua ditemukan polimorfik dan percobaan crossing telah menunjukkan bahwa mereka mematuhi hukum Mendel.

Penanda baru yang kemudian digunakan untuk mengevaluasi keragaman genetik 44 populasi liar (sesuai dengan 1.152 spesimen ikan). Nilai-nilai Fis multilokus mengungkapkan bahwa 5 spesies menunjukkan perbedaan yang signifikan dalam keseimbangan dan Hardy-Weinberg menyarankan kemungkinan adanya subpopulasi dibedakan secara genetik. Dikombinasikan dengan analisis filogenetik dari sitokrom oksidase gen I (IOC) dan pengamatan beberapa karakter morfologi diagnostik, 12 penanda mikrosatelit juga digunakan untuk mengkarakterisasi 8 spesies baru yang belum teridentifikasikan. Akhirnya, penanda mikrosatelit yang diterapkan untuk menganalisa dan membandingkan variabilitas genetik M. boesemani diperoleh dari enam peternakan sekitar Jakarta dengan dua populasi asli dari danau Ayamaru dan danau Uter (Papua Barat). Hasil penelitian menunjukkan bahwa semua strain yang telah di budidayakan berasal dari danau Ayamaru. Tidak ada kekurangan heterozigot kekurangan yang teramati, menunjukkan bahwa tidak ada perkawinan sedarah yang signifikan dalam strain budidaya tersebut. Analisis genotipe juga menunjukkan bahwa spesies M. boesemani yang dibudidayakan merupakan populasi yang berbeda. Pada akhirnya, hasil ini menunjukkan bahwa masalah yang dihadapi oleh pembudidaya ikan bukan karena perkawinan sedarah mungkin tapi yang paling mungkin terkait dengan faktor-faktor lain seperti manajemen budidaya yang tidak memadai dan / atau perairan pembibitan yang berkualitas buruk. Kesimpulannya, penanda mikrosatelit baru ini berguna untuk mengevaluasi struktur genetik dan keragaman spesies ikan pelangi, yang banyak mulai terancam punah. Hasil yang disajikan di sini di salah satu spesies paling terancam punah (M. boesemani) menunjukkan bahwa masih mungkin untuk mencegah kepunahan. Budidaya merupakan solusi untuk menekan penangkapan dihabitat alami. Ini membutuhkan manajemen yang lebih baik pada praktek pembudidayaan.

Les poissons arc-en-ciel (Melanotaeniidae) se distribuent entre la Nouvelle-Guinée et l'Australie. Ils sont très recherchés en aquariophilie en raison de leur coloration remarquable. Il en existe un très grand nombre d'espèces, dont certaines figurent sur la liste rouge des espèces menacées. L'espèce Melanotaenia boesemani, l'une des plus populaires au sein de cette famille, est en voie de disparition. L'aquaculture de cette espèce apparaît donc comme une solution prometteuse pour limiter la capture de spécimens sauvages. Pourtant, le nombre de fermes qui élèvent M. boesemani est très faible. Ceci est probablement dû aux problèmes rencontrés par les aquaculteurs, à savoir une proportion plus élevée de femelles par ponte, une perte de la coloration, un taux de croissance et une fécondité plus faibles, ainsi que l'apparition fréquente de malformations. Dans ce contexte, cette thèse visait à produire de nouvelles données génétiques en vue d'améliorer l'aquaculture et la conservation de cette famille. Plus précisément, les objectifs étaient: 1) de développer de nouveaux marqueurs microsatellites à partir d'ADN de M. boesemani, 2) d'évaluer la diversité génétique des populations sauvages de Melanotaenia et d'affiner leur taxonomie, 3) de définir l'origine géographique des souches de M. boesemani élevées en Indonésie, et d'évaluer la pression de consanguinité résultant de cette domestication. Par séquençage haut débit, 12 marqueurs microsatellites ont été développés et validés sur l'espèce M. boesemani. Les loci correspondant se sont tous révélés polymorphe et des expériences de croisement ont montré qu'ils se conformaient aux lois de Mendel.

Ces nouveaux marqueurs ont ensuite été mis en oeuvre pour évaluer la variabilité génétique de 44 populations sauvages (correspondant à 1.152 spécimens de poissons). Les valeurs de Fis multilocus ont révélé que 5 espèces présentaient des écarts significatifs à l'équilibre de Hardy-Weinberg et suggéré la présence possible de sous-populations génétiquement différenciées. Combinés à une analyse phylogénétique effectuée sur le gène de la cytochrome oxydase I (COI) et à l'observation de plusieurs caractères morphologiques diagnostic, les 12 marqueurs microsatellites ont également permis de caractériser 8 nouvelles espèces nonencore décrites. Enfin, ces marqueurs microsatellites ont été appliqués pour analyser et comparer la variabilité génétique d'échantillons de M. boesemani obtenus à partir de 6 fermes aquacoles autour de Jakarta avec celle des deux populations indigènes de cette espèce, à savoir des lacs Ayamaru et Uter (Papouasie occidentale). Les résultats ont indiqué que toutes les souches élevés provenaient du lac Ayamaru. Aucun déficit en hétérozygotes n'a été mis en évidence, suggérant qu'il n'y avait pas de consanguinité majeure dans ces souches d'élevage. L'analyse des génotypes a également suggéré que l'espèce M. boesemani représentait probablement une métapopulation constituée de populations génétiquement différenciées. En définitive, ces résultats indiquaient que les problèmes rencontrés par les aquaculteurs ne proviennent pas d'une éventuelle consanguinité mais sont plus surement liés à d'autres facteurs tels qu'une gestion inappropriée et / ou une mauvaise qualité des eaux d'élevage. En conclusion, ces nouveaux marqueurs microsatellites se sont avérés utiles pour évaluer la structure génétique et la diversité d'un grand nombre d'espèces de poisson arc-en-ciel, dont beaucoup sont en voie de disparition. Les résultats présentés ici sur l'une des espèces les plus menacées (M. boesemani) montrent qu'il est encore possible d'éviter son extinction. Ceci nécessite cependant d'augmenter sa production aquacole afin de soulager rapidement la pression de surpêche. Ceci passe par une meilleure gestion des pratiques d'élevage.

Rainbowfishes (Melanotaeniidae) are widely distributed throughout New Guinea and Australia. They are very famous for ornamental trade because of their vivid coloration. They display amazing species richness and some of them are on the red list of endangered species. The species Melanotaenia boesemani, one of the most popular within this family, is.facing great threats. Rearing of this species in aquaculture setups thus appears as a promising solution to limit capture of wild specimens. Yet, the number of farms that raise M. boesemani is very low. This is probably due to the problems reported by the farmers, i.e. higher proportion of females per spawning, loss of coloration, lower growth rate and fecundity, frequent morphological abnormalities. In this context, this study aimed at gathering new genetic information that would be useful for the aquaculture and conservation of the Melanotaeniidae family. Specifically, the objectives of the research were: 1) to develop new microsatellite DNA markers from the endangered M. boesemani, 2) to evaluate the genetic diversity of wild populations of Melanotaenia and refine their taxonomy, 3) to describe the geographic origins of M. boesemani reared by ornamental fish farmers in Indonesia, and evaluate the inbreeding pressure resulting from this domestication. Using next generation sequencing, 12 microsatellite DNA markers were developed and validated from M. boesemani. All microsatellite loci revealed polymorphic and cross-breeding experiments showed that they followed a Mendelian inheritance pattern.

These new markers were subsequently implemented to evaluate the genetic variability of 44 wild populations (corresponding to 1.152 fish specimens). Multilocus Fis values revealed that 5 species significantly departed from Hardy-Weinberg expectations and suggested the possible occurrence of genetically differentiated subpopulations. Combined with a phylogenetic analysis performed on the cytochrome oxydase I (COI) gene and with the observation of several diagnostic morphological characters, the 12 microsatellite markers also enabled to characterize 8 new species previously undescribed. Finally, these microsatellite markers were applied to analyze and compare the genetic variability of M. boesemani samples obtained from 6 aquaculture farms around Jakarta with that of the two native populations of this species , i.e. from Ayamaru and Uter Lakes (West Papua). Results indicated that all reared strains originated from Ayamaru Lake. No deficit in heterozygotes was evidenced, suggesting that there was no major inbreeding in these reared populations. Genotype analysis also suggested that M. boesemani species consists of a metapopulation composed of genetically differentiated populations. Altogether, these results indicated that the problems experienced by the farmers are obviously not due to inbreeding depression and are probably caused by other factors such as unsuitable management and/or poor water quality. In conclusion, these new microsatellite markers proved useful to evaluate the genetic structure and diversity of a large number of rainbowfish species, among which many are endangered. The results presented here on one of the most threatened species (M. boesemani) show that it is still possible to prevent its extinction. This, however, implies to increase its aquaculture production in order to quickly alleviate the overfishing pressure. This, in turn, involves a better management of rearing practices..

  addition to Gerald Allen, an Indonesian research team from the Ministry of Maritime Affairs and Fisheries (Sorong Fisheries Academy and Research and Development Center for Ornamental Fish Culture), in collaboration with French researchers (Institut De Recherche pour le Dévelopement -IRD), has conducted researches and species descriptions of this family through several expeditions conducted since 2007.

Figure 1 .

 1 Figure 1. Melanotaenia boesemani (photo: Dr. Laurent Pouyaud)

  Between 2007 and 2010, there were several expeditions conducted by IRD and Academy of Fishery Sorong in the frame of Dr. Kadarusman's PhD: Rainbowfish from West Papua (Melanotaeniidae): Evolution and Systematics. This study contributed to the revision of the taxonomy of rainbowfishes, especially by the identification of new morphological criteria and the use of mitochondrial (CO1, Cyt b and D-loop) and nuclear (S7) molecular markers. Six new species of Melanotaenia

Figure 2 .

 2 Figure 2. Map of the Melanotaeniidae distribution based on molecular phylogenies (Kadarusman 2012)

Figure 3 .

 3 Figure 3. Seven endangered Melanotaenia species from Bird's head peninsula (West papua) -(Photo: Dr. Laurent Pouyaud)

Figure 4 .

 4 Figure 4. Tree species rainbowfish, have been domesticated and cultivated in Indonesia, namely Glossolepis inicus weber 1908, Iriatherina werneri Meinken 1974 and Melanotaenia boesemani Allen & Cross 1980.

5 .

 5 Captive breeding of M. boesemani in Indonesian farms Rainbowfish aquaculture in Indonesia began in 1983 in Mr Gusi farm. He got founder population of M. boesemani in his farm from exporters of ornamental fish in Jakarta, Indonesia. In that decade, M. boesemani became very famous because of publications, especially that by

  generation sequencing technology released to the market, and circumvented the cloning requirement by taking advantage of a highly efficient in vitro DNA amplification method known as emulsion PCR. In emulsion PCR, individual DNA fragment-carrying streptavidin beads, obtained through shearing the DNA and attaching the fragments to the beads using adapters, are captured into separate emulsion droplets. The droplets act as individual amplification reactors, producing ∼10 7 clonal copies of a unique DNA template per bead. Each template-containing bead is subsequently transferred into a well of a picotiter plate and the clonally related templates are analyzed using a pyrosequencing reaction. The use of the picotiter plate allows hundreds of thousands of pyrosequencing reactions to be carried out in parallel, massively increasing the sequencing throughput. The pyrosequencing approach is a sequencing-by-synthesis technique that measures the release of inorganic pyrophosphate (PPi) by chemiluminescence. The template DNA is immobilized, and solutions of dNTPs are added one at a time; the release of PPi, whenever the complementary nucleotide is incorporated, is detectable by light produced by a chemiluminescent enzyme present in the reaction mix. The sequence of DNA template is determined from a "pyrogram," which corresponds to the order of correct nucleotides that had been incorporated. Since chemiluminescent signal intensity is proportional to the amount of pyrophosphate released and hence the number of bases incorporated, the pyrosequencing approach is prone to errors that result from incorrectly estimating the length of homopolymeric sequence stretches (i.e. indels).

Figure 6 .

 6 Figure 6. General flow chart for the development of simple sequence repeats (SSR) markers (reproduced from Zalappa et al., 2012).

Figure 7 .

 7 Figure 7. Allele reading of capillary electrophoresis with GeneMapper program.Illustration of a Gen Mapper view for several loci. The true allele is defined as the highest peak, and the bands before and after are considered to be stuttering bands. From top to bottom: loci Mb_tri 3, Mb_di 4, and Mb_tri4 and Mb_penta2. The 3 first ones appear homozygote in this fish specimen, while Mb_penta2 is heterozygote.

*

  Correspond to the 12 selected microsatellite loci They were first tested for their ability to amplify these loci in different Melanotaenia species. Therefore, in order to reduce the costs, they were not fluorescently labeled. Amplifications were carried out in a GeneAmp 2400 PCR cycler (Applied Biosystems), with the Fast-Start PCR kit (Roche). Each reaction contained 5 µl of 2x Master mix (Roche), 0.4 µM of forward and reverse primers and 0.5 µl of template DNA. Cycling conditions were as follow: initial denaturation at 95°C for 4 min followed by 30 cycles of 95°C for 20 s, 56°C for 20 s and 72 °C for 30 s, and a final elongation step of 7 min at 72°C. Amplification products were analyzed by gel electrophoresis with 2% (w/v) high-melt agarose (Dutscher Scientific) in 0,5% tris-acetate EDTA and visualized with SYBR Safe DNA gel stain (Invitrogen) under a blue light transilluminator in the presence of molecular weight markers (Trackit 1 kb plus DNA ladder, Invitrogen).Fragment size analysis was also examined by capillary electrophoresis. In this case, amplifications were performed with primers labeled with 4 different fluorescent dyes (Eurofins), in the same conditions as described above with two modifications: each reaction contained 0.1 µM of fluorescent forward primer and 0.4 µM of cold reverse primer, and the number of PCR cycles was lowered to 30. PCR products were diluted 1:50 in H2O, or sometimes 1:100 in the case of fluorescence saturation.One µl of diluted PCR from 4 distinct fluorescent couples of primers were mixed together with 13.8 µl of formamide and 0.2 µl of GeneScan™ 600LIZ® Size Standard (Applied Biosystems). Capillary electrophoresis was run in an Applied ABI Prism® 3500 XL 24-capillary sequencer belonging to the technical facilities of the labex "Centre Méditerranéen de l'Environnement et de la Biodiversité" (Montpellier).

Figure 8 .

 8 Figure 8. Example of agarose gel results. (a) Locus that was not amplified in all tested fish; (b) locus that could be amplified in all tested fish. Polymorphism can be visualized in panel (a) as the bands do not display the same molecular weight.

  the 7 genomes of M. boesemani used for sequencing.

Tabel 7 .Figure 9 .

 79 Figure 9. Factorial component analysis 5 mate-pairs of M. Boesemani and their progeny, = Cros 1, = Cross 2, = Cross 3, = cross 4, = Cross5

Kadarusman

  et al., (2012a) assessed the DNA barcoding diversity of 53 nominal species of Melanotaeniidae throughout the Indo-Australian archipelago. This study indicated the family contains three main clades corresponding to the three biogeographic regions of New Guinea and a fourth main clade restricted to Lengguru Range.In a recent study involving nuclear and mitochondrial markers,[START_REF] Unmack | Phylogeny and biogeography of rainbowfishes (Melanotaeniidae) from Australia and New Guinea[END_REF] confirmed that the three main clades correlated with the three main biogeographic regions of New Guinea, but failed to evidence the fourth clade restricted to Lengguru because of a lack of DNA samples (i.e. Melanotaenia mairasiAllen & Hadiaty, 2011).Kadarusman et al., (2012a) therefore confirmed that the Melanotaenia species from the Bird's Head region are monophyletic compared to the three other main clades and highlighted the presence of nearly twice as many evolutionary lineages among the 15 nominal species sampled in this area. These results revealed unexpected levels of cryptic diversity, demonstrating that the diversity of the family has been largely underestimated. The authors concluded that the Bird's Head region should include for Melanotaeniids the western side of Lengguru Range (i.e. all watersheds flowing to Arguni Bay), the Bomberai and the Bird's Head Peninsulas, the Bird's Head Isthmus joining both peninsulas and the 4 major Raja Ampat Islands (i.e. Misool, Batanta, Salawati, Waigeo). The Bird's Head region is a centre for rainbowfish diversity with 24 species currently described: Melanotaenia ajamaruensisAllen & Cross, 1980, M.

7

  min at 72°C. Amplicon size was analyzed by capillary electrophoresis, in the technical facilities of the labex "Centre Méditerranéen de l'Environnement et de la Biodiversité" (Montpellier). PCR products were diluted 1:50 in H 2 O (or 1:100 in the case of fluorescence saturation). One µl of diluted PCR from 4 distinct fluorescent couples of primers were mixed together with 13.8 µl of formamide and 0.2 µl of GeneScan™ 600LIZ® Size Standard (Applied Biosystems). Capillary electrophoresis was run in an Applied ABI Prism® 3500 XL 24 capillary sequencer. Allele sizing and genotyping were achieved with the Peak Scanner v1.0 and GeneMapper® v5.0 software (Applied Biosystems).

Figure 10 .

 10 Figure 10. Type localities of the 24 valid Melanotaenia species and the 8 new species from the Bird's Head region

Figure 11 .

 11 Figure 11. Factorial correspondence analysis of genotypes (255 individuals; 12 loci) from all the species belonging to the phylogenetic cluster "Central Ayamaru Plateau" and to M. arfakensis. Projection on axis 2 and axis 4 segregating the new species M. klasioensis, M. longispina and M. susii with other species.

Figure 13 .

 13 Figure 13. Factorial correspondence analysis of genotypes (186 individuals; 12 loci) from M. manibuii, M. arfakensis, M. multiradiata, M. fredericki, M. batanta, M. misoolensis, M. salawati. Projection on axis 3 and axis 4 segregating M. manibui sp. nov with other species

Figure 15 .

 15 Figure 15. Factorial correspondence analysis of genotypes (164 individuals; 12 loci) from the five new species belonging to COI "Northern Bird's Head" clade. Projection on axis 1 and axis 2 segregating the five new species M. klasioensis, M. longispina, M. manibuii, M. sembrae and M. susii.

18 4. 3 . 3 .

 1833 Mitochondrial DNA barcode and tree-based delimitation of West-Papuan species

  irianjaya, are morphologically closely related, although, they represent distinct species sorting in two different clades (i.e. frist clade for M. manibuii and second clade for M. irianjaya) and are separated by a high phylogenetic distances (K2P distance: 0.069 ± 0.009).

Figure 17 .

 17 Figure 17. Maximum likelihood tree for Bird's Head region Melanotaenia species based on analysis of partial COI sequences (650 bp).Bootstrap values obtained for ML on 500 replicates and for NJ (within brackets).

Figure 18 .

 18 Figure 18. Description and corresponding picture of the eight new species of Melanotaenia from West Papua. No Species name Picture 1

  field organized since 2007 represents approximately 20,000 km2, which corresponds to less than 10% of the global terrestrial surface of Western Papua and to less than 20% of karst landscapes. Therefore, there are still many unexplored areas that remain to be studied, with a special attention in the Central Bird's Head lowlands eastward of Ayamaru Plateau between southern Tambrauw and Arfak mountains and along the Northern foothills of Kumawa on Bomberai Peninsula.The Lengguru fold-and-thrust Belt (LFTB) combined with the Central Dividing Range proved to have played a key role in the diversification of Melanotaeniidae at the regional scale(Kadarusman et al., 2012a;[START_REF] Unmack | Phylogeny and biogeography of rainbowfishes (Melanotaeniidae) from Australia and New Guinea[END_REF]. Rapid tectonic uplift and subsidence raised in Mid-Miocene the radiation of 4 major monophyletic clades, each restricted to a specific biogeographic province with Western Papua Province probably being the center of origin of the genus Melanotaenia. The fact that all the species from Western Papua belonged to the same phylogenetic clade demonstrates their genetic relatedness and proves that dispersal with adjacent biogeographic provinces (i.e. Northern or Southern New Guinea) was interrupted since the geological formation of the LFTB. All the species of melanotaeniids from Western New Guinea are therefore endemic from

Figure 19 .

 19 Figure 19. Coloration patterns of Boeseman´s Rainbowfish Melanotaenia boesemani from Ayamaru and from Uter lake (Photo Dr. Laurent Pouyaud).

Figure 20 .

 20 Figure 20. Geographic localization of Ayamaru and Uter Lakes in West Papua, Indonesia.

  mg) of anal fin clip with the NucleoSpin® 96 Tissue kit (Macherey-Nagel), according to the manufacturer's instructions, using a Janus automated Workstation (Perkin Elmer). Forward primers were end-labeled with fluorescent dyes (5'FAM, 5'HEX, 5'ATO550, 5'ATO565) (Eurofins) and each reaction contained 5 µl of 2x Master mix (Fast-Start PCR kit, Roche), 0.1 µM of forward primer, 0.4 µM of reverse primer and 0.5 µl of template DNA. Cycling conditions consisted in an initial denaturation at 95°C for 4 min followed by 30 cycles of 95°C for 20 s, 56°C for 20 s and 72 °C for 30 s, and a final elongation step of 7 min at 72°C. Amplicon size was analyzed by capillary electrophoresis as described in the previous chapters, in the technical facilities of the labex "Centre Méditerranéen de l'Environnement et de la Biodiversité" (Montpellier). Allele sizing and genotyping were achieved with the Peak Scanner v1.0 and GeneMapper® v5.0 software (Applied Biosystems).

Figure 22 .

 22 Figure 22. Factorial Component Analysis based on the multilocus genotypes of Boeseman's Rainbowfish Melanotaenia boesemani individuals from the 2 wild populations of Ayamaru (n=28) and Uter (n=49) and the 6 Indonesian farms (n=29-34).

  identified the Bird's Head Peninsula as an independent biogeographic region characterized by important endemism rates. Beside most vertebrate groups, the Papuan freshwater fish fauna displays strong endemic rates including Rainbowfishes. For illustration, around 84% of the freshwater species are exclusively found in Papua island. Rainbowfish of the family Melanotaeniidae are the leading family of Papuan endemic species, especially in western New Guinea.Conservation biology was developed to understand the processes influencing extinction. Genetics has been an important focus of conservation biology because it helps determine the evolutionary context of endangered species and enables the development of better management strategies. Genetic variation interpreted in a population genetics context can be used to reconstruct the evolutionary history, examine the contemporary status, and predict the future of endangered species. Model organisms, habitat and ecosystem have sometimes been used to examine the bases of conservation management approaches. Overall, the framework of evolutionary genetics theory furnishes an elegant approach to interpret the measured amounts of genetic variation and predict the future effects of evolutionary factors and management strategies for endangered species.Conservation International Rapid Assessment Program (RAP) was created in 1990 to quickly provide biological information needed to catalyze conservation action and improve biodiversity protection. Papua is an important area for global conservation, because of its extraordinary biodiversity, unique ecosystem and many endemism and unique species / biotas. The combination of important levels of biodiversity with low human population density (2-6 inhabitants / km 2 ) led Conservation International to declare in 1997 New Guinea as the only "Major

  protection and climate regulation, which help maintain this biodiversity at a broader level. Biodiversity has to be considered at three levels, i.e. genetic, organism and ecosystem. Such conservation plans must result from collaborations between local governments and several ministries such as the Ministry of Marine Affairs and Fisheries, the Ministry of Forestry, the Ministry of Agriculture and the Ministry of Environment. Indonesian government has already promulgated several laws for conservation management of living resources and their ecosystem, but more efforts are still necessary in order to preserve this area. Indonesian regulation for species protection is now compatible with the rules of IUCN. Until now, 70 nature conservation areas currently exist in Papua, while only 29 have been granted legal status. According to Frettes (2007), 16 endemic species of rainbowfish are covered under protected areas, corresponding to a geographic coverage of only about 27% of their entire habitat. If Papua has recently attracted much conservation interest and investments from conservation organization, still a lot remains to do for protecting its wilderness.

  . Platforme Génomique Environnmentale du Labex Centre Méditerranéen Environnement Biodiversité Plateforme Génotypage -séquencage Université Montpellier II team (Dr. Fréderique Cerqueira, Dr. Erick Desmarais, Dr. Finally my heart felt thanks to all my big family, my parents, my uncles, my aunties, all my siblings and all my cousins.My hopefully this these will give many advantages particularly for Ichthyology, Aquaculture and Genetic Science.

	LIST OF PUBLICATION ASSOCIATED WITH THE THESIS
	1. Media Fitri Isma Nugraha, Laurent Pouyaud, Odang Carman, Kadarusman,
	Utut Widyastuti, Jean-Christophe Avarre. 2014. Development of twelve novel
	polymorphic microsatellite DNA markas for the Boeseman's rainbowfish
	(Melanotaenia boesemani) and test for their cross-utility in 21 rainbowfish
	species from West Papua (Indonesia). European Journal Wild Life Research.
	60:941-946. doi: 10. 1007/s10344-014-0868-2.
	2. Media Fitri Isma Nugraha, Kadarusman, Nicolas Hubert, Jean-Christophe
	Avarre, Renny Kurnia Hadiaty, Jacques Slembrouck, Odang Carman, Sudarto,
	Philippe Clair) 14. Thank you to my institution Ministry Marine and Fisheries Republic Indonesia -Balai Penelitian dan Pengembangan Budidaya Ikan Hias -Pusat Penelitian dan Pengembangan Perikanan budidaya -Badan Penelitian dan Pengembangan Kelautan dan Perikanan. 15. Thank you to all teachers and friends in BGF 2010 in Institut Francaise Indonesia. 16. My friends in PPI Montpellier and Franco-Indonesia family in Montpellier having given me much wonderful memory. Ma grand-mère Nurcaya Syam et Ma mère Asma Murni Ran Ogistira and Laurent Pouyaud. 2015. Eight new species of Rainbowfishes (Melanotaeniidae) from the Birds Head Region, West Papua, Indonesia. 17. A ma Mitochondrial Cybium 39(2): 99-130.

3. Media Fitri Isma Nugraha, Laurent Pouyaud, Odang Carman, Utut Widyastuti, M. Zairin Junior, Kadarusman, Jean-Christophe Avarre. 2015. Genetic diversity of Boeseman's rainbowfish Melanotaenia boesemani reared in Indonesian farms compared to endangered natural population. Tropical Conservation Science.Vol. 8(3): 796-805.

Table 1 .

 1 Rainbowfish classification according to ANGFA http://rainbowfish.angfaqld.org.au/Melano.htm (download 4 September 2015)

	A.	Melanotaeniidae
		a)	Cairnsichthys
			1. rhombosomoides (Nichols & Raven, 1928)
		b)	Chilatherina
			1. alleni Price, 1997
			2. axelrodi Allen, 1980
			3. bleheri Allen, 1985
			4. bulolo (Whitley, 1938)
			5. campsi (Whitley, 1956)
			6. crassispinosa (Weber, 1913)
			7. fasciata (Weber, 1913)
			8. lorentzi (Weber, 1908)
			9. pagwiensis Allen & Unmack, 2012
			10. pricei Allen & Renyaan, 1996
			11. sentaniensis (Weber, 1908)
		c)	Glossolepis
			1. dorityi Allen, 2001
			2. incisus Weber, 1908
			3. kabia (Herre, 1935)
			4. leggetti Allen & Renyaan, 1998
			5. maculosus Allen, 1981
			6. multisquamata (Weber & de Beaufort, 1922)
			7. pseudoincisus Allen & Cross, 1980
			8. ramuensis Allen, 1985
			9. wanamensis Allen & Kailola, 1979
		d)	Iriatherina
			1. werneri Meinken, 1974
		e)	Melanotaenia
			1. affinis (Weber, 1908)
			2. ajamaruensis Allen & Cross, 1980
			3. albimarginata Allen, Hadiaty, Unmack & Erdmann, 2015
			4. ammeri Allen, Unmack & Hadiaty, 2008
			5. angfa Allen, 1990
			6. arfakensis Allen, 1990
			7. arguni Kadarusman, Hadiaty & Pouyaud in Kadarusman,
			Hadiaty, Segura, Setiawibawa, Caruso & Pouyaud, 2012
			8. aruensis Allen, Hadiaty, Unmack & Erdmann, 2015
			9. australis (Castelnau, 1875)
			10. batanta

  are: 1) Infinite allele models (IAM), each mutation can create new alleles at random and assume that microsatellite mutations can create an infinite number of repeating units, and the sequence of the original alleles is not present in the population 2) Stepwise mutation models (SMM) is a gradual mutation when microsatellite mutates. They loss or gain of a single tandem repeat, and hence alleles may possibly mutate towards allele state already present in the population . 3) K-allele models (KAM): this is the classic model. In this model, all alleles have equivalent mutation probability because alleles in this model can mutate with equal probability to mutate to one of other alleles (K-1) (where in if

there are 8 replications, then the mutation that has occurred is 7 replications, in which the 7 replications is the new mutation allele), 4) Two phase models (TPM) is an extension of the SMM mutation. Microsatellite mutations occur because of the increased or reduced X replications. Opportunities to microsatellite mutations involve more than one unit

  This research was carried out from September 2012 to October 2013. The place of the research was in the laboratory of the 'Institut des Sciences de l'Evolution de Montpellier, Université de Montpellier, France.

	3.2.1. Origin of the fish
	Wild fish were captured during several expeditions in Western Papua that

took place between 2007 and 2010. Microsatellite isolation was achieved from seven individuals of Melanotaenia boesemani. Five animals belonging to 11 other Melanotaenia species distributed in Western New Guinea and Central Lengguru phylogenetic groups (Kadarusman et al., 2012b) were also used for cross-species validation: M. catherinae, M. synergos, M. parva, M. fredericki, M. misoolensis, M. salawati, M. batanta, M. veoliae, M. ammeri, M. mairasi and M. irianjaya. For microsatellite experimental validation, commercial Melanotaenia boesemani were purchased from the French retailer Botanic (Montpellier, France) and reared in the aquatic experimental facility of ISEM (Platax, Montpellier).

Table 2 .

 2 Sequence of the 61 selected microsatellite primer pairs.

	No	Sequence code	Forward Primer sequence (5'-3')	Reverse primer sequence (5'-3')	Repetition Motif
	1	HSOVYIT04H6V3A_A_0	CCTCAGCAGAGAACGACTGA	AACCCAAAATATTGTCTCGCA	(ac) 19
	2	HSOVYIT04I00V6_A_0	TTGGTAAAACCCAGTGAAGCA	AAAGCAATTTCCTTCGGGAT	(agac) 15
	3	HSOVYIT04I1G7Q_A_0*	TTTACTACACGTGGTCTTCACTTT	CGTTTTAGCCACAAACAGCC	(aatg)6
	4	HSOVYIT04I1U2C_A_0	TCCTCCACATGTAACGTAGCAA	CCACGCTCAGCTTCTGTAAA	(tg) 16
	5	HSOVYIT04I2DAU_A_0	GACCCAACTCTGGATAAGCG	TGGAAAGGTCCAAGGCTGTA	(tgga)9
	6	HSOVYIT04I3NVN_A_0	ACCTGGGTCTTTCACCCTGT	CAGCCACATCTTTGTCAGGA	(ca)16
	7	HSOVYIT04I6SQZ_A_0	TGTAATGTTCACTGACGGCTG	CAATAACATTTCGGTAGGGTCA	(ggat)9
	8	HSOVYIT04I76LQ_A_0	TTCCTTCTGTGTCCTTGTTAGC	CTGGATGCTTGAATTCCTCG	(taga)8
	9	HSOVYIT04I990W_A_0	AGGGCTCTCCGAAGGGATTA	GTGGGTTTCTGCTTGTGTTG	(ttcta)7
		HSOVYIT04I9AT3_A_0	GGTGTATTCAGCCTCTTGGC	CGCCACATTTACCCAGATG	(gatg)18
		HSOVYIT04I9MJJ_A_0	ATGCATGAGTTTCTTCTGTGGA	ATCGGACCCGTTAACACTGA	(atct)15
		HSOVYIT04IAD7N_A_0	CCTGGCTGTGGAGGAGTGT	CAGCTATTTACTGCAGAGGATCT	(ca)19
		HSOVYIT04IAEI4_A_0	CTTCACTATACGGCTGTTCATGT	CAGCAGGACCTGTGACTTAGA	(ac)17
		HSOVYIT04IDFWF_A_0	GAAACATGGCAGCATAAGCA	TCATGAATGTTACTTTGTACTGAGC	(ca)15
		HSOVYIT04IE9NT_A_0	TCTCCATGTTTTCTGTGGTGA	CACTGATCAGCCGGTCAAC	(tg) 16
		HSOVYIT04IH6B3_A_0	ACCATGCCACGTTTAAGTCA	GGTCACCTTGTTCACCTGCT	(tgt)10
		HSOVYIT04IHH5E_A_0	GGATCCCTTTGTAACCCAAC	GATTTCACGTACCTATCTTCAGCA	(ttatg)10
		HSOVYIT04IHKMX_A_0	TGTCATGAAAACCTCACAGAATG	CGTTTCAATCATGGCAGAGA	(gga)13
		HSOVYIT04IHULC_A_0	ACACTACACTGTAAAGTCACCAAGC	TGTGACTTGGACTTGCACAAA	(tatc)17
		HSOVYIT04IIQME_A_0	TAGGGCGAGCAGAGGTTAAA	ACATTTGACTGATCCCTGGC	(ggaga)8
		HSOVYIT04IK01K_A_0	TGCGATAGAAATTAGCGAACC	GGAGGGGTTGCGACCTAAG	(ccat)10
		HSOVYIT04ILEWK_A_0	CGGGGAGAAAATGAGAAACA	GACAAGCACTGCACCTTCAA	(atct)16
		HSOVYIT04ILLR5_A_0*	CAAGGCAGCAAAAGAACAAA	CAGGCCATTCCTGAGTCAAT	(ac)20
		HSOVYIT04INQTO_A_0	CAGACCTCCTGATGTCACCAT	GAAGAAGGGACCAGGGAAAG	(atcc)12
		HSOVYIT04INQX5_A_0	ATCGCAAACATGTGCCATAA	CAAGGGTTGCAGCCAAATAC	(tg)17
		HSOVYIT04IOCJB_A_0*	GAAGAGTATGAAGAAGCCTTAGTCTGA	CGTCCTGCAATCTACTGTGA	(catc)18
		HSOVYIT04IS8JB_A_0	TGAGTCAAGGGATGTCCAAA	GATGTCCCTCCATAGCCTGT	(ca)23
		HSOVYIT04ITBI1_A_0	CAAACCTCTGCTTCGTTACTCA	TCATTTGACGTCATGGCAAC	(taga)19
		HSOVYIT04IY6LI_A_0	TCCTACCAGATATGGGCTGC	CCACGGGACAATATTAACTGC	(gt)18
		HSOVYIT04IZ9GS_A_0	CACCAAGCCAGGATGTGAC	GCATTTTGTTTCAGCTGTGG	(catac)8
		HSOVYIT04IZTAQ_A_0	CTTCCCTGCTTAGGCAGCTT	TGAAACTTTAGAATCATGTTATTCAGC	(tgga)16
		HSOVYIT04J3U3K_A_0	AACAGAAGCAGTTGCAAGTTGA	CCCAAACATTTTCTTGCCTT	(gttag)7
		HSOVYIT04JDEPM_A_0	TCCTGCCAATGTGGTGAGTA	TAGCCTGGGTGAAGCAGAGT	(ac)18
		HSOVYIT04JJLCQ_A_0	TCAGTGATCGTGAAGAGCAG	AGCAGGTCAGGCAGTAAACG	(caa)10
		HSOVYIT04JNI1V_A_0	TTGACTGACGCTCCAGTTTG	CAGATGAAAATGCGGAAGGT	(atcc)19
		HSOVYIT04JNSDY_A_0	AGGAAGAAGAGCAGGTTGGA	ACCATGGCTACCACTGCTTC	(gag)10
		HSOVYIT04JP9IA_A_0*	TCCCACCATGCATTATTAACAC	GAACTGCAGGCTCAACACAT	(gt)18
		HSOVYIT04JPWFS_A_0	TGACAGTAACAGGGACGATGA	AAGGCAACCTAATGTGCTGT	(atg)12
		HSOVYIT04JQ6XV_A_0*	AAAGTGGTAAAATTTCAAAACCAA	CGATATCCACGTGTGAGGTG	(ca)23
		HSOVYIT04JRTLS_A_0*	TGTAACGAGTGAATTTCTCCACTG	TCCACAATATTTACTGGAACTGC	(tctaa)12
		HSOVYIT04JS706_A_0	TCTTTCCATTCAGTACCTGCG	TGACAAATTTATTTCTGTTCCACA	(gata)16
		HSOVYIT04JT3SY_A_0	TCACCAAGGATAGTTTGTTTTGG	CTTGTTCGTGAGCTTTGTTCA	(ac)16
		HSOVYIT04JVSN3_A_0	TCATCCACAGATCATGCACA	CACATTCACACTTCGGTGCT	(aggag)6
		HSOVYIT04JYXHB_A_0	TGCTCTAGCCCTTAGGGAGG	CGGCCACTCGTTCATGTAT	(ggaga)13
		HSOVYIT04JZHKE_A_0*	CTGTCAGAGTGCAGGACTGG	TCTTTTCCAGGCCAAGTGAC	(gga)8
		concat88_A_0*	TCCTTGAATACATTCCTGCCA	CAATAATTTCAAGGAAGAGCCTTT	(ac)16
		cons1004_2_A_0	CTGGTTGTATTGACCATGCG	CAGGCTTTGGATCAGAATCA	(ctgaac)5
		cons1260_2_A_0*	TGGAGGATTGTAGGTCTGGG	CATCAACATAGCAATCAGTGCC	(gt)16
		cons1347_2_A_0	TCCCCTTAAATACACCAGCG	CTGGTCTTCCTGCTCACCTT	(gag)9
		cons1501_4_A_0	TACAAGGAGAGGAGAGGCGA	CCATGGAATGAAGATGCTGA	(ggaga)10
		cons1666_3_A_0	CTAAAGCTAGCACACTAGCGTTCCTT	GAATAAACCTTATCTGAAAGAATGTGA	(ctt)18
		cons1716_2_A_0*	CGTGTCTATTCCTATCGTGCC	TGGATTCACATCCTCTTGAGTG	(gag)11
		cons220_2_A_0	TCATTTCTCACATGTCCCCA	CCTGCAGGTAGAGACCCTCA	(atc)8
		cons2338_2_A_0	AATGATGCAGGTGAGGCG	CACCCTCGAATCATCACAGA	(tg)16
		cons2619_3_A_0	GTGCATCCATGTGTTTCAGC	GCCTCTCCAGACTGACTTGC	(tg)17
		cons2669_2_A_0	TTTTCATACAGGGTGCCAAA	CGCTTTGGATAAAAGCGTCT	(ctatt)5
		cons452_2_A_0	CACTGCAGACAGAAGGCAGA	TGTACCATTCAGATGGTGGG	(gata)15
		cons469_2_A_0	TTCAGGAGATCCTGTGTCCC	TGAAATCTGAGCCAATCGGT	(atcc)13
		cons731_3_A_0	TGCTCCCGACCTACAGCTAC	ATGGATGGTTCTCACGGTGT	(ac)17
		cons881_2_A_0	ATATCTCAGGAAGCCATCGG	GAATGTGGGGCTATCTCTGG	(atg)12
		cons951_2_A_0	AACAACAGGCAGGGAGTCAC	CCAAGAGGTTGTGGTGTTCC	(atg)10

Table 3 .

 3 Crossing of M. boesemani for microsatellite marker validation.

	No	Crossing	Σ Male	Σ Female	Σ Progenies
	1	Crossing 1	1	1	24
	2	Crossing 2	1	1	40
	3	Crossing 3	1	1	23
	4	Crossing 4	1	1	32
	5

Table 4 .

 4 Name and main characteristics of the 12 microsatellite markers validated on 12 Melanotaenia species.

	Locus name	Gene bank accession	Primer Sequence 5'-3'	Repetition Motif*	Fluorescent label	PCR Mix
	Mb_di1

Table 5 .

 5 Allele frequencies for each breeding pair (crossing) and its corresponding offspring at the 12 microsatellite loci

	Allele						Crossing and corresponding offspring
		M1 F1	O1 M2 F2	O2 M3 F3	O3 M4 F4	O4 M5 F5	O5
	Mb_di1
	95	-	-	-	-	-	-	0.50 -	0.28 -	-	-	0.50 -	0.29
	97	-	-	-	-	-	-	-	-	-	-	-	-	0.50 -	0.21
	109	-	-	-	0.50 0.50 0.55 -	-	-	-	-	-	-	-	-
	113	1.00 0.50 0.67 -	0.50 0.21 -	1.00 0.50 1.00 0.50 0.70 -	0.50 0.24
	117	-	0.50 0.33 0.50 -	0.24 0.50 -	0.22 -	0.50 0.30 -	-	-
	119	-	-	-	-	-	-	-	-	-	-	-	-	-	0.50 0.26
	Mb_di2
	113	-	0.50 0.33 -	-	-	0.50 1.00 -	-	-	-	-	-	-
	123	-	-	-	0.50 -	0.35 -	-	-	0.50 -	0.22 -	-	-
	127	-	0.50 0.17 -	-	-	-	-	-	-	-	-	-	-	-
	129	-	-	-	-	0.50 0.20 -	-	-	0.50 1.00 0.78 -	0.50 0.26
	133	-	-	-	0.50 -	0.15 -	-	0.72 -	-	-	-	-	-
	135	-	-	-	-	-	-	-	-	-	-	-	-	0.50 -	0.23
	139	0.50 -	0.33 -	0.50 0.30 0.50 -	0.28 -	-	-	-	-	-
	141	-	-	-	-	-	-	-	-	-	-	-	-	0.50 0.50 0.51
	143	0.50 -	0.17 -	-	-	-	-	-	-	-	-	-	-	-
	Mb_di3
	153	-	1.00 0.50 -	-	-	0.50 0.50 0.46 0.50 0.50 0.48 -	-	-
	161	-	-	-	-	-	-	-	-	-	-	-	-	-	0.50 0.25
	163	0.50 -	0.25 1.00 0.50 0.80 0.50 0.50 0.54 -	-	-	1.00 -	0.50
	165	-	-	-	-	0.50 0.20 -	-	-	-	-	-	-	-	-
	171	0.50 -	0.25 -	-	-	-	-	-	0.50 0.50 0.52 -	0.50 0.25
	Mb_penta1
	160	-	-	-	-	-	-	0.50 1.00 0.65 -	-	-	0.50 -	0.29

Table 6 .

 6 Observed (Ho) and expected (He) heterozygosity calculated on all loci for each crossing population and associated Fis

		Crossing 1	Crossing 2	Crossing 3	Crossing 4	Crossing 5
	Ho ± SD	0.65 ± 0.27	0.69 ± 0.32	0.67 ± 0.30	0.56 + 0.26	0.70 ± 0.38
	He ± SD	0.52 ± 0.19	0.49 ± 0.21	0.47 ± 0.19	0.43 ± 0.18	0.49 ± 0.25
	Fis	NS	NS	NS	NS	NS

Table 10 .

 10 Characteristics of the microsatellite markers on Melanotaenia boesemani and on the 28 cross-amplified species.

	Locus name	Bank Gene	Primer sequences 5'-3'	Repetition	S b	S c	A d	A e
		code		Motif a
	Mb_di1	KF85688	F: TGAGTCAAGGGATGTCCAAA	(CA)23	089-139	085-151	14
			R: GATGTCCCTCCATAGCCTGT
	Mb_di2	KF856887	F: TCCCACCATGCATTATTAACAC	(GT)18	113-145	111-227	14
			R: GAACTGCAGGCTCAACACAT
	Mb_di3	KF856888	F: TGGAGGATTGTAGGTCTGGG	(GT)16	153-173	148-231	8
			R: CATCAACATAGCAATCAGTGCC
	Mb_penta1	KF856889	F: TGTAACGAGTGAATTTCTCCACTG	(TCTAA)12	160-200	150-240	8
			R: TCCACAATATTTACTGGAACTGC
	Mb_di4	KF856890	F: CAAGGCAGCAAAAGAACAAA	(AC)20	112-152	098-172	15
			R: CAGGCCATTCCTGAGTCAAT
	Mb_tetra1	KF856891	F: TTTACTACACGTGGTCTTCACTTT	(AATG)6	135-157	125-217	7
			R: CGTTTTAGCCACAAACAGCC
	Mb_tri1	KF856892	F: CTGTCAGAGTGCAGGACTGG	(GGA)8	107-119	101-128	4
			R: TCTTTTCCAGGCCAAGTGAC
	Mb_tri2	KF856893	F: CGTGTCTATTCCTATCGTGCC	(GAG)11	138-150	132-153	4	8
			R: TGGATTCACATCCTCTTGAGTG
	Mb_di5	KF856894	F: TCCTTGAATACATTCCTGCCA	(AC)16	085-099	077-129	8
			R: CAATAATTTCAAGGAAGAGCCTTT
	Mb_tetra2	KF856895	F: GAAGAGTATGAAGAAGCCTTAGTCTGA	(CATC)18	172-252	136-324	18
			R: CGTCCTGCAATCTACTGTGA
	Mb_tri3	KF856896	F: TGACAGTAACAGGGACGATGA	(ATG)12	133-154	111-163	3
			R: AAGGCAACCTAATGTGCTGT
	Mb_tri4	KF856897	F: AACAACAGGCAGGGAGTCAC	(ATG)10	099-114	098-132	4
			R: CCAAGAGGTTGTGGTGTTCC

a According to the M. boesemani DNA used to construct the genomic DNA library b Allele size range (bp) for M. boesemani c Allele size range (bp) for all 28 species d Allele number for M. boesemani e Allele number for all 28 species

Table 11 .

 11 Sample size, allele diversity and average observed (Ho) and expected (He) heterozygosity per population across loci

	Population name						Locus Mb_						Na	Ho	He	N
		di 1	di2	di3	penta1 di4	tetra1 tri1 tri2 di5	tetra2 tri3	tri4
	M. ajamaruensis	9	12	8	7	10	6	3	5	5	11	3	3	82	0.6699 0.6357	30
	M. ammeri	2	2	2	3	1	1	1	1	2	1	1	1	18	0.1046 0.1557	20
	M. angfa	1	2	2	2	1	3	1	1	3	2	1	1	20	0.2448 0.234	35
	M. arfakensis	6	3	12	9	1	5	2	1	2	11	6	1	59	0.4037 0.3975	45
	M. arguni	8	7	11	7	5	10	5	4	5	10	5	3	80	0.6769 0.6939	21
	M. batanta	1	8	1	1	1	2	1	1	1	9	1	1	28	0.1583 0.1402	21
	M. boesemani	14	14	8	8	15	7	4	4	8	18	3	4	107 0.6815 0.678	28
	M. catherinae	2	1	1	3	1	2	1	1	1	2	1	3	19	0.1464 0.1436	37
	M. ericrobertsi	1	1	1	2	1	1	1	1	1	1	1	1	13	0.042	0.042	2
	M. fasinensis	4	5	9	3	4	6	1	1	2	8	1	1	45	0.3646 0.3508	40
	M. fredericki	7	19	17	10	3	12	3	6	12	9	4	4	106 0.6568 0.7105	13
	M. irianjaya	13	14	17	9	6	14	8	5	10	12	7	5	120 0.7082 0.7546	24
	M. misoolensis	4	5	3	5	4	5	2	1	4	4	2	1	40	0.5611 0.5129	5
	M. multiradiata	13	10	23	9	19	13	3	3	4	18	7	2	124 0.6083 0.6127	40
	M. parva	5	5	8	6	1	5	2	2	3	4	2	1	44	0.3863 0.3796	40
	M. salawati	1	5	6	5	8	4	2	2	3	6	2	1	45	0.3	0.3	22
	M. synergos	2	1	1	3	1	3	1	1	2	3	2	1	21	0.1906 0.1863	39
	M. urisa	1	3	2	5	2	5	1	3	1	4	2	4	33	0.3762 0.3974	19
	M. veoliae	1	1	1	2	1	1	3	1	3	1	1	1	17	0.1267 0.1184	24
	M. wanoma	1	2	4	4	3	5	3	3	2	3	1	2	33	0.3869 0.3688	14
	M. klasioensis	1	15	6	2	3	4	2	1	2	3	1	1	41	0.3104 0.3046	40
	M. longispina	18	9	16	9	15	17	3	6	13	14	7	6	133 0.7433 0.7312	39
	M. naramasa	15	15	18	10	12	8	4	6	6	9	6	1	110 0.6377 0.6952	29
	M. rumberponensis	2	2	2	3	1	4	1	2	3	1	2	1	24	0.216	0.2099	17
	M. sembrae	2	15	7	5	2	5	4	2	3	13	2	6	66	0.5375 0.5117	15
	M. sikuensis	2	3	2	2	1	3	2	2	1	1	2	1	22	0.1491 0.1579	42
	M. susii	1	13	14	5	6	11	3	1	1	5	3	1	64	0.4325 0.4358	31
	M. manibuii	4	19	19	8	2	9	5	2	5	3	6	2	84	0.5652 0.6085	39
	Number allele per locus 141	211 221 147	130 171	72	69	108	186	82	60
	Na= Number of alleles ; N=number of samples

Table 12 .

 12 Locus by locus and multilocus Fis values for each population.

	Population	Mb_di1 Mb_di2 Mb_di3	Mb_penta1 Mb_di4 Mb_tetra1 Mb_tri1 Mb_tri2 Mb_di5	Mb_tetra2 Mb_tri3	Mb_tri4 Multilocus
	M. ajamaruensis	0.062	-0.046	-0.084	0.042	-0.104	-0.134	0.112	-0.128	-0.014	-0.036	-0.051	-0.072	-0.037
	M. ammeri

. Confirmation by the microsatellite markers of valid species and 8 new Melanotaenia species from West Papua

	4.3.2
		------	0.447** 0.274	-0.093	-0.056	-0.191	0.145	0.077	-0.083	-0.273	------	-0.040	-0.012
	M. klasioensis	------	-0.046	-0.046	-0.026	0.06	0.047	-0.164	------	0.093	-0.041	------	------	-0.006
	M. longispina	0.032	-0.06	-0.095	0.001	-0.013	-0.002	-0.118	0.118	-0.02	-0.055	0.124	-0.026	-0.003
	M. naramasae	-0.024	0.265** 0.131**	-0.068	0.006	0.02	-0.017	0.023	0.361**	0.384**	0.06	------	0.100***
	M. rumberponensis -0.151 0	-0.143	-0.076	------	-0.08	------	-0.103	0.477	------	-0.231	------	0.001
	M. sembrae	-0.077	0.085	-0.02	-0.096	-0.368	-0.117	-0.064	1.000	-0.05	-0.138	0.451	0.085	-0.015
	M. sikuensis	0.079	0.145	-0.025	0.027	------	-0.006	-0.025	0.071	------	------	-0.053	------	0.068
	M. susii	------	-0.046	0.042	0.143	0.052	-0.033	-0.039	------	------	-0.104	0.17	------	0.024
	M. manibuii	-0.145	0.015	-0.009	0.119*	0	-0.038	0.128*	-0.116	0.086	0.059	0.433**	0.316	0.084**
	-Indicates a monomorphic locus, *p<0.05, **p<0.01, **P<0.001

  The microsatellite data therefore demonstrated that the eight new species represent distinct genetic entities, since they differ from each other and also from closely related valid species. Their species description are available inNugraha et al., 2015 (Annex 2). Because no intermediate genotypes and no overlap were observed between these taxonomic entities, we therefore considered that these species represent new species. The morphological description of the new species was not part of my work (Dr. Kadarusman PhD Thesis, 2012), they are not presented in my thesis but can be found in the corresponding published paper presented in Annex 2. The new species were given the names as listed in Figure

		M. parva	M. angfa M. rumberponensi	M. naramasae
	M. sikuensis	0.63	0.739	0.714	0.485
	M. parva	-	0.46	0.545	0.245
	M. angfa	-	-	0.707	0.401
	M. rumberponensis	-	-	-	0.356

Tabel 15.

Pairwise Fst values between species composing the cluster "Bird's Neck".

  . The first clade "Northern Bird's Head" (bootstrap: ML 96%, NJ 94%) includes all Melanotaenia species from the Bird's , M. rumberponensis, M. sikuensis). The third clade "Waigeo" is supported by high bootstrap values (ML 100%; NJ 99%) and includes the two species M. catherinae and M. synergos known from the Northern Raja Ampat (Figure

	Head Peninsula (i.e. M. ajamaruensis, M. arfakensis, M. boesemani, M.
	ericrobertsi, M. fasinensis, M. fredericki, M. multiradiata) and from the Southern
	Raja Ampat Islands (i.e. M. batanta, M. misoolensis, M. salawati). It also
	includes 5 new species (i.e. M. klasioensis, M. susi, M. longispina, M. sembrae, M.
	manibuii). The second clade "Southern Bird's Head" (bootstrap: ML 98%, NJ
	94%) includes all the Melanotaenia species from the Bomberai Peninsula (i.e. M.

ammeri, M. arguni, M. irianjaya, M. urisa, M. veoliae, M. wanoma) and from the Bird's Neck (i.e. M. angfa, M. parva). It also includes 3 new species (i.e. M. naramasae

Table 16 .

 16 List of M. boesemani farmers in Jakarta and Bekasi.

	No Farmers name and addres	Year	Number of
			Cultivation	Samples
	1	Farm Gusi, Ciracas	1983	34
	2	Farm Sukri, Depok	2000	30
	3	Farm Hasan, Bekasi	1983	30
	4	Farm Yahya, Bekasi	2009	30
	5	Farm Didi, Tridayasakti, Bekasi	1986	29
	6

Table 17 .

 17 Number of alleles per locus for each population.

		Uter	Ayamaru	Gusi	Sukri	Hasan	Yahya	Didi	Warso
	Locus	Lake	Lake	farm	farm	farm	farm	farm	farm
		n=49	n=28	n=34	n=30	n=30	n=30	n=29	n=30
	Mb_di1	9	14	8	8	10	9	9	8
	Mb_di2	13	14	9	8	10	9	9	9
	Mb_di3	10	8	7	5	7	7	5	6
	Mb_penta1 6	8	8	7	7	7	6	6
	Mb_di4	10	15	10	8	11	12	7	13
	Mb_tetra1	6	7	3	4	6	5	2	3
	Mb_tri1	4	4	4	3	3	2	2	2
	Mb_tri2	3	4	4	4	4	5	3	4
	Mb_di5	6	8	8	9	8	7	6	7
	Mb_tetra2	10	18	10	11	12	6	6	7
	Mb_tri3	3	3	3	3	4	2	3	2
	Mb_tri4	2	4	5	4	4	4	4	4
	TNA*	82	107	79	74	86	75	62	71
	*TNA: total number of alleles

  The combination of important levels of biodiversity with low human population density (2-6 inhabitants / km 2 ) led Conservation International to declare in 1997 New Guinea as the only "Major Tropical Wilderness Area" remaining in Asia. For illustration, all the species of melanotaeniids from Western New Guinea are endemic from the area. Because many rainbowfish species have restricted distributions and are confined to specific habitats, such as isolated lacustrine environments or small parts of a single river system, they are highly vulnerable to environmental disturbance and over-harvesting. Ayamaru Lake is affected by the development of residential areas and ecotourism. As a result, forest clearance has increased channel obstructions by sediments and led to partial drying of the lake. Meanwhile, simultaneous over-catching of the endemic M.

molecular markers will save time and cost, and the potential results can be seen clearly; moreover, this can estimate the production profit clearly and precisely.Finally, forensics analysis in aquaculture is very important for identification of fish pedigree in selective breeding. Forensics in aquaculture aims to anticipate illegal acts like misuse of fisheries resources, misinterpretation of the content of fisheries products, illegal trading in fish or fisheries product and accidental or deliberate undesirable release or introduction into natural waters. Applications of forensics in aquaculture relate to the identification of species, populations and individuals. Microsatellite markers are very well suited to fulfill these needs[START_REF] Groblera | The application of microsatellite DNA markers for forensic analysis of koi carp (Cyprinus carpio)[END_REF]

devant le jury composé de JURY devant le jury composé de JURY government, thank you for the scholarship with "Bourse Government France

MATERIAL AND METHODS

This research was carried out from September 2012 to October 2013. The place of the research was in the laboratory of the 'Institut des Sciences de l'Evolution de montpellier, Université de Montpellier, France.

Origin of the investigated fish

The samples were collected during three expeditions conducted between 2007 and 2010 in West Papua by The Akademi Perikanan Sorong, Indonesia (APSOR-KKP) The Institut de Recherche pour le Développement, Montpellier, France (IRD) and the Balai Riset Budidaya Ikan Hias in Depok, Indonesia (BRBIH-KKP). A total of 32 populations from Papua-New Guinea were analyzed.

(Table 8).

Among these populations, 28 had previously been analyzed for their cytochrome oxydase I gene (COI) barcode by Kadarusman et al (2012a).

Sampling strategy consisted to collect each population at a type locality. For most of them, between 2 and 45 individuals were captured at a given location by using a landing net (Table 8).

Because of low population density, five species (i.e. M. ammeri, M. fredericki, M. irianjaya, M. manibuii sp. nov., M. naramasae sp. nov.) were sampled at various localities in the same river drainage, with geographic distances varying between 1 to 5 km.

RESULTS

Analysis of all the wild populations with the microsatellite markers

Twelve microsatellite loci were successfully amplified on 775 specimens (28 species) including those analyzed by their COI DNA barcode analysis (Table 9 Fig. 17). Characteristics of these loci are presented in table 10. All microsatellite loci revealed polymorphic with a number of alleles per-locus ranging between 8 (locus Mb_tri2) to 53 (Mb_di2). Total number of alleles per-species (Table 10) indicate that M. ericrobertsi and M. veoliae are the species with the lowest level of polymorphism (13 and 17 alleles) while M. multiradiata and the new species M. longispina are the most polymorphic species (124 and 133 alleles respectively).

Average observed heterozygosity per species ranged between 0.042 and 0.743 (Table 11). Most the species were at panmictic equibrium according to non significant multilocus FIS values (Table12). Only five species (i.e. M. ammeri, M. fredericki, M. irianjaya, M. manibuii, M. naramasa) showed a significant deviation from hardy-Weinberg equilibrium. No evidence for null alleles, scoring errors due to stuttering and for large allele dropout was revealed on the entire data by using Micro-Checker software. It is important to remind here that the five species presenting significant multilocus Fis values are a mix of individuals sampled at various locations along the same river drainage. These results therefore suggest the possible occurrence of genetically differentiated subpopulations patchily distributed in the same river and must be taken in consideration in future sampling strategies or population monitoring of Melanotaenia species. Tabel 13. Pairwise Fst values between species from the cluster "Central Ayamaru Plateau" and M. arfakensis. Tabel 14. Pairwise Although there is a lack of precise data, the number of Indonesian farms breeding this species seems to be extremely low, with no more than 10. They export more than 1000 males per week, at price of about 0,75 -2 US $ per individual, depending on its size, which is usually comprised between 1.75 and 2.5 inches (personal communication from the Jakarta fish quarantine). All the farms are located around Jakarta, and their production is not likely to account for the totality of exported fish. Melanotaenia boesemani has been domesticated and produced in Indonesian farms since 1983. At present, farmers claim a decrease of the production in terms of quantity and quality, i.e.: males are not as colored as in the wild; growth rate and fecundity are slower; the number of females per spawning is higher; many specimens display a different but typical morphology (which the producers call "perrot", figure 21). In this context, we implemented the new microsatellite markers in view of assessing the genetic variability of six different strains of M. boesemani reared in six Javanese farms, as well as to compare their genetic variability with that of wild populations for determining the geographic origin of the founder breeders and for quantifying a possible loss of variability.

2 MATERIAL AND METHODS

This research was held from December 2013 -August 2014, partly in Jakarta and its region (for fish sampling) and in the laboratory of the 'Institut des Sciences de l'Evolution de Montpellier, Université de Montpellier, France (for genetic analyses)

Origin and sampling of the investigated fish

This research used 183 captive M. boesemani specimens from six local cultivation farms located around Jakarta in West Java (Table 16). Based on a preliminary survey realized in 9 farms, these 6 farms have been cultivating M.

boesemani for many years (up to 30 years), do not practice out-crossings with other breeders and are facing difficulties such as a higher number of females in each harvest (while only males are suitable for sale), a smaller body size and a loss of color brightness. Approximately 30 individuals were collected from each farm. Fish were anaesthetized with 0.1 mL/L Eugenol and a ~1-cm 2 piece of anal fin was collected and stored in absolute ethanol for further DNA extractions. Fish were then allowed to recover from anesthetic and were released back into the ponds. In order to investigate the possible origin of these reared M. boesemani fish, we also analyzed two M. boesemani wild populations: one from the vicinity of Ayamaru lake (Tiwit tributary, 1°15.463'S and 132°14.939 'E, 28 specimens) and one from Uter lake (1°25.957'S and 132°23.258'E, 49 specimens), which are the only known localities where this species can be found. These wild specimens were collected during the 2007 Lengguru expedition.

Geographic origin of M. boesemani reared in Indonesian farms

A factorial correspondence analysis (FCA) was performed on all multilocus genotypes obtained for the 6 captive and the 2 wild populations (Figure 22). The projection made on axis 1 (5.99%) and axis 2 (2.47%) indicated that the wild population from Uter Lake could be genetically differentiated from that of Ayamaru Lake. Moreover, all the fish collected from the farms (i.e. polygon "pooled strains") were partly grouped with those of Ayamaru Lake (i.e. polygon "Ayamaru lake"). The third axis of the FCA did not enable to separate the farm samples from Ayamaru wild population. Likewise, when Uter population was removed, the Ayamaru and farm specimens still overlapped. These results suggest that all captive fish originate from the region of Ayamaru Lake.

ANNEXE