Philippe Borsa
email: philippe.borsa@ird.fr

Philippe Béarez
email: philippeborsa@yahoo.fr

Wei-Jen Chen

Gymnocranius oblongus, a new large-eye bream species from New Caledonia (Teleostei: Lethrinidae)

Keywords: Monotaxinae, Taxonomy, Molecular genotyping, Reproductive isolation, Cryptic species Monotaxinae, Taxonomie, Génotypage moléculaire, Isolement reproductif, Espèces cryptiques

Gymnocranius oblongus is described as a new species of the subfamily Monotaxinae (Sparoidaea : Lethrinidae), a group of commercially important fishes distributed throughout the Indo-West Pacific, from six specimens collected in New Caledonia. It is characterized by an oblong, fusiform body, slightly rounded snout, elongate tail with rounded tips and sub-horizontal, wavy blue lines or dashes on snout and cheeks. It is distinct from sympatric G. grandoculis by a more slender body which is also more symmetrical dorso-ventrally, and a more elongated caudal fin. Both mitochondrial-DNA and nuclear-DNA markers provide a genetic basis to the distinction of G. oblongus from G. grandoculis.

Résumé

Gymnocranius

oblongus, une nouvelle espèce de bossu blanc de la Nouvelle-Calédonie (Pisces: Lethrinidae). Gymnocranius oblongus est décrit comme nouvelle espèce de la sous-famille des Monotaxinae (Sparoidaea : Lethrinidae), un groupe de poissons d'importance commerciale distribué dans tout l'Indo-Pacifique ouest, à partir de six spécimens récoltés en Nouvelle-Calédonie. Il se caractérise par un corps oblong et fusiforme, un museau légèrement arrondi, une queue allongée aux extrémités arrondies, des lignes ou des tirets bleus sub-horizontaux et sinueux sur le museau et les joues. Il se distingue de l'espèce sympatrique G. grandoculis par un corps plus allongé et plus symétrique dorso-ventralement et une nageoire caudale plus allongée. Les marqueurs mitochondriaux et nucléaires confirment la distinction des deux espèces.

Introduction

Although most Lethrinidae (emperor fishes and large-eye breams) are of significant interest to fisheries in the Indo-Pacific region, research on their biology and fisheries is hampered by taxonomic confusion and difficulties in species identification [START_REF] Sato | A systematic review of the sparoid fishes of the subfamily Monotaxinae[END_REF][START_REF] Carpenter | FAO species catalogue, vol. 9, Emperor fishes and large-eye breams of the world (family Lethrinidae), An annotated and illustrated catalogue of lethrinid species known to date[END_REF]. The taxonomy of large-eye breams of the genus Gymnocranius (Lethrinidae: Monotaxinae) is notoriously difficult. The two most recent revisions, made only three years apart [START_REF] Sato | A systematic review of the sparoid fishes of the subfamily Monotaxinae[END_REF][START_REF] Carpenter | FAO species catalogue, vol. 9, Emperor fishes and large-eye breams of the world (family Lethrinidae), An annotated and illustrated catalogue of lethrinid species known to date[END_REF] disagreed on several points. Sato [START_REF] Sato | A systematic review of the sparoid fishes of the subfamily Monotaxinae[END_REF] recognised six species in the genus, whereas Carpenter and Allen [START_REF] Carpenter | FAO species catalogue, vol. 9, Emperor fishes and large-eye breams of the world (family Lethrinidae), An annotated and illustrated catalogue of lethrinid species known to date[END_REF] recognised 8, and some of the species recognised by both authors were ascribed different names, both [START_REF] Sato | A systematic review of the sparoid fishes of the subfamily Monotaxinae[END_REF] and [START_REF] Carpenter | FAO species catalogue, vol. 9, Emperor fishes and large-eye breams of the world (family Lethrinidae), An annotated and illustrated catalogue of lethrinid species known to date[END_REF] invoking the rules on nomenclature [START_REF]International Commission on Zoological Nomenclature, International code of zoological nomenclature[END_REF]. For instance, the name G. grandoculis (Valenciennes 1830) was not retained by Sato [START_REF] Sato | A systematic review of the sparoid fishes of the subfamily Monotaxinae[END_REF], who questioned the identity of the holotype of Cantharus grandoculis Valenciennes 1830.

Sato [START_REF] Sato | A systematic review of the sparoid fishes of the subfamily Monotaxinae[END_REF] instead used the name G. robinsoni (Gilchrist and Thompson 1909), but the latter has been discarded by Carpenter and Allen [START_REF] Carpenter | FAO species catalogue, vol. 9, Emperor fishes and large-eye breams of the world (family Lethrinidae), An annotated and illustrated catalogue of lethrinid species known to date[END_REF] as a junior synonym of G. grandoculis. Among the problems pending is the description of a species first erroneously identified as G. lethrinoides (non Bleeker 1850) [START_REF] Masuda | The fishes of the Japanese Archipelago[END_REF] and now widely referred to as "Gymnocranius sp." [START_REF] Sato | A systematic review of the sparoid fishes of the subfamily Monotaxinae[END_REF][START_REF] Carpenter | FAO species catalogue, vol. 9, Emperor fishes and large-eye breams of the world (family Lethrinidae), An annotated and illustrated catalogue of lethrinid species known to date[END_REF][START_REF] Randall | Fishes of the Great Barrier Reef and Coral Sea[END_REF][START_REF] Laboute | Poissons de Nouvelle-Calédonie[END_REF][START_REF] Carpenter | Lethrinidae -emperors (emperor snappers)[END_REF][START_REF] Béarez | First record of Scolopsis taeniopterus (Nemipteridae) and Gymnocranius elongatus (Lethrinidae) from New Caledonia[END_REF]. Agreement was reached by both [START_REF] Sato | A systematic review of the sparoid fishes of the subfamily Monotaxinae[END_REF] and [START_REF] Carpenter | FAO species catalogue, vol. 9, Emperor fishes and large-eye breams of the world (family Lethrinidae), An annotated and illustrated catalogue of lethrinid species known to date[END_REF] in recognising difficulties in the taxonomy of the genus, and on the need of further research.

Several Gymnocranius species regularly occur on the fish market in Noumea, including G. elongatus, G. euanus and G. grandoculis ([8]; P. Borsa pers. obs.). Between 2003 and 2008 we collected adult or sub-adult specimens of Lethrinidae to establish a reference genetic database from intron length polymorphisms and mtDNA (16S rDNA) single-strand conformation patterns [START_REF] Borsa | Les premiers stades de vie des poissons de Nouvelle-Calédonie : identification des habitats lagonaires pour les stades pré-et post-installation, Volet thématique 3 : identification des jeunes stades[END_REF]. The purpose of this database was to permit the identification of unknown Lethrinidae larvae. In the process, we had difficulties identifying to species a number of adult or sub-adult specimens of the genus Gymnocranius using the diagnoses provided by Sato [START_REF] Sato | A systematic review of the sparoid fishes of the subfamily Monotaxinae[END_REF] and Carpenter and Allen [START_REF] Carpenter | FAO species catalogue, vol. 9, Emperor fishes and large-eye breams of the world (family Lethrinidae), An annotated and illustrated catalogue of lethrinid species known to date[END_REF]. Multiplelocus genotype frequencies revealed three undescribed Gymnocranius species among our samples from New Caledonia [START_REF] Borsa | Les premiers stades de vie des poissons de Nouvelle-Calédonie : identification des habitats lagonaires pour les stades pré-et post-installation, Volet thématique 3 : identification des jeunes stades[END_REF]. Here, we describe one of these species, of which we also captured an early juvenile.

Materials and methods

The following specimens of Gymnocranius oblongus n. sp., all deposited at the Museum national Gymnocranius oblongus n. sp. individuals had been initially identified by us as G. grandoculis, as they fell out with that species in the identification keys of both Sato [START_REF] Sato | A systematic review of the sparoid fishes of the subfamily Monotaxinae[END_REF] and Carpenter and Allen [START_REF] Carpenter | FAO species catalogue, vol. 9, Emperor fishes and large-eye breams of the world (family Lethrinidae), An annotated and illustrated catalogue of lethrinid species known to date[END_REF]. For molecular genotyping, whole genomic DNA of individuals was extracted from fin clips preserved frozen or in ethanol, using either the classical phenol-chloroform protocol [START_REF] Sambrook | Molecular cloning: a laboratory manual[END_REF], or the"DNeasy® Tissue Kit" of Qiagen GmbH (Hilden, Germany) according to the manufacturer's instructions. DNA extracts were conserved in ultrapure water at -20°C. PCR amplification was done in 25 µl reaction mixture containing 1.5-2.0 mM MgCl 2 , 0.64 mM dNTP mix, 0.1-0.3 µM of each primer and 0.03-0.2 U Taq DNA polymerase (Promega, Madison WI, USA). The primers for the 16S rRNA locus (forward: 5'-GCC CAA CCA AAG ACA TTA GGG CAG-3'; reverse: 5'-GAC CCG TAT GAA TGG CAT AAC GAG-3') were designed from the alignment of Lethrinus ornatus, Lethrinus rubrioperculatus and Beryx splendens homologous sequences (GenBank AF247446, AF247447 and AY141406). The primers used to amplify an intron of the metallothionein gene (forward: 5'-ATG GAY CCY TGH GAC TGC TC-3'; reverse: 5'-RCA GGA TCC WCC GCA GYT GC-3') were designed in the flanking exons, from the alignment of homologous metallothionein genes of Carassius cuvieri, Dicentrarchus labrax, Takifugu rubripes and Salmo salar (GenBank AY165048, AF199014, CA847265 and BG935118). The primers designed to amplify the other introns have been published previously [START_REF] Chow | Intron length variation observed in the creatine kinase and ribosomal protein genes of the swordfish Xiphias gladius[END_REF][START_REF] Hassan | Seventeen new exon-primed intron-crossing polymerase chain reaction amplifiable introns in fish[END_REF][START_REF] Rohfritsch | Genetic structure of Indian scad mackerel Decapterus russelli: Pleistocene vicariance and secondary contact in the central Indo-West Pacific seas[END_REF]. PCRs were run in a RoboCycler Gradient 96 thermocycler (Stratagene, Cedar Creek TX, USA), with annealing temperature set to 50°C (CK), 51°C (16S rRNA) or 52°C (Aldo-B, GnRH-1, Met). Immediately before migration, 6 µl denaturing loading buffer (95% formamide, 10 mM NaOH, 84‰ bromophenol blue, 5% glycérol) was added to each well of the PCR plate and the mixture was heated for 5 min at 95°C. Amplified 16S rDNA fragments were subjected to single-strand conformation polymorphism analysis after electrophoresis of the DNA fragments in vertical, non-denaturing polyacrylamide gels (MDE 1X, FMC corporation, Rockland, USA) overnight at 2 W at ambient temperature (25°C). Introns were separated according to size by vertical electrophoresis in denaturing, 0.4-mm thick polyacrylamide gels [6% acrylamide:bis-acrylamide (29:1) solution, TBE 1X, 7 M urea] at 50 W. DNAs were stained using silver nitrate.

Comparative material thus included two

Results

Diagnostic description

Gymnocranius oblongus Borsa, Béarez and Chen, n. sp. (Figs. 123; Table 1); Gymnocranius sp. A [START_REF] Justine | Monogenea: Diplectanidae) from Gymnocranius spp. (Lethrinidae: Monotaxinae) off New Caledonia, with proposal of the new morphological group 'tubulicornis' within Lamellodiscus Johnston & Tiegs[END_REF][START_REF] Borsa | Identification par barcode (séquences nucléotidiques d'un fragment du gène du cytochrome b) des larves de Lethrinidae capturées en baies de Dumbéa et Ouano (Nouvelle-Calédonie)[END_REF]; Gymnocranius cf. grandoculis [17, in 16].

The following description of G. oblongus n. sp. is based on four specimens deposited at Museum A species of Lethrinidae with the following combination of characters: four rows of scales on cheek; ten soft rays in dorsal fin; ten soft rays in anal fin; body oblong (hence the species epithet) and fusiform, ratio of standard length to body depth between 2.6 and 2.8, increasing with size (Table 1); other measurements in Table 1; dorsal and ventral profiles almost similarly convex; tip of snout only slightly below axis of body; snout slightly rounded; tail elongated with rounded tips; posterior part of jaws reaching to about level of nostril; pored scales on lateral line: 48; scales between middle portion of spinous dorsal fin and lateral line: 6. Lower edge of eye slightly (in the smaller individuals examined) to well above a line from tip of snout to middle of caudal fin fork; horizontal or subhorizontal wavy blue lines or dashes on lower part of snout and on cheeks; pale blue speckles more or less visible on operculum. The lines or dashes become dark red or brown against paler background in preserved specimens; they do not extend up to the upper part of snout and their number slightly increases with size (Figs. 1,2). Forehead, snout and upper lip of fresh specimens can be bright yellow, matched by similar yellow colouration of margin of operculum (Fig. 2); loosely defined vertical dark bar crossing the eye (e.g. Fig. 1 B-D); on fresh animals, dorsal, pectoral, anal and caudal fins drab, brownish or yellowish, with reddish to vermilion edges.

Each lower jaw has a raw of three small, slender canines on each side of one large canine at front, and lateral row of 8-10 conical teeth; numerous villiform teeth form a brush behind the front canines; each upper jaw has a front row of 6-10 small, slender canines followed by 4-5 conical teeth and a patch of villiform teeth.

Habitat and distribution

The type locality of Gymnocranius oblongus n. sp. is New Caledonia, from where all six specimens on which this description is based originate. Those four specimens from the southern lagoon of New Caledonia were captured using gillnets deployed on sandy bottom at ~8 to 15 m depth, close to the shore of Grande Terre. Two individuals were seen by P. Borsa at ~1 m depth, swimming above the front of the northern fringing reef of Baie des Citrons, Noumea (22°17'S, 166°26'E; 14 June 2009).

An early juvenile (Fig. 3) was captured by light-trapping [START_REF] Borsa | Les premiers stades de vie des poissons de Nouvelle-Calédonie : diversité des espèces et rôle des zones côtières pour leur croissance et leur survie[END_REF] in Noumea's Grande Rade bay in January 2003. G. oblongus n. sp. was not found in several localities across the Indo-West Pacific, including Bali (Indonesia), Raja Ampat (West Papua) and Fiji, where other Gymnocranius species were sampled (P. Borsa, pers. obs.). Gymnocranius oblongus appears to occur from November to May at the Noumea fish market, although it is less abundant than G. euanus and G. grandoculis.

Other biological data

The stomach of specimen IRDN-20090410-A contained a broken fragment of bivalve shell with meat still partly attached to it; that of specimen IRDN-20090410-B contained a crushed Penaeid shrimp and bivalve meat (but no shell fragment). Specimen IRDN-20090410-A was a male with immature testicles; IRDN-20090410-B was a female, with empty gonads. Both specimens were captured in April 2009 at ~8 m depth on sandy bottom close to the shore of Nouville peninsula, Nouméa.

The two individuals sighted underwater in Baie des Citrons, Nouméa, had their whole body silvery to pale grey and the only noticeable colour patterns were the loosely defined vertical dark bar crossing the eye and the vermilion edges of the hind part of dorsal fin and of the caudal fin.

Comparison with G. grandoculis and assumed synonyms

Table 1 includes as G. grandoculis the type specimen of Cantharus grandoculis Valenciennes 1830 (Fig. 4A), in accord with [START_REF] Carpenter | FAO species catalogue, vol. 9, Emperor fishes and large-eye breams of the world (family Lethrinidae), An annotated and illustrated catalogue of lethrinid species known to date[END_REF], and that of Paradentex marshalli (Fig. 4B), in accord with [START_REF] Sato | A systematic review of the sparoid fishes of the subfamily Monotaxinae[END_REF] (under G. robinsoni) and [START_REF] Carpenter | FAO species catalogue, vol. 9, Emperor fishes and large-eye breams of the world (family Lethrinidae), An annotated and illustrated catalogue of lethrinid species known to date[END_REF]. Gymnocranius oblongus n. sp. is distinct from G. grandoculis by its ratio of standard length to body depth: for a given standard length, the body of G. grandoculis is higher (see Table 1). The head of G. oblongus is more symmetrical dorso-ventrally than that of G. grandoculis, the latter showing a higher profile above the tip of mouth than below. Four (16S rRNA, Aldo-B, CK-6, Met) out of five of the genetic loci scored in the present study were diagnostic (Table 2): any of those loci could be used to differentiate G. oblongus n. sp. from G. grandoculis. We concur with [START_REF] Sato | A systematic review of the sparoid fishes of the subfamily Monotaxinae[END_REF] in questioning the identity of both Pentapus curtus and P. dux with G. grandoculis. The identity of G. oblongus with P. dux (410 mm SL) certainly can be rejected on the basis of general body shape (Table 3). The general body shape of P. curtus (Table 3) also differs from that of G. oblongus but the small size of the only type specimen of P. curtus (150 mm SL) relative to the type material of G. oblongus (Table 1) precludes meaningful comparisons.

Discussion

Molecular markers demonstrated that Gymnocranius oblongus n. sp. is reproductively isolated from all other Gymnocranius species sampled in New Caledonia. Gymnocranius oblongus is unique among species of the genus Gymnocranius by the combination of three features, which are the subhorizontal, wavy blue lines or dashes on snout and cheeks, the fusiform body and the elongate tail.

Two other species, namely G. frenatus and G. grandoculis, also have wavy blue lines on the cheek [START_REF] Carpenter | FAO species catalogue, vol. 9, Emperor fishes and large-eye breams of the world (family Lethrinidae), An annotated and illustrated catalogue of lethrinid species known to date[END_REF], but of shapes generally different from those of G. oblongus. Gymnocranius frenatus' markings are oblique, running from the lower part of snout towards the eye, and they are thicker than those of G. oblongus; G. grandoculis' markings are more continuous and appear to cover a wider area of snout and cheek than those of G. oblongus, especially in larger individuals. Another species, G. microdon, has blue markings on the snout: those are dots or dashes, either sub-vertical [START_REF] Carpenter | FAO species catalogue, vol. 9, Emperor fishes and large-eye breams of the world (family Lethrinidae), An annotated and illustrated catalogue of lethrinid species known to date[END_REF] or sub-horizontal (P.Borsa, pers. obs.). The more-markedly elongated and fusiform body of G. oblongus actually makes it distinct from all other described Gymnocranius species, except G. microdon. However, smaller individuals of G. oblongus appear to be bulkier than G. microdon according to the measurements reported for the latter species [START_REF] Carpenter | FAO species catalogue, vol. 9, Emperor fishes and large-eye breams of the world (family Lethrinidae), An annotated and illustrated catalogue of lethrinid species known to date[END_REF][START_REF] Carpenter | Lethrinidae -emperors (emperor snappers)[END_REF]. Also, the forehead of G. microdon is distinctively prominent: the forehead of G. oblongus is moderately prominent and less steep than that of G. microdon. The tail of G. elongatus [START_REF] Senta | A new Sparoid fish, Gymnocranius elongatus, from the southern South China sea[END_REF] is similar in shape to that of the smaller specimens of G. oblongus examined here, but the tips of the caudal fin are pointed in G. elongatus whereas they are rounded in G. oblongus, especially in larger individuals. In all cases, the caudal fin of G. oblongus is not as indented and elongate as that of G. elongatus. It is also different from that of G. grandoculis, which is broad, with a subtle middle notch (Fig. 4C).

Large-eye breams of genus Gymnocranius are large-size, commercial fishes abundantly distributed throughout the tropical Indo-West Pacific [START_REF] Sato | A systematic review of the sparoid fishes of the subfamily Monotaxinae[END_REF][START_REF] Carpenter | FAO species catalogue, vol. 9, Emperor fishes and large-eye breams of the world (family Lethrinidae), An annotated and illustrated catalogue of lethrinid species known to date[END_REF][START_REF] Carpenter | Lethrinidae -emperors (emperor snappers)[END_REF] where they are sold on fish markets; their flesh is prized [START_REF] Sato | A systematic review of the sparoid fishes of the subfamily Monotaxinae[END_REF][START_REF] Carpenter | FAO species catalogue, vol. 9, Emperor fishes and large-eye breams of the world (family Lethrinidae), An annotated and illustrated catalogue of lethrinid species known to date[END_REF][START_REF] Coleman | Australian sea fishes north of 30[END_REF]. Therefore, the discovery of a new Gymnocranius species is a significant event: why this species and several other Gymnocranius species awaiting description [START_REF] Justine | Monogenea: Diplectanidae) from Gymnocranius spp. (Lethrinidae: Monotaxinae) off New Caledonia, with proposal of the new morphological group 'tubulicornis' within Lamellodiscus Johnston & Tiegs[END_REF][START_REF] Borsa | Identification par barcode (séquences nucléotidiques d'un fragment du gène du cytochrome b) des larves de Lethrinidae capturées en baies de Dumbéa et Ouano (Nouvelle-Calédonie)[END_REF] have been unnoticed up to now can be explained by several factors, including the rarity of well-preserved specimens in museum collections [START_REF] Carpenter | FAO species catalogue, vol. 9, Emperor fishes and large-eye breams of the world (family Lethrinidae), An annotated and illustrated catalogue of lethrinid species known to date[END_REF], the difficulties generally encountered for separating Gymnocranius species on the single basis of morphology [START_REF] Carpenter | FAO species catalogue, vol. 9, Emperor fishes and large-eye breams of the world (family Lethrinidae), An annotated and illustrated catalogue of lethrinid species known to date[END_REF], allometry in growth (present work), and the apparent lability of blue markings on snout and cheeks, which turn out to be one of the most helpful characters in identification to species ([2]; present work).

  d'histoire naturelle, Paris, were examined: MNHN 2009-0009 (Southern lagoon, New Caledonia, holotype), standard length (SL) 358 mm; MNHN 2009-0005 (Southern lagoon, New Caledonia, paratype), SL 220 mm; MNHN 2009-0007 (New Caledonia, paratype), SL 245 mm; and MNHN 2009-008 (New Caledonia, paratype), SL 226 mm. Two other specimens whose skull was preserved at Institut de recherche pour le développement (IRD), Nouméa (IRDN-20090410 A and B, from the Southern lagoon of New Caledonia), were examined fresh. Codes here follow [10] except IRDN, which refers to the specimens in the collections of IRD, Nouméa.

  national d'histoire naturelle, Paris [MNHN 2009-0009 (holotype), and MNHN 2009-0005, MNHN 2009-0007 and MNHN 2009-0008 (all paratypes)], and two individuals now preserved as skulls and tissue samples at Institut de recherche pour le développement, Nouméa, under reference numbers IRDN-20090410 A and B.

Figure 1 .

 1 Figure 1. Type material of Gymnocranius oblongus n. sp. (A) MNHN 2009-0009 (holotype, SL 358 mm); (B) MNHN 2009-0005 (paratype, SL 221 mm); (C) MNHN 2009-0007 (paratype, SL 245 mm); (D) MNHN 2009-0008 (paratype, SL 226 mm). Scale bar: 5 cm.

Figure 2 .

 2 Figure 2. Gymnocranius oblongus n. sp. Colour patterns of head, specimen no. IRDN-20090410B from the southwestern lagoon of New Caledonia.

Figure 3 .

 3 Figure 3. Gymnocranius oblongus n. sp. early juvenile from the southwestern lagoon of New Caledonia, SL 21.8 mm (composite photograph, courtesy of D. Ponton, IRD).

Figure 4 .

 4 Figure 4. Specimens of Gymnocranius grandoculis. (A) MNHN 0000-8811, holotype of Cantharus grandoculis Valenciennes, 1830, Seychelles, SL 186 mm; (B) QMI 5284, holotype of Paradentex marshalli Whitley, 1936, Queensland, SL 481 mm (photograph courtesy of the Queensland Museum, Brisbane); (C) MNHN 2009-0006, voucher, Northern Lagoon of New Caledonia's Grande Terre, SL 474 mm.

Table 1

 1 Measurements (in mm) and other parameters on specimens of Gymnocranius oblongus n. sp. and comparison with G. grandoculis specimens of a range of sizes.MNHN Museum national d'histoire naturelle, Paris; IRDN Institut de recherche pour le développement, Noumea; QMI Queensland Museum, Brisbane; SL standard length; BD body depth at dorsal-fin origin

	Parameter	G. oblongus

a from photograph courtesy by the Queensland Museum

Table 3

 3 Measurements (in mm) and other parameters on the holotypes ofPentapus curtus and P. dux. Same abbreviations as in Table1.

	Parameter	Species
		Pentapus curtus,	Pentapus dux,
		MNHN 0000-1317	MNHN 0000-2591
	Measurement (mm)
	SL	150	410
	BD	56	133
	Body depth at anal-fin origin	58	124
	Head length	48	117
	Snout length	18	53.5
	Eye diameter	16	32
	Inter-orbital width	16	40.5
	Predorsal length	51	133
	Prepelvic length	51	133
	Preanal length	94	245
	Other
	Ratio of SL to BD	2.68	3.11
	Pored scales on lateral line	48	47

Acknowledgements

We are grateful to Dominique Deschamps, fisherman and fish merchant at the Noumea fish market, for the provision of several specimens, including the holotype; to Jeff Johnson (Queensland Museum, Brisbane) for the photograph of specimen QMI 5284; to Laure Carassou and Dominique Ponton (IRD, Nouméa) for sorting and photographing the Lethrinidae larvae captured in light traps; to Torao Sato (Misaki Marine Biological Station, Miura) for helpful advice and encouragement; to David Parkhill (Queensland Museum) for bibliographic assistance; to Romain Causse and Patrice Pruvost (MNHN) for access to the ichthyological collections at MNHN and for curating the specimens deposited by us; to Adrien Rivaton (Adecal) and Pablo Chavance (ZoNéCo) for support. Funded in part by IRD-DRV, Fonds pacifique pour le développement and ZoNéCo. We declare no conflict of interest.

Table 2

Haplotype frequencies at four DNA loci in Gymnocranius oblongus n. sp., compared to those of G.

grandoculis. Haplotypes at three other Gymnocranius species sampled in New Caledonia [START_REF] Justine | Monogenea: Diplectanidae) from Gymnocranius spp. (Lethrinidae: Monotaxinae) off New Caledonia, with proposal of the new morphological group 'tubulicornis' within Lamellodiscus Johnston & Tiegs[END_REF][START_REF] Borsa | Identification par barcode (séquences nucléotidiques d'un fragment du gène du cytochrome b) des larves de Lethrinidae capturées en baies de Dumbéa et Ouano (Nouvelle-Calédonie)[END_REF]

a Includes one larva (Fig. 3) a posteriori identified to species by its multiple-locus DNA barcode


Sarah Lemer

Didier Aurelle

Laurent Vigliola

Jean-Dominique Durand

Philippe Borsa
email: philippe.borsa@noumea.ird.nc

Cytochrome b barcoding, molecular systematics and geographic differentiation in rabbitfishes (Siganidae)

The fish genus Siganus (Siganidae) is widely distributed in the coastal habitats of all the tropical Indo-Pacific, with 28 nominal species recognized so far, based on general morphology and coloration patterns. A mitochondrial phylogeny of 16 Siganidae species, based on the partial nucleotide sequences of the cytochome b gene, was produced. Individual haplotypes of given nominal species generally clustered at the extremity of long branches, thus validating the current taxonomy. However, S. lineatus haplotypes formed a paraphyletic group including S. guttatus, while S. fuscescens haplotypes were apparently splitted in two groups, calling for further investigation. S. woodlandi and S. argenteus formed a monophyletic group, as expected from their close morphological relatedness, although they were separated by a substantial, 14.5%-16.3% nucleotide distance. Among 8 species sampled from different locations across the Indo-West Pacific, S. argenteus and S. spinus showed the lowest degree of geographic differentiation, a result that correlated well with their extended pelagic larval stage. Fixation index estimates were high in all 6 other species tested (S. doliatus, S. fuscescens, S. lineatus, S. puellus, S. punctatus, S. vulpinus). The cytochrome b gene fragment chosen here proved useful as a barcode in Siganidae.

Résumé

Identification par code-barre, systématique moléculaire et différenciation géographique chez les picots (Siganidae), sur la base des séquences nucléotidiques du gène du cytochrome b. Les poissons du genre Siganus (Siganidae), caractéristiques des habitats côtiers de l'Indo-Pacifique tropical, comprennent 28 espèces nominales, définies sur la base de leur morphologie générale et de leurs patrons de coloration. Une phylogénie mitochondriale portant sur 16 espèces du genre a été produite à partir des séquences nucléotidiques d'un fragment du gène du cytochrome b. Les haplotypes d'une espèce donnée se regroupent généralement à l'extrémité de longues branches, validant de ce fait la taxinomie actuelle. Cependant, les haplotypes de S. lineatus forment un groupe paraphylétique avec S. guttatus et deux haplogroupes distincts sont observés chez S. fuscescens, résultats qu'il conviendrait d'approfondir. S. woodlandi et S. argenteus forment un groupe monophylétique, comme cela avait été inféré du fait de leur ressemblance morphologique, quoique ces deux espèces soient séparées par une distance nucléotidique relativement élevée (14.5%-16.3%). Des 8 espèces échantillonnées en différentes localités de la région Indo-Ouest Pacifique, S. argenteus et S. spinus montrent le plus faible degré de différenciation géographique. Ce résultat s'avère concorder avec la longue durée de leur phase larvaire pélagique. Les valeurs estimées de l'indice de fixation sont élevées chez les 6 autres espèces testées (S. doliatus, S. fuscescens, S. lineatus, S. puellus, S. punctatus, S. vulpinus). Le fragment du gène du cytochrome b utilisé pour la présente étude s'avère être un bon marqueur pour l'identification à l'espèce (ou codebarre) chez les Siganidae.

Introduction

Good systematics and taxonomy are desirable for understanding the biogeographical patterns of species diversity. Of all the marine living organisms of the Indo-West Pacific, Teleostean fishes probably are among the best-described, even though their systematics and taxonomy still need considerable research effort [START_REF] Knowlton | Molecular genetic analyses of species boundaries in the sea[END_REF][START_REF] Ward | DNA barcoding Australia's fish species[END_REF].

The fish family Siganidae ('rabbitfishes') comprises a single genus, Siganus, with 28 nominal species validated so far [START_REF] Woodland | Revision of the fish family Siganidae with description of two new species and comments on distribution and biology[END_REF][START_REF] Randall | Siganus woodlandi, new species of rabbitfish (Siganidae) from New Caledonia[END_REF]. Species richness in Siganidae decreases from the central Indo-West Pacific region, where a majority of the species are thought to originate from, to the periphery [START_REF] Woodland | Revision of the fish family Siganidae with description of two new species and comments on distribution and biology[END_REF][START_REF] Woodland | Zoogeography of the Siganidae: an interpretation of distribution and richness patterns[END_REF]. This pattern has been observed for other groups of marine organisms [START_REF] Palumbi | What can molecular genetics contribute to marine biogeography? An urchin's tale[END_REF].

Therefore, Siganidae constitute an interesting model-group to study speciation processes in the Indo-West Pacific, but their taxonomy first requires a precise reevaluation. Remarkable homogeneity in those characters that usually allow to diagnose species, such as numbers of fin spines and soft rays, tooth count and tooth shape and size, and broad across-species overlap in other characters, such as gill-raker and lateral-line scale counts [START_REF] Woodland | Revision of the fish family Siganidae with description of two new species and comments on distribution and biology[END_REF][START_REF] Randall | Siganus woodlandi, new species of rabbitfish (Siganidae) from New Caledonia[END_REF] have earlier led to taxonomic confusion in Siganidae. The present taxonomy of Siganidae, based on [START_REF] Woodland | Revision of the fish family Siganidae with description of two new species and comments on distribution and biology[END_REF] extensive revision [START_REF] Woodland | Revision of the fish family Siganidae with description of two new species and comments on distribution and biology[END_REF] relies on colour patterns and gross morphological features, such as body proportions and relative length of snout [START_REF] Woodland | Revision of the fish family Siganidae with description of two new species and comments on distribution and biology[END_REF][START_REF] Randall | Siganus woodlandi, new species of rabbitfish (Siganidae) from New Caledonia[END_REF]. Apart from an allozyme survey describing the genetic relationships among 10 species of the family, 9 of which were sampled from a localised region of the tropical West-Pacific [START_REF] Lacson | Genetic distances among fishes of the genus Siganus (Siganidae) from the western Pacific ocean[END_REF], little emphasis has been placed so far on validating the current classification using genetic markers.

Fishes of the family Siganidae occur in the coastal waters of the tropical Indo-Pacific. Two species occurring in the Red Sea, S. luridus and S. rivulatus, have recently expanded their area of distribution into the Mediterranean across the Suez Canal [START_REF] Tuvia | Two siganid fishes of Red Sea origin in the eastern Mediterranean, Bull. Sea Fish[END_REF]. The geographic distributions of rabbitfishes range from the entire tropical Indo-West Pacific except the easternmost Pacific islands to regional, presumably, in part, as a consequence of differences in the dispersal potential of juveniles [START_REF] Woodland | Revision of the fish family Siganidae with description of two new species and comments on distribution and biology[END_REF]. A proportion of species are originally endemic to restricted geographic areas such as the enclosed Red Sea (S. rivulatus) and Andaman Sea (S. magnificus), and isolated groups of islands or reefs (Maldives: S. puelloides; scattered reefs off Western Australia: S. trispilos; Fiji: S. uspi; Tonga: S. niger) [START_REF] Woodland | Revision of the fish family Siganidae with description of two new species and comments on distribution and biology[END_REF]. One species (S. unimaculatus) has an antitropical distribution [START_REF] Woodland | Revision of the fish family Siganidae with description of two new species and comments on distribution and biology[END_REF]. There might be a second example of antitropical distribution in Siganus: S. woodlandi [START_REF] Randall | Siganus woodlandi, new species of rabbitfish (Siganidae) from New Caledonia[END_REF] has so far been reported from New Caledonia only, but the sedakahana-aigo, an undescribed rabbitfish from Japan "that appears to be either S. woodlandi or a very close relative" ( [START_REF] Randall | Siganus woodlandi, new species of rabbitfish (Siganidae) from New Caledonia[END_REF], and references therein) might be its antitropical counterpart. Pairs of presumed sibling species have been reported [START_REF] Woodland | Revision of the fish family Siganidae with description of two new species and comments on distribution and biology[END_REF]. The latter have an essentially Indian / West Pacific distribution with some overlap in the central Indo-Malay region [START_REF] Woodland | Revision of the fish family Siganidae with description of two new species and comments on distribution and biology[END_REF]. Among these, morphologically intermediate individuals thought to be hybrids between S. doliatus and S.

virgatus have been reported [START_REF] Woodland | Revision of the fish family Siganidae with description of two new species and comments on distribution and biology[END_REF][START_REF] Randall | Reef and shore fishes of the South Pacific[END_REF], and hybrids between S. guttatus and S. lineatus are similarly expected [START_REF] Randall | Reef and shore fishes of the South Pacific[END_REF].

Here, we analysed the nucleotide sequences of a fragment of the cytochrome b gene in 16 Siganidae species sampled across the Indo-West Pacific. Our objectives were: (1) to evaluate the potential of the cytochrome b gene as a species-specific marker (a DNA-barcode) in Siganidae [START_REF] Ward | DNA barcoding Australia's fish species[END_REF] to quantify genetic differences between sibling species with parapatric distribution; (3) to estimate the degree of genetic differentiation among populations within a few species differing by the width of their geographic distribution.

Materials and methods

Two hundred sixty two rabbitfish belonging to 15 nominal species, and 22 unidentified rabbitfish larvae were collected from 7 locations in the Indo-West Pacific and the Mediterranean (Table 1). The fish were obtained from fishermen (all samples from Ouvéa, Makassar and Truk, and most samples from the southern lagoon of New Caledonia), or as sidesamples of ecological surveys (the S. spinus sample of Moorea, and some samples from the southern lagoon of New Caledonia including all larvae, which were collected using light traps [START_REF] Borsa | Les premiers stades de vie des poissons de Nouvelle-Calédonie: diversité des espèces et rôle des zones côtières pour leur croissance et leur survie[END_REF]). Voucher-specimens for all Siganidae species collected in the southern lagoon of New Caledonia and in Moorea were deposited at the Museum national d'histoire naturelle, Paris (MNHN 2006-1525 to 1537). Muscle tissue samples of S. luridus and S. rivulatus, preserved frozen at Station méditerranéenne de l'environnement littoral, Sète, were sent to us by F. Bonhomme. A piece of muscle tissue of a S. lineatus specimen from the Maldives, deposited at the Bishop Museum, Honolulu (BPBM 40390), was donated to us by J.E. Randall. For one more species (S. virgatus) we only had at our disposal cytochrome b gene sequences available from the GENBANK sequence database (http://www.ncbi.nih.gov/Genbank: accession nos. AY190546, AY190549, and AY190550). The samples from the Makassar Strait region (South Sulawesi, Indonesia) included a group of 9 individuals whose coloration patterns ranged from typical S. guttatus to typical S. lineatus [START_REF] Woodland | Revision of the fish family Siganidae with description of two new species and comments on distribution and biology[END_REF], through a range of intermediate patterns (for 7 individuals), indicating possible introgression between the two species.

DNA was extracted from ethanol-preserved fin clips or muscle fragments using either the standard phenol-chloroform extractions with Phase-Lock Gel TM tubes (Eppendorf, Le Pecq, France), or the DNeasy® kit (Qiagen GmbH, Hilden, Germany). Individual DNA extracts were subjected to PCR amplification of a 326-bp fragment of the cytochrome b gene, using a pair of degenerate primers (forward: 5'-TCMGAYATCGCCACAGCCTTCTC-3'; reverse: 5'-AGGAAGTGGAAKGCGAAGAA-3') designed from the alignment of the cytochrome b gene sequences of Siganidae available in GENBANK (accession nos. AY190542 to AY190555). The amplification reactions were done in 96-well plates with each well containing 20 µL reaction mixture [2 µL template DNA, 2.5 mM MgCl 2 , 0.48 mM dNTP mix, 0.5 µM of each primer and 0.5 U Taq polymerase (Promega, Madison WI, U.S.A.)] in a Robocycler TM thermocycler (Stratagene, La Jolla CA, U.S.A.). The PCR program consisted of 3 min DNA-denaturation (at 94°C) followed by 35 cycles of 1 min denaturation (94°C) / 1 min annealing (60°C) / 1 min elongation (72°C). The PCR products were shipped to GATC Biotech (Konstanz, Germany), or Macrogen (Seoul, South Korea) for nucleotide sequencing and the sequences were subsequently retrieved from the company's Internet website.

All nucleotide sequences, which were deposited in GENBANK (accession numbers DQ898027 to DQ898094), were aligned, together with their homologues from the Philippines (GENBANK AY190543 to AY190556), using BIOEDIT [START_REF] Hall | BIOEDIT: a user friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[END_REF]. The phylogenetic relationships among species in the genus Siganus were inferred from the matrix of haplotype sequences using three different algorithms: neighbour-joining (NJ [START_REF] Saitou | The neighbor-joining method: a new method for reconstructing phylogenetic trees[END_REF]), maximum parsimony (MP [START_REF] Fitch | Towards defining the course of evolution: minimum change for a specific tree topology[END_REF]), and maximum likelihood (ML [START_REF] Felsenstein | Maximum likelihood and minimum-steps methods for estimating evolutionary trees from data on discrete characters[END_REF]). NJ and MP analyses were done using MEGA 3.1 [START_REF] Kumar | MEGA 3: Integrated software for molecular evolutionary genetics analysis and sequence alignment[END_REF]. ML analysis was done using PHYLIP, version 3.65 [START_REF] Felsenstein | PHYLIP (phylogeny inference package) version 3.65[END_REF]. Nucleotide distances between haplotypes ( d ˆ) were estimated according to Kimura's 2-parameter model of nucleotide substitution ( [START_REF] Kimura | A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences[END_REF], implemented in MEGA 3.1). Gaps were treated as deletions in pairwise comparisons of sequences (option "Pairwise-Deletion" in MEGA 3.1). Haplotype frequency differences between populations were estimated using , Weir and Cockerham's fixation-index estimator, which apportions the genetic diversity among samples relative to the total diversity [START_REF] Weir | Estimating F-statistics for the analysis of population structure[END_REF]. Tests of significance were done by comparing the observed θ ˆθ ˆ-value with 1000 pseudo-θ ˆ produced by random permutations of haplotypes among samples using GENETIX 4.05 [START_REF] Belkhir | Genetix 4.05, logiciel sous WindowsTM pour la génétique des populations, Laboratoire Génome, Populations, Interactions[END_REF]. Taking into account the information harboured in the nucleotide sequences of each allele along with allele frequencies, we used analysis of molecular variance (AMOVA [START_REF] Excoffier | Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data[END_REF]) implemented in ARLEQUIN 3.01 [START_REF] Excoffier | ARLEQUIN ver. 3.0: an integrated software package for population genetics data analysis[END_REF] to estimate Φst, [START_REF] Excoffier | Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data[END_REF] fixation index for nucleotide sequence data [START_REF] Excoffier | Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data[END_REF]. The Φst-estimate produced by AMOVA is equivalent to a multiple-locus θ ˆ where each variable nucleotide site is considered as a different locus [START_REF] Michalakis | A generic estimation of population subdivision using distances between alleles with special reference for microsatellite loci[END_REF]. The significance of Φst-estimates was tested using the non-parametric permutation algorithm implemented in ARLEQUIN 3.01.

Results

The mitochondrial phylogeny of Siganidae, based on a 300-bp fragment of the cytochrome b gene, displayed clusters of haplotypes or haplogroups atop long branches, generally supported by high bootstrap scores (Fig. 1). In all cases, a haplogroup corresponded to a nominal species, except for the pair S. doliatus / S. virgatus, whose haplotypes formed a single, tight cluster. The homogeneity of S. fuscescens was questionable, as the haplotypes sampled for that species appeared to form two separate haplogroups, distant by d = 0.010-0.024. The haplogroup corresponding to S. guttatus was weakly supported and appeared to be nested within a S. guttatus / S. lineatus cluster, whose most external haplotype was the one found in S. lineatus from the Maldives. The The degree of population differentiation, as estimated by fixation indices at the scale of the Indo-West Pacific, was significant in seven of the eight species tested (Table 2). The high degree of genetic differentiation found among S. fuscescens populations however could be misleading in the case where two reproductively isolated populations had been sampled in New Caledonia (see above). Accordingly, we re-estimated the fixation-index values after considering

Discussion

Individual haplotypes of given nominal species generally formed tight clusters at the extremity of long branches, thus validating the current, morphologically-based taxonomy. While S. guttatus haplotypes appeared to cluster into a single clade, thus confirming its recognition as a separate species [in spite of apparent hybridization with S. lineatus (present results)], this group appeared to be nested within S. lineatus, suggesting that S. guttatus may be an offshoot of an ancestral proto-S. lineatus stock. The fact that the only haplotype sampled in the isolated S. lineatus population of the Maldives placed itself as outgroup to both S. guttatus and Indo-West Pacific S. lineatus, hints that the current taxon S. lineatus may include two geographically isolated populations with different evolutionary histories. Further insight into the genetic relationships of West-Pacific and Indian-Ocean populations of S. lineatus, requiring extensive sampling across the species' range is warranted, as this will inform us on the degree of genetic differentiation of Maldives S. lineatus vs. other S. lineatus populations. The two mitochondrial clades observed within S. fuscescens were separated from each other by an average nucleotide distance of the same order as that between S. guttatus and Indo-West Pacific S. lineatus, hinting at the possible occurrence of two distinct forms within the current taxon S. fuscescens in New Caledonia. To test that hypothesis requires further sampling of individuals, and the use of Mendelian markers to test for reproductive isolation.

Three pairs of presumed, sibling species with parapatric distribution were sampled for the present survey. Contrasted patterns of pairwise genetic differentiation were observed. The hypothesis of sibling species for the pair S. luridus / S. spinus was not supported. Letting aside the case of Maldives S. lineatus, the phylogenetic arrangement of S. guttatus / S. lineatus haplotypes was not inconsistent with the sibling-species hypothesis, but the fact that the two haplogroups did not appear as clearly separate clades also leaves open the possibility that the two species have not fully achieved reciprocal monophyly of their mitochondrial lineages. Nucleotide sequencing of a larger segment of the mitochondrial DNA may provide the necessary information to address that question. Last, there was no evidence of genetic differentiation of S. doliatus vs. S. virgatus. S. virgatus haplotypes were from individuals sampled in the Philippines, which border the zone of overlap with S. doliatus [START_REF] Woodland | Revision of the fish family Siganidae with description of two new species and comments on distribution and biology[END_REF]. Since the two species are believed to hybridize [START_REF] Woodland | Revision of the fish family Siganidae with description of two new species and comments on distribution and biology[END_REF][START_REF] Randall | Reef and shore fishes of the South Pacific[END_REF], it is possible that those individuals identified as S. virgatus [START_REF] Ochavillo | Identification and discrimination of late pelagic presettlement siganids: morphometic and mtDNA analyses[END_REF] harboured S. doliatus mitochondria as a result of introgression. In that case, further sampling of S. virgatus would be necessary, ideally outside the zone of overlap to sample 'true' S. virgatus mitochondria. Alternatively, the divergence between the two species may be recent, to the point that the sorting of their mitochondrial lineages is still not perceptible.

At the infra-specific scale, the degree of genetic differentiation was found to be lowest in S. argenteus and S. spinus, which of all Siganidae are the two species with the presumably longest pelagic, larval / prejuvenile stage [START_REF] Woodland | Revision of the fish family Siganidae with description of two new species and comments on distribution and biology[END_REF] and also the widest distribution. Geneticdifferentiation estimates in other species with more restricted ranges (S. doliatus, S. fuscescens, S. lineatus, S. puellus, S. punctatus, S. vulpinus) were all higher. Thus, distribution ranges, which primarily correlate with pelagic duration of larvae [START_REF] Woodland | Revision of the fish family Siganidae with description of two new species and comments on distribution and biology[END_REF], also correlated well with the degree of genetic homogeneity among populations. These observations indirectly validate Woodland's (1990) hypothesis that dispersal is higher in species with longer larval stage in Siganidae [START_REF] Woodland | Revision of the fish family Siganidae with description of two new species and comments on distribution and biology[END_REF].

The 300-bp cytochrome b gene fragment sequenced here proved a potential barcode for Siganidae, since the haplotypes sampled in a species generally clustered tightly, relative to the long intervals between species. In other terms, intraspecific pairwise nucleotide distances were distinctly lower than pairwise distances between haplotypes of different species, allowing efficient discrimination. This molecular marker allowed us to identify Siganus spp. larvae, all of which the current, morphology-based identification keys can only assign to family [START_REF] Leis | The larvae of Indo-Pacific coastal fishes: an identification guide to marine fish larvae[END_REF]. Most of the cytochrome b gene sequences that were available on GENBANK in late 2005 when we started the present survey were from an unpublished study by D.G. Ochavillo and coauthors [START_REF] Ochavillo | Identification and discrimination of late pelagic presettlement siganids: morphometic and mtDNA analyses[END_REF] whose objective was the identification and discrimination of juvenile Siganidae at the late pelagic presettlement stage. Our results allowed the barcode identification of two of the individuals sequenced by these authors, that is AY190543, initially identified as S. punctatissimus but which was found to be S. punctatus, and AY190556, which was identified as S. vermiculatus but here found to be S. lineatus. Barcoding of Siganidae will be useful in our investigations on the population and community ecology of shorefish larvae and juveniles, which require identification at the species level [START_REF] Borsa | Les premiers stades de vie des poissons de Nouvelle-Calédonie: diversité des espèces et rôle des zones côtières pour leur croissance et leur survie[END_REF][START_REF] Carassou | Les premiers stades de vie des poissons de Nouvelle-Calédonie: identification des habitats lagonaires pour les stades pré-et post-installation, volet thématique 1: stades pré-installation[END_REF][START_REF] Borsa | Les premiers stades de vie des poissons de Nouvelle-Calédonie: identification des habitats lagonaires pour les stades pré-et post-installation, volet thématique 3: Identification des jeunes stades[END_REF].

Table 2 Siganus spp. mitochondrial haplotype (partial sequence of cytochrome b gene) frequencies by sample. All sequence data were from the present survey, except for 10 Philippines specimens, whose sequences were retrieved from GENBANK (details in footnote). Degree of genetic differentiation estimated using Weir and Cockerham's [START_REF] Weir | Estimating F-statistics for the analysis of population structure[END_REF][START_REF] Belkhir | Genetix 4.05, logiciel sous WindowsTM pour la génétique des populations, Laboratoire Génome, Populations, Interactions[END_REF] and Excoffier et al.'s [START_REF] Excoffier | Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data[END_REF][START_REF] Excoffier | ARLEQUIN ver. 3.0: an integrated software package for population genetics data analysis[END_REF]. Nucleotide sites numbered from 1 to 300, with the first site homologous to site no. 14611 in Atlantic salmon, Salmo salar (GENBANK AF133701). NC: southern lagoon of New Caledonia (for all species except S. lineatus, sampled on the western coast and in the northern lagoon); MS: Makassar strait off Sulawesi Island (for all samples except where noted); *: ). Neighbour-joining tree (Kimura's 2-parameter nucleotide distances [START_REF] Kimura | A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences[END_REF]; MEGA 3.1 [START_REF] Kumar | MEGA 3: Integrated software for molecular evolutionary genetics analysis and sequence alignment[END_REF]) of nucleotide sequences of a 300-bp fragment of the cytochrome b gene (GENBANK DQ898028 to DQ898094 and AY190543 to AY190556). Homologous sequence in Acanthurus blochii (Acanthuridae) (GENBANK DQ898027) used as outgroup [START_REF] Tang | The phylogenetic relationships of the suborder Acanthuroidei (Teleostei: Perciformes) based on molecular and morphological evidence[END_REF]. Numbers at a node are percentage scores (>50%) after bootstrap resampling [START_REF] Felsenstein | Confidence limits on phylogenies: an approach using the bootstrap[END_REF] of nucleotide sites in the matrix of sequences [bold: Neighbourjoining, 1000 bootstraps; italics: maximum parsimony (MEGA 3.1), 1000 bootstraps; regular: maximum likelihood (PHYLIP 3.65 [START_REF] Felsenstein | PHYLIP (phylogeny inference package) version 3.65[END_REF]), 100 bootstraps; for the sake of clarity, the two latter percentages were figured on the main nodes only]. Scale bar: 2% nucleotide divergence estimate. Synonymous sequences: AY190549=AY190550; DQ898039= AY190546; DQ898047= AY190547; DQ898054=AY190555; DQ898068= AY190543.

  S. guttatus haplogroup was distant from Indo-West Pacific (i.e., Makassar Strait, Philippines, New Caledonia) S. lineatus by = 0.010-0.020 while the latter were separated from the Maldives S. lineatus haplotype by d = 0.024-0.027. S. luridus and S. spinus were separated by a high nucleotide distance ( = 0.145-0.163) and the topology of the tree provided little support to the hypothesis that they form sister-clades. S. vulpinus haplotypes formed a sub-clade of a strongly supported clade that also included S. puellus and S. punctatus. S. woodlandi haplotypes formed a monophyletic clade with S. argenteus, distant from it by = 0.090-0.106. dˆd dˆ The haplotypes of 7 individuals morphologically intermediate between S. guttatus and S. lineatus were equally distributed among the two respective haplogroups characteristic of each species. The haplotypes of the 22 unidentified larvae sampled in the southern lagoon of New Caledonia clustered with either S. lineatus or S. fuscescens.

  solely the S. fuscescens clade formed by haplotypes DQ898042, DQ898093, AY190545, and AY190551 to AY190553, represented in both New Caledonia and the Philippines, and found θ ˆ = 0.250 (P<0.01) and = 0.373 (P<0.01). The fixation indices could not be estimated in the other S. fuscescens clade (formed by haplotypes DQ898040, DQ898041, DQ898043 to st Φ DQ898045, and DQ898094) since the latter was represented in the New Caledonian sample only.

Fig. 1 .

 1 Fig.1. Mitochondrial phylogeny of Siganidae (Siganus spp.). Neighbour-joining tree (Kimura's 2-parameter nucleotide distances[START_REF] Kimura | A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences[END_REF]; MEGA 3.1[START_REF] Kumar | MEGA 3: Integrated software for molecular evolutionary genetics analysis and sequence alignment[END_REF]) of nucleotide sequences of a 300-bp fragment of the cytochrome b gene (GENBANK DQ898028 to DQ898094 and AY190543 to AY190556). Homologous sequence in Acanthurus blochii (Acanthuridae) (GENBANK DQ898027) used as outgroup[START_REF] Tang | The phylogenetic relationships of the suborder Acanthuroidei (Teleostei: Perciformes) based on molecular and morphological evidence[END_REF]. Numbers at a node are percentage scores (>50%) after bootstrap resampling[START_REF] Felsenstein | Confidence limits on phylogenies: an approach using the bootstrap[END_REF] of nucleotide sites in the matrix of sequences [bold: Neighbourjoining, 1000 bootstraps; italics: maximum parsimony (MEGA 3.1), 1000 bootstraps; regular: maximum likelihood (PHYLIP 3.65[START_REF] Felsenstein | PHYLIP (phylogeny inference package) version 3.65[END_REF]), 100 bootstraps; for the sake of clarity, the two latter percentages were figured on the main nodes only]. Scale bar: 2% nucleotide divergence estimate. Synonymous sequences: AY190549=AY190550; DQ898039= AY190546; DQ898047= AY190547; DQ898054=AY190555; DQ898068= AY190543.

Initially identified as S. vermiculatus (GenBank AY190556;[START_REF] Ochavillo | Identification and discrimination of late pelagic presettlement siganids: morphometic and mtDNA analyses[END_REF]) but barcode-identified as S. lineatus (present results);

Initially identified as S. punctatissimus (GenBank AY190543;[START_REF] Ochavillo | Identification and discrimination of late pelagic presettlement siganids: morphometic and mtDNA analyses[END_REF]) but barcode-identified as S. lineatus (present results).

Specimens from the Philippines identified as S. virgatus[START_REF] Ochavillo | Identification and discrimination of late pelagic presettlement siganids: morphometic and mtDNA analyses[END_REF];

From the Philippines[START_REF] Ochavillo | Identification and discrimination of late pelagic presettlement siganids: morphometic and mtDNA analyses[END_REF];

Specimen from the Philippines initially identified as S. vermiculatus (GENBANK AY190556;[START_REF] Ochavillo | Identification and discrimination of late pelagic presettlement siganids: morphometic and mtDNA analyses[END_REF]) but barcode-identified as S. lineatus (present results);

Includes one sequence of a Philippines specimen initially identified as S. punctatissimus (GENBANK AY190543;[START_REF] Ochavillo | Identification and discrimination of late pelagic presettlement siganids: morphometic and mtDNA analyses[END_REF]) but barcode-identified as S. punctatus (present results);

From the Philippines[START_REF] Ochavillo | Identification and discrimination of late pelagic presettlement siganids: morphometic and mtDNA analyses[END_REF]

Acknowledgements

A part of this work was included in S.L.'s M.Sc. memoir. Thanks to those who kindly provided samples: R.C. Anderson, F. Bonhomme, L. Carassou, J.-L. Justine, C. Mellin, G. Mou Tham, D. Lecchini, M. Leopold, J.E. Randall, P. Villard, and the Li-Khau family. Thanks to C. Rocher for technical help at the Dimar laboratory, and to P. Pruvost and L. Nandrin for curating voucher specimens at MNHN. Funded by the French government's Fonds Pacifique pour le développement, IRD-UR 128 "Biocomplexité des écosystèmes coralliens de l'Indo-Pacifique", Dimar, and ZoNéCo-Opération "Jeunes poissons".