Corinne Cruaud
email: cruaud@genoscope.cns.fr

Jean-Lou Justine
email: justine@mnhn.fr

Charlotte Schoelinck
email: schoelinck@mnhn.fr

Are all species of Pseudorhabdosynochus strictly host specific? -a molecular study

Species of the diplectanid monogenean genus Pseudorhabdosynochus are strictly host-specific (specialist), with the exception of P. cyanopodus, which was reported in New Caledonia, South Pacific, from two host species, Epinephelus cyanopodus and E. chlorostigma. We sequenced the COI gene of both host fish species and of their monogeneans. Morphological identification and pairwise distances showed that the two fish species were distinct (difference 6.1-6.6%), but that their monogeneans were not (difference 0-1.5%). A morphological study of sclerotised parts showed that specimens of P. cyanopodus are similar in both fish. Most species of groupers and their associated Pseudorhabdosynochus species are from warm surface waters, but the two groupers E. cyanopodus and E. chlorostigma are usually caught in deep-sea on the outer slope of the coral reef. This suggests that acquisition of a less strict host specificity is an adaptation of P. cyanopodus to deep-sea hosts.

Introduction

Species of the monogenean diplectanid genus Pseudorhabdosynochus are strictly host-specific. A study of more than 25 species of groupers [START_REF] Beveridge | An annotated list of parasites (Isopoda, Copepoda, Monogenea, Digenea, Cestoda and Nematoda) collected in groupers (Serranidae, Epinephelinae) in New Caledonia emphasizes parasite biodiversity in coral reef fish[END_REF] in the natural environment off New Caledonia, South Pacific, demonstrated that most grouper species harboured 1-8 species of Pseudorhabdosynochus, and that each of these monogenean species was "specialist", i.e. found on a single host species [START_REF] Beveridge | An annotated list of parasites (Isopoda, Copepoda, Monogenea, Digenea, Cestoda and Nematoda) collected in groupers (Serranidae, Epinephelinae) in New Caledonia emphasizes parasite biodiversity in coral reef fish[END_REF][START_REF] Justine | Species of Pseudorhabdosynochus Yamaguti, 1958 (Monogenea: Diplectanidae) from Epinephelus fasciatus and E. merra (Perciformes: Serranidae) off New Caledonia and other parts of the Indo-Pacific Ocean, with a comparison of measurements of specimens prepared using different methods, and a description of P. caledonicus n. sp[END_REF][START_REF] Justine | Pseudorhabdosynochus argus n. sp. (Monogenea: Diplectanidae) from Cephalopholis argus, P. minutus n. sp. and Diplectanum nanus n. sp. from C. sonnerati and other monogeneans from Cephalopholis spp. (Perciformes: Serranidae) off Australia and New Caledonia[END_REF][START_REF] Justine | Parasite biodiversity in a coral reef fish: twelve species of monogeneans on the gills of the grouper Epinephelus maculatus (Perciformes: Serranidae) off New Caledonia, with a description of eight new species of Pseudorhabdosynochus (Monogenea: Diplectanidae)[END_REF][START_REF] Sigura | Monogeneans of the speckled blue grouper, Epinephelus cyanopodus (Perciformes, Serranidae), from off New Caledonia, with a description of four new species of Pseudorhabdosynochus and one new species of Laticola (Monogenea: Diplectanidae), and evidence of monogenean faunal changes according to the size of fish[END_REF][START_REF] Sigura | Monogeneans of the malabar grouper Epinephelus malabaricus (Perciformes, Serranidae) off New Caledonia, with a description of six new species of Pseudorhabdosynochus (Monogenea: Diplectanidae)[END_REF][START_REF] Hinsinger | The 'Pseudorhabdosynochus cupatus group' (Monogenea: Diplectanidae) on Epinephelus fasciatus, E. howlandi, E. rivulatus and E. merra (Perciformes: Serranidae) off New Caledonia, with descriptions of Pseudorhabdosynochus cyathus n. sp. and P. calathus n. sp[END_REF][START_REF] Henry | Monogeneans from Epinephelus chlorostigma (Val.) (Perciformes: Serranidae) off New Caledonia, with the description of three new species of diplectanids[END_REF][START_REF] Schoelinck | Four species of Pseudorhabdosynochus (Monogenea: Diplectanidae) from the camouflage grouper Epinephelus polyphekadion (Perciformes: Serranidae) off New Caledonia[END_REF]. An exception was P. cyanopodus [START_REF] Sigura | Monogeneans of the speckled blue grouper, Epinephelus cyanopodus (Perciformes, Serranidae), from off New Caledonia, with a description of four new species of Pseudorhabdosynochus and one new species of Laticola (Monogenea: Diplectanidae), and evidence of monogenean faunal changes according to the size of fish[END_REF], reported both from its type-host, Epinephelus cyanopodus, and from E. chlorostigma; however, this report was based only on comparative measurements [START_REF] Henry | Monogeneans from Epinephelus chlorostigma (Val.) (Perciformes: Serranidae) off New Caledonia, with the description of three new species of diplectanids[END_REF]. In this paper, we provide additional morphological evidence and the first molecular confirmation of the presence of the same Pseudorhabdosynochus species on these two species of hosts, and we also confirm, with molecular methods, that the two host species are distinct.

Material and Methods

Fish were collected from off the barrier reef of New Caledonia [START_REF] Sigura | Monogeneans of the speckled blue grouper, Epinephelus cyanopodus (Perciformes, Serranidae), from off New Caledonia, with a description of four new species of Pseudorhabdosynochus and one new species of Laticola (Monogenea: Diplectanidae), and evidence of monogenean faunal changes according to the size of fish[END_REF][START_REF] Henry | Monogeneans from Epinephelus chlorostigma (Val.) (Perciformes: Serranidae) off New Caledonia, with the description of three new species of diplectanids[END_REF] or from the fish market of Nouméa, New Caledonia. Specimens of E. chlorostigma and E. cyanopodus were morphologically identified according to keys [START_REF] Heemstra | Groupers of the world (Family Serranidae, Subfamily Epinephelinae). An annotated and illustrated catalogue of the grouper, rockcod, hind, coral grouper and lyretail species known to date Rome: FAO[END_REF] and their identification was confirmed from photographs by confirmed ichthyologists. A photograph of specimen JNC3142 of E. chlorostigma was deposited in FishBase (www.fishbase.org). Parasites were collected [START_REF] Henry | Monogeneans from Epinephelus chlorostigma (Val.) (Perciformes: Serranidae) off New Caledonia, with the description of three new species of diplectanids[END_REF] from 2 specimens of each fish species.

Monogeneans were either processed for morphological examination [START_REF] Henry | Monogeneans from Epinephelus chlorostigma (Val.) (Perciformes: Serranidae) off New Caledonia, with the description of three new species of diplectanids[END_REF] or for molecular characterization; in the latter case, each monogenean was examined with a microscope for species identification and then destroyed in the process of DNA extraction.

Fish DNA was extracted from tissue samples using NucleoSpin 96 tissue kit (Macherey-Nagel, Düren, Germany). Approximately 654 bp were amplified from the 5' region of the Cytochrome Oxidase I (COI) gene from mitochondrial DNA using FishF1

(forward 5'-TCAACCAACCACAAAGACATTGGCAC-3') and FishR1 (Reverse 5'-TAGACTTCTGGGTGGCCAAAGAATCA3') [START_REF] Ward | DNA barcoding Australia's fish species[END_REF].

Monogenean DNA was extracted from the whole specimen using DNeasy® 96 Blood & Tissue kit (Qiagen). A fragment of 424 bp of COI gene was amplified using the specific primers COI-ASmit1

(forward 5'-TTTTTTGGGCATCCTGAGGTTTAT-3') and COI-ASmit2 (reverse 5'-TAAAGAAAGAACATAATGAAAATG-3') [START_REF] Littlewood | Parasite speciation within or between host species? -Phylogenetic evidence from site-specific polystome monogeneans[END_REF]. Each PCR reaction was performed in final 20 µl volume containing 1 ng of DNA, 1 × reaction buffer, 0.26 mM dNTP, 0,8 µM of each primer, 5% DMSO and 1.5 units of Taq DNA polymerase (Qiagen). Thermocycles consisted of an initial denaturation step at 94°C for 2', followed by 37 cycles of denaturation at 94°C for 30'', annealing at 48°C for 40'' and extension at 72°C for 1'. The final extension was conducted at 72°C for 10'. Purification and cycle-sequencing reactions were performed at the Génoscope (Évry, France), using the BigDye Terminator version 3 sequencing kit, the GeneAmp PCR System 9700 and a capillary ABI3730 DNA Analyser, all from Applied Biosystems.

Both DNA strands were sequenced for all PCR products. Sequences were edited and assembled using Sequencher 4.9 (Gene Codes Corporation, Ann Arbor, MI, USA). Sequences for this study were deposited as GenBank ID: JQ412500-JQ412503 (fish) and JQ400132-JQ400138 (monogeneans).

COI sequences were automatically aligned using ClustalW implemented in BioEdit version 7.0.5.3 [START_REF] Hall | BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[END_REF]. The accuracy of automatic alignments was confirmed by eye. Uncorrected pairwise distances were computed using Mega 5 [START_REF] Tamura | MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[END_REF]. Results were visualized on a pairwise difference matrix.

The identity of fish species was checked using BLAST in BOLD [START_REF] Ratnasingham | BOLD: The Barcode of Life Data System (www. barcodinglife. org)[END_REF]; issues in the specific attribution of sequences are outlined below (3.1).

Results

Analysis of fish COI sequences

Sequences of our specimens of E. cyanopodus and E. chlorostigma showed low intraspecific variability (respectively 0.2% and 0.5%), and high interspecific differences (6.1-6.6 %) (Table 1). COI sequences are considered reliable indicators for species delimitation [START_REF] Ward | DNA barcoding Australia's fish species[END_REF][START_REF] Hebert | Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species[END_REF], and thus we consider that the two fish species were distinct. Sequences of E. cyanopodus from our specimens matched with the three deposited sequences of this species in BOLD with 99.84-100% similarity (date: 11 January 2012; unpublished sequences in BOLD AMS513-08, EPINE003-11, EPINE003-12; no sequence in GenBank); we thus consider that molecular evidence confirms morphological identification.

Sequences of our specimens of E. chlorostigma matched with the six deposited sequences of this species in GenBank with only 95.04-95.37% (date: 11 January 2012; HQ149841 [START_REF] Asgharian | Cytochrome c oxidase subunit 1 barcode data of fish of the Nayband National Park in the Persian Gulf and analysis using meta-data flag several cryptic species[END_REF]; EF609514-5, EU39202-4 [START_REF] Lakra | DNA barcoding Indian marine fishes[END_REF]). According to Heemstra & Randall [START_REF] Heemstra | Groupers of the world (Family Serranidae, Subfamily Epinephelinae). An annotated and illustrated catalogue of the grouper, rockcod, hind, coral grouper and lyretail species known to date Rome: FAO[END_REF], two species, E. polylepis and E. gabriellae, were often misidentified as E. chlorostigma, but have different geographical distributions. On the basis of colour pattern and biogeographical distribution, we suggest that sequences previously deposited in GenBank as E. chlorostigma from the west coast of India [START_REF] Lakra | DNA barcoding Indian marine fishes[END_REF] and the Persian Gulf [START_REF] Asgharian | Cytochrome c oxidase subunit 1 barcode data of fish of the Nayband National Park in the Persian Gulf and analysis using meta-data flag several cryptic species[END_REF] correspond to E. polylepis (this species ranges from the west coast of India to the Gulf of Aden [START_REF] Heemstra | Groupers of the world (Family Serranidae, Subfamily Epinephelinae). An annotated and illustrated catalogue of the grouper, rockcod, hind, coral grouper and lyretail species known to date Rome: FAO[END_REF]). We provisionally consider that our specimens correspond to E. chlorostigma (the species is already recorded from New Caledonia [START_REF] Heemstra | Groupers of the world (Family Serranidae, Subfamily Epinephelinae). An annotated and illustrated catalogue of the grouper, rockcod, hind, coral grouper and lyretail species known to date Rome: FAO[END_REF][START_REF] Fricke | Checklist of the fishes of New Caledonia, and their distribution in the Southwest Pacific Ocean (Pisces)[END_REF]) but we urge caution because the type locality of E.

chlorostigma is the Seychelles in the Indian Ocean [START_REF] Heemstra | Groupers of the world (Family Serranidae, Subfamily Epinephelinae). An annotated and illustrated catalogue of the grouper, rockcod, hind, coral grouper and lyretail species known to date Rome: FAO[END_REF]; a molecular reappraisal of the status of E. chlorostigma and its close relatives is certainly needed, but is beyond the scope of this parasitological paper.

Morphology of monogeneans

Morphological examination of monogeneans (Fig. 1) demonstrated that the key structures which allow species differentiation [START_REF] Justine | Parasite biodiversity in a coral reef fish: twelve species of monogeneans on the gills of the grouper Epinephelus maculatus (Perciformes: Serranidae) off New Caledonia, with a description of eight new species of Pseudorhabdosynochus (Monogenea: Diplectanidae)[END_REF][START_REF] Henry | Monogeneans from Epinephelus chlorostigma (Val.) (Perciformes: Serranidae) off New Caledonia, with the description of three new species of diplectanids[END_REF][START_REF] Justine | A redescription of Pseudorhabdosynochus epinepheli (Yamaguti, 1938), the type-species of Pseudorhabdosynochus Yamaguti, 1958 (Monogenea: Diplectanidae), and the description of P. satyui n. sp. from Epinephelus akaara off Japan[END_REF], i.e. the sclerotised male copulatory organ (Fig. 1A,B)

and vagina (Fig. 1C-E), were similar in P. cyanopodus from E. chlorostigma to the corresponding structures found in specimens from E. cyanopodus (see Figures 234in [START_REF] Sigura | Monogeneans of the speckled blue grouper, Epinephelus cyanopodus (Perciformes, Serranidae), from off New Caledonia, with a description of four new species of Pseudorhabdosynochus and one new species of Laticola (Monogenea: Diplectanidae), and evidence of monogenean faunal changes according to the size of fish[END_REF]). The squamodiscs (Fig. 1J)

showed "spurs", a structure not mentioned in the original description of P. cyanopodus [START_REF] Sigura | Monogeneans of the speckled blue grouper, Epinephelus cyanopodus (Perciformes, Serranidae), from off New Caledonia, with a description of four new species of Pseudorhabdosynochus and one new species of Laticola (Monogenea: Diplectanidae), and evidence of monogenean faunal changes according to the size of fish[END_REF], but this is not a reliable systematic character [START_REF] Henry | Monogeneans from Epinephelus chlorostigma (Val.) (Perciformes: Serranidae) off New Caledonia, with the description of three new species of diplectanids[END_REF]. Measurements were similar (see Tables 2,3 in [START_REF] Henry | Monogeneans from Epinephelus chlorostigma (Val.) (Perciformes: Serranidae) off New Caledonia, with the description of three new species of diplectanids[END_REF]).

Analysis of monogenean COI sequences

Seven monogenean sequences were obtained. The difference matrix of monogenean COI sequences (Table 2) showed that variability ranged 0-0.5% for specimens from E. cyanopodus and 0.7-1.5% for specimens from E. chlorostigma. Variability between monogeneans from the two fish species did not exceed 1.5%. We consider that such small variability demonstrates that monogenean specimens from both fish species belong to a single species [START_REF] Smith | DNA barcoding for effective biodiversity assessment of a hyperdiverse arthropod group: the ants of Madagascar[END_REF][START_REF] Ward | DNA barcode divergence among species and genera of birds and fishes[END_REF][START_REF] Poisot | Morphological and molecular evolution are not linked in Lamellodiscus (Plathyhelminthes [sic], Monogenea)[END_REF].

Discussion

Host specificity is one of the basic problems of parasitology. Euzet & Combes [START_REF] Euzet | Les problèmes de l'espèce chez les animaux parasites[END_REF] discussed the problem of species in parasites and remarked that most monogeneans are strictly host-specific ("oioxenous specificity") but rejected the description of new species of parasites on the simple basis of their recovery from a new species of host. Several genera of monopisthocotylean monogeneans include many species: these include the gyrodactylid Gyrodactylus [START_REF] Bakke | Host specificity dynamics: observations on gyrodactylid monogeneans[END_REF], the dactylogyrids Anacanthorus [START_REF] Kritsky | Neotropical Monogenoidea. 17. Anacanthorus Mizelle and Price, 1965 (Dactylogyridae, Anacanthorinae) from characoid fishes of the central Amazon[END_REF] and Dactylogyrus [START_REF] Simkova | Co-existence of nine gill ectoparasites (Dactylogyrus: Monogenea) parasitising the roach (Rutilus rutilus L.): history and present ecology[END_REF], the ancyrocephalids Haliotrema [START_REF] Kritsky | Haliotrema abaddon n. sp. (Monogenoidea: Dactylogyridae) from the gills of wild and maricultured west australian dhufish Glaucosoma hebraicum (Teleostei: Glaucosomatidae) in Australia[END_REF][START_REF] Lim | Haliotrema banana sp. n. (Monogenea: Ancyrocephalidae) from Bodianus perditio (Perciformes: Labridae) off New Caledonia[END_REF], Haliotrematoides [START_REF] Kritsky | Dactylogyrids (Monogenoidea, Polyonchoinea) parasitizing the gills of snappers (Perciformes, Lutjanidae): Proposal of Haliotrematoides n. gen. and descriptions of new and previously described species from marine fishes of the Red Sea, the eastern and Indo-west Pacific Ocean, Gulf of Mexico and Caribbean Sea[END_REF] and Ligophorus [START_REF] Dmitrieva | Redescription of Ligophorus mediterraneus Sarabeev, Balbuena & Euzet, 2005 (Monogenea: Ancyrocephalidae) with some methodological notes[END_REF], and the diplectanids Lamellodiscus [START_REF] Poisot | Morphological and molecular evolution are not linked in Lamellodiscus (Plathyhelminthes [sic], Monogenea)[END_REF][START_REF] Diamanka | Lamellodiscus (Monogenea: Diplectanidae) parasites of Dentex macrophthalmus (Teleostei: Sparidae) from the North Atlantic coast of Africa, with a redescription of L. dentexi Aljoshkina, 1984, and description of three new species[END_REF][START_REF] Briand | Three new species, Lamellodiscus tubulicornis n. sp., L. magnicornis n. sp. and L. parvicornis n. sp. (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] Ogawa | Three species of Lamellodiscus (Monogenea Diplectanidae) from gills of Japanese Black Sea Bream, Acanthopagrus schlegeli (Bleeker)[END_REF] and Pseudorhabdosynochus [START_REF] Beveridge | An annotated list of parasites (Isopoda, Copepoda, Monogenea, Digenea, Cestoda and Nematoda) collected in groupers (Serranidae, Epinephelinae) in New Caledonia emphasizes parasite biodiversity in coral reef fish[END_REF][START_REF] Justine | Species of Pseudorhabdosynochus Yamaguti, 1958 (Monogenea: Diplectanidae) from Epinephelus fasciatus and E. merra (Perciformes: Serranidae) off New Caledonia and other parts of the Indo-Pacific Ocean, with a comparison of measurements of specimens prepared using different methods, and a description of P. caledonicus n. sp[END_REF][START_REF] Justine | Pseudorhabdosynochus argus n. sp. (Monogenea: Diplectanidae) from Cephalopholis argus, P. minutus n. sp. and Diplectanum nanus n. sp. from C. sonnerati and other monogeneans from Cephalopholis spp. (Perciformes: Serranidae) off Australia and New Caledonia[END_REF][START_REF] Justine | Parasite biodiversity in a coral reef fish: twelve species of monogeneans on the gills of the grouper Epinephelus maculatus (Perciformes: Serranidae) off New Caledonia, with a description of eight new species of Pseudorhabdosynochus (Monogenea: Diplectanidae)[END_REF][START_REF] Sigura | Monogeneans of the speckled blue grouper, Epinephelus cyanopodus (Perciformes, Serranidae), from off New Caledonia, with a description of four new species of Pseudorhabdosynochus and one new species of Laticola (Monogenea: Diplectanidae), and evidence of monogenean faunal changes according to the size of fish[END_REF][START_REF] Sigura | Monogeneans of the malabar grouper Epinephelus malabaricus (Perciformes, Serranidae) off New Caledonia, with a description of six new species of Pseudorhabdosynochus (Monogenea: Diplectanidae)[END_REF][START_REF] Hinsinger | The 'Pseudorhabdosynochus cupatus group' (Monogenea: Diplectanidae) on Epinephelus fasciatus, E. howlandi, E. rivulatus and E. merra (Perciformes: Serranidae) off New Caledonia, with descriptions of Pseudorhabdosynochus cyathus n. sp. and P. calathus n. sp[END_REF][START_REF] Henry | Monogeneans from Epinephelus chlorostigma (Val.) (Perciformes: Serranidae) off New Caledonia, with the description of three new species of diplectanids[END_REF][START_REF] Schoelinck | Four species of Pseudorhabdosynochus (Monogenea: Diplectanidae) from the camouflage grouper Epinephelus polyphekadion (Perciformes: Serranidae) off New Caledonia[END_REF][START_REF] Justine | A redescription of Pseudorhabdosynochus epinepheli (Yamaguti, 1938), the type-species of Pseudorhabdosynochus Yamaguti, 1958 (Monogenea: Diplectanidae), and the description of P. satyui n. sp. from Epinephelus akaara off Japan[END_REF][START_REF] Neifar | Five new species of Pseudorhabdosynochus (Monogenea: Diplectanidae) from the gills of Epinephelus costae (Teleostei: Serranidae)[END_REF][START_REF] Mendoza-Franco | Six new and one previously described species of Pseudorhabdosynochus (Monogenoidea, Diplectanidae) infecting the gills of groupers (Perciformes, Serranidae) from the Pacific coasts of Mexico and Panama[END_REF][START_REF] Schoelinck | Pseudorhabdosynochus quadratus n. sp. (Monogenea: Diplectanidae) from the white-streaked grouper Epinephelus ongus (Bloch) (Perciformes: Serranidae) off New Caledonia[END_REF][START_REF] Sigura | Pseudorhabdosynochus bacchus sp. nov. (Monogenea, Diplectanidae) from Epinephelus coeruleopunctatus (Perciformes, Serranidae) off New Caledonia[END_REF]. Strict or non-strict host specificity does not follow a regular pattern within genera; if

we limit examples to diplectanids, species of Calydiscoides [START_REF] Justine | Species of Calydiscoides Young, 1969 (Monogenea: Diplectanidae) from lethrinid fishes, with the redescription of all of the type-specimens and the description of C. euzeti n. sp. from Lethrinus rubrioperculatus and L. xanthochilus off New Caledonia[END_REF][START_REF] Brena | Calydiscoides limae sp. nov. (Monogenea, Diplectanidae) from Pentapodus aureofasciatus (Perciformes, Nemipteridae) off New Caledonia[END_REF][START_REF] Rascalou | Three species of Calydiscoides (Monogenea: Diplectanidae) from five Lethrinus spp. (Lethrinidae: Perciformes) off New Caledonia, with a description of Calydiscoides terpsichore sp. n[END_REF] and Lamellodiscus [START_REF] Poisot | Morphological and molecular evolution are not linked in Lamellodiscus (Plathyhelminthes [sic], Monogenea)[END_REF][START_REF] Diamanka | Lamellodiscus (Monogenea: Diplectanidae) parasites of Dentex macrophthalmus (Teleostei: Sparidae) from the North Atlantic coast of Africa, with a redescription of L. dentexi Aljoshkina, 1984, and description of three new species[END_REF][START_REF] Briand | Three new species, Lamellodiscus tubulicornis n. sp., L. magnicornis n. sp. and L. parvicornis n. sp. (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] Ogawa | Three species of Lamellodiscus (Monogenea Diplectanidae) from gills of Japanese Black Sea Bream, Acanthopagrus schlegeli (Bleeker)[END_REF] show either strict specificity or are found in a small number of host species, but species of Pseudorhabdosynochus have hitherto been considered strictly host-specific, or "specialists" [START_REF] Beveridge | An annotated list of parasites (Isopoda, Copepoda, Monogenea, Digenea, Cestoda and Nematoda) collected in groupers (Serranidae, Epinephelinae) in New Caledonia emphasizes parasite biodiversity in coral reef fish[END_REF][START_REF] Justine | Species of Pseudorhabdosynochus Yamaguti, 1958 (Monogenea: Diplectanidae) from Epinephelus fasciatus and E. merra (Perciformes: Serranidae) off New Caledonia and other parts of the Indo-Pacific Ocean, with a comparison of measurements of specimens prepared using different methods, and a description of P. caledonicus n. sp[END_REF][START_REF] Justine | Pseudorhabdosynochus argus n. sp. (Monogenea: Diplectanidae) from Cephalopholis argus, P. minutus n. sp. and Diplectanum nanus n. sp. from C. sonnerati and other monogeneans from Cephalopholis spp. (Perciformes: Serranidae) off Australia and New Caledonia[END_REF][START_REF] Justine | Parasite biodiversity in a coral reef fish: twelve species of monogeneans on the gills of the grouper Epinephelus maculatus (Perciformes: Serranidae) off New Caledonia, with a description of eight new species of Pseudorhabdosynochus (Monogenea: Diplectanidae)[END_REF][START_REF] Sigura | Monogeneans of the speckled blue grouper, Epinephelus cyanopodus (Perciformes, Serranidae), from off New Caledonia, with a description of four new species of Pseudorhabdosynochus and one new species of Laticola (Monogenea: Diplectanidae), and evidence of monogenean faunal changes according to the size of fish[END_REF][START_REF] Sigura | Monogeneans of the malabar grouper Epinephelus malabaricus (Perciformes, Serranidae) off New Caledonia, with a description of six new species of Pseudorhabdosynochus (Monogenea: Diplectanidae)[END_REF][START_REF] Hinsinger | The 'Pseudorhabdosynochus cupatus group' (Monogenea: Diplectanidae) on Epinephelus fasciatus, E. howlandi, E. rivulatus and E. merra (Perciformes: Serranidae) off New Caledonia, with descriptions of Pseudorhabdosynochus cyathus n. sp. and P. calathus n. sp[END_REF][START_REF] Henry | Monogeneans from Epinephelus chlorostigma (Val.) (Perciformes: Serranidae) off New Caledonia, with the description of three new species of diplectanids[END_REF][START_REF] Schoelinck | Four species of Pseudorhabdosynochus (Monogenea: Diplectanidae) from the camouflage grouper Epinephelus polyphekadion (Perciformes: Serranidae) off New Caledonia[END_REF].

Reports of species of Pseudorhabdosynochus found in two fish species were considered erroneous and attributed to insufficient morphological differentiation [START_REF] Justine | A redescription of Pseudorhabdosynochus epinepheli (Yamaguti, 1938), the type-species of Pseudorhabdosynochus Yamaguti, 1958 (Monogenea: Diplectanidae), and the description of P. satyui n. sp. from Epinephelus akaara off Japan[END_REF][START_REF] Dupoux | Resolution of the discrepant host-specificity of Pseudorhabdosynochus species (Monogenea, Diplectanidae) from serranid fishes in the tropical Indo-Pacific[END_REF] or to the fact that living fish of different species were kept together in a tank, thus allowing accidental host changes [START_REF] Justine | A redescription of Pseudorhabdosynochus epinepheli (Yamaguti, 1938), the type-species of Pseudorhabdosynochus Yamaguti, 1958 (Monogenea: Diplectanidae), and the description of P. satyui n. sp. from Epinephelus akaara off Japan[END_REF]. In wild conditions in New Caledonia, most species of Pseudorhabdosynochus have been found on a single host species only. The only exceptions are very small numbers of P. duitoe and P. huitoe [START_REF] Sigura | Monogeneans of the speckled blue grouper, Epinephelus cyanopodus (Perciformes, Serranidae), from off New Caledonia, with a description of four new species of Pseudorhabdosynochus and one new species of Laticola (Monogenea: Diplectanidae), and evidence of monogenean faunal changes according to the size of fish[END_REF][START_REF] Henry | Monogeneans from Epinephelus chlorostigma (Val.) (Perciformes: Serranidae) off New Caledonia, with the description of three new species of diplectanids[END_REF], and P. podocyanus, and the subject of the present study, P. cyanopodus.

The present study is the first molecular and morphological demonstration of infection of two species of naturally infected hosts, E. cyanopodus and E. chlorostigma, with the same species of Pseudorhabdosynochus. Our morphological [START_REF] Henry | Monogeneans from Epinephelus chlorostigma (Val.) (Perciformes: Serranidae) off New Caledonia, with the description of three new species of diplectanids[END_REF] and molecular (unpublished COI sequences) studies of the other species of Pseudorhabdosynochus found on the same hosts show that they are distinct (no molecular data for P. podocyanus, which is rare).

The question is thus what conditions led to this exception? Our study of COI genes showed that the two fish species are distinct, confirming an earlier phylogeny based on other genes [START_REF] Craig | A molecular phylogeny of the groupers of the subfamily Epinephelinae (Serranidae) with a revised classification of the Epinephelini[END_REF].

Although the depth recorded for both species is variable (4-300 m for E. chlorostigma, 2-150 m for E. cyanopodus [START_REF] Heemstra | Groupers of the world (Family Serranidae, Subfamily Epinephelinae). An annotated and illustrated catalogue of the grouper, rockcod, hind, coral grouper and lyretail species known to date Rome: FAO[END_REF][START_REF] Fricke | Checklist of the fishes of New Caledonia, and their distribution in the Southwest Pacific Ocean (Pisces)[END_REF]), in New Caledonian waters adults of both species are deep-sea fish, usually caught off the barrier reef at depths of 60-200 m. Parasite diversity is known to be much smaller in the deep sea than in surface waters [START_REF] Rohde | Gill Monogenea of deepwater and surface fish in southeastern Australia[END_REF][START_REF] Klimpel | Metazoan deep-sea fish parasites[END_REF][START_REF] Bray | Digenean parasites of deep-sea teleosts: a review and case studies of intrageneric phylogenies[END_REF][START_REF] Boxshall | Host specificity in copepod parasites of deep-sea fishes[END_REF], especially in comparison to such high diversity spots as coral reefs [START_REF] Beveridge | An annotated list of parasites (Isopoda, Copepoda, Monogenea, Digenea, Cestoda and Nematoda) collected in groupers (Serranidae, Epinephelinae) in New Caledonia emphasizes parasite biodiversity in coral reef fish[END_REF][START_REF] Justine | Parasites of coral reef fish: how much do we know? With a bibliography of fish parasites in New Caledonia[END_REF], where most groupers live and from where most Pseudorhabdosynochus species have been described. In Dactylogyrus species from cyprinids in Europe, it has been demonstrated [START_REF] Šimková | Molecular phylogeny of congeneric monogenean parasites (Dactylogyrus): a case of intrahost speciation[END_REF] that certain duplication events gave rise to generalist species (i.e. generalist parasite species are sometimes more derived than specialist species [START_REF] Šimková | Molecular phylogeny of congeneric monogenean parasites (Dactylogyrus): a case of intrahost speciation[END_REF]). If such a process also occurs in Pseudorhabdosynochus, we may hypothesize that the low specificity of P. cyanopodus is a secondary adaptation to deep-sea conditions, where hosts are rare and separated by wide areas, and that infesting two species of hosts helps perpetuating the parasite species. Interestingly, P. epinepheli, another species recorded from E.

chlorostigma [START_REF] Justine | A redescription of Pseudorhabdosynochus epinepheli (Yamaguti, 1938), the type-species of Pseudorhabdosynochus Yamaguti, 1958 (Monogenea: Diplectanidae), and the description of P. satyui n. sp. from Epinephelus akaara off Japan[END_REF] has a single host in New Caledonia but apparently several hosts in other localities [START_REF] Justine | A redescription of Pseudorhabdosynochus epinepheli (Yamaguti, 1938), the type-species of Pseudorhabdosynochus Yamaguti, 1958 (Monogenea: Diplectanidae), and the description of P. satyui n. sp. from Epinephelus akaara off Japan[END_REF][START_REF] Isshiki | Occurrence of a monogenean gill parasite Pseudorhabdosynochus epinepheli on red spotted grouper Epinephelus akaara and its experimental treatment by hydrogen peroxide bathing[END_REF][START_REF] Yamaguti | Studies on the helminth fauna of Japan. Part 53. Trematodes of fishes, XII[END_REF]; however, molecular evidence is unavailable for P. epinepheli. We do not know why this process did not affect the other diplectanids [START_REF] Sigura | Monogeneans of the speckled blue grouper, Epinephelus cyanopodus (Perciformes, Serranidae), from off New Caledonia, with a description of four new species of Pseudorhabdosynochus and one new species of Laticola (Monogenea: Diplectanidae), and evidence of monogenean faunal changes according to the size of fish[END_REF][START_REF] Henry | Monogeneans from Epinephelus chlorostigma (Val.) (Perciformes: Serranidae) off New Caledonia, with the description of three new species of diplectanids[END_REF] from E. cyanopodus and E. chlorostigma, or those from other deep-sea groupers studied in the same locality [START_REF] Justine | Two new species of Pseudorhabdosynochus Yamaguti, 1958 (Monogenea: Diplectanidae) from the deep-sea grouper Epinephelus morrhua (Val.) (Perciformes: Serranidae) off New Caledonia[END_REF][START_REF] Justine | Pseudorhabdosynochus inversus sp. nov. (Monogenea, Diplectanidae) from the halfmoon grouper Epinephelus rivulatus (Perciformes, Serranidae) off New Caledonia[END_REF], all of which appear to be strictly specific species.

Tables

Fig. 1 .

 1 Fig. 1. Pseudorhabdosynochus cyanopodus Sigura & Justine, 2008 from Epinephelus chlorostigma. A, B, quadriloculate male copulatory organ; C-D, sclerotised vagina; F-I, haptoral hard parts (F, lateral bar; G, ventral hamulus; H, dorsal hamulus; I, ventral bar); J, dorsal squamodisc (dorsal view). J, carmine; A, C, D, F-I, picrate; B, D, Berlese.

Table 1 .

 1 Difference matrix of uncorrected pairwise differences between COI sequences of specimens of the two fish species E. cyanopodus and E. chlorostigma (two specimens per species). JNC numbers refer to individual fish identification.

	Fish species and specimen	Sequence	[1]	[2]	[3]
	E. chlorostigma JNC3141 [1]	JQ412501
	E. chlorostigma JNC3142 [2]	JQ412500	0.005
	E. cyanopodus JNC3262 [3]	JQ412503	0.066	0.063
	E. cyanopodus JNC1625 [4]	JQ412502	0.065	0.061	0.002

Table 2 .

 2 Difference matrix of uncorrected pairwise differences between COI sequences of specimens of P. cyanopodus from two fish species. JNC numbers refer to individual fish identification.

	Host fish species and specimen	Monogenean	Sequence	[1]	[2]	[3]	[4]	[5]	[6]
		specimen
	E. cyanopodus JNC3262	[1]	JQ400132	0.000
	E. cyanopodus JNC3262	[2]	JQ400133	0.000
	E. cyanopodus JNC3262	[3]	JQ400134	0.000	0.000
	E. cyanopodus JNC3144	[4]	JQ400137	0.005	0.005	0.005
	E. chlorostigma JNC3141	[5]	JQ400135	0.007	0.007	0.007	0.002
	E. chlorostigma JNC3141	[6]	JQ400136	0.015	0.015	0.015	0.010	0.012
	E. chlorostigma JNC3142	[7]	JQ400138	0.009	0.010	0.009	0.005	0.007	0.015

Acknowledgements

The identification of the hosts was kindly confirmed (from photographs) by John E. Randall (Bishop Museum, Hawaii). Aude Sigura and Émilie Henry, students, participated in the parasitological survey. Claude Chauvet (UNC, New Caledonia) kindly provided fish. David Gibson (NHM, London) provided advice for the title. Rod Bray (NHM, London) kindly edited the English. Funded by ATM barcode (MNHN) and ATM Biodiversité actuelle et fossile (MNHN).