Perotrochus caledonicus (Gastropoda: Pleurotomariidae) revisited: descriptions of new species from the South-West Pacific

. Morphological (shell) and molecular examination of a large suite of specimens of pleurotomariids from around new caledonia and the coral sea reveals the existence of four species in the complex of Perotrochus caledonicus : Perotrochus deforgesi Métivier, 1990 and P. pseudogranulosus sp. nov. live allopatrically on the plateaus and guyots of the coral sea; Perotrochus caledonicus Bouchet & Métivier, 1982 and Perotrochus wareni sp. nov. live sympatrically - but essentially not syntopically - on the slopes of new caledonia, norfolk ridge and the loyalty ridge. All species live in the 300–500 m interval, and together form a significant component of the mollusc fauna living on hard bottoms in the SW Pacific, with individual dredge hauls containing up to 25 specimens of Perotrochus .


Introduction
Perotrochus caledonicus Métivier, 1982 andP. tangaroanus Bouchet &Métivier, 1982 were the first pleurotomariids described from the South Pacific, from New Caledonia and from the Lau Ridge between new Zealand and Fiji (Bouchet & Métivier 1982), respectively. since then, additional species have been documented from new caledonia (Perotrochus deforgesi Métivier, 1990 and Bayerotrochus
the type material of P. caledonicus from off southern new caledonia consists of specimens with a macroscopically non-pustulose shell, with rather smooth to weakly beaded spiral cords. soon after its description, it appeared that a more granulose or pustulose morph also occurred around new Caledonia. However, as such material appeared very similar in terms of size, color and general profile, it was considered to represent just a form of P. caledonicus. the two forms were reported in print (Anseeuw 1990;Anseeuw & goto 1996) and referred to as the "smooth form" and "pustulose form" of P. caledonicus, respectively. since then, dealers and shell collectors have maintained this distinction, using the expression "caledonicus pustulose form" or "caledonicus granulose form", with some even suggesting that the latter could warrant full recognition as a species (e.g., www.conchology.be).
new material from new caledonia and the coral sea has now allowed revisiting this issue, using an integrative taxonomic approach combining dnA sequencing and shell characters of both juveniles and adults. dnA sequences provide independent characters to test whether the different forms of P. caledonicus correspond to divergent lineages or not. the congruence of the different sets of characters analyzed revealed that three species were hidden under the name P. caledonicus, two of which are now described as new. in addition, Perotrochus deforgesi was found to be molecularly closely related to this complex -which was not suspected from shell characters alone -and in this paper we will designate these small Perotrochus from the SW Pacific together as the "Perotrochus caledonicus complex".  Métivier, 1990. C. Perotrochus caledonicus Bouchet & Métivier, 1982. D. Perotrochus pseudogranulosus sp. nov. stations with sequenced specimens in black.

Material
A total of 284 lots and 879 specimens, including 59 sequenced specimens, attributable to the Perotrochus caledonicus complex were collected in the new caledonia region, including the coral sea (table 1, Fig. 1) during research cruises of the tropical deep-sea Benthos program (Bouchet, héros, lozouet & Maestrati 2008). After 2002, live-taken specimens were specifically processed on board for molecular analyses. A piece of the foot was cut and placed in 95° ethanol; the remaining body and the shells were also preserved for further examination. All specimens are preserved at the Muséum national d'histoire naturelle (Mnhn); each sequenced specimen is linked to a unique Mnhn collection number. these specimens are registered in Bold, the Barcode of life database, and the corresponding sequences are also registered in genBank (table 1, Fig 1).

DNA sequencing
total dnA was extracted from the piece of foot using the 6100 nucleic Acid Prepstation system (Applied Biosystem) or the epmotion 5075 robot (eppendorf) following the manufacturer's recommendations.
The barcode fragment of the COI gene (658 bp) was tentatively amplified using the universal primers lco1490 and hco2198 (Folmer, Black, hoeh, lutz & Vrijenhoek 1994). however, the rate of success was very low (less than 10%), and several other protocols and primer pairs were tested. Finally, we were able to amplify most of the specimens, including several for each morphological species hypothesis, using the primers Fishr2 (steinke & hanner 2011) and 140F (Ketmaier, giusti & caccone 2006), and the following protocol: Pcr reactions were performed in 20 µl containing 3 ng of dnA, 10× reaction buffer containing 15 mM Mgcl 2 , 0.26 mM dntP, 0.3 µM of each primer, 5% dMso, 1 mg/ml BsA, and 1 unit of QBiotaq (MPBiomedicals). Amplification consisted of an initial denaturation step at 95°C for 5', followed by 38 cycles of denaturation at 95°c for 40", annealing at 50°c for 40", followed by extension at 72°C for 50'. The final extension was at 72°C for 3'. PCR products were purified and sequenced by the Eurofins sequencing facility. Both directions were sequenced to confirm accuracy of each sequence.

Phylogenetic analyses
in addition to the specimens of Perotrochus sequenced by us, coi sequences of Pleurotomariidae from genBank were included in the dataset, as well as a sequence of Haliotis tuberculata (haliotidae), used as outgroup (table 1). Phylogenetic analyses were performed using MrBayes (huelsenbeck, ronquist & Hall 2001), running two parallel analyses, consisting each of five Markov chains of 20,000,000 generations with a sampling frequency of one tree each 2,500 generations. the number of swaps was set to 3, and the chain temperature at 0.02. Parameters of the substitution model were estimated during the analysis (6 substitution categories, a gamma-distributed rate variation across sites approximated in four discrete categories and a proportion of invariable sites

Morphological analyses
with the exception of some juveniles, we could separate all the specimens of the Perotrochus caledonicus complex into four different morphotypes (table 2): one group presents shell characters consistent with the name-bearing holotype of P. caledonicus as described by Bouchet & Métivier (1982); the second group corresponds to the "rugose" or "pustulose" form, and is described below as P. wareni sp. nov.; a third group, with a geographically limited distribution within the coral sea, shows a microgranular ANSEEUW P. et al., New species of Pleurotomariidae sculpture and is described as P. pseudogranulosus sp. nov.; the fourth group is readily assignable to P. deforgesi.

Phylogenetic analyses
the phylogenetic analyses show that the P. caledonicus group is a well-supported clade (Posterior Probability PP = 1) within the Pleurotomariidae (Fig. 2). Although based on a single gene, the tree would also suggest that the genus Perotrochus is not monophyletic. Furthermore, some genBank sequences appear to be either misidentified or contaminated; for example, the sequence L78912.1, identified as Bayerotrochus teramachii (Kuroda, 1955), is almost identical to three sequences identified as B. midas (Bayer, 1965). within P. caledonicus s.l., the analysis of the coi gene diversity revealed the presence of four groups, each corresponding to a well supported clade in the phylogenetic tree (PP = 1 for each of them), and totally congruent with the morphological analysis. P. deforgesi is the sister-clade of a group that includes the specimens attributed to P. caledonicus and P. wareni sp. nov. the mean K2P genetic distance between P. wareni sp. nov. and P. caledonicus is 3.8%, which is similar to the distance European Journal of Taxonomy 134: 1-23 (2015) between P. deforgesi and P. caledonicus (3.7%) and greater than the distance between P. deforgesi and P. wareni sp. nov. (2.1%). This would tend to confirm that if P. deforgesi is considered a valid species -which is morphologically indisputable -then the two morphs (P. caledonicus s.s. and P. wareni sp. nov.), included until now in P caledonicus, should be ranked as species as well. More surprisingly, P. pseudogranulosus sp. nov. is the sister-group to a clade that includes the other three species, with a mean genetic distance of 9.9% to the other three species, confirming its validity as a separate species.

Etymology
this new species is named in honor of dr Anders warén of naturhistoriska riksmuseet, stockholm, in recognition of his lifetime interest in deep-sea exploration and his participation in expeditions around New Caledonia and elsewhere in the South Pacific, many of which yielded specimens used in this paper.

Description (holotype)
Shell of medium size, solid, thick, general profile rather conical, with weakly convex, rather straightsided whorls, with a diameter a little smaller than its height (h/d ratio = 1.08), numbering 10.5 teleoconch whorls, with a mean spire angle of 65° with weakly impressed suture, whorl surface dull. Protoconch glassy, rather obtusely depressed. teleoconch with heavily beaded spiral cords very early on. dominant ANSEEUW P. et al., New species of Pleurotomariidae teleoconch sculpture consisting of finely but strongly beaded spiral cords, intersecting less marked axial riblets. Periphery of basal disc crenulated due to a strongly marked spiral cord running at edge of disc. on last whorl, 13 spiral cords above selenizone, 4 spiral cords below, and 2 major cords in the selenizone itself. slit short, about 1/ 7 th the circumference of last whorl, situated below midwhorl, and very narrow. Aperture rectangularly depressed. Basal disc rather flat, depressed in its center and sharply edged at its periphery, with a very wide (extending over 40% of base diameter) callus pad which is finely ridged radially. inside aperture, inner slit lips only partially covered by nacre, leaving a V-shaped area uncovered (approximately 25% of surface of inner slit lip extremity in aperture uncovered by nacre, showing only porcellaneous layer). nacre coverage thick, no surface sculpture showing through it. Background color yellowish beige, with some faint axial orange-red flammulations, not really arranged into a distinct checker-board pattern; basal disc of same colour, with some faint orange axial flammulation reaching only to basal disc edge. operculum small, multispiral, circular, light yellowish.

Measurements
Maximum basal diameter (d) 46.75 mm, minimum diameter 43.9 mm. height (h) 50.27 mm. h/d = 1.08. length of slit at upper margin 26.7 mm, at lower margin 16.2 mm. slit width: 1.6 mm; slit length: 1/ 6.51 th of circumference of last whorl. weight of empty shell 26.3 g.

Discussion
one of the distinctive shell characters separating Perotrochus wareni sp. nov. from P. caledonicus is the well-marked beading on the teleoconch spiral cords, visible also on the earlier whorls, giving it at first glance its typical pustulose or granular appearance. The intensity of the beading varies between specimens, leading to "heavily beaded"/"very pustulose" specimens and to "weakly beaded" / "light pustulose" specimens, with all different intergrades. This variability may reflect environmental conditions, as the beading intensity is generally stable within one haul / lot and varies between hauls / lots. leaving aside beading intensity, shell characters are quite stable in P. wareni sp. nov. the general profile of the teleoconch, the outline of the aperture, the selenizone and slit width, and the extension of the callus on the basal disc, all show consistent differences between P. wareni sp. nov., P. caledonicus and P. pseudogranulosus sp. nov. (table 2). Perotrochus wareni sp. nov. also bears some resemblance to P. gotoi Anseeuw, 1990, and, in fact, a specimen of the granulated P. "cfr. caledonicus" ([i.e., P. wareni sp. nov.) had been used for comparison at the time of its original description (Anseeuw 1990). Perotrochus wareni sp. nov. can be separated from P. gotoi by its somewhat shorter slit length (1/6.51 th of basal diameter in wareni sp. nov. vs 1/ 6 th in gotoi), the larger number of spiral cords on adult specimens, the more irregularly banded checkerboard colour pattern in the area below the selenizone, the heavier and thicker shell (around 60-70% heavier at comparable shell sizes), the less extensive area uncovered by the nacreous layer inside the apertural inner slit lip extremities, and the umbilical callus occupying a much larger surface on the basal disc (45% in P. wareni sp. nov. vs 28% in P. gotoi). the two species, however, share (also with Mikadotrochus salmianus (rolle, 1899) the nacreous coverage of the inner slit lips in the aperture, a feature that separates them from Perotrochus caledonicus s.s. and P. pseudogranulosus sp. nov. (table 2, Figs 3-4, 7). other features, like a deeper, more intense colour pattern on the teleoconch and basal disc, fine microgranulosity on the spiral cords, a thin, light shell, and a more lustrous shell surface, further separate Perotrochus pseudogranulosus sp. nov. from P. wareni sp. nov. (table 2, Figs 3-6).

Etymology
The specific epithet emphasizes the beaded spiral sculpture of the species.

Description (holotype)
Shell of medium size, light, thin, general profile rather conical, with weakly convex to straight-sided whorls with weakly impressed suture, diameter significantly exceeding height (H/D = 0.78), numbering 8 teleoconch whorls, with a mean spire angle of 80°. Protoconch ivory white, depressed. dominant sculpture of teleoconch consisting of numerous lightly beaded spiral cords, with microsculptural pattern of fine radiating threads, giving the entire whorl surface a shiny metallic luster. On last whorl, 11 spiral cords above selenizone, 7 below and 3 major cords in the selenizone itself. slit long, about 1/ 5 th the circumference of the last whorl, situated below midwhorl, and rather narrow. Aperture depressed. Basal disc rather flattened, with angular edge, with a relatively narrow (extending over 30% of base diameter) light nacreous callus pad which is finely ridged radially and ends in a raised porcellaneous edge. Inside the aperture inner slit lips nearly completely covered by nacre, leaving a narrow area (approximately 15% of the surface) parallel to the inner slit lips uncovered, showing only porcellaneous layer. Background colour yellowish beige, with intense reddish crimson colour markings arranged in very regular checkerboard pattern, overall reinforcing color intensity; basal disc showing some contrasting reddish crimson flammulations, particularly visible at its periphery, and more yellowish tan towards the center. Operculum small, multispiral, circular, light yellowish (fallen off/missing in holotype).

Measurements
Maximum basal diameter (d) 64.9 mm, minimum diameter 59.1 mm. height (h) 50.7 mm. h/d = 0.78. depth of slit at upper margin 42.1 mm, depth of slit at lower margin 26.9 mm. slit width 3.1 mm. slit length: 1/ 5.64 th of circumference of last whorl. weight of empty shell 42.6 g.

Discussion
Perotrochus pseudogranulosus sp. nov. most closely resembles P. caledonicus (Fig. 7) at first glance, but is distinguished by its more conical and higher shell, a more flattened basal disc profile and a more intense and regular checkerboard colour pattern and more lustrous shell surface. it differs from P. wareni sp. nov. by its weakly beaded spiral cords with a microsculpture of fine radiating threads, its more intensely ANSEEUW P. et al., New species of Pleurotomariidae marked checkerboard colour pattern, with metallic luster, a much longer slit and a much smaller callus pad area on the basal disc. Finally, it differs from P. deforgesi (Fig. 7), the only other species occurring in the coral sea, by its general outline which is distinctly higher conical, its thin shell, its more intense checkerboard colour markings, its less granular spiral cords and smaller callus pad coverage on the basal disc.
some specimens of P. pseudogranulosus sp. nov. have over the years turned up in the shell trade as "P. cfr. caledonicus", supposedly originating from nw Australia or even from the south china sea (Anseeuw & goto 1996). however, based on the lack of precise and trustworthy locality data, the lack of more recent confirmation of those alleged findings and, most of all, the general unavailability of such material for study, we reject these localities as intentionally or unintentionally unproven and unverifiable.

Discussion
the geographical distribution within the new caledonia region (Fig. 1) is different for the four species. Many samples of Perotrochus caledonicus s.s. originate from the sw off ile des Pins, southern new caledonia, with scattered specimens from norfolk ridge, the loyalty ridge and the Far north of new caledonia (grand Passage); the average depth of occurrence, based on 135 lots, is 407 meters. the distribution of Perotrochus wareni sp. nov. includes norfolk ridge, the loyalty ridge, the Far north of new caledonia (grand Passage) and the lansdowne Plateau in the coral sea; the average depth of occurrence, based on 73 lots, is 344 meters. Perotrochus caledonicus and P. wareni sp. nov. thus co-occur locally in the south of new caledonia, norfolk ridge and grand Passage, where they have different bathymetric preferences, although they were sometimes found at the same stations (lithist sta. cP14 and sta. cP16; norFolK1 sta. dw1658 and sta. dw1709; MusorstoM4 sta. dw222; terrAsses sta. dw3101 and sta. dw3110). Perotrochus pseudogranulosus sp. nov. has a distribution restricted to the Chesterfield Plateau, with a mean depth of 360 meters based on 40 lots. Perotrochus deforgesi is also restricted to the coral sea, but occurs only on the Bellona Plateau and capel Bank, thus allopatrically with regard to P. pseudogranulosus sp. nov., but at similar depths (mean depth of 380 meters based on 36 lots). the morphological, molecular and microdistribution data, thus all converge to support the conclusion that there are four small Perotrochus in the new caledonia region, of which two have been described as new in the present paper.
At genus level, although Anseeuw & Poppe (2005) Askew, 1993, P. lucaya Bayer, 1965) that cluster with it in the tree are also from the western Atlantic. it thus appears that, although they are traditionally placed in Perotrochus (e.g., Anseeuw & goto 1996;Harasewych 2002), the small pleurotomariids from the Pacific are not congeneric with true Perotrochus from the western Atlantic, and a new genus will have to be established to classify them. overview of the programme and the acknowledgements to the captains, principal scientists and crew involved are applicable here. we thank Yoshihiro goto and guido Poppe, with whom we discussed the taxonomic hypotheses presented in this paper. Manuel tenorio and Javier conde attempted a morphometric analysis (results not shown). Barbara Buge, Virginie héros, Julien Brisset and Philippe Maestrati curated the material, Philippe Maestrati and Manuel caballer-gutiérrez provided the photos. this work was supported by the service de systématique Moléculaire (uMs 2700 cnrs-Mnhn), and we acknowledge the advice of M.G. Harasewych on DNA amplification in pleurotomariids.