Review of the Nassarius pauperus (Gould, 1850) complex (Nassariidae): Part 3, reinstatement of the genus Reticunassa , with the description of six new species

. In this review (third part), several species within the Nassarius pauperus complex from the eastern Indian Ocean and western Paci ﬁ c are treated, including a revised concept of Nassa paupera Gould, 1850, type species of the genus Reticunassa Iredale, 1936. In the most recent taxonomic revision, several species had been synonymized with Nassarius pauperus (Gould, 1850), despite distinctive differences among these species in shell morphology. We sequenced a fragment of the mitochondrial COI and the nuclear 28S genes of all available Nassarius pauperus complex species. Automatic barcode gap discovery and reciprocal monophyly were applied to propose species delimitation hypotheses and to support a new concept of the genus. Using morphological and molecular characters, Reticunassa is elevated to full genus rank. Six new species are described: Reticunassa visayaensis sp. nov., R. poppeorum sp. nov., R. annabolteae sp. nov., R. goliath sp. nov., R. intrudens sp. nov. and R. thailandensis sp. nov., while R. tringa (Souverbie, 1864) is recognized as a valid species.


Material and methods
Material was obtained from various natural history museums and private collections, listed in the acronyms section.Live-collected specimens used for molecular studies were anesthetized with MgCl 2 or microwaved (see Galindo et al. 2014).The bodies, preserved in 95% ethanol, were removed from the shells in the laboratory to prevent progressive deterioration of the shells by etching.The shells are kept dry and have been assigned the same registration number as the corresponding body or tissue in ethanol.
A stereo microscope was used to observe shell morphology and color patterns.A digital caliper (accurate to 0.1 mm) was used for measurements.Shells of the new species were photographed in dorsal, ventral and lateral views, using a Canon EOS 60D camera.Scanning electron microscope (SEM) images were obtained by backscattered electron diffraction on a VEGA II LSU TESCAN, under conventional high vacuum at 20 kV acceleration voltage (12 to 15 Pa chamber pressure) at the SEM-EDS of the 'Direction des Collections' at MNHN.In general, the terms and methods used for the morphological analysis follow part 1 of the review (Kool & Dekker 2006).Procedures from the Consortium for the Barcode of Life barcoding protocols (www.barcodeoflife.org)adapted to large biodiversity collections were followed (see Puillandre et al. 2012a).A piece of muscle from the foot, sufficient for approximately five DNA extractions, was stored in a 2D barcode tube as a reference tissue collection.Extraction was done with the NucleoSpin® 96 Tissue Core Kit adapted to a NucleoSpin® 96 Plasmid Binding Plate (Macherey-Nagel) using a liquid handling workstation (epMotion® 5075 VAC, Eppendorf).Purified DNA was stored in a second 2D tube.PCR reactions were performed in 20 μl, containing 2 μl of DNA, 1× reaction buffer, 2.5 mM MgCl 2 , 0.26 mM dNTP, 0.3 μM of each primer, 1 μl DMSO, 1 μl BSA (10 mg/l) and 1.5 units of Q-Bio Taq DNA polymerase (QBiogene, Carlsbad, California, USA).A fragment of 658 bp of the cytochrome oxidase I (COI) mitochondrial gene was amplified, using the universal primer set (LCO1490 and HCO2198) (Folmer et al. 1994) and a primer set specific for Nassariidae (5COIF: 5'ACAAATCATAAAGAYATTGGAAC-3', 492COIR: 5'CGCTCAAATTGTATTCCTCG-3' and 492COIRD: 5'CGYTCRAATTGTATYCCTCG-3') (Galindo et al. 2016).Annealing temperature was 50°C for 35 seconds.The first subunit of the 28S gene was amplified using the C1' and D2 primers of Chisholm et al. (2001).Annealing temperature was 58ºC for 40 seconds or a touchdown gradient from 66ºC to 56ºC for 8 seconds (plus 58ºC for 30 seconds) whenever double bands were present.Sequencing was carried out by the Centre National de Séquençage, Genoscope (Évry, France) and Eurofins Genomics (Paris, France).In all cases, both directions were sequenced to confirm the accuracy of each haplotype.

Acronyms
Sequence cleaning and alignment was done by using Codon Code Aligner software.GenBank accession numbers for all the sequences obtained are included in Table 1.We compiled a dataset of sequences available for several closely related species, identified in a phylogenetic tree, based on a large dataset of Nassariidae (Galindo et al. 2016) to compare with our new species hypotheses.The analysis involved 89 COI sequences: 72 of them belong to the Nassarius pauperus complex, 16 sequences to other species of Nassariidae and one to a distant outgroup.The Automatic Barcode Gap Discovery (ABGD) method using the default parameters (Puillandre et al. 2012b) was employed.It automatically detects, when present, a threshold (the barcode gap) in the pairwise distribution of the genetic distances calculated between each pair of specimens.
To test whether the species hypotheses proposed by ABGD corresponded to monophyletic groups, Bayesian analyses, consisting of two Markov chains (35 million generations each with a sampling Table 1

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frequency of one tree for every hundred generations) run in eight parallel analyses, was performed using MrBayes v. 3.2.6 (Ronquist & Huelsenbeck 2003) run on XSEDE.When the log-likelihood scores were found to stabilize, a consensus tree was calculated after omitting the first 25% trees as burn-in.The analyses were performed using the CIPRES Science Gateway (Miller et al. 2010).Kimura 2-parameter (K2P) distances, estimated with MEGA v. 6 (Tamura et al. 2011), were used to compare each new species with its relatives as defined above.
For type specimen selection, we attributed more importance to the sequenced specimens (genophores sensu Pleijel et al. 2008) than to shell features.All the material (type series and sequences) belongs to MNHN unless otherwise mentioned.Detailed information on the tropical deep sea surveys run by MNHN, from which we obtained most of the new species described herein, can be found in Bouchet et al. (2008) and the BasExp database at http://expeditions.mnhn.fr/.

Results
A comprehensive dataset on the Nassarius pauperus complex was gathered.For COI, 72 partial sequences were unambiguously aligned without gaps; 244 sites were variable positions, of which 204 were parsimony informative.For 28S, 51 sequences of the first subunit were successfully amplified.The 28S tree supported all the species hypotheses evidenced by COI.
The resulting trees, constructed after the analyses of COI and 28S markers separately (Fig. 1A-B), are in general characterized by strong support values (posterior probability, PP) of deep and terminal nodes.The Nassarius pauperus complex was recovered as a monophyletic group (PP = 1).Branch lengths (sequence divergences), similar to those found for other major clades within Nassariinae (i.e., Phrontis Adams & Adams, 1853, Nassarius, Tritia Risso, 1826and Naytia Adams & Adams, 1853), support our taxonomic decision to consider this clade as a separate genus.Reticunassa is the only available genus group name for this clade, because Nassarius pauperus, its type species, was included in it (see genus remarks for more details).The African clade Naytia is confirmed as the sister group (PP = 0.96, 0.73) of the Indo-Pacific Reticunassa.
The differences between the intra-and the interspecific genetic distances are shown in Fig. 1C.Among species of Reticunassa, the intraspecific genetic distances range between 0.000 and 0.029.However, no clear barcode gap (sensu Puillandre et al. 2012b) was found within this Reticunassa data set.Fig. 1C shows two main low frequency regions on the pairwise distance frequencies, one around 2% and a second near 5%.
ABGD was able to recover 13 groups (Fig. 1): seven correspond to species recognized as valid: G1 R. compacta, G2 R. rotunda, G3 R. hanraveni, G4 R. silvardi, G7 R. paupera, G8 R. simoni and G9 R. crenulicostata.G11 corresponds to a separate lineage represented by a single sequence for a specimen morphologically close to the original description of Nassa tringa Souverbie in Souverbie & Montrouzier, 1864, a species considered a synonym of Nassarius pauperus by Cernohorsky (1984).Four lineages (G5, G6, G10 and G13) potentially correspond to new species.Two of these, G10 and G13, could be distinguished by shell features.However, it was not possible to find differences in shell morphology for the other two species (G5 and G6) and formal descriptions of these await the discovery of informative characters.
The ABGD approach grouped specimens from both Mozambique (originally identified as R. neoproducta), the Philippines and Papua New Guinea into G12.Those samples formed two separate (and sister) clades, well supported by reciprocal monophyly in the COI (PP = 1) and the 28S (PP = 0.91) trees, and in accordance with the geographical distribution.
In this paper the genus Reticunassa is redefined, together with its type species.Additionally, six species are described as new to science.

Diagnosis
Morphological features of the protoconch are of great importance when recognizing species of Reticunassa.Several species have a multispiral protoconch, indicating a planktotrophic / planktonic larval development, resulting in a larger range of distribution.Others have a paucispiral protoconch, indicating a short, non-planktotrophic / direct larval development.All species of Reticunassa possess an unkeeled protoconch (Fig. 2A-B), as do other Nassariinae genera in the Atlantic Ocean (Galindo et al. 2016).However, Reticunassa lives in sympatry with the Indo-Pacific genus Nassarius s. str., characterized by a keeled protoconch.Protoconchs of Reticunassa can be rather broad and flattened; a few are pointed.They might possess microscopic sculpture, such as spiral ridges, axial striae and minute pustules (Fig. 2A-B), but frequently a much higher resolution is needed to see details of this sculpture.
Due to the obvious importance of protoconch characters, the identification of specimens in this genus lacking a protoconch becomes more difficult.
Species of Reticunassa have strongly or weakly developed continuous spiral cords, from 10 to 15 on the body whorl (Fig. 2D-F), crossing over the axial ribs.The space between these spiral cords, the intercordal surface, can be smooth, axially striated or can display a number of (occasionally extremely) fine spiral striae (Fig. 2F).This makes the sculpture of the intercordal surface important for identification purposes within Reticunassa.Fig. 1A-B shows that differences on these intercordal surfaces are useful synapomorphies to separate SC1 from SC2.
Species within Reticunassa have an axially-ribbed teleoconch (Fig. 2C).These axial ribs may become weaker or absent on the body whorl towards the base and/or the outer lip.Ribs can be round or flat.The number of ribs differs among species.Most species develop a strong varix near the outer lip.The suture is usually deeply impressed.The aperture is oval to sub-circular, the inside of the outer lip with a number of denticles or lirae (Fig. 2G-H).A columellar callus is present and varies in shape and sculpture.It can be limited to the columella or partly extend over the body whorl.
In some species, the color is variable; generally, it is cream-yellowish to orange, or white to dark brown.Banding with darker colors may also be present.Sometimes only some of the spiral cords are darker than the remaining part of the shell.
Reticunassa species are small, usually varying between 5 and 12 mm, but one is up to 16.5 mm (R. goliath sp.nov.).Most species live in littoral to sublittoral ecosystems, only empty shells having been found in waters deeper than 260 m.Their occurrence is in the Indo-Pacific Ocean, from the Miocene to the present.

Remarks
The genus name Reticunassa was introduced to group together "species around pauperus Gould" by Iredale (1936: 322).Cernohorsky (1984: 175) regarded Reticunassa as a synonym of the subgenus "Hima Leach in Gray, 1852", within the genus Nassarius Duméril, 1805.However, Hima is not an available name, but an incorrect subsequent spelling of Hinia Leach in Gray, 1847 (Gray 1847a: 269), since "Hima" included the same three species as in the original description of Hinia (during a period in which original descriptions only consisted of introducing new names).Nonetheless, "Hima" and not Hinia was listed by Adams & Adams (1853).Cossmann (1901) considered both Hinia and "Hima" as valid names, with "Hima" treated sensu Adams & Adams (1853), not Leach in Gray, 1852, and with "type species" Nassa incrassata "Müller".Woodring (1928) noted the misspelling and this was reiterated by Lee (2013).Despite this, the error in the use of "Hima" has persisted.On the other hand, Hinia Leach in Gray, 1847 is a junior objective synonym of Tritia Risso, 1826 as they have the same type species, Buccinum reticulatum Linnaeus, 1758, designated for both genera by Gray (1847b: 139).Molecular results of Galindo et al. (2016) indicated that species considered part of the Nassarius pauperus complex, including "Nassarius pauper", form a clade distant from Buccinum reticulatum (accepted as Tritia reticulata), the type species of Tritia (= Hinia), also shown in Fig. 1A.The only available genus group name for this pauperus clade is Reticunassa Iredale, 1936, which is herein reinstated with full generic status.

Neotype
Both the lectotype and the paralectotype have been affected by Byne's disease (Kool & Dekker 2006).However, remnants of microscopic spiral sculpture on the intercordal surface of both specimens are vaguely visible.Gould (1850) did not mention this feature in his original description.The protoconch of the lectotype is badly damaged and that of the paralectotype is completely absent.The protoconch, figured by Cernohorsky (1984: 5, fig. 12) cannot be the protoconch of either of the types, but most likely belongs to Reticunassa tringa, which he considered a synonym of Nassarius pauperus.The poor condition of both existing types calls for the designation of a neotype for this species.Among the specimens in the MNHN collection, there is one sequenced individual that is very similar to the original types of the species.A proposal to designate a neotype is in the process of being submitted to the International Commission on Zoological Nomenclature (ICZN).If accepted, the neotype will be MNHN IM-2007-31782, length 6.9 mm, width 3.6 mm (Fig. 3A-C   oPerculum.Whitish, serrated.

Remarks
Several species, previously synonymized with R. paupera by Cernohorsky (1984), have been taken out of synonymy in the past few years.A series of published reviews (Kool & Dekker 2006, 2007), including the present article, demonstrate that the Nassarius pauperus complex is composed of several valid species.Therefore, the synonymy given for Reticunassa paupera s. str.has become very short and caution is advised with citations of this species name in the literature.
The combination of the multispiral protoconch, the many pronounced, round ribs, the deep suture, the broad varix, the very fine spirals, the sharply-bordered columella and the columellar denticles not lirae characterize this species, one of the smaller within the genus.The differences between R. paupera and the other species of Reticunassa, described below, will be discussed in the remarks section in each case.
In the taxonomic history of Nassa paupera, several declensions on the specific epithet have been used.
In Latin, there are two different adjectives with the same meaning: the classical pauper (3 rd declension, nominative unchanged) and the post-classical (medieval, generally clerical) pauperus (1 st declension, paupera feminine, pauperum neuter), both meaning "poor."The second adjective is obviously a derivation of the first.The fact that Gould (1850) used paupera in his original description indicates that he was referring to the second meaning.Combined with Reticunassa, the new combination is R. paupera (Gould, 1850).

Habitat
Specimens were collected from the intertidal to a depth of 82 m, mostly from 0 to 25 m; one empty shell was obtained at 260 m (Fiji Islands).The proposed neotype was collected on sand with dead corals.Specimens from Guam (HD 12797) were found in rubble.

Distribution
Widely distributed in the western Pacific Ocean, from Indonesia and the Philippines to the Tonga Islands (Fig. 4).

Description
Protoconch.Whitish, paucispiral, consisting of 1.5-1.75whorls (Fig. 3I).SEM photograph shows very fine growth lines, and in addition minute pustules on part of last whorl.
SiPhonal canal.Narrow, with strong fasciole.Siphonal area with weak cords and in some specimens faint axial striae between cords.
color.Off white, yellowish to orange or brown.Banding very variable: some specimens with chestnut to dark brown, or nearly black bands; on penultimate whorl a sub-and/or a supra-sutural band; on body whorl occasionally one band directly below periphery and one at base.Occasionally, some or most spiral cords darker, especially between axial ribs.Aperture yellowish, showing outside banding, if present.

Remarks
Characteristics of this species are a paucispiral protoconch, low spiral cords, in combination with numerous, evenly-spaced intercordal spiral striae (Fig. 2E), axial ribs decreasing in height anteriorly on the ventral side of the body whorl, and the presence of lirae on the columellar callus.In wellpreserved specimens, spiral striae are also visible on top of the spiral cords.The intercordal spiral striae were not mentioned in the original description by Souverbie (in Souverbie & Montrouzier, 1864).However, Preston (1907) mentioned this sculpture in the description of Nassa mamillata.We agree with Cernohorsky (1984), that the latter is a synonym of Reticunassa tringa.
R. tringa can easily be distinguished from R. paupera by its paucispiral protoconch, probably figured by Cernohorsky (1984: 5, fig. 12).Cernohorsky did not discuss protoconch morphology or other conchological features shared by species within the Nassarius pauperus complex, resulting in numerous synonyms of R. paupera, followed by many authors.
R. tringa closely resembles R. visayaensis sp.nov.and R. poppeorum sp.nov.For the differences between R. tringa and these species, see respective Remarks sections.

Habitat
Intertidal to 72 m, more frequently within the first 10 m of depth.
color.Yellowish, most spiral cords reddish between ribs.

Remarks
The intraspecific variability is considerable.The number of ribs and spiral cords may vary, and the color is extremely variable, white to yellow and orange to brown, unicolor or with narrow or broad yellow, brown, dark brown, or grey bands on all whorls or only on the body whorl.
Reticunassa visayaensis sp.nov.has a broader, less pointed protoconch than R. paupera (Gould, 1850).R. visayaensis sp.nov.also differs from R. paupera by its larger size, its more bulbous shape and its lower ribs, especially on the body whorl, and by usually displaying dark bands.R. visayaensis sp.nov. is very similar to R. tringa.When the protoconch is missing, a positive identification is almost impossible.The protoconch of R. tringa is paucispiral with 1.5-1.75whorls, whereas R. visayaensis sp.nov.has a protoconch of 2.25-2.5 whorls.The protoconch of R. tringa is nipple-shaped, hence the name "mamillata" (Preston 1907); the protoconch of R. visayaensis sp.nov. is dome-shaped.Preston's description is accompanied by a drawing of the paucispiral protoconch.R. visayaensis sp.nov. is the Reticunassa species most commonly offered in the shell trade from the central Philippines, as well as R. crenulicostata (Shuto, 1969).The latter is smaller (5-7 mm) and has a large multispiral protoconch of 3.5 whorls (Cernohorsky 1984: pl. 38, figs 1-2; Martin 2008: pl. 354, figs 4-5).These features are the most distinguishing differences between R. crenulicostata and R. visayaensis sp.nov.Reticunassa tringa has a paucispiral protoconch.Identification based on geographical distribution alone (Fig. 6) may be possible in specimens lacking a protoconch or welldefined teleoconch sculpture.

Habitat
Intertidal to 150 m, commonly from 0 to 20 m.
color.Whitish to yellowish; some spiral cords light brown between ribs.Lower half of dorsal side of body whorl frequently with broad, orange-brown band (Fig. 3O).

Remarks
The color varies from white to yellow and orange; the spiral cords occasionally brown, sometimes only locally between the axial ribs.Some specimens have 2 brown bands on the body whorl, occasionally only visible on the varix.Some specimens, including the holotype, display a broad orange-brown band on the lower part of the body whorl.R. poppeorum sp.nov. is often recognizable by a broad, dark orange-brown band on the last half or the last third of the ultimate whorl, and is characterized by its more slender shape compared to other species in this genus.
Reticunassa poppeorum sp.nov.shows resemblance to R. tringa and R. visayaensis sp.nov.The greatest difference among the three species is the morphology of the protoconch.R. tringa has a paucispiral protoconch with 1.5-1.75whorls, R. visayaensis sp.nov.has a multispiral protoconch of 2.25-2.5 whorls, whereas the protoconch of R. poppeorum sp.nov.has 2.5-2.75 whorls.The axial ribs of R. poppeorum sp.nov.are more pronounced and are present on the entire body whorl, whereas they decrease in height on the ventral side of the body whorl in R. tringa and R. visayaensis sp.nov.The most noticeable difference between R. paupera on the one hand and R. tringa, R. visayaensis sp.nov.and R. poppeorum sp.nov. on the other, is the presence of lirae in the latter three species over the entire surface of the extended columella.Besides this sculptural feature, the shape of the columellar callus differentiates R. paupera from the other three species; the callus of R. paupera is practically limited to the columella, whereas the callus of the other three partly extends over the body whorl.
There are no morphological differences between R. neoproducta Kool & Dekker, 2007 and the closely related R. poppeorum sp.nov.(Fig. 3N-Q).However, we decided to considerer these two lineages as different species based on the reciprocal monophyly indicated by the taxonomic trees (COI and 28S separately).Furthermore, R. neoproducta is found in the western Indian Ocean and R. poppeorum occurs in the western Pacific Ocean.To date, no specimens of these two species have been found east of Sri Lanka or west of Sumatra, respectively.It seems very unlikely that these two allopatric lineages still share any gene flux.

Habitat
In sand and coral rubble, 0-40 m, mainly between 1 to 20 m.Some freshly dead specimens with operculum were collected on a hard bottom with small pockets of sediment and in coral sand at a depth of 4-6 m.Some specimens were collected in mangrove areas and in seagrass.

Etymology
The species is dedicated to Mrs Anna Bolte, the second author's partner and travel companion for more than 20 years.

Habitat
Intertidal to 70 m, frequently between 0 and 10 m.Some specimens were found living on rocky platforms with algae, associated to eunicid tubes, coarse sand, and rolling stones.

Etymology
This species is named after the biblical Goliath, the giant who fought against David.This name is chosen because Reticunassa goliath sp.nov. is to date the largest known species in the genus Reticunassa.

Remarks
Color and banding pattern variable; some specimens have light to dark brown bands and others lack banding.In banded specimens, one band occurs on the body whorl, with an additional band on the sutural area.In some specimens, the spiral cords may be colored orange/brown, but only between the axial ribs.Aperture white, inside faintly showing the outside bands if present.
The strong columellar callus, together with the thickening of the inside of the outer lip and the strong varix, gives the aperture a round, "open mouth" appearance, whereas the other species in this group have a more oval-shaped aperture.
R. goliath sp.nov.and R. tringa both have a paucispiral protoconch, but the former has a considerably heavier and larger shell.It is similar to R. tringa, R. visayaensis sp.nov.and R. poppeorum sp.nov. in having weak spiral cords.R. goliath sp.nov.differs from R. paupera in weight, in its larger size, in the number of whorls, and in the size of the aperture relative to the total shell length.

Etymology
The name of this species refers to the spiral cords that seemingly intrude and continue over part of the body whorl of the protoconch.
Shell.Teleoconch consisting of 5 strongly impressed whorls.Penultimate whorl and body whorl with 13 pronounced strong, round axial ribs, varix strong.SPiral cordS.Penultimate whorl with 7 and body whorl with 10 narrow, fine, equally wide spiral cords.color.Creamy-yellowish, some spiral cords darker.Whorls with light subsutural band, body whorl with 3 bands, darkest on the thick varix and at base of shell.adult Size.5.4-9.7 mm, usually 7.5-8.5 mm.

Remarks
The most characteristic feature of this species is the broad, flattened, paucispiral and axially striated protoconch with, on the last part of it, 1-5 spiral cords that intrude from the teleoconch (Fig. 8L).This character was also mentioned by Cernohorsky (1984) in his description of Nassarius (Hima) pauperus (Gould, 1850) and was treated as intraspecific variation.These features, the broad, flat paucispiral protoconch and the extremely fine intercordal spiral striae of Reticunassa intrudens sp.nov., make it easy to distinguish it from R. paupera and the other species described in this paper.Moreover, R. intrudens sp.nov.lacks the minute pustules present in some other species described herein and most of the others also have a wider distribution; R. intrudens sp.nov. is only known from New Caledonia.It is one of the smallest species within this group.Habitat 0-110 m in muddy sand, with the largest concentration of specimens collected at 45-60 m.

Distribution
Only known from New Caledonia (Fig. 11).

Etymology
This species is named after Thailand, the type locality.

Remarks
The color is variable.Well-preserved specimens are cream or orange to light brown, either unicolor or with several darker-colored spiral cords.Occasionally remnants of bands are only present on the varix and on the fasciole.
The main difference between Reticunassa intrudens sp.nov and R. thailandensis sp.nov. is to be found in the protoconch morphology: the former has a protoconch with axial striae, in the latter it is partly smooth.Both are paucispiral and bulbous, and lack the microscopic pustules found in the other species described in this paper.R. paupera (Gould, 1850), R. visayaensis sp.nov.and R. poppeorum sp.nov.have a multispiral protoconch.R. thailandensis sp.nov.has more well developed spiral cords than the other species described here.Furthermore, in some specimens the 2-3 most anterior spiral cords are more strongly developed.In general, the distribution of the species, as shown on Fig. 12, might help with the identification.

Habitat
This species lives in sand and between dead corals, in depths of 1-90 m, usually found between 15 and 20 m.

Distribution
Thailand, Singapore, Malaysia, Indonesia, and the Philippines, where it is common at Balabac I., but apparently rare in the central Visayas (Fig. 12).

Discussion
This paper presents the first review on the diversification of genus Reticunassa from a molecular perspective.An integrative approach permitted the match between DNA sequences and morphological features to define the taxa discussed here.Previous references to "Nassarius pauperus" must be treated with caution because other species of Reticunassa might remain masked under that concept.This problem is particularly challenging when working with collections where specimens are kept dry and protoconchs have been lost, considering the small morphological interspecific differences among the species of Reticunassa.
So far, the only available morphological characters to distinguish species of the genus are the features of the protoconch, the spiral cords, the intercordal surface and the columella.Table 2 is a comparison of all available characters to facilitate species identification.However, in this paper we provide COI sequences that could help future identification of molecular samples.Reliably identified hologenophores for previously described species are becoming accessible, with their COI sequences available in online public libraries.For our new species we have prioritized the choice of name-bearing types such that they are also hologenophores, whenever possible, in order to avoid the perpetuation of a molluscan taxonomy in which many type specimens do not fulfill their function as name bearers (Bouchet & Strong 2010).
As can be seen in Fig. 1A-B, the trees are congruent with our taxonomic hypotheses concerning the status of Reticunassa and the new species described in this paper, initially based on morphological observations.Our results are also consistent with what has been observed in other nassariids: the intraspecific variability is about 1.9% among species of Nassarius (Kool & Galindo 2014) and ranges from 1.1% to 2.5% within species of Tritia (Couceiro et al. 2012;Couceiro et al. 2007;Simon-Bouhet et al. 2006).In general, K2P genetic distances between gastropod species span from 4.2% to 5.9% (Li et al. 2010;Kantor et al. 2012;Fedosov et al. 2014;Lorenz & Puillandre 2015).
The absence of a discrete barcode gap in a dataset can be explained many ways.In this case, it can be related to poor intraspecific replication within some species, i.e., there is only one sequence each representing R. paupera and R. tringa.
The trees (Fig. 1A-B) reveal the existence of several lineages, with, so far, no morphologically recognizable characters (i.e., MNHN IM-2007-31778 andMNHN IM-2013-17562), preventing their description.Further studies on Reticunassa might disclose patterns to help assign names to those taxonomic units.
In this paper, no particular taxonomic ranking has been given to the two subclades found within Reticunassa.However, there are clear differences among them.SC1 is formed by species that lack spiral striae; instead, they have either axial striae (R. hanraveni and R. silvardi) or relatively smooth intercordal surfaces (R. compacta, R. rotunda).Species of SC1 are distributed from the western Pacific Ocean to the Marquesas Islands.On the contrary, species of SC2 show the presence of spiral striae on the intercordal surface, and they occur in the Indian Ocean and the western Pacific Ocean.This result supports Galindo et al.'s (2016) hypothesis that geographical distribution patterns play an important role in the diversification of Nassariidae.Kool & Dekker (2006) placed Nassarius gregarius (Grabau & King, 1928) in the N. pauperus complex, because it is similar in size and shape and had been treated as a synonym of N. pauperus by Cernohorsky (1984).However, because this species has a keeled protoconch, it should not be included in Reticunassa, but should continue to be included in the genus Nassarius.
Abbreviations l= length lv = live-collected specimen spm = specimen(s), in Material examined sections stn = station w = width Molecular sequencing was carried out at the Service de Systématique Moléculaire (MNHN UMS 2700).Procedures from the Consortium for the Barcode of Life barcoding protocols (www.barcodeoflife.org)adapted to large biodiversity collections were followed (seePuillandre et al. 2012a).A piece of muscle from the foot, sufficient for approximately five DNA extractions, was stored in a 2D barcode tube as a reference tissue collection.Extraction was done with the NucleoSpin® 96 Tissue Core Kit adapted to a NucleoSpin® 96 Plasmid Binding Plate (Macherey-Nagel) using a liquid handling workstation (epMotion® 5075 VAC, Eppendorf).Purified DNA was stored in a second 2D tube.PCR reactions were performed in 20 μl, containing 2 μl of DNA, 1× reaction buffer, 2.5 mM MgCl 2 , 0.26 mM dNTP, 0.3 μM of each primer, 1 μl DMSO, 1 μl BSA (10 mg/l) and 1.5 units of Q-Bio Taq DNA polymerase (QBiogene, Carlsbad, California, USA).A fragment of 658 bp of the cytochrome oxidase I (COI) mitochondrial gene was amplified, using the universal primer set (LCO1490 and HCO2198)(Folmer et al. 1994) and a primer set specific for Nassariidae (5COIF: 5'ACAAATCATAAAGAYATTGGAAC-3', 492COIR: 5'CGCTCAAATTGTATTCCTCG-3' and 492COIRD: 5'CGYTCRAATTGTATYCCTCG-3')(Galindo et al. 2016).Annealing temperature was 50°C for 35 seconds.The first subunit of the 28S gene was amplified using the C1' and D2 primers ofChisholm et al. (2001).Annealing temperature was 58ºC for 40 seconds or a touchdown gradient from 66ºC to 56ºC for 8 seconds (plus 58ºC for 30 seconds) whenever double bands were present.Sequencing was carried out by the Centre National de Séquençage, Genoscope (Évry, France) and Eurofins Genomics (Paris, France).In all cases, both directions were sequenced to confirm the accuracy of each haplotype.

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GenBank accession numbers for sequenced specimens of Reticunassa included in this paper.H = holotype; Hg = hologenophore; N = neotype; P = paratype; PNG = Papua New Guinea.[continued on next two pages]

Fig. 1 .
Fig. 1. A. Bayesian tree based on COI sequences of species of Reticunassa.B. Bayesian tree based on 28S sequences of species of Reticunassa.C. Frequency of pairwise distance (K2P model) values among COI sequences.In gray, intra-specific variation.In black, inter-specific variation.PNG = Papua New Guinea.
DL = number of denticles on the lip; N°LW = number of axial ribs on the on last whorl; N°PW = number of axial ribs on the penultimate whorl; N°W = number of whorls on the protoconch; SC = sculpture of columella; TP = type of protoconch.[continued on next page]
SPiral cordS.Continuous, flat and narrow, 7 cords on penultimate, and 11 on body whorl, peripheral one somewhat darker.intercordal SculPture.Approximately 6 very fine, evenly spaced striae between spiral cords.

Fig. 6 .
Fig. 6.Geographical and bathymetrical distribution of Reticunassa visayaensis sp.nov.Star indicates type locality.Each bar represents all lv or dd specimens.

Fig. 7 .
Fig. 7. Geographical and bathymetrical distribution of Reticunassa poppeorum sp.nov.Star indicates type locality.Each bar represents all lv or dd specimens.

Fig. 9 .
Fig. 9. Geographical and bathymetrical distribution of Reticunassa annabolteae sp.nov.Star indicates type locality.Each bar represents all lv or dd specimens.
color.Off white to yellow, narrow band just suture and broader band below periphery; columella and outer lip white, aperture yellowish.adult Size.8.2-16.5 mm, usually 13-15 mm.

Fig. 10 .
Fig. 10.Geographical and bathymetrical distribution of Reticunassa goliath sp.nov.Star indicates type locality.Each bar represents all lv or dd specimens.

Fig. 11 .
Fig. 11.Geographical and bathymetrical distribution of Reticunassa intrudens sp.nov.Star indicates type locality.Each bar represents all lv or dd specimens.

Fig. 12 .
Fig. 12. Geographical and bathymetrical distribution of Reticunassa thailandensis sp.nov.Star indicates type locality.Each bar represents all lv or dd specimens.