Pleurolucina from the western Atlantic and eastern Pacific Oceans: a new intertidal species from Curaçao with unusual shell microstructure (Mollusca, Bivalvia, Lucinidae)

Abstract A new shallow water species of the lucinid bivalve Pleurolucina is described from Curaçao in the southern Caribbean Sea and compared with known species of the genus from the western Atlantic and eastern Pacific Oceans. Although confused with the Floridian species Pleurolucina leucocyma, it is most similar to the eastern Pacific Pleurolucina undata. As in all studied lucinids, the new species possesses symbiotic bacteria housed in the ctenidia. The shell microstructure is unusual with repeated and intercalated conchiolin layers that have sublayers of ‘tulip-shaped’ calcareous spherules. Predatory drillings by naticid gastropods frequently terminate at the conchiolin layers.


Introduction
The tropical and subtropical western Atlantic is one of the major centres of marine molluscan diversity and bivalves in the speciose family Lucinidae, with an estimated 46 species in this ocean, have been the focus of many studies since the discovery of their chemosymbiosis with sulphide-oxidising bacteria (e.g. Giere 1985, Fisher and Hand 1984, Frenkiel and Mouëza 1995, Frenkiel et al. 1996, Gros et al. 1998, 1998, 2012. Nonetheless, new species from both shallow and deep water are still discovered and new genera identified Glover 2009, Taylor et al. 2013). Additionally, within the area there are several cryptic species with narrower ranges nestled among supposedly widespread species (Huber 2015, Taylor and Glover submitted. Distributional data for western Atlantic lucinids indicates that although some are widespread, others have more restricted ranges. A recurring pattern is of congeneric pairs, one largely restricted to the Gulf of Mexico and Florida and the other with a more southerly Caribbean range as exemplified by Lucinisca nassula and L. muricata (Taylor and Glover submitted). This dual distribution is similar to that proposed by Petuch (1982) as a relict of the Caloosahatchee-Gatunian pattern dating from the Pliocene but possibly inherited by present day taxa. Additionally, in the eastern Pacific, there are lucinids closely similar morphologically and genetically to those of the western Atlantic and presumably separated by the rise of the Central American Isthmus around 3.5 mya. Examples of these are the pair Radiolucina amianta (Atlantic) and R. cancellaris (Pacific) (see Garfinkle 2012), and the pair Ctena imbricatula (Atlantic) and Ctena mexicana (Pacific) (Taylor et al. 2011).
Pleurolucina (Dall, 1901) is a genus of small lucinids characterised by broad radial ribs. The type species, Lucina leucocyma Dall, 1886, first described from off the Florida Keys, is documented as having a distribution from North Carolina to Colombia including Yucatan Peninsula (Britton 1970, Vokes and Vokes 1983, Huber 2015. Two other species, P. hendersoni Britton, 1972 and P. sombrerensis (Dall, 1886), are known from the western Atlantic (Britton 1972), while three further species are recorded from the Eastern Pacific (Coan and Valentich-Scott 2012). During field sampling in shallow seagrass around Curaçao in May 2015 we collected a Pleurolucina that we recognised as similar to, but likely distinct from, P. leucocyma. Further research showed this to be an undescribed species more widely distributed in the southern Caribbean and confounded with P. leucocyma. An apparent high incidence of failed naticid drill holes focused attention on the shell microstructure revealing intercalated organic layers. Thought to be related to Lucina or Cavilinga (Britton 1972, Bretsky 1976) and included by Taylor et al. (2011) in the subfamily Lucininae, no Pleurolucina species has previously been included in molecular analyses.
We describe this new Pleurolucina from Curaçao in comparison with other western Atlantic and Eastern Pacific species, detail its phylogenetic position and illustrate its unusual shell microstructure with calcified conchiolin layers.

Material and methods
Samples of the new species were collected in southern Curaçao -location below. Details of ctenidia and sperm were studied using critical point dried glutaraldehyde-fixed specimens. Shells, microstructure and anatomy were imaged using a Quanta FEI 650 FEG scanning electron microscope. Comparative shell material was studied in USNM and NHMUK. Other material. 19 ethanol preserved specimens (NHMUK), same locality as holotype.

Institutional abbreviations FMNH
Description. Shell subovate, slightly anteriorly extended, L to 9.6 mm, H to 9.7 mm, H/L 0.99, moderately inflated, sculpture of flat, closely spaced commarginal lamellae, with four prominent, broad ribs with interspaces variable in width, but always narrower than ribs themselves; microsculpture of tight rows of shallow pits (Fig. 1  P). Umbones low, situated on midline. Anterior dorsal area arcuate. Protoconch: PI 217 µm, PI + PII 228 µm, PII a narrow rim with fine increments (Fig. 1 O). Lunule short, semicircular, slightly impressed. Ligament short, set in shallow resilifer. Hinge teeth: LV with two cardinal teeth; a robust anterior lateral tooth and smaller posterior lateral. RV with a single large cardinal tooth and anterior and posterior lateral teeth. Anterior adductor muscle scar short, broad, widely divergent from pallial line (60-70 µm) for about half of length ( Fig. 2 A), posterior scar ovate; pallial line entire, pallial blood vessel scar sometimes visible. Shell margin finely beaded, sinuate with anterior sinus deeper. Shell within pallial line often patchily eroded to expose inner shell layers. Colour grey-white.
Anatomy. General anatomy resembles most other described lucinids (Fig. 3). Mantle fusion ventral to the posterior apertures is very short. Foot short and broad when retracted but can be vermiform when extended (Fig. 3 A) with a small heel. Visceral pouches absent. Distinct mantle gills are absent but the inner mantle ventral to the anterior adductor muscle is thickened (Fig. 3 C) and may be a respiratory area with blood space as seen in other lucinids (Taylor and Glover 2000). Labial palps are very short. In common with all other studied Lucinidae, P. harperae has ctenidia comprising inner demibranchs only; these were pink in life, large, thick and occupying much of the mantle cavity (Fig. 3 B). Ctenidial filaments are approx. 40 µm thick and 380 µm deep with a narrow 45 µm ciliated zone and a deep bacteriocyte zone (Fig 3 D). Bacteriocytes were packed with 'potato-shaped' bacteria 3-5 µm long and 1.5-2.0 µm wide (Figs 3 G, H). The surface of the microvilli-covered bacteriocytes and intercalary cells were colonised by abundant spirochaetes 2.5 µm long and 0.2 µm wide ( Fig. 3 F) similar to those re-  ported by Ball et al. (2009) from Euanodontia ovum (Reeve, 1850). In comparison the symbiotic bacteria of Clathrolucina costata collected at the same time and same habitat were longer and rod shaped, 8-10 µm in length and approx. 1 µm wide.
The sperm of P. harperae were 9 µm long and 1.2 µm wide at the base, tapering and curved distally (Figs 3 J, K). From the same locality, sperm of Clathrolucina costata were shorter, 4.8-5 µm and 1-1.2 µm wide with blunt tips. Oocytes of P. harperae were approx. 200 µm in diameter (Fig. 3 I). Comparative sperm data is available for a few other western Atlantic lucinids ); sperm of Codakia orbicularis were 14-15 µm long, tapering with a width of 0.8 µm; Ctena orbiculata were cylindrical, slightly curved, 7.5 µm long and 1-1.2 µm wide at base and Lucina pensylvanica were 15.5 µm long, with curved tapering heads and 1.1 µm wide at the posterior.
Shell microstructure. Within a very thin (ca 1 µm) periostracum, Pleurolucina harperae has a basic four layered shell (Figs 4 A,B); an outer composite prismatic layer, followed inwards by a thin crossed-lamellar layer, then a thicker layer of irregular spherulitic prisms and within the pallial line a complex crossed-lamellar layer with sublayers of irregular prisms. The shell layers are interrupted by sheets of conchiolin around 20-90 µm in thickness, each with repeated sublayers of small discrete 'tulipshaped' calcified spherulites approx. 5 µm in diameter (Figs 4 D, F). Each spherulite is joined to those of the layer below with a narrow (0.5 µm) semicalcified channel through the conchiolin (Figs 4 E, F). At the shell surface, the conchiolin sheets correspond to major depositional halts (Fig. 4 A) visible as notches in the shell with the conchiolin appearing contiguous with the invaginated periostracum. In each shell there may be between 1-5 of such sheets.
Drill holes in Pleurolucina harperae produced by predatory naticid gastropods were observed with full penetration in 14 out of 114 single valves, but with 12 records of   incomplete drill holes that terminated at an internal conchiolin layer (Fig. 5). In one shell there were three failed drills and in another two failures before successful penetration. Incidences of apparent multiple completed drill holes in dead shells may have resulted from post-mortem degradation of organic layers in failed drill holes.
Similar conchiolin calcified sheets were identified in Pleurolucina hendersoni (Figs 6 A, B) and P. undata (Figs 6 C-E) but not in P. leucocyma (2 shells examined) or P. sombrerensis (2 shells examined). Also conchiolin sheets with multiple layers of calcareous spherules were observed in Lucina pensylvanica from the Florida Keys (Figs F,G), apparently confined to the inner shell layer within the pallial line. This is distinct from the calcified periostracum of this species (Fig. 6 H) as described by Taylor et al. (2004). No conchiolin sheets were observed in a single Cavilinga blanda examined. For comparison, the repeated conchiolin sheets reported in Cardiolucina species by Ishikawa and Kase (2007) were studied in C. quadrata from the Philippines. These sheets were approx. 10-15 µm thick and only lightly calcified with sporadic spherulitic crystal aggregations (Figs 6 I-K) with no multiple sub-layers.  Habitat. Pleurolucina harperae is an intertidal to shallow subtidal species collected from sand amongst seagrass rhizomes (largely Thalassia testudinum, Halodule sp.) in contrast to P. leucocyma that is usually recorded from deeper water, for example 30-180 m around the Florida Keys (Britton 1970  Etymology. Named for Elizabeth (Liz) Harper, University of Cambridge, bivalve researcher, colleague and friend, who helped collect the new species.
Comparison with other species. Pleurolucina leucocyma (Fig. 7) was thought to be widespread across the tropical Western Atlantic but we now consider it to be restricted to Florida and the Gulf of Mexico with the southern Caribbean records representing Pleurolucina harperae. The new species differs from P. leucocyma (mean L 6.2 mm, H 7.4 mm, H/L 1.13) in being larger, less inflated and usually longer than high in the adult (Fig. 8). The radial folds are usually lower and the anterior adductor muscle scar is shorter and more divergent from the pallial line (Fig. 2 B). In shape and sculpture, it is most similar to the somewhat larger Pleurolucina undata (Figs 9 E-G) (mean L 15.1 mm, H 15 mm, H/L 0.95) from the eastern Pacific, Gulf of California, intertidal zone to 60 m (Coan and Valentich-Scott 2012).
Other less similar species are: P. hendersoni (Figs 9 A, B) an offshore to deep water species (to 300 m) from the southern Caribbean (Cuba, Lesser Antilles) that reaches The shell reaches about 6-7 mm in length and is rounded in outline, with a shallow radial anterior sulcus and prominent close commarginal lamellae, sometimes separated by deep interspaces. It does not closely resemble other Pleurolucina but shares some shell features including dentition and adductor scar shape. The larger P. leucocymoides (Figs 9 H-J) is known from shallow water to 150 m and ranges from Baja California to Ec-uador and Galapagos Islands. The sculpture of broad prominent commarginal lamellae and absence of prominent radial folds distinguish it from other Pleurolucina. Lastly, P. taylori (Figs 9 K-M) is known from the intertidal zone to 183 m in the Gulf of California; it is distinguished by the highly inflated shell and closely spaced, low commarginal lamellae with four to five radial folds and resembles the extinct late Pliocene -mid-Pleistocene Floridian species P. amabilis.

Discussion
Pleurolucina is a genus of seven living species from the tropical to subtropical western Atlantic and eastern Pacific with none recognised from the eastern Atlantic or Indo-West Pacific. In that respect, it is similar to Radiolucina (Garfinkle 2012) and Lucinisca that share similar distributions. In the western Atlantic, the most similar species to the southern Caribbean Pleurolucina harperae is P. leucocyma from Gulf of Mexico and Florida. This distributional pattern of northern and southern species pairs is seen in Ctena (C. orbiculata and C. imbricatula), Lucinisca (L. nassula and L. muricata) and Lucina (L. pensylvanica and L. roquesana) (see Taylor and Glover submitted). Cognate pairs of bivalves have been recognised from morphology and/or molecules on either side of the central American Isthmus (Marko 2002, Marko andMoran 2009). Although molecular confirmation is lacking, Pleurolucina harperae is similar in shell form to P. undata, P. hendersoni resembles P. leucocymoides and perhaps P. leucocyma is a sister species to P. taylori.
An interesting and unusual feature of Pleurolucina harperae is the repeated conchiolin sheets that are calcified with layers of embedded spherules. A model of conchiolin sheet formation in another lucinid genus, Cardiolucina, was proposed by Ishikawa and Kase (2007 fig. 7). Periodically, normal shell secretion of outer, middle and inner shell layers stops and a conchiolin sheet is secreted across the inside of the shell from the margin and extending within the pallial line. This break in normal calcification is marked by a distinct notch at the shell surface. Calcification then resumes with secretion of normal shell layers. Conchiolin layer formation in Pleurolucina is essentially similar but each layer is thicker with repeated sublayers of aragonitic spherules. The narrow channels linking successive spherule layers suggest some sort of original tissue connection to the cells of the mantle surface.
Conchiolin layers within the shell have been recorded in several bivalve families but those in the Corbulidae have attracted most attention because of the supposed resistance to predation by drilling gastropods evidenced by the high incidence of failed borings that terminate at the organic layers (e.g. Lewy andSamtleben 1979, Harper 1994). Alternatively, organic layers may enhance resistance to shell dissolution, endolithic organisms or shell breakage (Anderson 1992, Harper 1994, Kardon 1998. In contrast to Pleurolucina where the conchiolin layers are secreted episodically, the layers in Corbulidae are secreted continuously as a sublayer of normal shell formation. In Corbula gibba the conchiolin layer is calcified with cone-shaped spher-ules approx. 8 µm in diameter (Lewy and Samtleben 1979 figs 5A-F). The organic layers of Pleurolucina harperae are similar in position and mode of formation to those recorded for species of Cardiolucina (Ishikawa and Kase 2007), but are much more highly calcified. Cardiolucina spp also show a high incidence of multiple drill holes with many terminating at the organic layers (Ishikawa and Kase 2007). Pleurolucina and Cardiolucina are not closely related among the Lucininae and the occurrence of conchiolin layers in other lucinids seems to be sporadic and certainly absent in many genera although no comprehensive study has been made. Nonetheless, calcified conchiolin layers do occur in some individuals of Lucina pensylvanica that is more closely related to Pleurolucina. It is tempting to regard the conchiolin layers as an adaptation conferring some resistance to shell drilling predation but, as argued in the case of Corbula (e.g. Kardon 1998), the layers may be an exaptation having first developed with some other function such as resistance to shell dissolution or enhancement of mechanical strength.