Tamilokus mabinia, a new, anatomically divergent genus and species of wood-boring bivalve from the Philippines

Here we describe an anatomically divergent wood-boring bivalve belonging to the family Teredinidae. Specimens were collected off the coast of Mabini, Batangas, Philippines, in February 2018, from sunken driftwood at a depth of less than 2 m. A combination of characteristics differentiates these specimens from members of previously named teredinid genera and species. Most notable among these include: an enlarged cephalic hood which extends across the posterior slope of the shell valves and integrates into the posterior adductor muscle; a unique structure, which we term the ‘cephalic collar’, formed by protruding folds of the mantle immediately ventral to the foot and extending past the posterior margin of the valves; a large globular stomach located entirely posterior to the posterior adductor muscle and extending substantially beyond the posterior gape of the valves; an elongate crystalline style and style sac extending from the base of the foot, past the posterior adductor muscle, to the posteriorly located stomach; calcareous pallets distinct from those of described genera; a prominently flared mantle collar which extends midway along the stalk of the pallets; and, separated siphons that bear a pigmented pinstripe pattern with highly elaborate compound papillae on the incurrent siphon aperture. We used Micro-Computed Tomography (Micro-CT) to build a virtual 3D anatomical model of this organism, confirming the spatial arrangement of the structures described above. Phylogenetic analysis of the small (18S) and large (28S) nuclear rRNA gene sequences, place this bivalve within the Teredindae on a branch well differentiated from previously named genera and species. We propose the new genus and species Tamilokus mabinia to accommodate these organisms, raising the total number of genera in this economically and environmentally important family to 17. This study demonstrates the efficacy of Micro-CT for anatomical description of a systematically challenging group of bivalves whose highly derived body plans are differentiated predominantly by soft tissue adaptations rather than features of calcareous hard-parts.


INTRODUCTION
The Teredinidae, commonly referred to as shipworms, are a group of wood-boring bivalves with wide-ranging economic and ecological impacts in coastal marine systems. Their ability to bore into and digest wood is estimated to cause billions of dollars in damage per year to coastal structures, such as piers, jetties, wharfs, fishing equipment and aquaculture pens (Turner 1966;Distel 2003). Yet this same ability allows them to play a fundamental role in carbon cycling by processing recalcitrant organic carbon trapped in wood and liberating nutrients and energy that would otherwise be less accessible to other organisms (Nishimoto et al., 2015;Charles et al., 2016).
The unique wood-boring life-style of the Teredinidae has led to several anatomical specialisations and as such, this family is believed to exhibit more variation in morphology than any other group in the Mollusca (Turner, 1988). In contrast to more typical bivalves, the shell is greatly reduced in size and has prominent pedal and posterior gapes, allowing the protrusion of the foot and elongate body respectively. The location of the shell valves at the extreme anterior end of the body covers little of the visceral mass and offers little or no protection to the animal. Instead, the shell, which features tiny denticulated ridges on its surface, functions as an effective drilling and grinding tool for burrowing into wood (Nair, 1966;Turner, 1966;Voight, 2015). The body is long, vermiform and enclosed by a calcareous tube that is secreted onto the burrow walls, surrounding the animal from the posterior edge of the valves to the burrow opening. The siphons are flanked by a pair of calcareous, paddle or feather-like structures, known as the pallets. These pallets, which are unique to Teredinidae, serve as watertight and predator-resistant plugs to seal the burrow entrance (Nair, 1966;Turner, 1966;Voight, 2015). Currently, there are 16 accepted genera composed of 70 species in the Teredinidae (Bulatov, 1933;Turner, 1966;Macintosh, 2012;Borges & Merckelbach, 2018;Velásquez & Shipway, 2018). However, it has been recently suggested that between 80-100 species likely exist (Huber, 2015;Shipway et al., 2016).
Using integrative taxonomic methods, including anatomical, morphological and molecular data (Borges et al., 2012;Shipway et al., 2016), we identify and describe a new genus and species of wood-boring teredinid from the Philippines, which we name Tamilokus mabinia.

Specimen collection
Nine specimens of Tamilokus mabinia were collected in February 2018 off the coast of Balayan Bay, Mabini, Batangas, Philippines (13.758 • N, 120.925 • E), at a depth of <2 m, from a log measuring approximately 2 m in total length, as part of the Philippine Mollusk Symbiont (PMS) International Collaborative Biodiversity Group (ICBG) expedition. The specimen location map ( Fig. 1) was produced and approximate coordinates were determined using the National Oceanographic and Atmospheric Administration Map Viewer (https://www.nauticalcharts.noaa.gov/ENCOnline/enconline.html). Specimens were carefully extracted from their burrows, photographed (Fig. 2), measured, fixed in 4% formaldehyde solution freshly prepared from paraformaldehyde (PFA), washed, and dehydrated through an ethanol series (30%, 50% and 70%, 30 min per wash, x2 washes) with final storage in 70% ethanol. Partial specimens, identifiable as T. mabinia based on the unique morphology of the pallets, siphons and cephalic collar, were dissected or were fixed in 100% ethanol for DNA preservation. Morphological features (Fig. 3) and calcareous structures ( Fig. 4) were imaged using the Keyence VHX-6000 Digital Microscope (Osaka, Japan). Specimens were directly compared with all described teredinid genera from the Harvard Museum of Comparative Zoology. Part of this work was completed under the supervision of the Department of Agriculture-Bureau of Fisheries and Aquatic Resources, Philippines (DA-BFAR) in compliance with all required legal instruments and regulatory issuances covering the conduct of the research. All Philippine specimens used in this study were obtained using Gratuitous Permit GP-0140-17 issued by DA-BFAR.

DNA extraction & amplification
DNA was extracted from the siphonal tissue and associated musculature from one specimen of Tamilokus mabinia (PMS-3943P), using the DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany) following manufacturer's protocol. Approximate purity, concentration and yield of DNA were determined by UV spectrophotometry. Genomic DNA was cryo-preserved at −80 • C and archived at the Ocean Genome Legacy Center of New England Biolabs, Northeastern University, Nahant, MA, USA (accession number in Table 1). The small (18S) and large (28S) subunit nuclear rRNA genes were amplified from the resultant DNA preparation by polymerase chain reaction (PCR). Amplification reactions were prepared using 12.5 µL of high-fidelity polymerase solution (OneTaq, New England Biolabs, Ipswich, Massachusetts), 0.5 µL of each primer (10 mM), 1-2 µL DNA template (10-20 ng/µL), brought to a total volume of 25 µL with purified water. Fragments of the small (18S) and large (28S) subunit nuclear rRNA genes were amplified using the primer pairs 18S EukF

Zoobank registration
The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix http://zoobank.org/. The LSID for this publication is: LSID urn:lsid:zoobank.org:pub:0235E48D-D7B7-4A4B-A078-4204C1907240. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central and CLOCKSS.  (3 specimens  Tamilokus is similar to Teredora Bartsch 1921 and Uperotus Guettard 1770 in that it possesses a U-shaped caecum that, after passing posteriorly from the stomach, doubles back upon itself towards the right and extends anteriorly, terminating near its origin on the right side of the stomach (Turner, 1966). However, Tamilokus can be easily distinguished from these genera based on pallet morphology (as previously described). Additionally, the gills of Teredora and Uperotus extend the entire length of the animal from the base of the siphons to the mouth, whereas those of Tamilokus terminate near the posterior end of the caecum; the labial palps in both Teredora and Uperotus are large and free, but are very small and attached in Tamilokus; the crystalline style in both Teredora and Uperotus is located anteriorly to the posterior adductor muscle, but extends from the base of the foot well beyond the posterior margin of the adductor muscle in Tamilokus; the stomach of both Teredora and Uperotus is located anterior to posterior adductor muscle, whereas the stomach of Tamilokus is located posterior to the posterior adductor muscle; the heart of both Teredora and Uperotus is positioned anteriorly, but is medially positioned in Tamilokus; the siphons of Teredora and Uperotus are united along their entire length, but are separate in Tamilokus; the incurrent siphons of both Teredora and Uperotus feature a single primary row of papillae, whereas the incurrent siphon of Tamilokus has an additional secondary row that includes compound branched papillae; and, the cephalic hood in both Teredora and Uperotus is inconspicuous, whereas the cephalic hood in Tamilokus is prominent and covers the posterior slope of the shell valves. Finally, Tamilokus may be recognized by the presence of the cephalic collar which is unique to this genus. Taxonomic characters differentiating Tamilokus from Teredora and Uperotus are summarized in Table 2. Etymology: Mabinia (noun in apposition), in honour of Apolinario Mabini, a Philippine national hero, and pertaining to the type location of the specimens from Mabini, Batangas, Philippines. Description: All characteristics of the genus, plus; dorsal anterior slope of shell valves highly elongated (Fig. 4B); incurrent and excurrent siphons ringed with papillae (Fig. 3F); incurrent siphon features outer ring of long papillae, and inner ring of shorter compound or branched papillae (Fig. 3G); siphon tips are ringed with pink to brownish red pigment that extends posteriorly in narrow closely spaced parallel stripes ( Figs. 2A-2C & 3A). Definition: Pallets elongate, composed of an unsegmented blade built upon a central stalk (Fig. 4A); blade triangular, single concave U-shaped cup on distal margin with slightly more pronounced curvature on outer face; periostracum absent; mantle collar flared, extending approximately half the length of the pallet; stalk and blade approximately equal in length; stalk translucent, ovate and flattened in both sagittal and transverse section; blade calcareous and white.

Phylogeny
Bayesian analysis of concatenated small (18S) and large (28S) nuclear rRNA gene sequences (Fig. S1) indicate a basal position for Tamilokus within Teredinidae, sharing a wellsupported node with Teredora, but separated from that taxon by a branch length comparable to those separating most accepted teredinid genera. A subtree excerpted from Fig. S1 is displayed in Fig. 6.

DISCUSSION
Herein, we describe a divergent member of the Teredinidae that, based on anatomical, morphological and molecular characteristics, is distinct from members of described genera. Among the distinguishing features of Tamilokus mabinia, the most striking are the posterior position of the stomach and the elongation of the crystalline style and style sac to accommodate this posterior location (Fig. 5A). This position of the stomach is found only in Tamilokus but is similar to that found in Kuphus, except that in the latter, the entire visceral mass is located posterior to the posterior adductor. The distinct positions of these two taxa on the phylogenetic tree of Teredinidae suggests that this similarity is due to convergence rather than homology. The extension of the crystalline style, from a position ventral to and near the anterior end of the foot to a position well posterior to the shell valves, is unique to Tamilokus (Turner, 1966). In other bivalves, cilia-mediated rotation of the crystalline style is thought to aid digestion by mixing enzymes and reducing food particle size through mechanical action (Robin et al., 2018;Nelson, 1918;Edmondson, 1920;Yonge, 1923;Lavine, 1946;Morton, 1952;Horiuchi & Lane, 1982;Alyakrinskaya, 2001;Sakamoto et al., 2008;Mackenzie & Marshall, 2014). However, the functional significance of the exceptionally large style of Tamilokus remains to be explained.  Tamilokus also features a characteristic structure absent in other genera, which we term the 'cephalic collar'. This feature is formed by protruding folds of the mantle and is located directly ventral to the foot and anterior adductor muscle and extends posterior to the shell valves on the ventral mantle surface (Figs. 2A-2B, 3A, 3D-3E & 5A). Figures 3D-3E show that the cephalic collar can be contracted or expanded, and Micro-CT scans reveal that the style sac is located within the cephalic collar (Figs. 5A-5B). These observations suggest that the cephalic collar may serve to accommodate the large size of the crystalline style and may facilitate its movement.
Another unusual feature of Tamilokus is the caecum, which folds sharply to the right at its midpoint, doubling back upon itself to form a compressed U-shape. Among the Teredinidae, only two described genera, Teredora and Uperotus share this feature (Turner, 1966). However, Teredora and Uperotus differ from Tamilokus in several important ways. In these genera, the gills extend almost the entire length of the animal from the base of the siphons to the mouth, proportionately the longest gills among the family (Turner, 1966). In addition to the extended gills, the labial palps, which help sort and direct filtered particles to the mouth, are prominent and detached. This observation led to suggestions that these genera are specialized for planktotrophy rather than wood feeding (Turner, 1966;Nair & Saraswathy, 1971). This hypothesis, however, preceded the discovery of cellulolytic symbionts in the gills of shipworms, providing the competing hypothesis that larger gills are adaptive for wood digestion (Distel et al., 2011).
In contrast to Teredora and Uperotus, the gills of Tamilokus extend only to the posterior end of the caecum (Fig. 5A) and the palps are small, fixed, and appear to be vestigial. Also, in contrast to these genera, the intestine of Tamilokus is extremely wide. In all specimens examined, the capacious intestine was tightly packed with light-coloured excavated wood fiber (Fig. 2B), matching the colour of the wood substrate in which the animals burrowed. No evidence of planktonic organisms could be discerned in caecum contents or faecal material by microscopic examination.
Siphon morphology also distinguishes Tamilokus from Teredora and Uperotus. The siphons of the former are separate while those of the latter two are united along their entire length. The presence of pink pinstripes on both the incurrent and excurrent siphons of Tamilokus (Figs. 2 and 3) are also unique among described species. Additionally, the siphons are tipped with two rows of papillae on the incurrent siphon aperture; the primary outer row is simple in structure, while the inner secondary row include shorter compound papillae with multiple branches (Figs. 3F-3G). Characteristics of the siphons have been suggested as potentially useful in teredinid identification (Turner, 1966;Nair & Saraswathy, 1971), but have been described for only a small number of species (Roch, 1940;Turner, 1966;Lopes & Narchi, 1998;De Moraes & Lopes, 2003). Although the degree of genetic, ontogenetic and ecophenotypic variation in teredinid siphons has yet to be documented (Turner, 1966), characterizing these features could prove valuable for field identification, as specimens could be studied in situ without the necessity of extracting the teredinid from its burrow.
Aside from its unique anatomical and morphological features, the classification of Tamilokus as a new genus is further supported by molecular phylogenetic data. Phylogenetic analysis based on concatenated small (18S) and large (28S) nuclear rRNA gene sequences reveal well supported relationships between Tamilokus and basal taxa within Teredinidae. However, the branch length of Tamilokus is among the longest in the family, indicating divergence comparable to or greater than those separating accepted teredinid genera (Fig. 6). Anatomical and morphological characters are also consistent with the basal position of Tamilokus. As in other basal members of the family, the stomach of T. mabinia is globular (Type 2 of Turner 1966(Turner, 1966), the intestine loops anteriorly over the crystalline style sac, and brooded larvae are absent in the gills of all specimens examined. This is consistent with the hypothesis that these characteristics are plesiomorphic and that the elongate stomach, absence of the anterior intestinal loop and complex reproductive strategies are apomorphic (Distel et al., 2011).
In this investigation, Micro-CT has been invaluable for elucidating soft tissue anatomy. This has been especially important in this systematically challenging group, which is characterised by highly derived body parts and few taxonomically informative calcareous features. Previously, Micro-CT has been used to determine quantitative measurements of wood degradation and boring rates of Teredinidae (Amon et al., 2015). Additionally, Micro-CT has been used to reveal exceptionally well-preserved fossilized wood-boring bivalves in mid-Cretaceous wood, with silicified soft parts and overall body-plan characteristic of the Teredinidae (Robin et al., 2018). Herein, we further demonstrate the power of this technique to reveal the relationships among soft tissue features in three dimensions, and in the evaluation of likely mode of nutrition and life history strategy, while minimizing distortion that can result from manual dissection.
The extent of diversity within the Teredinidae has long been a subject of debate. Historically, as many as 130 species have been recognized (Moll & Roch, 1931), but many were later synonymized in the first comprehensive work on the taxonomy of this family (Turner, 1966). Recently, it has been suggested that teredinid diversity may be greater than previously thought (Huber, 2015;Shipway et al., 2016). This view is supported by the recent description of one new genus and several new species of teredinid bivalves (Macintosh, 2012;Borges & Merckelbach, 2018;Velásquez & Shipway, 2018). In addition, further reports suggest several more species (Shipway et al., 2016;Treneman et al., 2018) and at least one more genus (Lozouet & Plaziat, 2008) remain to be described.

CONCLUSIONS
We describe a new and anatomically divergent genus and species of wood-boring bivalve (Teredinidae) from the Philippines, which we name Tamilokus mabinia. A detailed Micro-CT 3D render of T. mabinia reveals several anatomically divergent characters, including a new structure which we term the 'cephalic collar', a prominent crystalline style sac and crystalline style originating within the cephalic collar and extending beyond the posterior adductor muscle into the posteriorly located stomach, and a caecum (wood-storing organ) which doubles back upon itself. In addition, the unique pallets, the primary taxonomic character of this family, could not be placed in any existing genera. Phylogenetic analysis places T. mabinia in a separate taxon among other basal members of the Teredinidae. The addition of Tamilokus raises the total number of genera in this economically and environmentally important group of wood-boring bivalves to 17.