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

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.

Micro-computed tomography

Specimen PMS-4051L was stained for 20 days in 10% iodine prior to imaging. Imaging was performed using a SkyScan 1173 micro-CT scanner (Bruker Micro-CT, Kontich, Belgium) equipped with a Hamamatsu 130/300 tungsten X-ray source and a FlatPanel Sensor camera detector with 2,240 × 2,240 pixels. Scanning parameters were as follows: source voltage = 60 kV; source current = 100 µA; exposure time = 900 ms; frames averaged = 3; frames acquired over 180° = 960; filter = no; binning = no; flat field correction = activated; scanning time =(X3)00:28:14; number of connected scans= 3; and, random movement = ON (10). Reconstruction of the raw data was accomplished using the software provided with the scanner (NRecon 1.6.6.0, Bruker Micro-CT, Kontich, Belgium). The following settings were employed to enhance image contrast and to compensate for ring and streak artefacts; smoothing = no, ring artefact correction = 4, and beam hardening correction = activated. Reconstructed scans were then analyzed using CTVox (Bruker Micro-CT, Kontich, Belgium). A Micro-CT 3D rendered model of Tamilokus mabinia, including major anatomical structures and transverse cross sections of the model is shown in Fig. 5.

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 (5′–WAY-CTG-GTT-GAT-CCT-GCC-AGT–3′) and 18S EukR (5′–TGA-TCC-TTC-YGC-AGG-TTC-ACC-TAC–3′) (Medlin et al., 1988); 28S-NLF184-21 (5′–ACC-CGC-TGA-AYT-TAA-GCA-TAT–3′) and 28S-1600R (5′–AGC-GCC-ATC-CAT-TTT-CAG-G–3′) (Distel et al., 2011), resulting in amplicons of approximately 1,686 and 1,416 base pairs, respectively. The PCR amplification proceeded as follows: an initial denaturation step of 94 °C for three minutes, followed by 35 cycles with a denaturation step of 94 °C for 20 s, an annealing step of 64 °C for 40 s for the 18S and 63 °C for 30 s for 28S, an extension step of 68 °C for 60 s and a final extension of 68 °C for five minutes. All reactions were performed on a PTC-200 Thermal Cycler (MJ Research, Quebec, Canada).

For each template, three separate amplicons, all produced under identical conditions, were pooled, cleaned and concentrated using the Zymo Clean & Concentrator Kit (Irvine, CA). Resulting products were sequenced bidirectionally on a 3730xl DNA Analyzer (Life Technologies, Grand Island, NY) using the Big Dye Terminator 3.1 Cycle Sequencing Kit (Life Technologies, Grand Island, NY) at New England Biolabs (Ipswich, Massachusetts). Primary sequence data from the small (18S) and large (28S) subunit nuclear rRNA genes were submitted to GenBank (NCBI) under accession numbers MH974682 and MH974683, respectively.

Phylogenetic analysis

The 18S and 28S rRNA gene sequences from specimen PMS-3943P were concatenated and aligned as in Distel et al. (2011) with sequences representing 10 of 16 recognized genera (Bulatov, 1933; Turner, 1966) including: Bankia Gray 1842, Dicyathifer Iredale 1932, Kuphus Guettard 1770, Lyrodus Gould 1870, Nausitora Wright 1864, Neoteredo Bartsch 1920, Spathoteredo Moll 1928, Teredo Linnaeus 1758, Teredora Bartsch 1921, and Teredothyra Bartsch 1921. Phylogenetic analysis was performed using MrBayes version 3.2.6 (Ronquist & Huelsenbeck, 2003) implemented in Geneious version 10.2.2 (https://www.geneious.com) using GTR + I + Γ nucleotide substitution model. Placopecten magellanicus was specified as the outgroup, the chain length was set to 5 million, subsampling every 2,000 generations and discarding the first 20% of the results as burn-in.

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.

Systematics

Type species: Tamilokus mabinia sp. nov.

Type material: Holotype PMS-3916Y; paratypes PMS-3899P, PMS-3915X, PMS-3943P, PMS-3949Y, PMS-4037Y, and PMS-4051L. Smallest to largest specimens measured 6.2 cm–15.4 cm in total body length. The holotype is currently held at the Academy of Natural Sciences of Philadelphia (ANSP) and will be deposited in the National Museum of the Philippines in Manila pending completion of an ongoing reorganization. Paratypes are deposited at ANSP, at the Marine Science Institute of the University of the Philippines (MSI) and at the Ocean Genome Legacy Center of New England Biolabs, Northeastern University (OGL). For catalog numbers assigned by holding institutions see Table 1.

Type locality: Balayan Bay, off Mabini, Batangas Province, Philippines (coordinates 13.758°N, 120.925°E).

Comparative Material: The following material was examined: Teredora malleolus (Turton, 1822) MCZ-350541 (3 specimens); Teredora princesae (Sivickis, 1928) MCZ-232090 (5 specimens); Uperotus clava (Gmelin, 1791) MCZ-238009 (3 specimens); and, Uperotus panamensis (Bartsch, 1922) MCZ-357824 (4 specimens).

Diagnosis: pallets triangular-shaped with an ovate, flattened stalk, prominent cephalic hood and cephalic crest, crystalline style extends beyond posterior adductor muscle, caecum U-shaped doubling back upon itself, siphons with distinct pin-striped pigmentation with incurrent siphon featuring three rows of papillae

Etymology: Tamilokus (masculine), in recognition of the common name for shipworm in the Philippines, ‘tamilok’.

Habitat: Marine, wood-borer.

Description: Pallets very small in relation to body length (Fig. 2A), formed of a broad, flattened, translucent and ovate stalk, with a solid, white calcareous blade (Fig. 4A); cephalic hood prominent, extending to cover the posterior slope of the shell valves (Figs. 2A, 3A–3C & 5A, 5C); “cephalic collar”, formed from protruding folds of the mantle, extending along the ventral surface from the base of the foot to a point slightly posterior to the posterior margin of the shell valves (Figs. 2A–2B, 3A, 3D–3E, 5A–5B); large crystalline style and style sac originating within the cephalic collar and extending beyond the posterior adductor muscle into the posteriorly located stomach (Figs. 5A–5E); large globular stomach (Figs. 2B & 5A, 5E–5F) located posteriorly in relation to posterior adductor muscle and valve pedal gape, composed of three lobes, with muscular opening into caecum (Figs. 5A, 5F); large digestive glands (Figs. 2A–2C & Fig. 5A, 5C–5E); caecum (Figs. 2A–2C & 5A, 5D–5I) large, elongate, U-shaped, doubling back upon itself anteriorly towards the right (Figs. 2B & 5D–5I), lacking a typhlosole (Figs. 5D–5I); intestine unusually broad and voluminous (Fig. 2B), especially where it passes ventral to the caecum (Figs. 5G–5I), containing short ovoid faecal pellets packed in multiple rows across its width (Fig. 2B); intestine extends anteriorly, looping over crystalline style sac (Figs. 5C–5E), then doubles back posteriorly, looping under the caecum before extending anteriorly on the dorsal surface of the caecum, finally looping under the posterior adductor muscle before extending a short distance posteriorly and opening into the anal canal; anal canal is open and does not retain faeces; gonad located centrally (Figs. 5A, 5I–5K), beginning posterior to the caecum and ending at the heart; heart located medially (Figs. 5J–5K); gill extends from the siphons anteriorly to the posterior tip of the gonads (Figs. 5A, 5K–5L); prominently flared mantle collar around siphons and pallets (Figs. 2B, 3A & 5A).

Remarks: Tamilokus gen. nov may be easily differentiated from all other genera within the family Teredinidae based on pallet morphology. The simple triangular cup-shaped pallet and thick ovate stalk of Tamilokus may be distinguished from the pallets of: Bankia Gray 1842, Nausitora Wright 1864, Nototeredo Bartsch 1923, and Spathoteredo Moll 1928 by the lack of segmentation; from Lyrodus Gould 1870, Teredo Linnaeus 1758, Zachsia Bulatoff & Rjabtschikoff 1933, and Nivanteredo Velásquez & Shipway, 2018 by the absence of a periostracum and presence of a broad ovate stalk; from Teredothyra Bartsch 1921 by the presence of a single undivided cup; from Dicyathifer Iredale 1932 and Kuphus Guettard 1770 by the absence of a medial ridge; from Bactronophorus Tapparone Canefri 1877 by the absence of a dagger-like extension; from Neoteredo Bartsch 1920, Psiloteredo Bartsch 1922, Teredora Bartsch 1921 and Uperotus Guettard 1770 by the cup-rather than paddle-shaped blades, and from the latter three genera by the lack a distinct thumb-nail like depression bearing concentric or radiating ridges.

Additionally, Tamilokus may be easily distinguished from Bactronophorus Tapparone Canefri 1877, Dicyathifer Iredale 1932, Neoteredo Bartsch 1920 and Teredothyra Bartsch 1921 by the absence of a muscular sphincter at the posterior end of the anal canal; from Lyrodus Gould 1870, Teredo Linnaeus 1758 and Zachsia Bulatoff & Rjabtschikoff 1933 by the absence of brood pouches on the gill; from Neoteredo Bartsch 1920 by the absence of dorsal lappets; and from Kuphus Guettard 1770 by the presence of a caecum and the absence of a strong muscular collar surrounding the valves.

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 well-supported 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.