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Observations on the Life History and Geographic Range of the Giant Chemosymbiotic Ship worm Kuphus polythalamius (Bivalvia: Teredinidae).

Introduction

Since the initial misidentification of Kuphus polythalamius as a serpulid polychaete by Linnaeus in 1758, the biology of this species has remained enshrouded in mystery. This situation is at least partly explained by the fact that, until recently, this giant worm-like sediment-dwelling teredinid bivalve was known primarily from descriptions of calcareous hard parts (shells, tubes, and pallets) and isolated, sporadic, and often secondhand reports of the existence of living specimens (e.g., Griffiths, 1806; Wright, 1866; Caiman, 1927 quoted in Societies and Academies, 1927; Sivickis, 1928). In 1966, Turner provided the first and, to date, the only detailed anatomical description of this species, based on a single ethanol-preserved specimen collected in the Solomon Islands in 1933. In 2017, Distel et al., reported the collection of several large living individuals of K. polythalamius in Mindanao, Philippines, in marine sediments, the first described in peer-reviewed literature. These ranged in body length from 11 to 64 cm prior to preservation. To our knowledge, neither smaller living specimens of this species nor preserved soft tissues derived therefrom have been observed or described in peer-reviewed literature. As a result, little is known about the early life history and recent geographic range of this enigmatic species.

Kuphus polythalamius is the largest and among the most atypical members of the Teredinidae (Huber, 2015). Teredinid bivalves are commonly referred to as shipworms due to their worm-like appearance and habit of burrowing in and ingesting wood, including the timbers of wooden ships (Turner, 1966). A number of anatomical adaptations facilitate this unique habit, including shells that bear a dense array of microscopic teeth used for boring in wood; large posterior adductor muscles that enable the boring action of the shells; a long vermiform body that facilitates deep penetration into wood; secretion of a hard calcareous tube that lines the inner surface of the burrow and provides protection against predators and desiccation; a pair of calcareous paddle-like structures (pallets) that flank the siphons and serve to plug the entrance to the burrow; an organ, known as the cecum, that facilitates storage and digestion of excavated wood particles (Turner, 1966); and a unique relationship with intracellular cellulolytic bacterial symbionts that reside within the gills and provide enzymes that aid in the digestion of wood (Pop-ham and Dickson, 1973; Waterbury et al., 1983; Distel et al., 1991; O'Connor et al., 2014). This distinctive array of adaptations has allowed shipworms to become broadly distributed across the world's oceans, exploiting a wide range of woody plant materials in marine to brackish environments, from mangrove and seagrass rhizomes to naturally occurring driftwood and man-made structures such as piers, boats, fishing equipment, and sea defenses (Distel, 2003).

Although K. polythalamius is similar in general body plan to other more typical shipworms, the observed habitat, anatomy, and symbiotic bacteria differ substantially. In contrast to other teredinid species, which burrow in and ingest wood or woody plant materials (Distel, 2003), individuals of K. polythalamius described previously were found burrowing in marine sediments (Griffiths, 1806; Wright, 1866; Caiman, 1927 quoted in Societies and Academies, 1927; Sivickis, 1928). Kuphus polythalamius also grows far larger than other ship-worms, with some specimens exceeding 1.6 m in length and 7 cm in diameter (Wright, 1866). This unusual size places K. polythalamius among the largest of bivalve species (Huber, 2015). The burrow lining is far more stout and massive than that of other shipworm species, consistent with the need to protect and support the animal in the absence of rigid wood substrates. The anterior of the shell valves is smooth in appearance and is greatly reduced in size by comparison with the body. The posterior adductor muscle is reduced compared with typical wood-boring teredinids, suggesting a more limited ability to burrow in hard substrates. The digestive system is highly reduced, and the wood-storing caecum is not evident (Turner, 1966), suggesting diminished capacity for wood digestion (Distel et al., 2017). Finally, unlike other teredinid species, which harbor cellulolytic endosymbionts (Waterbury et al., 1983; Distel et al., 1991, 2017), the symbiont community of K polythalamius is composed of sulphur-oxidizing chemoautotrophic bacteria, implying that reduced sulphur compounds, rather than wood, provide the major source of nutritional energy for large sediment-dwelling individuals of this species (Distel et al., 2017).

However, whereas fully grown individuals of K. polythalamius are strongly differentiated from other shipworms in terms of habitat, anatomy, and trophic strategy, smaller specimens have not been observed; and so it is not clear whether the same is true at earlier points in their life history. Here we examine a recent collection of teredinid bivalves similar in appearance to K. polythalamius. Unlike previously described specimens of K. polythalamius, these were found burrowing in wood rather than sediment, and these included individuals smaller than any previously observed for this species. We use molecular and morphological evidence to determine the species identity of these specimens, to ask whether wooden substrates play a role in the life history of K. polythalamius, and to evaluate the current geographic range of this enigmatic and rarely observed species.

Materials and Methods

Collection

All specimen collection locations are shown in Figure 1. Sediment-dwelling individuals of Kuphus polythalamius (Linnaeus, 1767) were collected in Kalamansig, Sultan Kudarat, Mindanao, Philippines, in 2-3-m water depth. Wood-boring specimens were collected from a single large piece of partially decomposed wood (Fig. 2A), located in Mabini, Batangas, Philippines, at a water depth of about 2 m. Additional specimens from museum collections were examined, including a sediment-dwelling specimen collected in the Solomon Islands, currently housed at the Harvard Museum of Comparative Zoology (MCZ 229089) and the basis of the first and only published anatomical description of K. polythalamius (Turner, 1966); an incomplete (siphon, pallets, and tube) wood-boring specimen from Tingloy, Batangas, Philippines, housed at the California Academy of Sciences (CAS190664); and three whole wood-boring specimens collected in a lagoon near Bil-bil Island, Madang, Papua New Guinea, at water depths of 2-3 m on fine coral-sand bottom during Biodiversity Expedition Papua Niugini 2012-2013, housed at the Museum National d'Histoire Naturelle (MNHN120508, MNHN120509, and MNHN80001). Details of specimens, collection locations, and habitat type are provided in Table 1.

Morphological examination

Intact individuals of K. polythalamius were carefully removed from their calcareous tubes (Fig. 3A), photographed, and measured prior to preservation. Calcareous structures (pallets and shell valves) were removed by dissection and imaged using a Nikon Eclipse E800 compound microscope (Tokyo, Japan).

Scanning electron microscopy

Shell valves (Fig. 4) were removed and dehydrated in absolute ethanol, critical-point dried using the SAMDRI-PVT-3D Critical Point Dryer (Tousumis, Rockville, MD), mounted on a standard aluminum scanning electron microscope stub, and coated with platinum to a thickness of 5 nm, using the Cressington 208 HR High Resolution Sputter Coater(Cressington Scientific Instruments, Watford, United Kingdom). Images were produced on the Hitachi S-4800 field emission scanning electron microscope (Krefeld, Germany).

DNA extraction and amplification

DNA was extracted from siphonal tissue and associated musculature. Total genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). Concentration, yield, and purity of DNA were determined by UV spectrophotometry, estimating that 1 absorption unit at 260 nm was approximately equal to 50 ng [micro][L.sup.-1] double-stranded DNA. Genomic DNA was cryo-preserved at--80 [degrees]C and archived at the Ocean Genome Legacy Center, Northeastern University, Nahant, Massachusetts.

Partial small (18S) and large (28S) subunit nuclear ribo-somal RNA (rRNA) and mitochondrial cytochrome oxidase I (mt-COI) genes were amplified from the resulting DNA preparations by polymerase chain reaction (PCR). Amplification reactions were prepared using 12.5 [micro]L of high-fidelity polymerase solution (OneTaq, New England Biolabs, Ipswich, MA), 0.5 [micro]L of each primer (10 mmol [L.sup.-1]), 1-2 [micro]L of DNA template (10-20 ng [micro][L.sup.-1]), brought to a total volume of 25 [micro]L with ultra-pure water (18 mol [L.sup.-1] 0-cm, 25 [degrees]C). Fragments of the 18S and 28S subunit nuclear rRNA and COI subunit genes, of approximately 1686, 1416, and 591 bp, respectively, were amplified using the primer pairs shown in Table 2. All reactions were performed following the cycling parameters in Table 3 on a PTC-200 Thermal Cycler (MJ Research, Quebec, Canada).

Subsequently, PCR products were visualized and sized by electrophoresis in 1% agarose gels and purified using the Zymo Clean and Concentrate Kit (Irvine, CA), following the manufacturer's protocol. Resulting products were sequenced bi-directionally on a 3730x1 DNA Analyzer (Life Technologies, Grand Island, NY), using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Life Technologies) at New England Biolabs (Ipswich, MA). The mt-COI sequences for sediment-dwelling specimens were obtained from previously published gill metagenomic data (GenBank accession no. SAMN06338154).

Partial sequences for 18S rRNA, 28s rRNA, and mt-COI were aligned using MUSCLE (Edgar, 2004), with default parameters, trimmed to equal length; and pairwise distance matrices (Table 4) were constructed using the Kimura 2 Parameter (K2P) model as implemented in Geneious v8.0.5 (Biomatters Limited, Auckland, New Zealand). The phylogram in Figure 5 was constructed by neighbor joining, as implemented in the Geneious Tree Builder tool, Geneious v8.0.5 (Biomatters), using the Tamura-Nei genetic distance model and an alignment of 573 bp. The tree shown is a consensus of 10,000 bootstrap replicates. Sequences used in these analyses have been deposited in GenBank, and associated genomic DNA samples have been deposited at the Ocean

Genome Legacy Center, Northeastern University (all accession numbers in Table 5).

Results and Discussion

Often exceeding 1 m in body length, Kuphus polythalamius has been reported to occur in shallow coastal waters of the Indo-Pacific in proximity to mangrove forests and coral reefs and in substrates including mud, gravel, and sand (HedIey, 1895; Sivickis, 1928; Turner, 1966). Here we report the collection of several small living specimens matching the anatomical and morphological characteristics of this species (Figs. 2-4), found burrowing in partially decayed wooden substrates in Mabini, Batangas, Philippines, and in a decaying Pandanus trunk in Madang, Papua New Guinea, in 2012 at water depths of about 2-3 m (see Table 1; Figs. 1-4). In addition, we located and examined museum specimens of K. polythalamius that were collected live and for which preserved soft tissues were available. These include a single specimen collected in the Solomon Islands in 1933, upon which the sole published anatomical description of K. polythalamius was based (Turner, 1966). We also examined a single partial specimen collected in soft decaying wood in Tingloy, Batangas, Philippines, in 2012 and previously identified as Dicyathifer mannii.

The morphologically identified wood-dwelling individuals ranged in body length from 3.4 to 19.9 cm prior to preservation (Table 1). To our knowledge, this set of samples included specimens smaller than any living K. polythalamius previously described in the literature, as well as specimens larger than some previously observed burrowing in sediments. To determine whether these small wood-boring specimens are of the same species as the large sediment-dwelling specimens described previously, we compared partial sequences of three genes frequently used in species diagnosis: the 18S and 28S rRNA genes and mt-COI. We determined that all wood- and sediment-dwelling specimens examined were highly similar with respect to all three loci. Among two wood-boring specimens and one sediment-dwelling specimen examined, the 18S and 28S rRNA sequences were greater than 99.94% and 99.05% identical, respectively. The phylogram shown in Figure 5 illustrates that, based on mt-COI sequences, wood-boring and sediment-dwelling specimens examined in this study form a single well-supported clade that is well differentiated from otheraccepted teredinid species. Furthermore, evolutionary distances within this clade, and between this clade and members of other teredinid species, are comparable to distances observed within and between accepted teredinid species, respectively. For example, the lowest and highest pair-wise divergences observed among mt-COI sequences were 0.09% and 1.22%, respectively (Table 4). This difference compares with previously observed intraspecies variations of 0.0%--1.7% among specimens within 4 shipworm species (Bankia carinata, Lyrodus pedicellatus, Teredothyra dominicensis, and Neoteredo norvagica). In contrast, interspecies sequence divergences ranged from 19.3% to 33.8% among the same species (Borges et al., 2012; Shipway et al., 2014). On this basis we conclude that both the wood-boring and sediment-dwelling specimens examined here are members of the single teredinid species K. polythalamius.

Historical reports, based primarily on observation of calcareous hard parts and dating back more than 300 years, suggest a broad distribution of K. polythalamius across the Indo-West Pacific region (e.g., Rumphius, 1705; Griffiths, 1806; Caiman, 1927 quoted in Societies and Academies, 1927; Si-vickis, 1928; Haga, 2011). The data presented here confirm a recently (since 1933) observed geographic range for living K. polythalamius that extends at least 3000 miles from the northern Philippine island of Luzon to Papua New Guinea and the Solomon Islands (Fig. 1).

Observations on the anatomy, morphology, and behavior of the newly discovered specimens provide previously unknown or unconfirmed details regarding the reproductive strategy and life history of K. polythalamius. For example, one wood-boring specimen (4.2 cm in body length) was observed to release eggs upon removal from the wood, suggesting that this individual was a mature female and demonstrating that K. polythalamius reproduces by broadcast spawning, as do most Teredinidae. Although the viability of the gametes was not confirmed, the presence of an egg-bearing individual in wood suggests that this species may reach maturity and reproduce within wood, potentially completing the entire life cycle without entering sediments. We also suggest that the anatomy of the tubes likely rules out intromission, a form of reproduction that, although rare in Bivalvia, has been observed in the teredinid genera Bankia and Nausitora (Clapp, 1951 quoted in Turner, 1966; Hiroki et ah, 1994; Velasquez et al., 2011). In these taxa, the male inserts the excurrent siphon into the incurrent siphon of the female before releasing sperm, resulting in internal fertilization. We observed that, even among small specimens of K. polythalamius, the movement of the siphons is strictly limited by a septum dividing the tube and by bifurcating calcareous tube extensions (see Fig. 2B), likely preventing intromission. These observations are consistent with the previous hypothesis that broadcast spawning is an ancestral condition in Teredinidae, whereas more complex reproductive strategies, including internal fertilization, intromission, larvipary, and male dwarfism, are derived traits confined to the lineage containing the genera Bankia, Nausitora, Teredo, Lyrodus, and Zachsia (Distel et al., 2011; Ship way era/., 2016).

Although K. polythalamius has long been thought to be a soft bottom dweller (Turner, 1966), the early life history and potential association of this species with wood has remained controversial. In 1927, Caiman (quoted in Societies and Academies, 1927) postulated that K. polythalamius is, in fact, the mature form of the wood-boring species Dicyathifer mannii, the pallets of which are very similar to those of K. polythalamius. Sivickis (1928) later argued against a wood-boring stage for K. polythalamius, citing a large colony of these animals found near Puerto Galera, Mindoro, in which "young" and "old" animals lived near each other, with all of them being buried in sand. Savazzi (1982), like Caiman (1927 quoted in Societies and Academies, 1927), postulated, without evidence, that K. polythalamius initially settled on wood and subsequently transitioned to the sediment. However, to our knowledge, there are no verified observations of this species in wood in peer-reviewed literature prior to those presented in this investigation. Our discovery of small individuals of K. polythalamius burrowing in wood in four distant geographic locations confirms that the larvae of K. polythalamius can settle, metamorphose, and begin early development on wood. This pattern is consistent with the fact that all teredinid bivalves whose life histories have been described settle and metamorphose on wood (Turner, 1966, 1969).

We note, however, that the small size of specimens found on wood is not necessarily an indication that these specimens are younger than the larger specimens found in sediment. An alternate hypothesis is that individuals of K. polythalamius can settle and metamorphose on either wood or sediment, but that those settling on wood grow substantially more slowly and do not attain the very large sizes achieved by individuals settling on sediment. In other words, the small animals observed in wood may be stunted, rather than young.

Although direct evidence is lacking, several observations argue against this hypothesis. First, in Kalamansig. where large sediment-dwelling specimens are abundant, all specimens of K. polythalamius observed in sediment to date have been greater than 11 cm in length. However, in this same location, smaller wood-boring specimens and large sediment-dwelling specimens have been observed growing in close proximity, suggesting that recruitment in this location is to wood rather than to sediment. Additionally, the largest specimens of K. polythalamius found in wood are substantially larger than the smallest specimens found in sediments, indicating that individuals can achieve significant sizes in wood. Finally, in all observed locations, all individuals smaller than 11 cm in length have been found in wood, indicating that individuals on wood either are constrained by the size of the wooden substrate and cannot grow larger or may transition from wood to the sediment, where they may continue to grow after the wood is decayed or consumed. Given these observations, and in the absence of evidence of radically different growth rates of wood-boring and sediment-dwelling individuals, it seems safest to assume that the small wood-boring specimens are juveniles or young adults and that the reproductive cycle of K. polythalamius resembles that of most teredinid species: beginning with broadcast spawning of eggs and larvae, followed by a period of planktonic development, settlement on a woody substrate, metamorphosis, and subsequent sexual maturation. We suggest that the very large size achieved by sediment-dwelling individuals of K. polythalamius more likely reflects the fact that these specimens can continue to grow unconstrained by the limited physical dimensions of wooden substrates, rather than a radical difference in their growth rates.

In a previous investigation, the gill symbiont community of large sediment-dwelling specimens of K. polythalamius was shown to be comprised nearly entirely of sulfur-oxidizing chemoautotrophic (thioautotrophic) bacteria (Distel et al., 2017) rather than cellulolytic symbionts, as observed in other shipworm species (Distel et al., 1991, 2002; Luyten et al., 2006; O'Connor et al., 2014). It was proposed that these thioautotrophic symbionts oxidized reduced sulfur compounds produced by microbial degradation of wood as an energy source and fixed inorganic carbon via the Calvin-Benson-Bassham cycle. The resulting fixed carbon was proposed to provide a source of carbon nutrition to the host, instead of, or in addition to, carbon derived from organic sources. It has been shown previously that sulfate reduction on decaying wood can produce sufficient quantities of reduced sulfur compounds to support thioautotrophic metabolism and thioautotrophic symbioses (Laurent et al., 2009, 2013; Fagervold et al., 2012; Bienhold et al., 2013; Yiicel et al., 2013).

It remains an open question whether the small wood-dwelling specimens of K. polythalamius observed here also harbor thioautotrophic symbionts or whether these small wood-dwelling individuals harbor cellulolytic symbionts. In addition to thioautotrophic symbionts, which dominate the symbiont community of large sediment-dwelling specimens of K. polythalamius (Distel et al., 2017), Teredinibacter turnerae, a widespread cellulolytic symbiont of Teredinidae, was also isolated from these tissues. It is therefore likely that cellulolytic symbionts are also present in the small wood-boring specimens, suggesting the possibility that cellulolytic symbiosis may play an important role in metabolism during wood-dwelling stages. If so, this species may undergo a shift from cellulolytic symbiosis to thioautotrophic symbiosis during later development.

Although we cannot rule out the possibility that K. polythalamius may be capable of wood feeding at earlier stages in development, as are other shipworms species (Mann and Gal-lager, 1985), the specimens examined here did not appear to rely heavily on wood as a food source. First, as previously reported (Distel et al., 2017), a calcareous cap sealed the anterior end of the tubes of most of the wood-boring specimens examined (Fig. 2B, 2C). The same was true for most sediment-dwelling specimens (Fig. 2D). This cap forms a barrier between the valves and the excavation face of the burrow, thus precluding wood feeding by the wood-boring specimens, as well as ingestion of sediments by the large sediment dwellers, as long as the cap is in place. Although the tubes must be uncapped at least intermittently to facilitate burrowing and growth, it appears that the tubes are likely sealed during a significant portion of the life cycle. Second, the small wood-boring specimens, like their larger sediment-dwelling counterparts, have a greatly reduced digestive system. In both cases, a well-developed wood-storing cecum, thought to be critical to wood digestion in other Teredinidae, is not evident. Third, the posterior adductor muscles in the wood-dwelling specimens are reduced in size, as are those of the large sediment-dwelling specimens previously examined. This reduction has previously been interpreted as a lack of specialization for wood boring (Turner, 1966). Fourth, the shell valves of both wood-and sediment-dwelling specimens of K. polythalamius lack the small, sharp, and finely sculpted shell teeth that, in other teredinid species, are used to produce fine micron-scale particles that facilitate wood digestion (Fig. 4). Instead, the shell teeth of K. polythalamius are large, broad, blunt, triangular, or flattened protrusions. These may be suitable for burrowing in sediment or wood but would not be expected to produce fine particles similar to those made by wood-feeding species. Finally, we were unable to identify wood particles in the digestive system of the examined specimens. Taken together, the reduced digestive system, the reduced shell valves and musculature, the lack of fine shell dentition, the absence of a wood-storing organ, the intermittent capping of the burrow, and the absence of wood in the digestive tract suggest that wood likely played a limited role in the nutrition of both the wood-boring and sediment-dwelling specimens of K. polythalamius examined to date. Additional physiological, microbiological, genomic, and biochemical examination of these specimens, and of earlier life stages of this species, will be required to more fully address the extent to which reduced sulfur compounds, wood, or other particulate food sources such as phy-toplankton or sediments may contribute to the nutrition of this species.

Clearly, many aspects of the biology and life history of K. polythalamius remain to be uncovered. The description of small specimens of K. polythalamius, and the discovery that they burrow in wood, adds substantially to the sparse body of knowledge available for this rare and rarely observed species. These realizations point out the need to include woody substrates in the effort to uncover the geographic range, preferred habitats, life cycle, and early life history of this elusive species that has remained shrouded in mystery for more than 300 years.

Acknowledgments

We thank Terry Gosliner (California Academy of Sciences), Adam J. Baldinger (Harvard University Museum of Comparative Zoology), and both Philippe Bouchet and Nicolas Puil-landre (Museum National d'Histoire Naturelle [MNHN]) for providing access to specimens. We are also grateful to Barbara Buge (MNHN) for help in data management and curation. The MNHN Kuphus polythalamius specimens originate from the expedition conducted as part of the Our Planet Reviewed program with Pro-Natura International (Papua Niugini 2012, in partnership with University of Papua New Guinea and the National Fisheries College). This expedition was made possible by grants and supports from the Prince Albert II of Monaco Foundation, the Total Foundation, the Stavros Niarchos Foundation, and Papua New Guinea's National Fisheries Authority. The research reported in this publication was supported by Fogarty International Center of the National Institutes of Health Award U19TW008163 (to MGH, GPC, and DLD), by National Science Foundation Award IOS1442759 (to DLD), and by Japan Society for the Promotion of Science KAKENHI grant 237855 (to TH). 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 Permits GP-0054-11 and GP-0107-15 issued by DA-BFAR. The content of this manuscript is solely the responsibility of the authors and does not necessarily represent the official views of the National Science Foundation or the National Institutes of Health.

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J. REUBEN SHIPWAY (1), MARVIN A. ALTAMIA (2), TAKUMA HAGA (3), MARCEL VELASQUEZ (4) JULIE ALBANO (5), RANDE DECHAVEZ (5), GISELA P. CONCEPCION (2,6), MARGO G. HAYGOOD (7), AND DANIEL L. DISTEL (1*)

(1) Ocean Genome Legacy Center, Department of Marine and Environmental Science, Northeastern University, Nahant, Massachusetts 01908; 'Marine Science Institute, University of the Philippines, Diliman, Quezon City 1101, Philippines; (3) Department of Geology and Paleontology, National Museum of Nature and Science, Tsukuba, Ibaraki 305-0005, Japan; (4) Museum National d'Histoire Naturelle, Sorbonne Universites, 75005 Paris, France; ' Sultan Kudarat State University, Tacurong City 9800, Sultan Kudarat, Philippines; (6) Philippine Genome Center, University of the Philippines System, Diliman, Quezon City 1101, Philippines; and (7) Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah 84112

Received 6 April 2018; Accepted 21 August 2018; Published online 5 November 2018.

(*) To whom correspondence should be addressed. E-mail: d.distel@northeastern.edu.

Abbreviations: mt-COI. mitochondrial cytochrome oxidase I gene; PCR. polymerase chain reaction; rRNA. ribosomal RNA.
Table 1
Specimens used in this investigation

No. and          Holding
specimen ID      institution  Collection location

1. PMS-1656S     UP           Kalamansig, Sultan Kudarat, Philippines
2. PMS-1677P     UP           Kalamansig, Sultan Kudarat, Philippines
3. PMS-1671X     UP           Kalamansig, Sultan Kudarat, Philippines
4. PMS-2133X     LP           Kalamansig, Sultan Kudarat, Philippines
5. PMS-2132W     UP           Kalamansig, Sultan Kudarat, Philippines
6. PMS-2193M     UP           Kalamansig. Sultan Kudarat. Philippines
7. PMS-3404P     UP           Mabini, Batangas, Philippines
8. PMS-3405S     UP           Mabini, Batangas. Philippines
9. PMS-3457L     UP           Mabini, Batangas, Philippines
10. PMS-3477H    LP           Mabini, Batangas. Philippines
11. PMS-3495U    UP           Mabini, Batangas, Philippines
12. PMS-3664H    UP           Mabini. Batangas. Philippines
13. PMS-3696Y    UP           Mabini. Batangas. Philippines
14. PMS-3715U    UP           Mabini, Batangas. Philippines
15. PMS-3720K    L'P          Mabini, Batangas, Philippines
16. PMS-3721L    UP           Mabini, Batangas, Philippines
17. CAS 190664   CAS          Tingloy, Batangas, Philippines
18. MNHN120508   MNHN         Madang, Papua New Guinea
19. MNHN 120509  MNHN         Madang, Papua New Guinea
20. MCZ 229089   MCZ          Solomon Islands

No. and                                      Water depth  Tube length
specimen ID      Collection date  Substrate  (m)          (cm)

1. PMS-1656S     November 2010    Sediment   <3            92.4
2. PMS-1677P     November 2010    Sediment   <3            66.3
3. PMS-1671X     November 2010    Sediment   <3            94.0
4. PMS-2133X     December 2011    Sediment   <3           106.7
5. PMS-2132W     December 2011    Sediment   <3           115.1
6. PMS-2193M     December 2011    Sediment   <3            27.9
7. PMS-3404P     May 2016         Wood       <2             9.3
8. PMS-3405S     May 2016         Wood       <2            10.5
9. PMS-3457L     May 2016         Wood       <2            24.4
10. PMS-3477H    May 2016         Wood       <2            10.6
11.PMS-3495U     May 2016         Wood       <2            35.0
12. PMS-3664H    May 2016         Wood       <2
13. PMS-3696Y    May 2016         Wood       <2             9.5
14. PMS-3715U    May 2016         Wood       <2             5.4
15. PMS-3720K    May 2016         Wood       <2             5.3
16. PMS-3721L    May 2016         Wood       <2             6.0
17. CAS 190664   November 2012    Wood
18. MNHN120508   November 2012    Wood       <3
19. MNHN 120509  November 2012    Wood       <3
20. MCZ 229089   1933             Sediment

No. and          Body length
specimen ID      (cm)

1. PMS-1656S     61.7
2. PMS-1677P     47.8
3. PMS-1671X     51.7
4. PMS-2133X     63.5
5. PMS-2132W
6. PMS-2193M     10.8
7. PMS-3404P
8. PMS-3405S      4.2
9. PMS-3457L     14.1
10. PMS-3477H     5.1
11. PMS-3495U    19.9
12. PMS-3664H     5.8
13. PMS-3696Y     7.7
14. PMS-3715U     3.4
15. PMS-3720K
16. PMS-3721L
17. CAS 190664
18. MNHN120508
19. MNHN 120509
20. MCZ 229089

Table 2
Amplification and sequencing primers used in this study

Primer name              Primer sequence (5'--3')

18S EukF (forward)       WAYCTGGTTGATCCTGCCAGT
18S EukR (reverse)       TGATCCTTCYGCAGGTTCACCTAC
28S-NLF184-21 (forward)  ACCCGCTGAAYTTAAGCATAT
28S-NLR1600 (reverse)    AGCGCCATCCATTTTCAGG
COI-1498-F (forward)     AAT-CAT-AAA-GAA-ATT-GGT-ACT-TTG-TAT-TT
COI-854-R (reverse)      TCW-GGR-TGW-CCA-AAA-AAY-CAA-AA

Primer name              Target    Reference

18S EukF (forward)       18S rRNA  Medlin et al., 1988
18S EukR (reverse)       18S rRNA  Medlin et al., 1988
28S-NLF184-21 (forward)  28S rRNA  Distel et al., 2011
28S-NLR1600 (reverse)    28S rRNA  Distel et al., 2011
COI-1498-F (forward)     mt-COI    This study
COI-854-R (reverse)      mt-COI    This study

mt-COI. mitochondrial cytochrome oxidase I gene; rRNA, ribosomal RNA.

Table 3
Amplification cycling profiles

Primer target  1 cycle

18S rRNA       94 [degrees]C, 3 min
28S rRNA       94 [degrees]C, 3 min
mt-COl         94 [degrees]C, 3 min

Primer target  35 cycles

18S rRNA       94 [degrees]C for 20 s, 64 [degrees]C for 40 s, 68
               [degrees]C for 60 s
28S rRNA       94 [degrees]C for 20 s, 63 [degrees]C for 30 s, 68
               [degrees]C for 60 s
mt-COl         94 [degrees]C for 30 s, 51 [degrees]C for 90 s, 72
               [degrees]C for 60 s

Primer target  1 cycle

18S rRNA       68 [degrees]C, 3 min
28S rRNA       68 [degrees]C, 3 min
mt-COl         72 [degrees]C, 3 min

mt-COl, mitochondrial cytochrome oxidase 1 gene; rRNA, ribosomal RNA.

Table 4
Percent pairwise nucleotide identity matrix for mitochondrial
cytochrome oxidase I (mi-COl) genes of specimens from sediments and
wood

No. and specimen ID  Location and substrate  1    2      3      4

1. PMS-2133X         Philippines, sediment   ...  99.21  99.65  99.3
2. PMS-3664H         Philippines, wood       ...  ...    99.21  99.91
3. CAS 190664        Philippines, wood       ...  ...    ...    99.3
4. MNHN120508        Papua New Guinea, wood  ...  ...    ...    ...
5. MNHN120509        Papua New Guinea, wood  ...  ...    ...    ...
6. MNHN80001         Papua New Guinea, wood  ...  ...    ...    ...

No. and specimen ID  5      6

1. PMS-2133X         99.13  99.13
2. PMS-3664H         99.39  99.74
3. CAS 190664        98.78  99.13
4. MNHN120508        99.48  99.83
5. MNHN120509        ...    99.65
6. MNHN80001         ...    ...

Table 5
Genomic DNA and sequence accession numbers

Specimen ID  GenBank 18S  GenBank 28S  GenBank mt-COI  OGL no.

PMS-2132W    MH516332     MH516335     MH521028
PMS-3696Y    MH516333     MH516336     MH521029        A31831
PMS-3715U    MH516334     MH516337                     A31836
MNHN120508                             MH521030        A31832
MNHN120509                             MH521031        A31833
MNHN80001                              MH521032        A31834
MCZ 229089                             MH521033        A31835

mt-COI, mitochondrial cytochrome oxidase I gene; OGL, Ocean Genome
Legacy Center. Northeastern University.
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Author:Shipway, J. Reuben; Altamia, Marvin A.; Haga, Takuma; Velasquez, Marcel; Albano, Julie; Dechavez, Ra
Publication:The Biological Bulletin
Article Type:Report
Date:Dec 1, 2018
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