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New species of bone-eating worm Osedax from the abyssal South Atlantic Ocean (Annelida, Siboglinidae)

Abstract A new species of bone-eating annelid, Osedaxbraziliensissp. n., found in a sunken whale carcass at a depth of 4,204 m at the base of the São Paulo Ridge in the South Atlantic Ocean off the Brazilian coast is described. The organism was retrieved using the human-occupied vehicle Shinkai 6500 during the QUELLE 2013 expedition. This is the 26th species of the genus and the first discovery from the South Atlantic Ocean, representing the deepest record of Osedax worldwide to date. This species morphologically resembles Osedaxfrankpressi but is distinguished by the presence of a yellow bump or patch behind the prostomium and its trunk length. Molecular phylogenetic analysis using three genetic markers (COI, 16S, and 18S) showed that O.braziliensissp. n. is distinct from all other Osedax worms reported and is a sister species of O.frankpressi.


Introduction
Whale falls provide an extensive food supply to the oligotrophic deep-sea environments and harbour a unique biological assemblage, which is considered a "whale-fall ecosystem" (Smith et al. 1989(Smith et al. , 2015. This ecosystem is known to be chemosynthesis-based, similar to hydrothermal vent and hydrocarbon seep ecosystems, but dynamic succession has been reported (Smith and Baco 2003). Numerous scavengers such as deep-sea sharks, hagfish, and small crustaceans devour the soft whale tissues when the carcasses reach the deep-sea floor (mobile-scavenger stage) (Smith and Baco 2003). After consumption of most soft tissues, organically enriched sediments and exposed bones are colonised by dense assemblages of opportunistic polychaetes and crustaceans (enrichment opportunist stage) (Smith and Baco 2003). Reducing chemicals such as sulphide are produced through anaerobic bacterial decomposition of organic materials in bones and support chemoautotrophs and chemoautotrophic symbiont-harbouring invertebrates as energy sources of primary production (sulphophilic stage) (Smith and Baco 2003). After the depletion of organic materials in the whale bones, the exhausted bones are thought to act as colonisation substrata primarily for suspension feeders (reef stage) but have never been observed in situ (Smith and Baco 2003).
One of the most unique organisms that has appeared in the whale-fall environment is an annelid polychaete of genus Osedax Rouse, Goffredi & Vrijenhoek, 2004 (Annelida, Siboglinidae), commonly known as bone-eating worms, discovered in whale carcasses in Monterey Bay (Rouse et al. 2004). Unlike other siboglinids, Osedax lack a discrete trophosome, the organ housing symbiotic bacteria in vestimentiferans and pogonophorans (Rouse et al. 2004). Instead, female Osedax possesses a vascularised "root" system penetrating the bone marrow (Rouse et al. 2004). Osedax worms are believed to acquire nutrition from the bones through their roots (Tresguerres et al. 2013, Miyamoto et al. 2017. Intracellular heterotrophic bacteria localise in the roots, but their role remains unclear (Goffredi et al. 2014). Surprisingly, all the visible worms are female, and the males are dwarfs, with the exception of one species . Morphological characterisation of Osedax has been minimal thus far. The body size, palp colour, presence/absence of pinnules and their location on palps, presence/ absence of oviduct and its length, and root form are examples of the limited characteristics used for species identification (Vrijenhoek et al. 2009).
In 2013, the Iatá-Piúna Expedition, a collaborative scientific cruise between Japan and Brazil, was conducted within the framework of the around-the-world research cruise Quelle 2013 (Quest for the Limit of Life) of Japan Agency for Marine-Earth Science and Technology (JAMSTEC) using the HOV Shinkai 6500 (Sumida et al. 2016). A sunken whale carcass was discovered at a depth of 4,204 m at the base of the São Paulo Ridge in the South Atlantic Ocean (Sumida et al. 2016). This was the first record of a natural whale fall in the deep Atlantic Ocean (Sumida et al. 2016). Forty-one benthic taxa including many new species were documented from the carcass in which galatheid crabs, Rubyspira gastropods, and polychaete annelids were dominant (Silva et al. 2015, Sumida et al. 2016, Shimabukuro et al. 2017a, 2017b. The skeleton belonged to an Atlantic minke whale (Balaenoptera bonaerensis) and was composed of nine caudal vertebrae, four of which were colonised by Osedax worms (Sumida et al. 2016, Alfaro-Lucas et al. 2017. Vertebrae not colonised by Osedax were well preserved and in a highly sulphophilic stage with chemosynthetic bacterial mats and numerous epifaunal organisms that fed on them. In contrast, vertebrae colonised by Osedax were heavily degraded and did not exhibit evidence of a sulphophilic stage, harbouring a distinct epifaunal assemblage (Alfaro-Lucas et al. 2017). A molecular phylogenetic analysis using mitochondrial COI sequences showed that the Osedax species from the São Paulo Ridge did not match any other sequences previously reported; therefore, the specimen was thought to be a new species.
Here we report a new species of Osedax collected from the South Atlantic at the deepest point recorded for this genus. Morphological and molecular phylogenetic characteristics are described.

Specimen collection
Whale vertebrae harbouring Osedax worms were collected at a depth of 4,204 m at the base of the São Paulo Ridge (28°31.12'S, 41°39.41'W), southwest Atlantic Ocean during the HOV Shinkai 6500 dives on April 24, 2013 (dive #1334), and April 26, 2013 (dive #1336), in the YK13-04 leg1 cruise using R/V Yokosuka (Figs 1-2). Upon recovery, the bones were immediately transferred to fresh chilled seawater (4 °C). Osedax worms were carefully removed from the bones under an on-board microscope just after the bone retrieval.

Treatment for electron microscopic observation
Whole bodies of Osedax worms (n = 21) were fixed with 2.5% glutaraldehyde in filtered seawater for 24 h and preserved in filtered seawater with 10 mM sodium azide at 4 °C. Samples were then washed in filtered seawater and fixed with 2% osmium tetroxide in filtered seawater for 2 h at 4 °C. For scanning electron microscopic observation, each sample was rinsed with distilled water and incubated with 1% aqueous tannic acid (pH 6.8) for 1 h for conductive staining. These samples were again washed with distilled water and treated with 1% aqueous osmium tetroxide for 1 h. The worms were dehydrated in a graded ethanol series and critical point-dried using a JCPD-5 critical point dryer (JEOL, Akishima, Japan). The samples were coated with osmium using a POC-3 osmium plasma coater (MEIWAFOSIS Co., Osaka, Japan). The coated tissues were then observed using a JSM-6700F field-emission scanning electron microscope (JEOL) at an acceleration voltage of 5 kV.

DNA preparation
DNA was extracted from the root tissues of nine the Osedax worms. To reduce surface contaminants, each tissue sample was thoroughly washed in autoclaved and filtered (0.22 µm) seawater. DNA extraction from tissue samples was conducted separately using the DNeasy Tissue Kit (Qiagen Japan, Tokyo, Japan), following the instruction provided by the manufacturer.

Polymerase chain reaction (PCR) amplification, cloning, and sequencing
PCR amplifications were conducted using an Ex Taq PCR kit (TaKaRa, Kyoto, Japan) for three different molecular markers: cytochrome c oxidase subunit I (COI), 16S rRNA (16S), and 18S rRNA (18S). Two oligonucleotide primers (0.2 µM each) and <1 µg of DNA template were added to the reaction mixtures. Thermal cycling was performed as follows: denaturing at 96 °C for 20 s, annealing at 55 °C for 45 s, and extension at 72 °C for 2 min for a total of 35 cycles. The oligonucleotide primer sequences used for the PCR amplification are shown in Table 1. The molecular sizes of the PCR products were confirmed with 1.2% Agarose S (Nippon Gene, Toyama, Japan) gel electrophoresis. The PCR products were purified using the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA). The DNA sequencing reaction was performed using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Specific primers for each gene were used in sequencing reactions according to the manufacturer's recommended procedure (Table 1). Sequencing was performed using an ABI PRISM 3100 genetic analyser (Applied Biosystems).

Phylogenetic analysis
Partial sequences of the COI, 16S, and 18S genes were analysed using the gapped-BLAST search algorithm (Altschul et al. 1997, Benson et al. 2000 to estimate the degree of similarity to other related sequences. Additional sequences of siboglinids for phylogenetic analyses were obtained from the non-redundant nucleotide sequence database of the DNA Data Bank of Japan (Kodama et al. 2018) (Table 2). Sequences were aligned using CLUSTAL X (Larkin et al. 2007), followed by automatic editing of the resulting alignments using the GBLOCKS program for all the genetic markers under the options allowing smaller final blocks, gap positions within the final blocks, and less strict flanking positions (Castresana 2000, Talavera andCastresana 2007). The alignments (34 OTUs / 3,112 bp in total) are available upon request from the corresponding author. The maximum likelihood (ML) analysis was performed using the RAxML-VI-HPC program (Stamatakis 2006). Evolutionary models for each marker (GTR + γ) were separately estimated using KAKUSAN4 software (Tanabe 2007). The ML bootstrap analyses (1,000 replicates, -f option) were constructed as in the model and using the settings described earlier in this section.

COI genetic distance
Minimum genetic distances based on Kimura 2 parameters (K2P) model were calculated between Osedax species using MEGA7 software (Kumar et al. 2016). These distances were calculated using the COI alignment used in the phylogenetic analyses without gaps.    Description. Genetic data (COI, 16S, and 18S) deposited in DDBJ (LC106303, LC381421, LC381422, LC381424, and LC381765-LC381787). Trunk length up to 22 mm, width at collar 0.5 mm, reddish purple while alive and whitish after fixation ( Fig. 3A-C); gelatinous hemispherical tube encases trunk and base of palps, 1-2 mm thick, contains eggs and dwarf males (Fig. 3A, C). Prostomium whitish while alive, present at top of trunk. Yellow bump or patch present behind prostomium: this yellow bump or patch size varies among individuals, biggest bump reaches top of trunk, and is absent in some specimens (Fig. 3D-F). Crown consists of four palps; palps about 1.5 mm length, red colour while alive with two whitish stripes on the inner side, fused for about 30% of length; pinnules on inner margin of palps, about 50-250 µm, 7-8 pinnules in transverse rows (Figs 3A,C,4B,C). Oviduct free to base, adjoined to the trunk at opposite side of prostomium region, reaching up to 20-30% of palp length (Fig. 4C). Ovisac whitish; trunk-ovisac junction about 15% of trunk length, light green while alive (Fig. 3B). Root lobulated without branching, yellow greenish while alive and whitish after fixation; intracellular symbiotic bacteria in root tissue (Fig. 3B). Eggs about 150 µm diameter (n = 20), whitish while alive (Fig. 3B).
Etymology. This species is named after the type locality, Brazil. This name is an adjective used as a substantive in the genitive case.    Phylogenetic analysis. The final lengths of the aligned sequences were 1,004 bp (COI), 486 bp (16S), and 1,604 bp (18S). The phylogenetic position of O. braziliensis sp. n. determined from our ML analysis recovered, with total support, a distinct species from that of all other Osedax species reported (Fig. 5). The six Osedax clades proposed by Rouse et al. (2018) were recovered. The phylogenetic analysis showed that O. braziliensis sp. n. falls into Clade IV, and is a sister species of O. frankpressi known from Monterey Bay at depths between 1,820 m and 2,898 m (Fig. 5).
Remarks. This species resembles Osedax frankpressi in the pinnules distributed only at the inner margin of palps, lobulated root structure without branching, gelatinous hemispherical tube, and dwarf males (Rouse et al. 2004). However, it can be discriminated from O. frankpressi by the presence of the yellow bump or patch behind the prostomium, trunk length, and genetic data. In O. braziliensis sp. n., the yellow bump or patch was present in some specimens including holotype, and the trunk length is long (6-22 mm), whereas in O. frankpressi, the bump or patch is absent in all specimens, and the trunk length is shorter (4.5 mm). COI genetic distances between O. braziliensis sp. n. and O. frankpressi are 0.111-0.117, which are greater than intraspecific values in O. braziliensis sp. n. (0.001-0.006). Genetic distances between O. braziliensis and the rest of the Osedax taxa for the COI ranged from 0.117 to 0.236 (Table 3).