Description of a new Osedax (Annelida, Polychaeta, Siboglinidae) species colonizing cow bones in the South Atlantic Ocean

Thammy Gularte; Paulo Y. G. Sumida; Gilberto Bergamo; Greg W. Rouse

doi:10.3897/zookeys.1219.134005

Abstract

A new species of Osedax is described here using molecular and morphological data. It was found at the depth of 550 m off the Brazilian coast through experimental deployment of cow bones. Osedax nataliae sp. nov. is the second Osedax species from the Southwest Atlantic Ocean and had been previously reported as Osedax ‘BioSuOr-4’. Phylogenetic analysis of five concatenated genetic makers (28S rDNA, Histone H3, 18S rDNA, 16S rDNA, and cytochrome c oxidase I) placed Osedax nataliae sp. nov. within a well-supported Osedax Clade V, nested within a clade of Pacific Ocean Osedax though with poor support. The minimum interspecific COI distance between O. nataliae sp. nov. and another known Osedax was 13.92% (closest to O. ‘sagami-3’). The maximum intraspecific COI diversity (uncorrected) within O. nataliae sp. nov. sampled here was 2.44% and population structure was visualized via haplotype network analysis. Morphologically, O. nataliae sp. nov. is characterized by its reddish orange crown of palps and a ventral yellowish collar on the anterior trunk where it meets the base of the crown. Osedax nataliae sp. nov. shares features with other Clade V species, notably pinnules inserted on the outer margin of palps. Additionally, the presence of dwarf males within the tube lumen of females was documented. Further sampling and research in the Southern Hemisphere are needed to understand the diversity and biogeography of Osedax across the world’s oceans.

Key words

New species, phylogeny, taxonomy, whale falls

Introduction

Dead whale carcasses are one of the most remarkable energy sources for a plethora of deep-sea organisms as they sink and reach the deep seafloor, known as whale falls, becoming a habitat island for many species during decomposition, which can last for a few years to decades (Lundsten et al. 2010; Smith et al. 2015). The enormous input of organic matter from whale falls supports diverse ecosystems, rich in opportunistic species capable of tolerating the organic matter decomposition (Smith and Baco 2003; Gaudron et al. 2010). Still, as with vents and seeps, these ecosystems can have unique and specialized species and communities (Smith and Baco 2003; Braby et al. 2007; Lundsten et al. 2010; Smith et al. 2015).

The study of these communities revealed some remarkable new species, including the annelid genus Osedax Rouse, Goffredi & Vrijenhoek, 2004 (Siboglinidae), a group of organisms specialized to exploit bones and teeth of dead marine vertebrates (Rouse and Goffredi 2023). Osedax taxa lack a digestive system and have a particular endosymbiotic relationship with heterotrophic bacteria hosted in the branching root system that excavates the bone. The symbiotic bacteria are capable of metabolizing complex carbon compounds within the bones yielding nutrition to both symbionts and the Osedax hosts (Goffredi et al. 2005, 2007; Katz et al. 2010; Tresguerres et al. 2013). Osedax specimens also usually exhibit a marked sexual dimorphism, in which the females can reach 1–10 cm and the males are microscopic dwarfs (paedomorphic) and live in harems attached to the female trunk (Rouse et al. 2004, 2008; Vrijenhoek et al. 2008, 2009). The genus currently has 33 described species in all the ocean basins, with a wide bathymetric range, varying from 21 to 4204 meters (Rouse et al. 2018; Fujiwara et al. 2019; Eilertsen et al. 2020; Georgieva et al. 2023; Berman et al. 2024). More than half of the named species were first collected from the California margin, where whale-fall studies are most numerous and long-standing (Smith et al. 2015).

As in most deep-sea exploration areas of knowledge, the data on whale-fall associated species in the Global South is still limited, and it is predicted that many hundreds of whale-fall species remain to be discovered in these ocean regions (Smith et al. 2015). This prediction relies on the high abundance of large marine mammals in this region (Laws 1977) and their seasonal migratory routes between high-latitude feeding grounds and low-latitude breeding grounds (Dawbin 1966). This knowledge gap is particularly pronounced for Osedax species, as Osedax braziliensis Fujiwara, Jimi, Sumida & Kitazato, 2019 is the only described species for the South Atlantic Ocean (excluding Antarctic and subantarctic species) and it was found inhabiting the first reported natural whale carcass in the deep Southwest Atlantic Ocean at 4204 m depth during an expedition with the human-occupied submersible Shinkai 6500 in 2018 (Fujiwara et al. 2019).

Aiming to reduce the knowledge gap regarding the organic falls community diversity in the South Atlantic Ocean and its global connectivity, the project BioSuOr (“Biodiversity and connectivity of benthic communities in organic substrates in the deep southwest Atlantic”) was conducted between 2016 and 2017, implanting mammalian bones and wood samples in Brazilian deep waters. These samples were implanted at multiple sites across three different depths: 550, 1500 and 3300 m. The deployment of the organic falls was achieved using free-fall landers equipped with acoustic releases for recovery and were subsequently colonized by a variety of polychaetes, crustaceans and mollusks. Some of the results from this project in the Southwest Atlantic Ocean have been documented (Shimabukuro and Sumida 2019; Shimabukuro et al. 2019, 2022; Souza et al. 2021; Avila et al. 2023; Bergamo et al. 2024), reporting the occurrence of 24 animal species (Shimabukuro et al. 2022), including potential new species.

During the sampling of BioSuOr project material, numerous Osedax specimens were observed colonizing the implanted cow bones (Fig. 1). Four Osedax species were recovered from this project and their mitochondrial cytochrome c oxidase subunit I (COI) sequences were reported and lodged on GenBank as BioSuOr-1, 2, 3, and 4 (Shimabukuro and Sumida 2019). Here, we use molecular phylogenetic and morphological data to formally describe Osedax ‘BioSuOr-4’, which was recovered on bones at a depth of 550 m, along with an assessment of its intraspecific diversity. Additionally, we documented the occurrence of dwarf males in this species.

Figure 1.

Cow femur colonized by Osedax individuals in an implanted free-fall lander after recovery.

Materials and methods

Sample collection and morphological analysis

Osedax specimens investigated in this study were obtained through an in-situ experiment that involved implanting bovine bones using experimental autonomous structures (landers) equipped with acoustic releases (see Saeedi et al. 2019) in the Southwest Atlantic Ocean, off the Brazilian continental margin (26°36'13.44"S, 46°09'9.29"W) at 550 m depth (Fig. 2). Landers were deployed in July 2016 using the R/V Alpha Crucis and recovered in May 2017 with the R/V Alucia. Once onboard bones were placed in sea water at 4 °C to photograph living Osedax. Bones with worms were then fixed in 96% ethanol. Osedax specimens were later extracted at Laboratório de Mar Profundo (LAMP), Instituto Oceanográfico, Universidade de São Paulo, with 115 female specimens and tubes extracted using a stereomicroscope. The holotype and attached dwarf males were imaged from fixed material with Leica MZ12.5 (+ Canon Rebel T6i camera), or Leica M205C (+ Leica MC170HD camera) stereomicroscopes. A dwarf male was imaged with a Leica DMR compound microscope and Canon Rebel T6i camera.

Figure 2.

The yellow circle indicates exact position of the lander at 550 m depth off the Brazilian continental margin recovered in May 2017 with the R/V Alucia.

For scanning electron microscopy (SEM) analysis, female specimens were dehydrated in absolute ethanol. They were then rinsed in two 10-min baths in a solution consisting of 50% ethanol and 50% Hexamethyldisilazane (HMDS), followed by an additional two 10-min baths in HMDS alone. Specimens were left to dry overnight, then mounted on stubs using carbon adhesive tape, sputter-coated with gold, and examined and photographed under a Zeiss Sigma VP SEM at Laboratório de Microscopia Eletrônica, Instituto de Biociências, Universidade de São Paulo.

DNA preparation, amplification, and sequencing

DNA of 37 female specimens was extracted from their root regions using Zymo Research DNA-tissue miniprep kits, following the manufacturer’s provided protocol. Extracted DNA was utilized as template for the polymerase chain reaction (PCR) amplification of fragments of mitochondrial cytochrome c oxidase subunit I (COI) and 16S rRNA (16S) genes, and the nuclear 18S rRNA (18S), 28S rRNA (28S) and Histone H3 (H3) genes, using primers shown in Table 1. COI was sequenced for all specimens initially for species delimitation. Subsequently, other markers were sequenced from a single representative specimen.

Table 1.

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List of genes, primers and PCR temperature profiles used in the present study.

Gene	Primer set	Reference	Cycle
Cytochrome c oxidase subunit I (COI)	OsCO1r/OsCO1f	Glover et al. 2005	120s at 95 °C, 35 cycles of 60s at 94 °C, 60s at 50 °C and 60s at 72 °C, 420s at 72 °C
16S rRNA (16S)	16SarL/16SbrH	Palumbi 1996	180s at 95 °C, 35 cycles of 40s at 95 °C, 40s at 50 °C and 50s at 72 °C, 300s at 72 °C
18S rRNA (18S)	18S-1F/18S-5R	Giribet et al. 1996	180s at 95 °C, 40 cycles of 30s at 95 °C, 30s at 50 °C and 90s at 72 °C, 480s at 72 °C
	18S-a2.0/18S-9R	Giribet et al. 1996; Whiting et al. 1997	180s at 95 °C, 40 cycles of 30s at 95 °C, 30s at 50 °C and 90s at 72 °C, 480s at 72 °C
	18S-3F/18S-bi	Giribet et al. 1996; Whiting et al. 1997	180s at 95 °C, 40 cycles of 30s at 95 °C, 30s at 52 °C and 90s at 72 °C, 480s at 72 °C
28S rRNA (28S)	D1F/D3R	Brown et al. 1999	180s at 94 °C, 35 cycles of 60s at 94 °C, 30s at 55 °C and 110s at 72 °C, 240s at 72 °C
Histone H3 (H3)	H3F/H3R	Colgan et al. 1998	180s at 95 °C, 40 cycles of 30s at 95 °C, 45s at 53 °C and 45s at 72 °C, 300s at 72 °C

PCR amplification was conducted using a mixture consisting of 12.5 µl Apex^TM 2.0× Taq Red DNA polymerase Master Mix (Genesee Scientific), 1 µl of each appropriate forward and reverse primers (10 µM), 8.5 µl of ddH2O, and 2 µl of eluted DNA. PCR cycling was conducted in a thermal cycler following specific profiles and temperatures for each primer, as indicated in Table 1. Following confirmation of appropriate bands via gel electrophoresis, PCR products were purified using ExoSAP-IT following the manufacturer’s protocol, and sent to Eurofins Genomics Company (Louisville, Kentucky, USA) for sequencing. Resulting sequences were assembled and edited in Geneious Prime R11.5.1 (Kearse et al. 2012) before deposition in GenBank under accession numbers shown in Table 2.

Table 2.

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List of species and GenBank accession numbers for sequences in this study. New sequences in bold.

Taxa	Source/Authority	COI	16S	18S	28S	H3
Outgroup
Lamellibrachia columna	Webb 1969	DQ996645	FJ347646	FJ347679	MG264417	FJ347696
Riftia pachyptila	Jones 1981	KP119562	KP119573	KP119591	KP119582	KP119555
Sclerolinum brattstromi	Webb 1964	FJ347644	FJ347644	FJ347680	FJ347677	FJ347697
*Osedax*
O. antarcticus	Glover et al. 2013	KF444422	KF444418	KF444420	–	–
O. ‘BioSuOr-1’	Shimabukuro and Sumida 2019	MH616036	–	–	–	–
O. ‘BioSuOr-2’	Shimabukuro and Sumida 2019	MH616081	–	–	–	–
O. ‘BioSuOr-3’	Shimabukuro and Sumida 2019	MH616075	–	–	–	–
O. nataliae sp. nov.	This study; Shimabukuro and Sumida 2019	MH616012–MH616016, PP765811–PP765827, PP982821–PP982840	PP669598	PP669599	PP669600	PP766874
O. bozoi	Berman et al. 2024	ON357627	ON261606	ON261611	ON261610	ON254806
O. braziliensis	Fujiwara et al. 2019	LC381421	–	LC381424	–	–
O. bryani	Rouse et al. 2018	KP119563	KP119574	KP119597	KP119584	KP119561
O. byronbayensis	Georgieva et al. 2023	OQ801427	OQ820973	OQ803227	–	–
O. craigmcclaini	Berman et al. 2024; McClain et al. 2019	MN258704	ON217799	ON220153	ON226742	ON254807
O. crouchi	Amon et al. 2014	KJ598038	KJ598032	KJ598035	–	–
O. deceptionensis	Taboada et al. 2015	KF444428	KF444419	KF444421	MG264418	KT860546
O. docricketts	Rouse et al. 2018	FJ347626	FJ347650	FJ347688	FJ347666	FJ347710
O. estcourti	Berman et al. 2024	ON211943	ON217536	ON220129	ON220739	ON254809
O. fenrisi	Eilertsen et al. 2020	MT556178	–	MT556473	–	–
O. frankpressi	Rouse et al. 2004	FJ347607	FJ347658	FJ347682	FJ347674	FJ347705
O. jabba	Rouse et al. 2018	FJ347638	FJ347647	FJ347693	FJ347676	FJ347703
O. japonicus	Fujikura et al. 2006	FM998111	–	FM995535	–	–
O. knutei	Rouse et al. 2018	FJ347635	FJ347648	FJ347692	FJ347664	FJ347700
O. lehmani	Rouse et al. 2018	DQ996634	FJ347660	FJ347689	FJ347672	FJ347706
O. lonnyi	Rouse et al. 2018	FJ347643	FJ347651	FJ347695	FJ347663	FJ347699
O. ‘MB16’	Salathé and Vrijenhoek 2012	JX280613	KP119581	KP119592	KP119588	KP119560
O. ‘mediterranea’	Taboada et al. 2015	KT860548	KT860551	KT860550	KT860549	KT860547
O. mucofloris	Glover et al. 2005	AY827562	–	–	AY941263	–
O. nordenskjoeldi	Amon et al. 2014	KJ598039	KJ598033	KJ598036	–	–
O. packardorum	Rouse et al. 2018	FJ347629	FJ347661	FJ347690	FJ347673	FJ347707
O. priapus	Rouse et al. 2015	KP119564	KP119575	KP119594	KP119585	KP119556
O. randyi	Rouse et al. 2018	FJ347615	FJ347659	FJ347684	FJ347675	FJ347712
O. rogersi	Amon et al. 2014	KJ598034	KJ598037	KJ598040	–	–
O. roseus	Rouse et al. 2008	FJ347609	FJ347657	FJ347683	FJ347670	FJ347709
O. rubiplumus	Rouse et al. 2004	EU852488	FJ347656	FJ347681	FJ347671	FJ347704
O. ryderi	Rouse et al. 2018	KP119563	KP119574	KP119597	KP119584	KP119561
O. ‘sagami-3’	Pradillon et al. unpublished	FM998081	–	FM995537	–	–
O. ‘sagami-4’	Pradillon et al. unpublished	FM998082	–	FM995541	–	–
O. ‘sagami-5’	Pradillon et al. unpublished	FM998083	–	FM995539	–	–
O. sigridae	Rouse et al. 2018	FJ347642	FJ347655	FJ347694	FJ347669	FJ347711
O. talkovici	Rouse et al. 2018	FJ347621	FJ347654	FJ347685	FJ347668	FJ347698
O. tiburon	Rouse et al. 2018	FJ347624	FJ347653	FJ347687	FJ347662	FJ347702
O. traceyae	Berman et al. 2024	ON211990	ON212680	ON10988	ON220740	ON254808
O. ventana	Rouse et al. 2018	EU236218	FJ347652	FJ347686	FJ347665	FJ347701
O. westernflyer	Rouse et al. 2018	FJ347631	FJ347649	FJ347691	FJ347667	FJ347708
O. waadjum	Georgieva et al. 2023	OQ801430	OQ820974	OQ803228	–	–

Phylogenetic analysis

Assembled sequences were align using MAFFT (Katoh and Standley 2013; Rozewicki et al. 2019) in the program Mesquite (Maddison and Maddison 2019), including sequences from 41 Osedax named and unnamed species and members of three other Siboglinidae genera as outgroups (Table 2).

The most appropriate evolutionary model for each marker was recovered using ModelTest-NG (Darriba et al. 2020). The best models chosen (based on AICc) were COI = GTR+I+G4, 16S = TIM2+I+G4, 18S = GTR+I+G4, 28S = TIM3+I+G4, and H3 = TVMef+I+G4. A maximum likelihood (ML) phylogenetic tree using the concatenated sequences of all five markers was generated using the RAxML GUI program (Edler et al. 2021). Node support was assessed through bootstrapping with 1000 replicates. We chose not to conduct a Bayesian phylogenetic analysis of the data as it would yield very similar estimate of the molecular phylogeny as the maximum likelihood results. Also, as pointed out in Berman et al. (2024), missing data for many Osedax terminals is likely responsible for the lack of well-supported relationships rather than any particular analytical method.

Minimum genetic distance based on uncorrected p–distance of COI was calculated using PAUP* (Swofford 2002) between the sampled specimens and the other 44 sequences from reported Osedax and non-Osedax species. These distances were calculated using the COI alignment employed in the phylogenetic analyses.

To provide insights into the genetic relationships at the population level of the new Osedax species described here, a TCS haplotype network (Clement et al. 2000) using a 426 bp alignment of the COI marker was recovered for the 37 specimens sequenced using the program PopART (Leigh and Bryant 2015).

Results

Taxonomy

Siboglinidae Caullery, 1914

Osedax Rouse, Goffredi & Vrijenhoek, 2004

Osedax nataliae Gularte, Sumida, Bergamo & Rouse, sp. nov

https://zoobank.org/ED3DE09A-776C-46FF-8418-59881D751365

Figs 3, 4, 5

Osedax ‘BioSuOr-4’ sec. Shimabukuro and Sumida 2019.

Type material

Holotype : MZUSP 6201, Female, preserved in ethanol, derived from an experimentally deployed cow bones (Bos taurus) at a depth of 550 m, collected with R/V Alucia on the continental margin off São Paulo state, Brazil (26°36'13.44"S, 46°09'9.29"W) on 18 May 2017. Paratypes: MZUSP 6203–6204, all females (30), preserved in ethanol, collected on cow bones deployed at the same locality and date as the holotype. Two dwarf male (allotypes), fixed in ethanol from tube of holotype: MZUSP 6202, same date and locality as holotype.

Diagnosis and description

Holotype female (Fig. 3A–C); body length ~ 14.7 mm; gelatinous tube (removed) 0.2 wide, longer than trunk and crown; crown of palps, ~ 2.8 mm long; trunk length ~ 7.4 mm, width ~ 0.5 mm; root structure ~ 4.5 mm long; width ~ 2.4 mm; Crown of four pinnulated palps, with the pinnules arranged along the outer margin of the palps (Figs 3B, 4). Conspicuous oviduct shorter than palps (Figs 3B, 4A–D). Collar ventrally along the margin of anterior trunk, except for the dorsal portion (Figs 3B, 4B–E). Live specimens with palps bright red-orange distally, becoming yellow and then white proximally to the boundary with the trunk (Fig. 5). No obvious pigmentation on trunk or demarcation into upper and lower trunk. Root structure missing in holotype, bulbous or lobulate in paratypes. Ovisac an ellipsoidal mass contain oocytes at various stages of development (Fig. 3A). Dwarf male ~ 170 μm in length, fusiform, no appendage organs (Fig. 3C); posterior hooks present (Fig. 3D).

Figure 3.

Osedax nataliae sp. nov. Preserved female holotype (MZUSP 6201) A–C and male specimens (MZUSP 6202) D: A lateral view of the entire specimen B detail of palps and trunk C detail of trunk with male attached to the surface D light microscope of individual male (preserved). Abbreviations: c, collar; m, male; od, oviduct; ov, ovisac; p, palps; pp, pinnules; t, trunk; h, hooks; y, yolk.

Figure 4.

Osedax nataliae sp. nov. Scanning Electron Microscopy (SEM) of two paratypes. Paratype (MZUSP 6204) A dorsal view of palps and trunk end of paratype 1. Paratype (MZUSP 6205) B dorsal view of palps and trunk end C lateral view highlighting oviduct and collar position D detail of the base of the palps E lateral view of the collar F detail of the pinnules in the palps. Abbreviations: c, collar; od, oviduct; p, palps; pp, pinnules; t, trunk.

Figure 5.

Osedax nataliae sp. nov. Live specimens photographed alive onboard R/V Alucia after recovery from the lander. Red palps and trunk are partially extended in gelatinous tubes.

Distribution

Known from the continental margin off São Paulo state, Santos basin, Brazil, at a depth of 550 m; on experimentally deployed cow bones.

Molecular results

The final lengths of sequences for the different genetic markers were 482–600 bp (COI), 454 bp (16S), 1769 bp (18S), 997 bp (28S) and 309 bp (H3). Uncorrected intraspecific divergence of O. nataliae sp. nov. for COI was up to 2.44%. In terms of distance, the most closely related species to O. nataliae sp. nov. was O. ‘sagami-3’, with a minimum interspecific distance for COI of 13.92% (Suppl. material 1). The phylogenetic analysis of the concatenated dataset of the five markers placed Osedax nataliae sp. nov. in the well-supported Clade V (see Rouse et al. 2018) although relationships within the clade were poorly supported. The new species was recovered as sister species to the clade formed by O. ‘sagami-3’, known from NW Pacific at unknown depth, and Osedax roseus, from NW and NE Pacific at depths of 633 to 1820 m (Fig. 6). A total of 22 distinct haplotypes were recovered for the COI dataset (n = 38), with the most common one being shared by ten individuals (Fig. 7). Despite originating from a single experimental lander, the network reveals a central and more common haplotype surrounded by several closely related and some more distant haplotypes with numerous nucleotide substitutions.

Figure 6.

Osedax phylogenetic analysis. Maximum likelihood phylogenetic tree based on a partitioned concatenated dataset of COI, 16S, 18S, 28S, and H3 markers (MAFFT-aligned) for the data shown in Table 2. Bootstrap support values are indicated. Black star values were ≥ 95% (BS). Missing values indicate BS < 50%.

Figure 7.

Haplotype network using COI for 37 Osedax nataliae sp. nov. Circles are haplotypes and crosshatches are single nucleotide substitutions.

Remarks

Osedax nataliae sp. nov. is part of the Clade V according to the phylogenetic analysis (Fig. 6) and shares some important morphological features with the other taxa within this clade, such as pinnules inserted on the outer margin of palps (Fig. 4D, E) and a collar at the base of the crown (Figs 4B, 5B–E). The collar of Osedax nataliae sp. nov. and Osedax roseus (the closest species in molecular phylogeny that has a morphological description) are similar in shape and position, though more inflated in O. nataliae sp. nov. Some specimens of Osedax nataliae sp. nov. appear to lack a collar, which could be an artifact of fixation. The dwarf males of O. nataliae sp. nov., with a length of 170 μm, are notably smaller than the males of O. rubiplumus (400 μm–1.1 mm long) but similar in size to those of O. roseus (130–210 μm) and O. frankpressi (150–250 μm). The body size (length of crown + trunk) of Osedax nataliae sp. nov. females varied markedly among the individuals examined, ranging from 4 mm to 15 mm, with a mean value of 6.76 mm. When compared with groups from the same clade, the body size is like O. roseus, O. bryani, and O. fenrisi females but much smaller than O. rubiplumus. Osedax nataliae sp. nov. is not obviously distinguishable from its relatives on morphology. Its notable features such as the red-orange distal crown of pinnulate palps, yellowing towards the base, collar, and long trunk (Fig. 3) may occur in other species of Clade V, such as O. roseus. However, molecular data from both the phylogenetic analysis (Fig. 6) and COI distance (Suppl. material 1) confirm Osedax nataliae sp. nov. as a new species.

Etymology

This species is named after Natalia Gularte, mother of the first author, in recognition of her long and continued support in this research effort.

Discussion

This study formally describes a second species of Osedax from the South Atlantic Ocean, combining both morphological and molecular approaches. Osedax nataliae sp. nov. was previously reported under the informal epithet of ‘BioSuOr-4’ (see Shimabukuro and Sumida 2019) based on COI alone though its phylogenetic placement was not clear based on such limited data. With the additional data of five molecular markers, O. nataliae sp. nov. joins O. bryani, O. fenrisi, O. craigmcclaini, O. rubiplumus, O. ‘sagami-4’, O. roseus, and O. ‘sagami-3’, to supplement the membership of the well-supported Clade V (Vrijenhoek et al. 2009; Rouse et al. 2015, 2018; Eilertsen et al. 2020; Berman et al. 2024). However, relationships within clade V are poorly supported likely owing to the lack of DNA data for most of the molecular markers used here for terminals such as O. ‘sagami-3’, O. ‘sagami-4’, and O. fenrisi. The present results suggest O. nataliae sp. nov. is nested among a Pacific clade of Osedax (Fig. 5), but sampling of Osedax diversity is presently biased towards that ocean basin.

The migratory routes of many species of whales through the Atlantic Ocean, including the sub-Atlantic populations of humpback whale (Megaptera novaeangliae) which migrate from South Georgia Islands through Rio Grande Rise and northwards to Abrolhos Bank (Best et al. 1993; Zerbini et. al 2006; Santos et al. 2010; Wedekin et al. 2014) suggest that the deep-sea oceans in the Southern Hemisphere may provide a rich supply of whale carcasses to support Osedax species. The report of 16 whale-fall-associated new species, including four Osedax species from the BioSuOr project conducted in the Southwest Atlantic Ocean (Shimabukuro et al. 2022) supports this hypothesis.

The haplotype network for O. nataliae sp. nov. (Fig. 7) reveals high genetic diversity within the study area, with 22 haplotypes recovered from the 37 specimens sequenced. This network also shows no evidence of any population ‘bottleneck’, which would have been indicated by a much lower diversity of haplotypes for the number of specimens sequenced (Avise 2000). This diverse haplotype network aligns with observations seen previously in other Osedax species (Glover et al. 2005; Rouse et al. 2008; Amon et al. 2014).

As part of the BioSuOr project, O. nataliae sp. nov. was exclusively reported from cow bones deployed at a depth of 550 m. No information is available regarding the possibility of this species colonizing only small bones nor is there data on its depth range capability. Further research is needed to investigate the potential substrate preferences and depth range of O. nataliae sp. nov. to better understand its ecological niche and distribution in deep-sea ecosystems.

Acknowledgments

We are thankful to the captains and crews of R/V Alpha Crucis and R/V Alucia for the deployment and recovery of the lander. We would also like to extend our thanks to Mauricio Shimabukuro, Orlemir Carrerette, Daniel M. Couto, Avery S. Hiley, and Sonja Huč for their training contributions and assistance during sample analyses. Thanks also to reviewers Thomas Dahlgren and Sergio Taboada for their valuable critiques.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

The authors would like to acknowledge the funding support provided by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) through grants 2011/50185-1, 2022/04019-7 and 2022/12683-4, and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) through grant 301554/2019-6.

Author contributions

Conceptualization: TG, PYGS, GWR. Methodology: TG, GB, PYGS, GWR. Formal analysis: TG, GB, GWR. Investigation: TG, GB, GWR. Resources: PYGS, GWR. Writing - Original draft: TG, GB. Writing - Review and Editing: TG, PYGS, GB, GWR. Visualization: TG, GB, GWR. Supervision: PYGS, GWR. Funding Acquisition: PYGS, GWR.

Author ORCIDs

Thammy Gularte https://orcid.org/0000-0002-3121-1973

Paulo Y. G. Sumida https://orcid.org/0000-0001-7549-4541

Gilberto Bergamo https://orcid.org/0000-0001-5464-909X

Greg W. Rouse https://orcid.org/0000-0001-9036-9263

Data availability

All data generated during this study are included in this article. Sequences are published in GenBank and BOLD.

References

Amon DJ, Wiklund H, Dahlgren TG, Copley JT, Smith CR, Jamieson AJ, Glover AG (2014) Molecular taxonomy of Osedax (Annelida: Siboglinidae) in the Southern Ocean. Zoologica Scripta 43: 405–417. https://doi.org/10.1111/zsc.12057

Avila AKF, Shimabukuro M, Couto DM, Alfaro-Lucas JM, Sumida PYG, Gallucci F (2023) Whale falls as chemosynthetic refugia: a perspective from free-living deep-sea nematodes. Frontiers in Marine Science 10: 1111249. https://doi.org/10.3389/fmars.2023.1111249

Avise JC (2000) Phylogeography: the history and formation of species. Harvard University Press, Cambridge, USA, 447 pp. https://doi.org/10.2307/j.ctv1nzfgj7

Bergamo G, Carrerette O, Shimabukuro M, Santos CSG, Sumida PYG (2024) Revealing a new eyeless Nereis (Nereididae: Annelida) clade from deep-sea organic falls. Zoological Journal of the Linnean Society 201: 1–31. https://doi.org/10.1093/zoolinnean/zlad122

Berman GH, Hiley AS, Read GB, Rouse GW (2024) New species of Osedax (Siboglinidae: Annelida) from New Zealand and the Gulf of Mexico. Zootaxa 5443: 337–352. https://doi.org/10.11646/zootaxa.5443.3.2

Best PB, Payne R, Rowntree V, Palazzo JT, Both MDC (1993) Long-range movements of South Atlantic right whales Eubalaena australis. Marine Mammal Science 9: 227–234. https://doi.org/10.1111/j.1748-7692.1993.tb00451.x

Braby CE, Rouse GW, Johnson SB, Jones WJ, Vrijenhoek RC (2007) Bathymetric and temporal variation among Osedax boneworms and associated megafauna on whale-falls in Monterey Bay, California. Deep-Sea Deep-Sea Research Part I Oceanographic Research Papers 54: 1773–1791. https://doi.org/10.1016/j.dsr.2007.05.014

Brown S, Rouse G, Hutchings P, Colgan D (1999) Assessing the usefulness of histone H3, U2 snRNA and 28S rDNA in analyses of polychaete relationships. Australian Journal of Zoology 47: 499–516. https://doi.org/10.1071/ZO99026

Clement M, Posada D, Crandall KA (2000) TCS: a computer program to estimate gene genealogies. Molecular Ecology 9: 1657–1659. https://doi.org/10.1046/j.1365-294x.2000.01020.x

Colgan DJ, McLauchlan A, Wilson GDF, Livingston SP, Edgecombe GD, Macaranas J, Cassis G, Gray MR (1998) Histone H3 and U2 snRNA DNA sequences and arthropod molecular evolution. Australian Journal of Zoology 46: 419–437. https://doi.org/10.1071/zo98048

Darriba D, Posada D, Kozlov AM, Stamatakis A, Morel B, Flouri T (2020) ModelTest-NG: a new and scalable tool for the selection of DNA and protein evolutionary models. Molecular Biology and Evolution 37: 291–294. https://doi.org/10.1093/molbev/msz189

Dawbin WH (1966) 9. The seasonal migratory cycle of humpback whales. In: Whales, Dolphins, and Porpoises University of California Press, 145–170. https://doi.org/10.1525/9780520321373-011

Edler D, Klein J, Antonelli A, Silvestro D (2021) raxmlGUI 2.0: a graphical interface and toolkit for phylogenetic analyses using RAxML. https://doi.org/10.1101/800912

Eilertsen MH, Dahlgren TG, Rapp HT (2020) A new species of Osedax (Siboglinidae: Annelida) from colonization experiments in the Arctic deep sea. Frontiers in Marine Science 7: 443. https://doi.org/10.3389/fmars.2020.00443

Fujikura K, Fujiwara Y, Kawato M (2006) A New species of Osedax (Annelida: Siboglinidae) associated with whale carcasses off Kyushu, Japan. Zoological Science 23: 733–740. https://doi.org/10.2108/zsj.23.733

Fujiwara Y, Jimi N, Sumida PYG, Kawato M, Kitazato H (2019) New species of bone-eating worm Osedax from the abyssal South Atlantic Ocean (Annelida, Siboglinidae). ZooKeys 814: 53–69. https://doi.org/10.3897/zookeys.814.28869

Gaudron SM, Pradillon F, Pailleret M, Duperron S, Le Bris N, Gaill F (2010) Colonization of organic substrates deployed in deep-sea reducing habitats by symbiotic species and associated fauna. Marine Environmental Research 70: 1–12. https://doi.org/10.1016/j.marenvres.2010.02.002

Georgieva MN, Wiklund H, Ramos DA, Neal L, Glasby CJ, Gunton LM (2023) The annelid community of a natural deep-sea whale fall off eastern Australia. In: Kupriyanova EK, Gunton LM (Eds) RV Investigator—Abyssal Annelida. Records of the Australian Museum 75: 167–213. https://doi.org/10.3853/j.2201-4349.75.2023.1800

Giribet G, Carranza S, Baguna J, Riutort M, Ribera C (1996) First molecular evidence for the existence of a Tardigrada + Arthropoda clade. Molecular Biology and Evolution 13: 76–84. https://doi.org/10.1093/oxfordjournals.molbev.a025573

Glover AG, Källström B, Smith CR, Dahlgren TG (2005) World-wide whale worms? A new species of Osedax from the shallow north Atlantic. Proceedings of the Royal Society B: Biological Sciences 272: 2587–2592. https://doi.org/10.1098/rspb.2005.3275

Glover AG, Wiklund H, Taboada S, Avila C, Cristobo J, Smith CR, Kemp KM, Jamieson AJ, Dahlgren TG (2013) Bone-eating worms from the Antarctic: the contrasting fate of whale and wood remains on the Southern Ocean seafloor. Proceedings of the Royal Society B: Biological Sciences 280: 20131390. https://doi.org/10.1098/rspb.2013.1390

Goffredi SK, Orphan VJ, Rouse GW, Jahnke L, Embaye T, Turk K, Lee R, Vrijenhoek RC (2005) Evolutionary innovation: a bone‐eating marine symbiosis. Environmental Microbiology 7: 1369–1378. https://doi.org/10.1111/j.1462-2920.2005.00824.x

Goffredi SK, Johnson SB, Vrijenhoek RC (2007) Genetic diversity and potential function of microbial symbionts associated with newly discovered species of Osedax polychaete worms. Applied and Environmental Microbiology 73: 2314–2323. https://doi.org/10.1128/AEM.01986-06

Jones ML (1981) Riftia pachyptila Jones: Observations on the vestimentiferan worm from the Galápagos Rift. Science 213: 333–336. https://doi.org/10.1126/science.213.4505.333

Katoh K, Standley DM (2013) MAFFT Multiple sequence alignment software Version 7: improvements in performance and usability. Molecular Biology and Evolution 30: 772–780. https://doi.org/10.1093/molbev/mst010

Katz S, Klepal W, Bright M (2010) The skin of Osedax (Siboglinidae, Annelida): An ultrastructural investigation of its epidermis. Journal of Morphology 271: 1272–1280. https://doi.org/10.1002/jmor.10873

Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Meintjes P, Drummond A (2012) Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28: 1647–1649. https://doi.org/10.1093/bioinformatics/bts199

Laws RM (1977) Seals and whales of the Southern Ocean. Philosophical Transactions of the Royal Society of London. B, Biological Sciences 279: 81–96. https://doi.org/10.1098/rstb.1977.0073

Leigh JW, Bryant D (2015) PopART: full‐feature software for haplotype network construction. Methods in Ecology and Evolution 6: 1110–1116. https://doi.org/10.1111/2041-210X.12410

Lundsten L, Schlining KL, Frasier K, Johnson SB, Kuhnz LA, Harvey JBJ, Clague G, Vrijenhoek RC (2010) Time-series analysis of six whale-fall communities in Monterey Canyon, California, USA. Deep Sea Research Part I: Oceanographic Research Papers 57: 1573–1584. https://doi.org/10.1016/j.dsr.2010.09.003

Maddison WP, Maddison DR (2019) Mesquite Project. Mesquite: A modular system for evolutionary analysis. https://www.mesquiteproject.org/ [accessed 8 July2024]

McClain CR, Nunnally C, Dixon R, Rouse GW, Benfield M (2019) Alligators in the abyss: The first experimental reptilian food fall in the deep ocean. PLoS ONE 14: e0225345. https://doi.org/10.1371/journal.pone.0225345

Palumbi SR (1996) Nucleic acids II: The polymerase chain reaction. In: Hillis DM, Moritz C, Mable BK (Eds) Molecular Systematics. Sinauer Associates, Sunderland, Massachusetts, 205–247.

Rouse GW, Goffredi SK (2023) Osedax (Siboglinidae: Annelida) utilizes shark teeth for nutrition. Journal of the Marine Biological Association of the United Kingdom 103: e35. https://doi.org/10.1017/S0025315423000243

Rouse GW, Goffredi SK, Vrijenhoek RC (2004) Osedax : Bone-eating marine worms with dwarf males. Science 305: 668–671. https://doi.org/10.1126/science.1098650

Rouse GW, Worsaae K, Johnson SB, Jones WJ, Vrijenhoek RC (2008) Acquisition of dwarf male “harems” by recently settled females of Osedax roseus n. sp. (Siboglinidae; Annelida). The Biological Bulletin 214: 67–82. https://doi.org/10.2307/25066661

Rouse GW, Wilson NG, Worsaae K, Vrijenhoek RC (2015) A Dwarf male reversal in bone-eating worms. Current Biology 25: 236–241. https://doi.org/10.1016/j.cub.2014.11.032

Rouse GW, Goffredi SK, Johnson SB, Vrijenhoek RC (2018) An inordinate fondness for Osedax (Siboglinidae: Annelida): Fourteen new species of bone worms from California. Zootaxa 4377: 451–489. https://doi.org/10.11646/zootaxa.4377.4.1

Rozewicki J, Li S, Amada KM, Standley DM, Katoh K (2019) MAFFT-DASH: integrated protein sequence and structural alignment. Nucleic Acids Research: gkz342. https://doi.org/10.1093/nar/gkz342

Saeedi H, Bernardino AF, Shimabukuro M, Falchetto G, Sumida PYG (2019) Macrofaunal community structure and biodiversity patterns based on a wood-fall experiment in the deep South-west Atlantic. Deep Sea Research Part I: Oceanographic Research Papers 145: 73–82. https://doi.org/10.1016/j.dsr.2019.01.008

Salathé RM, Vrijenhoek RC (2012) Temporal variation and lack of host specificity among bacterial endosymbionts of Osedax bone worms (Polychaeta: Siboglinidae). BMC Evolutionary Biology 12: 189. https://doi.org/10.1186/1471-2148-12-189

Santos MC de O, Siciliano S, Vicente AF de C, Alvarenga FS, Zampirolli É, Souza SP de, Maranho A (2010) Cetacean records along São Paulo state coast, Southeastern Brazil. Brazilian Journal of Oceanography 58: 123–142. https://doi.org/10.1590/S1679-87592010000200004

Shimabukuro M, Sumida PYG (2019) Diversity of bone-eating Osedax worms on the deep Atlantic whale falls—bathymetric variation and inter-basin distributions. Marine Biodiversity 49: 2587–2599. https://doi.org/10.1007/s12526-019-00988-2

Shimabukuro M, Carrerette O, Alfaro-Lucas JM, Rizzo AE, Halanych KM, Sumida PYG (2019) Diversity, distribution and phylogeny of Hesionidae (Annelida) colonizing whale falls: new species of Sirsoe and connections between ocean basins. Frontiers in Marine Science 6: 478. https://doi.org/10.3389/fmars.2019.00478

Shimabukuro M, Couto DM, Bernardino AF, Souza BHM, Carrerette O, Pellizari VH, Sumida PYG (2022) Whale bone communities in the deep Southwest Atlantic Ocean. Deep Sea Research Part I: Oceanographic Research Papers 190: 103916. https://doi.org/10.1016/j.dsr.2022.103916

Smith CR, Baco AR (2003) Ecology of whale falls at the deep sea floor. Oceanography and Marine Biology: an Annual Review 41: 311–354. https://doi.org/10.1201/9780203180594.ch6

Smith CR, Glover AG, Treude T, Higgs ND, Amon DJ (2015) Whale-Fall Ecosystems: Recent Insights into Ecology, Paleoecology, and Evolution. Annual Review of Marine Science 7: 571–596. https://doi.org/10.1146/annurev-marine-010213-135144

Souza BHM, Passos FD, Shimabukuro M, Sumida PYG (2021) An integrative approach distinguishes three new species of Abyssochrysoidea (Mollusca: Caenogastropoda) associated with organic falls of the deep south-west Atlantic. Zoological Journal of the Linnean Society 191: 748–771. https://doi.org/10.1093/zoolinnean/zlaa059

Swofford D. L. (2002) PAUP*: Phylogenetic analysis using parsimony (*and other methods). https://paup.phylosolutions.com

Taboada S, Riesgo A, Bas M, Arnedo MA, Cristobo J, Rouse GW, Avila C (2015) Bone-eating worms spread: Insights into shallow-Water Osedax (Annelida, Siboglinidae) from Antarctic, Subantarctic, and Mediterranean Waters. PLoS ONE 10: e0140341. https://doi.org/10.1371/journal.pone.0140341

Tresguerres M, Katz S, Rouse GW (2013) How to get into bones: proton pump and carbonic anhydrase in Osedax boneworms. Proceedings of the Royal Society B: Biological Sciences 280: 20130625. https://doi.org/10.1098/rspb.2013.0625

Vrijenhoek RC, Johnson SB, Rouse GW (2008) Bone‐eating Osedax females and their “harems” of dwarf males are recruited from a common larval pool. Molecular Ecology 17: 4535–4544. https://doi.org/10.1111/j.1365-294X.2008.03937.x

Vrijenhoek RC, Johnson SB, Rouse GW (2009) A remarkable diversity of bone-eating worms (Osedax; Siboglinidae; Annelida). BMC Biology 7: 74. https://doi.org/10.1186/1741-7007-7-74

Webb M (1964) A new bitentaculate pogonophoran from Hardangerfjorden, Norway. Sarsia 15: 49–56. https://doi.org/10.1080/00364827.1964.10409528

Webb M (1969) Lamellibrachia barhami, gen. nov., sp. nov. (Pogonophora), from the northeast Pacific. Bulletin of Marine Science 19: 18–47.

Wedekin LL, Rossi-Santos MR, Baracho C, Cypriano-Souza AL, Simões-Lopes PC (2014) Cetacean records along a coastal-offshore gradient in the Vitória-Trindade Chain, western South Atlantic Ocean. Brazilian Journal of Biology 74: 137–144. https://doi.org/10.1590/1519-6984.21812

Whiting MF, Carpenter JC, Wheeler QD, Wheeler WC (1997) The Strepsiptera problem: Phylogeny of the holometabolous Insect orders inferred from 18S and 28S ribosomal DNA sequences and morphology. Farrell B (Ed). Systematic Biology 46: 1–68. https://doi.org/10.1093/sysbio/46.1.1

Zerbini AN, Andriolo A, Heide-Jørgensen MP, Pizzorno JL, Maia YG, VanBlaricom GR, DeMaster DP, Simões-Lopes PC, Moreira S, Bethlem C (2006) Satellite-monitored movements of humpback whales Megaptera novaeangliae in the Southwest Atlantic Ocean. Marine Ecology Progress Series 313: 295–304. https://doi.org/10.3354/meps313295

Supplementary material

Supplementary material 1

Minimum genetic distance based on uncorrected p–distance between species within Clade V

Thammy Gularte, Paulo Y. G. Sumida, Gilberto Bergamo, Greg W. Rouse

Data type: xlsx

Explanation note: The maximum intraspecific divergences among Osedax nataliae sp. nov. specimens are in bold.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

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﻿Abstract

Key words

﻿Introduction

﻿Materials and methods

﻿Sample collection and morphological analysis

﻿DNA preparation, amplification, and sequencing

﻿Phylogenetic analysis

﻿Results

﻿Taxonomy

﻿ Osedax nataliae Gularte, Sumida, Bergamo & Rouse, sp. nov