Branchinotogluma Pettibone, 1985 is the most species-rich genus within the subfamily Lepidonotopodinae Pettibone, 1983, comprising 18 valid species from chemosynthesis-based ecosystems in the Pacific and Indian Oceans. Here, we report a new distributional record of Branchinotogluma bipapillata Zhou, Wang, Zhang & Wang, 2018, at the hydrothermal vent sites on the northern Central Indian Ridge (nCIR). This record represents the northernmost occurrence of B. bipapillata in the Indian Ocean. We conducted a comparative study of the nCIR population and other documented populations using distributional information, morphological traits, and genetic markers (two mitochondrial [COI, 16S rRNA] and one nuclear [18S rRNA] genes). While most morphological characters of B. bipapillata were consistent with those found in the Southwest Indian Ridge (SWIR), variations were noted in the segment with the last branchiae. Molecular data revealed that all populations of B. bipapillata form a single clade, indicating a wide distribution from the SWIR to nCIR, covering ~4,000 km across various ridges in the Indian Ocean. This study presents extensive distribution of a vent species with well-connected populations throughout the Indian Ocean, distinguishing it from many other vent species affected by the dispersal barrier in the Indian Ocean.

16S rRNA, 18S rRNA, CO1, deep-sea, hydrothermal vent, northern Central Indian Ridge, polynoids

The subfamily Lepidonotopodinae Pettibone, 1983 consists of scale worms endemic to chemosynthesis-based ecosystems (Wu et al. 2023). Currently, seven species from the Indian Ocean are identified within this subfamily: three Branchinotogluma, two Branchipolynoe, and two Levensteiniella (Han et al. 2023). Branchinotogluma Pettibone, 1985, the most species-rich genus in the subfamily, comprises 18 species found in the Pacific and Indian Oceans (Han et al. 2023). Specifically, three Branchinotogluma species are distributed across different ridge systems in the Indian Ocean: B. bipapillata Zhou et al., 2018 in the Southwest Indian Ridge (SWIR) and southern Central Indian Ridge (sCIR), B. jiaolongae Han et al., 2023 in the SWIR and Carlsberg Ridge (CR), and B. kaireiensis Han et al., 2023 in the sCIR and CR (Zhou et al. 2018, 2022; Han et al. 2023).

Since the initial discovery of vent fields in the Indian Ocean in 2000, the Rodriguez Triple Junction, which links the SWIR, CIR, and Southeast Indian Ridge, was assumed to be a dispersal barrier for vent species within the Indian Ocean (Gamo et al. 2001; Chen et al. 2015). However, with the discovery of more vent fields and associated fauna, it now appears that the primary dispersal barriers lie within the ridge system itself, mainly due to ridge offsets, rather than between different ridge systems (Sun et al. 2020). For instance, the vent crab Austinograea rodriguezensis Tsuchida & Hashimoto, 2002 was absent from the southern SWIR (sSWIR) but was found in the northern SWIR (nSWIR) and showed panmixia with populations from other ridges like the sCIR. Similarly, the distribution of the hairy snail Alviniconcha species complex shows connectivity between different ridges, northern CIR (nCIR) and CR, but subdivisions between the sCIR and nCIR on the same CIR (Sun et al. 2020; Jang et al. 2023).

While B. bipapillata has been reported from vent fields on two ridge systems, the SWIR and CIR, morphological and genetic studies were previously only conducted on specimens from the sSWIR. In this study, we collected Branchinotogluma species from hydrothermal vent fields on the nCIR and compared morphological and molecular data with those from vent fields on the sSWIR.

Specimens of Branchinotogluma were collected from hydrothermal vents in the nCIR during the 2023 KIOST expedition aboard the R/V Isabu (Fig. 1, Table 1) using a suction sampler and scoop mounted on the ROV ROPOS (Canadian Scientific Submersible Facility). Upon collection, a piece of elytron or parapodium from each specimen was dissected and preserved in 99% ethanol for molecular analysis. The entire body of the specimens was preserved in either 10% neutral buffered formalin or 70% ethanol for morphological studies.

Figure 1. 

Map displaying the geographic distribution of Branchinotogluma bipapillata in the Indian Ocean. Red indicates sampling locations from this study and black indicates records of B. bipapillata from previous studies. Some closely situated sampling sites (< 10 km apart, such as Onbada and Onnare, Saero and Onnuri) are marked with a single square.

For determination of morphological characters, all specimens were examined under a stereomicroscope (Stemi 508; Carl Zeiss, Germany). Specimen photographs were captured using a color camera (Axiocam 208 color; Carl Zeiss, Germany) and a DSLR camera (EOS 5D Mark IV; Canon, Tokyo, Japan). Images were processed with ZEN 3.3 blue edition (Carl Zeiss, Germany) and Helicon Focus software (Helicon Soft Ltd., Kharkov, Ukraine), and further edited using Adobe Photoshop 2022 (Adobe, San Jose, CA, USA). Specimen morphology was recorded following characters and states listed in Zhou et al. (2018).

A small piece of elytron or parapodium was used for total genomic DNA extraction using the AccuPrep® Genomic DNA Extraction Kit (Bioneer, Daejeon, South Korea), following the manufacturer’s instructions. Partial cytochrome c oxidase subunit 1 (CO1) and 18S rRNA (18S) sequences were amplified following the protocols in Lee et al. (2021) and Jimi et al. (2021), respectively. For 16S rRNA (16S), the primers 16SA (5′-CGCCGTTTATCAAAAACAT-3′) and 16Sbr (5′-CCGGTYTGAACTCAGATCAYG-3′) (Palumbi et al. 1991; Palumbi 1996) were used. Polymerase chain reaction (PCR) was conducted using a SimpliAmp™ Thermal Cycler (Applied Biosystems, Life technologies) under the following conditions: initial denaturation at 94 °C for 2 min; 5 cycles at 95 °C for 10 s, 42 °C for 30 s, and 72 °C for 60 s; 35 cycles at 95 °C for 10 s, 48 °C for 30 s, and 72 °C for 60 s; with a final extension at 72 °C for 2 min. PCR products were sent to Macrogen (Seoul, Korea) for Sanger sequencing.

New sequences were aligned with those of other Lepidonotopodinae species from GenBank (Suppl. material 1: table S1) using Geneious Prime ver. 2023.0.1 (Biomatters, Auckland, New Zealand). Sequence divergence for the CO1 and 16S genes was calculated using the p-distance method in MEGA11 (Tamura et al. 2021). For phylogenetic analysis, the three genes were concatenated using Geneious Prime. The best evolutionary model, GTR+I+G, was selected using jModelTest ver. 2.1.8 (Darriba et al. 2012). The phylogenetic tree was constructed using the maximum-likelihood method with raxmlGUI 2.0 (Edler et al. 2021).

All specimens used in this study are deposited at the Korea Research Institute of Bioscience and Biotechnology.

Family Polynoidae Kinberg, 1856

Subfamily Lepidonotopodinae Pettibone, 1983

Genus Branchinotogluma Pettibone, 1985

Branchinotogluma bipapillata Zhou, Wang, Zhang & Wang, 2018: 528–533, figs 1–7; table 1.

Material examined

Indian Ocean • 2 ♂; Cheoeum; 12°37.1'S, 66°07.6'E; depth 3018 m; 28 Mar. 2023; W-K Lee leg.; hydrothermal vent; GenBank: PP600168– PP600169; KRIBB310101 to KRIBB310102 • 2 ♂, 2 ♀, 1 undetermined; Onnuri; 11°24.9'S, 66°25.4'E; depth 2009 m; 1–2 Apr. 2023; W-K Lee leg.; hydrothermal vent; GenBank: PP600170– PP600174; KRIBB310103 to KRIBB310107 • 1 ♀, 2 undetermined; Onnare; 9°47.4'S, 66°41.9'E; depth 2993 m; 3 Apr. 2023; W-K Lee leg.; hydrothermal vent; GenBank: PP600175– PP600177; KRIBB310108 to KRIBB310110 • 1 ♂, 1 ♀; Onbada; 9°48.9'S, 66°40.6'E; depth 2563 m; 4 Apr. 2023; W-K Lee leg.; hydrothermal vent; GenBank: PP600178– PP600179; KRIBB310111 to KRIBB310112 • 2 ♂, 2 ♀; Saero; 11°19.7'S, 66°26.9'E; depth 3256 m; 7 Apr. 2023; W-K Lee leg.; hydrothermal vent; GenBank: PP600180– PP600183; KRIBB310113 to KRIBB310116.

Description

Specimens relatively well preserved, with 21 segments, 12.0–51.0 mm in length and 5.0–16.6 mm in width. Body shape fusiform, tapered anteriorly and posteriorly (Fig. 2A, B, Table 2). Pairs of elytra on elytrophores on segments 2, 4, 5, 7, 9, 11, 13, 15, 17, and 19; elytra oval to subreniform, white, slightly transparent, with a smooth surface (Fig. 2C–E). Dorsal cirri on segments 3, 6, 8, 10, 12, 14, 16, 18, 20, and 21, extending beyond the tips of neurochaetae. Branchiae arborescent; grouped in two, one at base of the notopodia and another at base of dorsal tubercles or elytrophores; starting from segment 3 and ending between segments 18 or 21 (Table 2).

Table 2.

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Morphological comparison of Branchinotogluma bipapillata from the nCIR and sSWIR.

Region	Length (mm)	Sex (# of ind.)	Last segment with branchiae	Number of dorsal/ventral papillae on pharynx	9th to 10th elytrophore diatmeter ratio	Reference
nCIR	24.4–48.0	Male (5)	18	Not observed	2.25–2.64	This study
	20.5–51.0	Female (8)	18 or 21		1.08–1.46
	12.0–17.8	Undetermined (3)	18	5/4*	1.23–1.28
SWIR	23.3–32.3	Male (1)	18	5/5	N/A	Zhou et al. 2018
		Female (2)	19		N/A

*Observed in a single specimen (KRIBB310108; Fig. 2G).

Figure 2. 

Branchinotogluma bipapillata specimens collected from the nCIR A dorsal and ventral views of male (KRIBB310116) B dorsal and ventral views of female (KRIBB310105) C 1^st–8^th left elytra D 9^th–10^th left elytra of male (KRIBB310103) E 9^th–10^th left elytra of female (KRIBB310110) F head featuring prostomium, palps, tentacular cirri, and first parapodia on segment 2 (KRIBB310112) G everted pharynx with dorsal and ventral papillae (KRIBB310108). Anterior and posterior views of left parapodia on (H-I) segment 2 and (J-K) segment 11 (KRIBB310106). Scale bars: 5 mm (A–E); 0.5 mm (F, G); 1 mm (H–K).

Prostomium bilobed, triangular anterior lobes with slender frontal filaments (Fig. 2F). Median antennae on anterior notch, with a cylindrical ceratophore and subulate style; palps thick, smooth, and end in subulate tips; lateral antennae and eyes absent (Fig. 2F). Tentacular segment fused to prostomium, with pair of tentacular cirri on each side, and a small acicular lobe at the base of tentaculophore; tentacular cirri slender (Fig. 2F).

First segment not distinct, fused to prostomium. Pharynx with five dorsal and four ventral papillae in one immature individual, but not seen in others (Fig. 2G). Second segment with first pair of elytrophores, ventral cirri, and biramous parapodia. Third segment with ventral cirri and first pair of branchiae. Fourth to last segments with ventral cirri and biramous parapodia. Notopodia smaller than neuropodia; notochaetae stout, few, arranged in radiating bundles; neurochaetae slender, numerous, forming a fan shape (Fig. 2H–K).

Sexual dimorphism evident. In males, posterior segments modified (Fig. 3A) with 10^th elytra and elytrophores much smaller than 9^th (Figs 2D, 3A, Table 2); ventral papillae present on segments 12–13, long, tapering, with slender tips extending to next segment; ventral lamellae on segments 14–17, round (Fig. 3B). In females, posterior segments not modified (Fig. 3C), with 10^th elytra and elytrophores similar to 9^th (Figs 2E, 3C, Table 2); ventral papillae present on segments 11–15, short and blunt (Fig. 3D).

Figure 3. 

Sexually dimorphic characters of Branchinotogluma bipapillata A dorsal view of posterior segments B ventral view of segments 12–17 of male (KRIBB310116) C dorsal view of posterior segments D ventral view of segments 11–15 of female (KRIBB310105). Arrows point to 9^th and 10^th elytrophores (EP) pointed with arrows. Ventral papillae are outlined in red and ventral lamellae in blue. Scale bars: 2 mm (A, C); 1 mm (B, D).

Distribution

Indian Ocean (depth 1732–3256 m): Longqi and Duanqiao vent fields on the southern Southwest Indian Ridge; Tiancheng vent field on the northern Southwest Indian Ridge; Edmond vent field on the southern Central Indian Ridge; Onnare, Onbada, Saero, Onnuri, and Cheoeum vent fields on the northern Central Indian Ridge.

Remarks

Comparisons of key morphological characters between the geographically distant populations are present in Table 2. The key characters of the nCIR specimens of B. bipapillata largely correspond with those of the SWIR specimens (Zhang et al. 2018). However, the two populations differ in the last segment with branchiae in females (segment 19 in sSWIR compared with segment 18 or 21 in nCIR; Table 2).

Among the 16 specimens from the nCIR population, 10 specimens with body length greater than 20 mm were well-developed in all features indicating adult morphology, while characters of sexual dimorphism were not observed in 6 specimens shorter than 20 mm.

DNA barcoding and phylogenetic analysis

Partial sequences of CO1, 16S, and 18S were recovered from 16 specimens collected from the nCIR. As shown in Table 1, 48 newly obtained sequences have been deposited in GenBank.

In CO1, the mean intra-population variation was 0.56% for nCIR and 0.65% for SWIR, with an inter-population variation of 1.00% (Table 3). In 16S, the mean intra-population variation was 0.27% for nCIR and 0.33% for SWIR, with an inter-population variation of 0.39%. In 18S, the mean intra-population variation was 0.01% for nCIR and 0.00% for SWIR, with an inter-population variation of 0.004%.

Table 3.

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Sequence divergence (%) among three Branchinotogluma bipapillata populations based on partial CO1 gene (553 bp).

Populations	nCIR	sCIR	SWIR
(# of ind.; intra)
nCIR	–
(16; 0.56)
sCIR	0.70	–
(1; NC*)	(0.20–1.18)
SWIR	1.00	0.47	–
(5; 0.65)	(0.00–1.65)	(0.20–1.19)

*not calculated.

The interspecific variation between B. bipapillata and other congeners ranged from 18.63% to 21.88% in CO1, and from 13.11% to 19.08% in 16S (Suppl. material 1: table S2). In 18S, the interspecific variation ranged from 1.69% to 3.80%.

The maximum likelihood phylogenetic tree, constructed with concatenated sequences of CO1, 16S, and 18S (Fig. 4), shows the SWIR and nCIR populations of B. bipapillata clustering together as a single clade, indicating no significant divergence between populations from different ridges. Within the Branchinotogluma genus, B. bipapillata is closely related to a clade including B. kaireiensis, B. pettiboneae Wu et al., 2019, and B. robusta Wu et al., 2023.

Figure 4. 

Maximum-likelihood phylogenetic tree of Branchinotogluma species based on concatenated sequences of the CO1, 16S, and 18S genes. Branchinotogluma bipapillata species are highlighted with a gray box. Red and black squares represent nCIR and sSWIR populations, respectively. GenBank accession numbers of the CO1, 16S and 18S genes of the outgroup are noted next to the species names. Maximum-likelihood bootstrap support values > 60 are displayed next to the nodes.

The vent scale worm B. bipapillata is widely distributed in the Indian Ocean, but comprehensive morphological and molecular data are lacking across all deep-sea oceanic ridges, and specimens are rarely reported at each sampling site (Fig. 1; only five specimens of B. bipapillata from the SWIR were barcoded with CO1, and only two sequences of 16S and 18S are available from SWIR specimens). In this study, 16 individuals, including female, male, and immature specimens of B. bipapillata from the nCIR were observed, enriching descriptions of features such as all elytra, and improving the molecular description of this species with both nuclear and mitochondrial gene sequences. Key morphological characters, such as the presence of an acicular lobe on the tentaculophore and the position of segmental ventral papillae, showed general congruence between the sSWIR and nCIR populations. Additionally, CO1 barcode sequences revealed a mean intraspecific variation of 0.72% in B. bipapillata, which is within the variation range observed in other Branchinotogluma species (0.00–1.05%; Suppl. material 1: table S2).Thus, molecular data on genetic distances within and between populations showed no significant differences, and the phylogenetic analysis revealed a single clade of B. bipapillata, with no divergence between populations (Fig. 4, Table 3). Based on these morphological and molecular findings, the southernmost and northernmost populations appear to be well connected, forming a single genetic population with minimal morphological variability.

Other vent endemic species in the Indian Ocean, such as the mussel Bathymodiolus marisindicus Hashimoto, 2001, the snail Chrysomallon squamiferum Chen et al., 2015, the crab A. rodriguezensis, the barnacle Neolepas marisindica Watanabe et al., 2018, and the worm Ophryotrocha jiaolongi Zhang et al., 2017, all show a wide distribution range on the SWIR and CIR (Sun et al. 2020; Zhou et al. 2022). However, unlike the B. bipapillata populations in this study, most of these species exhibit low connectivity between populations, likely due to ridge offsets acting as dispersal barriers between the sSWIR and nSWIR, which do not seem to affect the connectivity of B. bipapillata (Sun et al. 2020; Zhou et al. 2022). Although the reproductive and larval development strategies of B. bipapillata are not fully understood, observations of other species within the same subfamily suggest that B. bipapillata likely have lecithotrophic larvae (Van Dover et al. 1999; Jollivet et al. 2000). This larval type, capable of traveling long distances in oligotrophic deep-sea environments, might partially explain the high connectivity of B. bipapillata populations across ~4,000 km of different ridges within the Indian Ocean.

Many studies have considered geological and hydrological features, along with the dispersal abilities of species, to explain the distribution of vent species (Slatkin 1987; Vrijenhoek 2010; Taylor and Roterman 2017; Perez et al. 2021). However, to fully understand the broad geographical distribution of these species, it is also crucial to consider their ability to adapt to diverse vent environments across different ridge systems. To further elucidate the strategies that enable species such as B. bipapillata to inhabit separate and geographically distant vent fields with no genetic differentiation, future studies should consider in vitro experiments for culturing as well as transcriptomic and genomic level data of populations.

We thank all scientists and crew members of the R/V Isabu from KIOST for their support in sampling and data collection.