Phylogeography of hydrothermal vent stalked barnacles: a new species fills a gap in the Indian Ocean 'dispersal corridor' hypothesis.

Phylogeography of animals provides clues to processes governing their evolution and diversification. The Indian Ocean has been hypothesized as a 'dispersal corridor' connecting hydrothermal vent fauna of Atlantic and Pacific oceans. Stalked barnacles of the family Eolepadidae are common associates of deep-sea vents in Southern, Pacific and Indian oceans, and the family is an ideal group for testing this hypothesis. Here, we describe Neolepas marisindica sp. nov. from the Indian Ocean, distinguished from N. zevinae and N. rapanuii by having a tridentoid mandible in which the second tooth lacks small elongated teeth. Morphological variations suggest that environmental differences result in phenotypic plasticity in the capitulum and scales on the peduncle in eolepadids. We suggest that diagnostic characters in Eolepadidae should be based mainly on more reliable arthropodal characters and DNA barcoding, while the plate arrangement should be used carefully with their intraspecific variation in mind. We show morphologically that Neolepas specimens collected from the South West Indian Ridge, the South East Indian Ridge and the Central Indian Ridge belong to the new species. Molecular phylogeny and fossil evidence indicated that Neolepas migrated from the southern Pacific to the Indian Ocean through the Southern Ocean, providing key evidence against the 'dispersal corridor' hypothesis. Exploration of the South East Indian Ridge is urgently required to understand vent biogeography in the Indian Ocean.

Introduction

Distribution range and phylogeography of organisms are important basic ecological traits for elucidating their evolutionary history and their successful conservation. Distribution ranges of deep-sea animals are poorly understood, with the exception of those associated with hydrothermal vents which have been relatively well studied (e.g. [1,2]). Historical migrations across a geological timescale, species distributional ranges and biogeographical provinces of hydrothermal vent animals have been discussed on the basis of faunal compositions in local communities [1-5], molecular phylogenetic analysis (e.g. [6,7]) and recently, physico-oceanographic modelling (e.g. [8]). These studies revealed the crucial influence of plate tectonics, geological structures and oceanic current systems to realized ranges of metapopulations, as well as the biogeography of vent animals.

The Indian Ocean hosts three oceanic ridges: the Central Indian Ridge (CIR), the South West Indian Ridge (SWIR) and the South East Indian Ridge (SEIR). These ridges were suggested to act as corridors of dispersal for vent animals between Atlantic and Pacific oceans [9]. Hydrothermal activities in the Indian Ocean were first detected on the SEIR, with vertical profiles of thermometer and nephelometer equipped on dredges and core samplers detecting hydrothermal plumes at ‘site 21’ near the Amsterdam--St Paul Plateau, and the dredge successfully collected a new species of vent-associated barnacle belonging to the genus Neolepas [10]. Morphological characteristics of this vent barnacle from the SEIR were given [11,12], but without a name or formal description. As then, although both CIR and SWIR have been explored by manned submersibles and remotely operated vehicles [13-16], hydrothermal vents on the SEIR have never been observed directly. The distribution ranges of hydrothermal fauna across the entire Indian Ocean ridge systems, therefore, have not been elucidated in its entirety.

Barnacles of the family Eolepadidae, which includes the genus Neolepas, have been widely reported from deep-sea chemosynthetic environments in the Indo-Pacific and Southern oceans (from East Scotia Ridge, which is in the South Atlantic) [17], but they are apparently absent from the central and northern Atlantic Ocean and also the Arctic Ocean [18,19]. The first eolepadid barnacle to receive a formal description, Neolepas zevinae Newman 1979 [20] was collected at 21° N on the East Pacific Rise (EPR), off Mexico. At that time, Neolepas was classified in the subfamily Lithotryinae under Scalpellidae, based on having eight capitular plates. This genus was later transferred to a new subfamily, Eolepadinae, under Scalpellidae [21]. The second species in the genus, Neolepas rapanuii Jones 1993 [22] was identified and described from the 23° S site on the EPR, off Easter Island. Subsequently, Eolepadinae was elevated to a full family, Eolepadidae. A new subfamily, Neolepadinae, was established for Neolepas and the other subfamily, Eolepadinae, currently only houses two fossil genera—Archaeolepas and Eolepas [23]. The third Neolepas species, Neolepas osheai Buckeridge 2000 [13], was described from the South West Pacific [24]; but was later transferred to a new genus, Vulcanolepas, in the light of the discovery of another new genus and species, Leucolepas longa Southward & Jones 2003 in Edison Seamount [12]. Additionally, a fossil species that probably belongs to Neolepas, ?Neolepas augurata Buckeridge & Grant-Mackie 1985 [25] has been recorded from the lower Jurassic of New Caledonia. Leucolepas remains monotypic to date, while Vulcanolepas now further includes Vulcanolepas parensis Southward 2005 [26] from the Pacific-Antarctic Ridge and Vulcanolepas scotiaensis Buckeridge & Linse 2013 [27] from the East Scotia Ridge (ESR), the Southern Ocean. Another species of Vulcanolepas has been found in Lau Basin vents in the Western Pacific [6], which is currently under description (BKK Chan 2018, personal communication). A final genus currently included in Neolepadinae is Ashinkailepas, with two species in the Western Pacific (Ashinkailepas seepiophila Yamaguchi, Newman & Hashimoto 2004 [28] and Ashinkailepas kermadecensis Buckeridge 2009 [29]) [30].

Despite these progresses in the systematics of Eolepadidae, several Neolepas populations in the Indian Ocean, including the dredged specimens from SEIR and populations from CIR and SWIR, remain undescribed [17,31]. Molecular phylogenetics of Eolepadidae showed that Neolepas populations from the CIR and the SWIR exhibited distinct sequence divergence from other described eolepadid species [6], indicating that the Indian Ocean taxa indeed represent an undescribed species (Neolepas sp. 1 sensu Herrera et al. [6]). This study aims to characterize and describe this new Neolepas species mainly using material from CIR but supported by evidence from SWIR and SEIR to consider its distributional range across all three oceanic ridges in the Indian Ocean, as well as the global phylogeography of living eolepadid barnacles.

Material and methods

Sampling sites

Eolepadid stalked barnacles were collected from Kairei and Solitaire hydrothermal vent fields on CIR using the Human Occupied Vehicle (HOV) Shinkai 6500 on-board R/V Yokosuka of Japan Agency for Marine-Earth Science and Technology (JAMSTEC), during research cruises YK09-13, YK13-02 and YK16-E02 (figures 1 and 2; for YK09-13 also see [14]).

Figure 1.

Map of Indian Ocean Ridges, showing the locations of the four hydrothermal vent fields; Kairei and Solitaire fields on the Central Indian Ridge, Longqi field on the South West Indian Ridge, and site 21 near Amsterdam--St Paul Plateau on the South East Indian Ridge.

Figure 2.

Habitats of Neolepas marisindica sp. nov. (a) Kairei hydrothermal vent field, CIR, (b) Solitaire hydrothermal vent field, CIR.

Morphological examination

The barnacles were dissected and the body, including six pairs of cirri, the oral cone, the caudal appendages and the penis, were examined by light microscopy (Zeiss Axio-scope and stereomicroscope Leica M80). The terminology used to describe eolepadid barnacles herein follows those in the previous studies [12,27], whereas the setal classification and description follow the more general terminology for barnacles overall [32]. Type and voucher specimens were deposited in the National Museum of Nature and Science, Tsukuba (NSMT) and the University Museum, the University of Tokyo (UMUT).

Comparison of capitular morphology between Kairei and Solitaire populations

To compare morphological differences between specimens taken from Kairei and Solitaire hydrothermal vent fields, the peduncle length, capitular height, height of rostrum and median latus, number of peduncular scales per whorl just below the capitulum region, width of scales (from three scales), size of scales projected from the peduncles (from three scales) were measured using a digital caliper (±0.1 mm). The angle of the tergal apex was measured from photographs showing the lateral view of the capitulum, using the image analysis software Sigma Scan Pro 5. For each specimen, the ratio of peduncle : capitulum length, the ratio of rostrum : median latus, the size of projecting scales and the tergal apex angle were obtained. Variation in each capitular character between the two populations was tested using either t-test or Wilcoxon Rank Sum test (when the normality assumption was violated).

Molecular phylogenetic analysis

Genomic DNA was extracted using DNeasy Blood & Tissue Kit (QIAGEN) from the adductor muscle of barnacle specimens. Partial sequence of the mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified by polymerase chain reaction (PCR) using universal primer sets (LCO1490 and HCO2198, COI-3 and COI-6 [33,34]) and the Premix ExTaq Hot Start (TaKaRa). PCR was carried out in the following steps: initial denaturation at 94°C for 3 min and 35 cycles of denature (94°C for 30 s), annealing (50°C for 30 s) and extension (72°C for 90 s). PCR products were purified using Exo-SAP-it (USB, Affimetrix), following standard protocols. After BigDye reaction with BigDye Terminator v. 3.1, the products were sequenced using an ABI3130 automated sequencer (Applied Biosystems, Thermo Fisher). Electrophenograms obtained were checked by eye and assembled by Geneious v. 9 (Biomatters Limited) and registered to DNA Data Bank of Japan, with accession numbers LC350007–LC350015.

The sequences obtained were aligned with eolepadid sequences available in the databases of the International Nucleotide Sequence Database Collaboration, using Clustal X included in MEGA v. 6.06 [35]. A total of 123 sequences from seven eolepadid taxa were used (4--45 individuals per taxa), with one sequence of the pollicipedid barnacle Capitulum mitella (Linnaeus [36]) as the outgroup. Electronic supplementary material, table S1 shows the full list of sequences used in this study. The model selection programme in the same software was applied to select the best model for the maximum-likelihood algorithm, which was the Tamura three-parameter + Gamma distribution model. MEGA v. 6.06 was also used to reconstruct the phylogenetic trees using the maximum-likelihood algorithm, with 2000 bootstrap replicates.

Results

Systematics

Superorder Thoracica Darwin [37].

Order Scalpelliformes Buckeridge & Newman [38].

Family Eolepadidae Buckeridge [21]

Subfamily Neolepdinae Yamaguchi et al. [28]

Genus Neolepas Newman [20]

Neolepas marisindica sp. nov. Watanabe, Chen & Chan

Figures 3–12

Figure 3.

Neolepas marisindica sp. nov. (a) Holotype from Kairei vent field on the CIR (NSMT-Cr 26832). (b) A specimen collected from Solitaire vent field on the CIR (NSMT-Cr 26833). Note the variation in peduncular scales between the two populations. (c) A juvenile on the stalk of a specimen collected from the Solitaire vent field.

Figure 12.

Neolepas marisindica sp. nov. A specimen collected from the Solitaire vent field (NSMT-Cr 26834). (a) Mandible (dorsal view), (b) second and third teeth (dorsal view), (c) mandibulatory palp, (d) labrum, (e–h) cutting edge of labrum.

Unnamed Indian Ocean Ridge species – Southward and Jones: 2003 [12], figs 18D, 19F, tables 7–9

Neolepas sp. – Hashimoto et al. [13], table 1

Table 1.

Neolepas marisindica sp. nov. Segment counts of cirri on anterior and posterior ramus.

	I	II	III	IV	V	VI
Kairei	(holotype)
anterior	29	30	43	45	51	50
posterior	25	29	39	45	49	48
Solitaire
anterior	31	25	44	52	52	49
posterior	26	25	41	50	53	55

Neolepas n. sp. – Van Dover et al. [2], fig. 2g

Leucolepas sp. – Nakamura et al. [14], fig. 4 and table 1

Figure 4.

Neolepas marisindica sp. nov. (a) Paratype #1 (NSMT-Cr 26833), (b) paratype #2 (UMUT RA32760); both are from Kairei vent field on the CIR.

‘Neolepas sp. 1. CIR Kairei’ – Herrera et al. [6], fig. 2; electronic supplementary material, table S1

‘Neolepas sp. 1. SWIR Dragon’ – Herrera et al. [6], fig. 2; electronic supplementary material, table S1

‘Neolepas sp. 1’ – Copley et al. [16], table 1

ZooBank Registration. http://zoobank.org/urn:lsid:zoobank.org:act:0C5ECE6C-DCF7-4647-92AB-A3F872B2DB2D

Type locality. Kairei vent field (Monju Chimney), Central Indian Ridge, 25°19.2265′ S, 70°02.4181′ E, 2422 m in depth.

Type materials. Holotype (figure 3a): NSMT-Cr 26832, Kairei vent field (Monju Chimney), Central Indian Ridge, 25°19.2265′ S, 70°02.4181′ E, 2422 m in depth, collected by a slurp gun, HOV Shinkai 6500 Dive #1175, R/V Yokosuka cruise YK09-13. Leg. 2 (principal scientist: Kentaro Nakamura), 13 November 2009, fixed and stored in 99.5% ethanol.

Paratypes. #1 (a cluster of 14 specimens; figure 2a), NSMT-Cr 26833, Kairei vent field (Monju Chimney), Central Indian Ridge, 25°19.2250′ S 70°02.4211′ E, 2426 m in depth, collected by a slurp gun in HOV Shinkai 6500 Dive 1450, R/V Yokosuka YK16-E02 cruise (principal scientist: Ken Takai), 14 February 2016, fixed and stored in 10% seawater-buffered formalin. #2 (a cluster of seven specimens), same data as Paratype #1, fixed and stored in 99.5% ethanol (UMUT RA32760; figure 2b).

Other materials examined. One lot of three specimens (NSMT-Cr 26834; figure 3b), Solitaire vent field, Central Indian Ridge, 19°33.398′ S 65°50.871′ E, 2621 m in depth, HOV Shinkai 6500 Dive #1327, R/V Yokosuka cruise YK13-02 (principal scientist: Manabu Nishizawa), 11 February 2013. One lot of five juvenile specimens (UMUT RA32761), same data as above. Further specimens used for measurements and DNA sequencing: nine specimens from Solitaire vent field (same data as above) and 10 specimens from Kairei vent field (same data as holotype or paratype #1).

Diagnosis. Neolepas with tridentoid mandibles. Second tooth without attached small elongated teeth. Tergum apex angle ranges from 60 to 65°.

Description (based on the holotype). Capitulum composed of eight fully calcified plates, including carina, rostrum, paired scutum, tergum, median latus (figures 3a and 5a). Peduncle to capitulum ratio 2 : 1 (figure 3a). All plates in capitulum with transverse growth ridges (figures 3a and 5). Tergum quadrangular with clear, sharp apical-basal ridge. Umbo apical, tergal apex angle 60° (figures 3 and 5). Basal angle of tergum located at capitulum-peduncle margin (figure 5a). Scutum quadrangular, tergal margin slightly concave, occludent margin slightly convex, apical-basal ridge slightly curved, scutum apex angle 33°, basal angle sharp, 56° (figure 5a). Median latus triangular, narrow, apex angle 30° (figure 5a). Height generally twice the width (figure 5a). Rostrum curved, scutal margin strongly curved, length of rostrum equal to height of median latus (figure 5b). Carina slightly curved, height of carina approximately 2/3 height of capitulum (figure 5c).

Figure 5.

Neolepas marisindica sp. nov. Holotype (NSMT-Cr 26832), showing (a) lateral, (b) carinal and (c) rostral views of the capitulum. T, tergum; S, Scutum; Ca, Carina; R, Rostrum; BA, basal angle of tergum; TA, tergal apex angle; ML, median latus. Ratio of rostrum: carina is a/b.

Peduncles with up to 26 peduncular scales per whorl, just below capitulum. Scales larger on the lower part of peduncle, becoming 15 per whorl by middle region of the peduncle. Scales approximately 0.7 mm wide, projecting 0.6 mm out of peduncle on lower region of peduncle.

Oral cone. Maxilla hatchet-shaped, margins with long simple setae, inferior angle protruded as blunt triangle (figure 6a). Simple setae present around margins of maxilla (figure 6b). Maxillule trapezoid, cutting edge straight with 24 large spines (figure 6c). Inferior and exterior margin with simple setae (figure 6d). Mandibles tridentoid, first tooth large and sharply pointed, cutting edges of second and third teeth denticulate (figure 6e,f,g). Lower margin and inferior angle with a number of spines (figure 6h). Dorsal view of mandibles reveals lack of small longitudinal teeth on second tooth (figure 7a,b,c) Mandibulatory palp elongated, with simple setae (figure 7d). Labrum cutting edge concaved, with one row of fine teeth (figure 7e,f).

Figure 6.

Neolepas marisindica sp. nov. Holotype (NSMT-Cr 26832). Oral cone. (a) Maxilla, (b) simple setae on the margin of maxilla, (c) maxillule, (d) spines on the cutting edge of maxillule, (d) spines on the cutting edge of maxillule, (e) mandibles (ventral view), (f) first tooth of mandible (ventral view), (g) second and third tooth (ventral view), (h) inferior angle of mandible (ventral view).

Figure 7.

Neolepas marisindica sp. nov. Holotype (NSMT-Cr 26832). (a) Mandible (dorsal view), (b) second and third teeth (dorsal view), (c) lower margin (dorsal view), (d) inferior angle of mandible (dorsal view), (e) second and third tooth (dorsal view), (f) mandibulatory palp, (g) labrum, (h) cutting edge of labrum.

Cirri. All six pairs of cirri are long and slender (figure 8). Cirral counts of anterior and posterior rami are given in table 1. Cirrus I, both anterior and posterior rami similar in length, protuberant at the last eight proximal segments (height approx. 3 times length), become antenniform starting from middle to distal region of ramus (figure 8a). Cirrus I bear simple type setae, setae become denser at proximal region of both rami (figure 9a). Cirrus II, anterior ramus and posterior ramus similar in length. Proximal 11 segments of both rami protuberant. Both rami become antenniform starting from middle to distal region of ramus (figure 8b). Setae in both rami simple (figure 9b,c). Proximal segments bear high density of setae (figure 8b). Intermediate segments of Cirrus II bear four pairs of long simple setae plus one pair of short simple setae (figure 9d) Cirri III to VI similar in morphology, both anterior and posterior rami similar in length. Intermediate segments of cirri III bear five pairs of long simple setae plus two pairs of short simple setae (figure 9e). Intermediate segments of cirri IV to VI bear five pairs of long simple setae plus one to two pairs of short simple setae (figure 9f–h). Length of long simple setae in cirri III to IV approximately 4–5 times length of an intermediate segment (figure 9). Caudal appendages unarticulate, short (figure 9g). Penis long, about half length of cirrus VI (figure 9h).

Figure 8.

Neolepas marisindica sp. nov. Holotype (NSMT-Cr 26832). (a) Cirrus I, (b) cirrus II, (c) cirrus III, (d) cirrus IV, (e) cirrus V, (f) cirrus VI, (g) caudal appendages, (h) penis. ant, anterior ramus, pos, posterior ramus.

Figure 9.

Neolepas marisindica sp. nov. Holotype (NSMT-Cr 26832). (a) Simple setae on proximal region of cirrus I, (b) simple setae on distal region of cirrus I, (c) simple setae on cirrus II, (d): intermediate segment of anterior ramus on cirrus II, (e) intermediate segment of anterior ramus on cirrus III, (f) intermediate segment of anterior ramus on cirrus IV, (g) intermediate segment of anterior ramus on cirrus V, (h) intermediate segment of anterior ramus on cirrus VI.

Juveniles. Some juvenile individuals were found attached on the lower part of the peduncle of the barnacles collected in the Solitaire vent field. Peduncle to capitulum ratio in five juveniles observed is about 1 : 1. The carina and rostrum in juveniles are relatively straight and apex extends beyond the margin of the capitulum (figure 3c).

Etymology. Latin, adjective (maris = sea; indica = Indian), named after its type locality and known distribution.

Distribution. Presently known from Kairei and Solitaire hydrothermal fields in the CIR (greater than 2500 m depth) and Longqi hydrothermal field in the SWIR (has also been referred to as the ‘Dragon vent field’ [6,16]). We consider the dredged material from 41° S site (site 21), SEIR [12] also represents the same species (see Discussion below).

Remarks: The present new species is placed in Neolepas, based on its ratio of rostrum to median latus (average 1.3 : 1 from Kairei and 1.45 : 1 from Solitaire populations) and approximately 20 peduncular scales per whorl. Presently, there are two other recognized species of Neolepas: N. zevinae and N. rapanuii. Although the present new species is clearly genetically distinct from both of these species, the genetic distance between N. zevinae and N. rapanuii seemed insufficient for separation at species level [6]. However, the capitular arrangement of these species exhibits difference, with the rostrum being as high as the median latus in N. rapanuii, about the same in N. marisindica sp. nov. and higher than the median latus in N. zevinae [22]. The main difference among these three species is seen in the morphology of the mandibles. Neolepas zevinae has a tridentoid mandible in which the second tooth of the mandible has elongated teeth (fig. 2i in [20]), N. rapanuii has a quadridentoid mandible with a small fourth tooth in-between the third tooth and the inferior margin (fig. 3d in [22]). In N. marisindica sp. nov., the mandible is tridentoid and without any small elongated teeth on the mandibular teeth. Among the three Neolepas species, the tergum of N. marisindica sp. nov. is the sharpest, having a mean apex angle of approximately 70°. The apex angles of both N. rapanuii and N. zevinae are approximately 75° [31].

Morphological variations. The Kairei field population specimens were with orange-coloured peduncle and the capitulum coated with dark brown mineral deposits (figures 3a and 4); the Solitaire field population was whitish and without mineral deposits (figure 3b). A specimen (6 K-1327-R2-1) from the Solitaire field was dissected to demonstrate the variation in the external morphology. Compared to the holotype, the specimen from Solitaire field had a wider tergal apex angle, at 73°. The ratio of rostrum to median latus was 1.3. Twelve peduncular scales present per whorl at the region below the capitulum. Scales were approximately 2.4 mm wide and projected 1.65 mm out of the peduncle (figure 3b).

Arthropodal characters from the Solitaire field specimens are similar to those of the holotype (Kairei field). Six pairs of cirri: cirral counts of both anterior and posterior rami of each cirrus are similar between the holotype and the Solitaire specimen concerned (figure 10 and table 1). Maxilla and maxillule of the specimen from the Solitaire field do not show great variation from the holotype (figure 11a–d). Both maxillule and maxilla with simple type setae. Mandibles tridentoid and without extra small elongated teeth on the second tooth (figures 11d–h and 12a,b). Mandibulatory palp elongated with simple setae (figure 12c), labrum with a single row of small teeth (figure 12d–h).

Figure 10.

Neolepas marisindica sp. nov. A specimen collected from Solitaire vent field (NSMT-Cr 26834). (a) Cirrus I, (b) cirrus II, (c) cirrus III, (d) cirrus IV, (e) cirrus V, (f) cirrus VI, (g) caudal appendages, (h) penis.

Figure 11.

Neolepas marisindica sp. nov. A specimen from Solitaire vent field (NSMT-Cr 26834). Oral cone. (a) Maxilla, (b) simple setae on the margin of maxilla, (c) maxillule, (d) spines on the cutting edge of maxillule, (e) mandibles (ventral view), (f) first tooth of mandible (ventral view), (g) second and third tooth (ventral view), (h) inferior angle of mandible.

Comparing variations in capitular morphological characters between the Kairei population (10 specimens) and the Solitaire population (9 specimens), both populations shared similar peduncle characters: capitulum ratio (3 in Kairei and 3.8 in Solitaire), rostrum to median latus ratio (1.34 in Kairei and 1.45 in Solitaire), tergal apex angle (68 in Kairei and 71 in Solitaire) and the number of scales per whorl (20 in both populations; table 2). However, the Kairei specimens had significantly smaller scales (scale width 0.8 mm) when compared with the Solitaire population (1.54 mm; t-test, t = 3.5, d.f. = 17, p < 0.05; table 2, also figure 3a,b).

Table 2.

Variation in morphological characters mean ± 1 s.d. (range) of Neolepas marisindica sp. nov. from Kairei and Solitaire hydrothermal fields.

	Kairei vents (n = 10)	Solitaire vents (n = 9)
capitulum height	15.8 ± 11 (6.9–25)	14.18 ± 5.8 (2.8–25)
peduncle: capitulum	3.08 ± 1.61 (1.7–6.0)	3.87 ± 1.94 (1.8–7.7)
R : ML	1.34 ± 0.25 (1.0–1.8)	1.45 ± 0.23 (1.1–1.7)
tergal apex angle	68.4 ± 6.1 (58–76)	71.3 ± 5.29 (66–81)
no. of scales per whorl	19.7 ± 4.7 (12–28)	20.3 ± 5.8 (14–30)
scale width	0.83 ± 0.14 (0.6–0.9)a	1.54 ± 0.32 (1.2–2.25)a
size of scales projected	0.83 ± 0.26 (0.5–1.16)	1.21 ± 0.2 (0.9–1.46)

aIndicates significant difference in t-tests, p < 0.05.

The reconstructed phylogenetic tree based on the maximum-likelihood algorithm is shown in figure 13. The relationships among eolepadid species were the same as previously shown [6], except for the additional OTUs of Vulcanolepas cf. parensis in Manus Basin [39], which was shown to share some haplotypes with L. longa in TOTO Caldera and Edison Seamount [6]. Neolepas marisindica sp. nov. from the three populations formed a single clade with previously reported sequences [6], which was sister to V. scotiaensis in the Southern Ocean. The N. marisindica sp. nov.–V. scotiaensis group is a sister group to EPR and Southern EPR populations of N. zevinae-rapanuii complex, whose outgroups consist of the undescribed Vulcanolepas species from the Lau Basin and the Tonga Arc [6] and V. osheai from the Kermadec Arc.

Figure 13.

Molecular phylogenetic tree of eolepadid barnacles constructed by maximum-likelihood algorithm with Tamura three-parameter + gamma distribution model. Numbers beside each branch indicate bootstrapping value (2000 replicates, numbers lower than 70 not shown).

Discussion

Morphological variation and distribution range of N. marisindica sp. nov.

This study characterized Neolepas marisindica sp. nov. from deep-sea hydrothermal vent fields of the Indian Ocean, showing its morphological variability and phylogeography. As briefly mentioned above, the two CIR populations from Kairei and Solitaire hydrothermal vent fields of Neolepas marisindica sp. nov. examined in this study exhibited some differences in morphologies of capitulum and scales on peduncle, despite a lack of distinct sequence divergence in the COI gene between the two populations. The Solitaire hydrothermal field population, where diffuse flow venting was dominant, had larger scales with width greater than 1 mm compared with those from the Kairei hydrothermal field, where vigorous venting from black-smoker chimneys was dominant, whose scale width was approximately 0.8 mm. This difference was supported by statistical significance (p < 0.05; table 2). Morphological variations in neolepadines were also reported for Vulcanolepas scotiaensis in hydrothermal vent fields in the East Scotia Ridge, Southern Ocean, which exhibit a ‘robust’ form with short peduncle of peduncle : capitulum ratio as 1 : 1 in the site with low hydrothermal activity and a ‘gracile’ form with long peduncle of peduncle : capitulum ratio up to 20 : 1 in the site with active diffuse venting, but molecular analysis could not detect differences between the two [27]. The peduncular length is also variable in Vulcanolepas parensis, compared with the congeneric V. osheai and L. longa [27]. In the recent revision of taxonomy of Eolepadidae [27], the size of peduncular scales was used to discriminate Vulcanolepas and Neolepas, and the angle of tergal apex was considered diagnostic for Leucolepas and Neolepas. The presently examined specimens of N. marisindica sp. nov. exhibit intermediate characters between Vulcanolepas and Leucolepas in these two characters, respectively. The peduncular scales in the Kairei population are projected less than 1 mm out from the peduncle (table 2), which is within the diagnostic range indicated for Vulcanolepas [27]. Some individuals of N. marisindica sp. nov. had tergal apex angles of approximately 60°, which is supposedly a characteristic of Leucolepas (diagnostic tergal apex angle in Neolepas is 75° [27]).

The ratio of rostrum to median latus, as a key diagnostic character, is said to be 1.5 for Neolepas and 1 for other genera [27]. In this study, we found variations in the rostrum to median latus ratio among different specimens of N. marisindica sp. nov., which ranged from 1.0 to 1.8. The ratio of lengths of different capitular plates is clearly a continuous variable and it is highly problematic to treat these as the only diagnostic character for genus or even species identification, unless the range of variation is taken into consideration. We, therefore, suggest that the diagnostics and identification of Neolepas species is best relied upon investigation of arthropodal characters including mandibles, while also carefully considering their plate arrangement (with the intraspecific variation in mind), coupled with molecular DNA barcode analysis.

The present phylogenetic analysis was consistent with previous molecular studies [6,27], showing a close relationship between V. scotiaensis and members of the genus Neolepas (figure 13). This is different from taxonomic assignments based solely on hard part morphology, where V. scotiaensis was placed close to other Vulcanolepas species such as V. osheai [27]. These two species are then, in turn, sister to a clade consisting of N. zevinae and N. rapanuii, which appear to be genetically indistinguishable, at least using COI barcodes. This means V. scotiaensis is nested within the genus Neolepas. In addition, the mandible morphology of V. scotiaensis is actually very similar to those of other Neolepas species, as it has none or only minute longitudinal teeth. These results combined provide strong evidence that V. scotiaensis, in fact, belongs to the genus Neolepas, and therefore it is here formally transferred to Neolepas, as Neolepas scotiaensis (Buckeridge et al. [27]) comb. nov.

In contrast with high plasticity in the hard parts, morphologies of arthropodal characters are relatively stable and well supported by molecular phylogenetics. In this study, mandibles of the dissected individuals exhibited very similar morphological characteristics (figures 6e, 7e, 11e, 12e), whereas their hard part morphologies were more different (figure 3). The morphology of mandibles of Neolepas from SEIR [12] was the characteristic of N. marisindica sp. nov., as it lacks small longitudinal teeth on the second tooth. Therefore, we here consider these specimens to represent a further population of N. marisindica sp. nov., extending its distribution to SEIR, at least to 41° S. The DNA barcoding sequences of Neolepas marisinsica sp. nov. collected from Kairei and Solitaire hydrothermal fields on the CIR could not be separated from the Longqi population previously reported from the SWIR [6], confirming the distribution of the present new species on the SWIR, at least as far as the Longqi field. Therefore, N. marisindica sp. nov. is the only species of vent animal so far confirmed to range across hydrothermal vents in all three Indian Ocean oceanic ridges—the CIR, the SWIR and the SEIR. The fact that the same haplotypes have been recovered multiple times from populations on the CIR and the SWIR indicates that N. marisindica sp. nov. probably has sufficiently high dispersal ability to contain a metapopulation connecting the CIR and the SWIR across the Rodriguez Triple Junction, while for the scaly-foot gastropod Chrysomallon squamiferum the triple junction is known to act as a dispersal barrier [40]. As no vent on the SEIR has been visited by a submersible, further investigation of vents on the SEIR and samples from there will certainly reveal valuable information on the biogeography of deep-sea hydrothermal vent fauna in the Indian Ocean.

Phylogeography of vent barnacles

The phylogenetic analysis of vent stalked barnacles here elucidated their historical migration patterns across a geological timescale. As V. cf. parensis in Manus Basin shared some haplotypes with L. longa in TOTO Caldera and Edison Seamount (figure 13), here we regarded V. cf. parensis in Manus Basin as misidentification of L. longa. Leucolepas longa from the Mariana Forearc and the Manus Basin diversified at the most basal branch in the neolepadines, subsequently Vulcanolepas osheai in Kermadec Arc, and then an undescribed Vulcanolepas from the Lau Basin diversified, and finally the monophyletic Neolepas (figure 13). This renders Vulcanolepas paraphyletic. A previous tree published by Herrera et al. [6] combined three genes (28S, H3 and COI), however, with a different pattern with the basal branching being between a monophyletic Neolepas and a Leucolepas–Vulcanolepas clade. This node was highly supported in their study (0.88 and 100 for Bayesian posterior probability and bootstrap value, respectively). Considering that in our tree the node splitting Leucolepas from Vulcanolepas–Neolepas was not statistically supported (less than 0.70 in bootstrap probability), we interpret that the branching pattern observed in Herrera et al. [6] is more reliable. We, therefore, consider Vulcanolepas and Neolepas to be separate genera, following Herrera et al. [6]. On the other hand, the branching pattern within Neolepas (i.e. N. zevinae/rapanuii complex in the southern EPR, then N. marisindica sp. nov. in the Indian Ocean and finally N. scotiaensis in the Southern Ocean) was supported by high bootstrap probabilities (greater than 95 in bootstrap probabilities; figure 13).

The branching pattern, indicating close relationships between the species in Indian and Southern oceans compared with those in the southern EPR, is consistent with the pattern reported for the ‘yeti crabs’, squat lobsters in the genus Kiwa [7]. Neither Neolepas nor Kiwa has been reported from the Atlantic Ocean (except on the ESR of the Southern Ocean, which is technically in the extreme southern Atlantic), and their distribution and historical migration may be similar. The divergence between N. marisindica sp. nov. and N. scotiaensis was 1.7 Ma (95% HPD: 0.4–3.8) and the divergence between N. marisindica sp. nov.–N. scotiaensis and N. zevinae/rapanuii complex was 6.4 Ma (95% HPD: 3.0–11.2) [6]. The divergence between Kiwa sp. SWIR and Kiwa tyleri Thatje 2015 in Thatje et al. [41] from ESR, Southern Ocean was 1.5 Ma (95% HPD: 0.6–2.3) and the divergence between these two Kiwa species and Kiwa hirsuta from the Pacific-Antarctic Ridge was 19.1 Ma (95% HPD: 13.4–25.9) [7]. Geological evidence including the formation of the ESR and the Drake Passage, changes in the intensity and latitude of the Antarctic Circumpolar Current, and the realignment in the spreading axis of the Chile Rise seemed to have acted as key species vicariance events for Kiwa [7]. Characterization of faunal composition of hydrothermal vent fields on the ESR suggested the effect of the high intensity of the Antarctic Circumpolar Current around the middle Miocene (approx. 13.8 Ma) was crucial in separating vent fauna in ESR from other regions due to the inhibition of larval dispersal, as is known for non-vent Antarctic / Southern Ocean fauna [5,42]. Therefore, the present results strengthen the evidence to reject the hypothesis that Indian Ocean ridges act as ‘corridors’ for dispersal of vent taxa connecting Atlantic and Pacific Oceans [9], and instead, support the hypothesis of migration of vent fauna from the Pacific Ocean to the Indian Ocean, through the Southern Ocean. The phylogeny results indicate that the Pacific Ocean origin for the group may be the southern EPR, but this is necessarily only deduction from the known living species. Considering that a Jurassic neolepadine fossil (?Neolepas augurata) has been recorded from New Caledonia [25], it is highly likely that the true origin for the neolepadines is in the southwest Pacific near New Caledonia, from where it diversified towards both the EPR and Indian Ocean–Southern Ocean. Additionally, this is also in line with the fact that the basal taxa in Eolepadidae such as Leucolepas longa are found in the southwest Pacific.

In summary, we characterized a new hydrothermal vent barnacle Neolepas marisindica sp. nov. widely distributed in Indian Ocean vents which is hitherto the only species known to be distributed across all three mid-oceanic ridges in the Indian Ocean. Phylogeography of eolepadid stalked barnacles, including the new species, provides another piece of evidence against the Indian Ocean ‘corridor’ hypothesis. Future explorations of SEIR vents are urgently needed to shed further light on the biogeography of deep-sea hydrothermal vent taxa in the Indian Ocean and beyond. Morphological characteristics of hard parts in eolepadid barnacles, as in barnacles in general, were shown to exhibit high plasticity. We, therefore, suggest that the genus and species diagnostic characters should be mainly based on arthropodal characters (such as mouth parts) coupled with DNA barcoding, while the arrangement of hard parts such as the capitulum or scales on the peduncle should be used carefully with their intraspecific variation in mind.