Reevaluation of the Systematic Status of Branchinotogluma ( Annelida , Polynoidae ), with the Establishment of Two New Species

,


Introduction
The deep-sea hydrothermal vents and cold seeps are typical extreme environments on Earth, sharing members of specialized macrofaunas, such as siboglinid tubeworms, bathymodiolin mussels, and vesicomyid clams [1]. Polynoidae is among the most diverse annelid families in the deep sea [2], particularly well represented in deep-sea hydrothermal vents, cold seeps, whale carcasses, and sunken wood [3].
Previous studies have indicated the paraphyly of Branchinotogluma [8,16,17]. The phylogenetic relationships among the species of Branchinotogluma, particularly the two "weird" species, B. segonzaci [7] and B. trifurcus [7] need to be reevaluated. Both species have an indistinctly bilobed prostomium with a pair of prominently cylindrical anterior lobes. In addition, females of B. segonzaci have no ventral papillae, and B. trifurcus has reduced parapodia resembling members of Branchipolynoe, while most species of Branchinotogluma have a bilobed prostomium with triangular anterior lobes, ventral papillae in females, and a uniform pattern for parapodia. Barcoding and phylogenetic analyses have showed that both species are clearly divergent from the congeneric species [10,16], further urging us to reevaluate the systematic status of the genus Branchinotogluma and related taxa.
Our examination of the specimens recently collected from the deep-sea cold seeps in the South China Sea revealed two undescribed species of Branchinotogluma which are herein described as B. nanhaiensis sp. nov. and B. robusta sp. nov. Thus, we reevaluated the systematic status of Branchinotogluma and related taxa based on four gene fragments (COI, 16S rRNA, 18S rRNA, and 28S rRNA). Moreover, we traced the evolution of the characters related to prostomium, segment, elytra, branchiae, and ventral papillae and established ancestral and derived characters of the main clades within the polynoids from chemosynthesisbased ecosystems.

Specimen Collection and Morphological Examination.
All specimens were obtained from five dives by the remotely operated vehicle (ROV) Fa Xian (Discovery) on board the R/V Ke Xue (Science) from a deep-sea cold seep (119°17 ′ E, 22°074N, 1118. 6-1121.8 m) in the South China Sea from July 2015 to August 2016. They were preserved in 80% (v/v) ethanol solution and later deposited in the Marine Biological Museum of the Chinese Academy of Sciences (MBMCAS), Institute of Oceanology, Chinese Academy of Sciences (IOCAS).
Aligned sequences for COI, 16S, 18S, and 28S were concatenated using PhyloSuite v1.2.2 [28]. Best partitioning models for the four predefined partitions were selected using the greedy algorithm and the corrected Akaike information criterion (AIC) in PartitionFinder2 [29]. The GTR+I+G model was selected for the COI sequences and both concatenated sequences. Maximum likelihood (ML) analysis was performed using IQ-TREE 1 [30] under Edge-unlinked partition model for 100,000 ultrafast [31] bootstraps. Bayesian inference (BI) analysis of the concatenated data were conducted using MrBayes 3.2.6 [32] under the partitioned models (2 parallel runs, 10,000,000 generations) with sampling every 1000 generations. The initial 25% of sampled data were discarded as burn-in and the remaining trees were used to construct the 50% majority rule consensus tree and to estimate the posterior probabilities (PP). The effective sample size (ESS) values for all sampled parameters were diagnosed by Tracer v1.6 [33] to make sure convergence was reached. Finally, the phylogenetic tree was annotated with the ML bootstrap percentages and the BI posterior probabilities.
2.4. Character Evolution. Eleven morphological characters correlated with prostomium, segment, elytra, branchiae, and ventral papillae ( Table 2) were summarized (Table S3) and then mapped onto a simplified BI tree. Ancestral state reconstruction (ASR) analyses were conducted using Mesquite v.3.6.1 [34] to examine the character evolutionary patterns and infer ancestral and derived characters within the studied polynoids. MK1 likelihood model was adopted because this model incorporates branch length information into the character transformation.  (Table S2). Genetic distances among the species of Brachinotogluma ranged from 0.084 to 0.282. Among these species, four pairs of species have relatively small interspecific distances, i.e., 0.084 between B. elytropapillata [8] and B. ovata [10], 0.084 between B. ovata and B. sagamiensis [6], 0.088 between B. japonicus [9] and B. nanhaiensis sp. nov., and 0.150 between B. pettiboneae [10] and B. robusta sp. nov. The affinities between B. elytropapillata and B. ovata, B. ovata and B. sagamiensis, B. japonicus and B. nanhaiensis sp. nov., and B. pettiboneae and B. robusta sp. nov. can also be supported by their closed resemblance in morphology ( [10]; this study). In contrast, the intraspecific distances within the species of B. japonicus, B. nanhaiensis sp. nov., B. pettiboneae, and B. robusta sp. nov. were very low (0.0004-0.0038, Table S2), supporting the establishment of the two new species.

Results and Discussion
The topologies of the concatenated phylogenetic trees reconstructed from both ML and BI analyses were identical in most clades ( Figures S1 and S2). The only difference is related to the polytomous branches which is resulted from the unresolved relationships between the four clades (Branchipolynoe and Clades1-4; Clade 5; Peinaleopolynoe; Clade 6). Thus, only the BI tree is presented with BI posterior probability and ML bootstrap scores indicated on each node (Figure 2). Phylogenetic trees support the establishment of the two new species and sister relationship between B. nanhaiensis sp. nov. and B. japonicus [9] and that between B. robusta sp. nov. and B. pettiboneae [10].  Hatch et al. [16] Branchipolynoe eliseae

Journal of Zoological Systematics and Evolutionary Research
Branchinotogluma is nonmonophyletic, as indicated by both the ML and BI analyses. However, the monophyly of the two closely related genera, Branchipolynoe and Peinaleopolynoe, is well-supported, and both genera are intermingled with the clades consisting of Branchinotogluma. Furthermore, the phylogenetic analysis indicates that the species of Branchinotogluma can be divided into 7 clades. Clade 1 (PP/BP = 1:00/100): Branchinotogluma marianus [5] and B. nikkoenisis [6] formed a well-supported clade and was recovered as the sister group to Branchipolynoe clade (PP/BP = 1:00/99).
Clade 4: Branchinotogluma hessleri [11] formed a separated clade and was the sister group to the clade consisting of all the above-mentioned clades with low supports (PP/BP = 0:61/54).
Our phylogenetic analyses also showed that the polynoids inhabiting deep-sea chemosynthetic environment can be divided into two main sister clades (Figures 2 and 3). Clade A consisted of clades 1-6, Branchipolynoe clade and Peinaleopolynoe clades. Clade B consisted of species of Levensteiniella, Lepidonotopodium, Bathykurila, Thermopolynoe, and B. segonzaci. The species Branchiplicatus cupreus [11] was recovered as a basal clade which is the sister to the large clade composed of Clade A and Clade B.
3.2. Character Evolution. BI molecular phylogeny was generated using the reduced concatenated dataset (Figure 3). Eleven morphological characters correlated with prostomium, segment, elytra, branchiae, and ventral papillae (Figure 4) for the 43 polynoid species were summarized (Table S3) and then mapped onto the simplified BI tree (Figures 5 and 6).
The ancestral state of shape of prostomium for the chemosynthesis-based polynoids was inferred to be distinctly bilobed prostomium with triangular anterior lobes (Figures 4(i) and 4(l)). The slightly bilobed or nonbilobed prostomium as well as cylindrical anterior lobes (Figures 4(j) and 4(k)) are derived characters (Figures 5(a) and 5(b)).
The number of elytra and the number of segments were closely linked according to the analyses. The ancestral state for Clade A was suggested to be 20-21 segments and 9-10 pairs of elytra. In contrast, most species of Clade B have 22-30 segments with 11 pairs of elytra, which were derived from 20 to 21 segments with 9-10 elytra. However, the ancestor for the chemosynthesis-based polynoids seems to have more than 30 segments with more than 11 pairs of elytra (Figures 5(c) and 5(d)). Almost all the polynoids except Branchipolynoe have large elytra covering dorsum, which was considered as an ancestral state for the studied polynoids ( Figure 3).
All the chemosynthesis-based polynoids except Branchiplicatus cupreus have ventral papillae in males, which was referred to be an ancestral state for the large clade composed of Clade A and Clade B (Figure 6(c)). The presence of ventral papillae in females only occurs in Clade A and was likely to be an ancestral state for Clade A (Figure 6(a)). In contrast, the absence of ventral papillae in females was inferred to be the ancestral state of Clade B (Figure 6(a)).
The ancestral state of the start of ventral papillae in males was inferred to be at segment 12 (Figures 4(a)-4(c), 4(g), and 4(h)) for both Clade A and Clade B. The start of ventral papillae from segment 11 (Figures 4(d)-4(f)) in males seems to be derived from the start from segment 12 ( Figure 6(d)). The ventral lamellae (Figures 4(a), 4(b), 4(g), and 4(h)) occur in males of Branchinotogluma and some species of Peinaleopolynoe. The ancestral state was arguably inferred to be presence of ventral lamellae in males for Clade A but absence of ventral lamellae in males for Clade B (Figure 6(b)). (1) Type Species. Branchinotogluma hessleri [11], by original designation.

Systematics. Class
(2) Diagnosis. Body fusiform or ovate, 20 or 21 segments, 9 or 10 pairs of elytra. Elytra rounded to subreniform, usually smooth. Branchiae arborescent, two pairs per segment, from segment 3. Prostomium usually bilobed with anterior lobes triangular with minute or digitiform frontal filaments. Pharynx with 4-9 terminal papillae and two pairs of jaws with or without denticles. Notopodial bracts present on segment 2 or 9 Journal of Zoological Systematics and Evolutionary Research all elytrigerous segments, enclosing or fused with acicular lobes. Notochaetae usually shorter and stouter than neurochaetae, either smooth or with widely spaced spines along edge. Neurochaetae more numerous, forming a fan-shaped bundle; spinous, tips hooked or tapered. Males with paired ventral papillae from segment 12, followed by rounded lamellae. Females with 0-7 paired small, rounded ventral papillae from segment 11. Males with modified parapodia on posterior segments. Pygidium small, rectangular, usually with two anal cirri.    (2) Etymology. The specific name is derived from Nanhai, the Chinese name of the South China Sea, where the species was collected.
Males with 21 segments and a pair of prominent ventral papillae on ventral side of segment 12 (Figure 8(d)), followed by five pairs of thin, semiovate ventral lamellae along segments 13-17. Females with 20 segments and two pairs of concave ventral papillae on segments 11 and 12 (Figure 8(c)).  Branchinotogluma nanhaiensis sp. nov. resembles B. elytropapillata, B. hessleri [11], B. japonicus [9], B. nikkoensis [6], B. ovata, B. segonzaci [7], and B. trifurcus [7] in having a pair of ventral papillae and five pairs of ventral lamellae in males. Among these species, B. nanhainensis sp. nov. is similar to B. hessleri, B. japonicus, and B. nikkoensis in having prominent and thick ventral papillae, which are short and tapered in the other species. Branchinotogluma nanhaiensis sp. nov. also resembles B. japonicus in having similar body size and shape, slender pharynx with two bundles of lateral tapered papillae and soft jaws without teeth. B. nanhainensis sp. nov. differs from B. japonicus in having 20 segments in females (vs. 21 segments) and presence of concave ventral papillae (vs. absent).
All parapodia biramous. Second segment bearing first pair of elytrophores, notopodia much shorter than neuropodia, and buccal cirri similar to ventral tentacular cirri, much thicker and longer than subsequent ventral cirri (Figure 11(a)); notopodium conical with bract fused with acicular lobe, posteriorly forming a pocket surrounding a small bundle of notochaetae; neuropodium with a long conical prechaetal lobe and a short subtriangular postchaetal lobe (Figure 11(a)). Following segments with notopodia shorter than neuropodia on anterior segments (Figures 11(b)-11(d)), becoming as long as neuropodia on posterior segments (Figures 11(e) and 11(f)). Notopodium with a projecting acicular lobe on lower side and numerous notochaetae forming a radiating bundle. Notochaetae much stouter than neurochaetae, straight or slightly curved with blunt bare tips, smooth in upper and middle fascicle, and serrated with two rows of widely spaced spines in lower positions (Figures 11(h)-11(j)). Neuropodium including a long, conical prechaetal lobe with a projecting acicular lobe, and a short subtriangular postchaetal lobe with a small projecting lobe on dorsal base (Figures 11(b)-11(d)). Neurochaetae numerous forming a fan-shaped bundle. Neurochaetae straight, tips hooked; spinous region extending to tips, with two rows of spines along a longitudinal furrow; spines short and close-set on distal part, becoming longer and widely spaced proximally  (5) Remarks. Branchinotogluma robusta sp. nov. highly resembles B. pettiboneae [10] in most main features, such as body size and shape, pharynx, ventral papillae, and parapodium. However, B. robusta sp. nov. can be distinguished from B. pettiboneae in having neurochaetae with hooked tips (vs. straight spinous tips on the neurochaetae).  Based on the newly generated data along with data downloaded from GenBank, we made a barcoding analysis for 16 species of Brachinotogluma. The interspecific genetic distances are much greater than the intraspecific distances, supporting the species delimitation which is based on morphological examination. In addition, phylogenetic analyses also support the establishment of the two new species. Both morphology and phylogenetic analysis supported the close relationship between B. nanhaiensis sp. nov. and B. japonicus [9] and that between B. robusta sp. nov. and B. pettiboneae [10]. Branchinotogluma japonicus and B. pettiboneae were found, respectively, from the hydrothermal vents of Okinawa Trough and Manus Basin. It is noteworthy that Lepidonotopodium okinawae [35] and Branchipolynoe pettiboneae [9], originally described from the vents of Okinawa Trough, also occur accompanying with the two new species (authors' examination), indicating the evolutionary links between hydrothermal vent and methane seep faunas.

Systematics and Phylogeny of Branchinotogluma.
The genus Branchinotogluma is expanded to contain 16 valid species after the description of two new species. It was recovered as paraphyletic with seven clades scattering in two main clades. Six of them formed a well-supported clade together with Branchipolynoe and Peinaleopolynoe. Branchinotogluma segonzaci [7] was recovered as the seventh clade, located in the other main clade composed by Bathykurila, Lepidonotopodium, Levensteiniella, and Thermopolynoe. However, we suggest preserving the stability of all these genera rather than synonymizing them as a single genus due to their distinct morphological difference and the lack of morphological and molecular data. Branchinotogluma trifurcus [7] was recovered as the sister group with high supports to the clade composed of Branchipolynoe, Clade 1, and Clade 2. The species is characterized in Branchinotogluma by having reduced parapodia and prechaetal and postchaetal lobes similar in shape [10], which are also characters of Branchipolynoe. The reduction of the parapodium in Branchipolynoe was considered as an ancestral character descended from B. trifurcus. In addition, B. trifurcus is characterized by a trapezoid pro-stomium and cylindrical anterior lobes without frontal filaments ( Figure 4). However, these characters seem to be derived from the bilobed anterior lobes with frontal filaments according to our analyses.
Phylogenetic analyses showed the unusual placement of B. segonzaci, which was separated from the other species of Branchinotogluma and recovered as the sister group to the clade formed by species of Levensteiniella, Lepidonotopodium, Thermopholynoe and Bathykurila. The separation of B. segonzaci from the other congeners is in consistent with the morphological differences between them. Branchinotogluma segonzaci is characterized by the prominently cylindrical anterior lobes with digitiform frontal filaments, which highly resemble the median antenna (Figure 4(j)), while the other species have delicate or no frontal filaments (Figures 4(i) and 4(l)). Among the species of Branchinotogluma, three species, viz. B. sandersi [11], B. burkensis [5], and B. trifurcus, have cylindrical anterior lobes. However, the frontal filaments are slender in B. sandersi and B. burkensis and absent in B. trifurcus (Figure 4(k)).
In addition, B. segonzaci is characterized for the lack of ventral papillae in females (Figure 4(g)), while almost all species of Branchinotogluma have various numbers of ventral papillae in females (Figures 4(a), 4(b), 4(g), and 4(h)). The females of B. marianus [5] and B. tunnicliffeae [12] have not been described so their ventral papillae in females are unknown (Figures 3 and 5). The absence of ventral papillae in females of B. japonicus [9] described by Zhang et al. [8,17] is uncertain because only a single female specimen with poor preservation was examined (Figures 3 and 5). It is interesting that the ventral papillae in females only occur in Branchipolynoe and Branchinotogluma. In contrast, all species in Clade B including B. segonzaci have no ventral papillae in females, which is a reason to explain why B. segonzaci is phylogenetically placed in the clade composed of Bathykurila, Lepidonotopodium, Levensteiniella, and Thermopolynoe ( Figure 2). However, B. segonzaci differs distinctly from these related genera in the numbers of segments and elytra, presence of modified segments and ventral lamellae, and ventral papillae in males starting at segment 12.

Character Evolution.
The polynoids inhabiting the deepsea hydrothermal vents and cold seeps exhibit diverse morphological characters. Most of them have bilobed prostomium with triangular anterior cirri. Only the species Branchinotogluma trifurcus [7] and Branchiplicatus cupreus [11] possess fused prostomium, which seems to be a derived feature. The cylindrical anterior cirri are also inferred to be derived from triangular ones. The minute frontal filaments on the anterior cirri were considered as lateral antennae by Hatch et al. [16]. As the lateral antennae have been used as a very important character in classifying different genera and subfamilies among the Polynoidae, we would suggest keeping the original term unless we have anatomical evidence to support the proposal.
The numbers of segments and elytra are also key characters for polynoids. Species of Clade A have stable numbers of segments (20-21, mostly 21) and elytra (9-10, mostly 10 pairs), while most species of Clade B have more segments 19 Journal of Zoological Systematics and Evolutionary Research (22)(23)(24)(25)(26)(27)(28)(29)(30) with a stable number of elytra (11 pairs). The exceptions are Bathykurila guaymasensis [5] and Branchinotogluma segonzaci [7], which have 15 segments with 7 pairs of elytra and 21 segments with 10 pairs of elytra, respectively. The characters of 20-21 segments and 9-10 pairs of elytra are considered as ancestral characters for the big clade composed of Clade A and Clade B. Almost all species have large elytra covering the whole dorsum, except the species of Branchipolynoe. The reduction of elytra in Branchipolynoe is a derived character, which is speculated to be an adaption to the parasitic lifestyle.
Polynoids inhabiting deep-sea chemosynthetic environments often exhibit sexual dimorphism, with different types of ventral papillae. Elongated ventral papillae are present in males of almost all the chemosynthesis-based polynoids and are inferred as a common character for the big clade composed of Clade A and Clade B (Figure 6(c)). Ventral papillae in Clade A start from segment 12 in males and segment 11 in females, while in Clade B, they usually start from segment 11 (except B. segonzaci with segment 12) in males and are absent in females. Ventral papillae starting from segment 12 in males is inferred to be an ancestral state for the big clade composed of Clade A and Clade B.