Morphology and phylogenetic analysis of two new deep-sea species of Chrysogorgia (Cnidaria, Octocorallia, Chrysogorgiidae) from Kocebu Guyot (Magellan seamounts) in the Pacific Ocean

Abstract Two new species of Chrysogorgia Duchassaing & Michelotti, 1864 collected from Kocebu Guyot in the Magellan seamounts of the Pacific Ocean are described and illustrated: Chrysogorgia ramificanssp. nov. collected from a depth of 1831 m and Chrysogorgia binatasp. nov. collected from a depth of 1669 m. Chrysogorgia ramificanssp. nov. belongs to the Chrysogorgia “group A, Spiculosae” with rods distributed in body wall and tentacles, and C. binatasp. nov. belongs to the “group C, Squamosae typicae” with rods and/or spindles not present but only scales. Chrysogorgia ramificanssp. nov. differs from congeners by its main stem with 2/5R branching sequence at the bottom forming two large bottlebrush-shaped branches with 1/3R branching sequence at the top. Chrysogorgia binatasp. nov. is similar to C. scintillans Bayer & Stefani, 1988, but differs by its larger polyps, larger sclerites in the body wall, and different scales in the upper part of polyps. The mtMutS genetic distances between C. ramificanssp. nov. and C. binatasp. nov. and congeners are in the range of 0.33%–2.28% and 0.33%–2.94%, respectively, while the intraspecific distances are in the range of 0–0.16%. Molecular phylogenetic analysis indicates that C. ramificanssp. nov. is clustered with C. monticola Cairns, 2007 and C. binatasp. nov. is clustered with C. chryseis Bayer & Stefani, 1988, both with high support indicating close relationships.


Introduction
Within the gorgonian family Chrysogorgiidae, the genus Chrysogorgia Duchassaing & Michelotti, 1864 is the largest and most common group, distributed worldwide including the Antarctic, ranging from 100 m to 3860 m water depth. In some colonies it is characterized by a spiralling main axis that branches sympodially giving off secondary branches that subdivide dichotomously, resulting in a bottle-brush colony shape. In others, the sympodial main axis does not spiral, resulting in fan-like planar or bi-flabellate colonies (Pante and Watling 2011;Cordeiro et al. 2015). To date, Chrysogorgia contains 70 species (Cairns 2018). Among them, 45 species are found only from the Pacific, 17 species only from the Atlantic and 7 only from the Indian Ocean (Cairns 2001(Cairns , 2007(Cairns , 2018Pante and Watling 2011;Cordeiro et al. 2015). Chrysogorgia flexilis Wright & Studer, 1889 occurs in both the Pacific and Indian Oceans (Wright and Studer 1889;Cairns 2001).
Based on the presence of rods or scales in the body wall and tentacles, Versluys (1902) divided Chrysogorgia species into three groups, which were summarized by Cairns (2001) as following: "group A, Spiculosae" (rods and/or spindles in body wall and tentacles) with 38 species, "group B, Squamosae aberrantes" (rods and/or spindles in tentacles but not in body wall) with 13 species, and "group C, Squamosae typicae" (rods and/or spindles not present; only scales) with 18 species. More recently, Cordeiro et al. (2015) described the species C. upsilonia, which possesses spindles in the body wall but not in the tentacles, and classified it as "group D, Spiculosae aberrantes". The separation of four groups was further recognized by Cairns (2018).
During the investigation of the Magellan seamount benthic diversity in the tropical Western Pacific, we obtained two golden gorgonians from the Kocebu Guyot using a remotely operated vehicle (ROV). Based on morphological and phylogenetic analyses, both species proved to be new species of Chrysogorgia and are described as C. ramificans sp. nov. and C. binata sp. nov., respectively. Their genetic distances and phylogenetic relationships within Chrysogorgia are discussed.

Specimen collection and morphological examination
Specimens were obtained by the ROV FaXian (Discovery) from the Kocebu Guyot in the Magellan seamounts in the tropical Western Pacific during the cruises of the R/V KeXue (Science) in 2018 (Fig. 1). The specimens were photographed in situ before sampled, photographed onboard, and then stored in 75% ethanol after collection. Small branches were cut off and stored at -80 °C for molecular study.
The morphological terminology follows Bayer et al. (1983). A stereo dissecting microscope was used to examine the general morphology and anatomy. The sclerites of the polyps and branches were isolated by digestion of the tissues in sodium hypochlorite, and then were washed with deionized water repeatedly. To investigate the structure of polyps and sclerites, they were air-dried and mounted on carbon double adhesive tape and coated for scanning electron microscopy (SEM) observation. SEM scans were obtained and the optimum magnification was chosen for each kind of sclerites by using TM3030Plus SEM at 5 kV.
The type specimens (registration numbers: MBM286307 and MBM286346) of the two new species have been deposited in the Marine Biological Museum of Chinese Academy of Sciences (MBMCAS) at Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China.

DNA extraction and sequencing
Total genomic DNA was extracted from the polyps of each specimen using the TIANamp Marine Animal DNA Kit (Tiangen Bio. Co., Beijing, China) following the manufacturer's instructions. PCR amplification for the mitochondrial genomic region 5'-end of the DNA mismatch repair protein -mutS -homolog (mtMutS) was conducted using primers AnthoC-orMSH (5'-AGGAGAATTATTCTAAGTATGG-3'; Herrera et al. 2010) and Mut-3458R (5'-TSGAGCAAAAGCCACTCC-3'; Sánchez et al. 2003). PCR reactions were performed using I-5 2 × High-Fidelity Master Mix DNA polymerase (TsingKe Biotech, Beijing, China). The amplification cycle conditions were as follow: denaturation at 98 °C for 2 min, followed by 32 cycles of denaturation at 98 °C for 20 s, annealing at 50 °C for 20 s, extension at 72 °C for 15 s, and a final extension step at 72 °C for 2 min. PCR purification and sequencing were performed by TsingKe Biological Technology (TsingKe Biotech, Beijing, China).

Genetic distance and phylogenetic analyses
All the available mtMutS sequences of Chrysogorgia spp. and the out-group species from related chrysogorgiidid genera were downloaded from GenBank, and those without associated publications or named Chrysogorgia sp. were omitted from the molecular analyses (see Table 2 and Fig. 8). The sequences were aligned using MAFFT v.7 (Katoh and Standley 2013) with the G-INS-i algorithm. Genetic distances, calculated as uncorrected "p" distances within each species and among species, were estimated using v.6 (Tamura et al. 2013).
For the phylogenetic analyses, only one sequence was randomly selected from the conspecific sequences without genetic divergence (see Table 2). The evolutionary model GTR+G was the best-fit model for mtMutS, selected by AIC as implemented in jModeltest2 (Darriba et al. 2012). Maximum likelihood (ML) analysis was carried out using PhyML-3.1 (Guindon et al. 2010). For the ML bootstraps, we consider values < 70% as low, 70-94% as moderate and ≥ 95% as high following Hillis and Bull (1993). Node support came from a majority-rule consensus tree of 1000 bootstrap replicates.
Bayesian inference (BI) analysis was carried out using MrBayes v3.2.3 (Ronquist and Huelsenbeck 2003) on CIPRES Science Gateway. Posterior probability was estimated using four chains running 10,000,000 generations sampling every 1000 generations. The first 25% of sampled trees were considered burn-in trees. For the Bayesian posterior probabilities, we consider values < 0.95 as low and ≥ 0.95 as high following Alfaro et al. (2003). The accession numbers of the mtMutS sequences are listed next to the species names in the phylogenetic tree (Fig. 8). Diagnosis. Chrysogorgia (tertiary "group A, Spiculosae" -rods or spindles in the tentacles and the body wall) with a short basal stem leading to a bottlebrush-shaped main stem, giving of a single major branch also bottlebrush-shaped. Minor branches subdivided dichotomously, up to fourth order, with the first branch internode 20-30 mm long. Branching sequence 1/3R in two large branches and 2/5R in the basal stem. Polyps 2-4 mm tall with a thin neck. Sclerites of polyp body of large and thick rods and spindles with many warts. Small scales and rods in tentacles with many warts. Scales in coenenchyme elongate with irregular edges and a few warts. Description. Specimen about 73 cm long with the holdfast not recovered. Main stem forming two large bottlebrush-shaped branches whose axis has a brown metallic luster. The larger branch is 49 cm long and the other 45 cm long. The basal stem about 24 cm long and 4 mm in diameter (Fig. 2C). Branching sequences differing from bottom to top, 2/5R in the basal stem and 1/3R in the two large branches. Branches subdivided dichotomously, up to fourth order, the first branch internodes 20-30 mm long, with the terminal branchlets up to 41 mm. Distance between adjacent branches 8-12 mm, and orthostiche intervals about 30 mm in the two large branches and 50 mm at the bottom. Polyps thin, about 2-4 mm long and 1.0-1.5 mm wide at bases, with a long neck less than 1 mm wide. Two to four polyps on the first internodes, up to eight on terminal branchlets (Fig. 2D). No polyps on main axis internodes.
Etymology. The Latin adjective ramificans (branching) refers to the ramous structure of the stem.
Distribution. Found only from the Kocebu Guyot with water depth of 1831 m. Habitat. Colony attached to a rocky substrate with a small, oval-shaped holdfast ( Fig. 2A).
Within the group A, Chrysogorgia ramificans sp. nov. is also similar to C. arborescens Nutting, 1908, C. tuberculata Cordeiro et al., 2015and C. terasticha Versluys, 1902. However, the new species differs from C. arborescens by its much longer interval of adjacent branches (8-12 mm vs. 3 mm), the higher number of polyps in the distal branchlets (up to 8 vs. 2), and usually regular sclerites (vs. irregular) (Nutting 1908). It differs from C. tuberculata by the larger orthostiche intervals (30-50 mm vs. 8-23 mm), rods with numerous coarse warts (vs. spindles with acute and sparse warts), rods present in tentacles (vs. only scales), and scales in coenenchyme with inconspicuous warts (vs. prominent) (Cordeiro et al. 2015). The new species can be easily distinguished from C. terasticha by its branching sequence (1/3R at top and 2/5R at bottom vs. 1/4L), larger orthostiche intervals (30-50 mm vs. no more than 8 mm), no nematozooids in coenenchyme (vs. many), larger polyps (2-4 mm vs. no more than 1.6 mm), larger rods with various ends (vs. smaller with rounded ends), and the absence of scales at the polyp base (vs. presence) (Versluys 1902). Diagnosis. Chrysogorgia ("group C, Squamosae typicae") with a biflabellate colony and a short main stem. Polyps 3-5 mm tall. Scales smooth and thin in the basal part of polyps body with various shape, up to 1 mm long. Scales in the upper part of polyps of various shapes, converged to form an inconspicuous and blunt point at the base of a naked tract below each tentacle. Scales bluntly lancet-shaped, often with numerous coarse granules, longitudinally arranged around both sides of each naked tract. Scales irregular and coarse, usually with lobed edges in the back of tentacles. Scales of coenenchyme slipper-shaped with a medial contraction. Nematozooids absent from coenenchyme.
Description. Specimen with two attached individuals of the crustacean genus Galathea Fabricius, 1793 (Fig. 4C). Main stem short with a principal bifurcation, forming two roughly parallel, fans (Fig. 4A, F). Calcareous holdfast small and white, about 7 mm in diameter (Fig. 4F). Major branches of each fan subdivided dichotomously or sympodially. Specimen about 16 cm long and 15 cm wide with a brilliant golden axis, and the stem about 1.5 mm in diameter at base (Fig. 4C). Internodes about 5-9 mm long, each with a single polyp except the terminal twigs, which may sometimes have two polyps. Polyps large and orange in situ, about 3-5 mm tall by 1-2 mm wide, with sclerites forming an inconspicuous blunt point at the base of a naked tract below each tentacle (Figs 4D, 5A). Terminal polyps usually with a long and narrow body (Fig. 4E).
In the basal part of the polyp body, the sclerites comprise transversally arranged, large, smooth scales. They represent a variety of shapes, a few with broad marginal lobes, length by width measuring 216-936 × 58-283 μm, with an average of 549 × 166 μm, (Figs 5B, 6A). There are also scales in the upper part of polyps of various shapes, sometimes thick and with a medial contraction, often sharp at one end , broad and lobed at the other, which combine to form an inconspicuous and blunt point at the base of a naked tract below each tentacle; measuring 275-635 × 77-254 μm, with an average of 451 × 151 μm (Figs 5B, 6B). Above these points are irregular, elongate or lancet-shaped scales mostly with coarse granules on surface, that are arranged longitudinally around the sides of each naked tract. The scales measure 337-650 × 45-85 μm with an average 431 × 70 μm (Figs 5B, 7A). The scales in the back of tentacles are coarse, of various shapes, mostly flat and lobed, and densely and transversally arranged, measuring 88-352 × 19-149 μm, with an average of 183 × 55 μm (Figs 5A, 7B). The scales in the pinnules are curved at an obtuse angle, and are sometimes thick with a few lobes on their edges; measuring 87-196 × 19-34 μm, with an average of 152 × 27 μm (Fig. 7D). The scales of the coenenchyme are generally slipper-shaped, some elongate elliptical, occasionally with indentations in their edges, and measure 138-361 × 40-87 μm, with an average of 222 × 56 μm (Fig. 7C). The coenenchyme is thin with no nematozooids. Etymology. The Latin adjective binatus (binate) refers to the biflabellate structure of the species.
Distribution. Found only from the Kocebu Guyot in the Magellan seamounts with water depth of 1669 m. Habitat. Colony attached to a rocky substrate with a small holdfast (Fig. 4B).
Both Chrysogorgia binata sp. nov. and C. electra have a biflabellate colony. However, the new species differs from the latter by its larger polyps (3-5 mm vs. generally 1.75-2.00 mm in height), eight short and blunt points beneath the tentacles (vs. inconspicuous), scales of various shapes with low and broad marginal lobes in the body wall (vs. elongate with narrow prominent marginal lobes), scales mainly slipper-shaped in coenenchyme (vs. elongate tapered with prominent marginal lobes) (Bayer and Stefani 1988). Chrysogorgia binata sp. nov. differs from C. scintillans by its larger polyps (3-5 mm vs. up to 2.8 mm in height), larger sclerites in the body wall (maximum length 0.93 mm vs. 0.65 mm), scales in the upper part of polyps (irregular and usually with sharp end vs. regular and usually with smooth end), scales around the sides of each naked tract (lancet-shaped and usually with coarse granules vs. twisted, flat and often lobed) Stefani 1988, Cairns 2018).

Genetic distance and phylogenetic analyses
Two mtMutS sequences of the two new species were obtained and deposited in Gen-Bank, and the accession number and the length are as follows: MK431863, 695 bp for C. ramificans sp. nov.; and MK431862, 690 bp for C. binata sp. nov. The alignment datasets each comprised 649 nucleotide positions. The mtMutS genetic distances among the species of Chrysogorgia range from 0.16% to 2.94%, while the intraspecific distances within C. binata sp. nov., C. tricaulis, C. artospira, C. averta, C. abludo and C. chryseis are in the range 0-0.16% (Table 2). Thus, there is no distinct barcoding gap between the intra-and interspecific distances. The genetic distances between the new species C. ramificans sp. nov. and the known sequences of the congeners range from 0.33%-2.28%, and those between C. binata sp. nov. and the congeners are in the range of 0.33%-2.94% (Table 2).
The ML and BI phylogenetic trees of the mtMutS gene were nearly identical in topology and thus were combined into a consensus tree with both support values (Fig. 8). All the Chrysogorgia species were separated into two main groups (Fig. 8).

Discussion
Chrysogorgia ramificans sp. nov. mostly resembles C. monticola Cairns, 2007, which is also strongly supported by the phylogenetic tree and their genetic distance. However, the two species can be easily separated, as discussed above. In the phylogenetic trees, C. binata sp. nov., C. cf. stellata Stefani, 1988 andC. chryseis Bayer &Stefani, 1988 formed a single clade with high support, indicating their close relationships (Fig. 8). However, C. binata sp. nov. belongs to the Chrysogorgia "group C, Squamosae typicae", while C. chryseis belongs to "group B, Squamosae aberrantes" (Table 1; Bayer and Stefani 1988). Bayer and Stefani (1988) also reported a specimen they recorded as C. cf. stellata Nutting, 1908, which was based only on some detached branches. Chrysogorgia binata sp. nov. differs from C. cf. stellata by its larger polyps (3-5 mm vs. about 2 mm), many elongate or lancet-shaped scales below the tentacle base (vs. one or two rods with coarse granules), short, squarish or polygonal scales in the polyp body wall (vs. narrow and long), regular slipper-shaped scales in coenenchyme (vs. relatively irregular) (Bayer and Stefani 1988). No sequences are available for Chrysogorgia stellata Nutting, 1908, a species possessing a multiflabellate colony form, while C. binata sp. nov. is biflabellate. The new species differs from C. stellata also in the shorter and more blunt points beneath the tentacles (vs. long and sharp), various shapes of scales in the upper part of the body wall (vs. only a single slenderly elongate shape), and generally slipper-shaped scales in coenenchyme (vs. elongate with more lobed margin).