Morphology and phylogenetic analysis of five deep-sea golden gorgonians (Cnidaria, Octocorallia, Chrysogorgiidae) in the Western Pacific Ocean, with the description of a new species

Abstract Explorations of seamounts in the Western Pacific Ocean and South China Sea resulted in collecting 18 specimens of golden gorgonians. Based on the morphology and the genetic analysis of mtMutS, they are described as one new species, Chrysogorgia carolinensissp. nov., and four known species, including Chrysogorgia dendritica Xu, Zhan & Xu, 2020, Metallogorgia melanotrichos (Wright & Studer, 1889), Metallogorgia macrospina Kükenthal, 1919, and Pseudochrysogorgia bellona Pante & France, 2010. Chrysogorgia carolinensis belongs to the Chrysogorgia “group A, Spiculosae” with rods or spindles distributed in the polyp-body wall and tentacles, and differs from all of its congeners except C. dendritica by the 1/3L branching sequence and amoeba-shaped sclerites at the basal polyp body. The mtMutS sequence of C. carolinensissp. nov. has six deletion mutations compared to those of its congeners, supporting the establishment of the new species. Although no genetic variability was observed between the closely related species C. dendritica and C. abludo Pante & Watling, 2012, the former is different from the latter by the apparently irregular sclerites in the polyp body wall. The two specimens of Metallogorgia melanotrichos match well with the original description except for relatively larger polyps, while the M. macrospina specimens have slightly smaller polyps than the holotype. The juvenile of Metallogorgia has an obvious morphological difference with the adults in the colony shape and branches, but they can be unified by the same polyps and sclerites as well as mitochondrial MutS sequences. Thus, the generic diagnosis of Metallogorgia is slightly extended to include the morphology of juveniles. The morphology of Pseudochrysogorgia bellona Pante & France, 2010, as a new record for the South China Sea, matches well with that of the original description. In the phylogenetic trees, the Chrysogorgia species are separated into two clades, and while Metallogorgia and Pseudochrysogorgia formed a sister clade.


introduction
Chrysogorgiids are found in all major oceans from Iceland to Antarctica, and they are conspicuous members of deep-water benthic assemblages . The family Chrysogorgiidae Verrill, 1883 currently contains 14 genera, with 12 genera having been analyzed by molecular phylogenetic methods with and six of them forming a monophyletic clade (called the Monophyletic Chrysogorgiidae Clade, or MCC), including Chrysogorgia Duchassaing & Michelotti, 1864, Pseudochrysogorgia Pante & France, 2010, Iridogorgia Verrill, 1883, Rhodaniridogorgia Watling, 2007, Radicipes Stearns, 1883and Metallogorgia Versluys, 1902(Watling et al. 2011. The genus Chrysogorgia Duchassaing & Michelotti, 1864 contains 75 species and is distributed worldwide (Watling et al. 2011;Cairns 2018;Xu et al. 2020). It is characterized by four branching forms of the colony, including a single ascending spiral (clockwise or counterclockwise, bottlebrush-shaped colony), a single fan (planar colony), two or more fans emerging from a short main stem (bi-or multi-flabellate colony) and a bush of branches perched on top of a long straight stem (tree-shaped colony) Cordeiro et al. 2015;Xu et al. 2020). Based on the shapes of rods or scales in the polyp-body wall and tentacles, four groups have been recognized for the separation of Chrysogorgia species, including "group A, Spiculosae" with 42 species, "group B, Squamosae aberrantes" with 13 species, "group C, Squamosae typicae" with 19 species and "group D, Spiculosae aberrantes" with only one species (Versluys 1902;Cairns 2001;Cordeiro, Castro and Pérez 2015;Xu et al. 2019).
During an investigation of the seamount benthic diversity in the Western Pacific Ocean and the South China Sea, we obtained 18 specimens of golden gorgonians. Based on morphological and phylogenetic analyses, they were described as one new species Chrysogorgia carolinensis sp. nov., and four known species, including Chrysogorgia dendritica Xu, Zhan & Xu, 2020, Metallogorgia melanotrichos (Wright & Studer, 1889), Metallogorgia macrospina Kükenthal, 1919 and Pseudochrysogorgia bellona Pante & France, 2010. Their phylogenetic positions are also discussed.

Specimen collection and morphological examination
One specimen of Chrysogorgia Duchassaing & Michelotti, 1864 was collected from a seamount (tentatively named as M2) near the Mariana Trench by the ROV (remotely operated vehicle) FaXian (Discovery) in the tropical Western Pacific during the cruises of the R/V KeXue (Science) in 2016. One specimen of Pseudochrysogorgia Pante & France, 2010 was initially collected from the Ganquan Plateau in the South China Sea in 2018, unfortunately only a few frozen fragments of this specimen and a picture were obtained. Ten specimens of Chrysogorgia and six specimens of Metallogorgia Versluys, 1902 were obtained from the seamounts (tentatively named as M5-M8) located on the Caroline Ridge during the cruises of the R/V KeXue (Science) in 2019. The Chrysogorgia and Metallogorgia specimens were photographed in situ before sampling, and photographed on board and then stored in 75% ethanol after collection. Small branches were detached and stored at -80 °C for molecular study.
The morphological terminology follows Bayer et al. (1983). The general morphology and anatomy were examined by using a stereo dissecting microscope. 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. Polyps and sclerites were air-dried and mounted on carbon double adhesive tape and coated for scanning electron microscopy (SEM) observation to investigate their structure. SEM scans were obtained and the optimum magnification was chosen for each kind of sclerite by using TM3030Plus SEM.
The type and voucher specimens have been deposited in the Marine Biological Museum of Chinese Academy of Sciences (MBMCAS) at Qingdao, China.

Genetic distance and phylogenetic analyses
The mtMutS gene in octocorals was selected for molecular identification and phylogenetic analyses. All the available mtMutS sequences of Chrysogorgia, Metallogorgia, Pseudochrysogorgia and the out-group species from related chrysogorgiid and plexaurid genera were downloaded from GenBank, and those from duplicate isolates or without associated publications or not identified to species level were omitted from the molecular analyses. To correct possible mistakes, all the selected sequences were visually inspected, and translated to amino acids (AA) to insure all the AA sequences did not include stop codons and suspicious substitutions. The nucleotide and AA sequences were aligned using MAFFT v.7 (Katoh and Standley 2013) with the G-INS-i algorithm. With the guidance of the AA alignment, the nucleotide alignment was refined using BioEdit v7.0.5 (Hall 1999), and only the nucleotide alignment was used in the subsequent analyses. Genetic distances, calculated as uncorrected "p" distances within each species and among species, were estimated using MEGA 6.0 (Tamura et al. 2013).
For the phylogenetic analyses, only one known sequence was randomly selected from the conspecific sequences without genetic divergence (see Table 4). The TPM1uf+G evolutionary model was the best-fitted 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. Convergence was assessed by checking the standard deviation of partition frequencies (< 0.01), the potential scale reduction factor (ca. 1.00), and the plots of log likelihood values (no obvious trend was observed over time). For the Bayesian posterior probabilities, we consider values < 0.95 as low and ≥ 0.95 as high following Alfaro et al. (2003). The museum voucher specimen and GenBank accession numbers of the mtMutS sequences were listed next to the species names in the phylogenetic trees ( Figure 21).  Xu et al. 2020). Colony branching usually sympodial, occasionally monopodial, arising from a single ascending spiral (clockwise or counterclockwise, bottlebrush-shaped colony), a fan (planar colony), two fans emerging from a short main stem (biflabellate colony), or an unbranched main stem forming a tree-shaped colony. Axis with a metallic shine, dark to golden in color. Branch subdivided dichotomously or pinnately. Most polyps relatively large to the size of the branches they sit on, few in number and well separated from one another. Sclerites in the form of spindles, rods, scales, and rare plates with little ornamentation. Extended diagnosis. Chrysogorgia "group A, Spiculosae" with 1/3L Branching sequence and a monopodial or a little zigzagging stem. Juvenile with a bottlebrush-like colony, while adult usually having a tree-shaped colony. Branches nearly perpendicular to stem, subdivided dichotomously. Polyps with a long neck and an expanded base. Rods and rare scales in tentacles, longitudinally arranged. Rods/spindles and elongate scales in polyp neck longitudinally arranged, coarse with many warts on surface. Scales and rare plates at the basal polyp body irregularly and alternately arranged, irregular and often amoeba-shaped. Scales in coenenchyme sparse and elongate, usually lobed with irregular edges.

Class
Description. For morphological measurements, see Table 1. Remarks. The four specimens match the holotype of Chrysogorgia dendritica Xu, Zhan & Xu, 2020 in having a monopodial stem and the same sclerite form, for example, rods and rare scales in tentacles, rods/spindles and elongate scales in polyp neck, irregular scales at the basal polyp body, and elongate scales in coenenchyme. Moreover, their mtMutS gene sequences are identical (see the genetic analysis below). Thus, we identified the four specimens as C. dendritica. The sclerites in the four  However, these differences are minor and not constant, and we thus treate as the conspecific variation.
The four specimens of C. dendritica showed a series of growth stages, from bottlebrush-like colony (juvenile) to tree-shaped colony (adult). Considering the diameter size of the stem base and scars on the stem, the specimen MBM286442 is likely a juvenile with a narrow stem and without scars, while the other specimens have wider stems and some old scars of the past branches (Table 1). The juvenile has a bottlebrushlike colony ( Figure 2B), while the adult has a long monopodial stem with branches occurring on the top, forming a tree-shaped colony ( Figures 1B, 3B). Compared to the changing colony shapes, the sclerite forms showed small variation in the growth stages and can be used as a main character to identify the species. Diagnosis. Chrysogorgia "group A, Spiculosae" with 1/3L branching sequence. Branches subdivided dichotomously, up to sixth order. Polyps only present in the end of terminal branchlets. Polyps large with pitcher shape, up to 8 mm long. Rods and spindles slender and coarse with many warts on surface in the back and base of tentacles. Scales amoeba-shaped, branched toward to any directions, irregularly and alternately arranged at basal polyp body. Scales rare, transversally arranged in coenenchyme.
Description. Specimen of holotype ca. 31 cm long and 14 cm wide excluding the holdfast ( Figure 5C). Colony bottlebrush-shaped, with branching sequence 1/3L. Stem golden with metallic luster, ca. 1 mm in diameter at base. Branches subdivided dichotomously, up to sixth order, with distance between adjacent branches 6-12 mm long and orthostiche interval 19-35 mm long in measurements from all specimens. Branches up to 6 cm with the first branch internodes 7-15 mm long and terminal branchlets up to 2 cm long. Polyps only present in the end of the terminal branchlets. Polyps large with pitcher shape, some of them contracted and narrow at the base of tentacles, average 5 mm long with the terminal one up to 8 mm long, and 1-3 mm wide ( Figure 5B, D, F). No polyps in axis internodes.
Rods and spindles slender and coarse with many warts on surface, some of them branched, rarely with one end a little flat, longitudinally arranged in the back of tentacles and usually forming eight distinct columns, and transversally or longitudinally arranged in the base of tentacles, measuring 107-814 × 10-78 μm (length × width,  the same below, Figures 5E, G, 6A). Rare sclerites extend into pinnules, but mostly pinnules free of sclerites. Scales amoeba-shaped, branched in any directions, irregularly and alternately arranged at the basal polyp body, and measuring 161-483 × 14-170 μm ( Figures 5H, 6B). Scales rare and elongate, some of them lobed with irregular edges, transversally arranged in coenenchyme, and measuring 139-221 × 29-67 μm ( Figures 5I, 6C). Type locality. A seamount (tentatively named as M8) located on the Caroline Ridge with a depth range of 1506-1832 m.
Etymology. Named after the type locality, the Caroline Ridge, where the species was discovered.
Distribution and habitat. Found only from a seamount located on the Caroline Ridge. Colony attached to a rocky substrate ( Figure 5A).
Remarks. Chrysogorgia carolinensis sp. nov. belongs to the "group A, Spiculosae" with an unusual branching sequence of 1/3L and bottlebrush-shaped colony, which is similar to C. midas Cairns, 2018 andC. abludo Pante &. However, the new species differs distinctly from these species by the presence of amoeba-shaped scales, which branch toward to any directions at the basal polyp body (vs. absence in both species). Chrysogorgia carolinensis sp. nov. is also similar to C. dendritica by 1/3L branching sequence and the conspicuously amoeba-shaped sclerites at the basal polyp body. However, the new species is easily separated by the bottlebrush-shaped colony (vs. tree-shaped), absence of polyps on internodes (vs. presence) and larger polyps up to 8 mm long (vs. no more than 5 mm) ( Table 1).

Genus Metallogorgia Versluys, 1902
Diagnosis (based on Versluys 1902; Nutting 1908; Kükenthal 1919). Main stem monopodial with a few branches occurring on the side. Axis round and solid with a smooth surface and strong metallic luster. Branches irregular, subdivided dichotomously with branchlets forming a sympodium. The coenenchyme usually thin with a few sclerites or not well differentiated this layer.
Type species. Metallogorgia melanotrichos (Wright & Studer, 1889) Distribution. Atlantic Ocean, Pacific Ocean and Indian Ocean, in a depth range of 567-2311 m (Watling et al. 2011, and the present study). (     In adults, main stem monopodial with a few large branches occurring on the distal end. Branching angle between these large branches usually obtuse. In each large branch, strong branchlets subdivided dichotomously and forming a sympodium pattern. In juveniles, main stem monopodial and gracile with branches producing on the lateral of the trunk randomly and subdivided dichotomously in multiple planes. Polyps conical or cylindrical, absent on the stem of adults but present in juveniles. Sclerites elongated and often crossed, arranged densely in polyps and relatively sparsely in coenenchyme. Rods longitudinally arranged in tentacles, covered with sparse fine warts. Rods longitudinally arranged on upper part of polyp body, and scales partially crosswise or transversely arranged on bottom, with nearly smooth surface. Scales transversely arranged in coenenchyme, usually with rounded ends and occasionally irregular edges. Nematozooids absent.

Metallogorgia melanotrichos
Description. For morphological measurements, see Table 2. Distribution. Central Indo-Pacific Ocean (Wright and Studer 1889;Versluys 1902), Western and Central Pacific (Nutting 1908;Pasternak 1981;present  Remarks. Metallogorgia melanotrichos (Wright & Studer, 1889) is characterized by its completely monopodial stem, with the branches in adults occurring on the distal end, and scales in both body wall and coenenchyme (Table 3). Furthermore, M. melanotrichos has a much more extensive distribution than its congeners, while other species have a relatively limited distribution. Our specimens match well with the original description in the sclerites, but possess a relatively larger polyp (most 2 mm, up to  Table 2). The branches of the specimen MBM286486 on one side is irregular and tend to form a fake spiral with branches subdivided dichotomously in multiple planes, while it is regular and forms a planar layer that nearly perpendicular to the trunk in the specimen MBM286485. Diagnosis (extended on the basis of Kükenthal 1919). In adults, main stem monopodial with branches forming a similar spiral on the top. Strong branches subdivided dichotomously, with branchlets forming a sympodium pattern in each plane. In juveniles, main stem monopodial and thin/gracile with branches occurring on the lateral of the trunk randomly and subdivided dichotomously in mul-  Wright and Studer 1889, Versluys 1902, Nutting 1908, Kükenthal 1919, Pasternak 1981, present study Verrill 1883, Versluys 1902, Kükenthal 1919, Deichmann 1936 Pasternak 1981   tiple planes. Polyp cylindrical, some of them with a slightly expanded base, absent on stem of adults but present in juveniles. Sclerites relatively coarse with many small warts on surface, cross-shaped occasionally. Rods relatively regular, longitu-  well differentiated with a layer of sclerites. In the adults (specimen MBM286487-286489), colony like a tree shape with branches forming a similar spiral on the top of the stem. Main stem monopodial and strong. Branches subdivided dichotomously with branchlets forming a sympodium pattern in each plane. Polyps cylindrical, some of them with a slightly expanded base, absent from the stem. Branch coenenchyme usually not well differentiated.

Metallogorgia macrospina Kükenthal, 1919
Sclerites with same forms and arrangement in juveniles and adults, both relatively coarse with many small warts on surface, cross-shaped occasionally. Rods relatively regular, longitudinally arranged in tentacles and the upper part of the polyp body, and partially crosswise or transversely arranged on the body bottom. Scales and rods elongated, usually coarse with serrated edges, transversely arranged in coenenchyme. For detailed morphological measurements, see Table 2.
Distribution. West Sumatra (Kükenthal 1919); the unnamed seamounts on the Caroline Ridge in the Western Pacific (present study); Southwest Pacific , 720-1280 m depth.
Remarks. According to Kükenthal (1919), the sclerites in the M. macrospina polyps contain rods and spindles, and those in coenenchyme are slender rods, some of them flat or irregular. The sclerites in our specimens match well with the original description as well as pictures. Therefore, we identify our specimen as M. macrospina.  Metallogorgia macrospina is similar to M. melanotrichos by the branchlets forming a sympodium pattern in the large branches. In the original description, Kükenthal (1919) pointed out that M. macrospina differed from M. melanotrichos by its more densely arranged branches, larger polyps, longer sclerites in coenenchyme and different color. However, the polyps of M. macrospina in our specimens are generally smaller than those of M. melanotrichos (Table 2), and there are no conspicuous differences in color (almost brown to black) among these colonies. Therefore, based on the morphological features of our specimens, M. macrospina can be separated from M. melanotrichos by its sympodial branching part forming a spiral on the colony top (vs. monopodial), only rods in the polyp-body wall (vs. rods and scales), and rods and scales in coenenchyme (vs. only scales).
The juvenile of Metallogorgia macrospina (specimen MBM286484) has significant differences in the branching pattern from the adult specimens MBM286487, MBM286488 and MBM286489 in morphology. It differs also by having a slightly bottlebrush-shaped colony (vs. similarly tree-shaped colony), gracile and flexible stem (vs. hard and strong), monopodial branching part (vs. sympodial), branchlets  in multiple planes (vs. forming a sympodium in one plane), and well differentiated coenenchyme with more sclerites (vs. not well differentiated and with relatively sparse sclerites). However, the same sclerites in polyps and coenenchyme, and particularly the mtMutS gene data analyzed below indicate these specimens belong to the same species.

Genus Pseudochrysogorgia Pante & France, 2010
Diagnosis (based on Pante and France 2010). Main stem monopodial or slightly zigzagging with branches subdivided dichotomously in multiple planes, forming a bottlebrush-shaped colony. Most polyps leaning distad and neck narrower than head. Sclerites slightly ornamented, in the form of plates, scales and rods. When polyp not leaning distad, sclerites arranged obliquely on the polyp body. When polyps leaning distad, sclerites mostly arranged longitudinally (parallel to branch) on the polyp body, placed obliquely in the area of neck, and arranged longitudinally on head and along the back of tentacles. Scales and plates in branch coenenchyme mostly parallel to main branch axis. Voucher specimen. MBM286490, the Ganquan Plateau in the South China Sea, 586-910 m.
Description. No whole colony was obtained, thus the description was based on a picture ( Figure 19A) and a few frozen fragments. Colony ca. 50 cm long and 11 cm wide with dendritic holdfast ca. 12 cm long. Main stem more zigzagging. Branching sequences unknown. Branches subdivided dichotomously, up to sixth order with distance between adjacent branches 10-25 mm. Polyps cylindrical, or with an expanded body base and obviously narrow neck, 1.0-2.5 mm long and 0.5-2.5 mm wide at bases, with tentacles up to 1 mm long ( Figure 19B-G).
Rods with many small warts on surface, usually with two round ends and relatively less ornamentation, longitudinally arranged along the back of tentacles, and measuring 20A). Sclerites in the polyp-body wall including rods, usually with one sharp end, and scales, slightly ornamented, some of them thick and elongated, and measuring 150-620 × 31-127 μm. When the polyp not leaning distad, rods and elongated scales arranged obliquely in the polyp-body wall. While polyps leaning distad, scales mostly arranged longitudinally (parallel to the branch) in upper of body, rods and scales placed obliquely in neck region, and arranged longitudinally on head and the base of tentacles (Figures 19I,20B). Scales slightly ornamented, mostly parallel to main branch axis in coenenchyme, and measuring 115-397 × 16-150 μm (Figures 19J, 20C).
Distribution. Bellona Plateau, Coral Sea and Otara Seamount, at southern tip of the Kermadec Ridge France 2010, Pante et al. 2012); and South China Sea (present study), 586-1462 m depth.
Remarks. Pseudochrysogorgia bellona Pante & France, 2010 resembles the species of Chrysogorgia Duchassaing & Michelotti, 1864 by having dichotomously subdivided branches arising from the main stem in a spiraling fashion and forming a bottlebrush-shaped colony. However, it differs in the monopodial or more zigzagging stem (vs. almost sympodial, except Chrysogorgia abludo C. dendritica Xu, Zhan &Xu, 2020) and obviously different polyps (Xu et al. 2020). Furthermore, P. bellona is similar to species of Metallogorgia Versluys, 1902 in its monopodial colony and has a close genetic distance as well. However, P. bellona can be separated by its polyps usually with obviously narrow neck (vs. none), dendritic holdfast (vs. discoid) and sclerites including plates, scales and rods with more ornamentation (vs. almost rods and scales with little ornamentation) (Pante and France 2010). In our specimen, the rods in tentacles are more regular usually with two round ends and less ornamentation. The elongated thick scales are more abundant in coenenchyme, and plates are scarcer than the type colony. We suggest that these differences are population-dependent.

Molecular sequences, genetic distances, and phylogenetic analyses
The conspecific sequences for each newly sampled species were identical, and only the holotype sequence was deposited in GenBank and analysed here. The accession number and the length are as follows: MT269889, 686 bp for Chrysogorgia carolinensis sp. nov., MT269888, 693 bp for C. dendritica, MT050468, 691 bp for M. macrospina; MT050469, 695 bp for M. melanotrichos, MT050470, 690 bp for P. bellona. The alignment dataset comprised 629 nucleotide positions. Compared to the other Chrysogorgia species, C. carolinensis sp. nov. has six-nucleotide deletion in the mtMust sequence ( Figure 21). There was no intraspecific variability, while the interspecific dis- tances ranged from zero to 2.42% (Table 4). The genetic distances between the new species C. carolinensis sp. nov. and its congeners were in range of 0-2.42%, and those between C. dendritica and its congeners were in the range of 0-1.63%. To date, only three species are available for the genetic distance and phylogenetic analysis for Metallogorgia and Pseudochrysogorgia. The mtMutS genetic distances between M. macrospina and M. melanotrichos was 0.16%, while there was no intraspecific variation within the two species and P. bellona (Table 4). The genetic distances between Metallogorgia and Pseudochrysogorgia were 1.94% and 2.10%.
The ML and BI phylogenetic trees are identical in topology, and thus only the former with the both support values is shown (Figure 21). In both trees, Metallogorgia, Pseudochrysogorgia, Iridogorgia, Radicipes and Chrysogorgia formed a "monophyletic Chrysogorgiidae clade'' (MCC) sensu  with moderate to high support (ML 86%; BI 0.95). The Chrysogorgia species were separated into two main clades (Clade I and II) with high support values, which is consistent with Xu et al. (2019Xu et al. ( , 2020. Clade I includes the sister species C. binata, C. cf. stellata and C. chryseis, and Clade II contains all the rest species. The species C. dendritica and C. abludo formed a sister subclade, followed by C. fragilis. Chrysogorgia carolinensis sp. nov. formed a sister subclade with the cluster C. ramificans + C. monticola, the group C. dendritica + C. abludo + C. fragilis and the rest species within Clade II. Metallogorgia melanotrichos and M. macrospina formed sister clade, followed by P. bellona with full node support ( Figure 21).

Discussion
Both the morphology and molecular phylogenetic analysis support the assignment of the new species to the genus Chrysogorgia Duchassaing & Michelotti, 1864. The barcoding analysis of mtMutS is considered as the first step in molecular identification of octocorals (McFadden et al. 2011;). However, the mtMutS genetic distances within Chrysogorgia are relatively low, and there is no barcoding gap (intraspecific zero vs. interspecific 0-2.42%) for species identification, as shown by Xu et al. (2020). Thus, the discrimination of the closely related species was mainly relied on other molecular evidence and morphological characters. Alternatively, single mutations on mtMutS can be used to separate Chrysogorgia species Pante et al. 2015;Xu et al. 2020). The mtMutS sequence of C. carolinensis sp. nov. has six deletion mutations compared to those of its congeners (Figure 20), supporting the establishment of the new species. Furthermore, the closely related species C. carolinensis sp. nov., C. artospira and C. pinnata are easily separated by the 1/3L branching sequence (vs. 2/5L and flabellate, respectively) and amoeba-shaped scales at the basal polyp body (vs. regular scales and rods, respectively) (Cairns 2007;. No genetic variability was observed between C. dendritica and the closely related species C. abludo. However, the former is morphologically distinctly different from the latter by the presence of irregular sclerites in the polyp-body wall   GQ868331, GQ868332 4.04% 4.04% 3.88% 3.55%. 3.25% 3.23% 3.23% 3.39% 3.55% 3.88% 3.88% 4.52% 4.52% 4.36% 1.94% 2.10% 0 (often branched and amoeba-shaped with more lobed edges vs. relatively regular with less lobed edges; Xu et al. 2020).
Colonial branching pattern was regarded as one of the diagnostic characters to distinguish chrysogorgiid octocorals ). However, it should be carefully applied to the identification of Metallogorgia species, since polymorphism of the colony shape is known for M. melanotrichos during its life cycle (Mosher and Watling 2009). In the present study, one juvenile and three adults of M. macrospina Kükenthal, 1919 were collected and identified. There are also obvious morphological differences between the juvenile and the adults in the colony shape and branch pattern (see before, Figures 11C, 15C). Nevertheless, like the case of M. melanotrichos, their conspecific assignment can be determined by the same polyps and sclerites, and the MutS gene sequences (Figures 11-18, 21). On the other hand, the M. melanotrichos and M. macrospina juveniles rather than the adults, are most similar to the Pseudochrysogorgia in having lateral branches subdivided dichotomously and not forming a sympodium, and a slightly bottlebrush-shaped colony, mirroring their sister relationship showed by the phylogenetic trees ( Figures 11C, 21). Seemingly, the colony branch morphogenesis may be useful to infer the phylogenetic relationship for chrysogorgiids, and the colonial branching pattern may be an apomorphy for chrysogorgiids.
To include the juvenile morphology, we slightly extend the diagnosis of the genus Metallogorgia on the basis of Versluys (1902) and Kükenthal (1919): Colonies with distinctly monopodial stem giving rise to a few lateral branches. Branches arising distally in adults, while arising on the lateral of stem randomly in juveniles. Branch subdivided dichotomously, with branchlets either forming a sympodium in one plane in adults, or in multiple planes in juvenile. Branching part monopodial forming an approximate planar layer or a slight bottlebrush shape, or sympodial forming a spiral or tree shape. Axis round, hard and strong with a smooth surface and an extremely pronounced metallic luster. Polyps cylindrical without obvious neck, relatively little to the size of branches they sit on and well separated from one another, covered with abundant and dense sclerites. Coenenchyme thin, most with a few sclerites or not differentiated well into this layer. Sclerites in the main form of rods, spindles and scales, with little ornamentation.
Furthermore, based on the present morphological descriptions, we provide a preliminary key to full grow grown specimens of the genus Metallogorgia Versluys, 1902. 1 Only