biology

Article Morphological and Molecular Characterization of Five Species Including Three New Species of Golden Gorgonians (Cnidaria: Octocorallia) from Seamounts in the Western Pacific

Yu Xu 1,2,3,†, Zifeng Zhan 1,† and Kuidong Xu 1,2,3,*

1 Laboratory of Marine Organism Taxonomy and Phylogeny, Shandong Province Key Laboratory of Experimental Marine Biology, Center for Ocean Mega-Science, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China; [email protected] (Y.X.); [email protected] (Z.Z.) 2 Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Zhuhai 519082, China 3 University of Chinese Academy of Sciences, Beijing 100049, China * Correspondence: [email protected]; Tel.: +86-0532-8289-8776 † Contributed equally to this work.

Simple Summary: Deep-water octocorals are main components of vulnerable marine ecosystems (VMEs) and play an important role in conservation and research. Iridogorgia Verill, 1883 is a distinct octocoral group characterized by a remarkably spiral structure and large size in deep sea, where the diversity of Iridogorgia is poorly known in the Western Pacific. Based on the collection of Iridogorgia


specimens from seamounts in the tropical Western Pacific, we described five species including three
 new species using an integrated morphological-molecular approach. We assessed the potential of Citation: Xu, Y.; Zhan, Z.; Xu, K. the mitochondrial genes MutS and COI, and the nuclear 28S rDNA for species delimitation and Morphological and Molecular phylogenetic reconstruction of Iridogorgia. The results suggest that the mitochondrial markers are Characterization of Five Species not able to resolve the species boundaries and deeply divergent relationships adequately, while 28S Including Three New Species of rDNA showed potential application in DNA barcoding and phylogenetic reconstruction for this Golden Gorgonians (Cnidaria: genus. This study will help to understand the Iridogorgia biodiversity in the Western Pacific and shed Octocorallia) from Seamounts in the more light on the screening of barcodes for octocorals. Western Pacific. Biology 2021, 10, 588. https://doi.org/10.3390/ Abstract: Members of genus Iridogorgia Verrill, 1883 are the typical deep-sea megabenthos with only biology10070588 seven species reported. Based on an integrated morphological-molecular approach, eight sampled

Academic Editor: Alberto specimens of Iridogorgia from seamounts in the tropical Western Pacific are identified as three new Teodorico Correia species, and two known species I. magnispiralis Watling, 2007 and I. densispicula Xu, Zhan, Li and Xu, 2020. Iridogorgia flexilis sp. nov. is unique in having a very broad polyp body base with stout Received: 29 April 2021 and thick scales. Iridogorgia densispiralis sp. nov. can be distinguished by rods present in both Accepted: 24 June 2021 polyps and coenenchyme, and I. verrucosa sp. nov. is characterized by having numerous verrucae Published: 26 June 2021 in coenenchyme and irregular spindles and scales in the polyp body wall. Phylogenetic analysis based on the nuclear 28S rDNA indicated that I. densispiralis sp. nov. showed close relationships Publisher’s Note: MDPI stays neutral with I. splendens Watling, 2007 and I. verrucosa sp. nov., and I. flexilis sp. nov. formed a sister with regard to jurisdictional claims in clade with I. magnispiralis. In addition, due to Rhodaniridogorgia fragilis Watling, 2007 nested into published maps and institutional affil- the Iridogorgia clade in mtMutS-COI trees and shared highly similar morphology to the latter, we iations. propose to eliminate the genus Rhodaniridogorgia by establishing a new combination Iridogorgia fragilis (Watling, 2007) comb. nov. and resurrecting I. superba Nutting, 1908.

Keywords: Anthozoa; Chrysogorgiidae; Iridogorgia flexilis; Iridogorgia verrucosa; Iridogorgia densispi- Copyright: © 2021 by the authors. ralis; taxonomy; morphology; phylogeny Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons 1. Introduction Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ Deep-water octocorals are main components of vulnerable marine ecosystems (VMEs) 4.0/). and represent one group of VME-indicator species, playing an important role in conser-

Biology 2021, 10, 588. https://doi.org/10.3390/biology10070588 https://www.mdpi.com/journal/biology Biology 2021, 10, 588 2 of 27

vation and research [1,2]. The octocorals usually have slow growth rates and long life, which suggest that full recovery from damage can take from decades to centuries, de- pending on the species [3]. The genus Iridogorgia is an easily recognized octocoral group which is characterized by a remarkably spiral structure and large size at water depths of 558–2311 m [4–7]. The species Iridogorgia magnispiralis Watling, 2007 is a representative, which is known as the world’s largest gorgonian reaching about 5.7 m tall [8]. Currently, only seven valid species are known in the genus Iridogorgia [7,9]. Among them, four species are found in the North Atlantic, especially on the seamounts of the Northwest Atlantic, including I. pourtalesii Verrill, 1883, I. magnispiralis Watling, 2007, I. splendens Watling, 2007 and I. fontinalis Watling, 2007 [4,10,11]. Two species are recorded off the coast of Hawaii: I. bella Nutting, 1908 and I. magnispiralis [4,8,11–13]. The diversity of Iridogorgia in the Western Pacific is still poorly known, I. magnispiralis was found in the Phoenix area and Northeast Australia [6,14], and I. densispicula Xu, Zhan, Li and Xu, 2020 and I. squarrosa Xu, Zhan, Li and Xu, 2020 are found in the seamounts near the Mariana Trench [7]. While studying the seamount benthic diversity in the tropical Western Pacific Ocean, we collected eight specimens of Iridogorgia from the seamounts located on the Caroline Ridge in 2019. Based on the integrated morphological-molecular approach, we described three new species including Iridogorgia flexilis sp. nov., I. verrucosa sp. nov. and I. densispiralis sp. nov., and reported two known species I. magnispiralis and I. densispicula. Meanwhile, we assessed the potential of the genetic distance of the mitochondrial genes MutS and COI, and the nuclear 28S rDNA for species discrimination of this genus. We also compared the resolving power of the mitochondrial and nuclear markers for the phylogenetic reconstruc- tion. The objective of this study was to help understand the Iridogorgia diversity in the Western Pacific and shed more light on the screening of barcodes for the octocorals.

2. Materials and Methods 2.1. Specimen Collection and Morphological Examination Eight specimens were obtained from the seamounts located on the Caroline Ridge by ROV FaXian (Discovery) in the tropical Western Pacific in 2019. These specimens were photographed in situ before being sampled, photographed on board, and then stored in 75% ethanol after collection. A few branches were stored at −80 ◦C for molecular research. Their morphology and anatomy were examined by using a stereo dissecting microscope. The preparation of sclerites for imaging follows Xu et al. (2020) [7]. SEM scans were obtained and the optimum magnification was chosen for each kind of polyps and sclerites by using TM3030Plus SEM. The morphological terminology used follows Bayer et al. (1983) [15]. The type specimen of the three new species has been deposited in the Marine Biological Museum of Chinese Academy of Sciences (MBMCAS) Qingdao, China. The three new species have been registered in ZooBank.

2.2. DNA Extraction and Sequencing Genomic DNA was extracted by a DNeasy Blood and Tissue Kit (Qiagen, Hilden, Ger- many) following the instructions. The mitochondrial regions of mutS homolog (mtMutS) and cytochrome oxidase subunit I (COI) and an approximately 800-nt fragment of the 28S nuclear ribosomal gene (28S rDNA) were selected for the phylogenetic analysis. To amplify mtMutS and COI, the primer pairs AnthoCorMSH (50-AGGAGAATTATTCTAAGTATGG- 30)[16] and Mut-3458R (50-TSGAGCAAAAGCCACTCC-30)[17], COI8414-F (50-CCAGGTA- GTATGTTAGGRGA-30; McFadden, unpublished) and HCO2198 (50- TAAACTTCAGGGTG- ACCAAAAAATCA-30)[18] were used, respectively. PCR reactions were conducted with the following conditions: 2 min at 98 ◦C followed by 35 cycles (98 ◦C for 15 s, 45–50 ◦C for 15 s, and 72 ◦C for 60 s) and a final extension at 72 ◦C for 2 min. We sequenced 28S rDNA using primers 28S-Far (50-CACGAGACCGATAGCGAACAAGTA-30) and 28S- Rar (50-TCATTTCGACCCTAAGACCTC-30)[19], and the same PCR protocol used for Biology 2021, 10, 588 3 of 27

mtMutS. PCR reactions were performed using I-5TM 2 × High-Fidelity Master Mix DNA polymerase, and sequencing was performed by TsingKe Biological Technology (TsingKe Biotech, Beijing, China).

2.3. Genetic Distance and Phylogenetic Analyses All the Iridogorgia sequences and the related chrysogorgiid genera and out-group species Plexaura kuna were downloaded from GenBank (Table1). The sequences were aligned using MAFFT v.7 [20] with the G-INS-i and Q-INS-i algorithms for the mitochon- drial and 28S rDNA regions, respectively. The nucleotide alignment was trimmed to equal length using BioEdit v7.0.5 [21]. The COI alignment showed that Iridogorgia magnispiralis DQ860111 and I. splendens DQ860112 shared few overlap positions (<100 bp) with the other Iridogorgia sequences, and therefore these two sequences were excluded from the analysis. Genetic distances of single loci and the concatenated region mtMutS-28S be- tween species/populations were calculated with MEGA 6.0 using Kimura 2-parameter model [22]. Phylogenetic analyses were performed on 28S rDNA and the concatenated mtMutS– COI. For the phylogenetic construction, when conspecific sequences showed no genetic variation, one was chosen randomly for analysis. Model selection and Maximum likelihood (ML) analyses were carried out using PhyML (ver. 3.0, see http://www.atgc-montpellier. fr/phyml/(accessed on 28 April 2021); [23]) on the online ATGC bioinformatic platform. The best-fitted model GTR + G was selected with the Akaike information criterion for both the mitochondrial and 28S rDNA alignments. Node support for the ML trees came from a majority-rule consensus tree of 1000 bootstrap replicates. Following Hillis and Bull (1993) [24], the ML bootstraps <70%, 70–94% and ≥95% were considered as low, moderate and high, respectively. Bayesian inference (BI) analysis was carried out using MrBayes v3.2.7a [25]. Posterior probability was estimated based on 10,000,000 Monte Carlo Markov Chain (MCMC) generations (×4 chains) sampling every 1000 generations (burn-in = 25%). Convergence of the MCMC was assessed using Tracer 1.4.1 [26]. Following Alfaro et al. (2003) [27], the Bayesian posterior probabilities <0.95 and ≥0.95 were considered as low and high, respectively.

Table 1. The sequences used in this study.

GenBank Accession Numbers Species Voucher Number Locatiom References mtMutS COI 28S rDNA Iridogorgia densispiralis sp. nov. MBM286454 10.37◦ N, 140.30◦ E present study MW841033 MW841036 MW841043 Iridogorgia flexilis sp. nov. MBM286453 10.40◦ N, 140.90◦ E present study MW841031 MW841035 MW841041 Iridogorgia verrucosa sp. nov. MBM286455 10.37◦ N, 140.04◦ E present study MW841034 MW840138 MW841044 Iridogorgia densispicula MBM286538 10.35◦ N, 140.07◦ E[7] MK431864 MW841037 MW841040 Iridogorgia magnispiralis MBM286450 10.50◦ N, 140.11◦ E present study MW841032 MW841039 MW841042 Iridogorgia squarrosa MBM286539 11.16◦ N, 139.25◦ E[7] MK431865 – – Iridogorgia fontinalis YPM 38584 34.81◦ N, 50.50◦ W[6] EU293802 GQ868321 – France, Iridogorgia magnispiralis YPM: IZ: 38580 – DQ860108 – – unpublished MNHN-Oct.0000- Iridogorgia magnispiralis 38.47◦ N, 27.9◦ W[6] GQ353316 – – 0576 Iridogorgia magnispiralis – 38.78◦ N, 63.96◦ W[6] EU268055 – – France & Pante, Iridogorgia magnispiralis – unknown – FJ268639 – unpublished Iridogorgia magnispiralis YPM 38580 38.78◦ N, 63.96◦ W[6] JN227997 – – Iridogorgia magnispiralis – 35.19◦ N, 47.68◦ W[6] GQ223116 GQ868318 – Iridogorgia magnispiralis YPM 38581 38.26◦ N, 60.55◦ W[6] GQ180141 – – Iridogorgia magnispiralis USNM 1092265 34.58◦ N, 56.84◦ W[6] GQ180142 – – Iridogorgia magnispiralis YPM 38582 38.86◦ N, 63.91◦ W[6] GQ180140 – – Iridogorgia magnispiralis – Gulf of Mexico [28,29] KC788263 KC788237 KX890214 Iridogorgia sp. – 21.32◦ N, 157.02◦ W[6] GQ868342 GQ868323 – Iridogorgia sp. YPM 28866 33.79◦ N, 62.59◦ W[6] DQ297422 – – Iridogorgia sp. type C – 23.05◦ N, 163.16◦ W[6] JN227919 – – Biology 2021, 10, 588 4 of 27

Table 1. Cont.

GenBank Accession Numbers Species Voucher Number Locatiom References mtMutS COI 28S rDNA MNHN-IC.2009- Iridogorgia sp. type A 9.15◦ S, 158.27◦ E[6] GQ180145 – – 0001 Iridogorgia splendens YPM 35397, 38586 38.85◦ N, 63.76◦ W[6] DQ860109 – – Iridogorgia splendens USNM 1092267 38.79◦ N, 64.13◦ W[6] JN227996 GQ868313 – Iridogorgia splendens YPM 38585 37.46◦ N, 59.95◦ W[6] JN228005 GQ868330 – Iridogorgia splendens – Gulf of Mexico [28] KC788271 KC788229 – Iridogorgia splendens – Gulf of Mexico [29] – – KX890215 Kelvin Seamount, Iridogorgia splendens USNM 1092267 [30] GQ180143 – – NW Atlantic Iridogorgia splendens YPM: IZ: 38585 37.46◦ N, 59.95◦ W[30] GQ180144 – – Rhodaniridogorgia fragilis YPM 38588 34.46◦ N, 56.73◦ W[6] JN228000 JN227954 – Chrysogorgia averta – Gulf of Mexico [28] KC788265 KC788235 KC788258 Chrysogorgia sp. – Gulf of Mexico [28] KC788268 KC788223 KC788240 Chrysogorgia sp. – Gulf of Mexico [29] – – KX890212 MNHN-IC.2008- Pseudochrysogorgia bellona 007 21.12◦ S, 158.5◦ E[6] GQ868332 GQ868310 – Paratype Pseudochrysogorgia sp. XMUB7697 15.52◦ N, 110.96◦ E[31] – – MW336977 Metallogorgia melanotrichos – 34.53◦ N, 47.79◦ W[6] GQ180158 FJ268633 – Metallogorgia macrospina NIWA15642 37.21◦ S, 177.24◦ E[6] JN228001 JN227952 – USNM: Radicipes stonei Alaska [32] MG986912 MG986961 MG980134 IZ:1418007 Stephanogorgia faulkneri NTM C014927 7.19◦ N, 134.32◦ E[6] GQ342485 GQ342406 JX203718 Chrysogorgiidae sp. – 41.37◦ S, 42.85◦ E[33] KP324387 KP678004 KP324611 RMNH Plexaura kuna South Africa [34] JX203807 JX203866 JX203748 Coel.40836 New sequences are in bold.

3. Results 3.1. Systematics Class Anthozoa Ehrenberg, 1834 [35]. Subclass Octocorallia Haeckel, 1866 [36]. Order Alcyonacea Lamouroux, 1812 [37]. Suborder Calcaxonia Grasshoff, 1999 [38]. Family Chrysogorgiidae Verrill, 1883 [10]. Genus Iridogorgia Verrill, 1883. Diagnosis (based on Watling, 2007 [4]). Chrysogorgiids with main axis monopodial and spiraling upward, and undivided branches emanating from one side of axis. Polyps uniserially arranged, when sexually mature with base expanded along upper side of branch. Sclerites rods, spindles, or scales, extending in tracts onto tentacles. Branch coenenchyme with sclerites oriented along branch or absent between polyps. Type species. Iridogorgia pourtalesii Verrill, 1883, by monotypy. Distribution. Pacific: tropical Western Pacific, Hawaii, Southwest Pacific; Atlantic: North and Central East Atlantic; Indian Ocean: Great Australian Bay, depths 558–2311 m [4–8,10–14,39,40]. Iridogorgia flexilis sp. nov. Figures1A–G and2; Table2. Biology 2021, 10, 588 5 of 27 Biology 2021, 10, x FOR PEER REVIEW 6 of 27

Figure 1. External morphology and polyps of Iridogorgia flexilis sp. nov. and different branch morphology of the Iridogorgia Figure 1. External morphology and polyps of Iridogorgia flexilis sp. nov. and different branch morphology of the Iridogorgia species in this passage (a–g). (A,B) The holotype in situ and after collection. Laser dots spaced at 33 cm used for measuring species in this passage (a–g). (A,B) The holotype in situ and after collection. Laser dots spaced at 33 cm used for measuring dimensions. (C,D) Single polyps under a light microscope. (E) A part of branch under SEM. (F) A single polyp under SEM. (dimensions.G) Sclerites in (C ,basalD) Single polyp polyps body underunder aSEM. light ( microscope.a–g) A part of (E branch) A part with of branch different under polyp SEM. size (F )and A single arrangement, polyp under followed SEM. by(G )I. Sclerites flexilis sp. in nov., basal I. polyp densispiralis body under sp. nov., SEM. I. verrucosa (a–g) A part sp. nov., of branch I. magnispiralis with different, I. densispicula polyp size (MBM286446, and arrangement, 286447, followed 286448). by ScalesI. flexilis = 20sp. cm nov., (B),I. 2 densispiralis cm (a–g in thesp. same nov., I.scale), verrucosa 2 mmsp. (C nov.,,D), 1I. mm magnispiralis (F), 500 μ,mI. densispicula(E,G). (MBM286446, 286447, 286448). Scales = 20 cm (B), 2 cm (a–g in the same scale), 2 mm (C,D), 1 mm (F), 500 µm (E,G).

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Table 2. Comparison of morphological and distributional characters of Iridogorgia species. “–” means missing data.

Characters/Species I. densispicula I. squarrosa I. bella I. fontinalis I. magnispiralis Each turn height (cm) 13–18 13–15 – 4 12–20 Helical diameter (cm) 3–4 4–5 – 2.5 1–9 Branch intervals (mm) 3–4 3–4 4 1–2 2–5 1 mm from branch to tentacle Polyp height and width (mm) 2–4/2–4 2–4/1–2 – 1–3/2–3 base Polyp intervals (mm) 2–7 2–6 7 5–9 2–7 Verrucae rare a few sparse abundant abundant spindles usually with two sharp spindles usually with two sharp spindles and a few scales: spindles usually with two sharp Sclerites in coenenchyme (µm) – ends: 203–967 × 11–65 ends: 180–840 × 23–60 397–1024 × 25–74 ends: 322–1032 × 21–81 spindles and scales coarse, usually lobed with irregular spindles thick and slender, nearly shape and rugged and ridged scales with coarse surface and Sclerites in polyp bodies (µm) needle-like or bar-shaped rods: 175–482 × 27–50 smooth or with many fine warts: surface in the upper part, and various shape: 72–569 × 24–150 224–383 × 23–57 regular and flat in the basal part: 80–627 × 14–110 rods: 325–433 × 26–43 scales: Sclerites in tentacles (µm) rods: 137–945 × 11–98 needle-like or bar-shaped shorter rods than bodies rods: 157–548 × 18–78 467–805 × 48–66 North Atlantic, near Hawaii, Distribution Western Pacific Western Pacific near Hawaii North Atlantic Western Pacific References [7], present study [7][6,7,11,13][4][4,8,11,14], present study continued Characters/Species I. pourtalesii I. splendens I. flexilis sp. nov. I. densispiralis sp. nov. I. verrucosa sp. nov. Each turn height (cm) – 5 13–15 2.5–3.0 3–4 Helical diameter (cm) – 1–2 3–4 0.5 1 Branch intervals (mm) 3–6 3–4 3 2–3 1–3 less than 1 mm from branch to Polyp height and width (mm) – 1–3/2–4 1–2/1–3 2/1–2 tentacle base Polyp intervals (mm) 5 6–8.5 4–6 3–9 4–6 Verrucae numerous numerous rare a few numerous rods and spindles usually with scales and spindles under polyps; spindles same as the body wall, spindles usually with two two rounded ends, sometimes Sclerites in coenenchyme (µm) lacking in the inter- polyps absent to rare in the inter-polyps: usually with two rounded ends: rounded ends: 322–892 × 25–72 sparse in the inter-polyps: 90–523 274–592 × 24–51 169–438 × 19–62 × 19–65 spindles, rods and a few spindles and scales stout and spindles and elongated scales scales with a constriction elongated scales often thick with Sclerites in polyp bodies (µm) spindles smooth: average 400 thick with nearly smooth surface: with sparse and fine warts and midway: 143–268 × 29–45 sparse and fine warts: 84–347 × 172–679 × 27–219 irregular edges: 116–450 × 21–65 18–60 Sclerites in tentacles (µm) sparse rods: 200–900 few rods: 169–274 × 27–39 rods: 261–532 × 19–58 rods: 95–442 × 11–52 rods: 160–459 × 17–73 Distribution North Atlantic North Atlantic Western Pacific Western Pacific Western Pacific References [4,10,11][4,6,11] present study present study present study Biology 2021, 10, 588 7 of 27

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Figure 2. Sclerites of Iridogorgia flexilis sp. nov. (A) Sclerites in tentacle rachis. (B) Sclerites in polyp body wall. (C) Sclerites Figure 2. Sclerites of Iridogorgia flexilis sp. nov. (A) Sclerites in tentacle rachis. (B) Sclerites in polyp body wall. (C) Sclerites in coenenchyme. Scales = 300 μm, all in the same scale. in coenenchyme. Scales = 300 µm, all in the same scale.

urn:lsid:zoobank.org:act:6BD2C9FC-9CD8-4604-971D-B2EC0BB108B8.urn:lsid:zoobank.org:act:6BD2C9FC-9CD8-4604-971D-B2EC0BB108B8. MaterialMaterial examined.examined. Holotype:Holotype: MBM286453, MBM286453, station station FX-Dive FX-Dive 222 222 (10 (10°4.7◦4.70 N,′ N,140 140°9.28◦9.280 E),′ E),a seamount a seamount (temporarily (temporarily named named as M5)as M5) on theon the Caroline Caroline Ridge, Ridge, 2016 2016 m, 10m, June10 June 2019. 2019. Diagnosis.Diagnosis. Colony Colony having having a a long long unbranched part part with branches producing on the top.top. Axis Axis loosely loosely coiled coiled on on the the top top with with helic helicalal turn 12–15 cm high. Polyps Polyps having a broad bodybody base base with with its its width width usually usually longer longer than than the theheight. height. Rods Rods in the in back the of back tentacle of tentacle rachis longitudinallyrachis longitudinally arranged, arranged, slender slender with more with or more less orwarts. less warts.Spindles Spindles and scales and in scales polyp in bodypolyp wall body transversely wall transversely or obliquely or obliquely arranged arranged,, stout and stout thick, and thick,occasionally occasionally crossed crossed with irregularwith irregular edges. edges. Spindles Spindles in coenenchyme in coenenchyme slender slender and thick, and thick,occasionally occasionally branched. branched. Ver- rucaeVerrucae rare rare in branches. in branches. Description.Description. Holotype Holotype is isincomplete incomplete colony colony with withoutout holdfast, holdfast, about about 85 cm 85 long cm (Fig- long ure(Figure 1B).1 BThe). Thedirection direction of grow of growthth clockwise. clockwise. Axis Axis about about 4 4mm mm in in diameter diameter at at base base with iridescentiridescent metallicmetallic luster.luster. The The branching branching part part about about 34 34 cm cm long long including including three three loosely loosely heli- helical turns with each helical turn 12–15 cm long and 3–4 cm in diameter. Branches ar- ranged along one side, about 3 mm apart, up to 28 cm long with 39 polyps counted. Polyps

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cal turns with each helical turn 12–15 cm long and 3–4 cm in diameter. Branches arranged along one side, about 3 mm apart, up to 28 cm long with 39 polyps counted. Polyps have a broad body base with its width usually longer than the height, forming a contraction at the base of the tentacular part (Figure1C,D). Polyps 4–6 mm apart, 1–3 mm tall, and 2–4 mm wide at base (Figure1a). Tentacular part about 1 mm long, 1.0–1.5 mm wide, composed of numerous rods usually forming eight obvious parallel columns terminating at its base. Polyps white after fixation in alcohol. Verrucae rare in branches. Rods in the back of tentacle rachis longitudinally arranged, slender with two round ends, surface usually with numerous small conical or ridge-like warts, occasionally with sparse warts or shallow cracks, measuring 261–532 × 19–58 µm (Figures1F and2A). Spin- dles and scales in polyp body wall transversely or obliquely arranged, stout and thick, occa- sionally crossed with irregular edges or various shapes, measuring 172–679 × 27–219 µm (Figures1G and2B). Their surface usually with sparse and fine warts, occasionally nearly smooth or with large protuberances and some shallow cracks. Spindles in co- enenchyme slender and thick, usually with two rounded ends, nearly smooth or with sparse and fine warts, and occasionally branched or with narrow or sharp ends, measuring 322–892 × 25–72 µm (Figures1E and2C). Type locality. A seamount (temporarily named as M5) located on the Caroline Ridge in the Western Pacific with water depth of 2016 m. Etymology. The Latin adjective flexilis (flexile) refers to the flexile axis of this species. Distribution and Habitat. Found only from a seamount located on the Caroline Ridge. Grown in a rocky bottom with the water temperature about 2.1 ◦C and salinity 36.5 (Figure1A). Remarks. Iridogorgia flexilis sp. nov. is characterized by its highly helical turns, broad polyp body base with stout and thick spindles and scales. It is similar to I. magnispiralis Watling, 2007 and I. densispicula Xu et al., 2020 in the helical turn, but differs distinctly from I. magnispiralis by the presence of scales in polyp body wall (vs. absent), from I. densispicula by the thick and relatively smooth sclerites in the polyp body wall (vs. very thin and coarse often with rugged and ridged surface) [4,7]. Iridogorgia densispiralis sp. nov. Figures1b,3 and4; Table2. urn:lsid:zoobank.org:act:026E2F14-7A66-4558-9D65-B7E3CC0EC8D0. Material examined. Holotype: MBM286454, station FX-Dive 225 (10◦36.750 N, 140◦3.850 E), a seamount (temporarily named as M8) located on the Caroline Ridge, 1574 m, 13 June 2019. Diagnosis. Colony relatively short. Axis have many close helical turns each 2.5–3.0 cm high. Polyps small with an expanded and conical body base. Rods in tentacle rachis longi- tudinally arranged, usually with many small warts. Spindles, rods and a few elongated scales in polyp body wall transversely or obliquely arranged, usually thick with sparse and fine warts. Rods and spindles in coenenchyme thick and nearly smooth or with sparse and fine warts. Branches with a few verrucae. Description. Holotype orange in situ, about 48 cm long with the holdfast not recovered (Figure3A,B). The direction of growth clockwise. Axis about 3 mm in diameter at base with branches producing nearly from the bottom to top and light iridescent metallic luster. Axis has 13 helical turns each 2.5–3.0 cm high and 0.5 cm wide. Branches usually grow upward, arranged along one side, 2–3 mm apart. Branches almost incomplete after collection and up to 11 cm long with 14 polyps counted. Polyps small, 3–9 mm apart, 1–2 mm tall and 1–3 mm wide at base (Figures1b and3C–E). Tentacular part about 1.0–1.5 mm long and wide, situated at an acute angle with the branch. Polyps white after fixation in alcohol. A few large verrucae present at polyp base and branches. Biology 2021, 10, 588 9 of 27 Biology 2021, 10, x FOR PEER REVIEW 9 of 27

Figure 3. External morphology and polyps of Iridogorgia densispiralis sp. nov. nov. ( A) The holotype in situ. ( B) The holotype after collection. (C) A single polyp under a light microscope. (D,E) Singles polyps under SEM. (F) Tentacular part under after collection. (C) A single polyp under a light microscope. (D,E) Singles polyps under SEM. (F) Tentacular part under SEM. (G) A part of branch under SEM. Scales = 20 cm (B), 1 mm (C–E), 500 μm (F,G). SEM. (G) A part of branch under SEM. Scales = 20 cm (B), 1 mm (C–E), 500 µm (F,G).

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Figure 4. ScleritesFigure of 4.Iridogorgia Sclerites densispiralisof Iridogorgiasp. densispiralis nov. (A) Sclerites sp. nov. in tentacle(A) Sclerites rachis. in ( Btentacle) Sclerites rachis. in polyp (B) Sclerites body wall. in polyp body wall. (C) Sclerites in coenenchyme. Scales = 300 μm, all in the same scale. (C) Sclerites in coenenchyme. Scales = 300 µm, all in the same scale.

urn:lsid:zoobank.org:acRods in the backt:026E2F14-7A66-4558-9D65-B7E3CC0EC8D0. of tentacle rachis longitudinally arranged, with many small warts and Materialtwo examined. rounded ends, Holotype: sometimes withMBM286454, deep cracks orstation a few warts,FX-Dive measuring 225 95–442(10°36.75× 11–52′ N, µm 140°3.85′ E),(Figures a seamount3F and (temporarily4A). Spindles, named rods and as M8) a few located elongated on the scales Caroline in polyp Ridge, body 1574 wall m, 13 June 2019.transversely or obliquely arranged, often thick with sparse and fine warts, occasion- ally branched or crossed with shallow cracks on surface, measuring 84–347 × 18–60 µm Diagnosis. Colony relatively short. Axis have many close helical turns each 2.5–3.0 (Figure4B) . Rods and spindles in coenenchyme, thick with two sharp or round ends, nearly cm high. Polypssmooth small or with with sparse an expanded and fine warts, and occasionally conical body crossed, base. measuring Rods in tentacle90–523 × rachis19–65 µm longitudinally(Figures arranged,3G and usually4C). Sclerites with sometimes many small sparse warts. in the Spindles, coenenchyme rods betweenand a few polyps. elon- gated scales in polypType locality. body Awall seamount transversely (tentatively or obliquely named as M8)arranged, located usually on the Caroline thick with Ridge in sparse and finethe Westernwarts. Rods Pacific and with spindles water depth in coenenchyme of 1574 m. thick and nearly smooth or with sparse and fine warts.Etymology. Branches Composite with a offew the verrucae. Latin adjectives densus (dense) and spiralis (spiral), Description.referring Holotype to the dense orange spirals in ofsitu, this about species. 48 cm long with the holdfast not recov- ered (Figure 3A,B).Distribution The direction and Habitat. of growth Found clockwise. only from a Axis seamount about located 3 mm on in the diameter Caroline Ridge.at Grown in a rocky bottom with branches in upper part of the colony incomplete (Figure3A ). base with branches producing nearly from the bottom to top and light iridescent metallic Remarks. Iridogorgia densispiralis sp. nov. is characterized by the relatively short luster. Axis colony,has 13 short helical and turns narrow each helical 2.5–3.0 turn, cm and high rods presentand 0.5 in cm both wide. polyps Branches and coenenchyme. usually It grow upward,is most arranged similar along to I. splendens one side,Watling, 2–3 mm 2007 apart. in the relativelyBranches short almost colony incomplete and helical after turn in collection and up to 11 cm long with 14 polyps counted. Polyps small, 3–9 mm apart, 1–2 mm tall and 1–3 mm wide at base (Figures 1b and 3C–E). Tentacular part about 1.0–1.5 mm long and wide, situated at an acute angle with the branch. Polyps white after fixation in alcohol. A few large verrucae present at polyp base and branches. Rods in the back of tentacle rachis longitudinally arranged, with many small warts and two rounded ends, sometimes with deep cracks or a few warts, measuring 95–442 × 11–52 μm (Figures 3F and 4A). Spindles, rods and a few elongated scales in polyp body

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wall transversely or obliquely arranged, often thick with sparse and fine warts, occasion- ally branched or crossed with shallow cracks on surface, measuring 84–347 × 18–60 μm (Figure 4B). Rods and spindles in coenenchyme, thick with two sharp or round ends, nearly smooth or with sparse and fine warts, occasionally crossed, measuring 90–523 × 19–65 μm (Figures 3G and 4C). Sclerites sometimes sparse in the coenenchyme between polyps. Type locality. A seamount (tentatively named as M8) located on the Caroline Ridge in the Western Pacific with water depth of 1574 m. Etymology. Composite of the Latin adjectives densus (dense) and spiralis (spiral), re- ferring to the dense spirals of this species. Distribution and Habitat. Found only from a seamount located on the Caroline Ridge. Grown in a rocky bottom with branches in upper part of the colony incomplete (Figure 3A). Biology 2021, 10, 588 Remarks. Iridogorgia densispiralis sp. nov. is characterized by the relatively short col-11 of 27 ony, short and narrow helical turn, and rods present in both polyps and coenenchyme. It is most similar to I. splendens Watling, 2007 in the relatively short colony and helical turn in adults, butadults, differs but by differs the pr byesence the presence of thick of thickrods rodsin coenenchyme in coenenchyme (vs. (vs. absent) absent) and and abun- abundant dant scleritessclerites in tentacles in tentacles (vs. sparse) (vs. sparse) [4]. [4]. Iridogorgia verrucosaIridogorgia sp. verrucosa nov. sp. nov. Figures 1c, Figures5, 6; Table1c,5 and2. 6; Table2.

Figure 5. ExternalFigure morphology 5. External and morphology polyps of Iridogorgia and polyps verrucosa of Iridogorgiasp. nov. (A verrucosa) The holotype sp. nov. in situ. (A) (TheB) The holotype holotype in after situ. collection. (C)(B A) singleThe holotype polyp under after a collection. light microscope. (C) A (singleD,E) Single polyp polyps under under a light SEM. microscope. (F) Tentacular (D,E part) Single under polyps SEM. under SEM. (F) Tentacular part under SEM. (G) Coenenchyme with many verrucae under a light (G) Coenenchyme with many verrucae under a light microscope. Scales = 20 cm (B), 1 mm (C–E,G), 500 µm (F). microscope. Scales = 20 cm (B), 1 mm (C–E,G), 500 μm (F). urn:lsid:zoobank.org:act:E388B7B2-936E-485C-AB38-E7F95DD4F0A4. Material examined. Holotype: MBM286455, station FX-Dive 225 (10◦36.770 N, 140◦04.050 E), a seamount (temporarily named as M8) located on the Caroline Ridge, 1397 m, 13 June 2019. Diagnosis. Colony relatively short with branches producing nearly from the bottom to top. Axis having many close helical turns each 3–4 cm in high. Polyps small with long tentacles. Rods in tentacle rachis longitudinally arranged and usually with many small warts. Spindles and elongated scales in polyp body wall transversely or obliquely placed, thick with irregular edges. Spindles in coenenchyme slender and thick with usually rounded ends. Branches with many cylindroid verrucae. Description. Holotype orange, about 51 cm long with the holdfast not recovered (Figure5B). Growth direction clockwise. Axis about 4 mm in diameter at base with branches producing nearly from bottom to top, composed of 14 helical turns each about 3–4 cm high and 1 cm wide. Branches usually growing upward, arranged along one side, Biology 2021, 10, 588 12 of 27

1–3 mm apart. Branches almost incomplete after collection, up to 10 cm long with 14 polyps counted. Polyps small, towards to the branch end and situated with an acute angle, 4–6 mm apart, average 2 mm tall, and 1–2 mm wide at base (Figures1c and5C–E). Tentacular part Biology 2021, 10, x FOR PEER REVIEW up to 2 mm long and 1 mm wide. Polyps white after fixation in alcohol. Numerous12 of 27 and

dense small cylindroid verrucae present in polyp base and branches (Figure5C,G).

Figure 6. ScleritesFigure of Iridogorgia6. Sclerites verrucosa of Iridogorgiasp. nov. verrucosa (A) Sclerites sp. innov. tentacle (A) Sclerites rachis. (B in) Sclerites tentacle in rachis. polyp body (B) Sclerites wall. (C) in Sclerites polyp in coenenchyme.body Scales wall. = ( 300C) Scleritesµm, all in in the coenenchyme. same scale. Scales = 300 μm, all in the same scale.

urn:lsid:zoobank.org:act:E388B7Rods in the back of tentacleB2-936E-485C-AB38-E7F95DD4F0A4. rachis longitudinally arranged, usually with many small Materialconical examined. warts and Holotype: round ends, MBM286455, sometimes with station deep cracksFX-Dive on surface, 225 (10°36.77 occasionally′ N, lobed × µ 140°04.05′ E),with a seamount irregular edges,(temporarily measuring named 160–459 as M8)17–73 locatedm on (Figures the Caroline5F and6A). Ridge, Spindles 1397 and elongated scales in polyp body wall transversely or obliquely placed, usually thick with m, 13 June 2019. sparse and fine warts and irregular edges, measuring 116–450 × 21–65 µm (Figure6B). Spin- Diagnosis.dles Colony in coenenchyme relatively very short similar with to branches those in polyp producing body wall, nearly almost from slender, the bottom measuring to top. Axis 169–438having ×many19–62 closeµm (Figurehelical6 C).turns each 3–4 cm in high. Polyps small with long tentacles. Rods inType tentacle locality. rachis A seamount longitudinally (temporarily arranged named and as M8) usually located with on the many Caroline small Ridge warts. Spindlesin the and Western elongated Pacific scales with waterin polyp depth body of 1397 wall m. transversely or obliquely placed, thick with irregularEtymology. edges. The Spindles Latin adjective in coenenchymeverrucosus (verrucose)slender and refers thick to the with numerous usually small rounded ends.verrucae Branches of this with species. many cylindroid verrucae. Description.Distribution Holotype orange, and Habitat. about Found 51 cm only long from with a seamount the holdfast located not on recovered the Caroline (Fig- Ridge. Grown in a rocky bottom with black coral and scleractinian coral living around (Figure5A ). ure 5B). Growth direction clockwise. Axis about 4 mm in diameter at base with branches Remarks. Iridogorgia verrucosa sp. nov. is characterized by the relatively short colony producing nearlyand narrow from helicalbottom turn, to irregulartop, comp spindlesosed of and 14 scaleshelical in polypturns bodyeach wallabout and 3–4 numerous cm high and 1 cmverrucae. wide. Branches It is similar usually to I. splendens growingand upward,I. densispiralis arrangedsp. nov. along in the one short side, colony 1–3 andmm close apart. Brancheshelix, almost but differs incomplete from I. splendens after bycollection, the abundant up rodsto 10 in cm tentacles long (vs.with sparse), 14 polyps sclerites in counted. Polypspolyp small, body walltowards with irregularto the branch edges (vs.end regular),and situated and numerous with an scleritesacute angle, in inter-polyp 4–6 mm apart, average 2 mm tall, and 1–2 mm wide at base (Figures 1c and 5C–E). Tentacular part up to 2 mm long and 1 mm wide. Polyps white after fixation in alcohol. Numerous and dense small cylindroid verrucae present in polyp base and branches (Figure 5C,G). Rods in the back of tentacle rachis longitudinally arranged, usually with many small conical warts and round ends, sometimes with deep cracks on surface, occasionally lobed with irregular edges, measuring 160–459 × 17–73 μm (Figures 5F and 6A). Spindles and elongated scales in polyp body wall transversely or obliquely placed, usually thick with sparse and fine warts and irregular edges, measuring 116–450 × 21–65 μm (Figure 6B). Spindles in coenenchyme very similar to those in polyp body wall, almost slender, meas- uring 169–438 × 19–62 μm (Figure 6C). Type locality. A seamount (temporarily named as M8) located on the Caroline Ridge in the Western Pacific with water depth of 1397 m.

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Etymology. The Latin adjective verrucosus (verrucose) refers to the numerous small verrucae of this species. Distribution and Habitat. Found only from a seamount located on the Caroline Ridge. Grown in a rocky bottom with black coral and scleractinian coral living around (Figure 5A). Remarks. Iridogorgia verrucosa sp. nov. is characterized by the relatively short colony and narrow helical turn, irregular spindles and scales in polyp body wall and numerous Biology 2021, 10, 588verrucae. It is similar to I. splendens and I. densispiralis sp. nov. in the short colony and13 of 27 close helix, but differs from I. splendens by the abundant rods in tentacles (vs. sparse), scle- rites in polyp body wall with irregular edges (vs. regular), and numerous sclerites in inter- polyp coenenchymecoenenchyme (vs. rare (vs. rareto absent) to absent) [4]. [ 4Iridogorgia]. Iridogorgia verrucosa verrucosa sp.sp. nov. cancan be be distinguished distin- guished fromfrom I. densispiralisI. densispiralis sp. sp.nov. nov. by the by theabsence absence of ofrods rods in incoenenchyme coenenchyme (vs. (vs. present) present) and and numerousnumerous and dense and verrucae dense verrucae in branches in branches (vs. sparse). (vs. sparse). Iridogorgia magnispiralisIridogorgia magnispiralis Watling, 2007.Watling, 2007. Figures 1d, 7,Figures 8; Table1d, 72. and 8; Table2.

Figure 7. ExternalFigure morphology 7. External and morphology polyps of Iridogorgia and polyps magnispiralis of IridogorgiaWatling, magnispiralis 2007. (A) The Watling, specimen 2007. MBM286451 (A) The speci- in situ. Laser dots spacedmen MBM286451 at 33 cm used in for situ. measuring Laser dots dimensions. spaced ( Bat) Close-up33 cm used of branches for measuring and polyps dimensions. in situ. (C) ( TheB) Close-up specimen MBM286451of after branches collection. and ( D)polypsA part in of situ. branch (C) withThe specimen three polyps MBM286451 under a light after microscope. collection. (E) A(D) single A part polyp of branch under a light microscope.with three (F) A singlepolyps polyp under under a light SEM. microscope. Scales = 20 ( cmE) A (C single), 2 mm polyp (D), 1 under mm (E ,aF light). microscope. (F) A single polyp under SEM. Scales = 20 cm (C), 2 mm (D), 1 mm (E,F). Iridogorgia magnispiralis Watling, 2007: 395–397, Figures2 and3. Material Examined. MBM286450 and MBM286451, station FX-Dive 216 (10◦5.150 N, 140◦11.120 E), a seamount (tentatively named as M5) located on the Caroline Ridge in the Western Pacific, 845 m, 4 June 2019. Diagnosis (based on the present specimens and Watling, 2007 [4]). Colony with branches producing on upper part, and helical turn each 12–20 cm high. Polyps erect or slightly inclined along branches. Rods in tentacle rachis longitudinally arranged, regular with many small warts. Spindles in polyp body wall transversely or obliquely arranged, slender and nearly smooth. Spindles in coenenchyme transversely arranged, slender with Biology 2021, 10, 588 14 of 27

Biology 2021, 10, x FOR PEER REVIEW two sharp ends and nearly smooth surface. Scales absent. Branches with14 of many 27 verrucae. Inter-polyp coenenchyme with numerous sclerites.

Figure 8. FigureSclerites 8. ofScleritesIridogorgia of Iridogorgia magnispiralis magnispiralisWatling, 2007 Watling, (MBM286451). 2007 (MBM286451). (A) Sclerites in(A tentacle) Sclerites rachis. in tentacle (B) Sclerites in rachis. (B) Sclerites in polyp body wall. (C) Sclerites in coenenchyme. Scales = 500 μm, all in the polyp body wall. (C) Sclerites in coenenchyme. Scales = 500 µm, all in the same scale. same scale. Description. Two specimens have similar external morphology and size, and the Iridogorgia magnispiralisspecimen MBM286451 Watling, 2007: was described395–397, Figures in detail 2 here.and 3. Colony grown in a rocky bottom Material Examined.with a small MBM286450 holdfast in situand (FigureMBM286451,7A). Specimen station aboutFX-Dive 63 cm 216 long (10°5.15 with the′ N, holdfast not 140°11.12′ E), a seamountrecovered (tentatively (Figure7C). Thenamed direction as M5) of located growth on clockwise the Caroline and the Ridge same in in the MBM286450. Western Pacific,Axis 845 aboutm, 4 June 2.5 mm 2019. in diameter at base with branches producing on the upper part, having Diagnosis (basedtwo helical on the turns present each about specimen 12 cms high and andWatling, 1 cm in 2007 diameter. [4]). Colony Branches with arranged along branches producingone side, on upper about part, 2 mm and apart helical and up turn to 13.5each cm 12–20 long cm with high. 21 polyps Polyps counted. erect or Polyps erect slightly inclinedor along slightly branches. inclined Rods along in the tentacle branches, rachis 2–4 longitudinally mm apart, 1–3 mmarranged, long, andregular 2–3 mm wide at with many smallbase warts. (Figures Spindles1d and in7 polypB,D–F). body Tentacular wall transversely part 1.0–1.5 or mm obliquely long and arranged, wide. Polyps white slender and nearlyafter smooth. fixation Spindles in alcohol. in Branchescoenenchyme with manytransversely low verrucae. arranged, slender with Rods in the back of tentacle rachis longitudinally arranged, regular with two rounded two sharp ends and nearly smooth surface. Scales absent. Branches with many verrucae. ends, often with many small conical warts, occasionally with some shallow cracks and Inter-polyp coenenchyme with numerous sclerites. Description. Two specimens have similar external morphology and size, and the specimen MBM286451 was described in detail here. Colony grown in a rocky bottom with a small holdfast in situ (Figure 7A). Specimen about 63 cm long with the holdfast not re- covered (Figure 7C). The direction of growth clockwise and the same in MBM286450. Axis about 2.5 mm in diameter at base with branches producing on the upper part, having two

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helical turns each about 12 cm high and 1 cm in diameter. Branches arranged along one side, about 2 mm apart and up to 13.5 cm long with 21 polyps counted. Polyps erect or slightly inclined along the branches, 2–4 mm apart, 1–3 mm long, and 2–3 mm wide at base (Figures 1d and 7B,D–F). Tentacular part 1.0–1.5 mm long and wide. Polyps white after fixation in alcohol. Branches with many low verrucae. Rods in the back of tentacle rachis longitudinally arranged, regular with two rounded ends, often with many small conical warts, occasionally with some shallow cracks and a

Biology 2021, 10, 588few warts on surface, measuring 157–548 × 18–78 μm (Figure 8A). Spindles in polyp15 of 27 body wall transversely or obliquely arranged, thick, slender and usually with two sharp ends, nearly smooth or with many fine warts, occasionally rugged or with a few large protuber- ances, measuringa few warts214–1054 on surface, × 28–65 measuring μm (Figure 157–548 8B).× 18–78 Spindlesµm (Figure in coenenchyme8A). Spindles in slender polyp with two sharp ends,body wallnearly transversely smooth, or obliquelyoccasionally arranged, with thick, a few slender large and irregular usually with protuberances, two sharp measuring 332–1032ends, nearly × smooth21–81 orμm with (Figure many fine8C). warts, Scales occasionally absent in rugged this orspecimen with a few (Figures large 7F protuberances, measuring 214–1054 × 28–65 µm (Figure8B). Spindles in coenenchyme and 8). Inter-polypslender withcoenenchyme two sharp ends, with nearly numerous smooth, sclerites. occasionally with a few large irregular Distribution.protuberances, The western measuring Corner 332–1032 Rise× 21–81 Seamountsµm (Figure to8C). the Scales New absent England in this specimenSeamounts to Kelvin Seamount,(Figures and7F and the8). Inter-polypLost City coenenchymesite on the Mid-Atlantic with numerous sclerites. Ridge, 1650–2400 m [4]; Ha- Distribution. The western Corner Rise Seamounts to the New England Seamounts waii, 1310–1366to Kelvin m [6,8]; Seamount, Phoenix and Islands the Lost Protected City site on theArea, Mid-Atlantic 1000–2000 Ridge, m [14]; 1650–2400 Northeast m [4]; Aus- tralia, 728–777Hawaii, m [6]; 1310–1366 a seamount m [6,8]; located Phoenix on Islands the ProtectedCaroline Area, Ridge, 1000–2000 845 m. m [14]; Northeast Remarks.Australia, The present 728–777 mspecimens [6]; a seamount match located well on thewith Caroline the Ridge,holotype 845 m.of Iridogorgia mag- nispiralis Watling,Remarks. 2007. The The presentsmaller specimens number match of the well helical with theturns holotype and the of Iridogorgia shorter mag-branches nispiralis Watling, 2007. The smaller number of the helical turns and the shorter branches suggest the twosuggest specimens the two specimens are in a are young in a young stage. stage. Iridogorgia densispiculaIridogorgia densispicula Xu, Zhan,Xu,Zhan, Li and Li andXu, Xu, 2020. 2020. Figures 1e–g,Figures 9, 10A–E,1e–g,9, 10 andA–E 11–13; and 11– Table13; Table 2.2 .

Figure 9. ExternalFigure morphology 9. External of Iridogorgiamorphology densispicula of IridogorgiaXu et al., 2020 densispicula specimens MBM286446 Xu et al., ( A2020,B), MBM286447 specimens (C ,DMBM286446) and MBM286448(A,B), (E MBM286447,F). (A,C,E) Specimens (C,D) inand situ. MBM286448 (B,D,F) Specimens (E,F after). (A collection.,C,E) Specimens Scales = 20 in cm situ (B,D. ,(FB).,D,F) Specimens after collection. Scales = 20 cm (B,D,F).

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Figure 10.FigurePolyps 10. and Polyps sclerites and arrangementsclerites arrangement of Iridogorgia of Iridogorgia densispicula densispiculaXu et al., 2020 Xu et (A –al.,E). 2020(A–C ()A Single–E). (Apolyps–C) under a light microscope,Single polyps specimens under a MBM286446,light microscope, MBM286447 specimens and MBM286446, MBM286448, MBM286447 respectively. and (D )MBM286448, Polyp body underre- SEM. spectively. (D) Polyp body under SEM. (E) Conenechyme and the end of basal body under SEM. (E) Conenechyme and the end of basal body under SEM. Scales = 1 mm (A–C), 300 µm (D,E). Scales = 1 mm (A–C), 300 μm (D,E). Iridogorgia densispicula Xu, Zhan, Li & Xu, 2020: 251–254, Figures2 and3. Iridogorgia densispiculaMaterial Xu, Examined. Zhan, Li & MBM286446, Xu, 2020: 251–254, station Figures FX-Dive 2 226and(10 3. ◦38.80 N, 140◦4.150 E), a Material Examined.seamount MBM286446, (tentatively named station as FX-Dive M8) located 226 (10°38.8 on the Caroline′ N, 140°4.15 Ridge,′ E), 1741 a sea- m, 14 June 2019. mount (tentativelyMBM286447, named as station M8) located FX-Dive on 227 the (10 Caroline◦37.880 N, Ridge, 140◦5.62 17410 E), am, seamount 14 June (tentatively2019. named MBM286447, stationas M8) FX-Dive located 227 on (10°37.88 the Caroline′ N, 140°5.62 Ridge,′ 1678 E), a m,seamount 15 June (tentatively 2019. MBM286448, named station FX- ◦ 0 ◦ 0 as M8) located onDive the 211Caroline (10 2.93 Ridge,N, 140 167810.48 m, 15E), June a seamount 2019. MBM286448, (tentatively station named asFX-Dive M5) located on the 211 (10°2.93′ N, 140°10.48Caroline Ridge,′ E), a seamount 1482 m, 29 (tentatively May 2019. named as M5) located on the Caro- line Ridge, 1482 m, 29Improved May 2019. diagnosis (based on the present specimens and Xu et al. 2020 [7]). Colony Improved diagnosisslender with (based branches on the producingpresent specimens on the upper and Xu part. et al. Axis 2020 having [7]). Colony helical turns each slender with branches12–19 cmproducinghigh. Polyps on the usually upper part. inclined Axis towards having distalhelical end turns of each branch. 12–19 Rods in tenta- cle rachis longitudinally arranged, regular with many small conical or ridge-like warts on cm high. Polyps usually inclined towards distal end of branch. Rods in tentacle rachis surface and two rounded ends. Spindles and scales in polyp body wall transversely or longitudinally arranged, regular with many small conical or ridge-like warts on surface obliquely arranged, elongated with irregular edges and often rugged and ridged surface. and two roundedSpindles ends. Spindles in coenenchyme and scales slender in polyp and usuallybody wall with transversely two sharp ends. or obliquely Branch surface nearly arranged, elongatedsmooth, with rarely irregular with verrucae.edges and often rugged and ridged surface. Spindles in coenenchyme slenderDescription. and usually The with three two specimens sharp ends. are similar Branch in surface external nearly morphology, smooth, and only the rarely with verrucae.specimen MBM286446 was descripted in detail here. Colony grown in a rocky bottom with Description.a The small three white specimens holdfast in are situ similar (Figure 9inA). external Specimen morphology, about 73 cm and long only with the holdfast not specimen MBM286446recovered was (Figure descripted9B). The in direction detail here. of growth Colony counterclockwise grown in a rocky (clockwise bottom in MBM286447, with a small whitecounterclockwise holdfast in situ in (Figure the bottom 9A). branching Specimen part about and 73 clockwise cm longin with the the upper hold- in MBM286448). fast not recoveredAxis (Figure about 9B). 2.5 mm The in direction diameter of at growth base with counterclockwise dark iridescent metallic(clockwise luster in and having MBM286447, counterclockwise in the bottom branching part and clockwise in the upper in MBM286448). Axis about 2.5 mm in diameter at base with dark iridescent metallic luster

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and having six helical turns, each turn 16–18 cm long and 3–4 cm in diameter. The branch- ing part about 19 cm long with branches growing upward and not arc-shaped. Branches arranged alongsix helicalone side, turns, 3–4 each mm turn apart, 16–18 and cm up long to and20 cm 3–4 long cm in with diameter. 29 polyps The branching counted. part Polyps usuallyabout inclined 19 cm longtowards with distal branches end growing of branch, upward 2–4 mm and notapart, arc-shaped. average Branches2.5 mm long, arranged along one side, 3–4 mm apart, and up to 20 cm long with 29 polyps counted. Polyps and 2–4 mm wide at base (Figures 1e and 10A). Tentacular part 1–2 mm long, 1 mm wide, usually inclined towards distal end of branch, 2–4 mm apart, average 2.5 mm long, and usually forming2–4 mm eight wide obvious at base parallel (Figures 1colue andmns 10A). terminating Tentacular partat its 1–2 base mm (Figure long, 1 mm10C). wide, Polyps sometimesusually with forming an expanded eight obvious body parallel basecolumns and golden terminating eggs present at its base and (Figure visible 10 underC). Polyps the microscopesometimes (Figure with 10A). an expandedPolyps became bodybase white and after golden fixation. eggs present Branch and surface visible nearly under the smooth, rarelymicroscope with a few (Figure verrucae. 10A). Polyps became white after fixation. Branch surface nearly smooth, Rods inrarely the back with of a fewtentacle verrucae. rachis longitudinally arranged, slender and regular with usually two roundRods ends in theand back many of tentaclesmall conical rachis or longitudinally ridge-like warts, arranged, occasionally slender and branched regular with and with a fewusually large two protuberances, round ends and measuring many small 137–519 conical × or 11–52 ridge-like μm (Figure warts, occasionally 11A). Spindles branched and with a few large protuberances, measuring 137–519 × 11–52 µm (Figure 11A). Spindles and scales in polyp body wall transversely or obliquely arranged, elongate and often with and scales in polyp body wall transversely or obliquely arranged, elongate and often with a slight midwaya slight constriction midway constriction and many fine and warts, many fineusually warts, lobed usually with lobed irregular with irregularshape and shape rugged and andridged rugged surface and in ridged the upper surface part in theof the upper body part wall of the (Figure body 10D), wall (Figure and regular 10D), and and flat in theregular basal and part flat (Figure in the basal 10E), part measuring (Figure 10 80–540E), measuring × 14–101 80–540 μm× (Figure14–101 µ11B).m (Figure Spin- 11B). dles in coenenchymeSpindles in slender, coenenchyme usually slender, with two usually sharp with ends two and sharp many ends andfine manywarts fine on wartssur- on face, occasionallysurface, nearly occasionally smooth nearly or with smooth a few or irregular with a few protuberances, irregular protuberances, measuring measuring 203– 967 × 11–65 μ203–967m (Figure× 11–65 11C).µm Inter-polyp (Figure 11C). coenenchyme Inter-polyp coenenchyme with numerous with sclerites. numerous sclerites.

Figure 11. ScleritesFigure of 11.Iridogorgia Sclerites densispicula of IridogorgiaXu etal. densispicula 2020 specimen Xu MBM286446.et al. 2020 specimen (A) Sclerites MBM286446. in tentacle rachis. (A) (ScleritesB) Sclerites in in polyp bodytentacle wall. (C rachis.) Sclerites (B) in Sclerites coenenchyme. in polyp Scales body = 500 wall.µm, ( allC) inSclerites the same in scale. coenenchyme. Scales = 500 μm, all in the same scale.

Distribution. Seamounts located on the Caroline Ridge, 1204–1741 m ([7]). Remarks. Our specimens match well with the previous description, and they all have a slender stem, high helical turn, oblique polyps, and the same sclerite forms in tentacles and coenenchyme (Figures 9–13). Considering only one holotype described before, here

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Distribution. Seamounts located on the Caroline Ridge, 1204–1741 m ([7]). Remarks. Our specimens match well with the previous description, and they all Biology 2021, 10, x FOR PEER REVIEW 18 of 27 have a slender stem, high helical turn, oblique polyps, and the same sclerite forms in tentacles and coenenchyme (Figures9–13). Considering only one holotype described before, here we make a supplementary description for this species, especially the varied scleriteswe make in a thesupplementary polyp body wall,descri includingption for this spindles species, and especially scales, which the varied are elongated sclerites andin oftenthe polyp with body irregular wall, edges including and ruggedspindles and and ridged scales, surfaces which are (Figures elongated 11B, and12B andoften 13 withB). It isirregular worth notingedges and that rugged the elongated and ridged scales surfaces are occasionally (Figures 11B, present 12B and in the 13B). tentacles It is worth of the specimennoting that MBM286448 the elongated (Figure scales 13 areA). occasional These specially present scales in maythe tentacles be an extension of the specimen from the bodyMBM286448 extending (Figure to the 13A). tentacles. These special Furthermore, scales may the be polyp an extension shapes infrom these the specimensbody extend- are variable,ing to the including tentacles. bud-like Furthermore, (holotype the andpolyp MBM286448, shapes in these Figure specimens 10C), slender are withvariable, elongate in- bodycluding (MBM286446, bud-like (holotype Figure 10andA), MBM286448, cylindrical with Figure long 10C), extended slender tentacles with elongate (MBM286447, body Figure(MBM286446, 10B). Such Figure difference 10A), cylindrical may be a processwith long of extended growth stages tentacles (mature (MBM286447, or immature) Figure and caused10B). Such by thedifference alcohol may preservation. be a process of growth stages (mature or immature) and caused by the alcohol preservation.

Figure 12. Sclerites of Iridogorgia densispicula Xu et al. 2020 specimen MBM286447. (A) Sclerites in tentacles. (B) Sclerites in Figure 12. Sclerites of Iridogorgia densispicula Xu et al. 2020 specimen MBM286447. (A) Sclerites in tentacles. (B) Sclerites in polyp body wall. (C) Sclerites in coenenchyme. Scales = 500 μm, all in the same scale. polyp body wall. (C) Sclerites in coenenchyme. Scales = 500 µm, all in the same scale.

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Figure 13. Sclerites of Iridogorgia densispicula Xu et al. 2020 specimen MBM286448. (A) Sclerites in tentacles. (B) Sclerites in Figure 13. Sclerites of Iridogorgia densispicula Xu et al. 2020 specimen MBM286448. (A) Sclerites in tentacles. (B) Sclerites in polyp body wall. (C) Sclerites in coenenchyme. Scales = 200 μm (A,B in the same scale), 500 μm (C). polyp body wall. (C) Sclerites in coenenchyme. Scales = 200 µm (A,B in the same scale), 500 µm (C). 3.2. Genetic Distance and Phylogenetic Analyses 3.2. Genetic Distance and Phylogenetic Analyses The new sequences were deposited in GenBank (Table 1). The alignments comprised The new sequences were deposited in GenBank (Table1). The alignments comprised 663,663, 537 537 and and 797 797 nucleotide nucleotide positions positions for for the the mtMutS, mtMutS, COI COI and and 28S 28S rDNA rDNA regions, regions, respec- respec- tively.tively. Based Based on on the aligne alignedd region of 28S rDNA, the interspecificinterspecific distance of Iridogorgia rangedranged from from 0.32% to 6.08% 6.08% (average (average value at at 3.10%), 3.10%), while the intraspecific intraspecific distance was 0.32%,0.32%, which was calculatedcalculated fromfrom onlyonly two two populations populations of ofIridogorgia Iridogorgia magnispiralis magnispiralis(Table (Table3). 3).For For the the concatenated concatenated region region mtMutS-28S, mtMutS-28S, the the interspecific interspecific distance distance range range is inis thein the range range of of0.31%–3.48%, 0.31%–3.48%, and and no intraspecificno intraspecific data data are availableare available (Table (Table3). For 3). the For mitochondrial the mitochondrial align- alignments,ments, the interspecific the interspecific and intraspecificand intraspecific distances distances of Iridogorgia of Iridogorgiaare in are ranges in ranges of 0–1.33% of 0– 1.33%and 0–0.33% and 0–0.33% for mtMutS, for mtMutS, and 0–0.39% and 0–0.39% and zero and for zero COI, for respectively COI, respectively (Tables 4(Tables and5). 4 The and 5).genetic The genetic distances distances between betweenRhodaniridogorgia Rhodaniridogorgiaand Iridogorgia and Iridogorgiaare in are ranges in ranges of 0.47%–1.41% of 0.47%– 1.41%for mtMutS, for mtMutS, and 0–0.39% and 0–0.39% for COI, for which COI, which partially partially and fully and overlap fully overlap with the with interspecific the inter- specificdistances distances of Iridogorgia of Iridogorgia, respectively, respectively (Tables4 (Tables and5). 4 and 5).

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Table 3. Genetic distances at 28S rDNA (lower left) and the concatenated mtMutS -28S (upper right) of Iridogorgia species.

1 2 3 4 5 6 7 1 Iridogorgia densispiralis sp. nov. MW841043 - 1.17% 0.31% 3.48% 1.64% - - 2 Iridogorgia flexilis sp. nov. MW841041 2.30% - 1.09% 2.76% 0.46% - - 3 Iridogorgia verrucosa sp. nov. MW841044 0.65% 2.13% - 3.40% 1.56% - - 4 Iridogorgia densispicula MW841040 6.08% 4.68% 5.90% - 3.24% - - 5 Iridogorgia magnispiralis MW841042 2.97% 0.65% 2.80% 5.37% - - - 6 Iridogorgia magnispiralis KX890214 3.30% 0.98% 3.13% 5.54% 0.32% - - 7 Iridogorgia splendens KX890215 0.32% 2.30% 0.98% 5.72% 2.97% 3.30% - New sequences are in bold.

Table 4. Interspecific and intraspecific distances at mtMutS of Iridogorgia and Rhodaniridogorgia species.

1 2 3 4 5 6 7 8 9 10 11 12 13 1 Iridogorgia densispiralis sp. nov. MW841033 - 2 I. verrucosa sp. nov. MW841034 0.00% - 3 I. densispicula MK431864 1.11% 1.11% - 4 I. flexilis sp. nov. MW841031 0.15% 0.15% 0.95% - I. splendens DQ860109, JN227996, JN228005, 5 0.16% 0.16% 0.95% 0.00% 0.00% KC788271, GQ180143 6 I. splendens GQ180144 0.47% 0.47% 1.33% 0.32% 0.33% - 7 I. squarrosa MK431865 0.32% 0.32% 1.11% 0.16% 0.16% 0.49% - 8 I. fontinalis EU293802 0.48% 0.48% 1.27% 0.32% 0.32% 0.66% 0.48% - I. magnispiralis MW841032 DQ860108, GQ353316, EU268055, JN227997, 9 0.47% 0.47% 1.27% 0.32% 0.32% 0.66% 0.47% 0.00% 0.00% GQ223116, GQ180141, GQ180142, GQ180140, KC788263 10 Iridogorgia sp. JN227919 0.00% 0.00% 1.11% 0.15% 0.16% 0.47% 0.32% 0.48% 0.47% - 11 Iridogorgia sp. GQ868342 0.30% 0.30% 1.11% 0.15% 0.16% 0.47% 0.00% 0.48% 0.47% 0.30% - 12 Iridogorgia sp. DQ297422, GQ180145 0.46% 0.46% 1.27% 0.30% 0.32% 0.63% 0.47% 0.00% 0.00% 0.46% 0.46% 0.00% 13 Rhodaniridogorgia fragilis JN228000 0.70% 0.70% 1.41% 0.47% 0.47% 0.70% 0.93% 0.93% 0.93% 0.70% 0.70% 0.93% - New sequences are in bold. Biology 2021, 10, 588 21 of 27

Table 5. Interspecific and intraspecific distances at COI of Iridogorgia and Rhodaniridogorgia species.

1 2 3 4 5 6 7 8 9 1 Iridogorgia densispiralis sp. nov. MW841036 - 2 Iridogorgia verrucosa sp. nov. MW840138 0 - 3 Iridogorgia flexilis sp. nov. MW841035 0 0 - 4 Iridogorgia splendens GQ868313, GQ868330, KC788229 0 0 0 0 5 Iridogorgia sp. GQ868323 0 0 0 0 - 6 Iridogorgia densispicula MW841037 0.20% 0.20% 0.20% 0.20% 0.20% - 7 Iridogorgia fontinalis GQ868321 0.19% 0.19% 0.19% 0.19% 0.19% 0.39% - Iridogorgia magnispiralis MW841039, FJ268639, 8 0.19% 0.19% 0.19% 0.19% 0.19% 0.39% 0 0 GQ868318, KC788237 9 Rhodaniridogorgia fragilis JN227954 0.20% 0.20% 0.20% 0.20% 0.20% 0.39% 0 0 0 New sequences are in bold. Biology 2021, 10, 588 22 of 27

The ML tree is nearly identical to the BI tree in topology for both the mtMutS-COI and 28S rDNA regions, and thus a single tree with both support values was shown for each of the markers (Figures 14 and 15). In the mtMutS-COI trees, the monophyly of Iridogorgia was not supported due to the Rhodaniridogorgia nested into the Iridogorgia clade, and clustered with the subclade of I. magnispiralis, I. fontinalis and Iridogorgia sp. GQ180145 with low to moderate support (Figure 14). Within Iridogorgia, Iridogorgia densispicula branched outside of the main clade including the rest of the Iridogorgia species. Like the subclade of magnispi- ralis/fontinali, I. densispiralis sp. nov., I. verrucosa sp. nov. and Iridogorgia sp. GQ868342 also formed a subclade due to no genetic variation among the them (Tables4 and5 and Figure 14). Although I. flexilis sp. nov., I. splendens, Iridogorgia sp. GQ868342 and the sub- clades magnispiralis/fontinalis and densispiralis/verrucosa branched together, their deeply divergent relationships were not resolved (Figure 14). In the 28S rDNA trees, I. densispicula branched early, and the rest of the Iridogorgia species were separated to two clades with low support (ML < 70%; BI < 0.90; Figure 15). Within Clade I, I. densispiralis sp. nov. and I. Biology 2021, 10, x FOR PEER REVIEW 22 of 27 splendens branched together, followed by I. verrucosa sp. nov. with high support. Iridogorgia flexilis sp. nov. and I. magnispiralis formed the Clade II with high support.

Figure 14. Bayesian inference (BI) tree constructed by the concatenated sequences of mtMutS Figure 14. Bayesian inference (BI) tree constructed by the concatenated sequences of mtMutS and and COI showing phylogenetic relationships among Iridogorgia and Rhodaniridogorgia species. The COI showing phylogenetic relationships among Iridogorgia and Rhodaniridogorgia species. The Max- Maximum likelihood (ML) tree is identical to the BI tree in topology. Node support is as follows: imum likelihood (ML) tree is identical to the BI tree in topology. Node support is as follows: ML ML bootstrap/BI posterior probability. The ML bootstrap <70% or BI posterior probability <0.90 is bootstrap/BI posterior probability. The ML bootstrap <70% or BI posterior probability <0.90 is not not shown. Newly sequenced species are in bold. The GenBank accession numbers of the mtMutS shown. Newly sequenced species are in bold. The GenBank accession numbers of the mtMutS se- sequences were listed next to the species names. quences were listed next to the species names.

Figure 15. Maximum likelihood (ML) tree constructed by 28S rDNA showing phylogenetic relation- ships among Iridogorgia species. The Bayesian inference (BI) tree is identical with the ML tree in topology. Node support is as follows: ML bootstrap/BI posterior probability. The ML bootstrap <70% or BI posterior probability <0.90 is not shown. Newly sequenced species are in bold. The Gen- Bank accession numbers of the 28S rDNA sequences were listed next to the species names.

4. Discussion The DNA barcoding analysis of mtMutS, COI and 28S rDNA is considered as one of the first steps in an integrative identification of octocorals [19,41,42]. In the present analy- sis, COI exhibited much less variation than the other two loci, and no genetic variability

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Figure 14. Bayesian inference (BI) tree constructed by the concatenated sequences of mtMutS and COI showing phylogenetic relationships among Iridogorgia and Rhodaniridogorgia species. The Max- imum likelihood (ML) tree is identical to the BI tree in topology. Node support is as follows: ML bootstrap/BI posterior probability. The ML bootstrap <70% or BI posterior probability <0.90 is not Biology 2021, 10, 588 shown. Newly sequenced species are in bold. The GenBank accession numbers of the23 ofmtMutS 27 se- quences were listed next to the species names.

FigureFigure 15. Maximum likelihood likelihood (ML) (ML) tree tree constructed constructed by 28S by rDNA28S rDNA showing showing phylogenetic phylogenetic relation- relation- shipsships among among IridogorgiaIridogorgiaspecies. species. The The Bayesian Bayesian inference inference (BI) tree(BI) istr identicalee is identical with the with ML the tree ML in tree in topology.topology. NodeNode support support is as is follows: as follows: ML bootstrap/BI ML bootst posteriorrap/BI posterior probability. probabi The MLlity. bootstrap The ML <70% bootstrap <70%or BI posterioror BI posterior probability probability <0.90 is <0.90 not shown. is not Newlyshown. sequenced Newly sequenced species are species in bold. are The in GenBank bold. The Gen- Bankaccession accession numbers numbers of the 28S of the rDNA 28S sequences rDNA sequences were listed were next listed to the speciesnext to names. the species names. 4. Discussion 4. DiscussionThe DNA barcoding analysis of mtMutS, COI and 28S rDNA is considered as one of the firstThe steps DNA in barcoding an integrative analysis identification of mtMutS, of octocorals COI and [19, 4128S,42 rDNA]. In the is present considered analysis, as one of theCOI first exhibited steps in much an integrative less variation identification than the other of two octocorals loci, and [19,41,42] no genetic. variabilityIn the present was analy- sis,observed COI exhibited among the much three less new variation species and thanI. splendensthe otherWatling, two loci, 2007 and (Table no genetic5). Conse- variability quently, COI is less informative as a species-specific marker for octocorals. For the mtMutS, there is no barcoding gap for delimitating Iridogorgia species due to no genetic variability observed between I. densispiralis sp. nov. and I. verrucosa sp. nov., and between I. fontinalis Watling, 2007 and I. magnispiralis Watling, 2007, indicating its limited usefulness for the species delimitation (Table4). In contrast, the 28S rDNA showed a higher level of genetic

variation (e.g., interspecific distances 0.32–6.08% with average value of 3.10% vs. 0–1.33% at mtMutS and 0–0.39% at COI). A threshold of 0.5% can delimit all available Iridogorgia species except for I. splendens KX890215 (the distance between it and I. densispiralis sp. nov. is 0.32%). Like the case of 28S rDNA, the concatenated region mtMutS-28S can separate the present five species with a commonly used threshold of 0.3% (Table3). Nonetheless, only seven Iridogorgia sequences of 28S rDNA are available, more data including both conspecific and congeneric sequences are needed to confirm 28S rDNA and mtMutS-28S as effective barcodes for the genus Iridogorgia. The genus Rhodaniridogorgia was established by Watling [4] to contain the species Rhodaniridogorgia fragilis Walting, 2007 and R. superba (Nutting, 1908) transferred from Iridogorgia. It has a similar external shape and sclerites with Iridogorgia and is distinct mainly by the axis shape (wavy vs. coiled; [4]). In the mtMutS-COI trees, Rhodaniridogorgia fragilis nested into the Iridogorgia clade, and showed a close relationship with I. magnispiralis and I. fontinalis with low to moderate support, and the genetic distance data do not support the separation of R. fragilis from Iridogorgia (Tables4 and5; Figure 14). Furthermore, the wavy shape of the axis can be seen as a specific coiled spiral, when the helical diameter is narrow. Therefore, based on the morphological and phylogenetic data, we suggest a new combination Iridogorgia fragilis (Watling, 2007) comb. nov., resurrect I. superba Nutting, 1908 and eliminate the genus Rhodaniridogorgia. Here, we slightly update the diagnosed Iridogorgia to include the fragilis-like species: Chrysogorgiids with main axis monopodial, wavy or coiled spiraling upward, and undivided branches emanating from one side of the axis. Polyps uniserially arranged, when sexually mature with base expanded along upper side of branches. Sclerites rods, spindles, or scales, sometimes branched or with Biology 2021, 10, 588 24 of 27

coarse sculptures. Branches coenenchyme with sclerites oriented along branches or without sclerites between polyps. The present molecular phylogenetic analysis supported the assignment of the new species to the genus Iridogorgia. However, the mitochondrial marker MutS-COI could not resolve the deeply divergent relationships among the available Iridogorgia species except I. densispicula Xu, Zhan, Li and Xu, 2020 (Figure 14). In contrast, 28S rDNA showed better resolution for the Iridogorgia phylogeny. In the 28S rDNA trees, I. densispiralis sp. nov. showed close relationships with I. splendens and I. verrucosa sp. nov. This is consistent with their morphological characters that all these species have a relatively short colony and close helical turn in adults with branches producing nearly from the bottom to top (Figures3B,5B and 15) . Iridogorgia flexilis sp. nov. formed a sister clade with I. magnispiralis, which is also consistent with the morphological data that all have a high colony and loosely helical turn with branches producing on the upper part of the colony (Figures1B,7C and 15). Now there are twelve species in the genus Iridogorgia, for their distributions see the Figure 16. Among these, six species were reported from the Western Pacific, including I. flexilis sp. nov., I. verrucosa sp. nov., I. densispiralis sp. nov., I. magnispiralis, I. densispicula and I. squarrosa Xu, Zhan, Li and Xu, 2020. The data indicate a high diversity of Iridogorgia Biology 2021, 10, x FOR PEER REVIEW 24 of 27 in Western Pacific and provide a potential reference for vulnerable marine ecosystems and seamount conservation.

FigureFigure 16. 16.TheThe distributions distributions of ofthe the species species of of IridogorgiaIridogorgia, ,based based on on the the data ofof referencesreferences [ 4[4–8,10–14,39,40,43]–8,10–14,39,40,43]. . In order to distinguish the Iridogorgia species better, a dichotomous key based on In order to distinguish the Iridogorgia species better, a dichotomous key based on mor- morphological features is given. Due to the incomplete holotype and vague original phologicaldescription, featuresI. bella isis given. temporarily Due to not the considered incomplete here. holotype and vague original descrip- tion, I. bella is temporarily not considered here. 1. Colony with a wavy axis……………………………………………………………………..2 – Colony with a coiled axis ……………………………………………………….…………....3 2. Sclerites with a wide range of shapes, including spindles and scales……….…..I. fragilis – Sclerites uniform in size and shape, including slender smooth rods……………I. superba 3. Colony with loose helix in adult, each helical turn ≥12 cm high…………...... ….…4 – Colony with close helix in adult, each helical turn ≤7 cm high…………….…...…...….. 7 4. Scales present in the polyp body wall…………………………………………...….….……5 –Scales absent in the polyp body wall………………………..…..……….……I. magnispiralis 5. Sclerites in the polyp body wall usually without large tuberculate warts; small lumpy sclerites absent…………………………………………………………………...………….……6 – Sclerites in the polyp body wall usually sculptured by numerous large tuberculate warts; small lumpy sclerites often present…………………………..……………..I. squarrosa 6. Sclerites in the polyp body wall relatively slender and thin, surface coarse and/or rug- ged……………………………………..……...……………………………….…... I. densispicula – Sclerites in the polyp body wall stout and thick, surface relatively smooth……………………………………………………………………………....….. I. flexilis sp. nov. 7. Sclerites rare to absent in the inter-polyps coenenchyme…………………………………8 – Sclerites sparse to numerous in the inter-polyps coenenchyme……………………..……9 8. Scales present in polyps…………….....………………………..……...…………I. splendens – Scales absent in polyps……………….....………………………………...….…. I. pourtalesii 9. Branches producing on the top of colony (umbrella-shaped) ……...... …..…I. fontinalis – Branches producing from nearly bottom to top or the upper half of colony (tree-shaped) …………………………………………………………………………………………..10 10. Rods present in coenenchyme…………………………………… I. densispiralis sp. nov. – Rods absent in coenenchyme………………………………….....…….. I. verrucosa sp. nov.

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Figure 16. The distributions of the species of Iridogorgia, based on the data of references [4–8,10–14,39,40,43].

Biology 2021, 10, 588 In order to distinguish the Iridogorgia species better, a dichotomous key based on 25mor- of 27 phological features is given. Due to the incomplete holotype and vague original descrip- tion, I. bella is temporarily not considered here.

1. Colony with a wavy axis……………………………………………………………………..2 – Colony with a coiled axis ……………………………………………………….…………....3 2. Sclerites with a wide range of shapes, including spindles and scales……….…..I. fragilis – Sclerites uniform in size and shape, including slender smooth rods……………I. superba 3. Colony with loose helix in adult, each helical turn ≥12 cm high…………...... ….…4 – Colony with close helix in adult, each helical turn ≤7 cm high…………….…...…...…..…7 4. Scales present in the polyp body wall…………………………………………...….….……5 –Scales absent in the polyp body wall………………………..…..……….……I. magnispiralis 5. Sclerites in the polyp body wall usually without large tuberculate warts; small lumpy sclerites absent…………………………………………………………………...………….……6 – Sclerites in the polyp body wall usually sculptured by numerous large tuberculate warts; small lumpy sclerites often present…………………………..……………..I. squarrosa 6. Sclerites in the polyp body wall relatively slender and thin, surface coarse and/or rug- ged……………………………………..……...……………………………….…... I. densispicula – Sclerites in the polyp body wall stout and thick, surface relatively smooth……………………………………………………………………………....….. I. flexilis sp. nov. 7. Sclerites rare to absent in the inter-polyps coenenchyme…………………………………8 – Sclerites sparse to numerous in the inter-polyps coenenchyme……………………..……9 8. Scales present in polyps…………….....………………………..……...…………I. splendens – Scales absent in polyps……………….....………………………………...….…. I. pourtalesii 9. Branches producing on the top of colony (umbrella-shaped) ……...... …..…I. fontinalis – Branches producing from nearly bottom to top or the upper half of colony (tree-shaped) …………………………………………………………………………………………..………10 10. Rods present in coenenchyme…………………………………… I. densispiralis sp. nov. – Rods absent in coenenchyme………………………………….....…….. I. verrucosa sp. nov.

5. Conclusions Based on an integrated morphological-molecular approach, eight sampled specimens of Iridogorgia from seamounts in the tropical Western Pacific are identified as three new species I. flexilis sp. nov., I. densispiralis sp. nov. and I. verrucosa sp. nov., and two known species I. magnispiralis Watling, 2007 and I. densispicula Xu, Zhan, Li and Xu, 2020. Phyloge- netic analysis based on the nuclear 28S rDNA, indicated that I. densispiralis sp. nov. showed close relationships with I. splendens Watling, 2007 and I. verrucosa sp. nov., and I. flexilis sp. nov. formed a sister clade with I. magnispiralis. The genetic analysis demonstrates that neither the mtMutS nor COI barcodes could resolve species boundaries adequately, while 28S rDNA showed potential application in DNA barcoding and phylogenetic reconstruc- tion for this genus. In addition, based on morphological characters and the mitochondrial gene data, we propose to eliminate the genus Rhodaniridogorgia by establishing a new combination Iridogorgia fragilis (Watling, 2007) comb. nov. and resurrecting I. superba Nutting, 1908.

Author Contributions: The research was conceived and designed by K.X., Y.X. and Z.Z. Sampling conducted by K.X. Morphology was studied by Y.X. Molecular analysis was performed by Z.Z. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Key Program of National Natural Science Foundation of China (No. 41930533), the Science & Technology Basic Resources Investigation Program of China (2017FY100804), the Strategic Priority Research Program of Chinese Academy of Sciences (XDB42000000) and the Senior User Project of RV KEXUE (KEXUE2018G27, E12414101Q). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The specimens described in this study are available at the Marine Biological Museum of Chinese Academy of Sciences (MBMCAS) at Institute of Oceanology, Qingdao, Biology 2021, 10, 588 26 of 27

China. Voucher IDs: Iridogorgia flexilis sp. nov.: MBM286453; I. densispiralis sp. nov.: MBM286454; I. verrucosa sp. nov.: MBM286455; I. magnispiralis Watling, 2007: MBM286450, MBM286451; I. den- sispicula Xu, Zhan, Li & Xu, 2020: MBM286446, MBM286447, MBM286448. The mtMuts sequences that support the findings of this study have been deposited in NCBI GenBank with the accession codes MW841031 (I. flexilis sp. nov.), MW841033 (I. densispiralis sp. nov.), MW841034 (I. verrucosa sp. nov.) and MW841032 (I. magnispiralis). The COI sequences that support the findings of this study have been deposited in NCBI GenBank with the accession codes MW841035 (I. flexilis sp. nov.), MW841036 (I. densispiralis sp. nov.), MW841038 (I. verrucosa sp. nov.), MW841039 (I. mag- nispiralis) and MW841037 (I. densispicula). The 28S sequences that support the findings of this study have been deposited in NCBI GenBank with the accession codes MW841041 (I. flexilis sp. nov.), MW841043 (I. densispiralis sp. nov.), MW841044 (I. verrucosa sp. nov.), MW841042 (I. magnispiralis) and MW841040 (I. densispicula). The new species registration of Iridogorgia flexilis sp. nov., I. densispiralis sp. nov. and I. verrucosa sp. nov. in Zoobank with LSID: urn:lsid:zoobank.org:act:6BD2C9FC-9CD8- 4604-971D-B2EC0BB108B8, urn:lsid:zoobank.org:act:026E2F14-7A66-4558-9D65-B7E3CC0EC8D0 and urn:lsid:zoobank.org:act:E388B7B2-936E-485C-AB38-E7F95DD4F0A4, respectively. The publication LSID: urn:lsid:zoobank.org:pub:273D89B3-0E5C-42C7-9EF7-4526330B92B1. Acknowledgments: We thank the assistance of the crew of R/V KeXue and ROV FaXian for sample collection. Special thanks go to Shaoqing Wang for taking the photos on board. Conflicts of Interest: The authors declare no conflict of interest.

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