Molecular phylogeny and morphological comparison of the deep-sea genus Alloptilella Li, Zhan & Xu, 2021 (Octocorallia, Pennatulacea)

ORIGINAL PAPER Molecular phylogeny and morphological comparison of the deep-sea genus Alloptilella Li, Zhan & Xu, 2021 (Octocorallia, Pennatulacea) Pablo J. López-González 1 Received: 13 December 2021 /Revised: 18 January 2022 /Accepted: 19 January 2022 #The Author(s) 2022 Abstract A previously described species and a new one belonging to the recently described sea pen genus Alloptilella Li, Zhan & Xu, 2021, are here described and illustrated based on a morphological and molecular study of materials collected in the Tasman Sea (SW Pacific) and at Puerto Rico (Caribbean Sea), respectively. The species, Alloptilella moseleyi comb. nov. (Kölliker, 1880)and Alloptilella williamsi sp. nov., are in overall agreement with the generic diagnosis of Alloptilella., based on the type species, Alloptilella splendida Li, Zhan & Xu, 2021. A single relatively large colony (55 to 95 cm in total length) is known for each of the three Alloptilella species. The transferred and the new species differ from the type species in having an opposite, rather than alternate, placement of polyp leaves along the rachis, colouration of autozooids, and mesozooids (in the case of A. moseleyi comb. nov.), and spicular features (e.g. maximum sizes in different parts of the colony, presence/absence of tentacular sclerites). Alloptilella williamsi sp. nov. is the first species of the genus recorded so far from the Atlantic Ocean, all other described species being western Pacific. A molecular comparison based on a set of concatenated sequences of four markers (three mitochondrial genes (mtMutS,ND2,andCOI) and a nuclear segment (28S)) relates the species here studied to the published sequences of Alloptilella splendida, within the named Clade II of previous general phylogenetic studies on the octocoral Order Pennatulacea. Alloptilella is a monophyletic grouping, sister group of the genus Scytalium Herklots, 1858. The available molecular information of the genus Ptilella Gray, 1870, is reinforced with sequences (mtMutS,ND2 and 28S) from specimens of Ptilella inflata (Kükenthal, 1910) collected off the Namibian coast (SE Atlantic). Keywords Biodiversity .Coral .Sea pen .Morphology .Molecular analyses .Integrated approach Introduction Recent molecular studies in pennatulacean octocorals pointed out the polyphyly of the genus Pennatula Linnaeus, 1758 (Dolan et al. 2013; Kushida and Reimer 2018; García- Cárdenas et al. 2020). Two additional contributions proposed nomenclatural acts to resolve that situation: (1) the sequencing of certain North Eastern Atlantic specimens identified as Pennatula grandis Ehrenberg, 1834, resulted in the restoration of the old synonymized genus, Ptilella Gray, 1870 (García- Cárdenas et al. 2019); and (2) the existence of a divergent lineage from the genus Pennatula (sequences of the type species Pennatula phosphorea Linnaeus, 1758,anditsclose relatives) was corroborated with the description of a new genus based on North Western Pacific material, Alloptilella Li, Zhan & Xu, 2021 (Li et al. 2021). In both cases, molecular comparisons helped segregate morphological features previously considered within the morphological variability of the genus Pennatula (Kükenthal 1915;Williams1995). The morphological description and molecular characterization of Alloptilella splendida Li, Zhan & Xu, 2021, force the morphological and molecular revision of controversial material that was preliminary identified as Pennatula sp. (GenBank accession numbers DQ302870 for mtMutS and DQ302943 for ND2) from the Tasman Sea (SW Pacific) deposited at the Northern Territory Museum (Darwin, NT, Australia). This latter material is closely allied (from a molecular point of view) to Alloptilella, and was the basis of different discussions concerning the polyphyly of the genus Pennatula. This article is registered in ZooBank under http://zoobank.org/ 1A5DEA8C-29C1-49DA-AB42-6F2F07AA16FD Communicated by B. W. Hoeksema *Pablo J. López-González [email protected] 1 Biodiversidad y Ecología Acuática. Departamento de Zoología, Facultad de Biología, Universidad de Sevilla, Seville, Spain https://doi.org/10.1007/s12526-022-01260-w / Published online: 3 August 2022 Marine Biodiversity (2022) 52: 41 An additional specimen with a similar morphology and colouration pattern of A. splendida was collected during a deep-sea expedition in the North Western Atlantic. This material was deposited at the United States National Museum (Smithsonian Institution, Washington, USA), and the preliminary morphological and molecular study also confirmed its relatedness to the genus Alloptilella. The goal of the present paper is the description of a previously discovered and a new species of the genus Alloptilella based on western South Pacific and western North Atlantic specimens, respectively. A morphological and phylogenetic comparison of this material with A. splendida was carried out. Material and methods The material described here was collected during two different cruises, one of them in the Tasman Sea (RV Tangaroa) and the second one in the Caribbean Sea (NOAA ship Okeanos Explorer). The specimen from the Caribbean Sea was collected by the ROV Deep Discoverer (NOAA). For comparative purposes, specimens of the morphologically similar genera Pennatula and Ptilella collected in different survey programmes from the Artic to the Antarctica were also examined: Subantarctic and Antarctic (EASIZ, CLIMANT, BIOROSS), North Eastern Atlantic-Arctic (BIOICE), North Eastern Atlantic (Scotia cruises, INDEMARES Chica), South Eastern Atlantic (BENGUELA VIII), and Mediterranean (INDEMARES Cap de Creus, INDEMARES Canal de Menorca, INDEMARES Alborán). The colonies were fixed on board in ethanol 95% for morphological examination and further molecular studies. Sclerites of different parts of the colonies were prepared for SEM study employing the standard methodology described by different authors (e.g. Bayer and Stefani 1988), and permanent mounts were made for light microscopy. Calyces, tentacles, and other selected fragments were prepared in semipermanent mounts using clove oil as mounting medium for corroboration of the presence of sclerites and examination of their arrangement in a MOTIC B3 light microscope (Li et al. 2020). Sclerites were mounted on stubs, coated with goldpalladium under a Leica ACE600, and observed with a Zeis EVO SEM at the General Research Services of Microscopy at the University of Seville. Colony and sclerite terminologies mainly follow Bayer et al. (1983) and Li et al. (2021). Total genomic DNA was extracted from the ethanol (EtOH)-preserved specimen using the E.Z.N.A. DNA kit (OmegaBiotech) following the manufacturer’s instructions. Three mitochondrial regions, mtMutS, a homologue of the bacterial DNA mismatch repair gene mutS (=msh1), the NADH-dehydrogenase subunits 2 (ND2) and the cytochrome c oxidase subunit I (COI), plus a nuclear region, the large subunit ribosomal RNA gene (28S) were sequenced. These four markers were concatenated, representing the largest multiloci segment used today in sea pen phylogenies, previous contributions used mtMutS+ND2 (Dolan et al. 2013;Kushida and Reimer 2018)ormtMutS+COI+28S (García-Cárdenas et al. 2020). The start of the mtMutS region was amplified using the primers ND42599F and MUT3458R (France and Hoover 2002; Sánchez et al. 2003). The start of the ND2 region was amplified using the primers 16S47F and ND2- 1418R (McFadden et al. 2004). The COI region was amplified using the primers COII8068F and COIOCTR (France and Hoover 2002; McFadden et al. 2004). The 28S nuclear ribosomal gene (28S rDNA) was amplified using the primers 28S- Far and 28S-Rar (McFadden and van Ofwegen 2013). Each PCR used 1 U of MyTaq Red DNA Polymerase (Bioline), 10 μMofeachprimer,approximately30ngofgenomic DNA, and was brought to a final volume of 25 μL with H 2 O for molecular biology (PanReac-AppliChem). MtMutS PCR was carried out using the following cycle profile: initial denaturation at 95 °C for 1 min, 35 cycles of denaturation at 95 °C for 15 s, annealing at 55 °C for 15 s, and extension at 72 °C for 10 s, and a final extension at 72 °C for 5 min. The ND2, COI, and 28S PCRs used the same cycle profile, but 51°C,58°C,and50°Casannealingtemperatures,respectively. PCR products were purified using ExoSAP–IT™ PCR Product Cleanup Reagent (ThermoFisher Scientific) following the manufacturer’s instructions, before strong amplifications were sent to Macrogen Spain for sequencing in both directions. All chromatograms were visualized and sequence pairs matched and edited using Sequencher v4.0. A preliminary Maximum Likelihood (ML) comparison based on mtMutS (ca. 400 pennatulacean sequences, not shown) placed the studied taxa here close to a controversial sequence initially attributed to Pennatula sp. (GenBank accession number DQ302870, Li et al. 2021, López-González 2021) and the recently proposed genus and species Alloptilella splendida (GB MZ198005), within an informally named Clade 2 (Dolan et al. 2013; Kushida and Reimer 2018). These initially identified main Clades were referenced using Roman numerals (as Clade II in this case) by García-Cárdenas et al. (2019,2020), López-González (2021), and López- González and Drewery (2022). Since that Pennatula sp. specimen is studied and identified here as Pennatula moseleyi Kölliker, 1880, it is now proposed to be ascribed to the genus Alloptilella (see below). This molecular placement is in agreement with morphological features observed in this material, and diagnosed by Li et al. (2021)forthegenus Alloptilella. Therefore, for the present study, we will only include sequences of Clade II in order to not repeat images and ideas already discussed earlier (Li et al. 2021; López-González 2021). The available molecular information concerning the genus Alloptilella covered only mtMutS and ND2 markers (see Dolan et al. 2013 and Kushida and Reimer 2018 as 41 Page 2 of 25 Marine Biodiversity (2022) 52: 41 Pennatula sp.2),butLietal.(2021) sequenced also COI and 28S when describing Alloptilella splendida. The set of new sequences obtained in this study and those homologous from GenBank (see Table 1) were aligned using MUSCLE, implemented in MEGA 6 (Tamura et al. 2013). The dataset included 43 mtMutS,35ND2,27COI,and16 28S pennatulacean sequences. According to previous cited molecular phylogenies, the basal resolution of the main sea pen clades is poor. Thus, sequences of two ellisellids from GenBank were selected as an out-group. In addition to the most variable mitochondrial marker (mtMutS), three multiloci datasets were analysed: mtMutS+ND2,mtMutS+ND2+COI, and mtMutS+ND2+COI+28S. All the aligned matrices had 45 sequences (43 pennatulaceans and two ellisellids), but varied in the total number of bases: 698 for mtMutS,1239for mtMutS+ND2, 2013 for mtMutS+ND2+COI, and 2846 for mtMutS+ND2+COI+28S. The phylogeny reconstruction was obtained by applying Maximum Likelihood and Bayesian inference (BI) methods. After alignment, pairwise genetic distances based on the Kimura 2-parameter (K2P) model of nucleotide substitution (Kimura 1980) were obtained in order to compare them with previous analyses at genus and family levels, following the comparisons of Pante and France (2010), Pante et al. (2012), and López-González (2020). The best nucleotide substitution model was selected using Modeltest implemented in MEGA6, according to Akaike Information Criterion (AIC) and hierarchical likelihood ratio test (hLRT) values. The Maximum Likelihood method was carried out in MEGA6 using the NNI (Nearest Neighbour Interchange) heuristic method and 1000 bootstrap replications. The selected nucleotide substitution model was GTR+G for all multiloci and mtMutS matrices. The Bayesian Inference was carried out with MrBayes v3.1.2 (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003), using the substitution model GTR+G (lset nst=6 rates=gamma) and 10 7 generations, and discarding 25% of the initial trees. According to Hillis and Bull (1993), for ML bootstraps, those values <70% should be considered as low, between 70 and 94% moderate, and ≥95% high. According to Alfaro et al. (2003), for Bayesian posterior probabilities, those values <0.95 should be considered as low and ≥0.95 as high. The stationarity of the chains and convergence of the two runs were monitored for each parameter by Tracer (v.1.7.1) (Rambaut et al. 2018), determining whether the effective sample size (ESS) of all parameters was larger than 200, as recommended. Results Subclass Octocorallia Haeckel, 1866 Order Pennatulacea Verrill, 1865 Family Pennatulidae Ehrenberg, 1834 Genus Alloptilella Li, Zhan & Xu, 2021 Diagnosis (slightly modified from Li et al. 2021:1792, modifications in bold) Colonies elongated and slender, pinnate. Axis circular in cross section, present throughout length of colony. Rachis bilaterally symmetrical, with a central dorsal track naked of zooids. Rachis-peduncle limit with a distinct thickening or swelling that forms an edged ring at the thickest point. Polyp leaves large and conspicuous, narrow to deltoid, opposite or alternately disposed and inserted obliquely, extending ventrally upward. Autozooids in a row along the ventral edge of polyp leaves. Anthocodiae retractile into permanent spiculiferous calyces alternatively placed (giving the false impression of being two series). Calyces tubular, with eight terminal teeth (sometimes of unequal development).Mesozooids in a ridge in continuation of the dorsal base of polyp leaves, and coordinated with siphonozooid patches, basally.Siphonozooids in welldefined patches (V-shaped to check mark-shaped) part on latero-dorsal side of rachis and part between polyp leaves, along the distal insertion of each polyp leaf, on rachis, not on polyp leaves surfaces. Sclerites as three-flanged needles on calyces, polyp leaves and rachis; inconspicuous three-flanged rods on peduncle and pharynx. Y-shaped, crossed and 5 or 6 radiates can be present in peduncle, minute bodies ~0.02 mm can also be present. Three-flanged needles on autozooid tentacles can be present or absent. Distribution and depth Our knowledge on the distribution of this genus is limited, due to its recent description, the scarce number of collected specimens, the few species currently known and its distant type localities. West Pacific (tropical (~9°N, Y3 seamount), western and central Tasman Sea) and West Atlantic (Puerto Rico, Caribbean Sea); 559 to ~1733 m depth (Kölliker 1880;Li et al. 2021; present account). Type species Alloptilella splendida Li, Zhan & Xu, 2021 (by monotypy). Nominal species Alloptilella splendida, Alloptilella moseleyi comb. nov., and Alloptilella williamsi sp. nov. Remarks All recent molecular studies failed to recover the monophyly of the family Pennatulidae (e.g. Dolan et al. 2013; Kushida and Reimer 2018; García-Cárdenas et al. 2020; Page 3 of 25 41Marine Biodiversity (2022) 52: 41 Table 1 Octocoral species involved in the molecular comparisons carried out in this paper. Specimens and sequences in bold have been obtained for this study Species name in the tree (specimen code) (or species code: 1, 2) Catalog Nos. (or additional information) Geographic area / provenance mtMutS (=msh1) ND2 COI 28S Acanthoptilum gracile (1) 34212-029 NWFSC-west coast JN866529 - KF874188 - Acanthoptilum gracile (2) 34213-027 NWFSC-west coast JN866544 - KF874205 - Actinoptilum molle RMNH Coel. 40822 n.d. GQ342491 - GQ342414 JX203738 Alloptilella moseleyicomb. nov. NTM-C014415 BECA (G-3646) Tasman Sea-Australia DQ302870 OL692425 DQ302943 OL692430 -- Alloptilella splendida MBM286413 Tropical W Pacific MZ198005 - MZ198007 MZ198009 Alloptilella williamsisp. nov. USNM 1550625 BECA (G-3491) Puerto Rico –NW Atlantic OL692426 OL692431 OL689028 OL689085 Distichoptilum gracile NMS.Z.2019.25.2 Whittard Canyon MK919657 MK919657 MK919657 - Echinoptilum macintoshi (1) Isolate YK22 NW Pacific MK133373 MK133568 - - Echinoptilum macintoshi (2) Isolate YK23 NW Pacific MK133374 MK133569 - - Gilibelemnon octodentatum BECA (OPEN-452) (G-81) Seymour Island -Antarctica MK603841 MW863001 MK603855 MK603851 Pennatula aculeata (1) NMS.Z.2019.25.7 Whittard Canyon MK919663 MK919663 MK919663 - Pennatula aculeata (2) n.d. Bay of Biscay MK919664 MK919664 MK919664 - Pennatula phosphorea (1) BECA (OPEN-453) (G-88) Sea of the Hebrides -NE Atlantic MK603848 MW863002 MK603858 MK882492 Pennatula phosphorea (2) BECA OPEN-454 (G-199) Gulf of Cadiz -NE Atlantic MK603850 MW863003 MK603861 MK882491 Pennatula sp. 1 BECA (OPEN-152) (G-122) Ross Sea, Antarctica MK603849 MW863004 MK603859 MK882493 Protoptilum carpenteri NMS.Z.2019.25.10 Whittard Canyon MK919667 MK919667 MK919667 - Protoptilum sp. (sp. 1) USNM 94465 Pacific Ocean, Hawaii DQ297431 EU293804 DQ297450 - - Protoptilum sp. (sp. 2) NHM 2010-20 Eastern Pacific - Monterrey KF313844 KF313818 - - Ptilella grandis (1) BECA (OPEN-143) (G-92) South Iceland -NE Atlantic MK603844 MW863005 MK603860 MK603854 Ptilella grandis (2) NMS.Z.2019.2.6 BECA (G-69) Hebridean Shelf -NE Atlantic MK603843 MW863006 MK882496 MK882494 Ptilella grandis (3) MZB 2018-0759 BECA (G-138) Rockall Bank -NE Atlantic MW862998 MW863007 MW858343 MW862995 Ptilella grayi (1) BECA (OPEN-340) (G-2591) Rockall Bank -NE Atlantic MW862999 MW863008 MW858344 MW862996 Ptilella grayi (2) NMS.Z.2019.2.2 BECA (G-20) Rockall Bank -NE Atlantic MK603846 MW863009 MK603856 MK603853 Ptilella grayi (3) NMS.Z.2019.2.1 BECA (G-140) Rockall Bank -NE Atlantic MK603847 MW863010 MK882497 MK882495 Ptilella inflata(1) OPEN-456 (G-124) Namibia -SE Atlantic OL692427 OL692432 - - Ptilella inflata(2) MZB 2016-0099 BECA (OPEN-651) (G-3691) Namibia -SE Atlantic OL692428 OL692433 - OL689086 Ptilella inflata(3) MZB 2016-0105 BECA (OPEN-653) (G-3693) Namibia -SE Atlantic OL692429 OL692434 - - Ptilella cf. inflata * NMS.Z.2019.25.8 Whittard Canyon MK919666 MK919666 MK919666 - Ptilosarcus gurneyi (1) 34213-020 NWFSC-west coast JN866540 - KF874201 - Ptilosarcus gurneyi (2) 34210-009 NWFSC-west coast JN866521 - KF874180 - Renilla muelleri n.d. n.d. JX023273 JX023273 JX023273 - Renilla sp. CSM-2010-UF4000 E Pacific, Gulf of Panama GQ342526 - GQ342455 - Scytalium herklotsi USNM 1550636 Puerto Rico -NW Atlantic MW863000 MW863011 MW858345 MW862997 Scytalium martensi (1) Isolate YK03 NW Pacific MK133361 MK133556 - - Scytalium martensi (2) Isolate YK24 NW Pacific MK133375 MK133570 - - Scytalium sp.1 (1) Isolate YK04 NW Pacific MK133362 MK133557 - - Scytalium sp.1 (2) Isolate YK06 NW Pacific MK133363 MK133558 - - Scytalium sp.2 (1) Isolate YK02 NW Pacific MK133360 MK133555 - - Scytalium sp.2 (2) Isolate YK91 NW Pacific MK133430 MK133625 - - Scytalium veneris MBM286417 Tropical W Pacific MZ198006 - MZ198008 MZ198010 Stachyptilum dofleini (1) Isolate YK51 NW Pacific MK133396 MK133591 - - Stachyptilum dofleini (2) Isolate YK52 NW Pacific MK133397 MK133592 - - Stylatula elongata n.d. n.d. JX023275 JX023275 JX023275 - OUT-GROUP Viminella sp. RMNH Coel.40032 CSM-2012 n.d. JX203794 - JX203852 JX203703 Junceella fragilis n.d. Taiwan KJ541509 KJ541509 KJ541509 AF263355 41 Page 4 of 25 Marine Biodiversity (2022) 52: 41 Fig. 1 Alloptilella moseleyi comb.nov. (NTM-C014415); a Whole colony, specimen fragmented in four parts; bdetail of lowermost part of rachis, latero-dorsal, showing dorsal naked track (ndt)denselyfilled with red sclerites, polyp leaves (pl) progressively decreasing in size, mesozooids (msz) in distinct ridges formed by two lines proximally, but only one distally; c detail of a single polyp leaf showing the relatively narrow lateral sides (pll), and alternate orientation of autozooids; dlateral view of polyp leaves insertion showing lateral view of polyp leaf (pll), mesozooid ridge (msr), siphonozooid fields (sph), underdeveloped autozooid calyces at the ventralmost polyp leaf edge (udau) Note that all GenBank sequences are considered here with the names as they appear in GenBank and their original publications (including numbers or letters). Annotations between parentheses (1, 2, …sp.1, sp.2) after the species name are included by the present author just to identify different specimens of a given species or different species under the same denomination in GenBank. Abbreviations: BECA, Biodiversidad y Ecología Acuática (Seville, Spain); CSM, Collection of Catherine S. McFadden; MBM, Marine Biological Museum of Chinese Academy of Sciences (Qingdao, China); MBM, Marine Biological Museum of Chinese Academy of Sciences at Qingdao, China; MZB, Museu de Zoologia in Barcelona (Spain); NWFSC, Northwest Fisheries Science Center (Seattle, USA); NHM, Natural History Museum in London; NMS, National Museum Scotland Smithsonian (Scotland, UK); NTM, Museum and Art Gallery of the Northern Territory (Darwin City, Australia); RMNH, Rijksmuseum van Natuurlijke Historie (Leiden, Netherlands); USNM, Smithsonian Institution, United States National Museum; YK, collection of Y. Kushida; - no data *This specimen is probably conspecific with Pt. grandis (see “Discussion”,Fig.12, and SMFigs.1and 2). Page 5 of 25 41Marine Biodiversity (2022) 52: 41 Fig. 2 Alloptilella moseleyi comb.nov. (NTM-C014415); aand bDetail of ventral track showing polyp leaves insertion upwardly directed, underdeveloped autozooid calyces (udau) and single line of siphonozooids between two consecutive polyp leaves; crachis, lateral view, showing the “check mark”shape of siphonozooids fields (msz), on the right to the dorsal naked track, and on the left those rows placed between consecutive polyp leaves; ddetail of an autozooid calyx with teeth (tth) and autozooid tentacles (te) with a narrow band of red sclerites (tsc); eterminal part of an autozooid cut longitudinally to observe the white tentacles (te) with tentacular sclerites (tsc), calycular teeth (dp), and pharynx with red sclerites (phs); fdetail of sclerites from peduncle (light microscopy) showing minute bodies (mb)androds(rd)(seealso Figure 5) 41 Page 6 of 25 Marine Biodiversity (2022) 52: 41 López-González 2021;Lietal.2021). The placement of the genus Alloptilella in the family Pennatulidae is here considered for practical reasons, to be in agreement with previous literature, and gross morphologies attributed to Pennatulalike forms, there being a need for a more global revision of the family delimitations of the pennatulacean genera reunited in the informally named Clade II. Alloptilella moseleyi comb.nov. (Kölliker,1880) http://zoobank.org/9D34019B-56A6-4A5F-9130-CBFC43A29772 Pennatula moseleyi Kölliker 1880:6-7, Plt. 2 Figs. 8,9. Kükenthal 1915:83(in text). Hickson 1916: 183 (in text). García-Cárdenas et al. 2019:266(in text). Examined material NTM C014415, Lord Howe Plateau, Tasman Sea, 34° 12.18′S 162° 41.18′E, R.V Tangaroa, 748–772 m depth, 26 May 2003. Morphological description Colony pinnate, elongated and erect, approximately 952 mm in length in preserved state, fragmented in four parts (Fig. 1a). Rachis bilaterally symmetrical, 854 mm in length (89.7% of overall length) and 9 mm in width (measured at mid-length of rachis, without polyp leaves), with a moderate thickening at rachis-peduncle limit (10 mm in width), more or less distinct in preserved state (Fig. 1a). Peduncle 98 mm in length (10.3% Fig. 3 Alloptilella moseleyi comb.nov. (NTM-C014415). SEM photographs of sclerites; a Calyx, basal parts; bcalyx, upper part and teeth; ctentacle; d pharynx; epolyp leaves; f tentacular sclerites, magnified; g pharyngeal sclerites, magnified Page 7 of 25 41Marine Biodiversity (2022) 52: 41 of total length). Rachis with a distinctive naked central dorsal track and a hidden ventral track occupied by insertions of polyp leaves. Rachis with approximately 126 pairs of nearly opposite polyp leaves (the fragmented and contracted states of the type make a more accurate count difficult), with the lowest pairs gradually decreasing in size (Fig. 1b), averaging 18 pairs per 100 mm of length (at midrachis length). Polyp leaves inserted obliquely, extending ventrally upward. Fully developed polyp leaves narrow in lateral view, maximum length 40 mm, and dorsal edge slightly shorter than ventral edge, maximum width 6 mm (Fig. 1b, c). Axis present throughout colony, circular in cross section, 2.7 mm in diameter measured 12 cm above rachis-peduncle limit (I preferred not to further fragment this specimen looking for the value at the rachis-peduncle limit zone). Autozooids with calyces, up to 28 in number in the largest polyp leaves, arranged in one row along the ventral edge of each developed polyp leaf (indicated by arrangement of gastrovascular cavities in transversal sections of the polyp leaves), but oriented alternately, appearing falsely to be placed in two rows (Fig. 1c). Anthocodiae retractile into permanent spiculiferous calyces. Calyces tubular, spiculate, armed with 8 strongly projecting teeth, up to 4 mm in length (including teeth) and 1.5 mm in maximum width at basis of teeth level (Figs. 1c and 2d, e), teeth up to 1.6 mm in length (usually around 1.1 mm). Four to six under-developed autozooids on the proximal ventralmost edge of each polyp leaf (Figs. 1d and Fig. 4 Alloptilella moseleyi comb. nov. (NTM-C014415). SEM photographs of sclerites; a Polyp leaves; bmesozooids; c siphonozooids; drachis, dorsal track at mid length; erachis, just above the rachis-peduncle limit 41 Page 8 of 25 Marine Biodiversity (2022) 52: 41 2a, b). Mesozooids distributed on the lateral side of rachis, in an elevated ridge of one line (two lines proximally) (Fig. 1b, c), along the rachis and proximal dorsal edge of polyp leaves, up to 14 in number, decreasing in size from polyp leaf to dorsal track, the last ones of similar size to siphonozooids, the largest one conical, ~1.4 mm in height and 0.8 mm in width, spiculiferous. Siphonozooids minute, 0.17–0.26 mm in diameter (average 0.22 mm, N=20), numerous, arranged in ~10 rows along the axillae of polyp leaves to the latero-dorsal sides of the rachis between polyp leaves, resembling a “check mark”where the long stroke is placed between consecutive polyp leaves (Figs. 1c, d, and 2c), appearing as a single line on the ventral side (Fig. 2b). Sclerites distributed in various parts of the colony. Densely placed in peduncle, autozooid calices, around mesozooids and siphonozooids openings, and dorsal naked tracks (Figs. 1b, c, and 2c), but widely spaced in lateral (actually they are distal and proximal) sides of polyp leaves (Fig. 2a). Along the aboral side of tentacular axis the sclerites are disposed in a narrow line of 2–3 sclerites (Fig. 2d). Overall, in the proximal part of the rachis, the spicules are more densely packed than in the distal part, and the mesozooid ridges are better defined (Fig. 1b, d). Sclerites from calyx body as three-flanged needles, up to 0.59 mm in length (Fig. 3a). Sclerites from calycular teeth as three-flanged needles, up to 0.97 mm in length (Fig. 3b). Sclerites from aboral sides of autozooid tentacle axis as three-flanged needles, up to 0.21 mm in length (Fig. 3c, e). Sclerites from autozooid’s pharynx as three-flanged needles, up to 0.06 mm in length (Fig. 3d, f). Sclerites from polyp Fig. 5 Alloptilella moseleyi comb.nov. (NTM-C014415). SEM photographs of sclerites; a Peduncle, just below the rachispeduncle limit; bpeduncle, midlength, notice minute bodies (*); c peduncle, lower end, notice minute bodies (*); dand edetail from two sclerites from c,athreeflanged rod with a minute body (*) and a 6-radiated twinning; f magnified minute bodies present in the lower part of peduncle, note also their three-flanged structure Page 9 of 25 41Marine Biodiversity (2022) 52: 41 contribution to our knowledge of pennatulacean diversity and worldwide distribution (Williams 2011). Geographical and depth distribution At present, Alloptilella williamsi sp. nov. is only known from the Caribbean Sea (NW Atlantic) from 559.10 m depth. Bootstrap (Bst) and Posterior Probability (PP) values (Bst) and Posterior Probability (PP) values Phylogenetic analyses In the mtMutS+ND2+COI+28S hypothesis (Fig. 12), the BI and ML topologies are identical. The multiloci sequences obtained for Allopitlella moseleyi comb.nov and A. williamsi sp. nov. were reunited in a distinct and well-supported clade (Bootstrap (Bst) 99%, Posterior Probability (PP) 1) with other multiloci sequences recently published as Alloptilella splendida, the type material of the type species of the genus Alloptilella.Alloptilella is then easily identifiable as a monophyletic genus, being the sister group of all sequences of another distinctly morphological and molecular genus, Scytalium Herklots, 1858.Alloptilella and Scytalium are in a well-supported clade (Bst 97%, PP 1) which is the sister group of a poorly supported Ptilella-Distichoptilum clade (Bst 29%, PP 0.51). The position of the genus Pennatula in this phylogenetic hypothesis is distinctly separated from Ptilella and Alloptilella, Ptilosarcus being the sister group of Pennatula,withalowto moderate support from the ML inference (Bst 68%), but a high support (PP 0.99) from the Bayesian inference. Different genera currently included in the families Renillidae (Renilla), Virgulariidae (Acanthoptilum,Actinoptilum), and Stachyptilidae (Gilibelemnon) are placed in between the Pennatula-Ptilosarcus and the Ptilella-Distichoptilum and Alloptilella-Scytalium subclades (see Fig. 12). Fig. 10 Alloptilella williamsi sp. nov. Holotype (USNM 1550625). SEM photographs of sclerites; a Peduncle, just below the rachispeduncle limit; bcross from a, magnified; cpeduncle, midlength; drods and cross from c, magnified 41 Page 16 of 25 Marine Biodiversity (2022) 52: 41 The topologies obtained using the BI and ML methods for the mtMutS,mtMutS+ND2,andmtMutS+ND2+COI hypotheses are almost identical (SMFigs. 1,2), and somewhat more resolved (but moderately supported) in the multiloci datasets. The three genera discussed here (Pennatula,Ptilella,and Alloptilella) are well supported in all BI trees (PP 1), while Bst values (ML trees) varied depending on the dataset used (98, 91, and 94% for Pennatula; 100, 99, and 100% for Ptilella; and 92, 99, and 96% for Alloptilella, for each of the above listed hypotheses, respectively). In the mtMutS tree (SMFig. 1, left), species distinction is not always observed for all Ptilella and Alloptilella species. The three Pennatula species used for this tree are clearly differentiated, while P. grandis and P. inflata share identical sequences, and only P. grayi is differentiated. Concerning Alloptilella species, A. moseleyi comb. nov. and A. williamsi sp. nov. share the same sequence, and then, only A. splendida is differentiated. As in the four markers hypothesis, Scytalium is the sister group of Alloptilella (PP 1, Bst. 96%). In the mtMutS+ND2 hypothesis (SMFig. 1, right), the topologies of the BI and ML trees are also very similar and are in general agreement with the mtMutS hypothesis. However, in this case, all the Ptilella and Alloptilella species are differentiated (more clearly visible in Ptilella, where at least three colonies per species are sequenced). Ptilella grandis and P. inflata are sister groups (PP 1, Bst 96%), and they are the sister group of P. grayi (PP 1, Bst 99%). A specimen identified as P. cf. inflata from eastern North Atlantic (GenBank MK919666, complete mitochondrial sequence) is here clearly aligned with P. grandis.Ptilella inflata is recognized as a southern West Indian species, also present in South Africa and Namibia (Kükenthal 1910; Williams 1995;López- González et al. 2001). The present sequenced colonies of P. inflata were collected off Namibian coasts, 418–439 m in depth (López-González et al. 2001). In this hypothesis, Alloptilella species are minimally differentiated from a molecular point of view (see “Discussion”) forming a polytomy. As in the mtMutS hyphothesis, Pennatula species are well defined. A phylogenetic hypothesis considering only mitochondrial markers (mtMutS+ND2+COI) have the same main topology, but is reinforced at species level by the additional differences in COI marker (SMFig. 2). Ptilella and Pennatula species remain well separated, while Alloptilella species also maintain the same polytomy observed in mtMutS and mtMutS+ND2 hypotheses. In a selected dataset considering only mtMutS,uncorrected p-distances indicate that the average genetic distance within Alloptilella species is 0.1% (having only the values of 0.0% or 0.2%). Alloptilella moseleyi comb. nov. is 0.0% and 0.2% distant from Alloptilella williamsi sp. nov. and A. splendida, respectively. Alloptilella williamsi sp. nov. is 0.2% distant from A. splendida. At the immediately higher taxonomic level (genus level) and now using the K2 nucleotide substitution model, Alloptilella is 3.3% (range 3.1–3.6%) distant from Ptilella, and 4.5% (range 4.3–4.7%) distant from Pennatula, while Pennatula is 3.6% (range 3.4–3.7%) distant from Ptilella. Finally, Alloptilella is 4.9% (range 3.7–5.4%) distant from Scytalium, its sister group. In order to provide additional comparison, the most common multiloci dataset previously analysed in pennatulacean phylogenetic papers (mtMutS+ND2 and mtMutS+COI+28S) hasalsobeenexploredusingtheK2nucleotidesubstitution model (see SMTables 1and 2). In the mtMutS+ND2 data matrix, the mean intraspecific genetic distances in Alloptilella,Ptilella,andPennatula are 0.08%, 0.33%, and 0.27% respectively, while Alloptilella is 2.22% distant from Ptilella and 3.61% distant from Pennatula,andPennatula is 2.96% distant from Ptilella (see SMTable 1for ranges). In the analysis of the mtMutS+COI+28S data matrix, the mean intraspecific genetic distances in Alloptilella,Ptilella,and Pennatula are also low at 0.02%, 0.39%, and 1.06%, respectively, while Alloptilella is 27.62% distant from Ptilella and 29.11% distant from Pennatula,andPennatula is 9.54% distant from Ptilella (see SMTable 2for ranges). A final analysis including the multiloci mtMutS+COI (SMTable 3)corroborates the idea that genetic distances at genus level are improved by the inclusion of the 28S segment, as overall genetic distances are similar and of the same order of magnitude as those obtained using the mtMutS+ND2 dataset. ` Artificial key to Alloptilella species (see also Table 2for a complete comparison of morphological characters) 1. Colony red in colour (spicules of distal part of calyces and mesozooids are also red in colour); with sclerites on the tentacular axis ………………….….….….….….….….….….….…………….….….…….………….Alloptilella moseleyi comb. nov. -Colony red and yellow in colour (spicules of distal part of calyces and mesozooids are yellow in colour); with or without sclerites on the tentacular axis .................................................................................................................................................... 2 2. Polyp leaves in an alternate disposition; with sclerites on the tentacular axis ….….….….…..........…... Alloptilella splendida -Polyp leaves in an opposite disposition; without sclerites on the tentacular axis …………….... Alloptilella williamsi sp. nov. Page 17 of 25 41Marine Biodiversity (2022) 52: 41 41 Page 18 of 25 Marine Biodiversity (2022) 52: 41 Discussion The sequences in GenBank identified as Pennatula sp. (DQ302870 for mtMutS and DQ302943 for ND2)belongto the same material here identified as Pennatula moseleyi Kölliker, 1880. According to the current knowledge, the assignation of this material to Kölliker’s species is reasonable, and is also the most conservative proposal. This species is transferred here to the genus Alloptilella as A. moseleyi comb. nov. Due to its controversial position in the published phylogenetic trees (e.g. Dolan et al. 2013; Kushida and Reimer 2018; García-Cárdenas et al. 2020; López-González 2021; Li et al. 2021), total DNA of that specimen was extracted and sequenced again for this paper; only the previously known segments (mtMutS and ND2) were successfully amplified and Fig. 11 In situ photographs of Alloptilella williamsi sp. nov. Holotype (USNM 1550625); aROV Deep Discoverer on the bottom during the captures of the type material of A. williamsi sp. nov.; bwhole extended colony (lasers are 10 cm apart), notice damage at the mid-rachis length; c detail of damage on the rachis; ddetail of the upper part of rachis, polyp leaves extended, notice the shape of siphonozooid fields, alternate orientation of autozooids, low density of sclerites on the polyp leaves’lateral surfaces, and poor development of mesozooid ridges, notice one or two mesozooids on the proximal dorsal edge of each polyp leaf (arrows); e detail of the expanded rachis-peduncle limit and lowest polyp leaves, notice the progressively decreasing size of polyp leaves, and the welldeveloped and yellow-coloured mesozooid ridges; fgeneral view of the distal part of rachis, showing the opposite disposition of polyp leaves; g precise moment of collection by the arm of the ROV Deep Discoverer. Photos by NOAA Office of Ocean Exploration and Research (see all photos associated with the registration code USNM 1550625 on the Web site “Search the Department of Invertebrate Zoology Collections” of the Smithsonian Institution, National Museum of Natural History (https://collections.nmnh.si.edu/search/iz/)) Fig. 12 Bayesian analysis showing the phylogenetic relationships of Alloptilella moseleyi comb. nov. and Alloptilella williamsi sp. nov. with Alloptilella splendida and other related genera and species of sea pens within Clade II. The present hypothesis is based on the concatenated set of sequences of four markers: mtMutS+ND2+ COI+28S. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Supporting values of nodes as Bootstrap (%)/Posterior Probability. See Table 2for complete list of species and GenBank accession numbers. *This specimen is probably conspecific with P. grandis (see “Discussion”, SMFigs.1and 2) Page 19 of 25 41Marine Biodiversity (2022) 52: 41 Table 2 Comparative table of Alloptilella species discussed in this paper. Alloptilella species are listed according to their date of description. Notes: n.d., no data; (1) Kölliker (1880:p.6);(2)Lietal. (2021: 1792); (3) this paper Characters / species A. moseleyi comb. nov. (1, 3) A. splendida (2) A. williamsi sp. nov. (3) Ventral track Naked, narrow, hidden occupied by insertions of polyp leaves Naked, narrow, hidden occupied by insertions of polyp leaves With a few siphonozooids from the line running between consecutive polyp leaves, easy to confuse with under-developed autozooids. Ventral track narrow, as a line, hidden occupied by insertions of polyp leaves Dorsal track Naked throughout length of rachis Naked throughout length of rachis Naked throughout length of rachis Maximum number of polyp-leaf pairs and distribution (colony size) 126 pairs of nearly opposite polyp leaves, averaging 18 pairs per 100 mm in length (952 mm) 65 pairs, inserted obliquely, alternately disposed, averaging 14.1 pairs per 100 mm in length (550 mm) 50 pairs of nearly opposite polyp leaves, averaging 8 pairs per 100 mm in length (620 mm) Distribution autozooids Arranged in one row along the ventral edge of each developed polyp leaf (arrangement of gastrovascular cavities in the polyp leaves), but oriented alternately, appearing to be placed in two rows Arranged in one row along the ventral edge of polyp leaves, sometimes crowdedly and alternately arranged so appear to be placed in two rows Arranged in one row along the ventral edge of each developed polyp leaf (arrangement of gastrovascular cavities in the polyp leaves), but oriented alternately, appearing to be placed in two rows Maximum number of autozooids per polyp leaf (colony size) 28 (952 mm) 26 (550 mm) 29 (620 mm) Maximum size of the autozooids (length × widthinmm) 4 × 1.5 4 × 2 4.1 × 1.7 Spiculate teeth in the autozooid’s calyces 8, often of irregular development up to 8 8, often of irregular development Development of polyp leaves (colony size) Narrow in lateral view, maximum length 40 mm, and dorsal edge slightly shorter than ventral edge, maximum width 6 mm (952 mm) Deltoid in lateral view, with the bases 18 mm in maximum length, the ventral edges curved with maximum length 23 mm, and dorsal edges slightly shorter than ventral edges (550 mm) Close to a right/obtuse triangle in lateral view, maximum length 50 mm, and dorsal edge slightly shorter than ventral edge maximum width 20 mm (620 mm) Bases of polyp leaves overlapping Yes Yes Yes Distribution of mesozooids On the dorsal side of rachis, in an elevated ridge of one or two lines, along the rachis and proximal dorsal edge of polyp leaves, up to 14 in number, decreasing in size from polyp leaf to dorsal track, the last ones of similar size to siphonozooids On the dorsal side of rachis near the dorsal base of polyp leaves On the dorsal side of rachis, in an elevated ridge of one or two lines, along the rachis and proximal dorsal edge of polyp leaves, up to 6 in number, decreasing in size from polyp leaf to dorsal track, the last ones of similar size to siphonozooids Distribution of siphonozooids Up to 0.26 mm in diameter, numerous, arranged in up to ~10 rows dorsally, running along the axillae of polyp leaves to the latero-dorsal sides of the rachis between polyp leaf insertions, resembling a “check mark”where the long stroke is placed between consecutive polyp leaves 0.2 mm in height and 0.3 mm in width, mostly conical with converging sclerites forming a pointed apex; arranged in 1–10 rows along the axillae of polyp leaves to the latero-dorsal sides of the rachis between polyp leaves Up to 0.3 mm in diameter, numerous, arranged in up to ~6–8 rows dorsally, rows along the axillae of polyp leaves to the latero-dorsal sides of the rachis between polyp leaf insertions, resembling a“check mark”. The line of siphonozooids running between consecutive polyp leaves can appear on the ventral track Cross section of axis Circular Circular Circular Sclerites along the aboral side of tentacular axis Three-flangedneedles,upto0.21mminlength Three-flanged needles, approximately up to 0.18 mm in length Absent Sclerites of the autozooids’calyces and teeth Calyces:three-flangedneedles,upto0.59mmin length. Teeth: three-flanged needles, up to Calyces: three-flanged needles, up to 0.73 mm in length. Teeth: three-flanged needles, up to Calyces: three-flanged needles, up to 0.8 mm in length. Teeth: three-flanged needles, up to 41 Page 20 of 25 Marine Biodiversity (2022) 52: 41 Table 2 (continued) Characters / species A. moseleyi comb. nov. (1, 3) A. splendida (2) A. williamsi sp. nov. (3) 0.97 mm in length 1.2 mm in length 1.2 mm in length Sclerites of autozooids’pharynx Three-flanged needles, up to 0.06 mm in length Small three-flanged needles, up to 0.054 mm in length Three-flangedneedles,upto0.06mminlength Sclerites of polyp leaves Three-flanged needles, up to 1.1 mm in length Three-flangedneedles,upto0.67mminlength Three-flangedneedles,upto0.75mminlength Sclerites of mesozooids Three-flanged needles, up to 1.0 mm in length Approximately up to 0.5 mm in length Three-flanged needles,upto0.95mminlength Sclerites of siphonozooid areas Three-flanged needles, up to 0.9 mm in length Three-flanged needles, up to 0.64 mm in length Three-flanged needles, upto0.57mminlength Sclerites of mid-rachis (dorsal tracks) Three-flanged needles, up to 0.38 mm mid-dorsal track n.d. Three-flanged needles,upto0.13mmmid-dorsal track Sclerites of lowermost part of rachis, above rachis-peduncle limit Three-flangedneedles,upto0.37mmatthelowest part n.d. Three-flanged needles, up to 0.14 mm at the lowest part, more robust (wider) than upper part (see Fig. 9D and E comparatively) Sclerites of peduncle Short three-flanged rods up to 0.2 mm in length (slightly shorter at mid-length), some crosses up to 0.15 mm in length, more abundant at the mid and basal parts (where they can be 5 or 6 radiated), and minute bodies ~0.02 mm in length, also more abundant at basal end Short three-flanged rods, up to 0.08 mm in length Short three-flanged rods (sometimes pointed, flanges mainly noticeable at the ends, often simply as slits) up to 0.06 mm in length (of similar size at mid length), Y-shaped sclerites, crosses up to 0.055 mm in length, are present, and become more abundant and even, and 5 or 6-radiated at the middle parts. Minute bodies ~0.02 mm in length, also more abundant at basal end, where rods become rare Colour of sclerites All rachis, polyp leaves, and polyp sclerites are red. Peduncle with light red to transparent sclerites Autozooids with needles from calycular teeth and upper part calyces yellow. Needles from tentacles, basal part of calyces, polyp leaves, siphonozooids, and rachis reddish. Mesozooids with yellowish sclerites. Rods from peduncle translucent to whitish Autozooids with needles from calycular teeth and upper part of calyces yellow. Needles from basal part calyces, polyp leaves, siphonozooids, and rachis reddish. Mesozooids with yellowish sclerites. Rods and crosses from peduncle reddish to translucent Development of lowermost polyp leaves Decrease gradually in size, without any distinct zone of under-developed polyp leaves Lowest seven pairs rudimentary Decrease gradually in size, without any distinct zone of under-developed polyp leaves Rachis-peduncle limit With a moderate thickening With a distinct swelling that forms an edged ring at the widest point With a moderate thickening (single available colony damaged at this part) Geographical and bathymetric distribution Tasman Sea (off Sydney and central areas), 748 to ~1737 m in depth Seamount Y3 near the Yap Trench in the Tropical Western Pacific, 879 m in depth Puerto Rico, Caribbean Sea (NW Atlantic), 559 m in depth Page 21 of 25 41Marine Biodiversity (2022) 52: 41 sequenced. Both obtained sequences were in agreement with the previously published ones. Alloptilella is identifiable as a monophyletic genus, being the sister group of all sequences of another distinct morphological and molecular genus recently revisited, the genus Scytalium (see Li et al. 2021; López-González 2021). The resurrection of the genus Ptilella (see García-Cárdenas et al. 2019) and the description of the genus Alloptilella resolve the polyphyletic situation of Pennatula observed in recent phylogenetic studies focused on the order Pennatulacea (e.g. Dolan et al. 2013; Kushida and Reimer 2018; García-Cárdenas et al. 2020). The realignment of Pennatula species in three different genera (based on molecular and morphological features) does not resolve the fact that the numerous named species of Pennatula are still in need of revision. Attending to the relatively high numbers of proposed species and the potential number of species recognizable as valid nowadays (Williams 1995,2011), this is a task to be carried out in the future with the help of all available taxonomic tools. Molecular information seems to be essential in that revision, or at least the re-description of the already supposedly wellknown species, such as Pennatula phosphorea Linnaeus, 1758,P. aculeata Danielssen, 1860,andP. rubra (Ellis, 1764), among others (see García-Cárdenas et al. 2019). The description of the genus Alloptilella was based on Alloptilella splendida, a species inhabiting seamounts in the Tropical Western Pacific at up to 879 m depth. The erection of anewPennatula-like genus from a molecular point of view was completely necessary. This species is morphologically and chromatically close to Pennatula naresi Kölliker, 1880 (see Kölliker 1880: 5, Plt. I, Figs, 1, 2). García-Cárdenas et al. (2019) suggested that the latter species could belong to the genus Ptilella, but more morphological and molecular studies were necessary. The original description of P. naresi included enough details on the distribution and colour of the sclerites in the autozooids, mesozooids (named ventral zooids), and siphonozooids (named lateral zooids). The gastrovascular cavities of autozooids are arranged in the polyp leaves in a single line, and the yellow calyces are oriented in alternate directions giving the impression of being arranged in two series. The mesozooids, armed with yellow sclerites, form a row of polyps decreasing in size “which begin at the ventral margin of the pinnule at a little distance from its attachment, run obliquely upon the sides of the rachis, and end with a longitudinal streak”. The siphonozooids occupy the space between mesozooid rows also running between consecutive polyp leaves. Kölliker (1880) stated that only autozooids and mesozooids (as ventral zooids) are armed with yellow sclerites, as A. splendida.Lietal.(2021) mainly differentiate A. splendida and P. naresi in the placement of mesozooids, restricted to the dorsal area in A. splendida and also occupying the proximalmost part of the polyp leaf edge dorsally. The taxonomic placement of P. naresi is controversial from a morphological point of view. The arrangements of autozooids, mesozooids, and siphonozooids agree with Alloptilella; hence, a complete morphological redescription of its type material is desirable. Identification by DNA sequencing of the three morphologically similar genera is well established, although molecular differentiations among the three Alloptilella species are low. The mtMutS sequences of the Caribbean (A. williamsi sp. nov.) and Tasman (A. moseleyi comb. nov.) species are identical, while A. splendida (Tropical Western Pacific) differs in a single base. The ND2 sequences of A. williamsi sp. nov. and A. moseleyi comb. nov. (not available for A. splendida)differ also in a single base. The COI sequences of A. splendida and A. williamsi sp. nov. differ in two bases (only 539 bases are known for A. splendida). Remarkably, the nuclear 28S differs in a single base between A. splendida (Tropical Western Pacific) and A. williamsi sp. nov. (Caribbean Sea) (only 677 bases are known for A. splendida), while no information is currently available for A. moseleyi comb. nov. A similar situation occurs among the three species of Ptilella (P. grandis,P. grayi, and P. inflata) (see Table 2, Fig. 12,SMFigs.1,2in this paper and García-Cárdenas et al. 2019). For this paper, two mitochondrial (mtMutS and ND2) segments and one nuclear (28S) segment from SW Atlantic specimens of Ptilella inflata have been sequenced. Ptilella grandis and P. inflata share an identical sequence of mtMutS,whileP. grayi shows differences in two bases. ND2 sequences of P. grandis differ in four bases with P. grayi,but only in two with P. inflata,whileP. grayi and P. inflata differ also in two bases. Two bases differentiate P. grandis and P. grayi COI sequences (not available for P. inflata). Surprisingly, differences in the nuclear 28S are also low, P. grayi differs in three bases with P. grandis, and in four bases with P. inflata, while P. grandis and P. inflata only differ in a single base. From a chromatic point of view, Alloptilella splendida and A. williamsi sp. nov. share the same distribution pattern of coloured sclerites. Autozooids and mesozooids have yellow sclerites, while the rest of the sclerites in the colony are red, being light red or translucent in the lower parts of the peduncle. In the case of A. moseleyi, sclerites in all parts of the rachis are red, having a similar colour pattern in the peduncular sclerites to its congeners. However, A. splendida and A. moseleyi have red three-flanged sclerites along the aboral side of the tentacles, while in A. williamsi sp. nov. tentacular sclerites are completely absent. From a morphological point of view (apart from the abovementioned chromatic differences), both western Pacific species (Allopilella moseleyi and A. splendida) have a similar appearance (see Table 2for a complete comparative overview). The polyp leaves seem to be placed in a more opposite than alternate arrangement in A. moseleyi.Polyp leaves seem to be narrowest and proportionally longer in 41 Page 22 of 25 Marine Biodiversity (2022) 52: 41 A. moseleyi than those in A. splendida. Differences in the number of polyp leaves (126 vs. 65 may be due to the different sizes of the available colonies), with A. moseleyi also having a slightly higher density of polyp leaves per 100 mm of rachis (18 vs. 14.1). Tentacular sclerites are slightly shorter in A. moseleyi (up to 0.18 vs. 0.21 mm), but sclerites from polyp leaves, mesozooids, siphonozooids, and peduncle are larger in A. moseleyi (Table 2). Finally, the lowest pairs of polyp leaves decrease gradually in size in A. moseleyi without any distinct zone of under-developed polyp leaves as occurs in A. splendida. The new proposed species, Alloptilella williamsi sp. nov., is from a chromatic point of view, close to the type species of the genus, A. splendida. However, the new species differs in an opposite disposition of polyp leaves, absence of sclerites in the tentacles, a reduced number of polyp leaves per 100 mm of rachis (8 vs. 14.1), larger polyp leaves (in preserved state), presence of 1–2 mesozooids on the proximal dorsal edge of polyp leaves, larger sclerites in mesozooids (0.95 vs. 0.5 mm), and peduncle three-flanged rods of different shape (Li et al. 2021: Fig. 2f and Fig. 10a, c of this paper), as well as additional type sclerites (Y-shaped, crosses, 5–6 radiates twinning) in the peduncle (minute bodies are common in the peduncle of sea pens, perhaps a detailed search in the holotype of A. splendida could discover also this type, so I will not use this feature in this comparison), and a different developmental pattern in the lowermost polyp leaves (Table 2). Despite the above-listed chromatic and morphological differences between the three Alloptilella species, they are very close from a molecular view (as occurs also between Ptilella species), at least when we compare the traditional markers used, the mitochondrial mtMutS,ND2,COI, and even the nuclear 28S. These differences have been detailed above, but in summary, they can be one or two bases per marker, and sometimes none (e.g. mtMuts sequences of the Tasman Sea A. moseleyi comb. nov. and the Caribbean A. williamsi sp. nov.). It is true that the molecular coverage of the four markers used is not complete for all species, but it seems also that, at least in Alloptilella (and also Ptilella), additional and more variable molecular markers (such as the nuclear ITS region, and SRP54, see Aguilar and Sánchez 2007; Grajales et al. 2007; Concepcion et al. 2008)shouldalsobeexploredinthe future. In the case of Alloptilella (and Ptilella), we are close to that percentage of cases (~ 25–30%) in which the considered most variable molecular marker in octocorals (mtMutS)provides little or no information (McFadden et al. 2014). García-Cárdenas et al. (2019) proposed the assignation of Pennatula bellissima Fowler, 1888, to the genus Ptilella, as well as the possible synonymy of Pennatula bayeri Castro & Medeiros, 2001, but also suggested that a molecular characterization of this species is needed, because the alternate orientation of autozooids along the ventral edge of polyp leaves is not in agreement with the autozooid’s arrangement of Ptilella grandis and other Ptilella species also discussed in this paper. However, the distribution of mesozooids in P. bellissima agrees with that described for Ptilella species. On the other hand, the presence of one or two mesozooids on the proximal dorsal edge of polyp leaves in Pennatula rubra (Ellis, 1764)(seeKükenthal1915; Altuna 2015; García- Cárdenas et al. 2019) resembles a feature present in Alloptilella. However, P. rubra does not present additional mesozooid polyps forming a ridge along the dorsal side of the rachis directed upwardly. Under the current typological criteria in the description of new species, and considering the importance of molecular knowledge in the differentiation of the species in some of pennatulacean genera, such as the three similar genera Pennatula,Ptilella,andAlloptilella, the development of economically viable ancient DNA molecular techniques would help in the complementary molecular characterization of the old preserved type material. This should be carried out with the necessary agreement and acceptance for tissue sampling by the major museums that house ancient collections such as the NHM in London, the MNHN in Paris, Naturalis Biodiversity Center in Leiden, and the National Museum of Natural History (Smithsonian Institution) in Washington, among others. Successful molecular exploration of old type materials will surely avoid continual hypothesis building and will reduce discussions when morphological features sometimes overlap or are controversial due to poor knowledge of inter- and intraspecific variability. The set of ancient types of Pennatula species described by Kölliker would be a perfect target for the application of the aforementioned molecular techniques, helping to achieve a final generic assignment and the molecular characterization of the species. Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/s12526-022-01260-w. Acknowledgements The author would like to express his gratitude to Gavin Dally (Museum and Art Gallery of the Norther Territory, Darwin, Australia) and to Stephen Cairns and the staff of the Smithsonian Institution (Washington, USA) for making the material of Alloptilella moseleyi comb.nov.andthetypespecimenofA. williamsi sp. nov. available for study, respectively. I also express my gratitude to the cruise leader, D. Wagner, and participants on board the Océano Profundo 2018 cruise for the collection of the type material of A. williamsi sp. nov. Francesc Uribe (Museu de Zoologia in Barcelona) is thanked for the meticulous tissue sampling for molecular study here carried out on some Ptilella inflata colonies deposited by the author ca. 20 years ago. I would like to thank numerous colleagues and cruise leaders who have worked on the campaigns during which different materials examined here were collected. These materials also helped to better define morphological genera as similar as Pennatula,Ptilella,andAlloptilella. These cruises and research programmes are BIOROSS, BIOICE, ANT XXIII/8, Scotia cruises, and INDEMARES-Chica, BENGUELA cruises. On these cruises, our special thanks are addressed to Wolf Arntz, Josep-Maria Gili, Francesc Pagès, Enrique Macpherson, Jim Drewery, Gudmundur Gudmundsson, Gudmundur Vidir, Jörundur Svavarsson, Stefano Page 23 of 25 41Marine Biodiversity (2022) 52: 41 Schiaparelli, Annenina Lortz, Julian Gutt, Enrique Isla, Victor Díaz-del- Río, and José Luis Rueda. The study of the Antarctic specimens and the final conception of this paper were carried out under the project CTM2017- 83920-P (DIVERSICORAL) funded by the Spanish Ministry of Economy, Industry and Competitiveness. The author also acknowledges José Martín Garrido and María Encarnación Rubio Pérez for their technical support in completing DNA extractions and amplifications of Ptilella specimens. Thanks are also due to Yang Li and Kuidong Xu (Institute of Oceanology, Chinese Academy of Sciences) for their kind comments and additional detailed graphic information on the type material of Alloptilella splendida. Mr. Tony Krupa is thanked for reviewing the English version. Finally, the author would like to thank the two anonymous reviewers and the Editorial Office of Marine Biodiversity for all their constructive comments and suggestions, which helped improve the quality of an early version of the manuscript. Funding Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. The collection of some of the specimens here studied was carried out thanks to the Spanish Projects POL2006- 06399/CGL (Polarstern ANT XXIII/8 - CLIMANT), and LIFE07/NAT/ E/000732 LIFE+INDEMARES. This research is supported by the project CTM2017-83920-P (DIVERSICORAL), Spanish Ministry of Economy, Industry and Competitiveness. Declarations Conflict of interest The author declares no competing interests. Ethical approval All applicable international, national, and/or institutional guidelines for animal testing, animal care, and use of animals were followed by the author. Sampling and field studies All necessary permits for sampling and observational field studies have been obtained by the author (or responsible researchers of the different research programmes) from the competent authorities and are mentioned in the acknowledgements. Data availability The data generated and analysed during this study are deposited in public repositories. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. References Aguilar C, Sánchez JA (2007) Phylogenetic hypotheses of gorgoniid octocorals according to ITS2 and their predicted RNA secondary structures. Mol Phylogenet Evol 43:774–786. https://doi.org/10. 1016/j.ympev.2006.11.005 Alfaro ME, Zoller S, Lutzoni F (2003) Bayes or Bootstrap? A simulation study comparing the performance of Bayesian Markov chain Monte Carlo sampling and bootstrapping in assessing phylogenetic confidence. 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