Hidden in the crowd: re-evaluation of the generic status of the Antarctic sea pen Kophobelemnon molanderi (Anthozoa: Octocorallia: Pennatuloidea), a molecular and morphological approach

Sample collection

The material studied was collected on the tip of the Antarctic Peninsula, South Shetland Islands, near the South Orkney Islands, the South Sandwich Islands and the Scotia Sea (Fig. 1) on board the R/V Polarstern on the cruises ANT XIX/5 (LAMPOS, 3 April to 5 May 2002) and ANT XXIX/3 (22 January to 18 March 2013) sponsored by the Alfred Wegener Institut für Polar- und Meeresforschung (Bremerhaven) under the auspices of the Scientific Committee for Antarctic Research.

Octocoral colonies were collected using Agassiz trawls in the Bransfield Strait and South Shetland Islands area (Fig. 1). The octocorals were fixed on board in hexamethylenetetramine-buffered 10% formalin-seawater for morphological examination, or directly in absolute ethanol for further molecular studies.

DNA extraction and PCR procedures

Total genomic DNA was extracted from ethanol (EtOH)-preserved material using the E.Z.N.A. DNA kit (OmegaBiotech) following the manufacturer’s instructions. The mtMutS, ND2 and COI mitochondrial regions and the nuclear 28S were sequenced. The start of the mtMutS region was amplified using the primers ND42625F and MUT3458R (McFadden et al. 2006McFaddenC.S., FranceS.C., SánchezJ.A., AldersladeP.A. 2006. A molecular phylogenetic analysis of the Octocorallia (Cnidaria: Anthozoa) based on mitochondrial protein-coding sequences. Mol. Phylogenet. Evol. 41: 513-527. 10.1016/j.ympev.2006.06.010; Sánchez et al. 2003SánchezJ.A. McFaddenC.S., FranceS.C., LaskerH.R. 2003. Molecular phylogenetic analyses of shallow-water Caribbean octocorals. Mar. Biol. 142: 975-987. 10.1007/s00227-003-1018-7). ND2 was amplified using the primers 16S647F and ND21418R (McFadden et al. 2004McFaddenC.S., TullisI.D., HutchinsonM.B., WinnerK., SohmJ.A. 2004. Variation in coding (NADH dehydrogenase subunits 2, 3, and 6) and noncoding intergenic spacer regions of the mitochondrial genome in Octocorallia (Cnidaria: Anthozoa). Mar. Biotechnol. 6: 516-526. 10.1007/s10126-002-0102-1). The COI region was amplified using the primers COII8068F and COIOCTR (McFadden et al. 2004McFaddenC.S., TullisI.D., HutchinsonM.B., WinnerK., SohmJ.A. 2004. Variation in coding (NADH dehydrogenase subunits 2, 3, and 6) and noncoding intergenic spacer regions of the mitochondrial genome in Octocorallia (Cnidaria: Anthozoa). Mar. Biotechnol. 6: 516-526. 10.1007/s10126-002-0102-1; France and Hoover 2002FranceS., HooverL.L.2002. DNA sequences of the mitochondrial COI gene have low levels of divergence among deep-sea octocorals (Cnidaria: Anthozoa). Hydrobiologia471: 149-155. 10.1023/A:1016517724749). The 28S nuclear ribosomal gene (28S rDNA) was amplified using the primers 28S-Far and 28S-Rar (McFadden and van Ofwegen 2013McFadden C.S., Ofwegen vanL.P. 2013. Molecular phylogenetic evidence supports a new family of octocorals and a new genus of Alcyoniidae (Octocorallia, Alcyonacea). Zookeys346: 59-83. 10.3897/zookeys.346.6270). Each PCR used 1 U of MyTaq Red DNA Polymerase (Bioline), 10 μM of each primer and approximately 30 ng of genomic DNA, being brought to a final volume of 25 µL with molecular grade H₂O. MtMutS PCR was carried out using the following cycle profile: initial denaturation at 94°C for 2 min, 35 cycles of denaturation at 94°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 10 s and a final extension at 72°C for 5 min. The ND2, COI and 28S PCR used the same cycle profile, but the corresponding annealing temperatures were 51°C, 50°C and 58°C respectively. The 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 Europe for sequencing in both directions. The purified products were electrophoresed on an ABI PRISM® 3730xl Genetic Analyzer.

Phylogenetic analyses

All chromatograms were visualized and sequence pairs were matched and edited using Sequencher v4.0. A preliminary BLAST search identified our sequences close to the genera Kophobelemnon, Funiculina and Gyrophyllum, all them within the informally named Clade III. This relationship was also in an ML comparison based only on mtMutS (~400 pennatulacean sequences, not shown). Therefore, for the present study, we will only include sequences of Clade III to avoid repeating trees and discussions between clades already included in other recently published studies (e.g. Dolan et al. 2013DolanE., TylerP.A., YessonC., RogersA.D. 2013. Phylogeny and systematics of deep-sea sea pens (Anthozoa: Octocorallia: Pennatulacea). Mol. Phylogenet. Evol. 69: 610-618. 10.1016/j.ympev.2013.07.018, Kushida and Reimer 2019Kushida Y., Reimer J.D. 2019. Molecular phylogeny and diversity of sea pens (Cnidaria: Octocorallia: Pennatulacea) with a focus on shallow water species of the northwestern Pacific Ocean. Mol. Phylogenet. Evol. 131: 233-244. 10.1016/j.ympev.2018.10.032, Kushida et al. 2022KushidaY., ImaharaY., Wee H.B. et al. 2022. Exploring the trends of adaptation and evolution of sclerites with regards to habitat depth in sea pens. PeerJ10: e13929. 10.7717/peerj.13929, García-Cárdenas et al. 2020García-CárdenasF.J., Núñez-FloresM., López-GonzálezP.J. 2020. Molecular phylogeny and divergence time estimates in pennatulaceans (Cnidaria: Octocorallia: Pennatulacea). Sci. Mar. 84: 317-330. 10.3989/scimar.05067.28A).

The set of new sequences and the homologous ones from GenBank belonging to related genera and species of sea pens within Clade III (Table 1) were aligned using MUSCLE (MEGA6, Tamura et al. 2013TamuraK., StecherG., PetersonD., FilipskiA., KumarS. 2013. MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30: 2725-2729. 10.1093/molbev/mst197). After alignment, pairwise genetic distances based on the mtMutS marker and the Kimura 2-parameter (K2P) model of nucleotide substitution (Kimura 1980KimuraM. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide-sequences. J. Mol. Evol. 16: 111-120. 10.1007/BF01731581) were obtained to compare them with previous analyses at genus and family levels, following the comparisons of Pante et al. (2012PanteE., FranceS.C., CoulouxA., et al. 2012. Deep-sea origin and in-situ diversification of chrysogorgiid octocorals. PLoS ONE7: E38357. 10.1371/journal.pone.0038357), López-González (2020López-González P.J. 2020. A new calcaxonian genus and family for Trichogorgia utinomii Cordeiro, 2019 (Octocorallia, Alcyonacea): new records of a scleriteless gorgonian species from Antarctica. Mar. Biodivers. 50: 96. 10.1007/s12526-020-01109-0) and López-González and Drewery (2022López-González P.J., DreweryJ. 2022. When distant relatives look too alike: a new family, two new genera and a new species of deep-sea Umbellula-like sea pens (Anthozoa, Octocorallia, Pennatulacea). Invertebr. Systemat. 36: 199-225. 10.1071/IS21040).

Fig. 10.- Bayesian analysis showing the phylogenetic relationships ofScotiabelemnon pauciflorum comb. nov. and other related genera and species of sea pens within Clade III. The present hypotheses are based on mtMutS (left) and the concatenated set of mitochondrial sequences mtMutS+ND2+COI (right). Bootstrap and posterior probability values (Bst/PP) are indicated in each node. Kophobelemnon lineages I and II are indicated in each species as “K.I” and “K.II”, respectively. Kophobelemnon lineage I is highlighted in light green, whileKophobelemnon lineage II is highlighted in dark green. See Table 1 for complete list of species and GenBank accession numbers. 

In accordance with previously published molecular phylogenies on pennatuloids (e.g. Dolan et al. 2013DolanE., TylerP.A., YessonC., RogersA.D. 2013. Phylogeny and systematics of deep-sea sea pens (Anthozoa: Octocorallia: Pennatulacea). Mol. Phylogenet. Evol. 69: 610-618. 10.1016/j.ympev.2013.07.018, Kushida and Reimer 2019Kushida Y., Reimer J.D. 2019. Molecular phylogeny and diversity of sea pens (Cnidaria: Octocorallia: Pennatulacea) with a focus on shallow water species of the northwestern Pacific Ocean. Mol. Phylogenet. Evol. 131: 233-244. 10.1016/j.ympev.2018.10.032, García-Cárdenas et al. 2020García-CárdenasF.J., Núñez-FloresM., López-GonzálezP.J. 2020. Molecular phylogeny and divergence time estimates in pennatulaceans (Cnidaria: Octocorallia: Pennatulacea). Sci. Mar. 84: 317-330. 10.3989/scimar.05067.28A, López-González and Drewery 2022López-González P.J., DreweryJ. 2022. When distant relatives look too alike: a new family, two new genera and a new species of deep-sea Umbellula-like sea pens (Anthozoa, Octocorallia, Pennatulacea). Invertebr. Systemat. 36: 199-225. 10.1071/IS21040), sequences of ellisellids from GenBank were selected as out-groups. Three data sets of sequences were analysed: mtMutS separately, the three mitochondrial genes mtMutS+ND2+COI, and the last one with the addition of the nuclear gene 28S. The matrix including only the mtMutS sequences had 689 bases. The concatenated matrix including only the three mitochondrial sequences had 2014 bases. The concatenated matrix for the mtMutS+ND2+COI+28S sequences had 2814 bases. All data matrices had 26 pennatuloid plus three ellisellid sequences.

The phylogenetic reconstructions were obtained by applying the BI and ML methods. The best nucleotide substitution model was selected using Modeltest (Darriba et al. 2012DarribaD., TaboadaGL., DoalloR., PosadaD. 2012. jModelTest 2: more models, new heuristics and parallel computing. Nat. Methods. 9: 772. 10.1038/nmeth.2109) according to the Akaike information criterion and hierarchical likelihood ratio test (hLRT) values (T92 for all the data matrices). ML was implemented in MEGA6 using the NNI heuristic method (nearest neighbour interchange) and 1000 bootstrap replications (Felsenstein 1985FelsensteinJ. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution. 39: 783-791. 10.1111/j.1558-5646.1985.tb00420.x). The BI was carried out in the MrBayes v3.1.2 program (Huelsenbeck and Ronquist 2001HuelsenbeckJ.P., RonquistF. 2001. MrBAYES: Bayesian inference of phylogenetic trees. Bioinformatics17: 754-755. 10.1093/bioinformatics/17.8.754; Ronquist and Huelsenbeck 2003RonquistF., HuelsenbeckJ.P.2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics19: 1572-1574. 10.1093/bioinformatics/btg180), using the model GTR+G (lset nst=6 rates=gamma) with 10⁷ generations, discarding 25% of the initial trees. The stationarity of the chains and the convergence of the two runs were monitored for each parameter by Tracer (v.1.7.1) (Rambaut et al. 2018Rambaut A., DrummondA.J., XieD., BaeleG., SuchardM.A. 2018. Posterior Summarization in Bayesian Phylogenetics Using Tracer 1.7. Syst. Biol. 67: 901-4. 10.1093/sysbio/syy032) to ensure that the effective sample size of all parameters was larger than 200, as recommended.

Morphological study

Colonies were dissected under a Motic MSZ168 stereomicroscope, and fragments from different part of the colonies were prepared for corroboration of the presence of sclerites and sclerite preparation for light microscopy and scanning electron microscope observation. Fragments of different parts (tentacles, anthocodia, etc.) were prepared in clove oil to observe sclerite arrangement in a Leica DMLB light microscope with an OPTIKA C-P20CC digital camera and the OPTIKA PROVIEW image software. The same procedure was carried out to observe sexual products and to sex selected dissected colonies. Sclerites (selecting the largest to smallest of all possible morphological types) from different parts of the colony were mounted on stubs, coated with gold-palladium under a Leica ACE600 and observed with a Zeis EVO SEM at the General Research Services of Microscopy at the University of Seville.

Deposition of materials

The material examined for this study has been deposited in the Museu de Zoologia in Barcelona (MZB) and in the collection of the Biodiversidad y Ecología Acuática (BECA) research group at the University of Seville.

Nomenclatural acts

This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank Life Science Identifiers (LSID) can be resolved, and the associated information can be viewed through any standard web browser by appending the LSID to the prefix “http://zoobank.org/”. The LSID for this publication is https://zoobank.org/References/D8EEA0C1-C8F5-4BE2-9454-EFA268199A39

Taxonomy

Remarks

McFadden et al. (2022McFaddenC.S., van OfwegenL.P., QuattriniA.M. 2022. Revisionary systematics of Octocorallia (Cnidaria: Anthozoa) guided by phylogenomics. BSSB. 1(3). 10.18061/bssb.v1i3.8735) recently proposed that the order Pennatulacea Verrill, 1865, which includes at least 15 families (see also López-González et al. 2022), should be abandoned in favour of a superfamily Pennatuloidea McFadden, van Ofwegen and Quattrini, 2022 in a new octocoral order named Scleralcyonacea McFadden, van Ofwegen and Quattrini, 2022. This proposal builds on previous analyses on mtMutS+ND2 (McFadden et al. 2006McFaddenC.S., FranceS.C., SánchezJ.A., AldersladeP.A. 2006. A molecular phylogenetic analysis of the Octocorallia (Cnidaria: Anthozoa) based on mitochondrial protein-coding sequences. Mol. Phylogenet. Evol. 41: 513-527. 10.1016/j.ympev.2006.06.010), further discussions on calcaxonian systematics (Williams 2019WilliamsG.C. 2019. A new genus and species of enigmatic gorgonian coral from the Ryukyu Archipelago, northwestern Pacific, with a discussion of calcaxonian systematics (Cnidaria, Anthozoa, Octocorallia). Zootaxa, 4701: 417-433. 10.11646/zootaxa.4701.5.2), and analyses of ultraconserved exons and loci and mtMuts (McFadden et al. 2022McFaddenC.S., van OfwegenL.P., QuattriniA.M. 2022. Revisionary systematics of Octocorallia (Cnidaria: Anthozoa) guided by phylogenomics. BSSB. 1(3). 10.18061/bssb.v1i3.8735). The consideration of the previous order Pennatulacea as the superfamily Pennatuloidea is retained here to avoid the paraphyly of the recently described order Scleralcyonacea (see McFadden et al. 2022McFaddenC.S., van OfwegenL.P., QuattriniA.M. 2022. Revisionary systematics of Octocorallia (Cnidaria: Anthozoa) guided by phylogenomics. BSSB. 1(3). 10.18061/bssb.v1i3.8735). However, from a nomenclatural point of view, once the taxonomic category order is proposed to be removed for this grouping, the subordinate category superfamily (a rank of the family group) is regulated under the ICZN (1999ICZN [International Commission on Zoological Nomenclature]. International Code of Zoological Nomenclature, 4th ed.; International Trust for Zoological Nomenclature: London, UK, 1999, i-xxix, 306 pp. https://www.iczn.org/the-code/the-code-online/) according to the Article 36.1 Statement of the Principle of Coordination applied to family-group names. “A name established for a taxon at any rank in the family group is deemed to have been simultaneously established for nominal taxa at all other ranks in the family group; all these taxa have the same type genus, and their names are formed from the stem of the name of the type genus [Art. 29.3] with appropriate change of suffix [Art. 34.1]. The name has the same authorship and date at every rank.” This means that the authorship of this superfamily corresponds to Ehrenberg, 1834, because it was he who created the family Pennatulidae based on the type genus Pennatula according to Article 36.1, following the Principle of Coordination.

Diagnosis (amended from Williams, 1990WilliamsG.C. 1990. The Pennatulacea of southern Africa (Coelenterata, Anthozoa). Annals of the South African Museum99(4), 31-119.: 64, 69)

Colonies clavate to whip-like; with authozooids arranged bilaterally along rachis, sometimes in somewhat oblique series, rarely distally reunited, with or without spiculiferous calyces; siphonozooids on the rachis, with or without calyces; axis, present, rarely absent; sclerites, when present, three-flanged, tuberculate rods and plates. According to the current molecular knowledge, with a distinctive combination of nucleotide sequence in mtMutS (position 148-C, insertion 177-185, 203-C, 208-T, 225-T, 255-C, 358-C, 626-G, 688-A), ND2 (432-C, 537-C), and COI (273-A, 388-G) within Clade III, where the family Gyrophyllidae is also included.

Type genus

Funiculina Lamarck, 1816.

Diagnosis

Funiculinid with a terminal cluster of a few (3-4) upwardly directed autozooids. Siphonozooids in two sets, in short longitudinal lines at the rachis-peduncle limit, and as a distal dome over autozooids bases. Axis absent. Sclerites mainly as large monoaxial rods with points and longitudinal furrows, scarcely tuberculate platelets, knobbly three-flanged rods, plus smooth to nearly smooth indistinctly tree-flanged needles (siphonozooid field), and ovals to oval-rods with irregular knobbly ornamentation (rachis and peduncle). According to the current molecular knowledge, with a distinctive combination of nucleotide sequence in mtMutS (position 72-T, 93-A, 117-A, 289-G, 438-G, 463-G), ND2 (209-C, 309-G), and COI (255-T, 387-T, 396-T) within Funiculinidae.

Type species

Kophobelemnon molanderi Pasternak, 1975 (=Kophobelemnon pauciflorum Molander, 1929MolanderA.R. 1929. Die Octactiniarien. Further Zoological Results of the Swedish Antarctic Exped. 1901-1903. 2(2): 1-86., name unavailable by homonymy with K. pauciflorum Hickson, 1916HicksonS.J. 1916. The Pennatulacea of the Siboga expedition, with a general survey of the order. Siboga Expedition Monographs14(77): 1-265.).

Nomenclatural statement

An LSID number was obtained for the new genus: https://zoobank.org/NomenclaturalActs/27f06b9c-6c7f-4e81-bb87-4187b4184c70

Etymology

The new genus is named by combining Scotia in reference to the Scotia Sea and surrounding archipelagos, the currently known range of distribution of the type species after the works of Molander (1929MolanderA.R. 1929. Die Octactiniarien. Further Zoological Results of the Swedish Antarctic Exped. 1901-1903. 2(2): 1-86.), Pasternak (1975PasternakF.A. 1975. New data on the specific composition and distribution of the deep-sea pennatularians (Octocorallia, Pennatularia) of the Peru-Chile region and South Atlantic. Trud. Inst. Okeanol. Akademiya nauk SSSR103: 101-118. [In Russian with English summary]), Kushida et al. (2022KushidaY., ImaharaY., Wee H.B. et al. 2022. Exploring the trends of adaptation and evolution of sclerites with regards to habitat depth in sea pens. PeerJ10: e13929. 10.7717/peerj.13929) and this paper, and the Greek word belemnon (dart or javelin), a common suffix used in naming sea pen genera after the general shape of the colony. Gender neuter.

Nominal species

Scotiabelemnon pauciflorum (Molander. 1929PasternakF.A. 1975. New data on the specific composition and distribution of the deep-sea pennatularians (Octocorallia, Pennatularia) of the Peru-Chile region and South Atlantic. Trud. Inst. Okeanol. Akademiya nauk SSSR103: 101-118. [In Russian with English summary]).

Remarks on nominal species in Scotiabelemnon gen. nov.

Specimens sequenced by Kushida et al. (2022KushidaY., ImaharaY., Wee H.B. et al. 2022. Exploring the trends of adaptation and evolution of sclerites with regards to habitat depth in sea pens. PeerJ10: e13929. 10.7717/peerj.13929) as Pennatulacea sp. are here considered as Scotiabelemnon sp. mainly because of molecular differences in the COI mitochondrial marker and some spicular differences (Kushida and Reimer, pers. comm.) (see also phylogenetic approach part in this paper). Paradoxically, Pasternak (1975PasternakF.A. 1975. New data on the specific composition and distribution of the deep-sea pennatularians (Octocorallia, Pennatularia) of the Peru-Chile region and South Atlantic. Trud. Inst. Okeanol. Akademiya nauk SSSR103: 101-118. [In Russian with English summary]) correctly executed a nomenclatural act denoting the homonymy between Kophobelemnon pauciflorum Molander 1929MolanderA.R. 1929. Die Octactiniarien. Further Zoological Results of the Swedish Antarctic Exped. 1901-1903. 2(2): 1-86. and Kophobelemnon pauciflorum Hickson, 1916, providing a new name to Molander species whose type material retained the consideration of the name bearer. However, the erection of the genus Scotiabelemnon gen. nov. and the transfer of K. molanderi (=K. pauciflorum Molander) to it make the use of K. molanderi Pasternak unnecessary, as there are no other species using the specific epithet pauciflorum in Scotiabelemnon gen. nov. Therefore, the correct name for the material examined here and the type species of this new genus should be Scotiabelemnon pauciflorum (Molander, 1929). The present paper suggests that the diversity of the genus Scotiabelemnon gen. nov. could include more than a single species. Molander’s and our present material was collected in shallower waters (200–402 m depth) than the abyssal-hadal and upper hadal (5650–6150 m depth) specimens described by Pasternak (1975PasternakF.A. 1975. New data on the specific composition and distribution of the deep-sea pennatularians (Octocorallia, Pennatularia) of the Peru-Chile region and South Atlantic. Trud. Inst. Okeanol. Akademiya nauk SSSR103: 101-118. [In Russian with English summary]) collected from South Sandwich Islands and the northern Scotia Sea, for which no molecular data are available. Moreover, even Pasternak (1975PasternakF.A. 1975. New data on the specific composition and distribution of the deep-sea pennatularians (Octocorallia, Pennatularia) of the Peru-Chile region and South Atlantic. Trud. Inst. Okeanol. Akademiya nauk SSSR103: 101-118. [In Russian with English summary]: 102–103) himself indicated that the deep-sea water colonies are significantly different from the shallow ones (они значительно отличаются от мелководных), and that tentacular sclerites are only present in the tentacular axis, unlike in Molander’s and our specimens described here, which also have sclerites in the pinnulae (see below). This is a diagnostic characteristic at the species level that is frequently used in sea pens. For these reasons, we prefer to keep the potential synonymy of Pasternak’s specimens with a question mark.

Kophobelemnon pauciflorum Molander, 1929MolanderA.R. 1929. Die Octactiniarien. Further Zoological Results of the Swedish Antarctic Exped. 1901-1903. 2(2): 1-86.: 80.

Kophobelemnon molanderi Pasternak, 1975PasternakF.A. 1975. New data on the specific composition and distribution of the deep-sea pennatularians (Octocorallia, Pennatularia) of the Peru-Chile region and South Atlantic. Trud. Inst. Okeanol. Akademiya nauk SSSR103: 101-118. [In Russian with English summary]: 102 (substitution name for K. pauciflorum Molander, 1929MolanderA.R. 1929. Die Octactiniarien. Further Zoological Results of the Swedish Antarctic Exped. 1901-1903. 2(2): 1-86. invalidated by homonymy).

(?) Kophobelemnon molanderi Pasternak, 1975PasternakF.A. 1975. New data on the specific composition and distribution of the deep-sea pennatularians (Octocorallia, Pennatularia) of the Peru-Chile region and South Atlantic. Trud. Inst. Okeanol. Akademiya nauk SSSR103: 101-118. [In Russian with English summary]: 102 (South Sandwich Islands and northern Scotia Sea specimens).

non Kophobelemnon pauciflorum Hickson, 1916HicksonS.J. 1916. The Pennatulacea of the Siboga expedition, with a general survey of the order. Siboga Expedition Monographs14(77): 1-265.: 72.

Material examined

Newly collected material: MZB 2024-3405, ANT XIX/5 – LAMPOS, Stn. 231-1, 22 Apr 2002, Agassiz trawl, 43º27.42’W 60º59.19’S, 399-402 m depth, 1 whole colony, 32 mm in length. BECA(OPEN-667), ANT XIX/5 – LAMPOS, Stn. 231-1, 22 Apr 2002, Agassiz trawl, 43º27.42’W 60º59.19’S, 399-402 m depth, 1 colony in two pieces,10 mm in length, male. BECA(OPEN-122, G-330), ANT XXIX/3 – ECOWED, Stn 240-3, 9 Mar 2013, Agassiz trawl, 62°7.05’S 60°34.12’ W 275-277 m depth, 1 whole colony, 36 mm in length. BECA(OPEN-121, G-337), ANT XXIX/3 – ECOWED, Stn 240-3, 9 Mar 2013, Agassiz trawl, 62°7.05’S 60°34.12’ W 275-277 m depth, 1 whole colony in two halves, 38 mm in length, female.

Description

Colonies varied from 10 to 38 mm in length (Figs 2A, B; 4B, D), with a terminal group of up to 4 autozooids (largest studied colony, Fig. 3A left), usually 3 autozooids from 10 mm in colony length (Figs 4C-D). Colonies without a clearly distinguishable symmetry pattern (Fig. 2B). Internal axis completely absent (Figs 3D, 4D). Peduncle (calculated in the largest colonies) 33%–40% of total colony length, not excessively bulbous in living and preserved state. Rachis composed of a stalk 60%–77% of total colony length, where relatively large autozooids appear distally (although gastrovascular cavities run internally along most of the rachis length, Fig. 3D). Lower part of the stalk cylindrical to conical widening distally. Terminal cluster of polyps distinctly swollen (Figs A–C, 3D, 4). Colony of 38 mm in total length developing numerous oocytes, up to 900 µm in diameter (Figs 3D, 5A). Colony of 10 mm in total length developing numerous spermatic cysts, up to 360 µm in diameter (Figs 4B, D, 5B, C).

Fig. 3.- Scotiabelemnon pauciflorum comb. nov. Colony BECA OPEN-121: (A), partial extended autozooids;(B), detail of a couple of tentacles dissected including folded introvert; (C), colony dissected longitudinally, showing the large gastrovascular cavities with developing oocytes, the distal dome of siphonozooids and the lack of internal axis; (D), Detail of distal dome with siphonozooids. Abbreviations: introvert (int), oocytes (oo), pinnulae (pi), siphonozooids (si). 

Fig. 4.- Scotiabelemnon pauciflorum comb. nov. (A), detail of the distal part of the colony BECA OPEN-122, showing sclerites (as white marks) of the aboral side of tentacles and siphonozooids distally; (B), the smaller collected colony (BECA OPEN-667) in lateral view, 10 mm in length, showing spermatic cysts because of transparency of the autozooid wall; (C), detail of the same colony as in (B) in apical view, showing distal dome of siphonozooids and the three autozooids; (D), the same colony as in (B) partially dissected, showing the gastrovascular cavity of one of the autozooids with numerous spermatic cysts. Abbreviations: pinnulae (pi), spermatic cysts (sc), siphonozooids (si), tentacular sclerites (ts). 

Fig. 5.- Scotiabelemnon pauciflorum comb. nov. (A), group of developing oocytes from colony BECA OPEN-121, showing nucleus limits and nucleolus; (B), spermatic cyst from colony BECA OPEN-667, showing the typical radial arrangement of developing sperm heads and also the central space where the tails of the mature spermatozoa accumulate; (C), sperm heads and tails of mature sperms obtained by squash preparations of spermatic cysts of colony BECA OPEN-667, Abbreviations: nucleous limit (nuc), nucleoulus (nul), sph sperm heads (sph), sperm tails (spt). 

Fig. 6.- Scotiabelemnon pauciflorum comb. nov. (BECA OPEN-121). (A) sclerites from tentacular axis, (A₁ ) monoaxial rods, and (A₂) knobbly three-flanged sclerites; (B) sclerites from pinnulae, (B₁) scarcely tuberculate platelets, (B₂)knobbly three-flanged rods, and (B₃) intermediate forms. Sclerites from the same colony part connected by a continuous white line. Different sclerite types from the same part of the colony separated by a short gap. 

Autozooids relatively short and robust (Figs 2A–C, 3B, 4), partially extended in preserved state (also in just collected material, Figs 2A–C), up to 4.5 mm in height (without tentacle length, probably much larger in fully extended state) and 4.0 mm in width, almost cylindrical, with an apparently smooth surface. Introvert folded in preserved state (Fig. 3C). Pharynx elongated and wrinkled. Tentacles relatively large and robust (Fig. 3B, C), up to 8 mm in preserved state. Pinnulae thick and relatively short, closely placed in a single line (Fig. 3B, C), up to 2.5 mm in length in preserved state, and up to 16 in number on each side of main tentacle axis.

Siphonozooids (0.2–0.5 mm in diameter) in two sets, at the basal part of rachis along intermittent longitudinal rows of 4–14 zooids (Fig. 2A, B, D), and numerous as a distal dome among the autozooid bases (Figs 2C, 4A, C).

Sclerites in all parts of the colony. Five kinds of sclerites: distinctly large monoaxial rods with numerous points and longitudinal furrows (Fig. 6A₁, 7A₁), scarcely tuberculate platelets (Fig. 8A₂), smooth to nearly smooth indistinctly tree-flanged needles (Fig. 8A₁), knobbly three-flanged rods (Figs 7B₂, C₂), and ovals to oval-rods with irregular knobbly ornamentation (Figs 8B, 9).

Fig. 8.- Scotiabelemnon pauciflorum comb. nov. (BECA OPEN-121). (A) sclerites from siphonozooid field, (A₁) smooth to nearly smooth indistinctly tree-flanged needles, (A₂) scarcely tuberculate platelets, and (A₃) short knobbly three-flanged rods; (B) sclerites from rachis. Sclerites from the same colony part connected by a continuous white line. Different sclerite types from the same part of the colony separated by a short gap. 

Fig. 9.- Scotiabelemnon pauciflorum comb. nov. (BECA OPEN-121). Sclerites from peduncle, ovals to oval-rods. 

Sclerites of main tentacular axis (Fig. 6A) in two types: large monoaxial rods up to 0.42 mm in length (Fig. 6A₁) and elongate knobbly three-flanged rods up to 0.19 mm in length (Fig. 6A₂). Pinnulae with scarcely tuberculate platelets (Fig. 6B₁), knobbly three-flanged rods (Fig. 6B₂) and intermediate forms (Fig. 6B₃), all three up to 0.21 mm in length.

Distal part of body of autozooid with large monoaxial rods up to 0.35 mm in length (Fig. 7A₁) and knobbly three-flanged rod, some of them nearly smooth, up to 0.18 mm in length (Fig. 7A₂). Lower part (introvert) with the same sclerome but slightly smaller, large monoaxial rods up to 0.29 mm in length (Fig. 7B₁) and knobbly three-flanged rods up to 0.18 mm in length (Fig. 7B₂). Pharyngeal tissue with the same composition as polyp body wall, but slightly thinner, large monoaxial rods up to 0.27 mm in length (Fig. 7C₁) and knobbly three-flanged rods (somewhat transitional in appearance to large monoaxial rods) up to 0.21 mm in length (Fig. 7C₂).

Fig. 7.- Scotiabelemnon pauciflorum comb. nov. (BECA OPEN-121). (A) sclerites from autozooid’s body distally, (A₁ ) monoaxial rods (A₂ ) knobbly three-flanged sclerites; (B) sclerites from autozooid’s body proximally, (B₁) monoaxial rods, and(B₂) knobbly three-flanged rods; (C) sclerites from autozooid’s pharynx, (C₁) monoaxial rod, and (C₂) knobbly three-flanged rods. Sclerites from the same colony part connected by a continuous white line. Different sclerite types from the same part of the colony separated by a short gap. 

Siphonozooids field among autozooids with smooth to nearly smooth indistinctly tree-flanged needles up to 0.4 mm in length (Fig. 8A₁), scarcely tuberculate platelets up to 0.24 mm in length (Fig. 8A₂) and short knobbly three-flanged rods up to 0.14 mm in length (Fig. 8A₃).

Rachis ovals to oval-rods with irregular knobbly ornamentation up to 0.18 mm in length (Fig. 8B). Peduncle sclerites similar to those from rachis but with smoother ornamentation, up to 0.16 mm in length (Fig. 9).

Colour

Living colonies dirty white to fleshy in colour (Fig. 2A, B); yellowish to orange colour in some colonies because of oocyte development. Siphonozooids as minute white spots (in both sets, those along the intermittent longitudinal rows and those at the distal dome); distal part of anthocodia (without tentacles) and pinnulae also distinctly whitish, while introvert is translucent probably because of a thinner body wall, allowing the observation by transparency of sexual products (Fig. 2B). Preserved specimens are milky to dirty white or yellowish in colour (Figs 3, 4)

Fig. 2.- Scotiabelemnon pauciflorum comb. nov. (A), two living colonies collected during ANT XXIX/3 cruise (BECA OPEN-121 on the left; BECA OPEN-122 on the right); (B), living colony collected during ANT XIX/5 cruise (MZB 2024-3405); (C), detail of the distal part of the colony BECA OPEN-121, showing three of the four partially extended autozooids and distal dome of siphonozooids;(D), detail of rachis peduncle limit of colony part of colony BECA OPEN-121, showing the short longitudinal rows of siphonozooids. Abbreviations: pinnulae (pi), rachis peduncle limit (rpl), siphonozooids (si). 

Distribution

Scotiabelemnon pauciflorum (Molander, 1929PanteE., FranceS.C., CoulouxA., et al. 2012. Deep-sea origin and in-situ diversification of chrysogorgiid octocorals. PLoS ONE7: E38357. 10.1371/journal.pone.0038357) comb. nov. is distributed along the tip of Antarctic Peninsula: Bransfiel Strait (Molander 1929MolanderA.R. 1929. Die Octactiniarien. Further Zoological Results of the Swedish Antarctic Exped. 1901-1903. 2(2): 1-86.), South Shetland island (this paper), and near South Orkney Islands (this paper), between 200 and 402 m depth.

Phylogenetic approach

In the hypothesis using only mtMutS (Fig. 10 left), Clade III showed a strongly supported family Gyrophyllidae (bootstrap [Bst] 99%, posterior probability [PP] 1) as the sister group of a moderately-supported polytomy grouping of taxa including Kophobelemnon s.l. and Funiculina sequences (Bst 77%, PP 0.79). Regardless of whether the BI or ML method was used, four groups were clearly differentiated: three of them correspond to species identified as belonging to the genus Kophobelemnon (Bst 96 to 100%, PP 0.99 to 1, see below), while the fourth is composed of Funiculina species (Bst 74%, PP 0.88 to 1). In this tree, Funiculina sequences are reunited with a set of Kophobelemnon sequences, here informally named Kophobelemnon I (Bst 91%, PP 0.99). Another set of Kophobelemnon sequences, here informally named Kophobelemnon II, was also distinctly separated (Bst 96%, PP 0.97 to 1), and the fourth grouping reunited sequences (Bst 99%, PP 1) of those colonies identified as Kophobelemnon pauciflorum (present study) and Pennatulacea sp. sequences (GenBank accession numbers ON603970 and ON603971), all these considered here within the genus Scotiabelemnon gen. nov.

In the mtMutS+ND2+COI hypothesis (Fig. 10 right), based on all of the mitochondrial markers examined here, Clade III again showed a strongly supported family Gyrophyllidae (Bst 100%, PP 1) as the sister group of the above grouping of taxa (here considered in the reformulated family Funiculinidae), including Kophobelemnon s.l. Scotiabelemnon gen. nov. and Funiculina sequences (Bst 77%, PP 1). Regardless of whether the BI or ML method was used, four groups were clearly differentiated in Funiculinidae: two of them correspond to species identified as belonging to the genus Kophobelemnon (lineages I and II), the third reunited Scotiabelemnon gen. nov. sequences, and the fourth is composed of Funiculina sequences.

In the third hypothesis including also the nuclear 28S markers (Fig. 11), the ML and BI methods showed initially different topologies. Concerning basal clades, the ML tree (Fig. 11 right) had a similar topology to that shown in the mtMutS tree (Fig. 2 left), and the genus Gyrophyllum arose from the more basal node of Clade III. However, the BI tree (Fig. 11 left) seemed to be more affected by the poor 28S coverage in Clade III, and the genus Funiculina arose from the more basal node of Clade III. 28S sequences are available only in 6 of the 22 species in Clade III, representing 7 of the 29 sequences in our analyses. Three 28S sequences are only available from the group informally called here Kophobelemnon II, two for one of the species of Scotiabelemnon, and none from Kophobelemnon I or Funiculina. Further efforts appear to be needed to complete the coverage of 28S and to discuss a more reliable four-marker hypothesis exploring possible internal relationships in Clade III. In any case the usefulness of 28S for resolving taxa at genus and family levels is debatable because of the observed mito-nuclear incongruences (see Discussion).

Fig. 11.- Bayesian inference (left) and maximum likelihood (right) analyses showing the phylogenetic relationships of Scotiabelemnon pauciflorum comb. nov. and other related genera and species of sea pens within Clade III. The present hypotheses are based on the concatenated dataset of mitochondrial sequences mtMutS+ND2+COI and nuclear 28S (those specimens in red). Posterior probability (PP) and bootstrap (Bst) values are indicated in each node. Kophobelemnon lineages I and II are indicated in each species as “K.I” and “K.II”, respectively. See Table 1 for complete list of species and GenBank accession numbers. 

Mean K2P genetic distances between groups were relatively homogeneous, Scotiabelemnon gen. nov. was 1.7% distant from Funiculina but 2.1% distant from all three other groupings (Kophobelemnon I, Kophobelemnon II and Gyrophyllum). The K2P mean genetic distances within groups were 0.0% (Gyrophyllum), 0.4% (Funiculina), 0.2% (Kophobelemnon I), 0.9% (Kophobelemnon II) and 0.0% (Scotiabelemnon gen. nov.).

The MtMutS sequence of Scotiabelemnon pauciflorum comb. nov. was 0.0% distant from the homologous sequence from colonies identified as Pennatulacea sp. (GenBank Accession numbers for mtMutS ON603970 and ON603971), denoting that, at least, they all belong to the same genus. Differences in the COI sequences suggest that more than one species of Scotiabelemon gen. nov. could be present in the study area.

Remarks on the family Kophobelemnidae

The family Kophobelemnidae was described by Gray (1860GrayJ.E. 1860. Revision of the family Pennatulidae, with some descriptions of some new species in the British Museum. Ann. Mag. nat. Hist. ser. 3, 5: 20-25. 10.1080/00222936008697169) as the tribe Kophobelemnonieae to accommodate club-shaped colonies with polyps in longitudinal rows on the ventral surface, at the time to include only the genus Kophobelemnon.

From a morphological point of view, the family Kophobelemnidae included three genera: Kophobelemnon Asbjørnsen, 1856, Sclerobelemnon Kölliker, 1872 and Malacobelemnon Tixier-Durivault, 1966 (Williams 1995Williams G.C. 1995. Living genera of sea pens (Coelenterata: Octocorallia: Pennatulacea): illustrated key and synopses. Zool. J. Linn. Soc. 114: 93-140. 10.1111/j.1096-3642.1995.tb00929.x, McFadden et al. 2024McFaddenC.S.; CordeiroR., WilliamsG., van OfwegenL. 2024. World List of Octocorallia. Kophobelemnon Asbjörnsen, 1856. Accessed through:World Register of Marine Species at: https://marinespecies.org/aphia.php?p=taxdetails&id=128492 on 2024-06-05). Because of the relative simplicity and poorly defined morphological characters (cylindrical to clavate colonies, with or without sclerites, autozooids in longitudinal rows with bilateral symmetry but not always clearly defined) the first molecular analyses that included more than the type genus (Kophobelemnon) suggested that this family is more of a taxonomic repository than a natural unit.

McFadden et al. (2006McFaddenC.S., FranceS.C., SánchezJ.A., AldersladeP.A. 2006. A molecular phylogenetic analysis of the Octocorallia (Cnidaria: Anthozoa) based on mitochondrial protein-coding sequences. Mol. Phylogenet. Evol. 41: 513-527. 10.1016/j.ympev.2006.06.010) first included sequences of Kophobelemnon and Sclerobelemnon species in a single tree (McFadden et al. 2006McFaddenC.S., FranceS.C., SánchezJ.A., AldersladeP.A. 2006. A molecular phylogenetic analysis of the Octocorallia (Cnidaria: Anthozoa) based on mitochondrial protein-coding sequences. Mol. Phylogenet. Evol. 41: 513-527. 10.1016/j.ympev.2006.06.010: Fig. 2), showing a large genetic distance between the two genera, the former aligned with Gyrophyllum (later named as Clade III) and the latter related to Pteroeides (later named as Clade I). Subsequent phylogenetic studies focusing on pennatulaceans added more sequences but showed the same scenario, a phylogenetic tree with four main clades in which these two genera are widely separated (Dolan et al. 2013DolanE., TylerP.A., YessonC., RogersA.D. 2013. Phylogeny and systematics of deep-sea sea pens (Anthozoa: Octocorallia: Pennatulacea). Mol. Phylogenet. Evol. 69: 610-618. 10.1016/j.ympev.2013.07.018, Kushida and Reimer 2019Kushida Y., Reimer J.D. 2019. Molecular phylogeny and diversity of sea pens (Cnidaria: Octocorallia: Pennatulacea) with a focus on shallow water species of the northwestern Pacific Ocean. Mol. Phylogenet. Evol. 131: 233-244. 10.1016/j.ympev.2018.10.032, García-Cárdenas et al. 2020García-CárdenasF.J., Núñez-FloresM., López-GonzálezP.J. 2020. Molecular phylogeny and divergence time estimates in pennatulaceans (Cnidaria: Octocorallia: Pennatulacea). Sci. Mar. 84: 317-330. 10.3989/scimar.05067.28A). Kushida and Reimer (2019Kushida Y., Reimer J.D. 2019. Molecular phylogeny and diversity of sea pens (Cnidaria: Octocorallia: Pennatulacea) with a focus on shallow water species of the northwestern Pacific Ocean. Mol. Phylogenet. Evol. 131: 233-244. 10.1016/j.ympev.2018.10.032) showed that Veretillid genera and Sclerobelemnon formed a clade, with robust support from analyses utilizing the mtMutS and ND2 region. Furthermore, López-González and Drewery (2022López-González P.J., DreweryJ. 2022. When distant relatives look too alike: a new family, two new genera and a new species of deep-sea Umbellula-like sea pens (Anthozoa, Octocorallia, Pennatulacea). Invertebr. Systemat. 36: 199-225. 10.1071/IS21040), in a four molecular marker phylogenetic analysis, stated that “Sclerobelemnon merges among veretilid genera, thus the family Veretilidae can only be considered monophyletic if Sclerobelemnon is included within it”, a placement that is also supported by phylogenetic hypothesis based on mtMutS and ND2 analysed separately (López-González and Drewery 2022López-González P.J., DreweryJ. 2022. When distant relatives look too alike: a new family, two new genera and a new species of deep-sea Umbellula-like sea pens (Anthozoa, Octocorallia, Pennatulacea). Invertebr. Systemat. 36: 199-225. 10.1071/IS21040: 218 and Fig. 15–16).

The genus Malacobelemnon Tixier-Durivault, 1966 is still a question mark. This genus was based on M. stephensoni Tixier-Durivalt, 1966, a species with few usable morphological characteristics that has never been collected again, and no molecular information of it is available. More recently, López-González et al. (2009López-González P.J., GiliJ.M., FuentesV. 2009. A new species of shallow-water sea pen (Octocorallia: Pennatulacea: Kophobelemnidae) from Antarctica. Polar Biol. 32: 907-914. 10.1007/s00300-009-0591-8) described a second species in this genus because of its morphological simplicity, and trying to be conservative in the systematics of sea pens, the species was included in this genus as M. daytoni López-González, Gili and Fuentes, 2009. However, the first amplifications of newly collected material of M. daytoni showed it to be highly divergent (López-González unpublished data), while a deeper whole mitochondrial study is in preparation (Figueroa and López-González, in prep.). Thus, even considering that M. daytoni was correctly assigned to the genus Malacobelemnon, the pertinence of this genus to the old family Kophobelemnidae or to the here reformulated Funiculinidae remains doubtful and tentative.

López-González et al. (2022) concluded Clade III to be composed of three families: Funiculinidae (with the genus Funiculina), Kophobelemnidae (including at that time only the genus Kophobelemnon) and Gyrophyllidae (with the genus Gyrophyllum). All these genera were the type genus of their respective families, although some sequences in the genus Kophobelemnon were distinctly divergent. According to the current molecular knowledge and available sequenced genera and species (Dolan et al. 2013DolanE., TylerP.A., YessonC., RogersA.D. 2013. Phylogeny and systematics of deep-sea sea pens (Anthozoa: Octocorallia: Pennatulacea). Mol. Phylogenet. Evol. 69: 610-618. 10.1016/j.ympev.2013.07.018, Kushida and Reimer 2019Kushida Y., Reimer J.D. 2019. Molecular phylogeny and diversity of sea pens (Cnidaria: Octocorallia: Pennatulacea) with a focus on shallow water species of the northwestern Pacific Ocean. Mol. Phylogenet. Evol. 131: 233-244. 10.1016/j.ympev.2018.10.032, García-Cárdenas et al. 2019García-CárdenasF.J., DreweryJ., López-GonzálezP.J. 2019. Resurrection of the sea pen genus Ptilella Gray, 1870 and description of Ptilella grayi n. sp. from the NE Atlantic (Octocorallia, Pennatulacea). Sci. Mar. 83: 261-276. 10.3989/scimar.04845.26A, López-González and Drewery 2022López-González P.J., DreweryJ. 2022. When distant relatives look too alike: a new family, two new genera and a new species of deep-sea Umbellula-like sea pens (Anthozoa, Octocorallia, Pennatulacea). Invertebr. Systemat. 36: 199-225. 10.1071/IS21040, López-González et al. 2022López-GonzálezP.J., DreweryJ., WilliamsG.C. 2022. A new family for the enigmatic sea pen genus Gyrophyllum Studer, 1891 (Octocorallia, Pennatulacea), a molecular and morphological approach. Eur. J. Taxon. 847: 73-103. 10.5852/ejt.2022.847.1983, Hogan et al. 2023HoganR.I., HopkinsK., WheelerA.J., YessonC., AllcockA.L. 2023. Evolution of mitochondrial and nuclear genomes in Pennatulacea. Mol. Phylogenet. Evol. 178: 107630. 10.1016/j.ympev.2022.107630), it seems that evolutionary hypotheses can be divided into at least four, this paper representing a fifth because of the introduction of Scotiabelemnon gen. nov. (Fig. 12):

Fig. 12.- Schematic diagram of hypotheses about the Clade III relationships in past studies. Each number with a circle indicates the hypothesis number. Names of each taxon expressed by the initials of genus name; S, Scotiabelemnon; K, Kophobelemnon (lineages I and II are also indicated); F, Funiculina; G, Gyrophyllum; B, Balticina; P, Pennatulacea sp. ; Ps, Pseudumbellula. (-) indicates low support of that clade; *, indicates a variant of a given hypothesis. BI or ML, indicate Bayesian inference and maximum likelihood, respectively. Each line under the initials indicates the memberships not within Clade III. 

Hypothesis 1 had Gyrophyllum as the basal taxon of Clade III and nested Kophobelemnon and Funiculina (Dolan et al. 2013DolanE., TylerP.A., YessonC., RogersA.D. 2013. Phylogeny and systematics of deep-sea sea pens (Anthozoa: Octocorallia: Pennatulacea). Mol. Phylogenet. Evol. 69: 610-618. 10.1016/j.ympev.2013.07.018, Kushida and Reimer 2019Kushida Y., Reimer J.D. 2019. Molecular phylogeny and diversity of sea pens (Cnidaria: Octocorallia: Pennatulacea) with a focus on shallow water species of the northwestern Pacific Ocean. Mol. Phylogenet. Evol. 131: 233-244. 10.1016/j.ympev.2018.10.032, García-Cárdenas et al. 2020García-CárdenasF.J., Núñez-FloresM., López-GonzálezP.J. 2020. Molecular phylogeny and divergence time estimates in pennatulaceans (Cnidaria: Octocorallia: Pennatulacea). Sci. Mar. 84: 317-330. 10.3989/scimar.05067.28A [ML]). When Kophobelemnon I and II can be differentiated (mainly thanks to the amount of sequenced specimens included in the analysis), Funiculina is usually grouped with Kophobelemnon I distally. This topology is observed when mtMutS is analysed alone (by BI or ML) and when mtMutS is part of a concatenated dataset of mitochondrial markers only (by BI).

Hypothesis 2 did not support the large Clade III (García-Cárdenas et al. 2019García-CárdenasF.J., DreweryJ., López-GonzálezP.J. 2019. Resurrection of the sea pen genus Ptilella Gray, 1870 and description of Ptilella grayi n. sp. from the NE Atlantic (Octocorallia, Pennatulacea). Sci. Mar. 83: 261-276. 10.3989/scimar.04845.26A [BI]). 28S is included in a concatenated four-marker data set analysed by BI.

Hypothesis 3 had Funiculina as the basal taxon of Clade III and nested Kophobelemnon and Gyrophyllum (López-González and Drewery 2022, López-González et al. 2022). Gyrophyllum is grouped with Kophobellemnon II sequences distally. This occurs when 28S is included as the fourth marker.

Hypothesis 4 had Gyrophyllum as the basal taxon of Clade III and nested Funiculina and Kophobelemnon sequences (López-González and Drewery 2022López-González P.J., DreweryJ. 2022. When distant relatives look too alike: a new family, two new genera and a new species of deep-sea Umbellula-like sea pens (Anthozoa, Octocorallia, Pennatulacea). Invertebr. Systemat. 36: 199-225. 10.1071/IS21040). This topology occurs when only the three mitochondrial markers are analysed. Kophobelemon I and II are distinct and located distally with respect to Funiculina. A derived version of this hypothesis (as 4* in Fig. 12) is in Kushida et al. (2022KushidaY., ImaharaY., Wee H.B. et al. 2022. Exploring the trends of adaptation and evolution of sclerites with regards to habitat depth in sea pens. PeerJ10: e13929. 10.7717/peerj.13929), where a couple of divergent sequences based on specimens from the South Sandwich Islands (Antarctic waters) as Pennatulacea sp. were included in the analysis (P in Fig. 12). Pennatulacea sp. are related to Kophobelemnon II distally. These sequences are considered in this paper to belong to Scotiabelemnon gen. nov.

Hogan et al. (2023HoganR.I., HopkinsK., WheelerA.J., YessonC., AllcockA.L. 2023. Evolution of mitochondrial and nuclear genomes in Pennatulacea. Mol. Phylogenet. Evol. 178: 107630. 10.1016/j.ympev.2022.107630) utilized only Kophobelemnon and Funiculina for phylogenetic trees utilizing mitochondrial genome, and it was difficult to discuss the phylogenetic hypothesis within their Clade III as Gyrophyllum and potential Scotiabelelmnon gen. nov. data were not available.

All phylogenetic trees in this study supported Hypothesis 1 (derived as 1* by the inclusion of Scotiabelemnon gen. nov. sequences). Gyrophyllum is the basal taxon of Clade III and nested Kophobelemnon s.l., Scotiabelemnon gen. nov. and Funiculina (this paper, see Figs 10, 12). Basal relationships of these last three genera are still uncertain, but Funiculina is always grouped with Kophobelemnon I distally, while it is poorly suggested that Scotiabelemnon gen. nov. could be their sister group.

The genera Funiculina and Gyrophyllum have an unstable location when 28S is included in the analyses, likely affected by evolutionary differences between the mito-nuclear region (see Quattrini et al. 2023QuattriniA.M., SnyderK.E., Purow-RudermanR., et al. 2023. Mito-nuclear discordance within Anthozoa, with notes on unique properties of their mitochondrial genomes. Sci. Rep. 13: 7443. 10.1038/s41598-023-34059-1, Hogan et al. 2023HoganR.I., HopkinsK., WheelerA.J., YessonC., AllcockA.L. 2023. Evolution of mitochondrial and nuclear genomes in Pennatulacea. Mol. Phylogenet. Evol. 178: 107630. 10.1016/j.ympev.2022.107630) and the poor coverage of this marker in our datamatrix (only 6 of the 26 in Clade III are available). The inclusion of nuclear 28S in the mitochondrial marker data set in combination with the inference model used resulted in different taxa combinations (BI placed as basal Gyrophyllum when 28S is not included, but Funiculina when 28S is incorporated in the data matrix, while ML usually places Gyrophyllum as the basal taxon in Clade III, with and without 28S in the data matrix). Incomplete taxonomic and molecular coverage, especially at the 28S marker, probably also affects the results for a reliable four-marker hypothesis (see above).

In this paper, which includes the broadest coverage of Kophobelemnon and Funiculina sequences, the pennate colony forms of the family Gyrophylliadae are clearly differentiated from the flagelliform and clavate colony forms (Funiculina and Kophobelemnon s.l.+Scotiabelemnon gen nov., respectively). The sister group of Gyrophyllidae is a polytomy (mtMutS) bringing together four subclades, three of them with relatively low basal resolution, which includes sequences from Kophobelemnon, Scotiabelemnon gen. nov. and Funiculina. Among these four groups, the sequences initially attributed to Kophobelemnon represent three of them, the fourth being Funiculina sequences. One of these Kophobelemnon clusters is described here as the genus Scotiabelemnon gen. nov., while the other two are simply named here as Kophobelemnon I and II.

The observed stronger support of Kophobelemnon I clade to the Funiculina clade (Bst 91, PP 0.99 in mtMutS; Bst 92, PP 0.93 in mtMutS+ND2+COI;) than to Kophobelemnon II and Scotiabelemnon gen. nov. raises additional discussions concerning the delimitation of the families involved. Surprisingly, Scotiabelemnon gen. nov. is closer to Funiculina spp. (in K2P genetic distance) than to other sequenced specimens attributed to the genus Kophobelemnon I or II. In any event, Gyrophyllidae and Funiculinidae become monophyletic, but Kophobelemnidae (Kophobelemnon s.l. + Scotiabelemnon gen. nov.) become paraphyletic, Kophobelemnon I being closer to Funiculina spp. that to other putative species of Kophobelemnon in Kophobelemnon II. The proposal of this paper to avoid paraphyly of Kophobelemnidae is the reunion of Funiculina, Kophobelemnon s.l. and Scotiabelemnon gen. nov. in a single family, Funiculinidae. It is therefore proposed to consider only two families in Clade III, Gyrophyllidae and Funicunilidae.

The family name, Funiculinidae Gray 1860 must be used, applying the principle of priority (ICZN 1999ICZN [International Commission on Zoological Nomenclature]. International Code of Zoological Nomenclature, 4th ed.; International Trust for Zoological Nomenclature: London, UK, 1999, i-xxix, 306 pp. https://www.iczn.org/the-code/the-code-online/: Article 52.3). In 1860 Gray inserted into our sea pen literature as tribe names Funiculininae (Gray 1860GrayJ.E. 1860. Revision of the family Pennatulidae, with some descriptions of some new species in the British Museum. Ann. Mag. nat. Hist. ser. 3, 5: 20-25. 10.1080/00222936008697169: 20) and Kophobelemninae (Gray 1860GrayJ.E. 1860. Revision of the family Pennatulidae, with some descriptions of some new species in the British Museum. Ann. Mag. nat. Hist. ser. 3, 5: 20-25. 10.1080/00222936008697169: 23), and these names were elevated to family rank by Gray in 1870, as Funiculinidae (Gray 1870GrayJ.E. 1870. Catalogue of sea-pens or Pennatulariidae in the collection of the British Museum. British Museum, London, 40 pp. 10.5962/bhl.title.11307: 12) and Kophobelemnidae (Gray 1870GrayJ.E. 1870. Catalogue of sea-pens or Pennatulariidae in the collection of the British Museum. British Museum, London, 40 pp. 10.5962/bhl.title.11307: 27), using the appropriate suffix to accommodate the original names to the new taxonomic category.

On the new reformulated family Funiculinidae

The current conception of this family is the result of the merge of the monotypic family Funiculinidae with the family Kophobelemnidae (see Williams 1990WilliamsG.C. 1990. The Pennatulacea of southern Africa (Coelenterata, Anthozoa). Annals of the South African Museum99(4), 31-119. for previous separate morphological diagnoses) because of the close molecular relationships of their type genera Funiculina, and Kophobelemnon s.l., as well as the new genus proposed here, Scotiabelemnon gen. nov. As molecular systematics comes to redefine the boundaries between taxonomic categories previously considered stable, morphological diagnoses of several octocoral families become less morphologically diagnostic than ever, sometimes being merely informative about the morphological diversity of the taxa included in them (see McFadden et al. 2022McFaddenC.S., van OfwegenL.P., QuattriniA.M. 2022. Revisionary systematics of Octocorallia (Cnidaria: Anthozoa) guided by phylogenomics. BSSB. 1(3). 10.18061/bssb.v1i3.8735).

In the present paper we propose a diagnosis for the family Funiculinidae that complements morphological information with those molecular characteristics that currently distinguish the two families considered within Clade III, Funiculinidae Gray 1860 and Gyrophyllidae López-González, Drewery and Williams 2022. Obviously, as with any type of characteristics included in the diagnosis of a taxon, morphological and molecular information must be updated to cover the variability of the taxa considered in it at any given time.

In the present study, we considered with certainty three nominal genera in this reformulated family Funiculinidae: Funiculina, Kophobelemnon and Scotiabelenon gen. nov. Considering that the genus Kophobelemnon is in urgent need of revision, and that at least an additional genus name (new or resurrected from old literature) will be added once a reliable revision of these species is carried out.

Other genera previously considered in Kophobelemnidae, such as the genera Sclerobelemnon and Malacobelemnon, have been discussed above. Sequences of Sclerobelemnon are strongly attracted to genera in the family Veretillidae Herklots, 1858 (see García-Cárdenas and López-González 2020García-CárdenasF.J., Núñez-FloresM., López-GonzálezP.J. 2020. Molecular phylogeny and divergence time estimates in pennatulaceans (Cnidaria: Octocorallia: Pennatulacea). Sci. Mar. 84: 317-330. 10.3989/scimar.05067.28A, López-González and Drewery 2022López-González P.J., DreweryJ. 2022. When distant relatives look too alike: a new family, two new genera and a new species of deep-sea Umbellula-like sea pens (Anthozoa, Octocorallia, Pennatulacea). Invertebr. Systemat. 36: 199-225. 10.1071/IS21040), so its morphological features are not considered in this reformulated family Funiculinidae. As discussed above, the inclusion of Malacobelemnon in the family Funiculinidae is tentative until reliable molecular information on its type species becomes available.

On the genera Kophobelemnon and Scotiabelemnon gen. nov.

The genus Kophobelemnon was erected by Asbjørnsen (1856AsbjørnsenP.C. 1856. Beskrivelse over Kophobelemnon mülleri, en ny Sofjaerslaegt. In: SarsM., KorenJ., DanielssenD.C. (eds) Fauna Littoralis Norvegiae2: 81-85.) for the species Kophobelemnon muelleri Asbjørnsen, 1856, a junior synonym of Pennatula stellifera Müller, 1776. Once diversity of pennatuloid genera was expanded during the 19th century, P. stellifera was subsequently transferred from Pennatula Linnaeus, 1758 to Veretillum Cuvier, 1798, Umbellularia Lamarck, 1801 and Funiculina, and was finally accommodated in Kophobelemnon (Kükenthal and Broch 1911Kükenthal W., Broch H. 1911. Pennatulacea. Wissenschaftliche Ergebnisse der Deutschen Tiefsee-Expedition “Valdivia” 1898-9913: 113-576.: 224, Kükenthal 1915KükenthalW. 1915. Pennatularia. Das Tierreich. 43:1-132. Verlag von R. Friedländer und Sohn, Berlin. 10.5962/bhl.title.1092: 29).

From a morphological point of view, the genus Kophobelemnon is characterized by the following characters: 1) colonies elongate and cylindrical to slightly clavate or short, stout and distinctly clavate; 2) distal end rounded and knob-like or distinctly pointed; 3) rachis with bilateral symmetry throughout, although not always distinctly so; 4) axis thin, round to slightly quadrangular in cross section; 5) polyp leaves absent, autozooids up to approx. 50 in number, arranged biserially along rachis; 6) anthocodiae mostly not retractile, calyces absent; 7) siphonozooids numerous, often with minute spiculated calyces, on areas of rachis not occupied by autozooids; and 8) sclerites densely set, spindles and rods, mostly three-flanged, sometimes ornamented with tubercles (Williams 1995Williams G.C. 1995. Living genera of sea pens (Coelenterata: Octocorallia: Pennatulacea): illustrated key and synopses. Zool. J. Linn. Soc. 114: 93-140. 10.1111/j.1096-3642.1995.tb00929.x: 108-109).

Obviously, there is a strong morphological similarity between the colonies of the two Kophobelemnon lineages (I and II), but only one can retain the genus name. In this case we prefer not to decide here which genus name must be used for each of these two Kophobelemnon groupings yet, because these nomenclatural acts should only be carried out after a deep morphological and molecular study in which morphological features are explored and segregated and molecular differences have been established by the analysis of solitary genes or concatenated gene datasets (see Fig 12; Dolan et al. 2013DolanE., TylerP.A., YessonC., RogersA.D. 2013. Phylogeny and systematics of deep-sea sea pens (Anthozoa: Octocorallia: Pennatulacea). Mol. Phylogenet. Evol. 69: 610-618. 10.1016/j.ympev.2013.07.018, Kushida and Reimer 2019Kushida Y., Reimer J.D. 2019. Molecular phylogeny and diversity of sea pens (Cnidaria: Octocorallia: Pennatulacea) with a focus on shallow water species of the northwestern Pacific Ocean. Mol. Phylogenet. Evol. 131: 233-244. 10.1016/j.ympev.2018.10.032; Kushida et al. 2022KushidaY., ImaharaY., Wee H.B. et al. 2022. Exploring the trends of adaptation and evolution of sclerites with regards to habitat depth in sea pens. PeerJ10: e13929. 10.7717/peerj.13929; López-González and Drewery 2022López-González P.J., DreweryJ. 2022. When distant relatives look too alike: a new family, two new genera and a new species of deep-sea Umbellula-like sea pens (Anthozoa, Octocorallia, Pennatulacea). Invertebr. Systemat. 36: 199-225. 10.1071/IS21040; this paper). In this case, the genus name Kophoblemenon must be retained when Kophobelemnon muelleri Asbjørnsen, 1856, the type species of the genus [today recognized as Kophobellemon stelliferum (Müller, 1776MüllerO.F. 1776. Zoologiae Danicae Prodromus, seu animalium Daniae et Norvegiae indigenarum characteres, nomina, et synonyma imprimis popularium. Havniae, 282 pp. 10.5962/bhl.title.63795)], is correctly recognized. Rather than being an easy task, this step is a challenge. Müller (1776MüllerO.F. 1776. Zoologiae Danicae Prodromus, seu animalium Daniae et Norvegiae indigenarum characteres, nomina, et synonyma imprimis popularium. Havniae, 282 pp. 10.5962/bhl.title.63795) described in his Zoologiæ danicæ prodromus, seu animalium daniæ et norvegiæ indigenarum the species Pennatula stellifera based on material from an imprecise locality from the northern seas, whereas in both main Kophobelemnon lineages (I and II) specimens from the northeastern Atlantic have been sequenced. At this time, the sequenced specimen Kophobelemnon sp. 1-ED seems to be the geographically closest to the type locality of K. muelleri (=Kophobellemon stelliferum), although this fact may not be a decisive criterion. This sequence (MutS GenBank accession number KF313837) is placed in the here unofficially named Kophobelemnon I lineage. This lineage is also present in other localities of the northeastern Atlantic and western Pacific, while the Kophobelemnon II lineage is present in the northeastern Atlantic and the western and eastern Pacific.

Morphological features of Scotiablemnon molanderi comb. nov. can be used for a diagnostic description of a new genus, even though these features were previously considered to be included within the morphological variability of the genus Kophobelemnon. Apart from the molecular differences, the genus Scotiabelemnon gen. nov. is differentiated from Kophobelemnon s.l. in the following set of characters: 1) siphonozooids in two sets, short longitudinal lines over rachis-peduncle limit and as a distal dome among the few autozooid bases; 2) autozooids placed distally and upwardly directed (including gastrovascular cavities), and 3) absence of axis. As chronologically described above, the genus Kophobelemnon was quickly recognized as a para- or polyphyletic taxon (Dolan et al. 2013DolanE., TylerP.A., YessonC., RogersA.D. 2013. Phylogeny and systematics of deep-sea sea pens (Anthozoa: Octocorallia: Pennatulacea). Mol. Phylogenet. Evol. 69: 610-618. 10.1016/j.ympev.2013.07.018, Kushida and Reimer 2019Kushida Y., Reimer J.D. 2019. Molecular phylogeny and diversity of sea pens (Cnidaria: Octocorallia: Pennatulacea) with a focus on shallow water species of the northwestern Pacific Ocean. Mol. Phylogenet. Evol. 131: 233-244. 10.1016/j.ympev.2018.10.032, García-Cárdenas et al. 2020García-CárdenasF.J., Núñez-FloresM., López-GonzálezP.J. 2020. Molecular phylogeny and divergence time estimates in pennatulaceans (Cnidaria: Octocorallia: Pennatulacea). Sci. Mar. 84: 317-330. 10.3989/scimar.05067.28A, López-González and Drewery 2022López-González P.J., DreweryJ. 2022. When distant relatives look too alike: a new family, two new genera and a new species of deep-sea Umbellula-like sea pens (Anthozoa, Octocorallia, Pennatulacea). Invertebr. Systemat. 36: 199-225. 10.1071/IS21040, López-González et al. 2022López-GonzálezP.J., DreweryJ., WilliamsG.C. 2022. A new family for the enigmatic sea pen genus Gyrophyllum Studer, 1891 (Octocorallia, Pennatulacea), a molecular and morphological approach. Eur. J. Taxon. 847: 73-103. 10.5852/ejt.2022.847.1983). Moreover, the last phylogenetic hypotheses by Kushida et al. (2022KushidaY., ImaharaY., Wee H.B. et al. 2022. Exploring the trends of adaptation and evolution of sclerites with regards to habitat depth in sea pens. PeerJ10: e13929. 10.7717/peerj.13929) included sequences of additional species attributed here to the genus Scotiabelemnon gen. nov. This fact suggests that the diversity of this last genus is still unknown, and its placement in the phylogenetic trees reinforces the current paraphyletic nature of the genus Kophobelemnon.

Final statement

In the current scenario, Linnaean taxonomy (understood as the discipline that regulates how to name a hierarchical set of subordinate taxonomic categories) and phylogenetic hypotheses (generated after the analysis of an ever-increasing amount of molecular information) should find a conciliatory space to achieve a better and useful view of the tree of life. Undoubtedly, the addition of new sequences, even when taxa are only identified at the genus level, greatly helps to outline the limits of higher taxonomic units and to recognize para- or polyphyletic taxa (species, genus, or families) previously named on the basis of morphological characters. The nomenclatural problems produced by the inconsistencies observed in the phylogenetic trees should be progressively resolved through the complete description of the affected taxa in a combined morphological and molecular study, which is ultimately called integrative taxonomy (Di Camillo et al. 2018Di CamilloC.G., GraviliC., De VitoD., PicaD., et al. 2018. The importance of applying Standardised Integrative Taxonomy when describing marine benthic organisms and collecting ecological data. Invertebr. Syst. 32: 794-802. 10.1071/IS17067, Gómez Daglio and Dawson 2019Gómez DaglioL., Dawson M.N.2019. Integrative taxonomy: ghosts of past, present and future. J. Mar. Biol. Ass. U. K. 99: 1237-1246. 10.1017/S0025315419000201).