INTRODUCTIONTop

The anthozoan fauna from deep-sea zones is still poorly known, despite recent international efforts having improved our general knowledge of the diversity and ecology of deep-sea benthic communities. Sea pens are a group of specialized and morphologically distinct octocorals found in all oceans, from intertidal areas to more than 6000 m in depth (Bayer 1956Bayer F.M. 1956. Octocorallia. In: Moore R.C. (eds), Treatise on invertebrate paleontology. Part F. Coelenterata. Geol. Soc. America Univ. Kansas Press. New York and Lawrence Kansas, pp. 166-231., Williams 2011Williams G.C. 2011. The Global Diversity of Sea Pens (Cnidaria: Octocorallia: Pennatulacea). PLoS ONE 6: e22747.). The order Pennatulacea includes more than 200 species in 35 genera and 14 families (López-González et al. 2001López-González P.J., Gili J.M., Williams G.C. 2001. New records of Pennatulacea (Anthozoa: Octocorallia) from the African Atlantic coast, with description of a new species and a zoogeographic analysis. Sci. Mar. 65: 59-74., López-González and Williams 2002López-González P.J., Williams G.C. 2002. A new genus and species of sea pen (Octocorallia: Pennatulacea: Stachyptilidae) from the Antarctic Peninsula. Invertebr. Syst. 16: 919-929., Williams 2015Williams G.C. 2015. A new genus and species of pennatulacean octocoral from equatorial West Africa (Cnidaria, Anthozoa, Virgulariidae). Zookeys 546: 39-50.). Sea pens constitute an important structural component in marine soft-bottoms communities, increasing the complexity of these environments in a role similar to that of other coral groups such as gorgonians and scleractinians, which are typically found on a more rocky substrata (Sale 1977Sale P.F. 1977. Maintenance of high diversity in coral reef fish communities. Am. Nat. 111: 337-359., Done 1999Done T.J. 1999. Coral Community Adaptability to Environmental Change at the Scales of Regions, Reefs and Reef Zones. Am. Zool. 39: 66-79.). The ecological importance of the recently discovered rockpens (Williams and Alderslade 2011Williams G.C., Alderslade P. 2011. Three new species of pennatulacean octocorals with the ability to attach to rocky substrata (Cnidaria: Anthozoa: Pennatulacea). Zootaxa 3001: 33-48.) is yet to be evaluated, although they could potentially also contribute similarly to scleractinians and gorgonians by providing yet another environmental niche in rocky seabeds. Moreover, it has been demonstrated that sea pens are often used as a refuge or nurseries for demersal fauna (Sammarco and Coll 1992Sammarco P.W., Coll J.C. 1992. Chemical adaptations in the Octocorallia: evolutionary considerations. Mar. Ecol. Prog. Ser. 88: 93-104., Baillon et al. 2012Baillon S., Hamel J.F., Warehem V.E., et al. 2012. Deep cold-water corals as nurseries for fish larvae. Front. Ecol. Environ. 10: 351-356.). The ecological importance of sea pen beds is internationally recognized, with these being included in red lists of marine environments threatened by anthropogenic activities (see OSPAR Commission 2010OSPAR Commission. 2010. Background Document for Sea pen and Burrowing megafauna communities. Biodiversity Series. Ospar Convention for the Protection of the Marine Environment of the Northeast Atlantic.).

Among sea pens, the genus Pennatula Linnaeus, 1758 was one of the first described octocoral genera. It is the type genus of its own family, Pennatulidae. Colonies assigned to this genus exhibit a high consistency of morphological characters, being typically pinnate (feather-like) in shape, with well-developed polyp leaves, with polymorphic zooids. Autozooids arranged along the ventral edge of the polyp leaves, as well as siphonozooids and sometimes mesozooids at the base of the polyp leaves or on the rachis, and with the sclerites along the colony mainly as three-flanged needles (see Kükenthal 1915Kükenthal W. 1915. Pennatularia. Das Tierreich. 43: 1-132. Verlag von R. Friedländer und Sohn, Berlin., Williams 1995aWilliams G.C. 1995a. Living genera of sea pens (Coelenterata: Octocorallia: Pennatulacea): illustrated key and synopses. Zool. J. Linn. Soc. 113: 93-140.).

Recent molecular studies in octocorals have identified the order Pennatulacea as a monophyletic group (McFadden et al. 2006McFadden C.S., France S.C., Sánchez J.A., et al. 2006. A molecular phylogenetic analysis of the Octocorallia (Cnidaria: Anthozoa) based on mitochondrial protein-coding sequences. Mol. Phylogenet. Evol. 41: 513-527., Dolan et al. 2013Dolan E., Tyler P.A., Yesson C., et al. 2013. Phylogeny and systematics of deep-sea sea pens (Anthozoa: Octocorallia: Pennatulacea). Mol. Phylogenet. Evol. 69: 610-618., Kushida and Reimer 2018Kushida Y., Reimer J.D. 2018. 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.). However, the monophyly of the various supra-familial and familial pennatulacean groupings that have been proposed historically has recently been questioned by these same molecular studies (McFadden et al. 2006McFadden C.S., France S.C., Sánchez J.A., et al. 2006. A molecular phylogenetic analysis of the Octocorallia (Cnidaria: Anthozoa) based on mitochondrial protein-coding sequences. Mol. Phylogenet. Evol. 41: 513-527., Dolan et al. 2013Dolan E., Tyler P.A., Yesson C., et al. 2013. Phylogeny and systematics of deep-sea sea pens (Anthozoa: Octocorallia: Pennatulacea). Mol. Phylogenet. Evol. 69: 610-618., Kushida and Reimer 2018Kushida Y., Reimer J.D. 2018. 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.).

Although Dolan et al. (2013Dolan E., Tyler P.A., Yesson C., et al. 2013. Phylogeny and systematics of deep-sea sea pens (Anthozoa: Octocorallia: Pennatulacea). Mol. Phylogenet. Evol. 69: 610-618.: 615) does not reject the possible monophyly of some genera such as Kophobelemnon, Pennatula is clearly identified as polyphyletic, suggesting the need for a redefinition of this genus. This will provide a solution to the current problem that morphological criteria used to identify members of the genus Pennatula have been found to only poorly resolve their phylogenetic relationships when compared with analysis based on DNA sequence data (see Dolan et al. 2013Dolan E., Tyler P.A., Yesson C., et al. 2013. Phylogeny and systematics of deep-sea sea pens (Anthozoa: Octocorallia: Pennatulacea). Mol. Phylogenet. Evol. 69: 610-618.: 614-615). More recently (Kushida and Reimer 2018Kushida Y., Reimer J.D. 2018. 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.), the genera Umbellula, Pennatula and Kophobelemnon were also shown as polyphyletic groups. When a polyphyletic or paraphyletic group is detected from molecular evidence, morphological characters are re-examined to identify those characters concordant with the molecular clades, and to solve potential homoplastic situations according to nomenclatural rules (Lowther et al. 2004Lowther P.E., Fraga R., Schulenberg T.S., et al. 2004. Nomenclatural solution for a polyphyletic Agelaius. Bull. Br. Ornithol. Club. 124: 171-175., Fleck et al. 2008Fleck G., Brenk M., Misof B. 2008. Larval and molecular characters help to solve phylogenetic puzzles in the highly diverse dragonfly family Libellulidae (Insecta: Odonata: Anisoptera): The Tetrathemistinae are a polyphyletic group. Org. Divers. Evol. 8: 1-6.). The delimitation of monophyletic groupings and the establishment of their relationships is a common problem in evolutionary biology research (e.g. Wheeler and Nixon 1990Wheeler Q.D., Nixon K.C. 1990. Another way of looking at the species problem: a reply to de Queiroz and Donoghue. Cladistics. 6: 77-81., Crisp and Chandler 1996Crisp M.D., Chandler G.T. 1996. Paraphyletic species. Telopea 6: 813-844., Brummitt 2002Brummitt R.K. 2002. How to chop up a tree. Taxon 51: 31-41., among many others).

The current conception of the cosmopolitan genus Pennatula includes at least 14 valid species (Williams 2011Williams G.C. 2011. The Global Diversity of Sea Pens (Cnidaria: Octocorallia: Pennatulacea). PLoS ONE 6: e22747.). However, some of these species have descriptions based on single and frequently poorly preserved specimens, or on specimens lacking those characteristics currently in use for reliable diagnosis (Kölliker 1880Kölliker R.A. 1880. Report on the Pennatulida dredged by H. M. S. Challenger during the years 1873-1876. Report of the Scientific Results of the Voyage of H. M. S. Challenger during the years 1873-76. Zoology 1: 1-41., Kükenthal 1915Kükenthal W. 1915. Pennatularia. Das Tierreich. 43: 1-132. Verlag von R. Friedländer und Sohn, Berlin., Hickson 1916Hickson S.J. 1916. The Pennatulacea of the Siboga Expedition, with a general survey of the order. Siboga Expeditie Monographs 14, Livr. 77: 1-265.).

In recent years the benthic component of a range of surveys carried out in the northeastern Atlantic by Marine Scotland Science (MSS) has provided an interesting collection of pennatulaceans, many of which have formed the basis for this study. The material examined was collected over the period 2007-2016 during various demersal trawl surveys carried out by MSS on board the research vessel MRV Scotia.

In this study, certain colonies initially attributed to two chromatic forms of the northeastern Atlantic species Pennatula grandis Ehrenberg, 1834 were collected. However, detailed morphological and molecular study of these specimens highlighted differences, suggesting further consideration of the current genus Pennatula as a polyphyletic grouping. The genus placement of Ehrenberg’s species and the need for the resurrection of a forgotten sea pen genus is discussed, and a new species is proposed.

METHODSTop

Sampling

The material studied was collected using a large demersal trawl (supplemented occasionally with a small ground gear net that was deployed directly underneath the main net deployed) on banks and terraces to the west of the Outer Hebrides, Scotland, during the Rockall Haddock Survey (2010), the OFFCON Rockall Survey (2011), the Rockall Anglerfish Survey (2010, 2013 and 2014) and the Deepwater Time Series (2008, 2009 and 2011), and to the North East of Scotland during the North Sea Anglerfish Survey (2012). Further occurrences from similar surveys over the period 2005-2016 are used to improve the known distribution (Fig. 1; Suplementary material Table S1). Overall, these surveys covered a depth range of 42 to 2145 m.

All material analysed from the Scotia cruises was collected using a demersal fish trawl with both the codend and the full body of the net being thoroughly examined for specimens after each deployment. Pennatulaceans of all types, including the new specimens studied here, were rarely recorded in the codend but commonly encountered meshed in the wings or belly of the trawl. Observations from a total of 1541 trawl deployments were used to provide distributional data on the genus. Temperature data close to the seabed were recorded using either a vertical deployment of the Seabird 19+ CTD profiler or a Star-Oddi DST logger mounted on the headline of the net. Both gave temperatures of the water within 3 to 5 m of the seabed. The relevant information on materials and sampling stations of these and other comparative material used in this paper is compiled in Table 1.

Pennatulacean colonies were sorted, labelled and fixed in buffered formalin (5% in seawater). After the fixation period, colonies were preserved in 70% ethanol. A certain number of colonies were directly fixed in 100% ethanol for further molecular studies.

For comparative purposes, the morphology of additional Pennatula species was also examined. These colonies were collected during various benthic surveys and over different geographical areas: Antarctica (BIOROSS), the northeastern Atlantic-Arctic (BIOICE), the northeastern Atlantic (Scotia cruises, INDEMARES Chica), the southeastern Atlantic (BENGUELA VIII), and the Mediterranean (INDEMARES Alborán). A subsample of these were sequenced (Table 2).

The material from the Scotia cruises referred to here is deposited in the National Museum of Scotland (NMS), in the Natural History Museum in London (NHM), in the Museu de Zoologia de Barcelona (MZB) and in the collection of the research group Biodiversidad y Ecología Acuática of the University of Seville (BECA).

External morphology and SEM study

Sclerites of different parts of the colonies were prepared for SEM study employing the standard methodology described by several authors (e.g. Bayer and Stefani 1988Bayer F.M., Stefani J. 1988. Primnoidae (Gorgonacea) de Nouvelle-Caledonie. Bull. Mus. Natl. Hist. Nat. Paris 10(A)3: 449-476.), and permanent mounts were made for examination using light microscopy. Sclerite dimensions and illustrations are based on the holotype or indicated lot. Colony and sclerite terminology follows Bayer et al. (1983)Bayer F.M., Grasshoff M., Verseveldt J. 1983. Illustrated trilingual glossary of morphological and anatomical terms applied to Octocorallia. E. J. Brill/ Dr. Backhuys, Leiden. 75 pp..

DNA extraction, PCR amplification and sequencing

Total genomic DNA was extracted from ethanol (EtOH)-preserved specimens using the EZNA DNA kit (OmegaBiotech), following the manufacturer’s instructions. Two mitochondrial regions, mtMutS (=msh1) and Cox1, plus a nuclear region, 28S ribosomal DNA, were sequenced. These three markers are concatenated as this has been considered an octocoral barcode (McFadden et al. 2014McFadden C.S., Brown A.S., Brayton C., et al. 2014. Application of DNA barcoding in biodiversity studies of shallow-water octocorals: molecular proxies agree with morphological estimates of species richness in Palau. Coral Reefs 33: 275-286.). The start of the mtMutS region was amplified using the primers ND42599F and MUT3458R (France and Hoover 2002France S.C., Hoover L.L. 2002. DNA sequences of the mitochondrial COI gene have low levels of divergence among deep-sea octocorals (Cnidaria: Anthozoa). Hydrobiologia 471: 149-155., Sánchez et al. 2003Sánchez J.A., McFadden C.S., France S.C., et al. 2003. Molecular phylogenetic analyses of shallow-water Caribbean octocorals. Mar. Biol. 142: 975-987.). The Cox1 region was amplified using the primers COII8068F and COIOCTR (France and Hoover 2002France S.C., Hoover L.L. 2002. DNA sequences of the mitochondrial COI gene have low levels of divergence among deep-sea octocorals (Cnidaria: Anthozoa). Hydrobiologia 471: 149-155., McFadden et al. 2004McFadden C.S., Tullis I.D., Hutchinson M.B., et al. 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.). The 28S nuclear ribosomal gene (28S rDNA) was amplified using the primers 28S-Far and 28S-Rar (McFadden and van Ofwegen 2013McFadden C.S., van Ofwegen L.P. 2013. A second, cryptic species of the soft coral genus Incrustatus (Anthozoa: Octocorallia: Clavulariidae) from Tierra del Fuego, Argentina revealed by DNA barcoding. Helgol. Mar. Res. 67: 137-147.). Each PCR used 0.5 U of DNA Stream Polymerase (BIORON), 0.2 mM of dNTPs, 0.3 µM of each primer and approximately 30 ng of genomic DNA, and was brought to a final volume of 25 µL with H2O. The 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 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, and a final extension at 72°C for 5 min. The Cox1 PCR used the same cycle profile with 58ºC as the annealing temperature and 40 s for extension duration on each of the 35 cycles. The 28S PCR used the same cycle as the Cox1 profile, but with 50ºC as the annealing temperature. PCR products were purified using the NucleoSpin® Extract II DNA Purification Kit, following the manufacturer’s instructions. Purified products were electrophoresed on an ABI PRISM® 3730xl genetic analyser, and sequence traces were edited using Sequencher™ v4.0.

According to the molecular phylogeny of Dolan et al. (2013)Dolan E., Tyler P.A., Yesson C., et al. 2013. Phylogeny and systematics of deep-sea sea pens (Anthozoa: Octocorallia: Pennatulacea). Mol. Phylogenet. Evol. 69: 610-618. and Kushida and Reimer (2018)Kushida Y., Reimer J.D. 2018. 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., Pennatula species were included in Clade II. As no clear basal relationships have been demonstrated among the three pennatulacean clades, for the present study sequences of two ellisellids from GenBank were selected as out-groups. The set of new sequences obtained in this study and those from GenBank (see Table 2) were aligned using MUSCLE and then implemented in MEGA5 (Tamura et al. 2011Tamura K., Peterson D., Peterson N., et al. 2011. MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol. Biol. Evol. 28: 2731-2739.). After alignment, the best nucleotide substitution model was selected using Modeltest implemented in MEGA 5 according to Akaike Information Criterion and hierarchical likelihood ratio test (hLRT) values. The phylogenetic reconstruction was obtained by applying maximum likelihood and Bayesian inference methods. The maximum likelihood method was carried out in MEGA 5 using the nearest neighbour interchange heuristic method and 1000 bootstrap replications. The selected nucleotide substitution model was T92+G+I for the concatenated mtMutS+Cox1+28S data set. The Bayesian inference was carried out with MrBayes v3.1.2 (Huelsenbeck and Ronquist 2001Huelsenbeck J.P., Ronquist F. 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17: 754-755., Ronquist and Huelsenbeck 2003Ronquist F., Huelsenbeck J.P. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572-1574.), using the substitution model GTR+G (lset nst=6 rates=gamma) and 107 generations, and discarding 25% of the initial trees.

RESULTSTop

Subclass OCTOCORALLIA Haeckel, 1866
Order Pennatulacea Verrill, 1865
Family Pennatulidae Ehrenberg, 1834
Genus Ptilella Gray, 1870

Ptilella Gray 1870Gray J.E. 1870. Catalogue of sea-pens or Pennatulariidae in the collection of the British Museum. British Museum. London, 40 pp.: 21; Koren and Danielssen 1874Koren J., Danielssen D.C. 1874. Bidrag til de ved den norske Kyst levende Pennatuliders Naturhistorie. Nyt Mag. Naturvid. 12: 422-427.: 422, 1877: 82.

Pennatula, Kölliker 1872Kölliker R.A. 1872. Morphologie und Entwickelungsgeschichte des Pennatulidenstammes nebst allgemeinen Betrachtungen zu Descendenzlehre. Christian Winter, Frankfurt am Main, 87 pp.: 136 (in part); Kölliker 1880Kölliker R.A. 1880. Report on the Pennatulida dredged by H. M. S. Challenger during the years 1873-1876. Report of the Scientific Results of the Voyage of H. M. S. Challenger during the years 1873-76. Zoology 1: 1-41.: 4 (in part); Grieg 1892Grieg J. 1892. Ovsersigt over Norges pennatulider. In: Bergens Museums Aarsberetning for 1891, pp. 1-22.: 10 (in part); Jungersen 1904Jungersen H.F.E. 1904. Pennatulida. Danish Ingolf-Expedition. 5:1-95.: 11 (in part); Kükenthal and Broch 1910Kükenthal W., Broch H. 1910. System und Stammesgeschichte der Seefedem. Zool. Anz. 36: 222-230.: 348 (in part); Broch 1913Broch H. 1913. Die Alcyonarien des Trondhjemsfjordes, III: Pennatulacea; IV. Biogeographische Übersicht. Norske Videnskabers Selskab, Trondheim. Skrifter 1912(10): 1-59.: 28 (in part); Kükenthal 1915Kükenthal W. 1915. Pennatularia. Das Tierreich. 43: 1-132. Verlag von R. Friedländer und Sohn, Berlin.: 81 (in part); Hickson 1916Hickson S.J. 1916. The Pennatulacea of the Siboga Expedition, with a general survey of the order. Siboga Expeditie Monographs 14, Livr. 77: 1-265.: 181 (in part), 1937Hickson S.J. 1937. The Pennatulacea. Scientific Rep. John Murray Expedition, 1933-v1934 4(5): 109-130.: 123; Williams 1995aWilliams G.C. 1995a. Living genera of sea pens (Coelenterata: Octocorallia: Pennatulacea): illustrated key and synopses. Zool. J. Linn. Soc. 113: 93-140.: 125 (in part); López-González et al. 2001López-González P.J., Gili J.M., Williams G.C. 2001. New records of Pennatulacea (Anthozoa: Octocorallia) from the African Atlantic coast, with description of a new species and a zoogeographic analysis. Sci. Mar. 65: 59-74.: 70 (in part); Altuna 2015Altuna A. 2015. Identificación de las especies ibéricas del género Pennatula L., 1758 (Octocorallia: Pennatulacea). Campañas Demersales, Ecomarg, Indemares y Medits. Insub, 11 pp.: 2 (in part).

Pennatula (Ptilella), Verrill 1883Verrill A.E. 1883. Report on the Anthozoa, and on some additional species dredged by the “Blake” in 1877-1879, and by the U. S. Fish Commission steamer “Fish Hawk” in 1880-82. Bull. Mus. Comp. Zool. 11: 1-72.: 3, 1885: 532 (in part).

Diagnosis (modified from Gray 1870Gray J.E. 1870. Catalogue of sea-pens or Pennatulariidae in the collection of the British Museum. British Museum. London, 40 pp.: 21). Colonies pinnate. Rachis bilaterally symmetrical throughout. Rachis with a central dorsal track naked of zooids. Rachis-peduncle limit with a distinct thickening or swelling, the thickening sometimes forming an edged ring at the thickest point. Axis circular in section, present throughout the entire colony. Polyp leaves large, conspicuous and fan-shaped. Autozooids in groups of 3-4 (occasionally 2) polyps on ventral edge of polyp leaves, groups of polyps in oblique lines. Anthocodiae retractile into spiculiferous, tubular and eight-toothed calyces. Siphonozooids numerous, on the axillae of polyp leaves. Mesozooids on rachis (on both sides of longitudinal naked dorsal track) and on the proximal 2/3 part of dorsal edge of polyp leaves. Dorsal edge of polyp leaves without zooids distally. Sclerites three-flanged and rods. Sclerites at the upper and lower parts of rachis-peduncle limit distinctly differentiated (three-flanged spindles in upper portion, but smaller rods in lower portion).

Type species. Pennatula borealis Sars, 1846 (=Pennatula grandis Ehrenberg, 1834), by monotypy.

Remarks. Four Atlantic species are here attributed to this genus: Ptilella grandis (Ehrenberg, 1834) comb. nov. (N Atlantic), Ptilella bellissima (Fowler, 1888), (NE Atlantic), Ptilella inflata (Kükenthal, 1910) n. comb. (W Indian and SE Atlantic), and Ptilella grayi n. sp. (NE Atlantic). Furthermore, the morphological descriptions of another four Pacific species, P. naresi Kölliker (1880Kölliker R.A. 1880. Report on the Pennatulida dredged by H. M. S. Challenger during the years 1873-1876. Report of the Scientific Results of the Voyage of H. M. S. Challenger during the years 1873-76. Zoology 1: 1-41.: 2), P. pearceyi Kölliker (1880Kölliker R.A. 1880. Report on the Pennatulida dredged by H. M. S. Challenger during the years 1873-1876. Report of the Scientific Results of the Voyage of H. M. S. Challenger during the years 1873-76. Zoology 1: 1-41.: 4), P. murrayi Kölliker (1880Kölliker R.A. 1880. Report on the Pennatulida dredged by H. M. S. Challenger during the years 1873-1876. Report of the Scientific Results of the Voyage of H. M. S. Challenger during the years 1873-76. Zoology 1: 1-41.: 5), and P. moseleyi Kölliker (1880Kölliker R.A. 1880. Report on the Pennatulida dredged by H. M. S. Challenger during the years 1873-1876. Report of the Scientific Results of the Voyage of H. M. S. Challenger during the years 1873-76. Zoology 1: 1-41.: 6), suggest that these could also belong to the genus Ptilella. They are here tentatively included in Ptilella, although revised and updated morphological and molecular information is necessary to establish this conclusively (see also Discussion). The molecular analysis carried out in this paper suggests the need for a re-evaluation of the previously considered morphological variability within the genus Pennatula. Our study reveals that an historical attempt to segregate some of these characters had already been undertaken by Gray (1870)Gray J.E. 1870. Catalogue of sea-pens or Pennatulariidae in the collection of the British Museum. British Museum. London, 40 pp. in describing the genus Ptilella. As this is in concordance with our molecular data, Ptilella is resurrected here.

Ptilella grayi n. sp.
(Figs 1, 2, 3)

http://zoobank.org/9844EDA5-AAEC-4884-A054-6CBDE378BE15

Holotype: NMS.Z.2019.2.1, Rockall Haddock Survey cruise 1011S (haul S11426), 56º32.63N 14º30.92W to 56º33.97N 14º28.78W, northeastern Atlantic, 198-202 m depth, 30 Aug 2011.

Paratypes: NMS.Z.2019.2.2, Rockall Anglerfish Survey 2010 cruise 0410S (haul S10150), 56º25.22N 15º14.67W to 56º22.91N 15º18.75W, northeastern Atlantic, 246-261 m depth, 6 Apr 2010. NMS.Z.2019.2.3, Rockall Haddock Survey cruise 1011S (haul S11426), 56º25.22N 15º14.67W to 56º22.91N 15º18.75W, northeastern Atlantic, 198-202 m depth, 30 Aug 2011. MZB 2018-0761, Rockall Haddock Survey cruise 1011S (haul S11426), 56º25.22N 15º14.67W to 56º22.91N 15º18.75W, northeastern Atlantic, 198-202 m depth, 30 Aug 2011.

See Table 1 for additional materials.

Description of the holotype. Colony elongate, pinnate, and erect (Fig. 2A), 457 mm in length in preserved state. Axis present throughout colony, rounded in cross section, 5 mm in maximum diameter. Rachis bilaterally symmetrical, 354 mm in length (77% of overall length) and 42 mm in width, with a distinctive naked dorsal and ventral track visible along the rachis. Rachis-peduncle limit with a prominent edged ring (Fig. 2A: er). Peduncle 103 mm in length (23% of overall length) and 36 mm in width at the widest point (the thickening). Rachis with 94 polyp leaves, inserted obliquely and extending ventrally upward, leaves gradually increasing in size along the rachis until the mid-zone, then decreasing in size towards the distal part. Autozooids numerous (approximately 50-60 per polyp-leaf), well developed (up to 5 mm in length, up to 2 mm in width), arranged in groups of 3-4 (occasionally 2), forming oblique rows along the polyp-leaf edge (Fig. 2B: az). Anthocodiae retractile into permanent spiculiferous calyces. Calyces densely spiculated and distinctly eight-toothed. Siphonozooids minute, about 0.35-0.50 mm in diameter, numerous, on the axillae of polyp leaves (Fig. 2C: sz). Mesozooids well developed, 0.50-0.75 mm in diameter, numerous, situated on the latero-dorsal surface of rachis and at the dorsal edge of polyp leaves (Fig. 2D: mz). Dorsal edge of polyp leaf with distinctive zone free of zooids distally (Fig. 2D:*).

Sclerites in calyces three-flanged rods, up to 1.20 mm in length (Fig. 3A). Sclerites absent in walls and tentacles of autozooids. Sclerites of mesozooids three-flanged rods, up to 0.80 mm in length (Fig. 3B). Sclerites of siphonozooids short, three-flanged rods, up to 0.45 mm in length (Fig. 3C). Sclerites of polyp-leaf surfaces three-flanged rods, up to 1.45 mm in length (Fig. 3D). Sclerites in dorsal track scarce, short three-flanged rods, up to 0.40 mm in length (Fig. 3E). Sclerites above edged ring in conspicuous three-flanged rods, up to 0.30 mm in length (Fig. 3F, upper row). Sclerites below edged ring short rods, up to 0.18 mm in length (Fig. 3F, lower row).

Variations. Colonies grow to a considerable size, as evidenced by living specimens being encountered up to 581 mm length and up to 312 g in weight (weighed wet with excess water blotted off).

The general colonial structure of the paratype colonies and additional material examined is similar to that of the holotype (see Figs S1 and S2). All preserved colonies examined ranged between 254 and 575 mm in length. Rachis bilaterally symmetrical with 64-98 polyp leaves. Autozooids numerous, arranged in oblique groups of 2-4 along the ventral edge of polyp leaves, 4.0-6.5 mm in length and 1.5-2.0 mm in width. Siphonozooids 0.35-0.50 mm in diameter, on axillar proximal areas of polyp leaves. Mesozooids 0.45-0.75 mm in diameter, numerous, on two lateral bands flanking a naked dorsal track. Axis rounded in cross section, up to 5.4 mm in max diameter. Sclerites as in the holotype: those of calyces up to 1.40 mm in length (Fig. S3A); those of the mesozooids up to 0.80 mm in length (Fig. S3B); those of the siphonozooids up to 0.50 mm in length (Fig. S3C); those of the polyp-leaf surfaces up to 1.40 mm in length (Fig. S3D); those of the dorsal track scarce, up to 0.40 mm in length (Fig. S3E); those close to the rachis-peduncle limit up to 0.35 mm in length above edged ring (Fig. S3F, upper row), and up to 0.17 mm in length below edged ring (Fig. S3F, lower row).

Living colonies range in colour from light yellow/orange (Fig. 4) through to a delicate salmon pink, the latter having been observed in some small specimens. Preserved examined colonies are a light fleshy to dirty white colour in ethanol. Sclerites are colourless.

Etymology. The name grayi is chosen in honour of Dr. John Edward Gray (1800-1875), in recognition of his contributions to the knowledge of sea pens and of his being the first to recognize and describe Ptilella as a different genus to Pennatula.

Distribution and associated fauna. At present, Ptilella grayi n. sp. is only known from the upper Rockall Bank area (west of the Outer Hebrides in Scotland) from a depth range of 145-389 m (Fig. 1).

The bottom temperature at these localities had a range of 8.40°C to 9.73°C over the survey period of April to October during the years 2005-2016.

The species is spread over the upper, shallow gradient area of Rockall Bank, but it is broadly centred within the Rockall Haddock Box (RHB) and spreading somewhat towards both the NE and SW. The RHB encloses the entirety of ICES statistical rectangle 42D5 and has been subject to restrictions on mobile commercial fishing since 2001 by agreement between the North East Atlantic Fisheries Commission (NEAFC) and the European Union in order to reduce mortality of stocks of juvenile haddock. To the south and west of the observed distribution of Pt. grayi n. sp. lie two further areas that have been closed by the NEAFC to all forms of commercial fishing: the North-West Rockall Bank, which has been closed since 2007, and the South-West Rockall Area (Empress of Britain Bank), which has been closed since 2013. These closed areas are in place to protect vulnerable marine ecosystems (VME) from anthropogenic degradation, the VME in these cases being aggregations of Lophelia pertusa. Whether the distribution of Pt. grayi n. sp. extends into these areas is, however, presently unknown. Despite an extensive sampling effort, the species has not been recorded in the vicinity of Rockall islet itself. Similarly, Pt. grayi n. sp. has not been recorded at all to date on the upper shelf to the east of the Rockall Trough, despite intensive sampling there over a lengthy timeframe (2010-2017 for the upper shelf and 2005-2016 for the shelf slope) and a wide depth range (42-2147 m).

The area of Pt. grayi n. sp. distribution, along with the other parts of the upper Rockall Bank, is a productive area for demersal gadoids, particularly haddock, but also prominent are blue whiting (Micromesistius poutassou), poor cod (Trisopterus minutus), Norway haddock (Sebastes viviparus), bluemouth (Helicolenus dactylopterus), lesser argentine (Argentina sphyraena) and silvery pout (Gadiculus argenteus thori). In addition, megrim (Lepidorhombus whiffiagonis), grey gurnard (Eutrigla gurnardus), anglerfish (Lophius ssp.) and blue skate (Dipturus flossada) are common (all data available at the public website http://www.ices.dk/marine-data/data-portals/Pages/DATRAS.aspx).

Invertebrates frequently recorded in the same hauls as Pt. grayi n. sp. include another sea pen commonly encountered on top of Rockall Bank, Funiculina quadrangularis, and the associated brittlestar Asteronyx lovenii. Others include the scleractinian Caryophyllia smithii, the holothurian Parastichopus tremulus, the crustacean Pagurus forbesii, the asteroids Hippasteria phrygiana, Stichastrella rosea, Astropecten irregularis and Luidia cilaris, the echinoids Gracilechinus spp., and various actinarian spp.

In contrast, Pt. grandis was observed on the Donegal continental slope, Rockall Plateau and Rosemary Seamounts over a depth range of 682-1605 m, with the associated fish and invertebrate assemblages varying greatly with depth. This study also records this species in the southern Norwegian Sea at a depth of 380-404 m, and to the South Iceland at a depth of 1595 m.

Phylogenetic analyses

In our mtMutS+Cox1+28S hypothesis (Fig. 5), the sequences obtained for Ptilella grayi n. sp. and Pt. grandis were reunited in a distinct and well-supported clade (Bst >90%, PP >0.9), far from those related to the available concatenated sequences of Pennatula species, which are also in a well-supported clade (Bst >90%, PP >0.9). In this phylogenetic hypothesis, species of different genera such as Actinoptilum, Acanthoptilum, Renilla, Ptilosarcus and Gilibelemnon are placed between Ptilella and Pennatula sequences with Ptilella diverging earlier than Pennatula.

Our results demonstrate that Ptilella and its closest morphological genus Pennatula are different by 19 substitutions in mtMutS, 27 in Cox1 and 110 in 28S. Moreover, 13 nucleotide mutations in mtMutS and 20 in Cox1 result in silent mutations, while a number of nucleotide mutations (6 in mtMutS and 7 in Cox1) imply a change of Aa (see Table S2).

An incomplete mtMutS sequence of Pt. inflata from Namibia was obtained during the molecular works carried out for this paper. Despite several amplifications, it was not possible to obtain a clean sequence that could be used in this study in order to characterize Pt. inflata. However, at least 17 of the 19 substitutions differentiating Ptilella from Pennatula in mtMutS were in agreement with its placement in Ptilella.

DISCUSSIONTop

Historical remarks on Ptilella and its type species

Pennatula grandis was succinctly described by Ehrenberg (1834Ehrenberg C.G. 1834. Beitrage zur physiologishcen Kenntniss der Corallenthiere im allgemeinen, und besonders des rothen Meeres, nebst einem Versuche zur physiologischen Systematik derselben. Abh. Königl. Akad Wiss Berlin. Aus dem Jahre. Erster Theil 1832: 225-380.: 290) based on an old specimen in the Museum of Berlin. Later on, Sars (1846Sars M. 1846. Beschreibung der Pennatula borealis, einer neuen Seefeder. In: Druck und Verlag von J. D. (eds), Fauna littoralis Norvegiae: oder Beschreibung und Abbildungen neuer oder wenig bekannten Seethiere, nebst Beobachtungen über die Organisation. Christiana. Lebensweise u. Entwickelung derselben. 1: 17-19.: 17) described and illustrated Pennatula borealis based on material collected in Norway. The latter nomenclature and species were widely accepted thereafter by most of Sars’s contemporary octocoral researchers, such as Koren and Danielssen (1856)Koren J., Danielssen D.C. 1856. Virgularia Christii K. and D. In: Sars M., Koren J., Danielssen D.C. (eds), Fauna Littoralis Norvegiae 2: 91-93., Milne Edwards and Haime (1857)Milne Edwards H., Haime J. 1857. Histoire naturelle des coralliaires ou polypes proprement dits. Librairie Encyclopédique de Roret, Paris. 326 pp., Herklots (1858)Herklots J.A. 1858. Notices pour servir à l’étude des polypiers nageurs ou pennatulidés. Bijdragen tot de Dierkunde. 7: 1-31., Gray (1860)Gray J.E. 1860. Revision of the family Pennatulidæ, with descriptions of some new species in the British Museum. J. Nat. Hist. 5: 20-25., Richiardi (1869)Richiardi S. 1869. Monografía della famiglia dei Pennatularii. Ser. 2, Archivio per la zoología, l’anatomia e la fisiología. Vol. 1, Fava e Garagnani, Bologna, 150 pp. and Kölliker (1869-72)Kölliker R.A. 1869-72. Anatomisch-Systematische Beschreibung der Alcyonararien. I. Die Pennatuliden. Abh. Senckenb. Naturforsch. Ges. 7-8: 1-458..

Milne Edwards and Haime (1857Milne Edwards H., Haime J. 1857. Histoire naturelle des coralliaires ou polypes proprement dits. Librairie Encyclopédique de Roret, Paris. 326 pp.: 211) pointed out that Ehrenberg’s description of Pennatula grandis accorded well with that of Sars’s Pennatula borealis, using this last nomenclature. Richiardi (1869Richiardi S. 1869. Monografía della famiglia dei Pennatularii. Ser. 2, Archivio per la zoología, l’anatomia e la fisiología. Vol. 1, Fava e Garagnani, Bologna, 150 pp.: 31) also considered Pennatula grandis a synonym of Pennatula borealis.

Gray (1870Gray J.E. 1870. Catalogue of sea-pens or Pennatulariidae in the collection of the British Museum. British Museum. London, 40 pp.: 21) erected the genus Ptilella based on Pennatula borealis to include a number of distinctive morphological features including “pinnules membranaceous, broad, rounded, fringed with three close parallel series of short polype-cells on the edge”.

Kölliker (1872Kölliker R.A. 1872. Morphologie und Entwickelungsgeschichte des Pennatulidenstammes nebst allgemeinen Betrachtungen zu Descendenzlehre. Christian Winter, Frankfurt am Main, 87 pp.: 136) had the opportunity to examine the original material of Pennatula grandis deposited in the Museum of Berlin. He provided a diagnosis of the species as P. borealis and synonymized Ehrenberg’s species without being aware of the description of the genus Ptilella. However, this act was contrary to the principle of priority. Kölliker (1872)Kölliker R.A. 1872. Morphologie und Entwickelungsgeschichte des Pennatulidenstammes nebst allgemeinen Betrachtungen zu Descendenzlehre. Christian Winter, Frankfurt am Main, 87 pp. also described an additional specimen of 350 mm in length deposited in the collection of the Museum of Copenhagen.

Koren and Danielssen (1874Koren J., Danielssen D.C. 1874. Bidrag til de ved den norske Kyst levende Pennatuliders Naturhistorie. Nyt Mag. Naturvid. 12: 422-427.: 422) summarized the discussion on the identity of P. grandis and P. borealis in favour of the former nomenclature by priority of Ehrenberg’s contribution. These authors also recognized the differences of P. borealis (as the type species of Ptilella) from all other Pennatula species. Their report is the first to use the nomenclature Ptilella grandis (Ehrenberg). This name was also adopted in a further contribution by the same authors (Koren and Danielssen 1877Koren J., Danielssen D.C. 1877. Contribution to the natural history of the Pennatulidae living on the Norwegian coast. In: Sars M., Koren J., et al. (eds), Fauna Littoralis Norvegiae 3: 82-102.: 82).

Kölliker (1880Kölliker R.A. 1880. Report on the Pennatulida dredged by H. M. S. Challenger during the years 1873-1876. Report of the Scientific Results of the Voyage of H. M. S. Challenger during the years 1873-76. Zoology 1: 1-41.: 4), however, did not recognize the genus Ptilella, considering that several rows of autozooids on the ventral border of the polyp leaves and the row of mesozooids on the dorsal edge of the polyp leaves could be included within the variability of the genus Pennatula. Verrill (1883Verrill A.E. 1883. Report on the Anthozoa, and on some additional species dredged by the “Blake” in 1877-1879, and by the U. S. Fish Commission steamer “Fish Hawk” in 1880-82. Bull. Mus. Comp. Zool. 11: 1-72., 1885)Verrill A.E. 1885. Results of the explorations made by the steamer Albatross off the northern coast of the United States in 1883. Ann. Rep. US Comm. Fish. 1883: 503-699. considered Ptilella a subgenus of Pennatula, as Pennatula (Ptilella) borealis Sars. Subsequent contributions by Grieg (1892Grieg J. 1892. Ovsersigt over Norges pennatulider. In: Bergens Museums Aarsberetning for 1891, pp. 1-22.: 10), Storm (1901)Storm V. 1901. Oversigt over Throndheimsfjordens fauna (med et kort). Trondhjems Biologiske Station, Meddelelser fra stationsanleggets arbeidskomite. H. Moe’s Bog & Accidentstrykkeri, Trondhjem, 20 pp., Jungersen (1904)Jungersen H.F.E. 1904. Pennatulida. Danish Ingolf-Expedition. 5: 1-95.. Kükenthal and Broch (1910)Kükenthal W., Broch H. 1910. System und Stammesgeschichte der Seefedem. Zool. Anz. 36: 222-230. and Broch (1913)Broch H. 1913. Die Alcyonarien des Trondhjemsfjordes, III: Pennatulacea; IV. Biogeographische Übersicht. Norske Videnskabers Selskab, Trondheim. Skrifter 1912(10): 1-59. do not consider Ptilella at all but simply mention the species as Pennatula grandis.

Despite this, Balss (1910Balss H. 1910. Japanische Pennatuliden. In: Doflein F. (eds), Beiträge zur Naturgeschichte Ostasiens: Abhandlungen der Mathematisch-Physischen Classe der Königlich Sächsischen Gesellschaft der Wissenschaften 10 suppl.: 1-106.: 54) started to form groups among the species in Pennatula that he considered valid, while Kükenthal and Broch (1911Kükenthal W., Broch H. 1911. Pennatulacea. Wissenschaftliche Ergebnisse der deutschen Tiefsee-Expedition “Valdivia” 13: 113-576.: 350) considered within the genus Pennatula a “Grandis-Gruppe” that brought together P. grandis, P. naresi and P. inflata by the presence of polyp leaves placed in oblique lines and polyps present along at least part of the dorsal margin of the polyp leaves. These authors described a poorly preserved specimen from the North Atlantic as Pennatula aff. inflata, thus clearly establishing the close relationship of P. inflata with P. grandis and P. bellissima. The intrageneric group “Grandis” (without taxonomic category) was not considered in subsequent important contributions (e.g. Kükenthal 1915Kükenthal W. 1915. Pennatularia. Das Tierreich. 43: 1-132. Verlag von R. Friedländer und Sohn, Berlin., Hickson 1916Hickson S.J. 1916. The Pennatulacea of the Siboga Expedition, with a general survey of the order. Siboga Expeditie Monographs 14, Livr. 77: 1-265.: 181). However, Hickson (1937Hickson S.J. 1937. The Pennatulacea. Scientific Rep. John Murray Expedition, 1933-v1934 4(5): 109-130.: 123) again mentioned a “Grandis-Group” when reporting P. inflata from the Maldive Archipelago, considering in that moment the possible synonym of P. borealis and P. bellissima as P. grandis, and even P. inflata as a synonym of the latter.

More recently, Ptilella was either considered a synonym of Pennatula (Williams 1995aWilliams G.C. 1995a. Living genera of sea pens (Coelenterata: Octocorallia: Pennatulacea): illustrated key and synopses. Zool. J. Linn. Soc. 113: 93-140.: 126) or disappeared from the scarce specialized literature that was published (e.g. Altuna 2015Altuna A. 2015. Identificación de las especies ibéricas del género Pennatula L., 1758 (Octocorallia: Pennatulacea). Campañas Demersales, Ecomarg, Indemares y Medits. Insub, 11 pp.). Thus, Pennatula grandis is reported in general contributions on North Atlantic benthic communities (e.g. Edinger et al. 2007Edinger E.N., Wareham V.E., Haedrich R.L. 2007. Patterns of groundfish diversity and abundance in relation to deep-sea coral distributions in Newfoundland and Labrador waters. Bull. Mar. Sci. 81: 101-122., Hamoutene et al. 2008Hamoutene D., Puestow T., Miller-Banoub J., et al. 2008. Main lipid classes in some species of deep-sea corals in the Newfoundland and Labrador region (Northwest Atlantic Ocean). Coral reefs 27: 237-246., Murillo et al. 2011Murillo F.J., Durán M.P., Altuna A., et al. 2011. Distribution of deep-water corals of the Flemish Cap, Flemish Pass, and the Grand Banks of Newfoundland (Northwest Atlantic Ocean): interaction with fishing activities. ICES J. Mar. Sci. 68: 319-332., Baker et al. 2012Baker K.D., Wareham V.E., Snelgrove P.V.R, et al. 2012. Distributional patterns of deep-sea coral assemblages in three submarine canyons off Newfoundland, Canada. Mar. Ecol. Prog. Ser. 445: 235-249., Baillon et al. 2012Baillon S., Hamel J.F., Warehem V.E., et al. 2012. Deep cold-water corals as nurseries for fish larvae. Front. Ecol. Environ. 10: 351-356.).

In the present contribution, the genus Ptilella is considered different to Pennatula because of the following set of characters:

1) the lack of zooids along the dorsal track; 2) the autozooids on the margins of polyp leaves being arranged in an oblique group of 3-4 (occasionally 2) polyps, giving the impression of several (3-4) series along the polyp-leaf border; 3) a distinctive thickening at the boundary between rachis and peduncle, often giving the appearance of an edged ring while exhibiting different sclerite morphology in the upper and lower part of this thickening; 4) the insertion of polyp leaves on the rachis being distinctly oblique and extending ventrally upward; and 5) DNA sequences (mMutS, Cox1 and 28S) being divergent from those of other pennatulacean genera (see phylogenetic analyses).

In accordance with the differences described, it follows that the diagnosis of Pennatula needs to be amended to include characters that separate it clearly from Ptilella.

Diagnosis of Pennatula (modified from Williams 1995a: 126)

Colonies pinnate. Symmetry of rachis bilateral throughout. Rachis-peduncle limit just under the lower polyp leaves, as a narrowing, without a distinct thickening or swelling with a prominent edged ring at the rachis-peduncle limit. Axis circular in section, present throughout length of colony. Polyp leaves present, usually large and conspicuous, deltoid, sickle-shaped or fan-shaped. Autozooids in one single row along the ventral edge of polyp leaves. Anthocodiae retractile into permanent spiculiferous calyces. Calyces tubular, with eight terminal teeth. Siphonozooids on rachis, and between polyp leaves. Mesozooids on rachis, or on basal dorsal margin of polyp leaves. Sclerites three-flanged needles on calyces, mesozooids, siphonozooids, inconspicuous three-flanged rods on surface of peduncle, and small ovals in interior of peduncle (mostly >0.1 mm long).

Although a revision of the species in the genus is needed, according to Williams (1995a)Williams G.C. 1995a. Living genera of sea pens (Coelenterata: Octocorallia: Pennatulacea): illustrated key and synopses. Zool. J. Linn. Soc. 113: 93-140. the following list of species can be tentatively considered valid in the genus Pennatula: P. aculeata Danielssen, 1860 (North Atlantic); P. delicata Tixier-Durivault, 1966 (Madagascar); P. fimbriata Herklots, 1858 (Japan, Philippines); P. indica Thomson and Henderson, 1906 (Indian Ocean); P. phosphorea Linneaus, 1758 (northeastern Atlantic and Mediterranean, Cosmopolitan ?); P. rubra (Ellis, 1761) (Mediterranean Sea); and P. prolifera Jungersen, 1904 (North Atlantic).

As we state above, our phylogenetic hypothesis based on mitochondrial and nuclear markers places the type species of Ptilella, (Pennatula grandis), far from those sequences from the type species of the genus Pennatula, (P. phosphorea), or at least, taking into account that the genus Pennatula is in need of revision from the sequenced species and specimens sharing the morphological characters defined for Pennatula phosphorea in this paper and the most relevant references (e.g. Kükenthal 1915Kükenthal W. 1915. Pennatularia. Das Tierreich. 43: 1-132. Verlag von R. Friedländer und Sohn, Berlin., Kükenthal and Broch 1911Kükenthal W., Broch H. 1911. Pennatulacea. Wissenschaftliche Ergebnisse der deutschen Tiefsee-Expedition “Valdivia” 13: 113-576.) (see Figs 5 and 6).

Comparison of Ptilella species

In this paper, four Atlantic species are considered to be in the genus Ptilella (Pt. grandis, Pt. grayi n. sp. Pt. inflata, and Pt. bellissima) on the basis of a number of morphological features. Corroborative molecular information is currently available for two of them (Pt. grandis, Pt. grayi), and partially so for Pt. inflata (see above). Molecular grade tissue is, however, lacking from the old single specimen of Pt. bellissima due to the preservation method used.

In the late 19th century, Professor Albert R. von Kölliker described four Pacific sea pen species and ascribed them to the genus Pennatula: P. naresi Kölliker (1880Kölliker R.A. 1880. Report on the Pennatulida dredged by H. M. S. Challenger during the years 1873-1876. Report of the Scientific Results of the Voyage of H. M. S. Challenger during the years 1873-76. Zoology 1: 1-41.: 2), P. pearceyi Kölliker (1880Kölliker R.A. 1880. Report on the Pennatulida dredged by H. M. S. Challenger during the years 1873-1876. Report of the Scientific Results of the Voyage of H. M. S. Challenger during the years 1873-76. Zoology 1: 1-41.: 4), P. murrayi Kölliker (1880Kölliker R.A. 1880. Report on the Pennatulida dredged by H. M. S. Challenger during the years 1873-1876. Report of the Scientific Results of the Voyage of H. M. S. Challenger during the years 1873-76. Zoology 1: 1-41.: 5), and P. moseleyi Kölliker (1880Kölliker R.A. 1880. Report on the Pennatulida dredged by H. M. S. Challenger during the years 1873-1876. Report of the Scientific Results of the Voyage of H. M. S. Challenger during the years 1873-76. Zoology 1: 1-41.: 6). The original descriptions of these species agree with some of the characters considered here for Ptilella: 1) the obliquely inserted polyp leaves on the rachis; 2) at least two series of autozooids on the ventral edge of the polyp leaves; 3) the dorsal track free of zooids; and 4) mesozooids on the proximal dorsal part of the polyp leaves. While taking into account of the current state of knowledge of these species, their morphological features thus support their tentative inclusion in Ptilella here. However, it is important that their position be reassessed following an improved morphological and molecular understanding of these species in the future.

Ptilella grandis and Pt. grayi n. sp. are clearly differentiated from Pt. bellissima and Pt. inflata by their more rigid and spiculose colonies (especially visible on the upper and lower polyp leaf surfaces), a higher overall number of polyp leaves and a higher number of autozooids per polyp leaf. Moreover, the two former species have a much stronger separation between rachis and peduncle with an edged ring (see Figs 2A, S1A, and S2E for Pt. grayi n. sp.; Figs 7A and 8C for Pt. grandis). This structure is much more marked in Ptilella grandis and Pt. grayi n. sp. than in Pt. bellissima (see Figs 7C and 8I) and Pt. inflata (see Figs 7B and 8F), where the upper and lower surfaces are more continuous in structure.

Ptilella bellissima was described by Fowler (1888)Fowler G.H. 1888. On a new Pennatula from the Bahamas. Proc. Zool. Soc. Lond. 1888: 135-140. based on a single specimen from the Bahamas (northwestern Atlantic) as Pennatula bellissima, now deposited in the Natural History Museum in London (BMNH 1880.6.28.1). More than a century later, Castro and Medeiros (2001Castro C.B., Medeiros M.S. 2001. Brazilian Pennatulacea (Cnidaria: Octocorallia). Bull. Biol. Soc. Wash. 10: 140-159.: 154) described a new species of a large Pennatula (up to 350 mm in total length) from the Brazilian coast as Pennatula bayeri. After the comparison of both original descriptions and the holotype of Pt. bellissima, we believe P. bayeri to be a junior synonym of Pt. bellissima. As mentioned by Fowler (1888)Fowler G.H. 1888. On a new Pennatula from the Bahamas. Proc. Zool. Soc. Lond. 1888: 135-140., Castro and Medeiros (2001)Castro C.B., Medeiros M.S. 2001. Brazilian Pennatulacea (Cnidaria: Octocorallia). Bull. Biol. Soc. Wash. 10: 140-159., and as illustrated in the present paper, Pt. bellissima differs from Pt. inflata by its slender triangular and less fleshy polyp leaves, and well-spaced (sometimes difficult to follow) groups of 2-3 autozooids along the ventral edge of the polyp leaves. Castro and Medeiros (2001Castro C.B., Medeiros M.S. 2001. Brazilian Pennatulacea (Cnidaria: Octocorallia). Bull. Biol. Soc. Wash. 10: 140-159.: 157) discussed the possibility of synonymy between Pennatula bayeri (=Pt. bellissima) and Pt. inflata, but we consider them as two clearly morphologically distinct taxa. It is important that future molecular studies (based on appropriately fixed material) should further explore the internal relationships of Ptilella species. Particular attention should be paid to further specimens of Pt. bellissima, in order to correctly evaluate the variability in the distribution of mesozooids around the insertion point of the polyp leaves and the autozooids.

The most immediately evident feature differentiating Ptilella grandis and Pt. grayi n. sp. is the colour: dark-red to red-brown both alive and preserved in the former but light fleshy to dirty white in the latter in the preserved state and light yellow to light pink in living specimens. In the examined material the maximum observed lengths of sclerites in Pt. grandis and Pt. grayi n. sp. tends to be overall slightly larger in the former species (calyces 1.70 vs. 1.40 mm; mesozooids 1.0 vs. 0.80 mm; siphonozooids 0.80 vs. 0.50 mm; polyp leaves 1.11 vs. 1.40 mm; dorsal track 0.50 vs. 0.40; rachis-peduncle limit 0.50 vs. 0.35 above edged ring and 0.16 vs. 0.17 mm below edged ring).

The molecular comparison of Pt. grayi n. sp. vs Pt. grandis revealed the following substitutions: at the positions 249 (A/C) and 672 (C/A) in mtMutS; at the positions 372 (A/G) and 624 (C/A) in Cox1; and at the positions 166 (C/A), 210 (A/G) and 519 (G/C) in 28S (see Table S3). In mtMutS, both substitutions imply the change of Aa (Met by Ile, and Phe by Leu, respectively), although in both cases all Aa are non-polar. In Cox1, both are silent substitutions. Although the genetic distance obtained in mtMutS did not exceed 0.30%, the more conserved mitochondrial marker Cox1 also showed differences between these species, which are additionally supported by the differences in the nuclear 28S.

According to our current knowledge, Pt. grayi n. sp. seems to have an overlapping but somewhat more geographically restricted and shallower bathymetric distribution than Pt. grandis (145-389 m and 90-2700 m in depth, respectively). Moreover, the new species is only known, for the moment, from the NE Atlantic, though Pt. grandis has been reported from both sides of the North Atlantic Ocean. The densities of Pt. grayi n. sp. on Rockall Bank are presently unknown and cannot be inferred from swept area estimates using numbers caught per haul, as it is likely that demersal trawl is an inefficient method of capturing large sea pens that are anchored in the sediment. Moreover, the exact design of trawl used varied with the survey that the specimens were obtained from.

On the polyphyletic family Pennatulidae and the familial relationships in Clade II

The traditional family Pennatulidae comprises six genera: Gyrophyllum, Pennatula, Ptilosarcus, Sarcoptilus, Crassophyllum and Pteroeides (Williams 1995aWilliams G.C. 1995a. Living genera of sea pens (Coelenterata: Octocorallia: Pennatulacea): illustrated key and synopses. Zool. J. Linn. Soc. 113: 93-140., 1995bWilliams G.C. 1995b. The enigmatic sea pen genus Gyrophyllum - A phylogenetic reassessment and description of G. sibogae from Tasmanian waters (Coelenterata: Octocorallia). Proc. Cal. Acad. Sci. 48: 315-328.). Dolan et al. (2013)Dolan E., Tyler P.A., Yesson C., et al. 2013. Phylogeny and systematics of deep-sea sea pens (Anthozoa: Octocorallia: Pennatulacea). Mol. Phylogenet. Evol. 69: 610-618. showed a consensus phylogenetic tree suggesting a non-monophyletic Pennatulidae. Although the monophyly of Pennatulidae was not formally rejected by an SH test, in that study this family formed a non-natural group, placing the genera Pteroeides, Pennatula, and Gyrophyllum into three different clades (Clades I, II and III, respectively). Clade II based on two mitochondrial markers (mtMutS+ND2) included the genera Pennatula, Renilla, Distichoptillum and Protoptilum, the two former genera being sister groups. Later, results offered by Kushida and Reimer (2018)Kushida Y., Reimer J.D. 2018. 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. based on those same mitochondrial (mtMutS+ND2) again confirmed the polyphyletic nature of the current Pennatulidae, showing the same distribution of the genera Pteroeides, Pennatula and Gyrophyllum. In that study, Clade II again included the genus Pennatula as the sister group of Renilla, and both related to genera of different families: Echinoptilum (Echinoptilidae), Stachyptilum (Stachyptilidae), Distichoptilum and Protoptilum (both Protoptilidae), and Scytalium and Stylatula (both Virgulariidae). In our present phylogenetic analyses for Clade II we present a more complete molecular coverage, including two mitochondrial (mtMutS, Cox1) markers and a nuclear marker (28S), showing a well-supported genus Pennatula with Ptilosarcus as the sister group, with Renilla being the sister group of Acanthoptillum (see Fig. 5). Pennatula species were well separated from the resurrected genus Ptilella, which diverges early. Future sea pen studies utilizing a more complete set of taxonomic units (families, genera and species) and molecular markers may completely change our previous understanding of the distribution of genera among all the possible familial units in this specialized group of octocorals. We recognise the current incomplete and unstable state of taxonomic knowledge of the Pennatulidae and our placement of Ptilella within this family is to be regarded as tentative. We consider this as pending a thoroughly comprehensive review that will also achieve the segregation of diagnostic morphological characters across those well supported familial units.

ACKNOWLEDGEMENTSTop

The authors would like to express their gratitude to Miranda Lowe (British Museum of Natural History) for the loan of the holotype of Pennatula bellissima. Many thanks to the colleagues and crew of the MRV Scotia over the many cruises involved and particularly to several who dedicatedly examined the net a great many times for specimens: Matthew Kinghorn, Hubert Wozniak, Peter Brown and Christopher Boagey. Our thanks are also addressed to the officers and crew and to many colleagues for facilitating access to the collections or for their help on board during the cruises Polarstern ANT XXIII/8 (Antarctic Peninsula and eastern Weddell Sea), BIOROSS Tangaroa 0402 (Ross Sea, Antarctica), BENGUELA VIII (Namibia, southeastern Atlantic), INDEMARES Chica (Gulf of Cadiz, NE Atlantic), and INDEMARES Alborán (Mediterranean), where part of the comparative materials used in morphological and molecular comparisons were collected. Part of the material of Pt. grandis consulted in this paper was provided by the BIOICE project. We express our gratitude to Drs Gudmundur Vidir, Gudmundur Gudmundsson and Jörundur Svavarsson, and to the staff of the BIOICE project for allowing us to study the BIOICE octocoral material at the Sandgerdi Marine Centre in Iceland. PJL-G acknowledges financial support for a visit to the Sandgerdi Marine Centre (Iceland) under the EC-funded TMR BIOICE Large-Scale Facility Programme. Antarctic specimens of Pennatula were consulted within the project CTM2017-83920-P (DIVERSICORAL) funded by the Spanish Ministry of Economy, Industry and Competitiveness. The authors are also grateful for the criticisms and suggestions provided by three anonymous referees and the editor of SM. The use of the Scanning Electron Microscopy facilities of the University of Seville were made possible thanks to a grant to PJL-G from VIPPIT-2017-1.5 (VI Plan Propio de Investigación de la Universidad de Sevilla, Vicerrectorado de Investigación).

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SUPPLEMENTARY MATERIAL

The following supplementary material is available through the online version of this article and at the following link: http://scimar.icm.csic.es/scimar/supplm/sm04845esm.pdf

Table S1. – Data associated with all hauls where Ptilella grayi n. sp. and Ptilella grandis were observed, including the number of colonies recorded at each.

Table S2. – Nucleotide substitution implying amino acid changes in the mitochondrial loci mtMutS and Cox1, between the sequences of Ptilella and Pennatula used in the molecular analyses, once homologous sites of a datamatrix including Ptilella and Pennatula sequences are aligned (see Table 2 and Fig. 5).

Table S3. – Nucleotide and amino acid differences in the mitochondrial loci mtMutS and Cox1, and nucleotide differences in the nuclear 28S between Ptilella grayi n. sp. and Pt. grandis. Based on a datamatrix including only Ptiella and Pennatula species (see Table 2 and Fig. 5).

Fig. S1. – Ptilella grayi n. sp. Paratype (NMS.Z.2019.2.3). A, whole colony; B, dorsal view of polyp leaves and naked dorsal track showing the location of mesozooids; C, detail of autozooids on the ventral edge of the polyp leaves; D, detail of oblique rows of autozooids, sectioned basally, stained with methylene blue to increase contrast.

Fig. S2. – Ptilella grayi n. sp. Paratype (NMS.Z.2019.2.3). A, polyp-leaf sectioned from the base, lateral view; B, detail of dorsal area of a polyp-leaf showing autozooids (az) and mesozooids (mz), and distal area free of zooids (*); C, detail of dorsal basal part of polyp leaves showing siphonozooids (sz) and mesozooids (mz); D, detail of arrangement of siphonozooids (sz) in pad at the base of a polyp leaf; E, distinctive thickening at the rachis-peduncle limit.

Fig. S3. – Ptilella grayi n. sp. Paratype (NMS.Z.2019.2.3). SEM photographs of sclerites. A, calyces; B, mesozooids; C, siphonozooids; D, polyp leaves; E, dorsal track; F, rachis-peduncle limit, above edged ring (upper row) and below edged ring (lower row).