Resurrection of the sea pen genus Ptilella Gray, 1870 and description of Ptilella grayi n. sp. from the NE Atlantic (Octocorallia: Pennatulacea)

Resurrection of the sea pen genus Ptilella Gray, 1870 and description of Ptilella grayi n. sp. from the NE Atlantic (Octocorallia: Pennatulacea) Francisco J. García-Cárdenas 1, Jim Drewery 2, Pablo J. López-González 1 1 Biodiversidad y Ecología Acuática, Departamento de Zoología, Facultad de Biología, Universidad de Sevilla, Reina Mercedes 6, 41012 Sevilla, Spain. (FJG-C) (corresponding author) E-mail: [email protected]. ORCID-iD: https://orcid.org/0000-0002-1503-9552 (PJL-G) E-mail: [email protected]. ORCID-iD: https://orcid.org/0000-0002-7348-6270 2 Marine Scotland Science, Marine Laboratory, 375 Victoria Road, Aberdeen, Scotland, UK, AB11 9DB. (JD) E-mail: [email protected]. ORCID-iD: https://orcid.org/0000-0003-4308-1798 Summary: The order Pennatulacea covers a group of specialized and morphologically distinct octocorals found in all oceans from intertidal areas to more than 6000 m in depth. Sea pens constitute an important structural component in marine softbottom communities by increasing the complexity of these environments. Despite being both morphologically distinctive and ecologically important, the taxonomy and systematics of sea pens is still poorly understood. Recent molecular studies have shown the existence of convergent morphological features, making the current familial distribution of genera unstable. The genus Pennatula Linnaeus, 1758 was one of the first described octocoral genera. It is the type genus of its family, Pennatulidae. Colonies of this genus have a characteristic morphology. Recent sampling efforts in the northeastern Atlantic have provided a number of colonies initially attributable to the genus Pennatula. Both morphological and molecular (mtMutS, Cox1 and 28S genes) study of this material supports the polyphyletic nature of this genus and the need to resurrect the genus Ptilella Gray, 1870 to accommodate these and other species. A new species, Ptilella grayi n. sp., is described and illustrated. The species Pennatula bayeri is proposed to be a junior synonym of Pennatula bellissima (here also considered in the genus Ptilella). Keywords: NE Atlantic; Pennatulacea; Ptilella grayi; sea pen; new species. Resurrección del género de pluma de mar Ptilella Gray, 1870, y descripción de Ptilella grayi n. sp. del Atlántico NE (Octocorallia: Pennatulacea) Resumen: El orden Pennatulacea abarca a un grupo de octocorales especializados y morfológicamente diferenciados, distribuidos por todos los océanos, desde las zonas intermareales hasta más de 6000 metros de profundidad. Las plumas de mar constituyen un importante componente estructural en las comunidades marinas de fondo blando, incrementando la complejidad de estos ambientes. A pesar de ser característicos morfológicamente, e importantes ecológicamente, la taxonomía y la sistemática de las plumas de mar es aún pobremente conocida. Recientes estudios moleculares han mostrado la existencia de características morfológicas convergentes, haciendo inestable la actual distribución familiar de los géneros. El género Pennatula Linnaeus, 1758, fue uno de los primeros géneros de octocorales descritos. Es el género tipo de su familia, Pennatulidae. Las colonias de este género tienen una morfología característica. Los recientes esfuerzos de muestreo en el noreste Atlántico han proporcionado un número de colonias inicialmente atribuibles al género Pennatula. Tanto el estudio morfológico como molecular (genes mtMutS, Cox1 y 28S) de este material respaldan la naturaleza polifilética de este género, y la necesidad de resucitar al género Ptilella Gray, 1870 para acomodar a éstas y otras especies. Una nueva especie, Ptilella grayi n. sp., es descrita e ilustrada. La especie Pennatula bayeri es propuesta como sinónimo reciente de Pennatula bellissima (aquí también considerada en el género Ptilella). Palabras clave: noreste Atlántico; Pennatulacea; Ptilella grayi; pluma de mar; nueva especie. Citation/Como citar este artículo: García-Cárdenas F.J., Drewery J., López-González P.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(3): 261-276. https://doi.org/10.3989/scimar.04845.26A LSID: http://zoobank.org/urn:lsid:zoobank.org:pub:2E460301-B5FC-43E5-8393-9D803E034D02 Editor: D. Vaqué. Received: August 22, 2018. Accepted: June 7, 2019. Published: July 9, 2019: Copyright: © 2019 CSIC. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) License. Scientia Marina 83(3) September 2019, 261-276, Barcelona (Spain) ISSN-L: 0214-8358 https://doi.org/10.3989/scimar.04845.26A 262 • F.J. García Cárdenas et al. SCI. MAR. 83(3), September 2019, 261-276. ISSN-L 0214-8358 https://doi.org/10.3989/scimar.04845.26A INTRODUCTION 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 1956, Williams 2011). The order Pennatulacea includes more than 200 species in 35 genera and 14 families (López-González et al. 2001, López-González and Williams 2002, Williams 2015). 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 1977, Done 1999). The ecological importance of the recently discovered rockpens (Williams and Alderslade 2011) 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 1992, Baillon et al. 2012). 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 2010). 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 1915, Williams 1995a). Recent molecular studies in octocorals have identified the order Pennatulacea as a monophyletic group (McFadden et al. 2006, Dolan et al. 2013, Kushida and Reimer 2018). 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. 2006, Dolan et al. 2013, Kushida and Reimer 2018). Although Dolan et al. (2013: 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. 2013: 614-615). More recently (Kushida and Reimer 2018), 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. 2004, Fleck et al. 2008). The delimitation of monophyletic groupings and the establishment of their relationships is a common problem in evolutionary biology research (e.g. Wheeler and Nixon 1990, Crisp and Chandler 1996, Brummitt 2002, among many others). The current conception of the cosmopolitan genus Pennatula includes at least 14 valid species (Williams 2011). 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 1880, Kükenthal 1915, Hickson 1916). 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. METHODS 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 Resurrection of the sea pen genus Ptilella • 263 SCI. MAR. 83(3), September 2019, 261-276. ISSN-L 0214-8358 https://doi.org/10.3989/scimar.04845.26A 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 1988), 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). 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. 2014). The start of the mtMutS region was amplified using the primers ND42599F and MUT3458R (France and Hoover 2002, Sánchez et al. 2003). The Cox1 region was amplified using the primers COII8068F and COIOCTR (France and Hoover 2002, McFadden et al. 2004). The 28S nuclear ribosomal gene (28S rDNA) was amplified using the primers 28S-Far and 28S-Rar (Mc- Fadden and van Ofwegen 2013). Each PCR used 0.5 U of DNA Stream Polymerase (BIORON), 0.2 mM Fig. 1. – Known distribution of Ptilella grayi n. sp. (violet symbols) and Ptilella grandis (red symbols) in the study area and hauls with no occurrences (black dots). Symbols represent approximate midpoints of hauls. Circles indicate sampling stations where the species was recorded only, while triangles indicate sampling stations where the species was both recorded and examined over the course of this study. Map © Crown copyright. 264 • F.J. García Cárdenas et al. SCI. MAR. 83(3), September 2019, 261-276. ISSN-L 0214-8358 https://doi.org/10.3989/scimar.04845.26A 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 Table 1. – Sampling data associated with the Atlantic material of Ptilella compared in this paper. Abbreviations: NMS, National Museum of Scotland; MZB, Museu de Zoologia de Barcelona; NHM, Natural History Museum in London; BECA, Biodiversidad y Ecología Acuática of the University of Seville; n.a., not available; i.c., incomplete colony. Species Status Registration code Cruise Cruise no. (haul) Area and coordinates Start - Stop Dept range (m) Date Nº colonies (total length; mm) Ptilella grayi n. sp. Holotype NMS.Z.2019.2.1 Rockall Haddock Survey 2011 1011S (S11426) Rockall Bank, NE Atlantic 56°32.63’N 14°30.92’W 56°33.97’N 14°28.78’W 198-202 30 Aug 2011 1 (474) Ptilella grayi n. sp Paratype NMS.Z.2019.2.2 Rockall Anglerfish Survey 2010 0410S (S10150) Rockall Bank, NE Atlantic 56°25.22’N 15°14.67’W 56°22.91’N 15°18.75’W 246-261 6 Apr 2010 1 (254) Ptilella grayi n. sp Paratype NMS.Z.2019.2.3 Rockall Haddock Survey 2011 1011S (S11426) Rockall Bank, NE Atlantic 56°32.63’N 14°30.92’W 56°33.97’N 14°28.78’W 198-202 30 Aug 2011 1 (471) Ptilella grayi n. sp Paratype MZB 2018-0761 Rockall Haddock Survey 2011 1011S (S11426) Rockall Bank, NE Atlantic 56°32.63’N 14°30.92’W 56°33.97’N 14°28.78’W 198-202 30 Aug 2011 1 (391) Ptilella grayi n. sp NMS.Z.2019.2.4 OFFCON 2012 0712S (S12287) Rockall Bank, NE Atlantic 57°22.33’N 13°54.87’W 57°21.36’N 13°57.73’W 146-152 24 Jul 2012 1 (305) Ptilella grayi n. sp NMS.Z.2019.2.5 Rockall Anglerfish Survey 2013 1413S (S13371) Rockall Bank, NE Atlantic 56°36.59’N 14°26.65’W 56°35.81’N 14°32.89’W 197-199 24 Oct 2013 1 (450) Ptilella grayi n. sp MZB 2018-0762 Rockall Anglerfish Survey 2013 1413S (S13372) Rockall Bank, NE Atlantic 56°55.42’N 14°31.33’W 56°58.65’N 14°28.61’W 179-188 24 Oct 2013 1 (450) Ptilella grayi n. sp BECA OPEN-335 Rockall Anglerfish Survey 2013 1413S (S13371) Rockall Bank, NE Atlantic 56°36.59’N 14°26.65’W 56°35.81’N 14°32.89’W 197-199 24 Oct 2013 1 (575) Ptilella grayi n. sp NHMUK 2019.1 Rockall Anglerfish Survey 2013 1413S (S13371) Rockall Bank, NE Atlantic 56°36.59’N 14°26.65’W 56°35.81’N 14°32.89’W 197-199 24 Oct 2013 1 (570) Ptilella grayi n. sp MZB 2018-0763 Rockall Anglerfish Survey 2013 1413S (S13371) Rockall Bank, NE Atlantic 56°36.59’N 14°26.65’W 56°35.81’N 14°32.89’W 197-199 24 Oct 2013 1 (545) Ptilella grayi n. sp BECA OPEN-338 Rockall Anglerfish Survey 2013 1413S (S13371) Rockall Bank, NE Atlantic 56°36.59’N 14°26.65’W 56°35.81’N 14°32.89’W 197-199 24 Oct 2013 1 (572) Ptilella grayi n. sp BECA OPEN-339 Rockall Anglerfish Survey 2014 0414S (S14141) Rockall Bank, NE Atlantic 56°25.40’N 14°57.37’W 56°26.56’N 14°54.75’W 206-215 14 Apr 2014 1 i.c. (115) Ptilella grayi n. sp BECA OPEN-340 Rockall Anglerfish Survey 2014 0414S (S14141) Rockall Bank, NE Atlantic 56°25.40’N 14°57.37’W 56°26.56’N 14°54.75’W 206-215 14 Apr 2014 1 i.c. (90) Ptilella grandis (Ehrenberg, 1834) NMS.Z.2019.2.6 Deepwater Time Series 2008 1108S (S08385) Hebridean Slope, NE Atlantic 55°13.79’N 10°08.87’W 55°08.49’N 10°10.66’W 1018- 1078 14 Sept 2008 1 (256) Ptilella grandis (Ehrenberg, 1834) MZB 2018-0759 OFFCON 2011 0711S (S11264) NE Rockall, NE Atlantic 57°36.37‘N 12°36.66‘W 57°35.94‘N 12°36.98‘W 1601- 1605 30 Jun 2011 1 (182) Ptilella grandis (Ehrenberg, 1834) NHMUK 2019. 2 North Sea Anglerfish Survey 2012 0412S (S12220) S Norwegian Sea, NE Atlantic 61°55.05’N 02°28.94’E 61°52.66’N 02°34.22’E 390-396 29 Apr 2012 1 (510) Ptilella grandis (Ehrenberg, 1834) BECA OPEN-143 BIOICE 516 (3507) South Iceland, NE Atlantic 61°54.95’N, 19°17.02’W 1595 3 Sept 2002 1 (114) Ptilella inflata (Kükenthal, 1910) NMS.Z.2019.2.7 Benguela VIII 67 Namibia, SE Atlantic 23°28.03’S 13°05.08’E 418 23 Jul 1985 1 (203) Ptilella inflata (Kükenthal, 1910) NHMUK 2019. 3 Benguela VIII 67 Namibia, SE Atlantic 23°28.03’S 13°05.08’E 418 23 Jul 1985 1 (207) Ptilella inflata (Kükenthal, 1910) MZB 2018-0760: Benguela VIII 67 Namibia, SE Atlantic 23°28.03’S 13°05.08’E 418 23 Jul 1985 1 (227) Ptilella bellissima (Fowler, 1888) Holotype BMNH (1888.6.28.1) n.a. n.a. Bahamas Islands, NW Atlantic n.a. 14 Feb 1888 1 (178) Resurrection of the sea pen genus Ptilella • 265 SCI. MAR. 83(3), September 2019, 261-276. ISSN-L 0214-8358 https://doi.org/10.3989/scimar.04845.26A 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) and Kushida and Reimer (2018), 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 outgroups. 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. 2011). 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 2001, Ronquist and Huelsenbeck 2003), using the substitution model GTR+G (lset nst=6 rates=gamma) and 107 generations, and discarding 25% of the initial trees. RESULTS Subclass OCTOCORALLIA Haeckel, 1866 Order Pennatulacea Verrill, 1865 Family Pennatulidae Ehrenberg, 1834 Genus Ptilella Gray, 1870 Ptilella Gray 1870: 21; Koren and Danielssen 1874: 422, 1877: 82. Pennatula, Kölliker 1872: 136 (in part); Kölliker 1880: 4 (in part); Grieg 1892: 10 (in part); Jungersen 1904: 11 (in part); Kükenthal and Broch 1910: 348 (in part); Broch 1913: 28 (in part); Kükenthal 1915: 81 (in part); Hickson 1916: 181 (in part), 1937: 123; Williams 1995a: 125 (in part); López-González et al. 2001: 70 (in part); Altuna 2015: 2 (in part). Pennatula (Ptilella), Verrill 1883: 3, 1885: 532 (in part). Diagnosis (modified from Gray 1870: 21). Colonies pinnate. Rachis bilaterally symmetrical throughout. Rachis with a central dorsal track naked of zooids. Rachispeduncle limit with a distinct thickening or swelling, the thickening sometimes forming an edged ring at the Table 2. – Pennatulaceans included in the molecular phylogenetic analyses. Species in bold are those sequenced for this study. Note that all GenBank sequences are presented here with the names as they appear in GenBank and their original publications. Family / Species Catalog numbers (or additional information) Geographic area mtMutS(=msh1)Cox1 28S Pennatulidae Ptilella grandis BECA OPEN-143 South Iceland, NE Atlantic MK603844 MK603860 MK603854 Ptilella grandis NMS.Z.2019.2.6 Hebridean Slope, NE Atlantic MK603843 MK882496 MK882494 Ptilella grayi n. sp. (Holotype) NMS.Z.2019.2.1 Rockall Bank, NE Atlantic MK603847 MK882497 MK882495 Ptilella grayi n. sp. (Paratype) NMS.Z.2019.2.2 Rockall Bank, NE Atlantic MK603846 MK603856 MK603853 Pennatula rubra BECA OPEN-139 Alborán, Mediterranean Sea MK603845 MK603857 MK603852 Pennatula phosphorea BECA OPEN-453 Sea of the Hebrides, NE Atlantic MK603848 MK603858 MK882492 Pennatula phosphorea BECA OPEN-454 Gulf of Cádiz, NE Atlantic MK603850 MK603861 MK882491 Pennatula sp. BECA OPEN-152 Ross Sea, Antarctica MK603849 MK603859 MK882493 Pennatula phosphorea* MBARI K2-06-070110-01 USA, Eastern Pacific KX9049755KX9049575 Pennatula phosphorea* NWFSC 34212-045 USA, Eastern Pacific JN8665312KF8741902 Ptilosarcus gurneyi NWFSC 34212-082 USA, Eastern Pacific JN8665362KF8741952 Ptilosarcus gurneyi NWFSC 34213-020 USA, Eastern Pacific JN8665402KF8742012 Virgularidae Acanthoptilum gracile NWFSC 34210-026 USA, Eastern Pacific JN8665252KF8741842 Acanthoptilum gracile NWFSC 34212-029 USA, Eastern Pacific JN8665292KF8741882 Echinoptilidae Actinoptilum molle RMNH Coel.40822 n.d. GQ3424913GQ3424143JX2037384 Protoptilidae Distichoptilum gracile NTM C14561 AUS, Tasman Sea, DQ3028661GQ3424543JX2037394 Stachyptilidae Gilibelemnon octodentatum BECA OPEN-452 Seymour Island, Antarctica MK603841 MK603855 MK603851 Renillidae Renilla muelleri SCF-FLA USA, Gulf of Mexico DQ2974321NC_0183781 Renilla sp. UF4000 Off California coast, Pacific GQ3425263GQ3424553 Outgroup (Ellisellidae) Viminella sp. RMNH Coel.40032 n.d. JX2037944JX2038524JX2037034 Viminella sp. RMNH Coel.40813 n.d. GQ3424933GQ3424163JX2037054 Abbreviations: NMS, National Museum Scotland; NHM, Natural History Museum (London, UK); MZB, Museu de Zoologia de Barcelona (Spain); BECA, Biodiversidad y Ecología Acuatica (Seville, Spain); MBARI, Monterey Bay Aquarium Research Institute (California, U.S.A.); NWFSC, Northwest Fisheries Science Center (Seattle, U.S.A.); NIWA, National Institute of Water and Atmospheric Research (Auckland, New Zealand); NOCS, National Oceanography Centre (Southampton, UK), RMNH, Rijksmuseum van Natuurlijke Historie (Leiden, Netherlands); NTM, Museum and Art Gallery of the Northern Territory (Darwin city, Australia); SCF, Collection of S.C. France (U.S.A.); UF, Florida Natural History Museum (Florida; U.S.A.). n.d.: no data.*: Both specimens are from the North Eastern Pacific. However, despite these specimens being originally identified as Pennatula phosphorea, according to the phylogenetic hypothesis presented in this paper (see Fig. 5) we consider these identifications as doubtful. We suggest these findings as indicative of a morphospecies and thus deserving of further research. References: (1) McFadden et al. 2006; (2) Elz et al. unpublished; (3) Brockman and McFadden 2012; (4) McFadden and van Ofwegen 2012; (5) Everett et al. 2016. 266 • F.J. García Cárdenas et al. SCI. MAR. 83(3), September 2019, 261-276. ISSN-L 0214-8358 https://doi.org/10.3989/scimar.04845.26A 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 (1880: 2), P. pearceyi Kölliker (1880: 4), P. murrayi Kölliker (1880: 5), and P. moseleyi Kölliker (1880: 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) in describing the genus Ptilella. As this is in concordance with our molecular data, Ptilella is resurrected here. Ptilella grayi n. sp. (Figs 1-3) 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:*). Fig. 2. – Ptilella grayi n. sp. Holotype (NMS.Z.2019.2.1). A, whole colony, showing the edged ring (er) betwen peduncle (pd) and rachis (re); B, detail of autozooids (az) in oblique rows, sectioned basally; C, dorsal view of polyp leaves showing the location of mesozooids (mz), siphonozooids (sz) and the distal part devoid of polyps (asterisk); D, area between two polyp leaves from latero-dorsal view showing the location of siphonozooids (sz), autozooids (az) on the ventral edge of the polyp-leaf, and the naked dorsal track (dt). Note that some parts in B to D are stained with methylene blue for increasing contrast. Resurrection of the sea pen genus Ptilella • 267 SCI. MAR. 83(3), September 2019, 261-276. ISSN-L 0214-8358 https://doi.org/10.3989/scimar.04845.26A 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 threeflanged 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 threeflanged 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 Fig. 3. – Ptilella grayi n. sp. Holotype (NMS.Z.2019.2.1). 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). Fig. 4. – Ptilella grayi n. sp. (NMS.Z.2019.2.4). Newly collected specimen, showing a more relaxed rachis-peduncle limit, but still with a distinct edged ring. A, dorsal view; B, ventral view. Both images © Crown copyright. 268 • F.J. García Cárdenas et al. SCI. MAR. 83(3), September 2019, 261-276. ISSN-L 0214-8358 https://doi.org/10.3989/scimar.04845.26A 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. DISCUSSION Historical remarks on Ptilella and its type species Pennatula grandis was succinctly described by Ehrenberg (1834: 290) based on an old specimen in the Museum of Berlin. Later on, Sars (1846: 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), Milne Edwards and Haime (1857), Herklots (1858), Gray (1860), Richiardi (1869) and Kölliker (1869-1872). Resurrection of the sea pen genus Ptilella • 269 SCI. MAR. 83(3), September 2019, 261-276. ISSN-L 0214-8358 https://doi.org/10.3989/scimar.04845.26A Milne Edwards and Haime (1857: 211) pointed out that Ehrenberg’s description of Pennatula grandis accorded well with that of Sars’s Pennatula borealis, using this last nomenclature. Richiardi (1869: 31) also considered Pennatula grandis a synonym of Pennatula borealis. Gray (1870: 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 (1872: 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) also described an additional specimen of 350 mm in length deposited in the collection of the Museum of Copenhagen. Koren and Danielssen (1874: 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 1877: 82). Kölliker (1880: 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 (1883, 1885) considered Ptilella a subgenus of Pennatula, as Pennatula (Ptilella) borealis Sars. Subsequent contributions by Grieg (1892: 10), Storm (1901), Jungersen (1904). Kükenthal and Broch (1910) and Broch (1913) do not consider Ptilella at all but simply mention the species as Pennatula grandis. Despite this, Balss (1910: 54) started to form groups among the species in Pennatula that he considered valid, while Kükenthal and Broch (1911: 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 1915, Hickson 1916: 181). However, Hickson (1937: 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 1995a: 126) or disappeared from the scarce specialized literature that was Fig. 5. – Bayesian analysis showing the phylogenetic relationships of Ptilella, Pennatula and other genera and species of sea pens (Clade II of Dolan et al. 2013, and Kushida and Reimer 2018) based on the concatenated mitochondrial sequences mtMutS+Cox1+28S. Sequences provided by this study are in bold. See Table 2 for a complete list of species and GenBank accession numbers. The asterisks indicate a pair of sequenced samples from the northeastern Pacific originally identified as Pennatula phosphorea. According to the results of the present analysis, which includes specimens of Pennatula phosphorea from the northeastern Atlantic [the original distribution area indicated by Linnaeus (1758: 818) and Ellis 1764: 420], the identity of the Pacific specimens is considered here as doubtful and deserving of additional morphological and molecular study. 276 • F.J. García Cárdenas et al. SCI. MAR. 83(3), September 2019, 261-276. ISSN-L 0214-8358 https://doi.org/10.3989/scimar.04845.26A https://doi.org/10.1006/zjls.1995.0004 Williams 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. Williams G.C. 2011. The Global Diversity of Sea Pens (Cnidaria: Octocorallia: Pennatulacea). PLoS ONE 6: e22747. https://doi.org/10.1371/journal.pone.0022747 Williams G.C. 2015. A new genus and species of pennatulacean octocoral from equatorial West Africa (Cnidaria, Anthozoa, Virgulariidae). Zookeys 546: 39-50. https://doi.org/10.3897/zookeys.546.6344 Williams 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. https://doi.org/10.11646/zootaxa.3001.1.2 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). Resurrection of the sea pen genus Ptilella Gray, 1870 and description of Ptilella grayi n. sp. from the NE Atlantic (Octocorallia: Pennatulacea) Francisco J. García-Cárdenas, Jim Drewery, Pablo J. López-González Supplementary material Scientia Marina 83(3) September 2019, S1-S6, Barcelona (Spain) ISSN-L: 0214-8358 S2 • F.J. García Cárdenas et al.. SCI. MAR. 83(3), September 2019, S1-S6. ISSN-L 0214-8358 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. Species Cruise Cruise no (haul) Area Coordinates Start - Stop Depth range (m) Date No. colonies Ptilella grayi n. sp. Rockall Haddock Survey 2005 1205S (S05357) Rockall Bank, NE Atlantic 56º35.49N 14º31.57W- 56º36.52N 14º28.72W 197-198 06-Sep-05 3 Ptilella grayi n. sp. Rockall Haddock Survey 2007 1207S (S07371) Rockall Bank, NE Atlantic 57º09.39N 14º12.16W- 57º09.59N 14º08.94W 166-170 13-Sep-07 1 Ptilella grayi n. sp. Rockall Haddock Survey 2007 1207S (S07386) Rockall Bank, NE Atlantic 56º32.78N 14º32.25W- 56º31.32N 14º34.00W 198-203 12-Sep-07 2 Ptilella grayi n. sp. Rockall Haddock Survey 2008 1108S (S08358) Rockall Bank, NE Atlantic 56º28.03N 14º41.91W- 56º27.82N 14º38.40W 199-202 06-Sep-08 5 Ptilella grayi n. sp. Rockall Haddock Survey 2008 1108S (S08360) Rockall Bank, NE Atlantic 56º35.43N 14º32.20W- 56º36.37N 14º29.54W 197-199 06-Sep-08 1 Ptilella grayi n. sp. Rockall Anglerfish Survey 2009 0409S (S09168) Rockall Bank, NE Atlantic 56º43.54N 14º34.52W- 56º46.84N 14º33.03W 187-195 5-May-09 1 Ptilella grayi n. sp. Rockall Anglerfish Survey 2009 0409S (S09169) Rockall Bank, NE Atlantic 56º37.05N 14º25.39W- 56º36.63N 14º31.29W 191-200 5-May-09 3 Ptilella grayi n. sp. Rockall Anglerfish Survey 2009 0409S (S09170) Rockall Bank, NE Atlantic 57º17.82N 14º14.45W- 57º14.44N 14º14.93W 170-178 6-May-09 3 Ptilella grayi n. sp. Rockall Anglerfish Survey 2009 0409S (S09172) Rockall Bank, NE Atlantic 57º22.21N 13º52.59W- 57º21.17N 13º58.32W 145-153 6-May-09 1 Ptilella grayi n. sp. Rockall Haddock Survey 2009 1209S (S09339) Rockall Bank, NE Atlantic 57º03.65N 14º08.69W- 57º02.74N 14º11.61W 167-191 07-Sep-09 1 Ptilella grayi n. sp. Rockall Haddock Survey 2009 1209S (S09347) Rockall Bank, NE Atlantic 56º36.11N 14º29.97W- 56º35.20N 14º32.73W 194-201 09-Sep-09 1 Ptilella grayi n. sp. Rockall Haddock Survey 2009 1209S (S09349) Rockall Bank, NE Atlantic 56º43.69N 14º30.18W- 56º44.51N 14º29.62W 192-198 09-Sep-09 2 Ptilella grayi n. sp. Rockall Haddock Survey 2009 1209S (S09350) Rockall Bank, NE Atlantic 56º47.90N 14º05.97W- 56º46.39N 14º08.07W 193-198 10-Sep-09 1 Ptilella grayi n. sp. Rockall Anglerfish Survey 2010 0410S (S10150) Rockall Bank, NE Atlantic 56º25.22N 15º14.67W- 56º22.91N 15º18.75W 238-262 06-Apr-10 1 Ptilella grayi n. sp. Rockall Haddock Survey 2011 0911S (S11426) Rockall Bank, NE Atlantic 56º32.63N 14º30.92W- 56º33.97N 14º28.78W 198-202 30-Aug-11 12 Ptilella grayi n. sp. Rockall Haddock Survey 2011 0911S (S11427) Rockall Bank, NE Atlantic 56º35.18N 14º24.27W- 56º36.48N 14º22.06W 204-209 30-Aug-11 2 Ptilella grayi n. sp. Rockall Haddock Survey 2011 0911S (S11430) Rockall Bank, NE Atlantic 56º42.54N 14º03.67W- 56º43.71N 14º01.29W 251-255 30-Aug-11 1 Ptilella grayi n. sp. Rockall Haddock Survey 2011 0911S (S11432) Rockall Bank, NE Atlantic 57º02.48N 13º52.08W- 57º03.75N 13º49.62W 182-186 30-Aug-11 1 Ptilella grayi n. sp. Rockall Anglerfish Survey 2012 0412S (S12141) Rockall Bank, NE Atlantic 55º51.10N 15º19.53W- 55º52.24N 15º13.35W 365-375 14-Apr-12 1 Ptilella grayi n. sp. Rockall Anglerfish Survey 2012 0412S (S12145) Rockall Bank, NE Atlantic 56º32.34N 14º55.91W- 56º35.17N 15º00.02W 195-225 15-Apr-12 3 Ptilella grayi n. sp. Rockall Anglerfish Survey 2012 0412S (S12146) Rockall Bank, NE Atlantic 56º53.19N 14º44.00W- 56º55.71N 14º39.29W 179-192 15-Apr-12 1 Ptilella grayi n. sp. OFFCON 2012 0712S (S12287) Rockall Bank, NE Atlantic 57º22.33N 13º54.87W- 57º21.36N 13º57.73W 146-152 15-Apr-12 1 Ptilella grayi n. sp. Rockall Haddock Survey 2013 1113S (S13304) Rockall Bank, NE Atlantic 56º43.65N 14º14.44W- 56º41.83N 14º14.62W 196-203 14-Sep-13 2 Ptilella grayi n. sp. Rockall Haddock Survey 2013 1113S (S13305) Rockall Bank, NE Atlantic 56º36.15N 14º22.46W- 56º34.79N 14º24.46W 206-209 14-Sep-13 1 Ptilella grayi n. sp. Rockall Haddock Survey 2013 1113S (S13306) Rockall Bank, NE Atlantic 56º36.05N 14º30.02W- 56º35.75N 14º33.40W 196-199 14-Sep-13 6 Ptilella grayi n. sp.Rockall Haddock Survey 2013 1113S (S13307) Rockall Bank, NE Atlantic 56º40.01N 14º39.58W- 56º39.38N 14º40.49W 195-198 14-Sep-13 2 Ptilella grayi n. sp. Rockall Anglerfish Survey 2013 1413S (S13371) Rockall Bank, NE Atlantic 56º36.59N 14º26.65W- 56º35.81N 14º32.89W 197-199 24-Oct-13 82 Ptilella grayi n. sp. Rockall Anglerfish Survey 2013 1413S (S13372) Rockall Bank, NE Atlantic 56º55.42N 14º31.33W- 56º58.65N 14º28.61W 179-188 24-Oct-13 2 Ptilella grayi n. sp. Rockall Anglerfish Survey 2013 1413S (S13373) Rockall Bank, NE Atlantic 57º10.17N 14º11.27W- 57º12.81N 14º06.85W 165-171 24-Oct-13 4 Ptilella grayi n. sp. Rockall Anglerfish Survey 2014 0414S (S14141) Rockall Bank, NE Atlantic 56º25.40N 14º57.37W- 56º26.56N 14º54.75W 206-212 14-Apr-14 2 Ptilella grayi n. sp.Rockall Anglerfish Survey 2014 0414S (S14144) Rockall Bank, NE Atlantic 56º41.94N 14º32.66W- 56º45.50N 14º32.42W 193-199 15-Apr-14 2 Ptilella grayi n. sp. Rockall Anglerfish Survey 2015 0415S (S15142) Rockall Bank, NE Atlantic 56º36.04N 14º14.27W- 56º32.91N 14º16.67W 260-299 11-Apr-15 1 Resurrection of the sea pen genus Ptilella • S3 SCI. MAR. 83(3), September 2019, S1-S6. ISSN-L 0214-8358 Species Cruise Cruise no (haul) Area Coordinates Start - Stop Depth range (m) Date No. colonies Ptilella grayi n. sp. Rockall Anglerfish Survey 2015 0415S (S15143) Rockall Bank, NE Atlantic 56º36.27N 14º26.94W- 56º35.43N 14º32.96W 194-205 12-Apr-15 17 Ptilella grayi n. sp. Rockall Anglerfish Survey 2015 0415S (S15144) Rockall Bank, NE Atlantic 56º29.97N 14º21.34W- 56º27.17N 14º24.64W 281-287 12-Apr-15 3 Ptilella grayi n. sp. Rockall Anglerfish Survey 2015 0415S (S15157) Rockall Bank, NE Atlantic 56º25.35N 14º56.29W- 56º27.76N 14º51.81W 204-214 14-Apr-15 1 Ptilella grayi n. sp. Rockall Anglerfish Survey 2015 0415S (S15159) Rockall Bank, NE Atlantic 56º34.02N 15º13.28W- 56º30.69N 15º15.78W 266-289 14-Apr-15 1 Ptilella grayi n. sp. Rockall Haddock Survey 2015 1115S (S15351) Rockall Bank, NE Atlantic 56º35.93N 14º31.35W- 56º36.48N 14º28.27W 196-198 31-Aug-15 1 Ptilella grayi n. sp. Rockall Haddock Survey 2015 1115S (S15352) Rockall Bank, NE Atlantic 56º36.95N 14º16.25W- 56º38.10N 14º13.72W 226-231 31-Aug-15 1 Ptilella grayi n. sp. Rockall Haddock Survey 2015 1115S (S15356) Rockall Bank, NE Atlantic 57º02.91N 13º25.79W- 57º01.49N 13º27.53W 270-278 01-Sep-15 1 Ptilella grayi n. sp. Rockall Anglerfish Survey 2016 0416S (S16137) Rockall Bank, NE Atlantic 57º36.48N 12º57.47W- 57º34.60N 12º57.59W 280-290 09-Apr-16 1 Ptilella grayi n. sp. Rockall Anglerfish Survey 2016 0416S (S16164) Rockall Bank, NE Atlantic 56º15.82N 16º14.36W- 56º12.51N 16º15.49W 358-389 13-Apr-16 2 Ptilella grayi n. sp. Rockall Haddock Survey 2016 1216S (S16365) Rockall Bank, NE Atlantic 56º33.53N 14º29.33W- 56º32.22N 14º31.75W 199-201 05-Sep-16 3 Ptilella grayi n. sp. Rockall Haddock Survey 2016 1216S (S16366) Rockall Bank, NE Atlantic 56º46.45N 14º27.75W- 56º44.81N 14º29.19W 186-193 05-Sep-16 3 Ptilella grayi n. sp. Rockall Haddock Survey 2016 1216S (S16368) Rockall Bank, NE Atlantic 56º47.22N 14º05.67W- 56º45.63N 14º07.23W 193-200 05-Sep-16 1 Ptilella grayi n. sp. Rockall Haddock Survey 2016 1216S (S16372) Rockall Bank, NE Atlantic 57º07.74N 13º23.21W- 57º06.31N 13º25.09W 215-223 06-Sep-16 1 Ptilella grandis Deepwater Time Series 2008 1108S (S08385) Donegal Slope, NE Atlantic 55º13.79N 10º08.87W- 55º08.49N 10º10.66W 1018-1045 14-Sep-08 1 Ptilella grandis OFFCON 2011 0711S (S11264) Rockall Basin, NE Atlantic 57º36.37N 12º36.66W- 57º35.94N 12º36.98W 1601-1605 30-Jun-11 1 Ptilella grandis North Sea Anglerfish Survey 2016 0412S (S12220) S Norwegian Sea, NE Atlantic 61º55.05N 02º28.94E- 61º52.66N 02º34.22E 380-404 29-Apr-12 1 Ptilella grandis MoreDeep 2014 1314S (S14379)Rosemary Seamount, NE Atlantic 59º26.55N 10º09.34W- 59º26.60N 10º02.14W 859-889 15-Sep-14 1 Ptilella grandis MoreDeep 2014 1314S (S14382)Rosemary Seamount, NE Atlantic 59º05.37N 09º54.16W- 59º06.40N 09º51.58W 1052-1075 16-Sep-14 1 Ptilella grandis Rockall Anglerfish Survey 2016 0416S (S16151) West Rockall Bank, NE Atlantic 56º57.12N 14º15.98W- 56º56.60N 14º21.78W 723-761 11-Apr-16 1 Ptilella grandis Rockall Anglerfish Survey 2016 0416S (S16155) West Rockall Bank, NE Atlantic 56º36.27N 14º26.94W- 56º35.43N 14º32.96W 682-727 11-Apr-16 1 Ptilella grandis MoreDeep 2016 1316S (S16379)Rosemary Seamount, NE Atlantic 59º06.16N 09º52.17W- 59º05.16N 09º54.75W 1061-1100 19-Sep-16 2 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). Loci Nucleotide positions Nucleotide triplet Ptilella / Pennatula Aa position Aa substitution Ptilella / Pennatula mtMutS 34 ATG / GTA(GTG)112 Met (M) / Val (V) 74 CCG / CAG 25 Pro (P) / Glm (Q) 164 GCT / GTT 55 Ala (A) / Val (V) 197 GGA / GAA 66 Gly (G) / Glu (E) 199 GTA / ATA 67 Val (V) / Ile (I 533 GCC / GTC 181 Ala (A) / Val (V) Cox1 226 TTG / ATG 76 Leu (L) / Met (M) 271 TCA / GCA 91 Ser (S) / Ala (A) 371 GCG(GCA)2 / GTA 124 Ala (A) / Val (V) 388 ATT / CTT 130 Ile (I) / Leu (L) 439 AGT / CTA(TTA) 147 Met (M) / Leu (L) 568 GTA / ATA 190 Val (V) / Ile (I) 574 GTT / ATT 192 Val (V) / Ile (I) 1 and 2, the third base of this triplet is a variable position within that genus (interspecific variability). The substitutions implying intergeneric differences occur in the first base of the triplet in 1, but in the second base in 2. S4 • F.J. García Cárdenas et al.. SCI. MAR. 83(3), September 2019, S1-S6. ISSN-L 0214-8358 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). Loci Nucleotide (position) Pt. grayi / Pt. grandis Amino acid (position) Pt. grayi / Pt. grandis mtMutS (249) A / C (83) Met / Ile (672) C / A (225) Phe / Leu Cox1 (372) A / G (124) Ala / Ala (624) C / A (208) Ile / Ile 28S (166) C / A (210) A / G (519) G / C 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. Resurrection of the sea pen genus Ptilella • S5 SCI. MAR. 83(3), September 2019, S1-S6. ISSN-L 0214-8358 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 and edged ring. B, C and D stained with methylene blue to increase contrast. S6 • F.J. García Cárdenas et al.. SCI. MAR. 83(3), September 2019, S1-S6. ISSN-L 0214-8358 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).