INTRODUCTIONTop

Anthozoan cnidarians are considered to be one of the most important marine bio-constructors, and they often dominate on rocky substrata (Roberts et al. 2006Roberts J.M., Wheeler A.J., Freiwald A. 2006. Reefs of the deep: the biology and geology of cold-water coral ecosystems. Science 312: 543-547., Mortensen et al. 2008Mortensen P.B., Buhl-Mortensen L., Gebruk A.V., et al. 2008. Occurrence of deep-water corals on the Mid-Atlantic Ridge based on MAR-ECO data. Deep-Sea Res. Part II 55: 142-152.). These structurally complex communities provide refuge and food for both larval and adult stages of a rich associated fauna by establishing numerous symbiotic relationships as well as trophic interactions (Sammarco and Coll 1992Sammarco P.W., Coll J.C. 1992. Chemical adaptations in the Octocorallia: evolutionary considerations. Mar. Ecol. Prog. Ser. 88: 93-104., Roberts et al. 2010Roberts J.M., Wheeler A.J., Freiwald A., et al. 2010. The biology and geology of deep-sea coral habitats. Oceanography 23: 226-227., 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.). Soft bottoms account for about 95% of ocean depths (Cognetti et al. 2001Cognetti G., Sarà M., Magazzù G. 2001. Biología marina. Ed. Ariel, Barcelona. 619 pp.), but they are an unstable substrate for the settlement of most of the anthozoan species and genera dominating in rocky areas (Chia and Crawford 1973Chia F.S., Crawford B.J. 1973. Some observations on gametogenesis, larval development and substratum selection of the sea pen Ptilosarcus guerneyi. Mar. Biol. 23: 73-82.). However, several anthozoans show adaptations (e.g. hooked basal parts in the antipatharian genus Schizopathes; tree-root bases in some species of the soft coral genus Anthomastus and the isidid Isidella; or elongated bodies in the order Ceriantharia) to life in these habitats (see Jungersen 1927Jungersen H.F.E. 1927. Anthomastus. The Danish Ingolf-Expedition. Vol. V. Bianco Luno. Copenhagen., Tiffon 1987Tiffon Y. 1987. Ordre des Cérianthaires. In: Grassé P (ed) Traité deZoologie: Anatomie, Systématique, Biologie - Cnidaires/Anthozoaires - Tome III. Masson, Paris, pp. 210-256., Opresko 2002Opresko D.M. 2002. Revision of the Antipatharia (Cnidaria: Anthozoa). Part II. Schizopathidae. Zool. Opresko Meded. Leiden 76: 411-442., among others). Among the octocorals, pennatulaceans seem to be the most specialized group, with significant morphological adaptations, such as the presence of a muscular peduncle serving as an anchor system on soft sediments (Herklots 1858Herklots J.A. 1858. Notices pour servir à l’étude des polypiers nageurs ou pennatulidés. Bijdragen tot de Dierkunde 7: 1-31., Tixier-Durivault 1965Tixier-Durivault A. 1965. Quelques octocoralliaires australiens. Bull. Mus. Natl. Hist. Nat. 4: 705-716., Williams 2011Williams G.C. 2011. The Global Diversity of Sea Pens (Cnidaria: Octocorallia: Pennatulacea). PloS ONE 6: e22747.), although a few rock-inhabiting sea pen species have been discovered, modifying the basal portion of the peduncle as a holdfast for attachment to rocky substrata (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.).

The order Pennatulacea contains more than 200 species considered valid, distributed in 37 genera and 14 families (Williams 2011Williams G.C. 2011. The Global Diversity of Sea Pens (Cnidaria: Octocorallia: Pennatulacea). PloS ONE 6: e22747., 2015Williams G.C. 2015. A new genus and species of pennatulacean octocoral from equatorial West Africa (Cnidaria, Anthozoa, Virgulariidae). Zookeys 546: 39-50., García-Cárdenas et al. 2019García-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: 261-276.). They are present in all oceans, with a bathymetric distribution ranging from intertidal zones to a depth of about 6100 m (Williams 2011Williams G.C. 2011. The Global Diversity of Sea Pens (Cnidaria: Octocorallia: Pennatulacea). PloS ONE 6: e22747.). Some sea pen species form extensive meadows modifying the habitat and increasing the local diversity because of the rich fauna associated with them (Hughes 1998Hughes D.J. 1998. Sea pens and burrowing megafauna (volume III). An overview of dynamics and sensitivity characteristics for conservation management of marine SACs. Scottish Association for Marine Science (UK Marine SACs Project). 105 pp., Baillon et al. 2014Baillon S., Hamel J.F., Mercier A. 2014. Diversity, distribution and nature of faunal associations with deep-sea pennatulacean corals in the Northwest Atlantic. PloS ONE 9: e111519., Clippele et al. 2015Clippele L.H., Buhl-Mortensen P., Buhl-Mortensen L. 2015. Fauna associated with cold water gorgonians and sea pens. Cont. Shelf. Res. 105: 67-78.). This important ecological role has been recognized by including pennatulaceans and their associated megafauna in the OSPAR list of threatened and/or declining species and habitats (Jones et al. 2000Jones L.A., Hiscock K., Connor D.W. 2000. Marine habitat reviews, a summary of ecological requirements and sensitivity characteristics for the conservation and management of Marine SAC’s. Peterborough: Joint Nature Conservation Committee (UK Marine SAC’s Project Report). 178 pp., Curd 2010Curd A. 2010. Background Document for sea pen and burrowing megafauna communities., Biodiversity Series. OSPAR Commission, Ospar Convention for the Protection of the Marine Environment of the Northeast Atlantic, 26 pp.).

Pennatulaceans are colonial organisms with a muscular peduncle anchoring the colony to soft substrata, and a polypary (or rachis) where zooids are found (Herklots 1858Herklots J.A. 1858. Notices pour servir à l’étude des polypiers nageurs ou pennatulidés. Bijdragen tot de Dierkunde 7: 1-31., Kükenthal 1912Kükenthal W. 1912. Der Stammbaum der Seefedem. Verhandlungen der international Zoologischen Kongress Jena 8: 563-570.). Both the colonial structure and common tissues are generated from the initial polyp, called the oozooid (Williams et al. 2012Williams G.C., Hoeksema B.W., van Ofwegen L.P. 2012. A fifth morphological polyp in pennatulacean octocorals, with a review of polyp polymorphism in the genera Pennatula and Pteroeides (Anthozoa: Pennatulidae). Zool. Stud. 51: 1006-1017.). The rest of the polyps arise by lateral budding of its body wall, and at least two kinds are distinguished: the autozooids (with a crown of eight collector tentacles, responsible for feeding and reproductive functions) and the siphonozooids (without a crown of collector tentacles, sometimes having just one with varying degrees of development) that are responsible for water exchange to and from the colonial interior (Tixier-Durivault 1965Tixier-Durivault A. 1965. Quelques octocoralliaires australiens. Bull. Mus. Natl. Hist. Nat. 4: 705-716., Williams et al. 2012Williams G.C., Hoeksema B.W., van Ofwegen L.P. 2012. A fifth morphological polyp in pennatulacean octocorals, with a review of polyp polymorphism in the genera Pennatula and Pteroeides (Anthozoa: Pennatulidae). Zool. Stud. 51: 1006-1017.). Two other types, mesozooids and acrozooids, can be present (the former in Pennatula and Ptilella and the latter in Pteroeides) (see Williams et al. 2012Williams G.C., Hoeksema B.W., van Ofwegen L.P. 2012. A fifth morphological polyp in pennatulacean octocorals, with a review of polyp polymorphism in the genera Pennatula and Pteroeides (Anthozoa: Pennatulidae). Zool. Stud. 51: 1006-1017., García-Cárdenas et al. 2019García-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: 261-276.).

The proposed classification for octocorals by Hickson (1930)Hickson S.J. 1930. On the Classification of the Alcyonaria. Proc. Zool. Soc. Lond. 100: 229-252., based on colonial forms and sclerite morphological diversity, was subsequently modified by Bayer (1981)Bayer F.M. 1981. Key to the genera of Octocorallia exclusive of Pennatulacea (Coelenterata: Anthozoa), with diagnoses of new taxa. Proc. Biol. Soc. Wash. 94: 902-947., who divided them into three main groups. Among them, pennatulaceans (O. Pennatulacea) are clearly distinguished from other octocorals [stolonate, soft corals and gorgonians (O. Alcyonacea) and blue corals (O. Helioporacea)] by the above-mentioned colonial structure (Williams 1995Williams G.C. 1995. Living genera of sea pens (Coelenterata: Octocorallia: Pennatulacea): illustrated key and synopses. Zool. J. Linn. Soc. 113: 93-140., Daly et al. 2007Daly M., Brugler M.R., Cartwright P. 2007. The phylum Cnidaria: a review of phylogenetic patterns and diversity 300 years after Linnaeus. Zootaxa 1668: 127-182., Pérez et al. 2016Pérez C.D., de Moura Neves B., Cordeiro R.T., et al. 2016. Diversity and distribution of Octocorallia. In: Goffredo S., Dubinsky Z. (eds). The Cnidaria, Past, Present and Future. Springer, Cham, Switzerland, pp. 109-123.). However, the first attempts to establish the possible phylogenetic relationships among the pennatulacean genera are attributed to Kölliker (1870)Kölliker R.A. 1870. Anatomisch-Systematische Beschreibung der Alcyonararien. I. Die Pennatuliden. Abh. Senckenb. Naturforsch. Ges. 7: 487-602., who used the morphological similarities and structural complexity of colonies to propose a common origin for the group (Kölliker 1880Kölliker R.A. 1880. Report of the Scientific Results of the Voyage of H. M. S. Challenger during the years 1873-76. Zoology 1: 1-41., Kükenthal and Broch 1911Kükenthal W., Broch H. 1911. Pennatulacea. Wissenschaftliche Ergebnisse der deutschen Tiefsee-Expedition “Valdivia” 13: 113-576.). Kükenthal (1915)Kükenthal W. 1915. Pennatularia. Das Tierreich. 43: 1-132. Verlag von R. Friedländer und Sohn, Berlin. proposed a classification in which the families were divided into two suborders: Sessiliflorae (polyps are directly located on rachis) and Subselliflorae (polyps are grouped forming high ridges or lateral leaves). Other proposals developed in the following decades (Hickson 1937Hickson S.J. 1937. The Pennatulacea. Scientific Rep. John Murray Expedition, 1933-v1934 4: 109-130., 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., 1981Bayer F.M. 1981. Key to the genera of Octocorallia exclusive of Pennatulacea (Coelenterata: Anthozoa), with diagnoses of new taxa. Proc. Biol. Soc. Wash. 94: 902-947.) continued to delve into details such as autozooid and siphonozooid distribution, as well as the shape and ornamentation of the sclerites (Williams 1995Williams G.C. 1995. Living genera of sea pens (Coelenterata: Octocorallia: Pennatulacea): illustrated key and synopses. Zool. J. Linn. Soc. 113: 93-140., Fabricius and Alderslade 2001Fabricius K., Alderslade P. 2001. Soft corals and sea fans. Australian Institute of Marine Science. Australia. 264 pp., 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.). The current classification of pennatulaceans is exclusively based on the aforementioned morphological set of characters (Williams 1997Williams G.C. 1997. Preliminary assessment of the phylogeny of Pennatulacea (Anthozoa: Octocorallia), with a reevaluation of Ediacaran frond-like fossils, and a synopsis of the history of evolutionary thought regarding the sea pens. Proc. 6th Int. Conf. Coel. Biol. 1997: 497-509.). More recently, molecular analyses have revealed possible homoplasies (e.g. the arrangement of autozooids in polyp leaves) and the consideration of non-monophyletic groupings within Pennatulacea (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. , García-Cárdenas et al. 2019García-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: 261-276.).

Hickson (1916)Hickson S.J. 1916. The Pennatulacea of the Siboga Expedition, with a general survey of the order. Siboga-Expeditie Monographs 14, Livr. 77: 265 pp., in agreement with Kükenthal (1912)Kükenthal W. 1912. Der Stammbaum der Seefedem. Verhandlungen der international Zoologischen Kongress Jena 8: 563-570. and Niedermeyer (1913)Niedermeyer A. 1913. Über einige histologische Befunde an Veretillum cynomorium. Zool. Anz. 43: 263-270., considered the veretillids (Veretillidae) to be the most primitive pennatulaceans, the order probably being derived from an alcyoniid ancestor with radial colony symmetry (related to the soft coral genus Anthomastus). These thoughts on the basal group of the pennatulaceans were considered feasible until the end of the 20^th century (Williams 1994Williams G.C. 1994. Biotic diversity, biogeography and phylogeny of pennatulacean octocorals associated with coral reefs in the Indo-Pacific. Proc. 7th Int. Coral Reef Symp. 1994: 739-745., 1997Williams G.C. 1997. Preliminary assessment of the phylogeny of Pennatulacea (Anthozoa: Octocorallia), with a reevaluation of Ediacaran frond-like fossils, and a synopsis of the history of evolutionary thought regarding the sea pens. Proc. 6th Int. Conf. Coel. Biol. 1997: 497-509.). The incorporation of molecular analysis to phylogenetic reconstruction in octocorals [based on mitochondrial markers msh1 (henceforth mtMutS) and ND2] has strongly supported gorgonians of the family Ellisellidae (one of the five families traditionally included in the Suborder Calcaxonia; see Grasshoff 1999Grasshoff M. 1999. The shallow water gorgonians of New Caledonia and adjacent islands (Coelenterata: Octocorallia). Senchenb. Biol. 78: 1-245.) as the sister group of the pennatulaceans (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.). Additional molecular markers, such as Cox1 and the nuclear 28S rDNA, corroborated the close relationship between ellisellids and pennatulaceans (McFadden et al. 2010McFadden C.S., Sánchez J.A., France S.C. 2010. Molecular phylogenetic insights into the evolution of Octocorallia: a review. Integr. Comp. Biol. 50: 389-410.). Consequently, Williams (2019)Williams G.C. 2019. A new genus and species of enigmatic gorgonian coral from the Ryukyu Archipelago, northwestern Pacific, with a discussion of calcaxonian systematics (Cnidaria, Anthozoa, Octocorallia). Zootaxa 4701: 417-433. proposed two alternative scenarios for the placement of pennatulaceans: 1) pennatulaceans must be included in the Calcaxonia along with the five previously recognized gorgonian families (Ellisellidae, Ifalukellidae, Primnoidae, Chrysogorgiidae and Isididae; see Grasshoff 1999Grasshoff M. 1999. The shallow water gorgonians of New Caledonia and adjacent islands (Coelenterata: Octocorallia). Senchenb. Biol. 78: 1-245.); and 2) ellisellids must be removed from the Calcaxonia and included together with pennatulaceans in a clade named Actinaxonia (sensu Williams 2019Williams G.C. 2019. A new genus and species of enigmatic gorgonian coral from the Ryukyu Archipelago, northwestern Pacific, with a discussion of calcaxonian systematics (Cnidaria, Anthozoa, Octocorallia). Zootaxa 4701: 417-433.). However, these hypotheses need to be tested using a more comprehensive molecular phylogeny of octocorals.

Although there were discrepancies regarding the sister group of pennatulaceans, both the morphological and molecular methodological approaches recognized the monophyletic origin of pennatulaceans which, according to the oldest undisputed fossil record of pennatulaceans, might have occurred in the Late Cretaceous (Reich and Kutscher 2011Reich M., Kutscher M. 2011. Sea pens (Octocorallia: Pennatulacea) from the Late Cretaceous of northern Germany. J. Paleontol. 85: 1042-1051.).

McFadden et al. (2014)McFadden 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. complemented their initial mitochondrial barcode for octocorals (Cox1+igr1+msh1, McFadden et al. 2011McFadden C.S., Benayahu Y., Pante E., et. al. 2011. Limitations of mitochondrial gene barcoding in Octocorallia. Mol. Ecol. Resour. 11: 19-31.) with a nuclear segment (28S rDNA), constituting this multilocus sequence (mtMutS+Cox1+28S rDNA), which has been considered a more accurate barcode for identifying species of octocorals and is useful in the identification of intra- and intergeneric relationships of a selected group of pennatulaceans (García-Cárdenas et al. 2019García-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: 261-276.).

Most recent phylogenetic studies in pennatulaceans based on mitochondrial genes (mtMutS and ND2) have proposed the existence of four main clades (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, given the low evolution rate of the mitochondrial genome in octocorals, and its uniparental inheritance, phylogenetic hypotheses relying solely on mtDNA could be biased, while the integration of both mitochondrial and nuclear markers (such as 28S) must be preferable (Bilewitch and Degnan 2011Bilewitch J.P., Degnan S.M. 2011. A unique horizontal gene transfer event has provided the octocoral mitochondrial genome with an active mismatch repair gene that has potential for an unusual self-contained function. BMC Evol. Biol. 11: 1-14., 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., Núñez-Flores et al. 2020Núñez-Flores M., Gomez-Uchida D., López-González P.J. 2020. Molecular and morphological data reveal three new species of Thouarella (Anthozoa: Octocorallia: Primnoidae) from the Southern Ocean. Mar. Biodivers. 50: 30.).

In the present contribution, a phylogenetic reconstruction on the internal relationships among pennatulacean taxa (suborders, families and genera) is carried out. A wide taxonomic coverage (providing 38 new pennatulacean sequences, 13 mtMutS, 13 Cox1, and 12 28S), based on the previously proposed concatenated barcode for octocorals (mtMutS, Cox1, and 28S) is used for that purpose. Phylogenetic relationships proposed in previous molecular studies are discussed, as well as the monophyletic or non-monophyletic nature of sea pen families and genera. Also included here for the first time is a divergence time estimation for pennatulaceans, which provides insights into the origination time of the different lineages comprising sea pens.

methodSTop

Sampling

The materials examined herein were collected during various surveys over different geographical areas and sampling programmes: the northeastern Arctic-Atlantic (BIOICE programme), the northeastern Atlantic (Scotia cruises, INDEMARES Chica), the Mediterranean Sea (INDEMARES Alborán, INDEMARES Cap de Creus), the southeastern Atlantic (BENGUELA VIII) and the Antarctic Peninsula and the eastern Weddell Sea (Polarstern cruises ANT XVII/3, ANT XIX/5, and ANT XXIII/8) (see Table 1).

During the different expeditions, the specimens were sorted and labelled on board. The colonies (or a tissue sample from each one) were directly fixed in 100% ethanol for further molecular studies. The remaining part of the colonies was fixed in hexamethylenetetramine-buffered 4% formalin-seawater or 70% ethanol. After the fixation period, all colonies were preserved in 70% ethanol. The sequenced voucher specimens are deposited in the Museu de Zoologia de Barcelona (MZB), in the Muséum National d’Histoire Naturelle (MNHN) in Paris and in the collection of the Biodiversidad y Ecología Acuática research group of the University of Seville (BECA).

Molecular analyses

DNA extraction and PCR profiles.

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 and Cox1) and a nuclear region (28S rDNA) were sequenced. 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. Molecular phylogenetic evidence supports a new family of octocorals and a new genus of Alcyoniidae (Octocorallia, Alcyonacea). Zookeys 346: 59-83.). 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 it 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. The PCR products were purified using the NucleoSpin® Extract II DNA Purification Kit, following the manufacturer’s instructions. The purified products were electrophoresed on an ABI PRISM® 3730xl genetic analyser, and sequence traces were edited using Sequencher™ v4.0.

Phylogenetic analyses

The new sequences were compared with homologous sequences obtained from GenBank (Table 1). Any sequences from GenBank showing doubtful identity were discarded for our analyses, especially some attributed to the genus Anthoptilum (already detected by 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 placement of this genus was considered according to a recent study revealing that Anthoptilum species (Anthoptilum grandiflorum MK91965 and Anthoptilum sp. 1 MK919656) have the same gene order as the bamboo corals Isididae sp. (EF622534) and Acanella eburnea (EF672731) (Hogan et al. 2019Hogan R.I., Hopkins K., Wheeler A.J., et al. 2019. Novel diversity in mitochondrial genomes of deep-sea Pennatulacea (Cnidaria: Anthozoa: Octocorallia). Mitochondr. DNA Part A. 30: 764-777.). According to previous molecular hypotheses (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., 2010McFadden C.S., Sánchez J.A., France S.C. 2010. Molecular phylogenetic insights into the evolution of Octocorallia: a review. Integr. Comp. Biol. 50: 389-410., McFadden and van Ofwegen 2012McFadden C.S., van Ofwegen L.P. 2012. Stoloniferous octocorals (Anthozoa, Octocorallia) from South Africa, with descriptions of a new family of Alcyonacea, a new genus of Clavulariidae, and a new species of Cornularia (Cornulariidae). Invertebr. Syst. 26: 331-356.), a set of ellisellid gorgonians was selected as an outgroup for the phylogenetic reconstruction (Brockman and McFadden 2012Brockman S.A., McFadden C.S. 2012. The mitochondrial genome of Paraminabea aldersladei (Cnidaria: Anthozoa: Octocorallia) supports intramolecular recombination as the primary mechanism of gene rearrangement in octocoral mitochondrial genomes. ‎Genome Biol. Evol. 4: 882-894., Everett et al. 2016Everett M.V., Park L.K., Berntson E.A., et al. 2016. Large-scale genotyping-by-sequencing indicates high levels of gene flow in the deep-sea octocoral Swiftia simplex (Nutting 1909) on the west coast of the United States. PloS ONE 11: e0165279., 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.).

Individual genes were tested for substitution saturation using the DAMBE software (Xia et al. 2003Xia X., Xie Z., Salemi M., et al. 2003. An index of substitution saturation and its application. Mol. Phylog. Evol. 26: 1-7., Xia and Lemey 2009Xia X., Lemey P. 2009. Assessing substitution saturation with DAMBE. In: Lemey P., Salemi M., et al. (eds) The Phylogenetic Handbook: A Practical Approach to DNA and Protein Phylogeny. Cambridge University Press, Cambridge, pp. 615-630.). MtMutS, Cox1 and 28S showed low levels of substitution saturation at the third position.

The set of new sequences obtained in this study (mtMutS, Cox1, 28S) and those from GenBank were aligned using the MUSCLE alignment method implemented in MEGA 6 (Tamura et al. 2013Tamura K., Stecher G., Peterson D. et al. 2013. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol. 30: 2725-2729.). The concatenated dataset involved 45 nucleotide mtMutS, 45 Cox1 and 27 28S sequences. The alignment was 704 bp for mtMutS (63% conserved positions), 775 bp for Cox1 (80% conserved), 790 bp for 28S (54% conserved) and 2323 bp for the concatenated mtMutS+Cox1+28S dataset. After alignment, the best nucleotide substitution model was selected using Modeltest implemented in MEGA 6, according to the Akaike information criterion and hierarchical likelihood ratio test values. The phylogenetic reconstruction was obtained by applying the maximum likelihood and Bayesian inference methods. The maximum likelihood method was carried out in MEGA 6 using the nearest neighbour interchange heuristic method and 1000 bootstrap replications. The selected nucleotide substitution model was T92+G for the concatenated mtMutS+Cox1+28S. 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. For comparative purposes and discussion, the clade designations I-IV used in previous phylogenetic studies (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.) were used here, although some of these might not be supported by our study.

In order to observe the effect of the mitochondrial or nuclear marker used, additional phylogenies were obtained based on the individual markers and the concatenated mtMutS+Cox1. The conditions of phylogenetic reconstruction were similar to those described above. The selected nucleotide substitution models used were T92+G+I for mtMutS, Cox1 and mtMutS+Cox1; and K2+G for 28S. Resulting trees were included in supplementary material (Figs S2-S5). When substitution saturation was detected, two additional Bayesian inferences were carried out in the same conditions, using (A) the 1st, 2nd and 3rd codon positions of mt-genes; and (B) only the 1st and 2nd positions (excluding possible saturated positions at the 3rd codon). The resulting trees were practically identical, only with slight differences in certain node support values (for example from 0.96 PP to 0.98 PP). This indicates that the little saturation detected with DAMBE does not influence the genus relationships showed here.

Analysis of time divergence

The analysis of time divergence was undertaken within a Bayesian framework in BEAST 2.5.0 (Bouckaert et al. 2014Bouckaert R., Heled J., Kühnert D., et al. 2014. BEAST 2: a software platform for Bayesian evolutionary analysis. PloS Comput. Biol. 10: e1003537.), using the 45 taxa from which at least two of the three loci were available. BEAST allows topologies to be considered “fixed” or estimated to accommodate for phylogenetic uncertainty (Bouckaert el al. 2014Bouckaert R., Heled J., Kühnert D., et al. 2014. BEAST 2: a software platform for Bayesian evolutionary analysis. PloS Comput. Biol. 10: e1003537., Drummond and Rambaut 2007Drummond A.J., Rambaut A. 2007. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol. Biol. 7: 214.). Here, we chose the second approach because the posterior node probabilities of Bayesian phylogeny were relatively low in few cases. Several works have emphasized the importance of the rigorous selection of an appropriate clock model (e.g. Duchêne et al. 2014Duchêne S., Lanfear R., Ho S.Y. 2014. The impact of calibration and clock-model choice on molecular estimates of divergence times. Mol. Phylogenet. Evol. 78: 277-289.), and to this end four models were compared: i) relaxed with an exponential local type distribution; ii) relaxed with a log-normal type distribution; iii) random local; and iv) strict. For each model, we estimated their marginal likelihoods using the nested sampling approach implemented in the NS package of BEAST 2 (Maturana et al. 2018Maturana R.P., Brewer B.J., Klaere S., et al. 2018. Model selection and parameter inference in phylogenetics using nested sampling. Syst. Biol. 68: 219-233.) and used the Bayes factor to obtain the best-supported model. In each of these models, the three partitions were treated as linked, and we used a pure-birth (Yule) tree prior (Bouckaert el al. 2014Bouckaert R., Heled J., Kühnert D., et al. 2014. BEAST 2: a software platform for Bayesian evolutionary analysis. PloS Comput. Biol. 10: e1003537., Drummond and Rambaut 2007Drummond A.J., Rambaut A. 2007. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol. Biol. 7: 214., Drummond and Bouckaert 2015Drummond A.J., Bouckaert R.R. 2015. Bayesian evolutionary analysis with BEAST. Cambridge University Press.). The Yule model is a simple model of speciation that is generally more appropriate for considering sequences from different species (Drummond and Bouckaert 2015Drummond A.J., Bouckaert R.R. 2015. Bayesian evolutionary analysis with BEAST. Cambridge University Press.). The absolute estimates of divergence times were calculated after one-fossil calibrations (see Fossil calibration section below). For the best-supported model of molecular clock, two independent runs of 100 million generations, sampling every 10000 generations, were performed. Runs were considered complete with effective sample sizes greater than 200 for all parameters (Rambaut et al. 2018Rambaut A., Drummond A.J., Xie D., et al. 2018. Posterior summarization in Bayesian phylogenetics using Tracer 1.7. Syst. Biol. 67: 901-904.). LogCombiner (Rambaut and Drummond 2010Rambaut A., Drummond A.J. 2010. TreeAnnotator version 1.6.1. University of Edinburgh, Edinburgh, UK.) was used to combine the log files from the independent BEAST runs. TreeAnnotator (Rambaut and Drummond 2010Rambaut A., Drummond A.J. 2010. TreeAnnotator version 1.6.1. University of Edinburgh, Edinburgh, UK.) was used to summarize resulting tree samples into a single consensus tree using the maximum clade credibility, mean height options and discarding 10% of trees as burn-in. The maximum clade credibility summarized the 95% highest posterior density (HPD) limits of the node age.

Fossil calibration. The oldest undisputed pennatulacean fossil comes from several Late Cretaceous beds in Europe and North America (Reich and Kutscher 2011Reich M., Kutscher M. 2011. Sea pens (Octocorallia: Pennatulacea) from the Late Cretaceous of northern Germany. J. Paleontol. 85: 1042-1051.). These forms were assigned to the genera ‘Graphularia’ (4 spp.) and Glyptosceptron (1 sp.; see details in Reich and Kutscher 2011Reich M., Kutscher M. 2011. Sea pens (Octocorallia: Pennatulacea) from the Late Cretaceous of northern Germany. J. Paleontol. 85: 1042-1051.), which are from the lower to upper Maastrichtian (66-72.1 Ma) of the Netherlands, Germany, Belgium, USA and Ukraine. The other well-recognized fossil record of pennatulaceans is from the Early to Middle Eocene (41.2-47.8 Ma) of Trinidad (Pointe-a-Pierre Formation) based on the recognition of Virgularia presbytes (Bayer, 1955), a genus with extant representatives. The age of the most recent common ancestor of the genus Virgularia (ca. 41.2 Ma) was set as an offset, while the mean and standard deviation of the log-normal distribution was set as M=2 and S=1. We avoid using the age of the oldest undisputed pennatulacean fossil in our time calibration analyses (Reich and Kutscher 2011Reich M., Kutscher M. 2011. Sea pens (Octocorallia: Pennatulacea) from the Late Cretaceous of northern Germany. J. Paleontol. 85: 1042-1051.) because it would certainly restrict the early origin of this poorly fossiliferous clade. In the following paragraphs we use the standard abbreviation ‘Ma’ (= million years ago) for the age of a specific moment in the geological past (Aubry et al. 2009Aubry M.P., Van Couvering J.A., Christie-Blick N., et al. 2009. Terminology of geological time: Establishment of a community standard. Stratigraphy 6: 100-105.).

ResultsTop

Phylogenetic analysis

General overview

Our phylogenetic analysis based on the concatenated mtMutS+Cox1+28S data sets showed the distribution of a set of sea pen genera into two related and well-supported clades [Clade I and Clade II, bootstrap (bst) >75%, posterior probability (PP) >0.90], and the rest of genera into two groupings whose relationships varied slightly depending on the phylogenetic method used (Fig. 1). The Bayesian inference method grouped the genera Gyrophyllum, Kophobelemnon and Halipteris, as well as a sequence attributed to a species of Umbellula (Umbellula sp. 2 MK919670) with strong support (0.98 PP), and not including the genus Funiculina, which formed a separate well-supported clade. The maximum likelihood method grouped the genera Gyrophyllum, Funiculina, and Kophobelemnon with strong support (96% bst) into what we have named Clade III, as a sister group of Clade I-Clade II, while Umbellula sp. 2 was reunited with the genus Halipteris, constituting a relatively poorly supported Clade IV (60% bst) (see Fig. 1, bottom box).

The distribution of genera obtained was not in agreement with the old commonly used suborders (Sessiliflorae and Subselliflorae). The genera with polyp leaves (previously under Subselliflorae) were located in different clades (Pteroeides and Virgularia within Clade I but Ptilella, Acanthoptilum, Ptilosarcus and Pennatula within Clade II), while species without polyp leaves (previously under Sessiliflorae) were found throughout all the groupings. As a result of this, the traditional suborders Sessiliflorae and Subselliflorae can no longer be recognized here as monophyletic groupings.

Colonies with radial symmetry (characterized by their cylindrical-clavate colonial shape and zooids without permanent calyces distributed evenly over the entire surface of rachis), represented here by the genera Cavernularia, Veretillum and Actinoptilum, were observed within Clades I and II (Fig. 1), while colonies with bilateral symmetry were widely distributed throughout the tree. These examples of morphological distribution illustrate the lack of concordance between molecular and traditional morphological groupings.

Status of traditional families

Of the 14 families currently in use in Pennatulacea, 12 were included in our phylogenetic analysis (Fig. 1, families are represented by symbols in the tree). Those families represented here by a single genus (such as Anthoptilidae, Funiculinidae, Stachyptilidae, Halipteridae, Kophobelemnidae, Renillidae and Echinoptilidae), most of them with more than a single species, showed a common origin, except for Umbellulidae (see below). Those families with two or more genera (such as Protoptilidae, Pennatulidae and Virgulariidae) were not recovered as monophyletic groups, except for Veretillidae, whose genera Cavernularia and Veretillum (including its type species V. cynomorium), were reunited within Clade I with strong support (bst=100%, PP=1).

The protoptilid genera Protoptilum and Distichoptilum, although located within the same Clade II, were not shown close to one another, the latter being the sister group of Ptilella (bst=49%, PP=1). The pennatulid genera were distributed within different clades: Pteroeides within Clade I with moderate support (bst=43%, PP=0.95); Pennatula and Ptilosarcus within Clade II with strong support (bst=92%, PP=0.98); and Gyrophyllum as the sister group of Kophobelemnon (PP=0.98). Ptilella (included tentatively in Pennatulidae) was placed within Clade II with strong support (bst=99%, PP=1) but not close to the other pennatulids Pennatula and Ptilosarcus. The two virgulariid genera (Virgularia and Acanthoptilum) were placed within two different clades: Virgularia within Clade I with moderate support (bst=43%, PP=0.95), and Acanthoptilum within Clade II with strong support (bst=99%, PP=1). The family Echinoptilidae represented here by the genus Actinoptilum was placed within Clade II, as the sister group of a clade including Renilla, Acanthoptilum, Ptilosarcus and Pennatula, with strong support (bst=99%, PP=1). The family Renillidae represented by the genus Renilla was located within Clade II with strong support (bst=100%, PP=1), as the sister group of the virgulariid genus Acanthoptilum. The family Umbellulidae represented by the genus Umbellula was placed within Clade I with strong support (bst=100%, PP=1), as the sister group of Anthoptilum spp. However, a sequence identified as Umbellula sp. 2 was related to Kophobelemnon, Gyrophyllum and Halipteris (PP=0.98). In the maximum likelihood hypothesis (see Fig. 1, bottom box), this sequence was only close to Halipteris spp. with moderate support (bst= 60%) (see Discussion). The family Funiculinidae, represented by the genus Funiculina with strong support (bst=100%, PP=1), had an unsupported location outside these previously described groupings, although in the maximum likelihood tree these sequences constituted the sister group of Kophobelemnon, with low support (bst=34%). Our results indicated that the placement of Funiculina spp. is mainly due to the addition of the nuclear segment 28S and not only due to the method used (Table S1). Phylogenies (maximum likelihood and Bayesian inference) based on mtMutS (Fig. S2), Cox1 (Fig. S3) and the combined mtMutS+Cox1 (Fig. S4) supported the inclusion of Funiculina within Clade III or IV, in contrast to the phylogeny based on 28S (Fig. S5) showing an unsupported placement outside the mentioned groupings (see Table S1 and Discussion). Finally, the family Anthoptilidae represented by the genus Anthoptilum was within Clade I with moderate support (bst=79%, PP=0.95) with Umbellula spp. as the sister group.

Clade classification in Pennatulacea

Our results show that supra-generic groupings distributed in clades resulting from molecular analyses are inconsistent with the traditional grouping of families based on morphological features, as there are genera with different colonial forms gathered in the same clade. Clade I with strong support (bts=98%, PP=1) is here formed by a mixture of bilateral, radial, elongated and clavate colony growth (Fig. 1).

Clade II (bts=99%, PP=1) gathered species belonging to six families of bilateral symmetry, except for Actinoptilum (radial symmetry). This clade includes genera with long-flagelliform colonies (e.g. Protoptilum, Distichoptilum), pansy-shaped (e.g. Renilla), with polyp leaves of moderate development (e.g. Acanthoptilum) and with well-developed polyp leaves (e.g. Ptilella). Among these genera, Protoptilum, Distichoptilum and Ptilella seem to be the earliest-diverging taxa, the Pennatula grouping being the most derived one.

The final grouping was made up of species belonging to four families covering different colony morphologies, including colonies with and without polyp leaves (e.g. Gyrophyllum and Kophobelemnon, respectively), or elongated and long-flagelliform forms (e.g. Halipteris). Finally, Funiculina sequences constituted a separate grouping, without a clear relationship to previously mentioned clades.

Time-tree analysis

The estimated marginal likelihood for the time-tree analysis under different clock models indicated that the relaxed log-normal is the best supported one (Table S2). The result of this model is represented in Figure 2 (and Fig. S1). The divergence dates inferred by the Bayesian relaxed clock analyses indicated Lower Cretaceous age (Berriasian age; inferred mean age of 144.3 Ma; HPD 95%: 65.7–249.1 Ma) for the origin of Pennatulacea. In addition, the four main lineages within Pennatulacea were originated soon after, with Clade I and II diverged during the Hauterivian age (Lower Cretaceous; inferred mean age of 132.4 Ma; 95% HPD: 64.1–229.9 Ma). The groupings obtained in the BEAST analysis were similar to those from MrBayes, even considering the uncertain affinities of the genus Funiculina (Figs 2, S1). The last genus appears to begin its diversification in the middle Miocene (inferred mean age of 15.6 Ma; 95% HPD: 2.2–36.4 Ma). The most recent common ancestor of the genera Halipteris, Kophobelemnon and Gyrophyllum is from the Coniacian age (Upper Cretaceous; inferred mean age of 89.3 Ma; 95% HPD: 35.2–161 Ma). The beginning of the diversification of most genera with more than one sequence, excepting Virgularia, Gyrophyllum and Ptilella, occurred during Oligocene and Miocene times (ranging from 33.9 to 5.3 Ma). Finally, veretillids (including Cavernularia and Veretillum), appear to have diversified only after the late Eocene (inferred mean age of 36.9 Ma; 95% HPD: 10.3-70 Ma).

DiscussionTop

Monophyly of basal divergences in pennatulaceans

Hickson (1916:131)Hickson S.J. 1916. The Pennatulacea of the Siboga Expedition, with a general survey of the order. Siboga-Expeditie Monographs 14, Livr. 77: 265 pp. had already highlighted the difficulty of carrying out systematic studies on Pennatulacea based on features such as colonial symmetry because of the wide range of variation of those characters overlapping among genera and species. As has happened before with other octocoral groups, there is an evident lack of agreement between the current systematics of pennatulaceans based on morphological characters (see Kükenthal 1915Kükenthal W. 1915. Pennatularia. Das Tierreich. 43: 1-132. Verlag von R. Friedländer und Sohn, Berlin., Hickson 1930Hickson S.J. 1930. On the Classification of the Alcyonaria. Proc. Zool. Soc. Lond. 100: 229-252., Williams 1997Williams G.C. 1997. Preliminary assessment of the phylogeny of Pennatulacea (Anthozoa: Octocorallia), with a reevaluation of Ediacaran frond-like fossils, and a synopsis of the history of evolutionary thought regarding the sea pens. Proc. 6th Int. Conf. Coel. Biol. 1997: 497-509., among others) and phylogenetic hypotheses generated from molecular data (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. , among others). The presence of homoplastic characters (e.g. convergence and reversals) is a serious concern in the formulation of a reliable evolutionary hypothesis of pennatulaceans based only on morphology (Landing et al. 2015Landing E., Antcliffe J.B., Brasier M.D., et al. 2015. Distinguishing Earth’s oldest known bryozoan (Pywackia, late Cambrian) from pennatulacean octocorals (Mesozoic-Recent). J. Paleontol. 89: 292-317., Pérez et al. 2016Pérez C.D., de Moura Neves B., Cordeiro R.T., et al. 2016. Diversity and distribution of Octocorallia. In: Goffredo S., Dubinsky Z. (eds). The Cnidaria, Past, Present and Future. Springer, Cham, Switzerland, pp. 109-123.).

However, several synapomorphies observed in sea pens have traditionally been used to differentiate pennatulaceans from other octocorals and support them as a monophyletic group (Hickson 1930Hickson S.J. 1930. On the Classification of the Alcyonaria. Proc. Zool. Soc. Lond. 100: 229-252., 1937Hickson S.J. 1937. The Pennatulacea. Scientific Rep. John Murray Expedition, 1933-v1934 4: 109-130., Williams 1994Williams G.C. 1994. Biotic diversity, biogeography and phylogeny of pennatulacean octocorals associated with coral reefs in the Indo-Pacific. Proc. 7th Int. Coral Reef Symp. 1994: 739-745.). In the last decades, despite the inclusion of unsuitable markers or doubtful sequences that questioned this common origin (see results of Berntson et al. 1999Berntson E.A., France S.C., Mullineaux L.S. 1999. Phylogenetic relationships within the class Anthozoa (phylum Cnidaria) based on nuclear 18S rDNA sequences. Mol. Phylogenet. Evol. 13: 417-433., 2001Berntson E.A., Bayer F.M., McArthur A.G., et al. 2001. Phylogenetic relationships within the Octocorallia (Cnidaria: Anthozoa) based on nuclear 18S rRNA sequences. Mar. Biol. 138: 235-246.), most of the molecular studies carried out have corroborated the monophyletic origin of pennatulaceans with strong support (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., Everett et al. 2016Everett M.V., Park L.K., Berntson E.A., et al. 2016. Large-scale genotyping-by-sequencing indicates high levels of gene flow in the deep-sea octocoral Swiftia simplex (Nutting 1909) on the west coast of the United States. PloS ONE 11: e0165279., 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 use of the two traditional suborders, Sessiliflorae and Subselliflorae (Kükenthal 1915Kükenthal W. 1915. Pennatularia. Das Tierreich. 43: 1-132. Verlag von R. Friedländer und Sohn, Berlin.), until the early 1990s (e.g. Hickson 1916Hickson S.J. 1916. The Pennatulacea of the Siboga Expedition, with a general survey of the order. Siboga-Expeditie Monographs 14, Livr. 77: 265 pp., Tixier-Durivault 1965Tixier-Durivault A. 1965. Quelques octocoralliaires australiens. Bull. Mus. Natl. Hist. Nat. 4: 705-716., Williams 1990Williams G.C. 1990. The Pennatulacea of southern Africa (Coelenterata, Anthozoa). Ann. S. Afr. Mus. 99: 1-120.) has been avoided in recent decades since Williams (1995: 136)Williams G.C. 1995. Living genera of sea pens (Coelenterata: Octocorallia: Pennatulacea): illustrated key and synopses. Zool. J. Linn. Soc. 113: 93-140. questioned the validity of these groupings. In monographic works the different families were simply listed from the most structurally simple colonies (radial forms such as veretillids) to the most structurally complex ones (bilaterals bearing well-developed polyp leaves such as pennatulids), the latter often being considered from a morphological point of view as the most derived forms (Williams 1997Williams G.C. 1997. Preliminary assessment of the phylogeny of Pennatulacea (Anthozoa: Octocorallia), with a reevaluation of Ediacaran frond-like fossils, and a synopsis of the history of evolutionary thought regarding the sea pens. Proc. 6th Int. Conf. Coel. Biol. 1997: 497-509., 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.). In the present study, using a molecular barcode for octocorals and a wider taxa sampling range, this traditional classification into suborders has been clearly shown as a non-monophyletic one, in agreement with previous 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. ). Therefore, although Sessiliflorae and Subselliflorae are still mentioned from a practical point of view (e.g. Yesson et al. 2012Yesson C., Taylor M.L., Tittensor D.P., et al. 2012. Global habitat suitability of cold-water octocorals. J. Biogeogr. 39: 1278-1292., Hogan et al. 2019Hogan R.I., Hopkins K., Wheeler A.J., et al. 2019. Novel diversity in mitochondrial genomes of deep-sea Pennatulacea (Cnidaria: Anthozoa: Octocorallia). Mitochondr. DNA Part A. 30: 764-777.), it is evident that they are non-monophyletic groupings, the internal morphological evolution of each clade still being difficult to understand.

Current status of traditional families

Morphological traits have been used to categorize the extant pennatulaceans into 14 families, 37 genera and approximately 200 considered valid species (Williams 2011Williams G.C. 2011. The Global Diversity of Sea Pens (Cnidaria: Octocorallia: Pennatulacea). PloS ONE 6: e22747., 2015Williams G.C. 2015. A new genus and species of pennatulacean octocoral from equatorial West Africa (Cnidaria, Anthozoa, Virgulariidae). Zookeys 546: 39-50., García-Cárdenas et al. 2019García-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: 261-276.) according to the most important contributions concerning the order Pennatulacea (e.g. Kükenthal and Broch 1910Kükenthal W., Broch H. 1910. System und Stammesgeschichte der Seefedem. Zool. Anz. 36: 222-230., 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: 265 pp., among many others). However, recent molecular studies have failed to recover the monophyly of some of these families, such as Umbellulidae, Pennatulidae, Virgulariidae, Protoptilidae, Scleroptilidae, Stachyptilidae and Kophobelemnidae (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. ). The present study, including seven genera not previously sequenced and additional mitochondrial sequences and molecular markers, confirmed the non-monophyletic nature of some of these families (such as Protoptilidae, Virgulariidae or Pennatulidae).

The family Veretillidae, which includes most of the structurally simplest and radial colonial forms observed in pennatulaceans, have been considered since the early 19th century as transitional forms between soft corals and pennatulaceans (Koch 1878Koch G. von. 1878. Notiz über die Zooide von Pennatula. Zool. Anz. 1: 103-104.). The idea that Veretillidae and Echinoptilidae were the earliest-diverging taxa in phylogenies based on morphology spread during the early (Kükenthal and Broch 1910Kükenthal W., Broch H. 1910. System und Stammesgeschichte der Seefedem. Zool. Anz. 36: 222-230., Niedermeyer 1913Niedermeyer A. 1913. Über einige histologische Befunde an Veretillum cynomorium. Zool. Anz. 43: 263-270., Hickson 1916Hickson S.J. 1916. The Pennatulacea of the Siboga Expedition, with a general survey of the order. Siboga-Expeditie Monographs 14, Livr. 77: 265 pp.) and late (Williams 1994Williams G.C. 1994. Biotic diversity, biogeography and phylogeny of pennatulacean octocorals associated with coral reefs in the Indo-Pacific. Proc. 7th Int. Coral Reef Symp. 1994: 739-745., 1997Williams G.C. 1997. Preliminary assessment of the phylogeny of Pennatulacea (Anthozoa: Octocorallia), with a reevaluation of Ediacaran frond-like fossils, and a synopsis of the history of evolutionary thought regarding the sea pens. Proc. 6th Int. Conf. Coel. Biol. 1997: 497-509.) 20th century and the transition to the 21st century (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.), before molecular approaches postulated a different origin and basal relationships of pennatulaceans from those of the rest of the octocoral taxa (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.). In contrast to that traditional idea, and following later molecular postulates, our phylogeny does not support the hypothesis that Veretillidae and Echinoptilidae are located at a basal position, exemplifying the ancestral sea pen morphology.

Recent phylogenies based on the mitochondrial markers ND2 and mtMutS including sequences of the veretillid genera Veretillum and Cavernulina showed them to be closely related to sequences of the genus Sclerobelemnon (Kophobelemnidae) and placed all of them within Clade I with moderate support (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. : 9). Our research includes new additional veretillid sequences belonging to Cavernularia (C. pusilla) and Veretillum (including the type species V. cynomorium Pallas, 1766). In our phylogeny these veretillids (Cavernularia and Veretillum) are gathered in a clade with strong support (bst= 100%, PP= 1) within Clade I (Fig. 1). An additional exploratory maximum likelihood analysis based on mtMutS including all available mtMutS sequences from GenBank (Fig. S2) revealed that sequences attributable to the Pacific species Veretillum sp. 1 (MK133435) and Veretillum sp. 2 (MK133539, MK133545, MK133526) from Kushida and Reimer (2018) (although falling into the same Clade I) were not close to our sequence of the type species V. cynomorium, but rather close to Cavernulina and Sclerobelemnon sequences, in a different grouping. Thus, the inclusion of Pacific and Mediterranean (type species) sequences in a single genus would result in a paraphyletic taxon. This fact suggests the need for a detailed morphological study of Pacific colonies, as they could belong to a different genus, despite their possible similar morphological appearance. In short, with current information, the family Veretillidae seems to be a monophyletic grouping within Clade I (Fig. 1). The gathering of further molecular data on the remaining veretillid genera (such as Lituaria or Amphibelemnon) would be desirable in order to better delineate the monophyletic nature of the current list of genera included in the family Veretillidae.

The family Echinoptilidae (including Actinoptilum and Echinoptilum) was traditionally considered the other earliest-diverging taxon in the phylogeny of pennatulaceans, mainly because of its radially symmetrical rachis (or at least in its distal portion) (Kükenthal and Broch 1911Kükenthal W., Broch H. 1911. Pennatulacea. Wissenschaftliche Ergebnisse der deutschen Tiefsee-Expedition “Valdivia” 13: 113-576., Niedermeyer 1913Niedermeyer A. 1913. Über einige histologische Befunde an Veretillum cynomorium. Zool. Anz. 43: 263-270., Williams 1992Williams G.C. 1992. Biogeography of the octocorallian coelenterate fauna of southern Africa. Biol. J. Linn. Soc. 46: 351-401.). 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. considered radial symmetry only for those species included in Veretillidae (Veretillum, Cavernulina) within Clade I, the rest of taxa in Clade I and all taxa within the Clades II, III and IV being bilaterals (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. :5). Nevertheless, both echinoptilid genera exhibit radial symmetry in colonies to different degrees (Williams 1995Williams G.C. 1995. Living genera of sea pens (Coelenterata: Octocorallia: Pennatulacea): illustrated key and synopses. Zool. J. Linn. Soc. 113: 93-140.). The phylogeny proposed by these authors showed the genus Echinoptilum as a derived taxon with strong support within Clade II, with Renilla-Pennatula as the sister group. In our study, the sequence attributable to the other echinoptilid genus, Actinoptilum molle, was also located as a derived taxon within Clade II as the sister group of a [(Renilla-Acanthoptilum)-(Ptilosarcus-Pennatula)] clade.

The family Protoptilidae, including the genera Protoptilum and Distichoptilum, is here recognized as a non-monophyletic taxon, in agreement with 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. . The family Renillidae, constituted by the genus Renilla, was not close to Veretillidae (as was suggested from morphology, see Williams 1997Williams G.C. 1997. Preliminary assessment of the phylogeny of Pennatulacea (Anthozoa: Octocorallia), with a reevaluation of Ediacaran frond-like fossils, and a synopsis of the history of evolutionary thought regarding the sea pens. Proc. 6th Int. Conf. Coel. Biol. 1997: 497-509., Pérez and Ocampo 2001Pérez C.D., Ocampo F.C. 2001. Cladistic analysis of the pennatulacean genus Renilla Lamarck, 1816 (Coelenterata, Octocorallia). J. Nat. Hist. 35: 169-173.), supporting the results 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. . In our phylogeny, Renilla is the sister group of Acanthoptilum (Virgulariidae), and both are a sister group to Ptilosarcus-Pennatula (Pennatulidae). The family Anthoptilidae, represented here by Anthoptilum sp. 1 MK919656 and A. grandiflorum MK919655, was placed within Clade I as the sister group of Umbellula, and not occupying an ancestral position as was suggested by Dolan et al. (2013, Fig. 1)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.. As commented above, the two best-represented families of pennatulaceans Pennatulidae and Virgulariidae (Williams 1995Williams G.C. 1995. Living genera of sea pens (Coelenterata: Octocorallia: Pennatulacea): illustrated key and synopses. Zool. J. Linn. Soc. 113: 93-140.) are here recognized as non-monophyletic groupings, in agreement with 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. .

The family Umbellulidae is shown here as a non-monophyletic grouping, as observed in previous works (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. ) (Figs 1, 2, S2). As the type species of Umbellula, U. encrinus (Linnaeus, 1758), falls into Clade I, that set of sequences has to be considered as the genus Umbellula. Although the genus Umbellula was involved in old hypotheses postulating the deep-sea origin of pennatulaceans (Kölliker 1880Kölliker R.A. 1880. Report of the Scientific Results of the Voyage of H. M. S. Challenger during the years 1873-76. Zoology 1: 1-41.), other authors (Marshall 1883Marshall A.M. 1883. Report on the Pennatulida dredged by H.M.S. Triton. Trans. R. Soc. Edinb. 32: 119-152.) recognized Umbellula as a highly modified form. In the 20th century, the Umbellulidae was considered as a highly derived family among the authors who supported the shallow-water origin of pennatulaceans (Kükenthal and Broch 1911Kükenthal W., Broch H. 1911. Pennatulacea. Wissenschaftliche Ergebnisse der deutschen Tiefsee-Expedition “Valdivia” 13: 113-576., Williams 1997Williams G.C. 1997. Preliminary assessment of the phylogeny of Pennatulacea (Anthozoa: Octocorallia), with a reevaluation of Ediacaran frond-like fossils, and a synopsis of the history of evolutionary thought regarding the sea pens. Proc. 6th Int. Conf. Coel. Biol. 1997: 497-509., among others). Our research supports a later divergence of Umbellula within Clade I (Fig. 2).

The inclusion in our study of the nuclear marker 28S was useful in order to reinforce the support of Clades I and II. However, it also highlighted the unstable location of the genera Funiculina and Halipteris (see Figs 1, S2-S5, and Table S1) depending on the phylogenetic approach used. Clade III and Clade IV identified 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. were recovered in our study (with high or relatively low support, bst 96% and 60%, respectively) only when the maximum likelihood method was used, while Bayesian inference indicated a different phylogenetic hypothesis. Therefore, the inclusion of the 28S locus allows us, on the one hand, to reinforce the support of Clades I and II, but also provides evidence of the instability in clades proposed by previous studies, probably requiring greater taxonomic and molecular sampling.

In general, our results, based on a combination of mitochondrial and nuclear segments (mtMutS+Cox1+28S), indicate the monophyletic origin of most of sea pen genera with strong support (1 PP). This is one of the few common points in which morphology and molecular studies agree.

Time divergence estimation in pennatulaceans

Very little is known about the time frame of pennatulacean evolution, and our study is the first attempt to build a time-calibrated phylogeny based on Bayesian relaxed molecular clock analysis. The Lower Cretaceous (Berriasian, ~144 Ma) estimation for the divergence of the Pennatulacea from their sister group Ellisellidae is slightly older than those recently inferred (estimated age of its most recent common ancestor as 82-125 Ma; see Bilewitch 2014Bilewitch J.P. 2014. The roles of morphological diversification, depth range expansions and a novel gene in the evolution of the Octocorallia. Unpublished Phd thesis, The University of Queensland, Australia.). However, it is highly consistent with previous studies regarding estimations of the divergence time of Calcaxonia (with which pennatulaceans have been related; 120-300 Ma; Park et al. 2012Park E., Hwang D.S., Lee J.S., et al. 2012. Estimation of divergence times in cnidarian evolution based on mitochondrial protein-coding genes and the fossil record. Mol. Phylogenet. Evol. 62: 329-345.), as well as with the age of the oldest undisputed pennatulacean fossils so far recovered (Reich and Kutscher 2011Reich M., Kutscher M. 2011. Sea pens (Octocorallia: Pennatulacea) from the Late Cretaceous of northern Germany. J. Paleontol. 85: 1042-1051.).

The Cretaceous is well-known as a greenhouse period caused largely by increased CO₂ from elevated global volcanic activity (e.g. Takashima et al. 2006Takashima R., Nishi H., Huber B.T., et al. 2006. Greenhouse world and the Mesozoic ocean. Oceanography 19: 64-74., Prokoph et al. 2008Prokoph A., Shields G.A., Veizer J. 2008. Compilation and time-series analysis of a marine carbonate δ¹⁸O, δ¹³C, ⁸⁷Sr/⁸⁶Sr and δ³⁴S database through Earth history. Earth-Science Reviews 87: 113-133.), but it also is the last stage in the Gondwana break-up, and displays high rates of seafloor spreading, high sea levels (as much as 260 m above the present) and high ocean temperatures (surface waters >35°C and deep-ocean water >20°C), as well as evidence for changes in global ocean circulation (Haq et al. 1987Haq B.U., Hardenbol J., Vail P.R. 1987. Chronology of fluctuating sea levels since the Triassic. Science 235: 1156-1167., Pearson et al. 2001Pearson P.N., Ditchfield P.W., Singano J., et al. 2001. Warm tropical sea surface temperatures in the Late Cretaceous and Eocene epochs. Nature 413: 481-487., Friedrich et al. 2012Friedrich O., Norris R.D., Erbacher J. 2012. Evolution of middle to Late Cretaceous oceans-a 55 my record of Earth’s temperature and carbon cycle. Geology 40: 107-110.). The global climatic conditions during the Berriasian, when the origin of pennatulaceans likely occurs, were generally arid and the sea level was rather low (Föllmi 2012Föllmi K.B. 2012. Early Cretaceous life, climate and anoxia. Cretaceous Res. 35: 230-257.). The Berriasian also coincides with an extinction phase (Tithonian/Berriasian), which, however, apparently affected continental life more strongly than marine life (Föllmi 2012Föllmi K.B. 2012. Early Cretaceous life, climate and anoxia. Cretaceous Res. 35: 230-257.). In addition, during the Lower Cretaceous several oceanic anoxic events occurred, representing time intervals of usually relatively short duration (<1 Ma) in which intermediate and bottom-water masses became depleted in oxygen (Föllmi 2012Föllmi K.B. 2012. Early Cretaceous life, climate and anoxia. Cretaceous Res. 35: 230-257.).

Our results therefore reinforce the notion that the Cretaceous was a pivotal time in octocoral evolution, because several clades appear to begin their diversification in this epoch (e.g. primnoids; Taylor et al. 2013Taylor P.D., Berning B., Wilson M.A. 2013. Reinterpretation of the Cambrian ‘Bryozoan’ Pywackia as an Octocoral. J. Paleontol. 87: 984-990.). The main clades recovered here appear to have diverged quickly (Hauterivian-Berriasian) after their initial pennatulacean origins (Fig. 2), and this finding indicates that the early phase of their diversification took place in a greenhouse period and especially in periods of dramatic environmental changes (Prokoph et al. 2008Prokoph A., Shields G.A., Veizer J. 2008. Compilation and time-series analysis of a marine carbonate δ¹⁸O, δ¹³C, ⁸⁷Sr/⁸⁶Sr and δ³⁴S database through Earth history. Earth-Science Reviews 87: 113-133., Föllmi 2012Föllmi K. B. 2012. Early Cretaceous life, climate and anoxia. Cretaceous Res. 35: 230-257.). Consequently, changes in sea temperature and oceanic anoxic events would have played a role in forcing pennatulacean diversification, and probably also vertical displacement (submersion and emersion of lineages) throughout the complete colonizable bathymetry, as it has been demonstrated that sea pens are one of the zoological groups present over the widest bathymetric range (0-6100 m depth) (Williams 2011Williams G.C. 2011. The Global Diversity of Sea Pens (Cnidaria: Octocorallia: Pennatulacea). PloS ONE 6: e22747.). Our results also provide further support for a late origination of veretillids (late Eocene), contrasting with the early notion based on morphological features previously discussed.

Finally, it is evident that the single available fossil calibrations used on the present work have an impact on the quality of the resulting chronogram. We thus stress that this chronogram must be regarded as the first attempt to produce a time-calibrated sea pen tree. Only after achieving a wider sampling of molecular information will we be able to refine or even test the interpretations offered in the present work.

ACKNOWLEDGEMENTSTop

Part of this study was included in an MSc thesis for the master’s degree in Evolutionary Biology at the University of Seville. We would like to thank numerous colleagues and cruise leaders who have worked on the campaigns during which the material examined here was obtained: BIAÇORES, GUINEA BISSAU, BENGUELA XV, ANT XVII/3, BIOICE, ANT XIX/5, BIOROSS, ANT XXIII/8, ARCO, INDEMARES I (Menorca Channel-Cap de Creus), the Scottia cruises, INDEMARES-Alborán and INDEMARES-Chica. On these cruises, our special thanks are addressed to Francesc Pagès, Wolf Arntz, Josep-Maria Gili, Jim Drewery, Gudmundur Gudmundsson, Gudmundur Vidir, Jörundur Svavarsson, Stefano Schiaparelli, Annenina Lortz, Julian Gutt, Enrique Isla, Helmut Zibrowius, Victor Díaz-del-Río, José Luís Rueda, Serge Gofas, Ángel Luque and César Megina. This research was partially supported by the Spanish projects REN2001-4929-E/ANT (LAMPOS, ANT XIX/5), POL2006-06399/CGL (CLIMANT, ANT XXIII/8), LIFE07/NAT/E/000732 LIFE+INDEMARES (INDEMARES-Cap de Creus, INDEMARES-Alborán and INDEMARES-Chica) and CONICYT–PCHA/Doctorado Nacional/2017–21170438 (NFM). The study of the Antarctic specimens and the final conception of this paper were carried out under the project CTM2017-83920-P (DIVERSICORAL) funded by the Spanish Ministry of Economy, Industry and Competitiveness. The authors would also like to thank R. Santos Gally (Department of Plant Biology and Ecology, University of Seville) and G. Gutiérrez Pozo (Department of Genetics, University of Seville) for their assistance during the molecular analyses. Mr Tony Krupa is thanked for reviewing the English version.

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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/sm05067esm.pdf

Fig. S1. – Calibrated tree from time divergence analysis. Phylogenetic relationships were based on Bayesian inference methods for combined regions mtMutS+Cox1+28S. Bars indicate the 95% highest posterior density with the inferred mean age. Red dot indicates fossil calibration point.

Fig. S2. – Phylogenetic relationships in the order Pennatulacea based on the maximum likelihood method for the marker mtMutS. Posterior probability and bootstrap supporting values are indicated on the different nodes. See Table S3 for species and GenBank accession numbers used in this tree.

Fig. S3. – Phylogenetic relationships in the order Pennatulacea based on the maximum likelihood method for the marker Cox1. Posterior probability and bootstrap supporting values are indicated on the different nodes. See Table 1 for species and GenBank accession numbers used in this tree.

Fig. S4. – Phylogenetic relationships in the order Pennatulacea based on the maximum likelihood method for the concatenated mitochondrial markers mtMutS+Cox1. Posterior probability and bootstrap supporting values are indicated on the different nodes. See Table 1 for species and GenBank accession numbers used in this tree.

Fig. S5. – Phylogenetic relationships in the order Pennatulacea based on the maximum likelihood method for the nuclear marker 28S. Posterior probability and bootstrap supporting values are indicated on the different nodes. See Table 1 for species and GenBank accession numbers used in this tree.

Table S1. – Different composition of Clade III according to the markers and phylogenetic methods used. G, Gyrophyllum; K, Kophobelemnon; F, Funiculina; H= Halipteris; ML, maximum likelihood method; BI= Bayesian inference.

Table S2. – Results from the molecular clock model comparisons.

Table S3. – GenBank accession number of mtMutS sequences used in Figure S2.