Molecular Phylogeny of the Astrophorida (Porifera, Demospongiaep) Reveals an Unexpected High Level of Spicule Homoplasy Paco Ca´rdenas1*¤, Joana R. Xavier2,3, Julie Reveillaud4,5, Christoffer Schander1,6, Hans Tore Rapp1,6 1 Department of Biology, University of Bergen, Bergen, Norway, 2 CIBIO – Research Centre for Biodiversity and Genetic Resources, CIBIO-Azores, Biology Department, University of the Azores, Azores, Portugal, 3 CEAB – Center for Advanced Studies of Blanes (CSIC), Blanes, Spain, 4 Marine Biology Section, Biology Department, Ghent University, Ghent, Belgium, 5 CeMoFE, Center for Molecular Phylogeny and Evolution, Ghent, Belgium, 6 Centre for Geobiology, University of Bergen, Bergen, Norway Abstract Background: The Astrophorida (Porifera, Demospongiaep) is geographically and bathymetrically widely distributed. Systema Porifera currently includes five families in this order: Ancorinidae, Calthropellidae, Geodiidae, Pachastrellidae and Thrombidae. To date, molecular phylogenetic studies including Astrophorida species are scarce and offer limited sampling. Phylogenetic relationships within this order are therefore for the most part unknown and hypotheses based on morphology largely untested. Astrophorida taxa have very diverse spicule sets that make them a model of choice to investigate spicule evolution. Methodology/Principal Findings: With a sampling of 153 specimens (9 families, 29 genera, 89 species) covering the deep- and shallow-waters worldwide, this work presents the first comprehensive molecular phylogeny of the Astrophorida, using a cytochrome c oxidase subunit I (COI) gene partial sequence and the 59 end terminal part of the 28S rDNA gene (C1-D2 domains). The resulting tree suggested that i) the Astrophorida included some lithistid families and some Alectonidae species, ii) the sub-orders Euastrophorida and Streptosclerophorida were both polyphyletic, iii) the Geodiidae, the Ancorinidae and the Pachastrellidae were not monophyletic, iv) the Calthropellidae was part of the Geodiidae clade (Calthropella at least), and finally that v) many genera were polyphyletic (Ecionemia, Erylus, Poecillastra, Penares, Rhabdastrella, Stelletta and Vulcanella). Conclusion: The Astrophorida is a larger order than previously considered, comprising ca. 820 species. Based on these results, we propose new classifications for the Astrophorida using both the classical rank-based nomenclature (i.e., Linnaean classification) and the phylogenetic nomenclature following the PhyloCode, independent of taxonomic rank. A key to the Astrophorida families, sub-families and genera incertae sedis is also included. Incongruences between our molecular tree and the current classification can be explained by the banality of convergent evolution and secondary loss in spicule evolution. These processes have taken place many times, in all the major clades, for megascleres and microscleres. Citation: Ca´rdenas P, Xavier JR, Reveillaud J, Schander C, Rapp HT (2011) Molecular Phylogeny of the Astrophorida (Porifera, Demospongiaep) Reveals an Unexpected High Level of Spicule Homoplasy. PLoS ONE 6(4): e18318. doi:10.1371/journal.pone.0018318 Editor: Dirk Steinke, Biodiversity Insitute of Ontario - University of Guelph, Canada Received November 3, 2010; Accepted March 3, 2011; Published April 8, 2011 Copyright: ß 2011 Ca´rdenas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Participation of P. Ca´rdenas at the ‘Molecular Evolution Workshop’ (Woods Hole, 2009) was made possible by the Bergen Forskningsstiftelse (‘‘Mohn Grant’’, to C. Schander). Visit to the Zoological Museum in Amsterdam (P. Ca´rdenas) received support from the SYNTHESYS Project which is financed by European Community Research Infrastructure Action under the FP6 ‘‘Structuring the European Research Area’’ Program. J.R. Xavier is funded by the Science and Technology Foundation (FCT-Portugal, grant no. SFRH/BPD/62946/2009). J. Reveillaud has received funding from the European Community’s Seventh Framework Program (FP7/2007-2013) under the HERMIONE project, grant agreement no. 226354. This work was supported by a grant from the ‘Norwegian Deep Sea Program’ and by the Norwegian Research Council (project number 158923/S40, to H.T. Rapp). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] ¤ Current address: Muse´um National d’Histoire Naturelle, De´partement Milieux et Peuplements Aquatiques, UMR ‘‘BOREA’’ 7208, Paris, France Introduction monophyletic [1,3,4,5]. The Astrophorida is geographically and bathymetrically widely distributed around the world, and Demospongiaep Sollas, 1885 [Borchiellini et al., 2004] make up represent around 660 extant species (van Soest et al. 2010[6]; this 85% of all living sponges, and is today subdivided in 13 extant study). In tropical and parts of warm temperate waters orders. Based on molecular results, Demospongiaep are subdivided in Astrophorida species are common at quite shallow depths, while four clades: G1/Keratosap [Borchiellini et al., 2004], G2/Myx- in boreal/antiboreal and Arctic/Antarctic waters they are usually ospongiaep [Borchiellini et al., 2004], G3/Haplosclerida and G4/ deep-water species. Astrophorida species have colonized hard- as Democlavia [1,2]. The Astrophorida Sollas, 1888 are found within well as soft-bottoms from various depths. In gravely hard-bottom the Democlavia clade and represent one of the few sponge orders habitats on the outer shelf and upper slope, Astrophorida can to have been consistently and with strong support, shown to be dominate ecosystems in terms of abundance and biomass forming PLoS ONE | www.plosone.org 1 April 2011 | Volume 6 | Issue 4 | e18318 Astrophorida Phylogeny sponge grounds [7,8]. Astrophorida species display a wide array of monophyly i) of the Euastrophorida/Streptosclerophorida sub- external morphologies (massive to thin encrusting, subspherical-, orders and ii) of the Astrophorida families and genera. Our second fan-, cup- or irregularly-shaped) and external colors (Fig. 1a–d), aim was to revise the taxonomy of this order using both the and they range in size from a few millimeters to more than a meter classical rank-based nomenclature (i.e. Linnaean classification) and in diameter. There is no single morphological synapomorphy of the phylogenetic nomenclature following the PhyloCode, indepen- the Astrophorida. They are nonetheless well characterized by the dent of taxonomic rank. To be clear, names established under the simultaneous presence of star-shaped microscleres (small spicules PhyloCode are always in italics and will be identified with the symbol called ‘asters’) and tetractinal megascleres (large spicules called ‘p’ (e.g. Demospongiaep). Authors of PhyloCode names are between ‘triaenes’) (Fig. 1e–g). Star-shaped microscleres may be euasters — square brackets (e.g. Demospongiaep Sollas, 1885 [Borchiellini et al., asters in which the rays radiate from a central point (e.g. oxyasters, 2004]). Finally, our third aim was to investigate the evolution of strongylasters, spherasters, sterrasters) or streptasters — asters in Astrophorida megascleres and microscleres in order to evaluate which the rays proceed from an axis that can be straight the importance of homoplastic spicule characters in this order. (amphiasters) or spiral (e.g. spirasters, metasters, plesiasters). According to the latest major revision of the Astrophorida [9], Materials and Methods five families are included: Ancorinidae Schmidt, 1870, Calthro- pellidae Lendenfeld, 1907, Geodiidae Gray, 1867, Pachastrellidae Ethics statement Carter, 1875, and Thrombidae, Sollas, 1888. Thirty-eight genera This study has been approved by the University of Bergen and two subgenera are currently distributed in those families. In through the acceptance of a Ph.D. project proposal. an effort to incorporate some lithistids in the Astrophorida, the sub-orders Euastrophorida Reid, 1963 (Astrophorida with eua- Sponge sampling sters) and Streptosclerophorida Dendy, 1924 (Astrophorida/ Most of our collecting was done in the Northeast Atlantic. lithistids with streptasters) were erected [10,11], but in spite of Sampling in the Korsfjord (60u109N, 05u109E), Langenuen molecular evidence confirming their incorporation within the (59u539N, 05u319E) and the Hjeltefjord (60u249N, 05u059E) Astrophorida [5,12,13], lithistids have been kept apart in the (Western Norway, south of Bergen) were carried out using a Systema Porifera [14]. Other taxa such as the boring sponges Alectona triangular dredge and a bottom trawl between 40 and 500 meters and Neamphius have also been suggested to be derived Astrophor- (between the years 2005 and 2009). Southern Norway samples ida species, based on morphological [15], molecular [16] and (58u139N, 08u359E) were dredged during the BIOSKAG 2006 larval data [17,18], but they are still considered to belong to the cruise. Northern Norway samples were collected during the order Hadromerida in the Systema Porifera [19]. Polarstern ARK-XXII/1a 2007 cruise with large boxcores and the The Astrophorida is an order with one of the most diverse Jago manned-submersible. Localities sampled were Sotbakken spicule repertoire among the Demospongiaep. For