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J. Mar. Biol. Ass. U.K. (2007), 87, 1585–1598 doi: 10.1017/S0025315407058237 Printed in the United Kingdom On the phylogenetic relationships of hadromerid and poecilosclerid sponges Kord M. Kober*† and Scott A. Nichols*‡ *Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA. †Present address: Department of Ecology and Evolutionary Biology, University of California, Santa Cruz, CA, USA. ‡Corresponding author, e-mail: [email protected] Recent phylogenetic analyses of demosponges have suggested that the order Poecilosclerida is monophyletic and nested within the paraphyletic ‘order’ Hadromerida. Until now, this result has rested upon very limited taxon sampling of SSU sequences and partial LSU sequences. We collected and analysed additional full- length SSU and LSU sequences to test the validity and position of the poecilosclerid/hadromerid clade within demosponges, and we sampled a short segment of the LSU from diverse hadromerids to explore the internal relationships of Hadromerida. Our data strongly support the existence of a hadromerid/poecilosclerid clade that is sister to a poorly characterized group of halichondrid and agelasid species (‘Clade C’). We find support for the monophyly of the hadromerid families Polymastiidae, Placospongiidae and Timeidae, and conditional support for the family Suberitidae. Furthermore, both LSU and SSU data support a clade that includes a mixture of species assigned to the families Tethyidae and Hemiasterellidae (TETH/HEM) and a mixed clade including members of the families Clionaidae and Spirastrellidae (CLIO/SPIR). The family Placospongiidae is reconstructed as sister to the clade CLIO/SPIR and the family Timeidae is supported as sister to the clade TETH/HEM. The order Poecilosclerida is most closely allied with the Placospongiidae/CLIO/SPIR clade. INTRODUCTION One novel phylogenetic association that has emerged from the study of small and large ribosomal subunit (SSU Sponges are globally distributed, inhabit nearly every and LSU, respectively) data from demosponges is the aquatic environment, and exhibit tremendous biodiversity. close relationship between poecilosclerid and hadromerid As many as 8179 valid species are currently recognized sponges. Specifically, the order Poecilosclerida is supported (van Soest et al., 2005) and this number is certainly a as monophyletic (with the caveat that it has been poorly gross underestimate, as many sponge faunas are poorly sampled), but is reconstructed as being nested within a characterized (even in densely populated coastal regions) paraphyletic assemblage of hadromerid taxa (Borchiellini et and many species are cryptic in habitat and/or morphology al., 2004; Nichols, 2005); the order Hadromerida as a whole or have bathymetric distributions that are not amenable has been reconstructed as polyphyletic when genera such as to study. Sponge systematics is a notoriously challenging Hemiasterella are considered (Nichols, 2005). In general, both endeavour due to the relative simplicity and plasticity of the hadromerids and poecilosclerids are diverse and speciose sponge body-plan and has recently started to rely heavily taxa and it is likely that increased sampling for molecular upon molecular phylogenetic approaches. Two general phylogenetic analyses will continue to reveal hidden diversity patterns that have emerged from these studies are that and novel clades. Currently, 13 hadromerid families and 25 ‘morphospecies’ frequently mask deep phylogenetically poecilosclerid families are recognized as valid (Hooper & diversity on sympatric and allopatric scales (Sole-Cava van Soest, 2002c). & Thorpe, 1986; Boury-Esnault et al., 1992; Sole-Cava et The three main objectives of this study are to: (1) al., 1992; Klautau et al., 1994, 1999; Muricy et al., 1996; corroborate the existence and position of a hadromerid/ Wörheide et al., 2003; Nichols & Barnes, 2005), and that poecilosclerid clade within demosponges; (2) use a broader standard classification schemes based largely upon skeletal sampling of hadromerid taxa to test fine-scale hadromerid and reproductive characteristics are often inaccurate at relationships; and (3) test the position of poecilosclerid every rank; neither subclasses, orders, families, or genera are sponges relative to specific hadromerid lineages. To address monophyletic (Chombard et al., 1998; Alvarez et al., 2000; these aims we collected and analysed full-length SSU and McCormack et al., 2002; Borchiellini et al., 2004; Erpenbeck full-length and partial LSU sequences from a diversity of et al., 2005; Nichols, 2005; Boury-Esnault, 2006; Redmond et sponges representing the order Hadromerida, the order al., 2007). Nevertheless, phylogenetic studies, while still vastly Poecilosclerida, and relevant outgroups. These data incomplete, robustly support new relationships between taxa satisfactorally address our first two aims, but do not fully that promise to provide a framework for re-interpreting resolve the position of the order Poecilosclerida within sponge biology and evolution. ‘Hadromerida’. Journal of the Marine Biological Association of the United Kingdom (2007) 1586 K.M. Kober and S.A. Nichols Hadromerid and poecilosclerid phylogeny MATERIALS AND METHODS 2. Partial LSU (local) Sampling In order to explore the tree-shape within ‘Hadromerida’ as a guide to selecting taxa for further sequencing we analysed Specimens included in this study either were acquired on a short section of the LSU from 69 putative hadromerid, loan from the institutions listed in Table 1 or were collected poecilosclerid and outgroup taxa. This step permitted using SCUBA. Upon collection, samples were immediately the identification of well-supported clades from which we placed in 95–100% EtOH and vouchers were deposited selected exemplars for further sequencing (complete LSU at the University of California Museum of Paleontology sequences were technically challenging to obtain due to (UCMP), Berkeley, CA. Taxonomic identification was their length and sequence variability). performed by one of us (S.A.N.) or by specialists at the museums from which material was borrowed. Order and 3. Complete LSU/SSU (local) family-level assignments follow Systema Porifera (Hooper & In order to test the internal relationships between van Soest, 2002c). the reconstructed hadromerid clades and the order Poecilosclerida we conducted independent and combined analyses of full-length sequences from ingroup taxa and DNA extraction, amplification and sequencing their closest demosponge outgroup. By narrowing the focus Genomic DNA was extracted as described by Nichols of our analyses we were able to optimize our alignments and (2005). The LSU and SSU were amplified using Taq exclude fewer data. polymerase (New England Biosystems) and touchdown PCR (95°C, 30 s/ (95°C, 30 s/ 58°C, 60 s/ 72°C, 2.5 min) ×19 – 1°C per cycle/ (94°C, 30 s/ 52°C for 60 s/ 72°C, 2.5 min) ×9/ Sequence analysis and phylogeny reconstruction 72°C 10 min). The PCR primers that were used are listed Each data group identified in Table 3 was aligned separately in Table 2. Multiple sets of primers were used to amplify using the default settings in CLUSTAL W v.1.83 (Jeanmougin overlapping 700–1100 bp regions of the LSU: 5.8SF/LR6, et al., 1998) or MAFFT (Katoh et al., 2005) and manually SN47F/SN47R, NL2F/NL2R, NL4F/NL4R, LF3/LR3, edited in BioEdit (www.mbio.ncsu.edu/BioEdit/bioedit.html) LF5/R3264. Two sets of primer pairs were used to amplify and SE-AL (v.2.0a11, http://evolve.zoo.ox.ac.uk/software. complete SSU sequences: 18SF/18SR and PRIMER A/ html?name=Se-Al).Ambiguously aligned regions were PRIMER B. identified and excluded using Gblocks v.0.91b (Castresana, The PCR products were either gel-extracted (Bio-Rad 2000) with default settings implemented and gap positions Freeze-N-Squeeze, Zymo Research Zymoclean Gel DNA allowed. The LSU and SSU partitions of concatenated Recovery Kit) or column-purified (Qiagen QIAquick PCR datasets were aligned prior to concatenation. All datafile type Purification Kit, Promega Wizare SV Gel and PCR Clean- conversion was done using Readseq (v.2.1.21, D. Gilbert, Up System) and directly sequenced or cloned. All LSU http://iubio.bio.indiana.edu/soft/molbio/readseq/java/). fragments were cloned into pCR-Blunt II-TOPO vector We implemented both maximum parsimony (MP) and using the Zero Blunt TOPO PCR Cloning Kit (Invitrogen); Bayesian inference (BI) optimality criteria in all phylogenetic colonies were picked, cultured, and screened using standard analyses under the assumption that clades reconstructed methods. Sequencing was performed by the UC Berkeley using these disparate methods must be well supported by DNA Sequencing Facility and the resulting chromatograms the data. Furthermore, we explored the effect of model were trimmed and assembled into contigs using Sequencher selection in our BI analyses by independently implementing v. 4.6 (Gene Codes Corp.) and Vector NTI Advance 10 both single and mixed nucleotide substitution models for (Invitrogen). Sequence identities were confirmed using predicted ‘stem’ and ‘loop’ structural regions (hereafter BLAST against the NCBI nr database. referred to as BI ‘singlet’ and BI ‘mixed’, respectively; see The SSU and LSU sequences of Amphimedon queenslandica below for details). The BI ‘mixed’ analysis incorporated all were compiled from the raw genome reads (Joint Genome aligned nucleotide positions to allow for proper secondary Institute, Walnut Creek) deposited at the GenBank trace structure prediction. archives.