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’.

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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. We used MrModeltest2 (v. 2.2, J. Nylander, http://www. abc.se/∼nylander/) and PAUP* 4.0b10 (Swofford, 2002) to evaluate the best-fit nucleotide substitution model based on Datasets the Akaike information criterion (AIC). We compiled LSU and SSU sequences from 96 and The MP analyses were conducted using PAUP* 4.0b10 88 species, respectively (Table 1). Of these sequences 14 and consisted of heuristic searches with 100,000 replicates complete SSU and 14 complete LSU sequences are new. of random stepwise addition and TBR branch swapping. Three groupings of six alignments were created to address Non-parametric bootstrapping was not computationally the following aims: possible, so 500,000 ‘fast-bootstrap’ pseudo-replicates were substituted (Mort et al., 2000). 1. Complete LSU/SSU (global) The BI ‘singlet’ analyses were conducted with the program In order to confirm the position of Poecilosclerida within MrBayes v. 3.1 (Ronquist & Huelsenbeck, 2003). No initial Hadromerida, and to identify a close demosponge outgroup values were assigned to the model parameters and empirical for this clade, we independently analysed complete SSU and nucleotide frequencies were used. Each dataset was run with LSU data from diverse sponge and animal and non-animal four Markov chains for one million generations and sampled outgroups. every 100 generations and each analysis was run four times.

Journal of the Marine Biological Association of the United Kingdom (2007) Hadromerid and poecilosclerid phylogeny K.M. Kober and S.A. Nichols 1587

Table 1. Classification and database accession numbers of taxa sampled. The clade names and classification for sponges were based on Hooper & van Soest (2002). The prefix QM G- indicates samples from the Queensland Museum, Brisbane, the prefix ZMA Por- indicates the Zoological Museum of Amsterdam, the prefix WAMZ- indicates the Western Australia Museum and the prefix UCMPWC- indicates the University of California Museum of Paleontology. Sequences from this study have been submitted to GenBank (indicated in bold text).

Museum GenBank accession Group and family Taxon accession SSU LSU

PORIFERA DEMOSPONGIAE Agelasida Agelasidae Agelas oroides AY348886 – Agelasidae Agelas sp. UCMPWC1026 EF654520 AY561929 Agelasidae Agelas dispar AY737640 – Agelasidae Agelas conifera AY734443 – Agelasidae Agelas clathroides AY769087 – Agelasidae Astrosclera willeyana UCMPWC1070 – AY561928 Agelasidae Agelas sp. UCMPWC1063 – AY561926 Chondrosida Chondrillidae Chondrosia reniformis AY348876 – Dendroceratida Darwinellidae Aplysilla sulfurea AF246618 – Dictyoceratida Dysideidae Pleraplysilla spinifera AF246617 – Spongiidae Spongia officinalis AY348888 – Irciniidae Ircinia felix AY734448 – Hadromerida Clionaidae Cliona celata ZMA Por14091 – AY626276, AY561891 Clionaidae Cliona delitrix UCMPWC931 – AY626277, AY561889 Clionaidae Cliona sp. UCMPWC1066 – AY626288 Clionaidae Cliona sp. UCMPWC1078 – AY561886 Clionaidae Cliona sp. WAMZ1303 – AY626291, AY626332 Clionaidae Cliona sp. UCMPWC 958 – AY626289, AY626331 Clionaidae Cliona sp. UCMPWC946 – AY626324, AY626342 Clionaidae Cliona sp. UCMPWC1045 – AY626325 Clionaidae Cliona sp. UCMPWC930 – AY626284, AY626328 Clionaidae Cliona sp. WAMZ3927 – AY626281, AY561897 Clionaidae Cliona varians UCMPWC934 – AY626383 Clionaidae Cliona mussae ZMA Por16915 – AY626278, AY626327 Clionaidae Pione sp. QM G315210 – AY262280 Clionaidae Pione velans WAMZ3196 – AY626279, AY561890 Clionaidae Spheciospongia peleia QM G301247 – AY626287 Clionaidae Spheciospongia vesparium AY734440 – Hemiasterellidae Adreus micraster ZMA Por06837 – AY626306, AY561903 Hemiasterellidae Axos cliftoni QM G300111 EF654523 AY626308 Hemiasterellidae Hemiasterella sp. QM G315767 – AY561901 Hemiasterellidae Hemiasterella sp. QM G304645 – AY626310 Hemiasterellidae Hemiasterella sp. WAMZ12383 – AY561947 Placospongiidae Placospongia sp. ZMA Por11818 – AY626298, AY626338 Placospongiidae Placospongia sp. UCMPWC865 EF654527 AY626299 Polymastiidae Polymastia invaginata QM G315011 – AY561922 Polymastiidae Polymastia pachymastia UCMPWC932 EF654528 AY561924 Polymastiidae Polymastia sp. WAMZ12384 – AY626282, AY561923 Spirastrellidae Cervicornia cuspidifera UCMPWC928 – AY626290, AY561888 Spirastrellidae Diplastrella megastellata UCMPWC997 EF654525 AY561893 Spirastrellidae Diplastrella spiniglobata ZMA Por10636 – AY626275, AY561894 Spirastrellidae Spirastrella hartmani UCMPWC861 – AY561895 Spirastrellidae Spirastrella aff. inconstans ZMA Por10391 – AY626396 Spirastrellidae Spirastrella cf. inconstans WAMZ3122 – AY626286, AY626330 Spirastrellidae Spirastrella cf. vagabunda QM G303463 – AY626292, AY626333 Spirastrellidae Spirastrella purpurea WAMZ11980 – AY626297, AY626337

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Table 1. (Continued.)

Spirastrellidae Spirastrella sp. ZMA Por14672 – AY626274, AY626326 Spirastrellidae Spirastrella sp. ZMA Por10786 – AY626293, AY626334 Spirastrellidae Spirastrella sp. WAMZ12385 – AY626294, AY626335 Spirastrellidae Spirastrella sp. ZMA Por13106 – AY626295, AY626336 Spirastrellidae Spirastrella vagabunda ZMA Por11613 – AY626285, AY626329 Suberitidae Aaptos aaptos QM G320130 – AY626314 Suberitidae Aaptos cf. lithophaga ZMA Por8789 – AY626312, AY561909 Suberitidae Aaptos sp. WAMZ3615 – AY626311, AY561908 Suberitidae Aaptos sp. QM G313447 – AY626313 Suberitidae Caulospongia biflabellata WAMZ16503 – AY626318, AY626341 Suberitidae Prosuberites laughlini UCMPWC524 EF654529 AY6263220 Suberitidae Prosuberites sp. QM G306290 – AY626319 Suberitidae Pseudosuberites sp. UCMPWC1069 EF654530 AY561917 Suberitidae Pseudosuberites sp. UCMPWC956 – AY626317, AY561913 Suberitidae Rhizaxinella sp. QM G319653 EF654531 AY561910 Suberitidae Suberites domuncula – AJ620113 Suberitidae Suberites ficus AF100947 AY026381 Suberitidae Suberites sp. UCMPWC859 – AY626315, AY561912 Suberitidae Terpios aurantiaca ZMA Por10080 – AY626316, AY561911 Tethyidae Laxotethya dampierensis WAMZ11871 – AY626307, AY561905 Tethyidae Stellitethya ingens WAMZ11947 – AY561920 Tethyidae Tethya actinia AY878079 – Tethyidae Tethya sp. UCMPWC957 EF654532 AY626300 Tethyidae Tethytimea stellagrandis ZMA Por15727 – AY561920 Timeidae Timea cf. centrifera WAMZ9801 – AY626304, AY561906 Timeidae Timea lowchoyi QM G303681 – AY561871 Timeidae Timea sp. QM G313459 EF654533 AY626303 Timeidae Timea sp. QM G313973 – AY561907 Trachycladidae Trachycladus laevispirulifer WAMZ1186 EF654534 AY626305 Halichondrida Axinellidae Axinella polypoides APU43190 – Axinellidae Axinella damicornis AY348887 – Axinellidae Axinella sp. QM G315196 EF654522 AY561925 Axinellidae Pseudaxinella lunaecharta AY734442 – Axinellidae Ptilocaulis gracilis AY737638 – Dictyonellidae Dictyonella incisia AY348880 – Dictyonellidae Scopalina ruetzleri UCMPWC992 AY561872 – Dictyonellidae Liosina arenosa UCMPWC1062 – AY561904 Halichondriidae Spongosorites genitrix AY348885 – Halichondriidae Halichondria melanodocia AY737639 – Haplosclerida Niphatidae Niphates sp. DQ927312 – Niphatidae Amphimedon queenslandica EF654521 EF654518 Petrosiidae Xestospongia muta AY621510 – Phloeodyctyidae Aka mucosum DQ927322 – Phloeodictyidae Calyx podatypa AY734447 – Spongillidae Ephydatia muelleri AF121110 – Spongillidae Spongilla lacustris AF121112 – Homosclerophorida Plakinidae Plakortis simplex AY348884 – Plakinidae Oscarella carmela EF654526 EF654519 ‘Lithistid’ Corallistidae Corallistes sp. AY737636 – Poecilosclerida Crambeidae Crambe crambe UCMPWC933 EF654524 AY561883 Crambeidae Monanchora arbuscula UCMPWC955 – AY626301, AY561882 Crellidae Crella elegans AY348882 – Hymedesmiidae Phorbas tenacior AY348881 – Iotrochotidae Iotrochota birotulata UCMPWC969 AY737641 AY561884 Microcionidae Clathria (Microciona) prolifera L10825 – Mycalina Mycale fibrexilis AF100946 AY026376

Journal of the Marine Biological Association of the United Kingdom (2007) Hadromerid and poecilosclerid phylogeny K.M. Kober and S.A. Nichols 1589

Table 1. (Continued.)

Mycalina Mycale sp. AY737643 – Tedaniidae Tedania ignis UCMPWC975 AY737642 AY626309, AY561878 Spirophorida Tetillidae Cinachyrella sp. AF062604 – Verongida Aplysinidae Aplysina aerophoba AY591799 – CALCAREA Leucosoleniidae Leucosolenia sp. AF100945 AY026372 Clathrinidae Clathrina cerebrum U42452 – Leucaltidae Leucaltis clathria AF452016 – Petrobionidae Petrobiona massiliana AF452026 – HEXACTINELLIDA Hexasterophora Rhabdocalyptus dawsoni AF100949 AY026379 Oopsacas minuta AF207844 – Farrea occa AF159623 – BILATERIA MOLLUSCA Aplysia californica AY039804 AY026366 PLATYHELMINTHES Dugesia tigrina AF013157 U78718 ECHINODERMATA Arbacia punctulata AH001568 AY026367 UROCHORDATA Styela plicata M97577 AF158724 CHORDATA Xenopus borealis K01373 X59733 CHOANOZOA MINISITERIIDA Ministeria vibrans AF271998 – CHOANOFLAGELLATEA Monosiga brevicolis AF100940 AY026374 CHOANOFLAGELLATEA Salpingoeca infusionum AF100941 AY026380 NUCLEARIIDA Nuclearia simpleax AF484687 – CNIDARIA ANTHZOA Montastraea franski AY026382 AY026375 Antipathes galapagensis AF100943 AY026365 HYDROZOA Hydra circumcincta AF358080 AY026371 Nectopyramis sp. AF358068 AY026377 Aglauropsis aeora AY920754 – SCYPHOZOA Atolla vanhoeffeni AF100942 AY026368 Chrysaora sp. AY920769 – STAUROZOA Haliclystus octoradiatus AY845346 – CUBOZOA Chironex fleckeri AF358104 – CTENOPHORA Beroe ovata AF293694 AY026369 Beroe forskalii AF293698 – Mnemiopsis leidyi AF293700 AY026373 Pleurobrachia bachei AF293677 AY026378 Mertensia ovum AF293678 – FUNGI ARCHEMYCOTA Chytriomyces hyalinus M59758 – ASCOMYCOTA Saccharomyces cerevisiae M27607 J01355 BASIDIOMYCOTA Tricholoma matsutake U62538 U62964 ZYGOMYCOTA Mucor racemosus AJ271061 AJ271061 MESOMYCETOZOA Ichthyophonus hoferi U25637 AY026370 PLACOZOA Trichoplax sp. AY652581 AY652584 Trichoplax adhaerens – AY303975

The first 2500 trees from each run were discarded so that The BI ‘mixed’ analyses were performed using the the final consensus tree was based on the combination program PHASE (v. 2.0, V. Gowri-Shankar & J. Jow, http:// of accepted trees from each run (a total of 30,004 trees). www.bioinf.man.ac.uk/resources/phase). We predicted a Convergence between the four runs was tested by examining consensus secondary structure from each alignment using the potential scale reduction factors (PSRF) produced by the program RNAalifold (Hofacker & Stadler, 1999). We the ‘sump’ command in MrBayes. Support for nodes was then constructed a PHASE data file by converting the determined using posterior probabilities (PP, calculated by alignment file to a modified PHYLIP format, replacing the MrBayes). unsupported IUPAC Nucleotide symbols ‘W’,‘H’,‘K’,‘M’

Journal of the Marine Biological Association of the United Kingdom (2007) 1590 K.M. Kober and S.A. Nichols Hadromerid and poecilosclerid phylogeny

Table 2. The PCR and sequencing primers used in this study.

Primer name Sequence (5'–>3') Source Gene

PCR 5.8SF TCG AGT CTT TGA ACG CAA AT (Medina et al., 2001) LSU LR6 GGC ATA GTT CAC CAT CTT TCG This study LSU SN47F GGA GGA AAA GAA ACT AAC AAG GAT TC This study LSU SN47R GGT CCC AAC AGA TGT GCT CT This study LSU NL2F TAC CGT GAG GGA AAG GTG AAA This study LSU NL2R CGG AGG GAA CCA GCT ACT AGA This study LSU NL4F GAC CCG AAA GAT GGT GAA CTA (Nichols, 2005) LSU NL4R ACC TTG GAG ACC TGA TGC G (Nichols, 2005) LSU LF3 TTG AAA CAC GGA CCA AGG AGT (Nichols, 2005) LSU LR3 TAC CAC CAA GAT CTG CAC CA (Nichols, 2005) LSU LF4 TGG TGC AGA TCT TGG TGG TA This study LSU LR4 TTT GAC ATT CAG AGC ACT GGG This study LSU LF5 TGA CGC AAT GTG ATT TCT GC (Nichols, 2005) LSU R3264 TTC YGACTT AGA GGC GT CAG (Medina et al., 2001) LSU 18SF CTG GTT GAT CCT GCC AGT AGT This study SSU 18SR GCA GGT TCA CCT ACA GAA ACC This study SSU PRIMER A CCG AAT TCG TCG ACA ACC TGG TTG ATC CTG CCA GT (Medlin et al., 1988) SSU PRIMER B CCC GGG ATC CAA GCT TGA TCC TTC TGC AGG TTC ACC TAC (Medlin et al., 1988) SSU Sequencing SSU_1170F GGT CGC AAG GCT GAA ACT TA This study SSU SSU_1158F TAA AGG AAT TGA CGG AAG GG This study SSU SN26R TCT GGA CCT GGT GAG TTT CC This study SSU

Table 3. The data partitions used for phylogenetic reconstruction. The alignment length prior (Full) to Gblocks, the resulting alignment size of conserved sites (Gblocks), the maximum parsimony (MP) constant sites and MP informative sites are listed.

Alignment length MP MP Dataset Genes Taxa (n) Full Gblocks Const. Inform.

Partial LSU (local) Partial LSU 69 1776 1582 1013 481

SSU (local) SSU 29 1857 1673 1410 159 LSU (local) LSU 16 4186 3503 2682 511 LSU+SSU (local) SSU+LSU 15 5608 5110 4147 624

SSU (global) SSU 87 2445 1723 713 739 LSU (global) LSU 41 5070 2813 1363 1050

Table 4. Comparison of 2ln Bayes factors results for the Bayesian partitioning strategies within each dataset.

Model likelihood1 Bayes factor2 Evidence 3 Dataset lnf(X|M1)lnf(X|M2)lnB12 2lnB12 for M1

Partial LSU (local) −14,454.17 −−−−

SSU (local) −5,625.92 −5,535.00 −90.92 −181.84 Negative (very strong) LSU (local) −13,571.18 −14,856.39 1,285.21 2,570.42 Very strong LSU+SSU (local) −17,873.31 −18,927.81 1,054.5 2,109 Very strong

SSU (global) −27,576.37 −32,080.12 4,502.75 9,007.5 Very strong LSU (global) −29,905.86 −56,709.44 26,803.58 52,607.16 Very strong

1 , M1 refers to the un-partitioned analysis under GTR+I+G. M2 refers to the mixed model analysis using REV (GTR) for loops and RNA7D for stems. The model likelihood is estimated by calculating the harmonic mean of the likelihood values from the stationary samples. These were obtained directly for the PHASE output, and via the ‘sump’ output of MrBayes; 2, the Bayes factor support for 3 model 1 over model 2. B12 is given as the ratio f(X|M1)/f(X|M2); , evidence for M1 follows Nylander et al., 2004.

Journal of the Marine Biological Association of the United Kingdom (2007) Hadromerid and poecilosclerid phylogeny K.M. Kober and S.A. Nichols 1591

Figure 1. The SSU vs LSU (global). The 50% majority rule consensus phylogram of the stationary trees obtained from the Bayesian inference analysis of (A) SSU and (B) LSU under a GTR+I+G model in MrBayes. Branches of major clades are labelled as follows from top to bottom: (top) posterior probabilities from an unpartitioned ‘singlet’ analysis (GTR+I+G), (middle) posterior probabilities from a partitioned ‘mixed’ analysis of inferred stems (RNA7D) and loops (REV), and (bottom) bootstrap values that were reconstructed in the maximum parsimony strict consensus tree. Branches not reconstructed in the partitioned or maximum parsimony analysis are denoted with the notation ‘^’ or ‘–’, respectively. Long branches that have been shortened are denoted by a dashed line. Support values are not shown for all minor branches. and ‘S’ with ‘N’ and adding the predicted consensus representing the other ten less frequent pairs. We used the secondary structure. RNA7D (Tillier & Collins, 1998) because it is a biologically We used different substitution models to analyse predicted plausible and analytically tractable restriction of the time ‘stem’ and ‘loop’ regions (Yang, 1996). For the loop regions we reversible seven state RNA7A model (Savill et al., 2001). To use the general time-reversible model REV (Tavare, 1986). model the variation in the substitution rates at different sites, For the stem regions we used a model with seven states, six of we implemented the discrete-gamma model with six categories which represent the most common base pairs (AU, GU, GC, to approximate the Γ-distribution with no invariant sites UA, UG and CG), plus a composite mismatch state (MM) allowed. We performed 1.5 million sampling iterations with a

Journal of the Marine Biological Association of the United Kingdom (2007) 1592 K.M. Kober and S.A. Nichols Hadromerid and poecilosclerid phylogeny

Figure 2. Partial LSU (local). The 50% majority rule consensus phylogram of the stationary trees among 69 demosponges from the orders Hadromerida and Poecilosclerida, rooted with ‘Clade C’ (Nichols, 2005), based on Bayesian inference analysis of partial LSU data. Branches are labelled above with the posterior probabilities from the ‘singlet’ analysis (GTR+I+G) and below with maximum parsi- mony bootstrap values. The MP bootstrap values less then 50 are represented by ‘–’. Taxa selected for full LSU and SSU sequencing are highlighted.

Journal of the Marine Biological Association of the United Kingdom (2007) Hadromerid and poecilosclerid phylogeny K.M. Kober and S.A. Nichols 1593 sampling period of 150 and burn-in iterations of 750,000. The data support the order Poecilosclerida and the hadromerid remaining parameters follow Hudelot and colleagues (2003). families Timeidae, Polymastiidae and Placospongiidae as To evaluate the relative strengths of the different Bayesian monophyletic. Family Suberitidae would be monophyletic partitioning methods, we used the Bayes factor (Kass & if not for the taxa Prosuberites laughlini, Rhizaxinella sp. QM Raftery, 1995). The model likelihood was estimated by G319653 and Hemiaseterella sp. WAMZ12383. However, calculating the harmonic mean of the likelihood values from note that P. laughlini is not confidently assigned to the genus the stationary samples (Brandley et al., 2005). These were Prosuberites (R.M.W. van Soest, personal communication) obtained directly from the PHASE sampling of the ‘mixed’ and was originally described as Erylus laughlini (order analysis via a Perl program using the Statistics-Descriptive Poecilosclerida). Furthermore, full-length LSU and SSU (v. 2.6, C. Kuskie, http://search.cpan.org/dist/Statistics- data strongly support an alliance between Rhizaxinella Descriptive/) module and from the ‘singlet’ sampling via and other members of the family Suberitidae. We did not the MrBayes ‘sump’ command (Table 4). The traditional verify the identity of the borrowed specimen identified criterion of 2ln Bayes factor ≥10 was accepted as very strong as Hemiasterella, but this genus appears to be polyphyletic evidence for the given hypothesis (Nylander et al., 2004). based upon these and previous analyses (Nichols, 2005). Other representatives of the family Hemiasterellidae that were included in this analysis were interspersed with RESULTS members of family Tethyidae. The families Clionaidae Alignments and model selection and Spirastrellidae also form an internally mixed Results of the Clustal X and MAFFT alignments, monophyletic clade that is strongly supported as sister to conserved block totals identified by Gblocks, and parsimony- family Placospongiidae. The family Trachycladiidae was informative characters totals are summarized in Table 3. represented by a single specimen and showed weak affinity For all six datasets, MrModelTest indicated that the most for any other group. appropriate model of nucleotide substitution is one that has For purposes of discussion we will hereafter use the six substitution rates, an assumed proportion of invariant following notation to identify the clades: sites, and gamma-shape parameter (GTR+I+G). • TIM. Family Timeidae. Singlet vs mixed Bayesian performance • POLY. Family Polymastiidae. • PLAC. Family Placospongiidae. The Bayes factor test results (Table 4) between the ‘singlet’ • SUB*. Dubious family Suberitidae. and ‘mixed’ Bayesian analyses show ‘very strong’ support • TRACH. Family Trachycladiidae. for the ‘singlet’ partitioning under the GTR+I+G given • TETH/HEM. Mixed families Tethyidae+Hemiasterellidae. each dataset except SSU (local) where the ‘mixed’ model • CLIO/SPIR. Mixed families Clionaidae+Spirastrellidae. has ‘very strong’ support. Hence, for simplicity we used the • POEC. Order Poecilosclerida. reconstructed 50% majority rule consensus phylogram from the ‘singlet’ analysis as the figure tree for all analysis. Hadromerid/poecilosclerid relationships: complete SSU/LSU (local) Outgroup determination: complete SSU/LSU (global) Figure 3 illustrates the relationships between hadromerid In order to test the close relationship between hadromerid and poecilosclerid sponges as reconstructed using and poecilosclerid sponges (Borchiellini et al., 2004; Nichols, concatenated SSU and LSU (full-length) sequences. The 2005) and to identify their closest outgroups we sampled results of these datasets analysed separately are provided in from the full diversity of complete SSU and LSU sequences Figure 4. The concatenated results corroborate the SSU- available for sponges and their animal and non-animal only tree with respect to the branching order of the families outgroups. The broad nature of our sampling was designed Polymastiidae (POLY) and Suberitidae (SUB*), and the to compensate for the diversity at the base of the Metazoan LSU-only tree with respect to the branching order of family tree as well as the possible paraphyly of Porifera (Medina et Placospongiidae (PLAC), the mixed clade of Clionaidae and al., 2001; Sperling & Peterson, in press). A clade with high Spirastrellidae (CLIO/SPIR), and the order Poecilosclerida support is defined as having posterior probability (PP) of (POEC). >95 and a bootstrap value (BV) >70. Furthermore, a clade The SSU and LSU data, analysed separately or together, is considered highly supported if it is present (or at least not support the monophyly of family Suberitidae (SUB*; contradicted) in the optimal trees reconstructed using both including Rhizaxinella sp. QM G319653). Furthermore BI and MP optimality criteria. Figure 1 illustrates that both the mixed group TETH/HEM that includes the families SSU and LSU datasets independently support a hadromerid/ Tethyidae and Hemiasterellidae is consistently allied with poecilosclerid clade sister to the clade previously identified families Timeidae and Trachycladiidae. Finally, the mixed as ‘Clade C’ (Nichols, 2005). clade including families Clionaidae and Spirastrellidae (CLIO/SPIR) is consistently supported as sister to family Hadromerid tree ‘shape’: partial LSU (local) Placospongiidae. The phylogenetic affinities of the order Rather than assume that hadromerid families are Poecilosclerida and the families Polymastiidae (POLY), monophyletic, we analysed a short LSU segment from a Suberitidae (SUB*) and Trachycladidae (TRACH) vary diverse set of hadromerid sponges to explore the internal tree- dramatically depending upon the dataset considered. The shape of the ‘order’ (Figure 2). Rooted with ‘Clade C,’ these only hadromerid taxa that are consistently supported as sister

Journal of the Marine Biological Association of the United Kingdom (2007) 1594 K.M. Kober and S.A. Nichols Hadromerid and poecilosclerid phylogeny

Figure 3. Concatenated SSU and LSU (local). The 50% majority rule conensus phylogram of the stationary trees reconstructed in the Bayesian inference analysis of the concatenated LSU and SSU data under a single model of evolution (GTR+I+G) for 14 demosponges, rooted with three members of ‘Clade C’ (Nichols, 2005). Branches are labelled as follows from top to bottom: (top) posterior probabilities from an unpartitioned ‘singlet’ analysis (GTR+I+G); (middle) posterior probabilities from a partitioned ‘mixed’ analysis of inferred stems (RNA7D) and loops (REV); and (bottom) bootstrap values that were reconstructed in the maximum parsimony strict consensus tree. Branches not reconstructed in the partitioned or maximum parsimony analysis are denoted with the notation ‘^’ or ‘–’, respectively.

Journal of the Marine Biological Association of the United Kingdom (2007) Hadromerid and poecilosclerid phylogeny K.M. Kober and S.A. Nichols 1595

Figure 4. The SSU vs LSU (local). Comparison of Bayesian inference 50% consensus phylogram of SSU and LSU trees (A and B, respectively) under the GTR+I+G model. The value above the branch correspond to posterior probability values of the singlet (GTR+I+G) model. Branches are labelled as follows from top to bottom: (top) posterior probabilities from an unpartitioned ‘singlet’ analysis (GTR+I+G); (middle) posterior probabilities from a partitioned ‘mixed’ analysis of inferred stems (RNA7D) and loops (REV); and (bottom) bootstrap values that were reconstructed in the maximum parsimony strict consensus tree. Branches not reconstructed in the partitioned or maximum parsimony analysis are denoted with the notation ‘^’ or ‘–’, respectively.

to the order Poecilosclerida are the family Placospongiidae position of calcareous and homoscleromorph sponges is a and the clade CLIO/SPIR. matter of debate (Peterson & Butterfield, 2005; Wang & Lavrov, 2007; Sperling & Peterson, in press). DISCUSSION With respect to resolving internal relationships of Phylogenetic utility of the LSU demosponges, only partial LSU sequences have been evaluated. In an LSU based study of inter-order relationships The large ribosomal subunit (LSU) is nearly twice the of demosponges (Nichols, 2005), major subgroups were size of the small ribosomal subunit (SSU) and has a greater adequately identified at roughly the ‘order’-level, but mixture of conserved and variable regions. The size these data performed substantially worse than SSU and heterogeneity of sequence conservation of the LSU data (Borchiellini et al., 2004) at resolving inter-‘order’ renders it potentially useful for phylogeny reconstruction of relationships. Erpenbeck and colleagues (2005) similarly both distantly and closely related taxa. Indeed, LSU data found that partial LSU sequences were incapable of fully have been shown to improve the resolution of SSU based resolving the relationships of halichondriid sponges. An studies of between- and within-phylum animal relationships emerging hazard of using partial LSU sequences is that (Medina et al., 2001; Collins et al., 2006). However, when clearly erroneous relationships can receive high support. For analysed separately, phylogenies produced using LSU data example, two species of the astrophorid genus Geodia were are typically less consistent with morphologically based allied with poecilosclerids in a study by Nichols (2005); this hypotheses of relationship at deep phylogenetic levels than result is highly dubious given the complexity of characters are SSU data. For example, LSU data in this study and that ally these species with other geodiids. Similarly, in the others (Medina et al., 2001) support demosponges as sister present study Rhizaxinella sp. is allied with the outgroup clade to bilaterians whereas SSU data support demosponges and that includes Agelas and Axinella based upon partial LSU data. hexacintellid sponges as sister to other animals (Borchiellini We believe that this result is erroneous given that complete et al., 2001; Medina et al., 2001), a result that is more LSU data (and SSU data) strongly support Rhizaxinella within consistent with current views of animal evolution. The the hadromerid family Suberitidae (where it is classified).

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In the present study, patterns of congruence and conflict structural predictions or little sequence variation (Hofacker between LSU and SSU data on ‘global’ and ‘local’ scales (i.e. et al., 2002). deep and shallow phylogenetic levels) are complex. On the global scale, both datasets support a monophyletic group of Hadromerid and poecilosclerid relationships demosponges (sensu Borchiellini et al., 2004) that is sister to As reviewed by Hooper & van Soest (2002c), de class Hexactinellida, and a sister relationship between homo- Laubenfels (1936) was probably the first to speculate about scleromorphs and calcareans (albeit with low support in the the relationships of the ‘order’ Hadromerida to other sponge LSU tree). These data also support the monophyly of ma- orders. While de Laubenfels emphasized the morphological jor groups such as Bilateria, Cnidaria and Ctenophora, but similarities between the Hadromerid and ‘tetraxon’ orders disagree about the interrelationships between these groups. [corresponding to the existing orders Astrophorida, Of course, disagreement about basal animal relationships is Spirophorida, Chondrosida and many lithistids (Hooper & not unique to ribosomal data and is hypothesized to result van Soest, 2002a)], he also suggested a possible relationship from short internodes between ancient branches that radi- between the hadromerids and poecilosclerids. Notably, de ated early in animal history (Rokas et al., 2005). Laubenfels’ hypotheses about hadromerid relationships At the local scale (i.e. within the hadromerid/poecilosclerid were based upon his concept of the order that only clade), clades that were highly supported by both Bayesian included the modern families Spirastrellidae, Timeidae, inference and maximum parsimony analyses of the LSU Suberitidae, Polymastiidae, Placospongiidae, Clionaidae, dataset are completely congruent with the independently and Gastrophanellidae (for a small group of lithistids). More analysed SSU dataset. When analysed together, SSU and recent morphologically-based analyses have supported a LSU data confirm these groupings. Other relationships relationship between poecilosclerids and haplosclerids (van supported by the concatenated dataset variously support Soest, 1987, 1991). An argument against the hadromerid/ the independent SSU or LSU trees, but are interpreted as poecilosclerid hypothesis is that they have different highly suspect. reproductive characteristics; hadromerids are oviparous and poecilosclerids are mostly viviparous (Hooper & van Soest, Phylogenetic effects of structural partitions 2002a). Nevertheless, it now seems that viviparity is derived Functional ribosomal RNA transcripts are folded into within the order Poecilosclerida. To fully understand the secondary structures that can be described as having ‘stem’ origin of viviparity within this clade it will be essential to and ‘loop’ regions that are characterized by paired and sample the enigmatic viviparous families Raspaliidae and unpaired nucleotides, respectively. As a result of these folding Rhabderemiidae. To fully understand the morphological patterns, mutations in stem regions require compensatory transition between hadromerids and poecilosclerids it will changes in complementary positions in order to maintain be essential to further resolve the internal relationships of the structural characteristics of the molecule. Phylogenetic both clades. Presently, the vast diversity within these clades, approaches that apply independent models to stem and combined with ambiguities about their internal relationships loop regions can account for character-state dependencies precludes a detailed analysis of their character evolution. at complementary nucleotide positions and show promise In general, that the order Poecilosclerida nests within for improving metazoan phylogeny reconstruction (Telford hadromerid sponges highlights the antiquity of major et al., 2005; Erpenbeck et al., 2007). demosponge clades overall. The earliest fossil traces of Inferring the secondary structure of rRNA becomes poecilosclerids date to the Permian (Reitner & Wörheide, more challenging as the phylogenetic distance between taxa 2002). This fact, considered along with the phylogenetic increases. In this study, we developed a simple automated placement of poecilosclerids, suggests that the major extant method to infer consensus secondary structural patterns and sponge clades radiated early and may extend far into to generate stem and loop partitions with defined doublet early animal ancestry. The tremendous age of the internal pairs. By automating the pairing, we retained consistency demosponge lineages no doubt contributes to the challenge in prediction across analyses and datasets. Surprisingly, we of reconstructing their relationships. found that our data were robust to the effects of character Comparatively, the order Poecilosclerida remains poorly dependencies that were not addressed in ‘singlet’ analyses sampled (considering that it is perhaps the most speciose (i.e. analyses without stem and loop partitions). The demosponge group) but all analyses to-date support its nearly universal result of applying ‘mixed doublet’ models monophyly. Nevertheless, it is plausible that increased to structurally partitioned datasets was the lessening of sampling will reveal that some taxa are erroneously posterior probability support compared to singlet analyses, assigned to this order. While the ‘order’ Hadromerida is albeit to negligible extents. This pattern likely reflects an not monophyletic, it is encouraging that many family-level overall reduction in character number in ‘mixed’ analyses. hadromerid taxa are supported as monophyletic (or nearly Because the algorithm implemented is primarily intended for so). Indeed, the families Placospongiidae, Polymastiidae, closely related taxa, it would be informative to evaluate the Timeidae and Suberitidae largely withstand the scrutiny of performance of automating secondary structural prediction increased sampling of ribosomal data. Futhermore, the clades using the alidot (Hofacker et al., 1998) and pfrali (Hofacker containing mixed assemblages of clionaids/spirastrellids and & Stadler, 1999) algorithms, as they are appropriate for tethyids/hemiasterellids can be comfortably reconciled given less conserved molecular structure. The drawback of this the spicule morphology of members of these groups. Members approach is that conservative predictions minimize false of the families Clionaidae and Spirastrellidae share similar positives by excluding regions of ambiguous thermodynamic spirasters as microscleres, and the families Tethyidae and

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Hemiasterellidae (and the closely related family Timeidae) Borchiellini, C., Chombard, C., Manuel, M., Alivon, E., have asterose microscleres in common. It is anticipated that Vacelet, J. & Boury-Esnault, N., 2004. Molecular phylogeny further morphological support for these clades will be found of Demospongiae: implications for classification and scenarios upon closer re-examination of their constituent taxa. That of character evolution. Molecular Phylogenetics and Evolution, 32, 823–837. some members of the genus Hemiasterella are not included in Borchiellini, C., Manuel, M., Alivon, E., Boury-Esnault, N., the hadromerid/poecilosclerid clade is not surprising upon Vacelet, J. & Le Parco, Y., 2001. Sponge paraphyly and the consideration that some members of this genus are similar origin of Metazoa. Journal of Evolutionary Biology, 14, 171–179. to the astrophorid genus Jaspis (Hooper, 2002). Boury-Esnault, N., 2006. Systematics and evolution of The challenge of sponge systematics derives from the Demospongiae. Canadian Journal of Zoology, 84, 205–224. great diversity of species globally, the paucity and plasticity Boury-Esnault, N., Sole-Cava, A.M. & Thorpe, J.P., 1992. Genetic and of available morphological characters, and the dearth of cytological divergence between colour morphs of the Mediterranean available information about the cell and developmental sponge Oscarella lobularis Schmidt (Porifera, Demospongiae, biology of most species. Systema Porifera (Hooper & van Oscarellidae). Journal of Natural History, 26, 271–284. Brandley, M., Schmitz, A. & Reeder, T., 2005. Partitioned Bayesian Soest, 2002c) created a framework that has accelerated the analyses, partition choice, and the phylogenetic relationships of molecular phylogenetic study of sponge relationships, and scincid lizards. Systematic Biology, 54, 373–390. the development of robust molecular phylogenies promises Castresana, J., 2000. Selection of conserved blocks from multiple to create a framework for re-interpreting many aspects of alignments for their use in phylogenetic analysis. Molecular Biology sponge biology and evolution. and Evolution, 17, 540–552. The emerging relationship between poecilosclerid and Chombard, C., Boury-Esnault, N. & Tillier, S., 1998. Reassessment hadromerid sponges highlights the necessity of characterizing of homology of morphological characters in tetractinellid sponges the broad-scale relationships between sponges before based on molecular data. Systematic Biology, 47, 351–366. attempting to resolve fine-scale relationships at the ‘family’- Collins, A., Schuchert, P., Marques, A., Jankowski, T., Medina, M. & Schierwater, B., 2006. Medusozoan phylogeny and character or ‘genus’- level. In fact, we promote the indiscriminate evolution clarified by new large and small subunit rDNA data accumulation of voucher-verified SSU sequences as a means and an assessment of the utility of phylogenetic mixture models. to gain perspective on the composition of major sponge Systematic Biology, 55, 97–115. clades before any revision of the existing classification is Erpenbeck, D., Breeuwer, J.A.J. & Soest, R.W.M. van, 2005. undertaken. Implications from a 28S rRNA gene fragment for the phylogenetic relationships of halichondrid sponges (Porifera: Demospongiae). CONCLUSIONS Journal of Zoological Systematics and Evolutionary Research, 43, 93–99. Erpenbeck, D., Nichols, S.A., Voigt, O., Dohrmann, M., Degnan, The focus of this study was on hadromerid sponges and B.M., Hooper, J.N. & Worheide, G., in press. Phylogenetic their relationships to the monophyletic order Poecilosclerida. analyses under secondary structure-specific substitution models It was beyond the scope of this study to sample broadly outperform traditional approaches—case studies with diploblast from within Poecilosclerida, but this step is warranted to LSU. Journal of Molecular Evolution. further test the monophyly of this clade and its position Hofacker, I.L., Fekete, M., Flamm, C., Huynen, M.A., Rauscher, within ‘Hadromerida’. Furthermore, the association of S., Stolorz, P.E. & Stadler, P.F., 1998. Automatic detection of Halichondria melanodocia with suberitids in our ‘global’ SSU conserved RNA structure elements in complete RNA virus genomes. Nucleic Acids Research, 26, 3825–3836. analysis and Liosina arenosa with the mixed clade of tethyids Hofacker, I.L., Fekete, M. & Stadler, P.F., 2002. Secondary and hemiasterellids (TETH/HEM) alludes to the potential structure prediction for aligned RNA sequences. Journal of relationships between hadromerid and halichondrid Molecular Biology, 319, 1059–1066. demosponges (see Erpenbeck et al., 2005). We conclude Hofacker, I.L. & Stadler, P.F., 1999. Automatic detection of that, in general, the composition of major demosponge conserved base pairing patterns in RNA virus genomes. Computers clades remains inadequately explored and that the existing and Chemistry, 23, 401–414. classification system can guide, but should not limit sampling Hooper, J.N.A., 2002. Family Hemiasterellidae Lendenfeld, 1889. strategies until a more robust ‘global’ phylogeny is available. In Systema Porifera: a guide to the classification of sponges (ed. J.N.A. Hooper and R.W.M. van Soest), pp. 186–195. New York: Kluwer Thanks to E. Beglinger, J. Cook, J. Fromont and R. van Soest for Academic/Plenum Publishers. sending museum samples and R. van Soest for identifying several Hooper, J.N.A. & Soest, R.W.M. van, 2002a. Order Hadromerida. In Systema Porifera: a guide to the classification of sponges (ed. J.N.A. specimens. We would also like to acknowledge the UC Museum of Hooper and R.W.M. van Soest), pp. 169–172. New York: Kluwer Paleontology and C. Hickman, D. Lindberg, and N. King for their Academic/Plenum Publishers. support of this project. Work done in the laboratory of N. King Hooper, J.N.A. & Soest, R.W.M. van, 2002b. Order Poecilosclerida. was supported by the Pew Scholars Program and the UC Berkeley In Systema Porifera: a guide to the classification of sponges (ed. J.N.A. Center for Integrative Genomics (CIG). S.A.N. was supported by Hooper and R.W.M. van Soest), pp. 403–408. New York: the American Cancer Society. This study utilized the resources of Kluwer Academic/Plenum Publishers. the Computational Biology Service Unit from Cornell University, Hooper, J.N.A. & Soest, R.W.M. van, 2002c. 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Journal of the Marine Biological Association of the United Kingdom (2007)