Molecular Phylogenetics and Evolution 40 (2006) 830–843 www.elsevier.com/locate/ympev Non-monophyly of most supraspeciWc taxa of calcareous sponges (Porifera, Calcarea) revealed by increased taxon sampling and partitioned Bayesian analysis of ribosomal DNA Martin Dohrmann a, Oliver Voigt a, Dirk Erpenbeck a,b, Gert Wörheide a,¤ a Department of Geobiology, Geoscience Centre Göttingen, Goldschmidtstr. 3, D-37077 Göttingen, Germany b Queensland Museum, South Brisbane, Qld., Australia Received 23 January 2006; revised 21 March 2006; accepted 4 April 2006 Available online 30 April 2006 Abstract Calcareous sponges (Porifera, Calcarea) play an important role for our understanding of early metazoan evolution, since several molecular studies suggested their closer relationship to Eumetazoa than to the other two sponge ‘classes,’ Demospongiae and Hexacti- nellida. The division of Calcarea into the subtaxa Calcinea and Calcaronea is well established by now, but their internal relationships remain largely unresolved. Here, we estimate phylogenetic relationships within Calcarea in a Bayesian framework, using full-length 18S and partial 28S ribosomal DNA sequences. Both genes were analyzed separately and in combination and were further partitioned by stem and loop regions, the former being modelled to take non-independence of paired sites into account. By substantially increasing taxon sampling, we show that most of the traditionally recognized supraspeciWc taxa within Calcinea and Calcaronea are not monophyletic, challenging the existing classiWcation system, while monophyly of Calcinea and Calcaronea is again highly supported. © 2006 Elsevier Inc. All rights reserved. Keywords: Porifera; Calcarea; Phylogeny; Ribosomal DNA; Bayesian inference; Bayes factors; Doublet-model; Data-partitioning 1. Introduction imply that the last recent common ancestor of (Eu)metazoa was a sponge-like organism or, alternatively, the sponge Sponges (Porifera Grant, 1836) are sessile, aquatic Wlter bauplan evolved twice, Calcarea play an important role in feeders that are considered to be the earliest branching met- the reconstruction of early animal evolution, making a azoans (e.g., Ax, 1995). Monophyly of Porifera has been well-resolved and supported phylogeny of this group questioned by a number of molecular studies (e.g., Adams clearly desirable. et al., 1999; Borchiellini et al., 2001; Cavalier-Smith et al., The calcareous sponges are represented by about 500, 1996; Collins, 1998; Kruse et al., 1998; Lafay et al., 1992; exclusively marine species distributed in all oceans (Manuel Medina et al., 2001; Zrzavy et al., 1998)—albeit usually et al., 2002). While the mineral skeleton of Demospongiae with low statistical support—with the calcareous sponges and Hexactinellida consists of intracellularly formed sili- (Calcarea Bowerbank, 1864) being more closely related to ceous spicules, Calcarea is characterized by the intercellular eumetazoans than to the other two classically recognized formation of spicules composed of calcium carbonate, major sponge lineages Demospongiae Sollas, 1885 and which is an autapomorphic character of this group (Ax, Hexactinellida Schmidt, 1870, which are commonly 1995; Böger, 1988; Manuel, 2006; Manuel et al., 2002). The grouped together as Silicispongia or Silicea. As this would monophyly of calcareous sponges is also supported by ribosomal DNA (rDNA) data (Borchiellini et al., 2001; Manuel et al., 2003, 2004). * Corresponding author. Fax: +49 551 397918. Cytological and embryological characters and features E-mail address: [email protected] (G. Wörheide). of spicule morphology strongly suggest a division of the 1055-7903/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2006.04.016 M. Dohrmann et al. / Molecular Phylogenetics and Evolution 40 (2006) 830–843 831 Calcarea into the subtaxa Calcinea and Calcaronea (Bid- Erpenbeck et al., unpublished data). The aims of this study der, 1898; Borojevic et al., 1990, 2000; Manuel, 2006; Man- were to evaluate the validity of classically recognized calci- uel et al., 2002). Another character distinguishing these two nean and calcaronean supraspeciWc taxa, for most of which groups is the ratio of diVerent carbon isotopes that are no clear statements about potential morphological apo- incorporated into the spicules during biomineralisation morphies can be found in the literature, and to re-evaluate (Reitner, 1992; Wörheide and Hooper, 1999). Although the earlier Wndings (Manuel et al., 2003, 2004) in the light of Calcinea and Calcaronea are very well characterized by substantially increased taxon sampling and a more Xexible these features, there still remains the possibility that some approach of inferring phylogenies. While distinction of the character states in one of the groups represent symplesio- classically recognized ‘subclasses’ Calcinea and Calcaronea morphies, rendering the respective group paraphyletic with is highly supported by our analyses, our results suggest that regard to the other (Manuel et al., 2002; but see Manuel, the majority of ‘orders’ and ‘families,’ as well as some ‘gen- 2006). As rDNA studies (Borchiellini et al., 2001; Manuel era,’ such as the species-rich Clathrina and Leucandra are et al., 2003, 2004) do support monophyly of Calcinea and not monophyletic. Calcaronea, this scenario seems rather unlikely, however. In contrast, phylogenetic relationships within Calcinea 2. Materials and methods and Calcaronea remain largely unclear, because the existing classiWcation of calcareous sponges (Borojevic et al., 1990, Species, collection sites, sample-numbers of the Queens- 2000, 2002a,b,c; Vacelet et al., 2002a,b) is primarily typo- land Museum (QM), South Brisbane (Australia), where logic, and a phylogenetic system of this group has not been most vouchers are deposited, and GenBank accession num- proposed so far (but see Reitner, 1992). Because of the bers of the sequences generated in this study, as well as apparent high level of morphological homoplasy (Manuel those retrieved from GenBank (http:// et al., 2003), such a system would be diYcult or impossible www.ncbi.nlm.nih.gov/), are given in Table 1; for full to base on the available morphological data alone. There- nomenclature of ingroup-taxa see Supplementary Table 1. fore, molecular data provide the most promising means to resolve this branch of the tree of life. 2.1. DNA-extraction, -ampliWcation, and -sequencing So far, only two studies (Manuel et al., 2003, 2004) explicitly addressed the question of phylogenetic relation- Genomic DNA was extracted from ethanol-preserved ships within Calcarea, applying maximum parsimony (MP) or silica-dried samples with the DNEasy Tissue Kit of and maximum likelihood (ML) methods to infer trees from Qiagen (Hilden, Germany), following the manufacturer’s 18S and 28S rDNA sequences and morphological character protocol. To avoid contamination with epibiontic organ- data of 17 calcareous sponge species, representing 15 ‘gen- isms, tissue from the interior of the sponges was used era,’ 13 ‘families’ and three out of Wve ‘orders.’ An impor- whenever possible. Full-length 18S rDNA was ampliWed tant result of these studies was the placement of Petrobiona by polymerase chain reaction (PCR) with primers 18S1 massiliana Vacelet and Lévi, 1958 in Baerida Borojevic and 18S2 (Manuel et al., 2003; see Supplementary Table 2) et al., 2000 instead of Lithonida Vacelet, 1981, which is also (2 min/94 °C; 34 cycles [1 min/94 °C; 1 min/50–58 °C; 2 min/ supported by some spiculation features such as the occur- 72 °C]; 7 min/72 °C). Partial 28S rDNA (domain D2 to rence of microdiactines and pugioles (dagger-shaped tetrac- helix 36; nomenclature of Michot et al., 1990) was ampli- tines). Furthermore, monophyly of Leucosolenida Wed with primers from Medina et al. (2001) and Nichols Hartman, 1958, Grantiidae Dendy, 1892, and Sycon Risso, (2005) (see Supplementary Table 2) (10 min/95 °C; 34 1826, was not supported. However, taxon sampling was still cycles [1 min/95 °C; 1 min/50–58 °C; 1–4 min/72 °C]; 7 min/ too sparse, especially with respect to Calcinea, to make fur- 72 °C). Reaction mixes contained 2.5 l of 10£ NH4 PCR- V ther inferences about higher-level relationships within the bu er (Bioline, Luckenwalde, Germany), 1.0–1.5 l MgCl2 two major groups of calcareous sponges. (50 mM), 1 l of each primer (10 M), 0.5 l dNTPs With this study, we extend the set of available calcarean (10 mM each), 0.05 l Taq-DNA-Polymerase (5 u/l; Bio- 18S and 28S rDNA sequences to 44 (mostly Indo-PaciWc) line, Luckenwalde, Germany) and 0.5–5 l template. Bands species, representing 27 ‘genera’, 18 ‘families’ and all Wve of expected size were cut out from agarose gels and puri- currently recognized ‘orders’ of Calcarea. Taxon sampling Wed following Boyle and Lew (1995). Both strands of the of Calcinea is increased from four (Manuel et al., 2003, amplicons were sequenced directly with BigDye Termina- 2004) to 20 species. From 31 species we also sequenced tor 3.1 chemistry and an ABI Prism 3100 Genetic Analyser »750 additional base pairs (bp) of the 28S rRNA gene. We (Applied Biosystems). Sequencing primers are given in analyzed both genes separately and in combination in a Supplementary Table 2. Intragenomic length variation did Bayesian framework that accounts for diVerent evolution- not allow direct sequencing of Eilhardia schulzei and Plec- ary constraints of stem and loop regions and non-indepen- troninia neocaledoniense, so PCR products were cloned dence of paired