Contributions to Zoology, 76 (2) 95-102 (2007) Affi nities of the family Sollasellidae (Porifera, Demospongiae). II. Molecular evidence Dirk Erpenbeck1, 2, 3, John N.A. Hooper1, Sue E. List-Armitage1, Bernard M. Degnan3, Gert Wörheide2 and Rob W.M. van Soest4 1 Biodiversity Program, Queensland Museum, PO Box 3300, South Brisbane, Qld, 4101, Australia, derpenb@gwdg. de; 2 Dept. of Geobiology, Geoscience Centre Göttingen, Goldschmidtstr. 3, 37077 Göttingen, Germany; 3 School of Integrative Biology, The University of Queensland, St Lucia, Qld, 4072, Australia; 4 Zoological Museum, Univer- sity of Amsterdam, P.O. Box 94766, 1090 GT Amsterdam, The Netherlands Key words: sponges, classifi cation, Raspailiidae, Sollasella, Raspailopsis, 28S rDNA, molecular systematics Abstract mann (1914), and consequently assigned more recently as incertae sedis in the order Hadromerida based on its This is the second part of a revision and re-classifi cation of the cortex and radiating skeleton (Hooper and Van Soest, demosponge family Sollasellidae, and an example of a success- 2002). However, the recent collection and examination ful use of combined morphological and molecular data. Solla- of a morphologically similar sponge from Oman identi- sella had been a poorly known, long forgotten taxon, placed fi ed as Raspailopsis cervicornis Burton, 1959 (= Ras- incertae sedis in the order Hadromerida in the last major revision of the demosponges. It has recently been suggested to belong to pailia (Parasyringella) cervicornis sensu Hooper, Raspailiidae in the order Poecilosclerida due to striking morpho- 2002b), raised evidence for a placement of Sollasella in logical similarities. The present analysis verifi ed this re-classi- the family Raspailiidae (order Poecilosclerida, Van Soest fi cation using molecular markers. Comparing 28S rDNA frag- et al., 2006). After further morphological analyses, R. ments of Sollasella cervicornis, a newly described species S. cervicornis was merged with Sollasella forming the moretonensis and a representative set of raspailiid and hadromer- twentieth valid genus in the Raspailiidae (Van Soest et id samples. In our analyses Sollasella clearly clusters inside the al., 2006). In the same publication Van Soest and col- Raspailiidae clade, and distantly from hadromerid taxa. Support- leagues describe a similar species from subtropical ing morphological hypothesis of Van Soest et al. (2006), that Sollasella is a raspailiid sponge. Australia, Sollasella moretonensis, which shares the characteristic polygonal perforation-like surface pattern of S. cervicornis and S. digitata, in addition to other features. These polygonal surface structures remain a Contents distinguishing feature for the genus. The aim of our analysis is to test the hypothesis that Introduction ...................................................................................... 95 Material and methods ..................................................................... 96 Sollasella is more closely related to poecilosclerid taxa Results ............................................................................................... 96 as concluded recently based on morphologic data in the Discussion ...................................................................................... 101 family Raspaillidae than to hadromerid sponges (Van Acknowledgements ...................................................................... 101 Soest et al., 2006). Among candidate hadromerid taxa References ...................................................................................... 102 are Polymastiidae, with which Sollasella shares the presence of a cortex (particularly the genus Pseudotra- chya, with which Sollasella shares the combination of Introduction choanosomal styles and ectosomal oxeas), and Suber- itidae, with which Sollasella shares the stalked habit A morphological re-examination of material of the and axially condensed skeleton (genera Homaxinella, demosponge family Sollasellidae has shed a new light Plicatellopsis, Rhizaxinella, see Van Soest, 2002a). on its classifi cation (Van Soest et al., 2006). This mono- There is also an apparent slight affi nity with Stylocor- specifi c, previously poorly known family, with the type dylidae (Van Soest, 2002b). locality in East Australia (Sollasella digitata Lendenfeld, Reliability of morphological systematics in sponges 1888), had been nearly completely forgotten since Hall- is hampered by the paucity of complex characters, many 96 D. Erpenbeck et al. – Affi nities of sponge family Sollasellidae of which are also prone to homoplasies. The family mM), 3.25 μl MgCl2 (25 mM), 2.5 μl 10× HotMaster Raspailiidae, as in many other demosponge taxa, does PCR Buffer, 2.5 μl BSA (100 mM, Sigma), 0.5 μl not possess unique autapomorphies, and is defi ned by primer (10 mM) and 4 μl HotMaster polymerase (Ep- a combination of characters that may each be found also pendorf). The BSA was used only on the Sollasella in some other demosponge orders and families (see moretonensis DNA extracts and replaced with ddH2O Hooper, 2002b). DNA sequence data should provide for all other samples. Cycle sequencing of both strands independent evidence to test morphological hypotheses. was performed with BigDye terminator v1.1 (ABI) and However, sponge molecular systematics has pitfalls of a capillary sequencer (ABI). MacClade 4.06 (Maddison its own such that phylogenies arising from these analy- and Maddison, 1992) was used for sequence manage- ses need to be cautiously interpreted pending better ment and manual alignment. The sequences were in- insight into demosponge molecular evolution. Molecu- corporated into two modifi ed alignments based on lar data occasionally generate hypotheses that confl ict secondary structure information. They consisted of the dramatically with phylogenies based on morphological full length D3-D5 data set as published in Erpenbeck characters (e.g., McCormack et al., 2002; Erpenbeck et et al. (2005) and a second, shorter data set with addi- al., 2006). Improved algorithms may aid to fi lter the tional overlapping sequences of Nichols (2005). As this correct phylogenetic signal from random noise and overlap data set is comparatively short - it resulted in provide explanations for the occasionally ‘odd’ phyl- an alignment with 441 characters - we restricted our- ogenies. Nevertheless, sponge molecular systematics selves to incorporate only those sequences of Nichols has repeatedly been shown to outcompete morphologi- (2005) with a complete 5’ region (see Table 1) and used cal approaches (e.g. Erpenbeck et al., 2006). it for comparative purposes only. In our present approach we generate and analyse Phylogenetic relationships were reconstructed with 28S rDNA data from specimens of Sollasella cervi- Bayesian inference methods (BI) using MrBayes 3.1 cornis (sensu Van Soest et al., 2006) from Oman, (Ronquist and Huelsenbeck, 2003). Potential overpa- Sollasella moretonensis from northeastern Australia, rameterization does not infl uence the correctness of BI and several species of other Raspailiidae. The se- reconstructions in MrBayes (Huelsenbeck et al., 2001). quences are compared with data sets from Erpenbeck Therefore, the GTR+G+I model was chosen for all BI et al. (2005) and partially from Nichols (2005), which analyses of the D3-D5 data set and non-pairing sites in is currently the most comprehensive 28S rDNA data secondary structure specifi c analyses with the Nichols’ set with a strong representation of hadromerid se- (2005) data set (SH for paring sites, see Erpenbeck et quences. al. 2007 for details). Results were compared with Minimum-evolution (ME) reconstructions under Maximum-likelihood (ML) distances. Modeltest 3.06 Material and methods (Posada and Crandall, 1998) estimated the relatively best-fi tting ML-model. Bayesian inference analyses The list of specimens used in this analysis is given in consisted of two runs of four Markov chains each for Table 1. We chose the 28S D3-D5 fragment for which maximal 10,000,000 generations. Runs were stopped a comprehensive taxon set has been sequenced. How- automatically when the average standard deviation of ever, no amplifi able DNA could be obtained from split frequencies dropped below 0.01. All other phylo- material of the type specimen of the family Sollaselli- genetic analyses were performed with PAUP*b10 dae, Sollasella digitata, from the Australian Museum (Swofford, 2002). The ME bootstrap values were cal- in Sydney (AM G9107). Sequences of Sollasella cer- culated on 1000 replicates on maximum likelihood vicornis, S. moretonensis and a representative set of distances. Different phylogenetic hypotheses were Raspailiidae have been recently generated and are tested using the COUNSEL 0.942 package (Shimodaira deposited in GenBank (see table 1). Their DNA was and Hasegawa, 2001) under default settings. extracted with Qiagen DNeasy kits. PCR primers em- ployed were taken from McCormack et al. (2002) (RD3A: GACCCGTCTTGAAACACGA and RD5B2: Results ACACACTCCTTAGCGGA, temperature regime: 94°C 2 min, 35 cycles at 94°C 30 sec; 47°C 20 sec; The different phylogenetic reconstruction methods of 65°C 30 sec, followed by 65°C 10 min). PCR amplifi - both data sets displayed identical results on the position cations contained 11.25 μl ddH2O, 4.15 μl dNTP (10 of the Sollasella cervicornis sequence. In the longer Contributions to Zoology, 76 (2) – 2007 97 data set this sequence clusters inside a clade compris- bootstrap probabilities (BI: 100, ME: 94). Similarly, ing all the other Raspailiidae