Evolutionary Relationship of Porifera Within the Eukaryotes
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Hydrobiologia (2006) 568(S):167–176 Ó Springer 2006 G. Coulter, O. Timoshkin, L. Timoshkina & K. Martens (eds), Species and Speciation in Ancient Lakes DOI 10.1007/s10750-006-0318-6 Evolutionary relationship of Porifera within the eukaryotes Werner E.G. Mu¨ ller*, Isabel M. Mu¨ ller & Heinz C. Schro¨ der Institut fu¨r Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universita¨t, Duesbergweg 6, 55099 , Mainz, Germany (*Author for correspondence: E-mail: [email protected]; Tel: +6131-3925910; Fax: +6131-3925243) Key words: phylogeny, COI, Metazoa Abstract Molecular data, gathered in our laboratory, strongly support the view that all metazoan phyla, including Porifera, are of monophyletic origin. More recently, a hypothetical common ancestor for all Metazoa, the Urmetazoa, had be formulated. The Urmetazoa possessed besides the key structures of an extracellular matrix also immune molecules and signal transduction molecules that are characteristic to Metazoa. In addition, we could establish that all Metazoa share a common ancestry with the Fungi and Plantae, while the unicellular eukaryotes are only distantly related. With respect to the origin of freshwater sponges, especially the species found in the Lake Baikal as a representative for an ‘‘old’’ lake, have been investigated. Sequence comparisons were performed with the ubiquitously distributed freshwater sponge Spongilla la- custris (family Spongillidae) as well as with one marine sponge, Suberites domuncula. The sequence com- parison of the mitochondrial COI gene revealed a monophyletic grouping of the endemic baikalian sponges with S. lacustris as the most related species to the common ancestor. The sequences from Baikalospongia recta, B. intermedia, B. bacillifera and Lubomirskia baicalensis were found to be identical and separated from that of Swartschewskia lacustris and S. papyracea. In a further approach the exon/intron sequences framing the intron-2 of the sponge tubulin gene were chosen for the phylogenetic analysis. Data analyses revealed again a monophyletic grouping with S. lacustris as the closest related species to the common ancestor. It is concluded that the baikalian sponges, which have been studied here, are of monophyletic origin. Furthermore, the data suggest that the endemic species S. papyracea is the phylogenetic oldest still extant endemic baikalian sponge species. Introduction mate receptor (Perovic et al., 1999), as well as (iii) homologs/modules of an primordial immune sys- With the introduction of molecular data based on tem, e.g. immunoglobulin-like molecules (Scha¨ cke analyses of nucleotide [nt] sequences, coding for et al., 1994a), SRCR- and SCR-repeats (Pancer proteins, it became evident that the phylum Porif- et al., 1997b) or the Rhesus system (Seack et al., era (sponges) is with the other metazoan phyla of 1997), and (iv) cell surface receptor molecules, e.g. monophyletic origin. The molecules which most receptor tyrosine kinases (Scha¨ cke et al., 1994b). strongly suggest that Porifera have to be included The cDNAs have been isolated – and some of them in the Metazoa are those which are constituent also expressed – from all classes of Porifera, mainly elements (i) of the basal lamina, e.g. integrin from the Demospongiae Geodia cydonium and receptor (Pancer et al., 1997a), fibronection (Pahler Suberites domuncula, but also from Calcarea, Sy- et al., 1998) and polypeptides, rich in scavenger con raphanus and from Hexactinellida, Rhabdoca- SRCR and SCR modules (Pahler et al., 1998), (ii) lyptus dawsoni. Based on these molecular data it is of neuronal transmission, e.g. metabotropic gluta- reasonable to the accept that multicellular animals 168 evolved only once (monophyly), as first published the position of sponges. Amino acid [aa] sequences in 1994 (Mu¨ ller et al., 1994) and later in detail found in all representatives of these subkingdoms, (Mu¨ ller, 1995, 1998). This conclusion drawn from one heat shock protein, the serine/threonine (Ser/ analyses of protein-coding nt sequences has been Thr) kinase domain of protein kinases, b-tubulin recently supported also by rRNA ribosomal data and calmodulin were analyzed together with those (Cavalier-Smith et al., 1996). deduced from sponge cDNAs. Unlike with Metazoa the monophyletic origin First, polypeptides grouped to the heat shock of Plantae appears to be established since a longer proteins [HSP] of the 70-kDa class, the HSP70, time; they can be traced back to the Chlorophyta were investigated. Heat shock proteins are highly (reviewed in: Margulis & Schwartz, 1995). The conserved throughout living kingdoms. These ancestry of the third group of multicellular molecules act as molecular chaperones under eukaryotes, the Fungi, is less clear; monophyletic physiological and stress conditions; the chaper- and polyphyletic evolution of these spore forming ones of prokaryotic organisms, which are related and amastigote organisms are discussed (in Mar- to the eukaryotic HSP70 multigene family are gulis & Schwartz, 1995). termed DnaK. They are divided according to We addressed for the first time the phylogenetic their different inducibility into (i) the group of relationship of the three multicellular eukaryotic constitutively expressed heat shock proteins, subkingdoms to unicellular eukaryotes, putting the which are also present under nonstressed condi- main emphasis on the position of sponges. Deduced tions, and (ii) the group of HSP70 polypeptides, amino acid [aa] sequences of cDNAs from sponges which are induced under temperature shock and coding for proteins, found in all representatives of other specific stress situations. The inducible, these subkingdoms have been analyzed. The data cytoplasmic HSP70 amino acid (aa) sequences presented (Schu¨ tze et al., 1999) show that the were selected for the analysis (Koziol et al., 1996, sponge molecules, especially those obtained from 1998). the classes Demospongiae and Calcarea form the Second, the serine/threonine (Ser/Thr) kinase basis of the metazoan branch. Furthermore, the domain found in protein kinases from Fungi and results reveal that the Metazoa appeared later dur- Metazoa, but not in Plantae or unicellular ing evolution from the unicellular eukaryotes than eukaryotes (Hardie & Hanks, 1995; Kruse et al., the Fungi and the Plantae (Viridiplantae). 1997) was used for the analyses. These enzymes are Focusing on freshwater sponges a similar clar- essential for fungal or metazoan organisms to ification of the evolutionary relationship remains recognize extracellular signals and to initiate to be determined. An interesting, approach to intracellularly appropriate adaptive biological solve the evolutionary origin of the freshwater responses. sponges are studies on endemic freshwater sponges Third, sequences of b-tubulin, one major ele- especially from geologically old biotopes. Lake ment of microtubuli (intracellular structures that Baikal is famous because of its high biodiversity, are employed for a number of functions in especially with respect to its freshwater sponges eukaryotes, including flagellar motility and chro- (Rezvoi, 1936). This lake is the oldest (>24 mil- mosome aggregation as well as cell maintenance of lion years), deepest (1637 m) and most volumi- cellular morphology) were analyzed (Schu¨ tze nous lake on earth, comprising one fifth of the et al., 1999). world’s unfrozen freshwater; it contains more than Finally, calmodulin, a protein ubiquitous in 1500 endemic species (Stewart, 1990). eukaryotes, was selected for phylogenetic analysis. It is a Ca2+ binding protein of approximately 150 aa residues that is involved in a wide range of Phylogenetic position of sponges: the Urmetazoa intracellular signaling pathways. The molecule binds four Ca2+ ions in a cooperative fashion The aim of a previous study (Schu¨ tze et al., 1999) during which it undergoes a conformational was the evaluation of the phylogenetic relationship change. The calmodulin sequence from G. cydo- of the three multicellular eukaryotic subkingdoms nium was identified for the purpose of the phylo- to unicellular eukaryotes, with the main interest in genetic analysis (Schu¨ tze et al., 1999). 169 The data on b-tubulin are summarized. Metazoa has been termed Urmetazoa (Mu¨ ller, Homology searches of the sponge deduced aa b- 2001). The major novelties, which can be attrib- tubulin sequence TBB_GEOCY revealed highest uted to the Urmetazoa, have been summarized similarity to the sequence from Caenorhabditis (Mu¨ ller, 2001). elegans (96% similar aa). Phylogenetic analysis was performed which showed that the b-tubulin molecules from Metazoa are more closely related to the tubulin sequences from Fungi or Plantae The phylogenetic position of the three sponge classes than to unicellular eukaryotes (Schu¨ tze et al., 1999); Figure 1. The rooted tree strikingly shows Until our studies the phylogenetic position of the that the sponge sequence forms with the other class Hexactinellida within the phylum Porifera metazoan molecules one branch (Fig. 1). The ro- [sponges] could not be resolved by molecular data, bust grouping separates the Metazoa from the because no cDNA encoding a protein from the Viridiplantae, the Fungi and the unicellular sponge class Hexactinellida was available. We eukaryotes. As an outgroup for this analysis the have isolated and characterized the cDNA bacterial cell-division protein FtsZ was used. encoding a protein kinase C belonging to the C In conclusion, phylogenetic analyses of the four subfamily (cPKC) from the hexactinellid sponge deduced