Molecular phylogeny restores the supra-generic subdivision of homoscleromorph sponges (Porifera, Homoscleromorpha)

Abstract

Background: Homoscleromorpha is the fourth major sponge lineage, recently recognized to be distinct from the Demospongiae. It contains <100 described species of exclusively marine sponges that have been traditionally subdivided into 7 genera based on morphological characters. Because some of the morphological features of the homoscleromorphs are shared with eumetazoans and are absent in other sponges, the phylogenetic position of the group has been investigated in several recent studies. However, the phylogenetic relationships within the group remain unexplored by modern methods.

Methodology/principal findings: Here we describe the first molecular phylogeny of Homoscleromorpha based on nuclear (18S and 28S rDNA) and complete mitochondrial DNA sequence data that focuses on inter-generic relationships. Our results revealed two robust clades within this group, one containing the spiculate species (genera Plakina, Plakortis, Plakinastrella and Corticium) and the other containing aspiculate species (genera Oscarella and Pseudocorticium), thus rejecting a close relationship between Pseudocorticium and Corticium. Among the spiculate species, we found affinities between the Plakortis and Plakinastrella genera, and between the Plakina and Corticium. The validity of these clades is furthermore supported by specific morphological characters, notably the type of spicules. Furthermore, the monophyly of the Corticium genus is supported while the monophyly of Plakina is not.

Conclusions/significance: As the result of our study we propose to restore the pre-1995 subdivision of Homoscleromorpha into two families: Plakinidae Schulze, 1880 for spiculate species and Oscarellidae Lendenfeld, 1887 for aspiculate species that had been rejected after the description of the genus Pseudocorticium. We also note that the two families of homoscleromorphs exhibit evolutionary stable, but have drastically distinct mitochondrial genome organizations that differ in gene content and gene order.

Figure 1. A few relevant morphological characters in the Homoscleromorpha.

(a, b) spicules of the Homoscleromorpha (SEM). (a): c - calthrop of Plakina trilopha (Marseilles, Mediterranean); d – diod of Plakina trilopha (Marseilles, Mediterranean); t – triod of Plakortis simplex (Marseilles, Mediterranean); (b): hc – heterolophose calthrop (candelabra) of Corticium candelabrum (Adriatic Sea); tc – tetralophose calthrop of Plakina weinbergi (Mediterranean, Lebanon). (c) – Oscarella kamchatkensis (Avacha Bay, Bering Sea, Russia), transverse semi-thin section showing the sylleibid aquiferous system with eurypylous choanocyte chambers (cc), fine ectosome (ec) and an ostium (o); (d) – Pseudocorticium jarrei (Marseilles, Mediterranean) transverse semi-thin section showing the leuconoid aquiferous system with diplodal choanocyte chambers (cc). (e) – Oscarella malakhovi (Japan Sea, Russia) SEM micrograph of the flagellated exopinacoderm with ostium (o). (f) – Oscarella viridis (Marseilles, Mediterranean) TEM micrograph showing basement membrane (arrow heads) underlining the endopinacocytes (en) and choanocytes (ch) and endobiotic bacteria (b) in the mesohyl. (g) – Corticium candelabrum (Marseilles, Mediterranean) transverse semi-thin section of cinctoblastula larva, showing anterior (ap) and posterior (pp) poles. (h) – Plakina trilopha TEM micrograph of the cross-striated ciliar rootlet (arrow) close to a nucleus (n) in cinctoblastula larva. (i) – Oscarella microlobata (Marseilles, Mediterranean) TEM micrograph of cell junctions (zonula adhaerens) between the ciliated cells of cinctoblastula larva (arrows).

Figure 2. Phylogram showing the relationships among the six genera of Homoscleromorpha based on 18S rDNA analyses.

The topology presented corresponds to the ML analysis. Outgroups are Calcarea (AM180965, AM180976, AF100945) and Demospongiae (AY737637, AY737638) species. The Homoscleromorpha species are split into two robust clades: A and B. The numbers are from top to bottom: posterior probabilities for BI and bootstrap values (>50) for ML and MP respectively. Bayesian and MP analyses recovered slightly different phylogenies (Fig. S2). The black square points out the node corresponding to Homoscleromorpha.

Figure 3. Phylogram showing the relationships among the six genera of Homoscleromorpha based on 28S rDNA analyses.

The topology presented corresponds to the ML analysis. Outgroups are Calcarea (AM180995, AM181007, AY026372) and Demospongiae (AY864741, AY864743) species. The Homoscleromorpha species are split into two robust clades: A and B. The numbers are from top to bottom: posterior probabilities for BI and bootstrap values (>50) for ML and MP respectively. Bayesian and MP analyses recovered slightly different phylogenies (Fig. S3). The black square points out the node corresponding to Homoscleromorpha.

Figure 4. Mitochondrial genome organization in Plakinidae and Oscarellidae.

Protein (green) and ribosomal (blue) genes are atp6, atp8–9: subunits 6, 8, and 9 of F0 adenosine triphosphatase (ATP) synthase; cob: apocytochrome b; cox1–3: cytochrome c oxidase subunits 1–3; nad1–6 and nad4L: NADH dehydrogenase subunits 1–6 and 4L; rns and rnl: small and large subunit rRNAs; tatC: twin-arginine translocase component C. tRNA genes (black) are identified by the one-letter code for their corresponding amino acid. Genes outside the main circle are transcribed clock-wise, inside – counter clock-wise. Variations within each genome organization are shown in red and explained in corresponding boxes.

Figure 5. Homoscleromorph relationships based on the analyses of concatenated sequences from 14 mitochondrial protein genes.

Bayesian tree obtained from the analysis of 3278 aligned amino acid positions for the 45 taxa with the CAT+GTR model is shown. Identical relationships within Homoscleromorpha were inferred using Bayesian analyses with the CAT+GTR model and Maximum Likelihood analyses of the small (35 taxa) amino acid dataset. Bayesian analyses with the CAT model as well as ML analysis of the nucleotide dataset and of the 45 taxa amino acid dataset resulted in slightly different phylogenies (Fig. S4). Asterisks indicate nodes within Homoscleromorpha with maximum support values in all analyses. For other nodes within this group, support values represent (from left to right): posterior probabilities in Bayesian analysis using CAT+GTR model with 45/35 taxa, bootstrap support values for the ML analyses of amino-acid datasets with 45/35 taxa, and bootstrap support values for the ML analysis of the 35 taxa nucleotide dataset. Two unstable nodes are shown in red with a minus sign indicating that the node was not recovered in the analysis. For nodes outside Homoscleromorpha, only posterior probability values for the Bayesian analysis with the CAT+GTR model and 45 taxa are shown.

Figure 6. Simplified consensus tree showing the genera relationships in Homoscleromorpha based on molecular phylogenies.

Morphological characters that are diagnostics of these clades are mapped. I: Absence of spicules in clade A. II: Specific gene arrangement in mitochondrial genome in clade A (see Fig. 4). III: Presence of spicules (lophate and alophate) in clade B. IV: Specific gene arrangement in mitochondrial genome in clade B (see Fig. 4). V: Presence of a candelabra (heterolophose calthrop) specific to Corticium genus, clade B1. VI: Presence of alophose spicules and absence of lophose spicules in clade B2. VII: Presence of tetralophose calthrop and of a well developed mesohyl in clade B3.