Other species with various levels of distance from the in-group were chosen: these include representatives of other demosponge subclasses - one Ceractinomorpha species (Halichondria panicea) and two Tetractinellida species (Cinachyrella sp.
Primers used were as follows (specificity, orientation, and position of primers in the aligned sequences of Figure 1 follow each sequence): ITS3 5[prime]-GTCGATGAAGAACGCAGC-3[prime], universal, forward, external 5[prime]; Ep1b[prime] 5[prime]-GTGGCCGGGAGAGGCAGC-3[prime], part of Demospongiae not Tetractinellida, forward, 257-274; Ep2 5[prime]-CTYYGACGTGCCTTTCCAGGT-3[prime], Demospongiae, reverse, 303-323; D2 5[prime]-TCCGTGTTTCAAGACGGG-3[prime], universal, reverse, external 3[prime].