Codosiga spp. was identified by life observations and scanning electron microscopy as shown (A). Figure 2 Abundance of heterotrophic nanoflagellates (light grey) and relative abundance of naked choanoflagellates (dark grey) in redoxclines of Gotland Deep in 2008 (GD 2008) and 2009 (GD 2009) and Landsort Deep

2009 (LD 2009) based on epifluorescence microscopy. The horizontal dashed line represents the first appearance of hydrogen sulfide (chemocline). Note the changes in the scale of some axis between the two years. Phylogenetic reconstructions using ribosomal gene sequences Nearly complete 18S rRNA gene sequences were obtained for both strain IOW73 (1748 base pairs in length), and strain IOW94 (1783 base pairs). Additionally, we generated partial 28S rRNA sequences for both strains to enable comparison with Codosiga gracilis from GenBank (the 18S rRNA sequence is missing learn more Temozolomide chemical structure for this unique Codosiga culture, see [6]). The 28S sequences obtained, including the divergent D1-D6 regions, possessed a length of 1620 and 1612

base pairs for strain IOW73 and strain IOW94, respectively. Strains IOW73 and IOW94 belong to the Salpingoecidae according to [6] and branched off with clade 1 by Carr et al. [5], and clade A by del Campo & Massana [16]. The 18S rRNA tree (Figure 3) additionally contains environmental sequences from different habitats closely related to clade A. The Codosiga sequences form a well supported clade with sequences from hypoxic habitats

such as the Baltic Sea (Gotland Deep), Framvaren Fjord, the Black Sea and Sagami Bay, Japan. The only exceptional sequence in this clade, that was not isolated from hypoxic environment, is AJ402325 from the Pacific [27] which forms the basal branch. We were able to establish cultures for two further strains, IOW74 (Gotland Deep, 208 m) and IOW75 (Landsort Deep, 260 m), whose short 18S rRNA sequence fragments are identical to strain IOW73 (data not shown). Figure 3 Phylogenetic relationships of choanoflagellate strains isolated within this study to environmental sequences from hypoxic habitats based on partial 18S rRNA sequences using MrBayes. New species are presented in white mafosfamide bold characters; environmental clonal sequences of hypoxic habitats are shown in bold face letters. Posterior probability and bootstrap values above 0.5 and 50 are indicated. Values above 0.99 and 99 are presented as bold face branches. Scale bar represents 0.1 mutations per position. Amoebidium parasiticum (Ichthyosporea) was used as outgroup representative. The phylogenetic tree based on partial 28S rRNA gene sequences, excluding the highly divergent D2 region, shows a well established branching order in the Craspedida and Acanthoecida (Figure 4). Sequences of our new isolates are closely related to Codosiga gracilis ATCC50454, rendering the genus Codosiga monophyletic. Strain IOW94 is more closely related to C. gracilis (p-distance 4.8%) than IOW73 (p-distance to C. gracilis 11.6%).