Fig. 5. Cell morphologies and molecular signatures of novel species Bathycoccus calidus, formerly Bathycoccus RCC716, as compared to Bathycoccus prasinos.

A Transmission electron micrographs of B. calidus isolate RCC716, reveal cell characteristics including, n nucleus, c chloroplast, m mitochondria, and arrows external scales. B The second internal transcribed spacer (ITS2) of Bathycoccus calidus (i.e., Bathycoccus Clade BII, KY563784) has ‘universal’ helices 1-4 and an additional helix (between helices 3 and 4), characteristic for the family Bathycoccaceae (i.e., Bathycoccus and Ostreococcus genera [103]). Helices of B. calidus were compared with homologous ITS2 helices from B. prasinos (Clade BI, JX625115) by highlighting compensatory base changes (CBCs), hemi-CBCs, base pairing/dissociation events (dotted lines), as well as hypothetical insertions/deletions (indels). A few regions, which were divergent and showed no intermediate evolutionary stages, were labelled as ‘not comparable’ (n.c.). Note that while the two Clade BII isolates have identical ITS sequences, among 13 Bathycoccus environmental rRNA/ITS sequences annotated as Bathycoccus Clade BII [12], four from tropical Pacific and Atlantic (KY382370, KY382373, KY382374, KY368637) present polymorphisms (green shading) at one nucleotide (helix 2, bp 16 reverse, G instead of A) as well as one deletion (helix 4, bp 13, bp not present). Tracing base pair evolution in the stem regions (i.e., helices) by CBCs and hemi-CBCs revealed several molecular signatures for B. calidus in helices 2 and 4, i.e., without homoplasies in the Bathycoccaceae [19], most of which evolved as hemi-CBCs. Signatures for B. calidus are indicated by orange shading. Base pairs 16 and 17 of helix 4 (corresponding to bp 18 and 19 in B. prasinos) were selected for the taxonomic diagnosis. A single-stranded nucleotide position in the conserved linker between the additional helix and helix 4 revealed another distinctive character of B. calidus.