Ns within diplomonads [4,8,9]. In this study we focus on Spironucleus barkhanus and Spironucleus salmonicida. Both species were previously known as S. barkhanus. However, parasites causing systemic infections in Atlantic salmon Salmo salar, Chinook salmon Oncorhynchus tshawytscha and Arctic char Salvelinus alpinus [10], are now classified as S. salmonicida [11]. These parasites have caused severe problems for fisheries [12,13]. The closely related S. barkhanus is a commensal in wild freshwater populations of Arctic char and grayling Thymallus thymallus [11]. These Staurosporine morphologically indistinguishable organisms are defined as separate species on the basis of their ecology (commensal and parasite) and that they form two clades in gene trees [11]. The genetic divergence is between 8 and 30 for the ribosomal RNA, alpha-tubulin and glutamate dehydrogenase genes [11]. The ATCC 50377 strain of S. salmomicida is the most studied diplomonad on the genetic level outside the Giardia genus [14], although there is an ongoing genome project on Spironucleus vortens [15]. The genome projects on S. salmonicida and G. intestinalis revealed reduced and compact genomes with few, ifany, genes with introns [14,16]. There are also variations between the diplomonad genomes. A number of lineagespecific genes obtained from bacteria were detected in S. salmonicida and the codon usages are drastically different [14]. In addition, all hexamitinid diplomonads (including Spironucleus) utilize an alternative genetic code, whereas the canonical code is used by members of the Giardia genus [4,17] The level of sequence divergence within diplomonad cells also vary extensively. Both nuclei in the vegetative trophozoite stage of G. intestinalis are diploid [18]. In situ hybridization studies of G. intestinalis genotype A have shown that each nucleus contains at least one complete copy of the genome and that the two nuclei are partitioned equationally at cytokinesis [19]. Allelic variations are expected to accumulate in the absence of genetic exchange between the two nuclei [20]. Nevertheless, a very low frequency of sequence heterozygosity is present in the G. intestinalis genotype A (WB) genome [21] and no allelic sequence variation was reported from the S. salmonicida genome [14]. Genetic exchange might occur between the nuclei, suggesting a mechanism that maintains a low sequence divergence [22]. However, the efficiency of such a mechanism appears to vary between closely related lineages; the draft G. intestinalis GS (genotype B) genome revealed an overall allelic sequence divergence of 0.5 [7]. We have performed a comparative genomic project on S. barkhanus and S. salmonicida, including the generation of 5000 expressed sequence tag (EST) sequences from S. barkhanus and an estimation of genome sizes of both species. The goal was to increase the understanding of the tempo and mode of genomic structure and content evolution within the diplomonads. The present study present several unexpected observations such as large differences of diplomonad genome size and codon usage, and a high level of allelic sequence variation in S. barkhanus.Results and Discussion S. barkhanus and S. salmonicida are two PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/17139194 morphologically indistinguishable protists which have recently been classified as two distinct species based on ecological and genetic data from a few genes [11]. We isolated S. barkhanus from the wild freshwater salmonid grayling (Thymallus thymallus) to further study the genetic.