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The cpGenome size ranged from 65,523 bp in Eutreptia viridis to 144,166 bp in

Strombomonas acuminata. This noteworthy heterogeneity in size can be explained in different

ways. First, there are the rRNA genes encoded in operons in each cpGenome sequenced so far. The operon codes for the 16S, 23S and 5S genes are interrupted by intergenic spaces. The operon configuration is specific for most chloroplast rRNA genes in algae and higher plants and has a characteristic prokaryotic gene order (Bogorad & Vasil 1991), supporting the insight that the primary endocytobiosis event occurred just once, followed by several secondary endocytobiosis events (Archibald 2015, Keeling 2010) and that the phototrophic euglenoids are the result of the latter with a green alga. As recent studies have shown remarkable differences in number and organization on the cpGenomes among different

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species, it was of main interest to figure out, if the increasing number of investigated species will offer an evolutionary pathway on rRNA operon evolution.

For instance, comparing the cpGenomes of the genus Euglena shows for E. gracilis strain Z that the operon structure is tandemly repeated three times plus a fourth 16S rRNA gene (Hallick et al. 1993). This alone resulted in over 21,000 bp, which yield a 15 % share of the full cpGenome. In contrast, the cpGenome of E. mutabilis (SAG 1224-9b) contains only one rRNA operon with a length of 4,640 bp, which represents only 5 % of the total cpGenomes size (Dabbagh & Preisfeld 2017). It is still unknown how and why these differences occur. Fourteen out of the 19 cpGenomes of phototrophic euglenoids sequenced, comprise only one rRNA copy, including the uncircularized cpGenomes of Et. viridis. Though for the latter the sequencing coverage was more than twice that of single copy protein-coding genes, implicating strongly that at least two copies should be present (Wiegert et al. 2012). The two basally branching Eutreptiella species contain two rRNA operons, whereby the cpGenome of

Etl. gymnastica contains two incomplete copies with the 5S rRNA genes missing (Hrdá et al.

2012). Another peculiarity regarding the rRNA operons of Etl. gymnastica is that one of the rRNA operons is divided by protein-coding genes and thus not arranged like a typical operon (Dabbagh et al. 2017, Hrdá et al. 2012). The cpGenome of S. acuminata shows one complete and one incomplete copy, missing the second 16S rRNA gene (Wiegert et al. 2013). Only the circularized cpGenomes of both E. gracilis strains contain three copies of the rRNA operon arranged in tandem repeat units. Beside that a further 16S rRNA copy is found in the cpGenome of E. gracilis strain Z.

Concludingly, the three tandemly repeated rRNA copies of the two Euglena species are unique so far and when mapping rRNA operon features onto the phylogenomic tree of the phototrophic euglenoids one can assume an evolutionary pattern (Results, Chapter III, Fig. 3, p. 100). The most basally branching euglenoids of the genus Eutreptiella both contain two rRNA copies, which correspond most closely to the cpGenomes of green algae (Dabbagh et al. 2017, Hrdá et al. 2012). The rRNA operons of the psychrophilic Etl. pomquetensis exhibit the fewest evolutionary changes towards the rRNA operons of Pyramimonadales and consequently to the resulting genome structure. Nevertheless, the two copies are identified in all three Eutreptiales as the closest relatives to green algae chloroplasts. In all other euglenoids chloroplast genomes only one copy was identified, albeit with two exceptions. A probable scenario could be, that during the diversification of the Euglenales the number of the rRNA copies was reduced to one. Only in S. acuminata and both E. gracilis species, a second

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incomplete copy and two further copies of the rRNA operon were acquired independently, because the gene arrangement in these three taxa differs significantly. The Euglena strains show a tandem structure and Strombomonas, which is not yet experimentally circularized, has two operons coded next to each other on different strands. The operons are encoded adjacently tail to tail (Bleidorn 2017).

As to the function of different numbers and organization of the RNA operon one can only speculate. Theoretically, the more copies of genes are encoded in a genome, the faster expression can generally proceed (Elowitz et al. 2002), but it is still highly dependent on regulatory dynamics, transcription rates and many other genetic factors controlling expression as Eberhard et al. (2002) ascertained for the chlorophyte Chlamydomonas reinhardtii. For euglenoids cpGenomes the function is hard to investigate, because euglenoids do not reproduce by sex, only by cell division (Gillott & Triemer 1978, Leedale 1967), thus mutation and altered expression of genes cannot be monitored easily. One possible explanation to understand the heterogeneous organization of euglenoids cpGenomes could be the low control of mutation in these early eukaryotes that can also be found in ribosomal genes of the nucleus, especially in osmotrophic, but also in all other euglenoids (Busse & Preisfeld 2002b, Busse et al. 2003). Yet, the advantages of varying operon structures and copy numbers in euglenoids are unknown.

A second reason for the size variance between the euglenoids cpGenomes is the heterogeneous intergenic space (IGS). For instance, the IGS between the two closely related

Eutreptiella species is strikingly different. The IGS of Etl. pomquetensis occupied more than

23 kb, which was more than twice that of Etl. gymnastica, showing only an intergenic space of approximately 11 kb (Dabbagh et al. 2017, Hrdá et al. 2012). It is conspicuous, that the largest cpGenomes comprised the highest intergenic space, with S. acuminata encompassing an intergenic space of more than 25 kb, with E. gracilis strain Z for more than 24 kb and with

Etl. pomquetensis 23 kb (Dabbagh et al. 2017, Hallick et al. 1993, Wiegert et al. 2013). By

contrast, the two smallest cpGenomes Et. viridis (cpGenomes of 65,523 bp) and P. orbicularis (cpGenomes of 65,992 bp) also have the smallest intergenic space with approximately 7,9 kb (Kasiborski et al. 2016, Wiegert et al. 2012). In total, the average intergenic space of euglenoid cpGenomes amounts to 13,9 kb. But still the last two reasons are not alone and not the only once and not foremost accountable for the size differences among the cpGenomes.

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The third and main reason for the size differences is a very unequal number of introns. Especially these introns were of remarkable interest in this study. Correspondingly questions regarding intron evolution occupy large parts of the analysis of changes on the intrageneric level as well as concerning all investigated phototrophic euglenoids. It was a matter of concern to investigate, if trends were detectable within single chloroplast genomes, between different species or even within the whole phototrophic lineage. The highest number of introns was located in the two sequenced E. gracilis strains. Euglena gracilis var. bacillaris contains 134 introns, while E. gracilis strain Z encompasses a total of over 150 introns (Bennett & Triemer 2015, Hrdá et al. 2012). These numbers result in over 60 % of protein- coding genes of the cpGenomes, which contain at least 1 intron. In contrast E. mutabilis contains ‘only’ 76 introns within protein-coding genes (Dabbagh & Preisfeld 2017). Here again, it can only be surmises that a low genetic control allows so many introns to invade the genome. It is also known that introns are of utmost importance in stimulating gene expression or regulation, called intron-mediated enhancement (IME) (Mascarenhas et al. 1990). Some of the possible functions of introns may be related to facilitating protein evolution for example by exon-shuffling (Rose et al. 2008). Additionally, it was shown that the signal for splicing can enhance the transcription by triggering the RNA polymerase (Le Hir et al. 2003). Though the advantage of a facilitated gene expression is a high price to be paid for inserting and removing the introns and to suppress mistakes. But anyway, in case of the euglenoid chloroplast, that was taken over from a green alga, it might have been necessary to support the regular genetic apparatus, for which communication with a chloroplast was a new development, by regulatory help from introns. Afterwards these introns stayed in the cpGenome, although gene transfer between the adopted plastid and the new nuclear genome had already been accomplished. The question as to whether such regulatory functions also apply to the euglenoids as a basal eukaryotic group remains to be answered.

Summarizing the reasons for noticeable size differences in euglenoid cpGenomes, it becomes clear, that the introns are the main factor, followed by organization of RNA operons and intergenic spaces.

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