Tetmemena sp. and E. woodruffi were both propagated in laboratory culture from single cells. The E. woodruffi MAC genome was sequenced and assembled from paired-end Illumina reads from whole cell DNA, which is mostly MAC-derived. For comparative analysis, the MAC genome of E. woodruffi was assembled using the same pipeline previously used for Tetmemena sp. (Chen et al., 2015). Because MIC DNA is significantly more sparse than MAC DNA in individual cells (Prescott, 1994), MIC DNA was enriched before sequencing (see Methods); however, this leads to much lower sequence coverage of the MIC than the MAC. Third-generation long reads (Pacific Biosciences and Oxford Nanopore Technologies) were combined with Illumina paired-end reads (Methods, see genome coverage in Supplementary file 1) to construct hybrid genome assemblies for Tetmemena sp. and E. woodruffi. Though the final genome assemblies are still fragmented, often due to transposon or other repetitive insertions at boundaries (Figure 2—figure supplement 1), the current draft assemblies cover most (>90%) MDSs for 89.1% of MAC nanochromosomes in Tetmemena, and for 90.0% of MAC nanochromosomes in E. woodruffi. This allowed us to establish near-complete rearrangement maps for the newly assembled genomes of Tetmemena and E. woodruffi, at a level comparable to the published reference for O. trifallax (Chen et al., 2014), which is appropriate for comparative analysis.

Table 1 shows a comparison of genome features for the three species. The three MAC genomes are similar in size, with most nanochromosomes bearing only one gene. The size distributions of MAC chromosomes are similar for the three species, though slightly shorter for E. woodruffi, consistent with prior observation via gel electrophoresis (Prescott, 1994, Figure 2—figure supplement 2). Like O. trifallax (Swart et al., 2013), the maximum number of genes encoded on one chromosome is 7–8 (Table 1). Surprisingly, the MIC genome sizes differ substantially: the Tetmemena MIC genome assembly is 237 Mbp, nearly half that of Oxytricha. The E. woodruffi MIC genome assembly is even smaller, approximately 172 Mbp (Table 1).

The expansion of repetitive elements in the Oxytricha lineage may contribute to the difference in MIC genome sizes (Figure 2A–C). Oxytricha has a variety of tranposable elements (TEs) in the MIC, with telomere-bearing elements (TBEs) of the Tc1/mariner family the most abundant (Chen et al., 2014; Chen and Landweber, 2016, Supplementary file 2). A complete TBE transposon contains three open reading frames (ORFs). ORF1 encodes a 42kD transposase with a DDE-catalytic motif. Though present only in the germline, TBEs are so abundant in hypotrichs that some were partially recovered and assembled from whole cell DNA (Chen and Landweber, 2016). The Oxytricha MIC genome contains ~10,000 complete TBEs and ~24,000 partial TBEs, which occupy approximately 15.20% (75 Mbp) of the genome (Figure 2A, Supplementary file 3; Chen et al., 2014; Chen and Landweber, 2016). Tetmemena, on the other hand, has many fewer TBE ORFs and only 48 complete TBEs (Supplementary file 3), comprising 1.83% (4.3 Mbp) of its MIC genome (Figure 2B). E. crassus has also been reported to have an abundant transposon family called Tec elements (Transposon of Euplotes crassus). Like TBEs, each Tec consists of three ORFs, and ORF1 also encodes a transposase from the Tc1/mariner family (Baird et al., 1989; Krikau and Jahn, 1991; Jahn et al., 1993; Jahn et al., 1989; Klobutcher and Herrick, 1997). The ~57 kD ORF2 encodes a tyrosine-type recombinase (Doak et al., 2003), and the 20kD ORF3 has unknown function (Jahn et al., 1993). Using the three ORFs of Tec1 and Tec2 as queries for search, we identified 74 complete Tec elements in E. woodruffi. Collectively, Tec ORFs occupy 3.6 Mbp, corresponding to only 2.1% of the MIC genome (Figure 2C). Notably, the transposase-encoding ORF1 is more abundant than the other two TBE/Tec ORFs in all three ciliates (Supplementary file 3), consistent with its proposed role in DNA cleavage during genome rearrangement in Oxytricha (Nowacki et al., 2009).

Oxytricha contains three families of TBEs. TBE3 appears to be the most ancient among hypotrichs, based on previous analysis of limited MIC genome data (Chen and Landweber, 2016). We constructed phylogenetic trees using randomly subsampled TBE sequences for all three ORFs from Oxytricha and Tetmemena (Figure 2D–F). This confirmed that only TBE3 is present in the Tetmemena MIC genome, as proposed in Chen and Landweber, 2016. This also suggests that TBE1 and TBE2 expanded in Oxytricha after its divergence from other hypotrichous ciliates. As illustrated in Figure 2—figure supplement 1, the MIC genome contexts of TBEs in Oxytricha and Tetmemena are similar, with many TE insertions within IESs, consistent with either IESs as hotspots for TE insertion or with the model (Klobutcher and Herrick, 1997) that some TE insertions may have generated IESs, as demonstrated in Paramecium (Sellis et al., 2021; Feng and Landweber, 2021). Subsequent sequence evolution at the edges of IES/MDS pointers (DuBois and Prescott, 1995) can give rise to boundaries that no longer correspond precisely to TBE ends. For further discussion of the conservation of TBE locations, see the section, ‘Oxytricha and Tetmemena share conserved rearrangement junctions’ below.

Additionally, Repeatmodeler/Repeatmasker identified that Oxytricha has more MIC repeats in the ‘Other’ category than Tetmemena or E. woodruffi (Figure 2, subcategories of repeat content in Supplementary file 2). 214 Mbp of the Oxytricha MIC genome (43%, which is greater than 35.9% reported in Chen et al., 2014 that used earlier versions of the software) is considered repetitive (including TBEs, tandem repeats, and other repeats in Figure 2), versus 31.7 Mbp for Tetmemena (13.4%) and 28.5 Mbp (16.8%) for E. woodruffi. Oxytricha’s additional ~180 Mbp in repeat content partially explains the significantly larger MIC genome size of Oxytricha versus the other spirotrich ciliates.

We used the genome rearrangement annotation tool, Scrambled DNA Rearrangement Annotation Protocol (SDRAP, Braun et al., 2022) to annotate the MIC genomes of Oxytricha, Tetmemena, and E. woodruffi (Methods). Consistent with their close genetic distance, the genomes of O. trifallax and Tetmemena have similarly high levels of discontinuity (Figure 3A). We annotated over 215,299 MDSs in Oxytricha and over 215,624 in Tetmemena with similar MDS length distributions (Figure 3A). By contrast, E. woodruffi MDSs are typically longer, which indicates a less interrupted genome (Figure 3A). We compared the number of MDSs between single-copy orthologs for single-gene MAC chromosomes across the three species and found that the orthologs have similar coding sequence (CDS) lengths (Figure 3—figure supplement 1A–B). There is a strong positive correlation between number of MDSs for orthologous genes in Oxytricha and Tetmemena (R²=0.75, Figure 3B). There is no correlation among number of MDSs between orthologs of E. woodruffi and Oxytricha (R²=0.003, Figure 3C), since E. woodruffi orthologs typically contain fewer MDSs.

The E. woodruffi genome is generally much less interrupted than that of Oxytricha or Tetmemena. 39.9% of MAC nanochromosomes in E. woodruffi lack IESs (IES-less nanochromosomes) compared to only 4.1 and 4.4% in Oxytricha and Tetmemena, respectively. The sparse IES distribution (as measured by plotting pointer distributions) in E. woodruffi displays a curious 5’ end bias on single-gene MAC chromosomes, oriented in gene direction (Figure 3E). A weak 5’ bias is also present in Oxytricha (Figure 3D) and Tetmemena (Figure 3—figure supplement 1C). In addition, E. woodruffi IESs preferentially accumulate in the 5’ UTR, a short distance upstream of start codons (Figure 3F). Notably, the median distance between the 5’ telomere addition site and the start codon in E. woodruffi is just 54 bp for single-gene chromosomes, approximately half that of Oxytricha (Swart et al., 2013).

Scrambled genome rearrangements exist in all three species, which we report here for the first time in Tetmemena and the early diverged E. woodruffi. Previous studies have described scrambled genes with confirmed MIC-MAC rearrangement maps for a limited species of hypotrichs (Chen et al., 2014; Chen et al., 2015; Hogan et al., 2001; Chang et al., 2005; Wong and Landweber, 2006; DuBois and Prescott, 1995) and Chilodonella (Katz and Kovner, 2010; Gao et al., 2014) but not in Euplotes. Consistent with the phylogenetic placement of Euplotes as an earlier diverged outgroup to hypotrichs (Lynn, 2008; Gao et al., 2016), the E. woodruffi genome is scrambled, but it contains approximately half as many scrambled genes (2429 genes encoded on 1913 chromosomes, or 7.3% of genes), versus 15.6% scrambled in O. trifallax (3613 genes encoded on 2852 chromosomes) and 13.6% in Tetmemena (3371 genes encoded on 2556 chromosomes). The E. woodruffi lineage may therefore reflect an evolutionary intermediate stage between ancestral genomes with only modest levels of genome scrambling and the more massively scrambled genomes of hypotrichs.

We infer that many genes were likely scrambled in the last common ancestor of Oxytricha and Tetmemena, because these two species share approximately half of their scrambled genes (Supplementary file 4). Furthermore, most scrambled genes are not new genes, since they possess at least one ortholog in other ciliate species (Supplementary file 4, Supplementary file 5).

Notably, scrambled genes in all three species generally have more paralogs (Figure 4). We identified orthogroups containing genes derived from the same gene in the last common ancestor of the three species (Methods). For each species, orthogroups with at least one scrambled gene are significantly larger than those containing no scrambled genes (p-value <1e−5, Mann-Whitney U test, Figure 4A–C). This association suggests a possible role of gene duplication in the origin of scrambled genes.

Scrambled pointers are generally longer than nonscrambled ones in all three species (Figure 3—figure supplement 2), consistent with prior observations (Chen et al., 2014) and the possibility that longer pointers participate in more complex rearrangements, including recombination between MDSs separated by greater distances (Landweber et al., 2000). Scrambled and nonscrambled IESs also differ in their length distribution (Figure 3—figure supplement 2). Curiously, scrambled ‘pointers’ in E. woodruffi can be as long as several hundred base pairs (median 48 bp, average 212 bp) unlike the more typical 2–20 bp canonical pointers. These long ‘pointers’ in E. woodruffi are more likely partial MDS duplications (Figure 4—figure supplement 1A). We also identified MDSs that map to two or more paralogous regions within the same MIC contig (Supplementary file 6), therefore representing MDS duplications and not alleles. Such paralogous regions could be alternatively incorporated into the rearranged MAC product. Moreover, we find that, for all three species, there are significantly more scrambled chromosomes than nonscrambled MAC chromosomes that contain at least one paralogous MDS (chi-square test, p-value <1e−10; Supplementary file 6). An example is shown in Figure 4—figure supplement 1A (MDS 7 and 7').

The presence of paralogous MDSs can contribute to the origin of scrambled rearrangements, as proposed in an elegant model by Gao et al., 2015; illustrated in Figure 4—figure supplement 1B. The model proposes that initial MDS duplications permit alternative use of either MDS copy into the mature MAC chromosome. As mutations accumulate in redundant paralogs, cells that incorporate the least decayed MDS regions into the MAC gene would have both a fitness advantage and a better match to the template RNA (Nowacki et al., 2008) that guides rearrangement, thus increasing the likelihood of incorporation into the MAC chromosome. The paralogous regions containing more mutations would gradually decay into IESs, and scrambled pointers eventually be reduced to a shorter length. The extended length ‘pointers’ that we identified in E. woodruffi may reflect an intermediate stage in the origin of scrambled genes (Figure 4—figure supplement 1B).

This model may generally explain the abundance and expansion of ‘odd-even’ patterns in ciliate scrambled genes (Landweber et al., 2000; Burns et al., 2016). As illustrated in Figure 4—figure supplement 1A, the even- and odd-numbered MDSs for many scrambled genes derive from different MIC genome clusters. The model predicts that the IES between MDS n−1 and n+1 often derives from ancestral duplication of a region containing MDS n (Figure 4—figure supplement 2A). To test this hypothesis explicitly, we extracted from all odd-even scrambled loci in the three species all sets of corresponding MDS/IES pairs that are flanked by identical pointers on both sides, i.e., all pairs of scrambled MDSs and IESs, where the IES between MDS n−1 and n+1 is directly exchanged for MDS n during DNA rearrangement (S1 and S2 in Figure 4—figure supplement 2A). To exclude the possibility of alleles confounding this analysis, MDS and IES pairs were only considered if they map to the same MIC contig. In E. woodruffi, the lengths of these MDS/IES pairs strongly correlate (Spearman correlation ρ=0.755, p<1e−5, Figure 4—figure supplement 2B). Moreover, many MDS and IES sequence pairs also share sequence similarity, consistent with paralogy: for 248 MDS-IES pairs of similar length, 90.3% share a core sequence with ~97.5% identity across 8–100% of both the IES and MDS length. The lowest end of these observations is also compatible with an alternative model (Chang et al., 2005) in which direct recombination between IESs and MDSs at short repeats can lead to expansion of odd-even patterns. For Oxytricha and Tetmemena, the MDS and IES lengths for such MDS/IES pairs also display a weakly-positive correlation (p-values and Spearman correlation ρ shown in Figure 4—figure supplement 2D–E). Remarkably, the odd-even-containing loci that are species-specific, and therefore became scrambled more recently, have the strongest length correlation (Figure 4—figure supplement 2C–E) and more pairs that display sequence similarity (Supplementary file 7) relative to older loci (scrambled in two or more species). This result is consistent with an evolutionary process in which mutations accumulate in one copy of the MDS, gradually obscuring its sequence homology and ability to be incorporated as a functional MDS, and eventually its ability to be recognized by the template RNAs that guide DNA rearrangement. This analysis also suggests that most of the odd-even scrambled loci in E. woodruffi arose recently, because there is greater sequence similarity between MDSs and the corresponding IESs that they replace. Conversely, we infer that most loci that are scrambled in both Oxytricha and Tetmemena became scrambled earlier in evolution, since they display weaker sequence similarity between exchanged MDS and IES regions.

Scrambled and nonscrambled genes display nearly identical expression support (the presence of at least one read in all three replicates) in both Oxytricha (Supplementary file 8) and Tetmemena. E. woodruffi has slightly more expression support for nonscrambled vs. scrambled genes (Figure 4—figure supplement 3), which could be explained by more recent acquisition of thousands of scrambled loci in E. woodruffi. In some of those cases the nonscrambled paralogs may still contribute the major function. The distribution of expression levels is similar for scrambled vs. nonscrambled genes in all three species, supporting their authenticity (Figure 4—figure supplement 3), although in a Mann-Whitney U test, the average expression level of three replicates is significantly higher in nonscrambled genes for Oxytricha and E. woodruffi, but not significant for Tetmemena.

To understand the conservation of genome rearrangement patterns, we developed a pipeline guided by protein sequence alignment to compare pointer positions for orthologous genes between any two species (Methods, Figure 5A). We compared pointers for 2503 three-species single-copy orthologs. 4448 pointer locations are conserved between Oxytricha and Tetmemena on 1345 ortholog pairs (Supplementary file 9), representing 38.3% of pointers in these orthologs in Oxytricha and 30.9% in Tetmemena. For Oxytricha/E. woodruffi and Tetmemena/E. woodruffi comparisons, 56 and 58 pointer pairs are conserved, respectively. We also identified 23 pointer locations shared among all three species (Supplementary file 9, Figure 5B, Figure 5—source data 1).

To test if these pointer locations are genuinely conserved versus coincidental matching by chance, we performed a Monte Carlo simulation, as also used to study intron conservation (Rogozin et al., 2003). We randomly shuffled pointer positions on CDS regions 1000 times and counted the number of conserved pointer pairs expected for each simulation (Methods). Of the 1000 simulations, none exceeded the observed number of conserved pointer pairs between Oxytricha and Tetmemena (p-value <0.001), suggesting evolutionary conservation of pointer positions (Supplementary file 9). A similar result was obtained for pointers conserved in all three species (Supplementary file 9). However, the numbers of pointer pairs conserved between Oxytricha/E. woodruffi and Tetmemena/E. woodruffi is similar to the expectations by chance (Supplementary file 9). The low level of pointer conservation of either hypotrichs with E. woodruffi may reflect the smaller number of IESs in E. woodruffi; hence, most pointers would have arisen in the hypotrich lineage. Furthermore, E. woodruffi is genetically more distant from the two hypotrichs; hence, the accumulation of substitutions would obscure protein sequence homology, which we used to compare pointer locations. For ortholog pairs between Oxytricha and Tetmemena, scrambled pointers are significantly more conserved than nonscrambled ones (chi-square test, p-value <1e−10, Supplementary file 10). We also find that most pointer sequences differ even if the positions are conserved (Figure 5B, Figure 5—source data 1, Supplementary file 11), suggesting that substitutions may accumulate in pointers without substantially altering rearrangement boundaries.

Oxytricha and Tetmemena both contain a high copy number of TBE transposons (Chen et al., 2014; Chen and Landweber, 2016; Supplementary file 3). We investigated the level of TBE conservation between these two species. To identify orthologous insertions, we focus on TBE insertions in nonscrambled IESs on single-copy orthologs, which include 1706 Oxytricha TBEs inserted in 1296 nonscrambled IESs (multiple TBEs can be inserted into an IES) and 180 Tetmemena TBEs inserted into 170 nonscrambled IESs. We refer to the pointer flanking a TBE-containing IES as a TBE pointer. No TBE pointer locations are conserved between two species. This suggests that TBEs might invade the genomes of Oxytricha and Tetmemena independently, or still be actively mobile in the genome. Only 27 Oxytricha TBE pointers (containing 36 TBEs) are conserved with non-TBE pointers in Tetmemena (Figure 5—source data 2, Figure 5C). No Tetmemena TBE pointer is conserved with an Oxytricha non-TBE pointer. This suggests that TBE insertions may preferentially produce new rearrangement junctions instead of inserting into an existing IES.

Ciliate genomes are generally intron-poor. Oxytricha averages 1.7 introns/gene, Tetmemena has 1.1, and E. woodruffi has 2.2. Among three-species orthologs, intron locations sometimes map near pointer positions (within a 20-bp window, Figure 5B, Figure 5—figure supplement 1). IESs and introns are both noncoding regions that are removed from mature transcripts, though at different stages. A previous single-gene study observed that an IES in Paraurostyla overlaps the position of an intron in Uroleptus, Urostyla, and also the human homolog (Chang et al., 2005). This observation suggested an intron-IES conversion model in which the ability to eliminate non-CDS regions as either DNA or RNA provides a potential backup mechanism. Such interconversion has also been observed between two strains of Stylonychia (Möllenbeck et al., 2006). In the present study, we identified 174 potential cases of intron-IES conversion in the three species (Figure 5—figure supplement 1, Supplementary file 12): 103 (59.2%) E. woodruffi introns map near Oxytricha/Tetmemena pointers. We used a 20-bp window for this analysis, since one would only expect the boundaries of introns and IESs to coincide precisely if they were recent evolutionary conversions. A Monte Carlo simulation for these intron-IES comparisons (Supplementary file 12) revealed that p<0.001 for most three-species comparisons. For two-species comparisons, we identify 306 cases where an intron boundary in one species precisely coincides with a pointer sequence in another species, with strongest statistical support for the comparison between Oxytricha intron positions and Tetmemena IES junctions (p=0.008) (Supplementary file 13). Notably, Tetmemena intron locations rarely coincide with Oxytricha IESs (Supplementary file 13), suggesting a possible bias in the direction of intron-IES conversion during evolution.

The observation that E. woodruffi has the most introns but the smallest number of IESs per gene (Figure 3) is consistent with removal of intragenic non-CDS regions as either DNA or RNA. The intron-sparseness of ciliates is compatible with a hypothesis that it is advantageous to eliminate noncoding regions earlier at the DNA level, with intron deletion sometimes providing an opportunity for repair if they fail to be excised as IESs (Chang et al., 2005).

Genome rearrangements in the Oxytricha lineage can include overlapping and nested loci, with MDSs for different MAC loci embedded in each other (Chen et al., 2014; Braun et al., 2018). When multiple gene loci are nested in each other, these have been called Russian doll loci (Braun et al., 2018). Oxytricha contains two loci with five or more layers of nested genes (Braun et al., 2018). Oxytricha and Tetmemena display a high degree of synteny and conservation in both Russian doll loci. In the first Russian doll gene cluster, one nested gene (green) is present in Oxytricha but absent in Tetmemena (Figure 6A, Figure 6—figure supplement 1, Figure 6—figure supplement 2), confirmed by PCR (Methods). Oxytricha also has a complete TBE3 insertion in the green gene (Figure 6A, Figure 6—figure supplement 1A), hinting at a possible link between transposition and new gene insertion. In addition, a two-gene chromosome in Oxytricha (orange) is present as two single-gene chromosomes in Tetmemena (Figure 6A, Figure 6—figure supplement 1). In Oxytricha, seven orange MDSs ligate across two other loci via an 18-bp pointer (TATATCTATACTAAACTT) to form a two-gene nanochromosome. However, in Tetmemena, telomeres are added to the ends of both gene loci instead, forming two independent MAC chromosomes (Figure 6A, Figure 6—figure supplement 1). The second Russian doll locus has an example of a long, conserved pointer (orange dotted line) that bridges three other loci (the green and blue scrambled loci and one nonscrambled locus, Figure 6B). Close to this region is a decayed TBE insertion (769 bp) in Oxytricha. None of the E. woodruffi orthologs of both Russian doll loci maps to the same MIC contig, which suggests that the Russian doll clusters arose after the divergence of Euplotes from the common ancestor of Oxytricha and Tetmemena.

Tetmemena sp. (strain SeJ-2015; Chen et al., 2015) was isolated as a single cell from a stock culture and propagated as a clonal strain via vegetative (asexual) cell culture. Cells were cultured in Pringsheim media (0.11 mM Na₂HPO₄, 0.08 mM MgSO₄, 0.85 mM Ca(NO₃)₂, 0.35 mM KCl, pH 7.0) and fed with Chlamydomonas reinhardtii, together with 0.1%(v/v) of an overnight culture of non-virulent Klebsiella pneumoniae. Macronuclei and micronuclei were isolated using sucrose gradient centrifugation (Lauth et al., 1976). Genomic DNA was subsequently purified using the Nucleospin Tissue Kit (Takara Bio USA, Inc). Macronuclear DNA was sequenced and assembled in Chen et al., 2015. Micronuclear DNA was further size-selected via BluePippin (Sage Science) for PacBio sequencing, or via 0.6% (w/v) SeaKem Gold agarose electrophoresis (Lonza) for Illumina sequencing. Micronuclear DNA purification and sequencing protocols are described in Chen et al., 2014.

E. woodruffi (strain Iz01) was cultured in Volvic water at room temperature and fed with green algae every 2–3 days. We fed cells with C. reinhardtii for MAC DNA collection, and switched to Chlorogonium capillatum for MIC DNA collection. In order to remove algal contamination, cells were starved for at least 2–3 days before collection. Cells were washed and concentrated as in Chen et al., 2014. Because MAC DNA is predominant in whole cell DNA, we used whole cell DNA (purified via NucleoSpin Tissue kit, Takara Bio USA, Inc) for MAC genome sequencing. Paired-end sequencing was performed on an Illumina Hiseq2000 at the Princeton University Genomics Core Facility.

MIC DNA was enriched from whole cell DNA and sequenced via three sequencing platforms (Illumina, Pacific Biosciences, and Oxford Nanopore Technologies). We used conventional and pulse-field gel electrophoresis (PFGE) to enrich MIC DNA:

We assembled the MAC genome of E. woodruffi using the same pipeline for Tetmemena sp. (Chen et al., 2015) for comparative analysis: two draft genomes were assembled by SPAdes (Bankevich et al., 2012) and Trinity (Grabherr et al., 2011), and were then merged by CAP3 (Huang and Madan, 1999). Trinity, which is a software developed for de novo transcriptome assembly (Grabherr et al., 2011), has been used to assemble hypotrich MAC genomes (Chen et al., 2015) because their nanochromosome genome structure is similar to transcriptomes, including properties such as variable copy number and alternative isoforms (Lindblad et al., 2019). Telomeric reads were mapped to contigs by BLAT (Kent, 2002), and contigs were further extended and capped by telomeres when at least five reads pile up at a position near ends by custom python scripts (https://github.com/yifeng-evo/Oxytricha_Tetmemena_Euplotes/tree/main/MAC_genome_telomere_capping) (Feng, 2022a). The mitochondrial DNA was removed if the contig has a TBLASTX (Camacho et al., 2009) hit on the Oxytricha mitochondrial genome (Genbank accession JN383842.1 and JN383843.1) or two Euplotes mitochondrial genomes (Euplotes minuta GQ903130.1, E. crassus GQ903131.1). Algal contigs were removed by BLASTN to all C. reinhardtii nucleotide sequences downloaded from Genbank. Non-telomeric contigs were mapped to bacterial NR by BLASTX to remove bacterial contaminations. The genome was further compressed by CD-HIT (Fu et al., 2012) in two steps: (1) contigs <500 bp were removed if 90% of the short contig can be aligned to a contig ≥ 500 bp with 90% similarity (-c 0.9 -aS 0.9 -uS 0.1); (2) then the genome was compressed by 95% similarity (-c 0.95 -aS 0.9 -uS 0.1). Contigs shorter than 500 bp without telomeres were removed. Nine contigs, likely Tec contaminants from the MIC genome, were also excluded (Tblastn, ‘-db_gencode 10 -evalue 1e-5’), and they could be assembled due to the high copy number in the MIC genome (47, 48, Genbank accessions of Tec ORFs are AAA62601.1, AAA62602.1, AAA62603.1, AAA91339.1, AAA91340.1, AAA91341.1, AAA91342.1).

Three biological replicates of total RNA was isolated from asexually growing E. woodruffi and Tetmemena sp. cells using TRIzol reagent (Thermo Fisher Scientific) and enriched for the poly(A)+fraction using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). Stranded RNA-seq libraries were constructed using the ScriptSeq v2 RNA-seq library preparation kit (Epicentre) and sequenced on an Illumina Nextseq500 at the Columbia Genome Center. For E. woodruffi, the transcriptome was assembled by Trinity (Grabherr et al., 2011), and transcript alignments to the MAC genome were generated by PASA (Haas et al., 2003).

We followed the gene prediction pipeline developed by the Broad institute (https://github.com/PASApipeline/PASApipeline/wiki); using EVidenceModeler (EVM, Haas et al., 2008) to generate the final gene predictions. EVM produced gene structures by weighted combination of evidence from three resources: ab initio prediction, protein alignments, and transcript alignments (the weight was 3, 3, and 10 respectively). Ab initio prediction was generated by BRAKER2 pipeline (Brůna et al., 2021). Protein alignments for EVM were generated by mapping Oxytricha proteins to the E. woodruffi MAC genome by Exonerate (Slater and Birney, 2005). EVM predicted 33,379 genes on MAC chromosomes with at least one telomere.

We assessed MAC genome completeness using three methods: (1) 28,294 (80.6%) of the 35,099 E. woodruffi MAC contigs have at least one telomere. (2) In the E. woodruffi genes predicted on telomeric contigs, 88.8% of BUSCO (Simão et al., 2015; Manni et al., 2021) genes in the lineage database alveolata_odb10 were identified as complete. Within the 171 BUSCO genes, 135 are complete and single-copy, 17 are complete and duplicated, 7 are fragmented, and 12 are missing. This represents the best Euplotes MAC genome assembly available. (3) We identified 51 tRNA genes encoding all 20 amino acids by tRNAscan-SE (Lowe and Eddy, 1997) in the MAC genome, including two suppressor tRNAs of UAA and UAG.

The MIC genome of Tetmemena was assembled with a hybrid approach to combine reads from different sequencing platforms. Tetmemena Illumina reads were first assembled by SPAdes (77, parameters ‘-k 21,33,55,77,99,127 –careful’). PacBio reads were error corrected by FMLRC (Wang et al., 2018) using Illumina reads with default parameters. Corrected PacBio reads were aligned to both the MAC genome and the Illumina MIC assembly with BLASTN. Reads were removed if they start or end with telomeres or are aligned better to the MAC. The remaining reads were assembled with wtdbg2 (Ruan and Li, 2020, parameters ‘-x rs’). The PacBio assembly was polished by Pilon (Walker et al., 2014) with the ‘--diploid’ option. The Illumina and PacBio assemblies were merged by quickmerge (Chakraborty et al., 2016) with the ‘-l 5000’ option.

The MIC genome of E. woodruffi was assembled using a similar procedure as described above for Tetmemena. E. woodruffi reads were filtered to remove bacterial contamination, including abundant high-GC-content contaminants, possibly endosymbionts (Boscaro et al., 2019). Nanopore reads with GC content ≥55% were assembled by Flye (Kolmogorov et al., 2019) with the parameter ‘--meta’ for metagenomic assembly of bacterial contigs. We used kaiju (Menzel et al., 2016) to identify bacteria taxa for these contigs. 9 of 10 top-covered contigs derive from Proteobacteria, from which many Euplotes symbionts derive (Boscaro et al., 2019). Bacterial contamination was removed from Illumina reads if perfectly mapping to these metagenomic contigs by Bowtie2 (Langmead and Salzberg, 2012). The cleaned Illumina reads were then assembled by SPAdes with ‘-k 21,33,55,77,99,127’ (Bankevich et al., 2012). Pacbio raw reads and Nanopore raw reads with GC content <55% were aligned to a concatenated database containing both the MAC genome and the Illumina MIC assembly with BLASTN. Reads were removed if they start or end with telomeres or align better to the MAC. Remaining PacBio/Nanopore reads were assembled by Flye with ‘--meta’ mode. The PacBio-Nanopore assembly was polished by Pilon with the ‘--diploid’ option. Illumina and PacBio-Nanopore assemblies were merged by quickmerge with the ‘-l 10000’ option. Contigs shorter than 1 kb were removed.

The draft MIC genome of Tetmemena was first mapped to telomeric MAC contigs by BLASTN. MIC contigs containing MDSs were included in the final assembly. The rest of the MIC contigs were filtered by a decontamination pipeline: (1) contigs were aligned to the K. pneumoniae genome, C. reinhardtii genome, and the Oxytricha mitochondrial genome by BLASTN to remove contaminants; (2) the remaining contigs were then searched against the bacteria NR database and a ciliate protein database (including protein sequences annotated in Tetrahymena thermophila: http://www.ciliate.org/system/downloads/tet-latest/4-Protein%20fasta.fasta; Paramecium tetraurelia: http://paramecium.cgm.cnrs-gif.fr; and O. trifallax: https://oxy.ciliate.org) by BLASTX. Contigs with higher bit score to bacteria NR or G+C >45% were removed. The E. woodruffi MIC genome was decontaminated, similarly, with addition of all Chlorogonium sequences (the algal food source) on NCBI and the two Euplotes mitochondrial genomes (E. minuta GQ903130.1, E. crassus GQ903131.1) to filter contaminants.

The repeat content in the MIC genomes was identified by RepeatModeler 1.0.10 (Smit and Hubley, 2008) and RepeatMasker 4.0.7 (Smit et al., 2013) with default parameters.

Representative Oxytricha TBE ORFs (Genbank accession AAB42034.1, AAB42016.1, and AAB42018.1) were used as queries to search TBEs in the Oxytricha and Tetmemena MIC genomes by TBLASTN (-db_gencode 6 -evalue 1e-7 -max_target_seqs 30000). Tec ORFs were similarly detected by using E. crassus Tec1 and Tec2 ORFs as queries (-db_gencode 10 -evalue 1e-5 -max_target_seqs 30000, Genbank accessions of Tec ORFs are AAA62601.1, AAA62602.1, AAA62603.1, AAA91339.1, AAA91340.1, AAA91341.1, AAA91342.1). Complete TBEs/Tecs were determined by custom python scripts when three ORFs are within 2000 bp from each other and in correct orientation (https://github.com/yifeng-evo/Oxytricha_Tetmemena_Euplotes/tree/main/TBE_ORFs/TBE_to_oxy_genome_tblastn_parse.py, Chen and Landweber, 2016). 30 TBE ORFs with >70% completeness were subsampled from each species for phylogenetic analysis (except for the 57 kD ORF in Tetmemena, for which 21 were subsampled). The subsampled TBE ORFs were aligned using MUSCLE (Edgar, 2004), and the alignments were trimmed by trimAl ‘-automated1’ (Capella-Gutiérrez et al., 2009). Phylogenetic trees were constructed using PhyML 3.3 (Guindon et al., 2010).

SDRAP (Braun et al., 2022) was used to annotate MDSs, pointers, and MIC-specific regions (minimum percent identity for preliminary match annotation = 95, minimum percent identity for additional match annotation = 90, minimum length of pointer annotation = 2). SDRAP requires MAC and MIC genomes as input. For the SDRAP annotation of Oxytricha, we used the MAC genome from Swart et al., 2013 instead of the latest hybrid assembly that incorporated PacBio reads (Lindblad et al., 2019), because the former version was primarily based on Illumina reads, similar to the MAC genomes of Tetmemena (7, Genbank GCA_001273295.2) and E. woodruffi which are also Illumina assemblies. Oxytricha and Tetmemena MAC genomes were preprocessed by removing MAC contigs with TBE ORFs, considered MIC contaminants (Chen and Landweber, 2016). SDRAP is a new program that can output the rearrangement annotations with minor differences from Chen et al., 2014, but most annotations are robust (Figure 3—figure supplement 2). Scrambled and nonscrambled junctions/IESs were annotated by custom python scripts (https://github.com/yifeng-evo/Oxytricha_Tetmemena_Euplotes/tree/main/scrambled_nonscrambled_IES_pointer).

Each MIC genome region is assigned to only one category in Figure 2A–C, even if it belongs to more than one category. The assignment is based on the following priority: MDS, TBE/Tec, MIC genes (only available for Oxytricha, which has developmental RNA-seq data), IES, tandem repeats, other repeats, and non-coding non-repetitive regions. For example, an MIC region can be a TBE in an IES, and it is only considered as TBE in Figure 2A–C.

Orthogroups of genes on telomeric MAC contigs were detected by OrthoFinder with ‘-S blast’ (Emms and Kelly, 2019). Single-copy orthologs were aligned by Clustal Omega (Sievers et al., 2011). Protein alignments were reversely translated to CDS alignments by a modified script of pal2nal (Suyama et al., 2006, https://github.com/yifeng-evo/Oxytricha_Tetmemena_Euplotes/tree/main/Ortholog_comparison/pal2nal.pl). Two modifications were made in the script: (1) the modified script allows pal2nal to take different genetic codes for three sequences (-codontable 6,6,10); (2) the script also fixed an error in the original pal2nal script in which codontable 10 for the Euplotid nuclear code was the same as the universal code. Visualization of pointer positions and intron locations on orthologs was implemented by a custom python script (https://github.com/yifeng-evo/Oxytricha_Tetmemena_Euplotes/blob/main/Ortholog_comparison/visualization_of_ortholog_comparison.py). Pointer positions or intron locations are considered conserved if they are within a 20-bp alignment window on the CDS alignment. Protein domains were annotated by HMMER (Finn et al., 2011). We performed Monte Carlo simulations by randomly shuffling pointer locations on the CDS but keeping their original position distribution. This was implemented by a custom python script, which transforms the CDS to a circle, rotates pointer positions on the circle, and outputs the shuffled position on the re-linearized CDS (https://github.com/yifeng-evo/Oxytricha_Tetmemena_Euplotes/blob/main/Ortholog_comparison/shuffle_simulation.py). The null hypothesis of the Monte Carlo test is that pointer positions are conserved by chance. p-Value of Monte Carlo test is given by N_{expected>observed}/N_total (N_{expected>observed} is the number of simulations when there are more conserved pointers in the simulation than the observation from real data, N_total = 1000 in this study).

The complex Russian doll locus on MIC contig TMEMEN_MIC_21461 in Tetmemena was validated by PCR to confirm the Tetmemena MIC genome assembly. Tetmemena micronuclear DNA was purified as described previously and used as template for PCR using PrimeSTAR Max DNA polymerase (Takara Bio). 11 primer sets (Supplementary file 14) were designed to amplify products between 3 kb and 6 kb in length, with overlapping regions between consecutive primer pairs. The resulting PCR products were visualized through agarose gel electrophoresis, and bands of the expected size were extracted using a Monarch DNA Gel Extraction Kit (New England Biolabs). The purified gel bands were cloned using a TOPO XL-2 Complete PCR Cloning Kit (Invitrogen), transformed into One Shot OmniMAX 2 T1R E. coli cells (Invitrogen), and individual clones were grown and their plasmids harvested with a QIAprep Spin Miniprep Kit (QIAGEN). The plasmid ends were Sanger sequenced, as well as the region where the Oxytricha MIC assembly contains inserted MDSs (Genewiz). Sanger sequencing reads were mapped to the Tetmemena MIC contig TMEMEN_MIC_21461 and visualized using Geneious Prime 2021.1.1 (https://www.geneious.com).

Custom scripts are public on https://github.com/yifeng-evo/Oxytricha_Tetmemena_Euplotes, (Feng, 2022b copy archived at swh:1:rev:fd66a0efeaf9feb2d79e183313192d641b4e5400). DNA-seq reads and genome assemblies are available at GenBank under Bioprojects PRJNA694964 (Tetmemena sp.) and PRJNA781979 (E. woodruffi). Genbank accession numbers for genomes are JAJKFJ000000000 (Tetmemena sp. Micronucleus genome), JAJLLS000000000 (E. woodruffi Micronucleus genome), and JAJLLT000000000 (E. woodruffi Macronucleus genome).

Three replicates of RNA-seq reads for vegetative cells are available at GenBank under accession numbers of SRR21815378, SRR21815379, and SRR21815380 for E. woodruffi and SRR21817702, SRR21817703, and SRR21817704 for Tetmemena sp.

MDS annotations for three species are available at https://doi.org/10.5061/dryad.5dv41ns96 and https://knot.math.usf.edu/mds_ies_db/2022/downloads.html (please select species from the drop-down menu).