The macronuclear genomic landscape within Tetrahymena thermophila

Abstract

The extent of intraspecific genomic variation is key to understanding species evolutionary history, including recent adaptive shifts. Intraspecific genomic variation remains poorly explored in eukaryotic micro-organisms, especially in the nuclear dimorphic ciliates, despite their fundamental role as laboratory model systems and their ecological importance in many ecosystems. We sequenced the macronuclear genome of 22 laboratory strains of the oligohymenophoran Tetrahymena thermophila, a model species in both cellular biology and evolutionary ecology. We explored polymorphisms at the junctions of programmed eliminated sequences, and reveal their utility to barcode very closely related cells. As for other species of the genus Tetrahymena, we confirm micronuclear centromeres as gene diversification centres in T. thermophila, but also reveal a two-speed evolution in these regions. In the rest of the genome, we highlight recent diversification of genes coding for extracellular proteins and cell adhesion. We discuss all these findings in relation to this ciliate's ecology and cellular characteristics.

Keywords: centromere evolution; ciliate genomics; macronuclear polymorphism; programmed DNA elimination.

Fig. 1.

The MAC polymorphism landscape of T. thermophila. After exchange and fusion of the two parental meiotic products, two mitoses of the newly formed MIC in each conjugant cell (represented on the left) give rise to four nuclei, two remain MICs (in black, one is later eliminated), and the two others are processed into new MACs (in blue), after which parental MACs degenerate (in grey) (see the complete conjugation process in fig. 10 of [75]). MAC development relies on intensive genome remodelling giving rise to a mosaic of SNPs with different origins. For simplicity, we illustrate here a unique MIC locus composed of two MAC Destined Sequences (MDS1 and MDS2, horizontal blue bars) separated by one Internal Eliminated Sequence (IES, horizontal black bar). This locus is composed of two parental alleles characterized by a point mutation (blue and yellow vertical bars=MIC SNPs). A first endoreplication round in young MACs increases the genomic copy number to four, without remodelling. During the second round of endoreplication, IES excision, a highly imprecise process in T. thermophila, can give rise to up to eight variants [76, 77]. From top to bottom for copies with the blue allele (first four copies): complete excision of the IES, small incomplete IES excision, small incomplete IES excision plus excision of a large part of MDS2, and small incomplete IES excision plus excision of a small part of MDS1. From top to bottom for copies with the yellow allele (last four copies): complete excision of the IES, large incomplete IES excision, complete IES excision plus excision of a large part of MDS2, and retention of the full IES. These variants are then propagated throughout MAC development, which continues after conjugant separation, i.e. during the juvenile phase of the vegetative cycle [78]. The whole process of MAC development notably includes breakage of the five MIC chromosomes into smaller chromosomes and stabilization of the MAC content to ~45 copies (except the chromosome encoding 35S rRNA). During the asexual phase, diploid MICs propagate mitotically, while MACs propagate amitotically [5]. MAC genomes are therefore subject to phenotypic assortment, which leads to the random fixation (or loss) of SNPs derived from the MICs (yellow bar in the right ‘MAC’ panel=MIC-derived-SNPs). Phenotypic assortment also retains one variant among those potentially created by imprecise excision of IES. Throughout the asexual phase, de novo MAC mutations (red vertical bars=MAC-de novo-SNPs) will segregate as well according to the phenotypic assortment process. The purple vertical bar represents a de novo MIC-SNP that can only be transmitted to an MAC after another conjugation event.

Fig. 2.

Evolutionary distances. On the left, neighbour-joining tree based on pairwise Jukes–Cantor distances between MAC genome assemblies (scale = percentage of substitutions per site). On the right, heatmap of the number of nucleotide differences with SB210 per 100 kb per MAC chromosome, with strains in rows and MAC chromosomes in columns (chr 1 to chr 181). Red vertical dashes indicate the approximate position of the centromeres of the five MIC chromosomes (named I to V), and black vertical dashes the limit between these five MIC chromosomes.

Fig. 3.

Polymorphism at the MDS junctions. (a) Numbers of SNPs and indels per position with respect to the MDS junction positions across the 22 strains. (b) Heatmap of presence/absence of indels at MDS junctions (±20 bp), insertions and deletions as compared with SB210 being shown in different colours. Each column represents an MDS junction. Note that the heatmap is truncated since all the 9973 MDS cannot be represented in a small figure.

Fig. 4.

Nucleotide diversity. (a) Nucleotide diversity (π) at the 3′ UTRs (last 50 bp of the CDS and first 300 bp of the intergenic region) and 5′ UTR (last 300 bp of the intergenic region and first 50 bp of the CDS). (b) Ratio of π (CDS)/π (non-CDS), ‘non-CDS’ including introns and intergenic regions, for each MAC chromosome (chr 1 to chr 181 on the x-axis). MAC chromosomes having fewer than 15 genes were not included (eight chromosomes). Red vertical dashed lines indicate the approximate position of the centromeres of the MIC chromosome (named I to V). (c) Distribution of pN/pS ratios. (d) Median of gene pN/pS per MAC chromosome.