Genomic insights into the phylogeny and biomass-degrading enzymes of rumen ciliates

Abstract

Understanding the biodiversity and genetics of gut microbiomes has important implications for host physiology and industrial enzymes, whereas most studies have been focused on bacteria and archaea, and to a lesser extent on fungi and viruses. One group, still underexplored and elusive, is ciliated protozoa, despite its importance in shaping microbiota populations. Integrating single-cell sequencing and an assembly-and-identification pipeline, we acquired 52 high-quality ciliate genomes of 22 rumen morphospecies from 11 abundant morphogenera. With these genomes, we resolved the taxonomic and phylogenetic framework that revised the 22 morphospecies into 19 species spanning 13 genera and reassigned the genus Dasytricha from Isotrichidae to a new family Dasytrichidae. Comparative genomic analyses revealed that extensive horizontal gene transfers and gene family expansion provided rumen ciliate species with a broad array of carbohydrate-active enzymes (CAZymes) to degrade all major kinds of plant and microbial carbohydrates. In particular, the genomes of Diplodiniinae and Ophryoscolecinae species encode as many CAZymes as gut fungi, and ~80% of their degradative CAZymes act on plant cell-wall. The activities of horizontally transferred cellulase and xylanase of ciliates were experimentally verified and were 2-9 folds higher than those of the inferred corresponding bacterial donors. Additionally, the new ciliate dataset greatly facilitated rumen metagenomic analyses by allowing ~12% of the metagenomic sequencing reads to be classified as ciliate sequences.

Fig. 1. Generation of genome catalog of rumen ciliates.

Light micrographs of the 22 rumen ciliate morphospecies and two cryptic species examined in this study (A) and an assembly-and-identification pipeline for recovering single-cell genomes and gene prediction (B). a Iso. prostoma. c Iso. intestinalis. e Das. ruminantium. f Ent. longinucleatum. g Ent. bursa. h Ent. caudatum. i Dip. anisacanthum. j Dip. dentatum. k Dip. flabellum monospinatum. l Dip. flabellum aspinatum. m Eno. triloricatum. n Met. minomm. o Ere. rostratum. p Ost. gracile. q Ost. venustum. r Ost. mammosum. s Pol. multivesiculatum. t Epi. caudatum. u Epi. cattanei. v Oph. bicinctus. w Oph. caudatus. x Oph. purkynjei. Iso. sp. YL-2021a (b) and Iso. sp. YL-2021b (d) were two cryptic species of Isotrichidae. Micrographs n and p show stained skeletal plates. Scale bars, 50 µm (a-x). More details of the rumen ciliates are shown in Fig. S1.

Fig. 2. Genome-based taxonomy and phylogeny of rumen ciliates.

a A heatmap showing the average nucleotide identity (ANI) among the 52 SAGs. The scanning electron microscopy images of 22 morphospecies and two cryptic species are shown. Synonymic morphospecies are wrapped in red boxes. Morphospecies and its cryptic species are wrapped in blue boxes. b ANI distributions among the 52 SAGs. c Rank normalization through relative evolutionary divergence (RED). The RED interval for each rank is shown by two vertical black lines, median ± 0.1. The reassigned taxa are indicated in red dot. d The maximum likelihood phylogenetic tree of 19 rumen ciliate species and T. thermophila (as the outgroup) based on 113 concatenated single-copy proteins. All nodes have a 100% bootstrap support. The size and number of skeletal plates and the extensively fragmentation (EF) or non-EF of genome are labeled for ciliate species.

Fig. 3. Genome characteristics of rumen ciliates.

a Chromosome length (the red triangles indicate N50) and b Genome size distributions across families/subfamilies. c Functional divergence based on the clans of Pfam (p-value of permutational multivariate analysis of variance <0.001). d Rank curves showing gene number divergence in the families/subfamilies-shared gene families. e Gene family divergence: From left to right: a time phylogenetic tree of rumen ciliates with gene family expansions and contractions; numbers of gene families in each of the 19 species shared within or specific to a taxon; a heat map showing the top 10 categories of domains found in order-specific gene families of each species. MRCA most recent common ancestor, DO Diplodiniinae and Ophryoscolecinae.

Fig. 4. CAZyme profiles of rumen ciliates.

a Maximum amino acid sequence similarities of the CAZymes identified in the rumen ciliate SAGs compared to the public databases for seven classes of CAZyme proteins: GH glycoside hydrolase, GT glycosyl transferase, PL polysaccharide lyases, CE carbohydrate esterases, AA auxiliary activities, CBM carbohydrate-binding modules, SLH S-layer homology modules. The numeric values in parentheses refer to the numbers of genes of each class. b Number of CAZyme-coding genes in the representative genomes of rumen ciliate families/subfamilies and gut fungi. c A PCoA plot showing the CAZyme profiles across the five ciliate families/subfamilies (the color schemes are the same as in b), and the p-value of permutational multivariate analysis of variance is <0.001. d, e The mean number and prevalence of degradative CAZymes (with the same EC number and within the same family) encoded in the genomes of rumen bacteria (n = 2405), gut fungi (n = 8), non-gut Alveolata (n = 13), and the five families/subfamilies of rumen ciliates (n = 19). The mean number of each type of degradative CAZymes of rumen bacteria was set as 1 in d. More details of degradative CAZymes encoded in each genome are presented in Table S6. f An example for the xylanase (EC 3.2.1.8 in GH10) of rumen ciliates likely acquired via horizontal gene transfer (HGT) from rumen bacteria. g, h Structural and activity divergences of cellulase and xylanase between rumen ciliates and its likely bacterial donors. *** represents a statistical significance of p-value <0.001. i Inhibition zone assays of one ciliate lysozyme in GH19.

Fig. 5. Classification rates of ciliate reads in rumen metagenomes.

a The classification rates of ciliate reads in rumen metagenomes across different hosts fed a diet with <80% concentrate (726 of the 901 metagenomes). b Improvement of classification rates of total metagenomic reads in two previous studies [2, 95] enabled by the ciliate dataset. c Classification rates of ciliate sequences in the rumen metagenomes of sheep suffering from subacute rumen acidosis (SARA, n = 8) or control sheep without SARA (CON, n = 8), and dairy cows in confinement (house, n = 23) or on pasture (grazing, n = 20). The metagenomes were from two previous studies [91, 93]. d Relative abundance of rumen ciliates as represented by the percentage of families/subfamilies sequences in the total ciliate sequences.