Abstract Plant metabolism heavily relies on plastids, derived from once free-living organisms. However, how two distinct organisms merged into one remains currently only partially understood. Protein-mediated metabolite exchange across the double-membrane plastid envelope is essential for plant cell function. Here, we investigate the evolutionary origins of plastid outer envelope proteins (OEPs) involved in these transport processes. The mosaic nature of the nuclear genome and the deep evolutionary distance since plastid acquisition are major challenges. To address them, we combine sequence-based analyses with emerging structure-based tools, which together enable more sensitive evolutionary comparisons than traditional methods alone. To uncover distinct evolutionary trajectories, we focused on 27 OEPs from metabolite transport and protein import, with diverse structures. We found that the β-barrel proteins involved in metabolite transport were recruited in a stepwise manner and structural homologs of some OEPs point to early recruitment via endosymbiotic gene transfer (EGT). Protein import is sequence-wise conserved across Archaeplastida, although refinements occur across Chloroplastdia and structural turnover is implicated. Notably, OEP40 shows a recent structural rearrangement, lacking clear structural homologs in plants other than Arabidopsis, yet retaining sequence conservation across all major land-plant lineages. The JASSY-like family is found across plastid-bearing species, while true JASSY orthologs emerged in embryophytes likely via a stable recruitment of the characteristic lipid-binding domain. Overall, our findings highlight the dynamic nature of the plastid outer envelope and show how new functions could evolve through structural reshaping and novel domain recruitment.
Abstract Protein complexes are central to cell biology and typically verified via a combination of interaction data, complete genome sequencing and comprehensive protein-coding gene predictions for reference eukaryotes. However, this information is lacking for non-reference eukaryotes. Protein complexes can be predicted in species for which no interaction data is available by mapping orthology of verified protein complex components from reference eukaryotes to predicted proteomes. Studies that map conservation of protein complex components by orthology are often limited to a small number of protein queries, an under-representation of non-reference, microbial eukaryotes and are scattered across the literature. Here, I integrate orthology and protein interaction data by mapping proteins of experimentally verified complexes to orthogroups of proteins spanning 31 diverse eukaryotes. Proteins within complex-harbouring orthogroups are retained and distributed more evenly across taxa than non-complex orthogroups. I identified 184 universal orthogroups that included orthologs of known protein complex components from all 31 eukaryotes, consistent with a conserved core repertoire, likely present in the last eukaryotic common ancestor (LECA). I generated the protein complex orthology cartographer (PCOC) suite to find significant duplications and reductions of proteins in universal orthogroups across and between eukaryotes. This revealed both multi-copy and notably single-copy proteins, in all queried species, from the exosome, spliceosome, proteasome, small-ribosomal processome, tRNA synthetases, MCM complexes and RNA polymerase III. Case analyses of Naegleria gruberi and Guillardia theta highlight taxon-specific expansions and show how broader protist inclusion improves domain-wide inference of eukaryotic protein-complex evolution.
Abstract Some invertebrate lineages are known to have lost their ability to methylate DNA cytosines. These lineages have often lost the enzymes responsible for cytosine methylation (DNMT1 and 3), as well as other enzymes that interact with methylated cytosines. Previous studies showed that myxosporeans, a group of parasitic cnidarians, lack recognisable DNMTs and other canonical methylation enzymes, and observed a lack of cytosine methylation in two myxosporeans using whole genome bisulfite sequencing. Recently, a study of five myxosporean samples using Oxford Nanopore Technologies (ONT) sequencing reported high levels of methylation in two samples and low levels in the others and the presence of methylation-related genes in their assemblies. Here, we provide evidence that the two assemblies for which high levels of methylation were reported are heavily contaminated with fish-host sequences. In the other three samples, the lower apparent methylation signal resembles that observed in unmethylated negative controls and can be explained by ONT-background noise. Moreover, the CpG observed-to-expected ratios of the five assemblies differ from those of well-curated myxosporean assemblies. Additionally, the reported methylation profiles do not exhibit the typical distribution seen in invertebrates, possibly reflecting the presence of host contaminants. Finally, we found that most of the genes analysed are not canonical components of cytosine methylation pathways, and that the few candidate methylation-related genes reported in myxosporeans are more consistent with bacterial contamination than with genuine myxosporean genes. Together, these observations underscore important caveats in inferring the presence or absence of cytosine methylation in parasite genomes, particularly when using emerging technologies, and suggest that there is currently no robust evidence for substantial cytosine methylation in myxosporeans.
Abstract Assessing genetic diversity at loci putatively involved in fitness, such as immune genes, could provide important insights into the survival and vulnerability of endangered species. Research on immune genes in wild species has traditionally focused on the Major Histocompatibility Complex (MHC) genes, which represent only a small proportion of the immune gene repertoire, thereby limiting our understanding of immunity in non-model species. Here, we investigate five families of non-MHC immune genes - Tumor Necrosis Factors, interleukins, Toll-like Receptors, Leukocyte Immunoglobulin-Like Receptors, and chemokines - involved in adaptive and innate immunity in tiger Panthera tigris, an endangered carnivore known to have experienced historical bottlenecks and inbreeding. By leveraging 107 publicly available whole genomes, this study provides a comparative, multi-subspecific assessment of non-MHC immunogenetic diversity in tigers and identifies populations of conservation concern. We found higher genetic variation in immune receptor genes than in neutral regions and immune signalling genes. Site frequency spectra suggest balancing selection on Toll-like receptor genes. Further, tiger populations with histories of bottlenecks and inbreeding, such as South China tigers and North-western Indian tigers, exhibit reduced genetic diversity and higher mutation load in immune genes compared to large, connected tiger populations. Overall, we find that genetic drift is likely the predominant evolutionary force shaping genetic variation in tigers at both neutral and immune loci. However, some immune polymorphisms persist despite drift, thereby maintaining elevated variation, indicating that tigers have not been entirely stripped of their immunogenetic resilience. Such immunogenetic resilience may prove vital as spillover threats intensify.
