Convergent extreme reductive evolution in ancient planthopper symbioses

Domain: neuroscience
Published: 2026-02-07
Editorial outcome: minor_revision

Abstract
Strictly heritable endosymbiotic bacteria that provide limiting nutrients to sap-sucking hemipteran insects are known for their highly reduced genomes conserved in organization and function. Here, we show how in ancestral endosymbionts of planthoppers, Sulcia and Vidania, which have been gradually losing genes during ~263 my of co-diversification with hosts, co-infections by additional microbes and host ecological switches coincided with more dramatic genomic changes. At its extremes, this has resulted in the smallest non-organellar bacterial genomes known, at barely 50-52 kb. Such minuscule Vidania genomes evolved convergently in two planthopper superfamilies, and are strikingly similar in gene contents, including the ability to produce a single amino acid (phenylalanine) for the host. Losing many additional cell-function genes places them very close to organelles of symbiotic origin in the level of host dependence, further blurring the bacteria-organelle boundary.

Introduction
The smallest artificial, carefully designed bacterial genome capable of axenic growth in a rich medium is only 531 kilobases [kb], an order of magnitude less than that of the familiar Escherichia coli 1. However, for some two decades, we have known that the genomes of obligatorily intracellular insect-symbiotic bacteria that rely on host support mechanisms can be much smaller 2. Specifically, the genomes of ancient, strictly heritable bacterial symbionts in diverse lineages of sap-sucking hemipteran insects have convergently reduced to only encode genes involved in essential nutrient biosynthesis and some key cellular functions, and sometimes barely exceed 100 kb 3–9. Nevertheless, after the presumed early period of extensive genomic degeneration, several symbiont lineages have reached the stage where the remaining genes are indispensable, and their further loss slows to nearly a halt 9. This is demonstrated by the conservation of gene sets and order among divergent strains of Buchnera or Tremblaya, which have co-diversified with their respective hemipteran hosts for over 200 my 10,11. However, stability has its limits, and surveys encompassing a greater range of host lineages have revealed lost genes and occasional rearrangements 11,12 or more dramatic processes such as the extreme and rapid degeneration of the symbiont Hodgkinia in some cicadas 5,13. These unusual genome evolutionary processes in hemipteran symbionts provide a unique natural framework for studying the boundaries of cellular life and the long-term consequences of obligate intracellular existence. Yet, although the overall patterns of gene loss and functional streamlining are established 6,9, it remains unclear how far this process can proceed, what factors govern it, and to what extent different symbiont lineages follow parallel evolutionary trajectories. Addressing these questions requires systems that encompass deep evolutionary timescales, ancient symbiotic associations, and multiple independent changes to microbial communities and to host ecology.

Here, we investigate the evolutionary dynamics of symbiont genomes in planthoppers (hemipteran infraorder Fulgoromorpha), a phylogenetically diverse, ecologically significant, and economically important clade of sap-feeding insects that originated ~263 million years ago. Planthoppers host a broad array of heritable nutritional endosymbionts, including ancient associations with two strictly vertically transmitted bacterial lineages with highly reduced genomes—the Bacteroidetes symbiont Candidatus Sulcia muelleri (hereafter Sulcia) and the Betaproteobacterium Candidatus Vidania fulgoroidea (Vidania) 12,14,15. In many planthopper clades, these ancestral partners have been complemented or replaced by other bacteria or fungi. This diversity provides an unparalleled opportunity to examine how extreme reductive evolution proceeds across related yet independently evolving symbioses and to test general principles governing the structure, stability, and functional limits of nutritional endosymbiont genomes. Here, using metagenomic data from 149 planthopper species representing 19 families 14, we identify the processes, limits, and drivers of genomic evolution of their ancient symbionts.

In this work, we show that the ancient bacterial symbionts Sulcia and Vidania of planthoppers are generally highly stable in their genomic organization and contents, yet some lineages exhibit unprecedented genome reduction, yielding the smallest bacterial genomes known to date, at only ~50–52 kilobases. We demonstrate that these ultra-small genomes, which have evolved convergently in distinct planthopper lineages, can encode nearly identical sets of essential genes. Comparative analyses indicate that host ecological shifts and the replacement or supplementation of ancient symbionts by additional microbial partners are the primary drivers of extreme genome erosion. Together, our results delineate the lower boundaries of bacterial genomic complexity and illuminate the evolutionary forces that shape the transitions of ancient, specialized symbioses to even more reduced and host-integrated forms.

Results
Sulcia and Vidania have co-diversified with planthoppers for ~263 my
Sulcia and Vidania infections are broadly distributed across planthopper taxonomic diversity (Fig. 1). Based on rRNA sequences reconstructed from metagenomes, we have detected Vidania, usually accompanied by Sulcia, in 87 species of 149 sequenced, representing 15 of 19 surveyed families, on both sides of the deepest split in the planthopper phylogeny (Fig. 1, and Supplementary Data 1). Indeed, Sulcia infection is known to have pre-dated the divergence of planthoppers from their sister clade, Cicadomorpha, which includes cicadas, spittlebugs, leafhoppers, and treehoppers 16. In turn, the initial infection with Vidania seems to have occurred after these clades separated 12. Sulcia and Vidania have co-diversified strictly with their planthopper hosts (Supplementary Fig. 1), as expected given their transovarial transmission 17. Where both Sulcia and Vidania are missing, we always observe Hypocreales fungi—members of an insect-pathogenic clade known to have repeatedly replaced ancient bacterial nutritional endosymbionts of diverse Auchenorrhyncha, including planthoppers 18–20.

Further, we often find Gammaproteobacteria (Sodalis, Arsenophonus, Symbiopectobacterium) and Alphaproteobacteria (Wolbachia, Tisiphia, Rickettsia, Bartonellaceae, Acetobacteraceae), acquired independently by different host lineages 17,19,21,22. These diverse symbionts are known to provide vitamins and other complementary nutrients 17,19,21–23 but also influence host reproduction, defense, or other traits 24,25. Together, our data show the relative stability of ancient bacterial associations while highlighting the evolutionary dynamics of more recently acquired microbes.

Sulcia and Vidania have tiny genomes
Of 131 complete genomes of planthopper symbionts (63 Sulcia, 67 Vidania) that we used for content comparisons, most were circular. The two exceptions included the genome of Sulcia from Trypetimorpha occidentalis, previously confirmed to comprise two distinct circularly mapping contigs 21, and Sulcia from Brixia sp., with the same organization. Analysis of read mapping and the remaining contigs in the assemblies confirmed the completeness of genomes, as did synteny comparisons. Specifically, all but one of Vidania genomes, including the two smallest, were syntenic relative to the ancestral state 12 (Supplementary Fig. 2). In Sulcia, one or more rearrangements relative to the ancestral state were seen in 13/63 (20.6%) genomes representing six families (Supplementary Fig. 3). We note that some of the Sulcia and Vidania genomes that we could not fully assemble using short-read data, with insufficient DNA amount and quality for long-read sequencing, also appear to have undergone structural changes.

The reconstructed Sulcia genomes ranged in size from 137,729 bp to 180,379 bp, and Vidania ranged from 50,141 bp to 136,554 bp (Supplementary Data 2). Several newly assembled Vidania genomes are much smaller than the tiniest bacterial genomes characterized so far, including Nasuia from leafhoppers (≥107.8 kb), previously characterized Vidania (≥108.6 kb), or Tremblaya from mealybugs (≥139.0 kb) 7,10,21,26. Correspondingly, many Vidania strains encode fewer protein-coding genes than almost any other symbiont—as few as 62 in one case (Fig. 2A). Smaller gene sets have so far only been reported from Hodgkinia that form unique complexes of cytologically and genetically distinct but complementary lineages that apparently exchange gene products 5,13,27, and from organelles: mitochondria and some of the most reduced chloroplasts 28,29. Vidania genomes were much more variable in size, gene set, and GC content than those of Sulcia (Fig. 2B).

Variable rate of symbiont gene loss during co-diversification with planthoppers
The contents comparison among these unusual and, in the case of Vidania, extremely rapidly evolving genomes (Supplementary Note 1) enabled the reconstruction of their ancestral gene sets. In the last common ancestor of extant planthoppers, Sulcia must have encoded ≥164 total genes with identified functions. This includes 9 genes involved in the biosynthesis of three essential amino acids (Leu, Ile, Val) plus one gene for Phe biosynthesis, 132 genes involved in genetic information processing, and 18 involved in metabolism. The ancestral Vidania must have encoded ≥169 total genes, including 43 genes for the biosynthesis of seven amino acids (Met, Arg, Thr, Trp, Phe, Lys, His), 113 genes for genetic information processing, and 13 metabolism-related (Supplementary Figs. 4 and 5). Additionally, across all Sulcia and Vidania genomes, we identified 40 and 86 open reading frames with no significant similarity to any records within the Uniprot database. While some of them were conserved in groups of species, suggesting functionality (Supplementary Data 3 and 4, and Supplementary Note 1), we did not include them in subsequent analyses.

We found no cases of apparent gene gain by any lineage. However, gene losses were common during the evolution of both Sulcia and Vidania, with a notably higher loss rate in the latter (Fig. 3). In both symbionts, the patterns suggest a gradual loss of genetic information processing and metabolism genes during host-symbiont co-diversification. Some genomes lost <5% of the reconstructed ancestral gene set, but we observe several instances of much more substantial losses involving also nutrient biosynthesis pathways (Fig. 3A, B, and Supplementary Figs. 4 and 5). For Vidania, this includes 49 gene losses on the branch leading to Saccharosydne sp. (Delphacidae), 40 losses in the lineage leading to the sole characterized Achilixiidae, Bebaiotes sp., 40 in the common ancestor of Derbidae, 32 in the ancestor of Meenoplus skotinophilus (Meenoplidae), and 25 in the ancestor of Cixidia pilatoi (Achilidae). Strikingly, four of the five lineages where Vidania lost most genes (all listed above except Bebaiotes) have also lost their Sulcia symbiont entirely, with only one additional recorded case of Sulcia-but-not-Vidania loss through apparent fungal replacement in Issus coleoptratus (Issidae) lineage. In turn, the branch leading to Trypetimorpha occidentalis (Tropiduchidae) and the ancestral branch of Tettigometridae exemplify cases where both Sulcia and Vidania lost multiple genes (Fig. 3A, B, and Supplementary Figs. 4 and 5).

We also found major differences among gene functional categories in the overall level of conservation (Fig. 3C). In Sulcia, ancestral biosynthesis genes are always retained, with one exception: aspC, involved in phenylalanine biosynthesis, is found in Sulcia from seven species, whereas in most other planthoppers this gene is encoded within the host or Vidania genome instead (Supplementary Figs. 4 and 5). In Vidania, despite overall conservation, every biosynthetic pathway has been lost from at least one genome, with the Phe pathway most consistently retained. In both symbionts, rRNA genes are always retained, and ribosomal proteins together with genes involved in protein-folding and degradation are highly conserved. In contrast, aminoacyl-tRNA synthetases are the functional category most frequently lost.

Convergence in the evolution of extremely reduced genomes
Planthopper symbionts include the smallest bacterial (non-organellar) genomes described to date: that of Vidania strain VFSACSP1 from Saccharosydne sp. (Delphacidae) has 50,141 bp, and that of VFMALBOS from Malenia bosnica (Derbidae) has 52,460 bp (Fig. 4A). VFSACSP1 encodes just 68 protein-coding genes with identified functions, and VFMALBOS 62. Notably, the extremely reduced state represented by these two genomes has evolved independently in two superfamilies separated by ~263 my of evolution 14.

Despite their independent evolution from an ancestor which must have had a genome approximately three times their size, these smallest genomes exhibit striking similarity in organization and gene contents (Fig. 4A, B, and Supplementary Figs. 2, 5 and 6). The set of genes involved in essential cellular processes comprises rRNA genes, a set of 35 or 38 ribosomal proteins, 8 or 6 DNA and RNA polymerase subunits, 15 other genes involved in genetic information processing, and one or two genes related to metabolism in VFSACPS1 and VFMALBOS, respectively. Both these strains encode the entire ancestral set involved in phenylalanine biosynthesis, while pathways for the biosynthesis of six other essential amino acids have become entirely lost (Fig. 4A, and Supplementary Figs. 5 and 6). Interestingly, neither genome retains any aminoacyl-tRNA synthetases. As in all other Vidania genomes, in VFMALBOS, but not in VFSACSP1, tRNAscan-SE identified some tRNA genes, although extreme rates of sequence evolution likely complicated their detection (Supplementary Data 4).

In contrast, the smallest genome from the third recognized planthopper superfamily, of Vidania VFMEESKO from a cave-dwelling Meenoplus skotinophilus (Meenoplidae), is substantially larger at 84,795 bp and retains an expanded set of genetic information processing and metabolism genes (Fig. 4A). It preserves the complete pathway for lysine biosynthesis and three genes from the arginine and tryptophan pathways, but interestingly, this is the only Vidania in our collection that has lost the phenylalanine biosynthesis pathway (Figs. 3B, 4A, and Supplementary Figs. 5 and 6). Notably, all planthoppers hosting these highly reduced Vidania strains have also lost Sulcia: they represent the few clades in our dataset where Sulcia was lost while Vidania remained (Figs. 3 and 5).

The organization and ultrastructure of Vidania-hosting bacteriome tissue of M. bosnica—the tiny-genome species with several specimens available for microscopy—differed from that of planthoppers studied before 17,19,21 by exhibiting an unusually high density of mitochondria (Fig. 4C). Additionally, in some specimens we observed signs of cellular degeneration, including vacuolization of the bacteriocyte cytoplasm and higher bacterial cytoplasm density (Fig. 4C). These features were also observed in the Vidania bacteriomes of other adult planthoppers where this symbiont has reduced gene set and Sulcia is absent, including the derbids Proutista sp. and Panmendanga sp., as well as the achilid C. pilatoi (Supplementary Fig. 7). In the bacteriome, we did not detect any other adjacent bacteria that could serve as an obvious source of proteins targeted to Vidania to replace the functions it lost 30.

Nutritional functions explain the evolution of symbiont genomes and multi-partite symbioses
In hemipteran nutritional symbioses, the set of functions related to nutrient biosynthesis is generally conserved at the level of symbiosis, even when microbial partners change. In previously characterized planthopper symbioses, Sulcia and Vidania were thought to fulfill their hosts’ nutritional needs by jointly encoding most genes for the biosynthesis of all ten essential amino acids, with genes encoded on the host genome though to complete the pathways (Fig. 5). However, we also find amino acid biosynthesis genes in other bacteria present in these metagenomes, including Sodalis, Arsenophonus, Symbiopectobacterium, Wolbachia, or Tisiphia (heritable endosymbionts previously detected in planthopper tissues 19), or Serratia, Klebsiella, Sphingomonas, or Pseudomonas (likely less specialized gut associates). The genes they encode often overlap with those present in Sulcia or Vidania, and in many species that had lost Sulcia, or some biosynthesis pathways from Vidania genomes, they often at least partially fill the resulting gaps (Fig. 5, and Supplementary Data 5). This is most evident in the two planthopper species with the tiniest Vidania genomes - Saccharosydne sp. and M. bosnica - where Tokpelaia and Sphingomonas, respectively, encode largely complete sets of amino acid biosynthesis genes 22. However, in assemblies for most other derbids, Issus coleoptratus, and others, we did not find alternative copies for a large share of genes lost from ancient genomes. For example, for M. skotinophilus, none of the genes from the nine amino acid biosynthesis pathways lost were present in the 695 Mba assembly for its dissected abdomen. We note, however, that functions encoded by microbes residing outside of the tissues used for metagenomics, or sequenced to insufficient coverage, could have been missed. Likewise, the reliable identification of biosynthesis genes encoded within the host genomes requires their more complete assembly.

The biology of most of the studied species is barely known, but mapping significant biological transitions onto the phylogeny suggests the role of host ecology in structuring nutritional demands. Specifically, Derbidae and Achilidae feed on fungal hyphae during their immature stages 31, species in the genus Tettigometra engage in obligate trophobiotic associations with ants 32, and Meenoplus skotinophilus has adapted to life in caves 33 (Fig. 5). The resulting alterations in nutrient availability and nutritional demands in these clades are likely to explain changes in biosynthetic gene sets.

Methods
Insects
Individuals of 149 specimens representing 19 planthopper families were collected from their natural habitats around the globe between 2004 and 2021 (Supplementary Data 1). Sampling was conducted in accordance with all applicable international and local permitting requirements. This research on non-protected, non-regulated invertebrate species complied with all relevant ethical regulations and institutional guidelines, and required no specific approvals. After collection, insects were identified based on morphological features and preserved whole in ethanol and stored at −20 °C, or in some cases, partially dissected and fixed in a 2.5% glutaraldehyde solution and stored at 4 °C until further analyses.

Metagenomic library preparation and sequencing
DNA from separated abdomens or dissected bacteriomes (symbiont-containing tissue) was extracted using the commercially available kits (Supplementary Data 1) and used for metagenomic library preparation using the NEBNext Ultra II DNA Library Prep kit for Illumina (New England BioLabs) or Novogene NGS DNA Library Prep Set, with the target insert length of 350 bp. Due to the known issue of index swapping that occurs during cluster formation and sequencing on Illumina platforms 58,59 and which can lead to cross-contamination among samples in multiplexed lanes, as a means of reducing such cross-talk we used dual-unique indexes for all but six earliest-processed samples. The library pools were sequenced in several batches on Illumina platforms: HiSeq 2500 in a Rapid Run mode (2×250 bp), HiSeq X or NovaSeq 6000 S4 (2×150 bp reads) (Supplementary Data 1).

Metagenome assemblies and characterization
Metagenomic raw reads were quality-filtered and adapter-trimmed using TrimGalore v0.6.4 (settings: –length 80 -q 30; https://github.com/FelixKrueger/TrimGalore). High-quality filtered reads were checked using FastQC v0.11.9 (https://github.com/s-andrews/FastQC). Contigs were assembled using Megahit v1.2.9 (maximum k-mer size = 255, min contig size = 1000) 60. Contigs were validated, and their coverage was estimated by read mapping using Bowtie 2 61. For the six libraries that were not double-uniquely indexed, we filtered the resulting assemblies for cross-contamination by discarding all contigs that had more than 10X (and typically >200X) greater coverage based on strictly mapped reads from another library with an overlapping index than based on reads from the same library.

Symbiont contigs were taxonomically identified through BLASTN and TBLASTX searches against the curated collection of Sulcia and Vidania genomes and the NCBI nt database. The circularity and contiguity of Sulcia and Vidania contigs were confirmed by read mapping and visualization using Tablet v. 1.20.12.24 62, and based on the presence of overlapping ends. The circular Sulcia and Vidania genomes were rearranged so that they had the same orientation and start position (Sulcia - gene lipB; Vidania - tufA). Genomes of symbionts other than Sulcia and Vidania were represented by multiple contigs and were or will be presented in separate manuscripts 22.

Sulcia and Vidania genome annotation
Because of the rapid evolution rates of Sulcia and especially Vidania genomes and the extreme genetic distance from reference sequences, standard tools such as Prokka or Interproscan struggle to detect and consistently label genes, leaving annotation gaps and unannotated or hypothetical proteins. Therefore, Sulcia and Vidania genomes were annotated using a custom Python script modified from our previous publication 13. The script extracts all Open Reading Frames (ORFs) and their amino acid sequences from each genome. It then searches these ORFs recursively using HMMER v3.3.1 63 against custom databases containing manually curated sets of protein-coding, rRNA, and non-coding RNA (ncRNA) gene alignments from previously characterized Sulcia or Vidania lineages. These references were updated after every round of annotation. rRNA and ncRNA genes were searched with nhmmer (HMMER v3.3.1) 63, and tRNAs were identified with tRNAscan-SE v2.0.7 64. Based on the relative length compared to the reference genes, protein-coding genes were classified as putatively functional (≥60% of the reference alignment length) or truncated-likely pseudogenes (<60%). These cutoffs were based on our previous work 12,13,17, and we confirmed that key functional domains were preserved in several cases above the cutoff. However, as explained in the section “Annotation challenges and unusual observations in fast-evolving symbiont genomes” (Supplementary Note 1), the situation was not always clear-cut, and the decisions were made on a case-by-case basis. Genomes for Fig. 4 were visualized using Proksee 65. Comparative synteny plots were generated using a custom Python and Processing workflow modified from previous work 13. Genome content tables were visualized using Processing v3.5.4 and edited with Inkscape v1.4.2.

Phylogenomic analyses
As the host phylogenomic framework, we used the recently published genome-based planthopper phylogeny 14. The phylogenies of Sulcia and Vidania symbionts were reconstructed based on 1291 and 97 protein-coding genes, respectively (Supplementary Data 6). We used Sulcia and the betaproteobacterial symbiont Nasuia from two species in the Membracoidea superfamily (a treehopper Entylia carinata and a leafhopper Macrosteles quadrilineatus) as outgroups for planthopper-associated Sulcia and Vidania, respectively. The single-copy orthologs were identified using OrthoFinder v2.5.5 66 based on the curated proteome resulting from the annotation pipeline above. Orthologs retained by >75% of samples (i.e., by >48 Sulcia lineages and by >53 Vidania lineages) were used for phylogenomic analyses. Nucleotide alignments of each ortholog were generated by MAFFT v7.526 67. We kept only the 1st and 2nd codon positions in the alignments to avoid saturation effects from the 3rd codon position. Alignments were then concatenated and partitioned by genes. Phylogenies were inferred with the Maximum Likelihood (ML) approach in IQ-TREE 2 v2.3.6 68. To decide on the partitioning scheme and the substitution model, we asked IQ-TREE 2 to perform extended model selection on each gene with free rate heterogeneity and subsequently merge genes until the model fit does not increase any further (setting: -m MFP+MERGE 69. The best partitioning scheme and the best-fit models were selected based on the highest Bayesian Information Criterion scores. Bootstrapping was conducted using the approximate likelihood ratio test (SH-aLRT) and ultrafast bootstrap methods with 1000 replicates 70,71. Phylogenetic trees were visualised using iTOL 72.

Analysis of symbiont contribution to amino acid biosynthesis pathways
The presence of bacterial amino acid biosynthesis genes in genomes of symbionts other than Sulcia and Vidania was assessed through HMMER searches within six-frame translated assemblies using HMM profiles from the NCBI Protein Family Models database. We taxonomically classified the identified proteins through BLAST-based comparisons against the nt database and against curated Sulcia and Vidania genomes. At that stage, we also filtered out genes of the known DNA extraction reagent-derived contaminant in our data, Cellulosimicrobium. The resulting hits were filtered based on alignment length and sequence similarity, with a large subset manually verified for gene identity and bacterial taxonomic assignment through blastn searches against the NCBI nt database. The results were visualized using a custom Processing script and edited in Inkscape v1.4.2.

Microscopic analyses
Histological and ultrastructural analyses. The whole insect abdomens or dissected bacteriomes were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) at 4 °C. The fixed material was then rinsed three times in the same buffer with the addition of sucrose (5.8 g/100 ml) and postfixed in 1% osmium tetroxide for 2 h at room temperature. After post-fixation, samples were dehydrated in a graded series of ethanol (30–100%) and acetone, embedded in epoxy resin Epon 812 (Merck, Darmstadt, Germany), and cut into sections using Reichert-Jung ultracut E microtome. Semithin sections (1 µm thick) were stained in 1% methylene blue in 1% borax and analyzed and subsequently photographed under a Nikon Eclipse 80i light microscope (LM). Ultrathin sections (90 nm thick) were contrasted with uranyl acetate and lead citrate and examined and photographed at 80 kV using a Jeol JEM 2100 electron transmission microscope (TEM) at the Institute of Zoology and Biomedical Research, Faculty of Biology, Jagiellonian University.

Fluorescence in situ hybridization. Fluorescence in situ hybridization was performed using fluorochrome-labelled oligonucleotide probes targeting 16S rRNA of Sulcia: Sulc644 5′Cy3-CCMCACATTCCAGYTACTCC3′ 73 and Vidania: Bet940 5′Cy5-TTAATCCACATCATCCACCG3′ 74. Ethanol-preserved insects were rehydrated and then postfixed in 4% paraformaldehyde for 2 h at room temperature. Next, the material was dehydrated again by incubation in increased concentrations of ethanol (30–100%) and acetone, embedded in Technovit 8100 resin (Kulzer, Wehrheim, Germany), and cut into semithin sections (1 µm thick). These sections were then incubated overnight at room temperature in a hybridization buffer containing the specific sets of probes with a final concentration of 100 nM. After hybridization, the slides were washed in PBS three times, dried, covered with ProLong Gold Antifade Reagent (Life Technologies), and examined using a confocal laser scanning microscope Zeiss Axio Observer LSM 710 at the Institute of Zoology and Biomedical Research, Faculty of Biology, Jagiellonian University.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript “The tiniest genomes shrink much further: extreme reductive evolution in planthopper symbionts” by Michalik and coworkers presents a comparative analysis of the genomes of an extensive group of Sulcia and Vidania strains, obligate endosymbionts of planthoppers, starting from material that have been collected in a broad survey over the world during many years. This is the result of an impressive amount of work and represents a very comprehensive study that provides some expectable results but also interesting surprises in the evolutionary path of these bacteria.\nThe way the experiments have been conducted is very appropriated. It is a pity they have not included current long-read sequencing technologies, which would have helped to solve some of the questions that remain unanswered.\nThe manuscript is well organized, clear and highly informative. I only have a few suggestions that might help improve it:\nAbstract, Lines 28-29. Although I agree that the losses undergone by the bacteria with these tiny genomes further blurs the bacteria-organelle boundary, I think it is a bit exaggerated to say that it places them among mitochondria. Due to the yet considerable differences between present endosymbionts of insects and eukaryotic mitochondria, I rather prefer to consider them “symbionelles” as suggested by Reyes-Prieto and coworkers back in 2014 (doi:10.1111/1462-2920.12220).\nNevertheless, I must admit that the loss of all aminoacyl-tRNA synthetases is amazing and also common to mitochondria. Do you have any clue about the possibility of the genes encoding them having been transferred to the nucleus of the insect or they are expected to be provided by other bacterial symbionts? Is there any other endosymbiotic model with this feature?\nFigure 1. There is no indication of why the five insects depicted in panel A and the one in panel B have been chosen among all species included in the phylogeny.\nLines 390 and 393: There must be an error in the description of the outgroups. As it is written, it indicates that Sulcia ENCA is used as the outgroup for Sulcia phylogeny.\nSupplementary Information\nJust driven by curiosity, regarding the example provided in pages 9-10 about the rplJ gene: In the figure it appears that rplA, which is upstream of rplJ in Vidania from Akotropis, does not appear in Vidania from Brixia. Could be rplJ2 the consequence of a gene fusion followed by degeneration?\nRegarding the broken ORFs (pages 11-12), have you analyzed if the original functional gene encoded a protein with several domains so that the broken version, no matter if it has retained less than 60% of the known functional ortholog, could still be functional? I recall at least one case in an endosymbiont of aphids regarding the annotation of the gene encoding ligase that was considered a pseudogene due to a big truncation that just removed the regulatory domain.\n(Remarks on code availability)\nI haven't reviewed the code extensively, but it's fully available via GitHub, including a README page.", "reviewer_2": "This paper was a pleasure to read – it is extremely clear and succinct, both in terms of the writing and figures. I should point out that I’m a little jaded by the “race to the bottom” (of symbiont genome size) that has transpired over the past decade which has yielded many publications in high profile journals that tend to have little content beyond reporting an incrementally smaller genome size. However, this work encompasses a very thorough survey and answers several important outstanding questions in the field, rationalizing concepts involving symbiont replacement, compartmentalization of functions and idiosyncrasies in mutation patterns and rates of degeneration. I therefore think that it merits publication in a high impact journal and will be of interest to a broad readership. To that end, I’ll state again that it is written in such a way that it is very clear and broadly accessible. The authors are also appropriate cautious regarding their interpretations and therefore I believe that the results and conclusions are well supported by the observations and data analyses presented in this paper. The figures and tables fit well with the flow and content of the manuscript and are easy to interpret for a broad audience. The methodology is robust and well documented.\nI will state that I was really struck by the very limited inventories of genes encoding DNA replication components that are maintained in these small genomes, particularly in light of the fact that they are proposed to replicate circular genomes. It appears to me that many components of the replisome that are specifically involved in “handling” circular chromosome replication (particularly dealing with initiation, termination and dimer resolution are absent, implying that there is a non-canonical method of replication in play. Free-living bacteria that lack such genes tend to replicate by cSDR, but these organisms also don’t seem to be capable of initiating cSDR. I would urge the authors to look into this in detail and aim to make some predictions relating to the mode of replication (or at least to outline what is lacking). There are many papers explaining how circular prokaryotic replication systems work and it should be possible to propose a model for operation. This is a very important and interesting question/opportunity, particularly in the light of current work engineering minimal synthetic cells. It could markedly increase the scope for citation of this work.\nSome structural suggestions:\nLine 35: change to “are much smaller”\nLine 54: change to “determine the processes”\nLines 89-90: synteny/syntenic (not synthetic)?\nLine 254: change to “those characterized previously”\nOne query:\nLine 259: What evidence supports the notion that changes are detrimental but not lethal? I think that’s an assumption derived from theory rather than empirical evidence and it should be explained as such. Perhaps also mention that these associations have endured for very long periods of time, suggesting that host-level selection is sufficiently potent to ensure that they are not replaced, which is clearly possible, but (IMHO) more likely catalyzed by a changing functional mandate rather than mutational meltdown, as supported by the comparative functional analyses documented in this paper.", "reviewer_3": "NCOMMS-25-58613\nThe tiniest genomes shrink much further: extreme reductive evolution in planthopper symbionts\nAnna Michalik, Diego C. Franco, Junchen Deng, Monika Prus-Frankowska, Adam Stroiński, Piotr Łukasik\nGeneral comment\nWe found this work to be a valuable contribution to the field of insect-microbe symbiosis and bacterial genome evolution. The authors have compiled a series of comprehensive analyses on symbiont genomics across a vast number of planthopper species (149 species representing 19 families!). The discovery of new ultra-small 50–52 kb Vidania symbiont genomes is striking, which revised our predisposition on the reductive symbiont genome evolution. The data presented are robust and the methods appear sound. Overall, the manuscript is well-written and provides a framework for driving future studies. On the other hand, there appear to be a few areas where the paper’s messages could be strengthened. We provide several comments for making the manuscript more appealing, although those amendments are not mandatory.\nMajor comments\n1. A critical point is that the central question raised in this paper seems somewhat vague, which is a common challenge with this type of exploratory genomic approach. As is common in genomic studies, the initial hypothesis can sometimes become less defined as a result of a data-driven approach. While this is natural, we suppose that the authors could enhance the paper’s flow by providing hypothesis uniquely testable in this taxon, or add a clear rationale for their chosen model system. For instance, there is no explicit explanation in the Introduction as to why the authors selected the infraorder Fulgoromorpha to provide a comprehensive view (Line 46-48). A convincing explanation for why planthoppers are uniquely suited for this study would strengthen the paper’s main argument.\n2. A potential weakness of this study is that it might be perceived as a finding specific only to planthoppers, thereby limiting its broader appeal. A possible way to address this is to provide a strong rationale for why a comprehensive analysis of this specific group was needed (this is related to Major comment 1). While the authors’ discussion already hints at this (Lines 246-249, 269-272, 300-310), we suggest to clearly state why planthoppers were the ideal model for this research in Introduction. This will help readers understand that the findings are not just about a single insect taxon, but provide valuable insights into the fundamental processes of genomic evolution.\n3. It is interesting but elusive how the extremely tiny-genome bacterium like Vidania can function as a living cellular entity. We would like to see authors’ hypothesis/opinion/perspective on the topic in Discussion in a more explicit manner.\nMinor Comments\nLines 75-82 (Fig. 1): Please provide more detailed caption for Fig. 1. In Fig. 1A, the color coding for the circles needs to be explained either within the figure or in the caption. For instance, we can assume yellow represents a third obligate symbiont, gray a facultative-like symbiont, and the golden-brown a yeast-like symbiont, but this is not explicitly stated. Additionally, the caption for the confocal microscopy image (Fig. 1C) should specify what the blue color represents.\nLines 104-105 (Fig. 2A): To ensure consistency, please unify the terminology between the figure and its caption. The caption refers to “the very smallest insect symbionts (pink)” and “the smallest bacteria (blue),” but the figure itself uses “insect symbiotic bacteria” and “other bacteria.”\nLine 124: The authors state the they found no cases of gene gain in any lineage. Please clarify the data that supports this statement. Does this mean that no lineage-specific orphan genes were found?\nLines 169-170: To avoid misinterpretation, please use a more precise expression than a range. For the two tiniest genomes, VFSACSP1 and VFMALBOS, it would be clearer to state the number of ribosomal proteins as “35 or 38, respectively.” The same applies to the “3-4 ribosomal DNA and RNA polymerase subunits.”\nLines 186-192: The imaging results for the tiniest Vidania are highly interesting, but the derived observations are open to interpretation. Please clarify the number of samples on which the statements regarding mitochondrial density and vacuolization are based. Additionally, providing context on the host insect’s condition (e.g., larva vs. adult) would be valuable. Ideally, a comparison with a closely related insect hosting a non-tiniest Vidania would make these findings more conclusive.\nLine 188, 190: Fig. 4C?\nLine 203: mitochondrium -> mitochondrion\nLine 215: Please remove the comma after “Pseudomonas.”\nLine 355-356: Please state the specific software used for read mapping. This detail is essential for the reproducibility of your analysis.\nLine 386: Please add details for the “Processing” and “Inkscape” software, as their specific versions should be specified for reproducibility.\nComments to SI\nLines SI 197-201: We acknowledge the difficulty of accurately annotating highly divergent genes. However, we are not fully convinced by the conclusion that highly truncated genes are functional simply based on their conservation across lineages. Please provide a clearer rationale or discuss other evidence that supports their functionality.\nLines SI 192, 208, 26, 246: We noticed distracting red lines under scientific names in the figures. Please remove them, as they appear to be from an active spell-check.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "RESPONSE TO REVIEWERS’ COMMENTS\nReviewer #1 (Remarks to the Author):\nThe manuscript “The tiniest genomes shrink much further: extreme reductive evolution in\nplanthopper symbionts” by Michalik and coworkers presents a comparative analysis of the\ngenomes of an extensive group of Sulcia and Vidania strains, obligate endosymbionts of\nplanthoppers, starting from material that have been collected in a broad survey over the world\nduring many years. This is the result of an impressive amount of work and represents a very\ncomprehensive study that provides some expectable results but also interesting surprises in the\nevolutionary path of these bacteria.\nThe way the experiments have been conducted is very appropriated. It is a pity they have not\nincluded current long-read sequencing technologies, which would have helped to solve some\nof the questions that remain unanswered.\nThe manuscript is well organized, clear and highly informative. I only have a few suggestions\nthat might help improve it:\n>>> Our response: We thank the reviewer for this valuable comment. We fully agree that\nlong-read sequencing could further resolve ancient symbiont genome organization and\nevolution - especially for systems where we could not assemble complete Sulcia and Vidania\ngenomes. Unfortunately, obtaining sufficient amounts of DNA for long-read sequencing has\nproven highly challenging for these small insects, and due to sample age and varying storage\nconditions, the DNA has often been degraded and thus unsuitable for long-read technology.\nThankfully, for the large majority of ancient symbiont strains analyzed here, the short-read data\nwere sufficient to yield complete, circularly mapping genomes, allowing robust comparative\nanalyses.\nWe have added the following explanation to the Results: “We note that some of the Sulcia and\nVidania genomes that we were not able to fully assemble based on short-read data, with\ninsufficient DNA amount and quality for long-read sequencing, also appear to have undergone\nstructural changes.” (lines 127-129 in the manuscript version with changes tracked in the\nmanuscript).\nWe are currently implementing long-read sequencing and complementary approaches such as\nsingle-cell sequencing in ongoing projects that resolve fragmented symbiont genomes in a\nsmall number of hemipteran species sampled more easily, including two from this study.\nHowever, as these projects have a distinct scope and are still in progress, we plan to present\ntheir results in separate publications.\nAbstract, Lines 28-29. Although I agree that the losses undergone by the bacteria with these\ntiny genomes further blurs the bacteria-organelle boundary, I think it is a bit exaggerated to say\nthat it places them among mitochondria. Due to the yet considerable differences between\npresent endosymbionts of insects and eukaryotic mitochondria, I rather prefer to consider them\n“symbionelles” as suggested by Reyes-Prieto and coworkers back in 2014 (doi:10.1111/1462-\n2920.12220).\n>>> Our response: Thank you for this thoughtful comment. We agree that the term\n“symbionelle” captures well the highly reduced but still bacterial nature of these intracellular\nentities. However, as this term has not been widely accepted, we have updated the final\nsentence of the Abstract using a different wording. The sentence now reads, “Losing many\nadditional cell-function genes places them very close to organelles of symbiotic origin in the\nlevel of host dependence, further blurring the bacteria-organelle boundary.” (lines 28-30).\nNevertheless, I must admit that the loss of all aminoacyl-tRNA synthetases is amazing and also\ncommon to mitochondria. Do you have any clue about the possibility of the genes encoding\nthem having been transferred to the nucleus of the insect or they are expected to be provided\nby other bacterial symbionts? Is there any other endosymbiotic model with this feature?\n>>> Our response: Thank you for this excellent point. The loss of all aminoacyl-tRNA\nsynthetases and other genes essential for translation has indeed been observed in several other\ninsect symbioses, including Sulcia–Nasuia/Hodgkinia in leafhoppers and cicadas, Tremblaya–\nMoranella in mealybugs, and Buchnera in aphids. In these systems, the missing functions are\nlikely complemented by host-encoded genes, often of bacterial origin, that are expressed in\nsymbiont-containing tissues and whose products are imported into the symbiont cells (although\nother possibilities exist, including import of proteins encoded in other symbiont genomes, or\nof charged tRNAs into symbiont cells). We anticipate a mechanism where host-derived\nenzymes likely substitute for missing symbiont functions in planthopper symbioses. However,\nbecause the host genomic data available for this study are low-coverage and highly fragmented,\nwe were not able to confidently identify horizontally transferred symbiont-support genes.\nWe have clarified this point in the Discussion and incorporated aminoacyl-tRNA synthetases\ninto the section addressing host support. The revised text reads: “Similarly, the frequent loss of\naminoacyl-tRNA synthetases, key components of the translation process, combined with the\nretention of tRNAs, suggests that they are likely provided by the host 48. This functional\nintegration between host and symbiont genetic information processing machineries would\nparallel the situation in mitochondria, where all core replication and translation proteins are\nencoded by the host and imported into the organelle 49 “ (lines 340-345).\nFigure 1. There is no indication of why the five insects depicted in panel A and the one in panel\nB have been chosen among all species included in the phylogeny.\n>>> Our response: In Figure 1, which provides a general overview of the diversity of both\nplanthoppers and their symbionts, we aimed to present representatives from various\nplanthopper families that differ in morphology and microbiome composition. The final\nselection was influenced by the availability of high-quality photographs of the planthoppers.\nAkotropis quercicola was chosen for Figure 1B based on the quality and availability of FISH\nimages showing the bacteriomes that host the ancestral planthopper symbionts Sulcia and\nVidania. Our prior work has demonstrated that the symbiont morphology and symbiotic tissue\norganization highly resemble those of other lineages of Sulcia and Vidania. We have modified\nthis portion of the legend to read: “Sulcia (green) and Vidania (red) live within the cytoplasm\nof dedicated cells (bacteriocytes) that form specialized organs (bacteriomes) within the host\nbody cavity. Here, Akotropis quercicola (Achilidae) exemplifies the conserved symbiont\nmorphology and bacteriome organization across taxonomically diverse planthoppers” (lines\n103-108).\nLines 390 and 393: There must be an error in the description of the outgroups. As it is written,\nit indicates that Sulcia ENCA is used as the outgroup for Sulcia phylogeny.\n>>> Our response: There is no error; indeed, Sulcia and Nasuia from treehopper Entylia\ncarinata (ENCA) and the leafhopper Macrosteles quadrilineatus (ALF) were used as\noutgroups for the phylogenies of planthopper-associated Sulcia and Vidania strains. However,\nwe have indeed not explained that we used non-planthopper Sulcia strains to root the\nplanthopper-Sulcia tree, and we suspect this may have led to confusion.\nWe have rewritten that portion of the Results so that it now reads “We used Sulcia and the\nbetaproteobacterial symbiont Nasuia from two species in the Membracoidea superfamily (a\ntreehopper Entylia carinata and a leafhopper Macrosteles quadrilineatus) as outgroups for\nplanthopper-associated Sulcia and Vidania, respectively.” (lines 466-469) We also removed\nthe branches corresponding to the outgroups from the trees shown in Supplementary Fig. 1 to\nimprove visibility. We have added this information to the figure legend.\nSupplementary Information\nJust driven by curiosity, regarding the example provided in pages 9-10 about the rplJ gene: In\nthe figure it appears that rplA, which is upstream of rplJ in Vidania from Akotropis, does not\nappear in Vidania from Brixia. Could be rplJ2 the consequence of a gene fusion followed by\ndegeneration?\n>>> Our response: Thank you for your question. We have identified the variant of the rplJ\ngene that we labeled rplJ2 in five species from different families. In four of them, the rplA gene\nwas also present. The length of the rplJ2 gene is variable, ranging from 145 to 174 nucleotides.\nAdditionally, the rplJ2 variant of the rplJ gene does not show similarity to the rplA gene.\nHowever, it should be noted that detecting such homology is very difficult due to the very high\nsequence divergence in these ancient symbionts and the minimal similarity even among\nvariants of the same gene. To address potential further concerns, we have added another\nexample (Cixius nervosus) to the Supplementary Information, where we describe this issue in\nthe context of genome annotation (Supplementary Text, page 10).\nRegarding the broken ORFs (pages 11-12), have you analyzed if the original functional gene\nencoded a protein with several domains so that the broken version, no matter if it has retained\nless than 60% of the known functional ortholog, could still be functional? I recall at least one\ncase in an endosymbiont of aphids regarding the annotation of the gene encoding ligase that\nwas considered a pseudogene due to a big truncation that just removed the regulatory domain.\n>>> Our response: We thank the reviewer for this valuable suggestion. We agree that domain-\nlevel analyses can help identify truncated genes that may retain partial functionality. While we\ndid not conduct a systematic domain-level survey, we have now examined the ribosomal\nprotein rplI, for which two variants differing in length were detected among Sulcia genomes.\nThe truncated variant retains the key N-terminal domain, suggesting that the encoded protein\nremains functional. This analysis has been added to the Supplementary Information\n(Supplementary Text, pages 11-12).\nOur custom annotation pipeline for Sulcia and Vidania uses length thresholds relative to full-\nlength reference genes to classify loci as functional or pseudogenized. We have clarified this\nin the Methods, adding an explanation that: “These cutoffs were based on our previous\nwork12,13,17, and we confirmed that key functional domains were preserved in several cases\nabove the cutoff”. (lines 454-455). We note that, unlike in cicada symbionts, in planthoppers,\nin the vast majority of cases, there was no doubt: the gene is either virtually intact or represented\nby small fragments unlikely to retain function.\nReviewer #1 (Remarks on code availability):\nI haven't reviewed the code extensively, but it's fully available via GitHub, including a\nREADME page.\n>>>Our response: We appreciate the comment. Indeed, all custom scripts used in this\nmanuscript are fully described and available on GitHub.\nReviewer #2 (Remarks to the Author):\nThis paper was a pleasure to read – it is extremely clear and succinct, both in terms of the\nwriting and figures. I should point out that I’m a little jaded by the “race to the bottom” (of\nsymbiont genome size) that has transpired over the past decade which has yielded many\npublications in high profile journals that tend to have little content beyond reporting an\nincrementally smaller genome size. However, this work encompasses a very thorough survey\nand answers several important outstanding questions in the field, rationalizing concepts\ninvolving symbiont replacement, compartmentalization of functions and idiosyncrasies in\nmutation patterns and rates of degeneration. I therefore think that it merits publication in a high\nimpact journal and will be of interest to a broad readership. To that end, I’ll state again that it\nis written in such a way that it is very clear and broadly accessible. The authors are also\nappropriate cautious regarding their interpretations and therefore I believe that the results and\nconclusions are well supported by the observations and data analyses presented in this paper.\nThe figures and tables fit well with the flow and content of the manuscript and are easy to\ninterpret for a broad audience. The methodology is robust and well documented.\n>>> Our response: We sincerely thank the reviewer for this generous and encouraging\nassessment of our work. From the outset, our goal was to reconstruct the broader patterns and\nevolutionary drivers underlying the extreme genome reduction observed in planthopper\nsymbionts through comparisons among independent instances of reduction, rather than simply\nreport very small genomes. We are glad that our approach and presentation have conveyed this\nperspective clearly. At the same time, we agree that the discovery of independently evolved\ngenomes as small as ~50–52 kb adds additional interest and relevance to the study.\nI will state that I was really struck by the very limited inventories of genes encoding DNA\nreplication components that are maintained in these small genomes, particularly in light of the\nfact that they are proposed to replicate circular genomes. It appears to me that many\ncomponents of the replisome that are specifically involved in “handling” circular chromosome\nreplication (particularly dealing with initiation, termination and dimer resolution) are absent,\nimplying that there is a non-canonical method of replication in play. Free-living bacteria that\nlack such genes tend to replicate by cSDR, but these organisms also don’t seem to be capable\nof initiating cSDR. I would urge the authors to look into this in detail and aim to make some\npredictions relating to the mode of replication (or at least to outline what is lacking). There are\nmany papers explaining how circular prokaryotic replication systems work and it should be\npossible to propose a model for operation. This is a very important and interesting\nquestion/opportunity, particularly in the light of current work engineering minimal synthetic\ncells. It could markedly increase the scope for citation of this work.\n>>> Our response: We thank the reviewer for this insightful comment. Our analyses indeed\nrevealed a striking reduction of replication-associated genes in Vidania. Of the >30 genes\ntypically involved in E. coli DNA replication, only eight were detected in any Vidania genome,\nand the smallest ~50 kb genomes retain only dnaE and dnaQ. Genes essential for canonical\nreplication initiation (dnaA, dnaC) and for alternative mechanisms such as cSDR or rolling-\ncircle replication (recA, priA, priB, dnaT, rep) are absent, suggesting that Vidania cannot\nsustain autonomous replication.\nBecause endosymbionts with highly reduced genomes are known to depend on host-derived\nproteins for essential functions, Vidania replication likely proceeds through a host-assisted\nprocess analogous to that of mitochondria. We have expanded the Discussion to reflect this\ninterpretation (lines 335-345). This section now reads:\n“For example, in Vidania genomes, of >30 genes involved in DNA replication in Escherichia\ncoli 44 only between eight and two remain. The loss of canonical initiator and helicase\ncomponents such as dnaA and dnaC, and key components of constitutive stable DNA\nreplication (cSDR)45 or rolling-circle replication (RCR)46,47, indicates that genome replication\nis likely initiated or regulated by host-encoded factors, while elongation proceeds through the\nfew polymerase subunits that Vidania retained (incl. dnaE, dnaQ). Similarly, the frequent loss\nof aminoacyl-tRNA synthetases, key components of the translation process, combined with the\nretention of tRNAs, suggests that they are likely provided by the host 48. This functional\nintegration between host and symbiont genetic information processing machineries would\nparallel the situation in mitochondria, where all core replication and translation proteins are\nencoded by the host and imported into the organelle 49.”\nSome structural suggestions:\nLine 35: change to “are much smaller”\nLine 54: change to “determine the processes”\nLines 89-90: synteny/syntenic (not synthetic)?\nLine 254: change to “those characterized previously”\n>>> Our response: Thank you, the suggested changes have been made.\nOne query:\nLine 259: What evidence supports the notion that changes are detrimental but not lethal? I think\nthat’s an assumption derived from theory rather than empirical evidence and it should be\nexplained as such. Perhaps also mention that these associations have endured for very long\nperiods of time, suggesting that host-level selection is sufficiently potent to ensure that they are\nnot replaced, which is clearly possible, but (IMHO) more likely catalyzed by a changing\nfunctional mandate rather than mutational meltdown, as supported by the comparative\nfunctional analyses documented in this paper.\n>>> Our response: We agree that the assumption that many accumulated changes are\n“detrimental but not lethal” derives from theoretical predictions rather than direct experimental\nevidence. We have now revised this section to clarify this point and to emphasize that long-\nterm persistence of these symbioses likely reflects the stabilizing influence of host-level\nselection, which can mitigate the consequences of mutational accumulation. The revised and\nexpanded section (lines 311-318) now reads:\n“These processes lead to the gradual loss of non-essential genes, and—according to population-\ngenetic theory, including Muller’s ratchet—the fixation of mildly deleterious but non-lethal\nchanges. At the same time, the very long-term persistence of these associations suggests that\nhost-level selection is sufficiently strong to prevent the symbionts’ complete mutational\nmeltdown and halt the gene loss, including the retention of the symbionts’ critical cellular\nfunctions and the biosynthetic capacity essential to the host. When further gene losses do occur,\nthey are more likely to reflect changes in the host biology or ecological context than purely\nstochastic accumulation of deleterious mutations. “\nReviewer #3 (Remarks to the Author):\nGeneral comment\nWe found this work to be a valuable contribution to the field of insect-microbe symbiosis and\nbacterial genome evolution. The authors have compiled a series of comprehensive analyses on\nsymbiont genomics across a vast number of planthopper species (149 species representing 19\nfamilies!). The discovery of new ultra-small 50–52 kb Vidania symbiont genomes is striking,\nwhich revised our predisposition on the reductive symbiont genome evolution. The data\npresented are robust and the methods appear sound. Overall, the manuscript is well-written and\nprovides a framework for driving future studies. On the other hand, there appear to be a few\nareas where the paper’s messages could be strengthened. We provide several comments for\nmaking the manuscript more appealing, although those amendments are not mandatory.\n>>> Our response: We thank the Reviewer for the positive and encouraging assessment of\nour work. We appreciate the recognition of the study’s scope, data robustness, and significance\nfor understanding symbiont genome evolution. We have carefully considered all of the\nReviewer’s suggestions and implemented clarifications and additions throughout the revised\nmanuscript to further strengthen the presentation and accessibility of our results.\nMajor comments\n1. A critical point is that the central question raised in this paper seems somewhat vague, which\nis a common challenge with this type of exploratory genomic approach. As is common in\ngenomic studies, the initial hypothesis can sometimes become less defined as a result of a data-\ndriven approach. While this is natural, we suppose that the authors could enhance the paper’s\nflow by providing hypothesis uniquely testable in this taxon, or add a clear rationale for their\nchosen model system. For instance, there is no explicit explanation in the Introduction as to\nwhy the authors selected the infraorder Fulgoromorpha to provide a comprehensive view (Line\n46-48). A convincing explanation for why planthoppers are uniquely suited for this study\nwould strengthen the paper’s main argument.\n>>> Our response: Thank you for this valuable comment. We agree that clarifying the\nrationale for focusing on the infraorder Fulgoromorpha strengthens the framing of our study.\nWe have now comprehensively revised and expanded the second part of the Introduction to\nemphasize that planthoppers represent a uniquely powerful model for studying the limits of\nsymbiont genome reduction and replacement (lines 47-69)\n2. A potential weakness of this study is that it might be perceived as a finding specific only to\nplanthoppers, thereby limiting its broader appeal. A possible way to address this is to provide\na strong rationale for why a comprehensive analysis of this specific group was needed (this is\nrelated to Major comment 1). While the authors’ discussion already hints at this (Lines 246-\n249, 269-272, 300-310), we suggest to clearly state why planthoppers were the ideal model for\nthis research in Introduction. This will help readers understand that the findings are not just\nabout a single insect taxon, but provide valuable insights into the fundamental processes of\ngenomic evolution.\n>>> Our response: Thank you for this thoughtful suggestion. As noted above, we have\nsubstantially expanded the second part of the Introduction. In addition to clarifying why\nplanthoppers provide an exceptional system for studying the limits of symbiont genome\nreduction, we now emphasize that insights from this group extend beyond a single insect\nlineage and inform general principles of genome evolution and host–microbe integration.\n3. It is interesting but elusive how the extremely tiny-genome bacterium like Vidania can\nfunction as a living cellular entity. We would like to see authors’\nhypothesis/opinion/perspective on the topic in Discussion in a more explicit manner.\n>>> Our response: We appreciate this thoughtful comment. We agree that understanding how\nsuch extremely reduced symbionts remain functional is a key question. We have clarified this\npoint in the revised Discussion, emphasizing that Vidania likely relies extensively on host-\nderived factors for essential cellular functions, including DNA replication (lines 335-345), and\nthat this integration parallels the host control observed in mitochondria. We believe that this\naddition now explicitly conveys our perspective on how Vidania can persist as a living yet\nhighly host-dependent cellular entity.\nMinor Comments\nLines 75-82 (Fig. 1): Please provide more detailed caption for Fig. 1. In Fig. 1A, the color\ncoding for the circles needs to be explained either within the figure or in the caption. For\ninstance, we can assume yellow represents a third obligate symbiont, gray a facultative-like\nsymbiont, and the golden-brown a yeast-like symbiont, but this is not explicitly stated. Additionaly,\nthe caption for the confocal microscopy image (Fig. 1C) should specify what the blue color represents.\n>>> Our response: Thank you for your comment. The colored circles in the figure have been\nlabeled as Sulcia, Vidania, Gammaproteobacteria, Alphaproteobacteria, and Hypocreales.\nHowever, we agree that an additional note in the legend improves clarity, and we have therefore\nadded a concise explanation of the color coding. We have also specified in the caption that the\nblue color in panel B represents DAPI staining (lines 98-108).\nLines 104-105 (Fig. 2A): To ensure consistency, please unify the terminology between the\nfigure and its caption. The caption refers to “the very smallest insect symbionts (pink)” and\n“the smallest bacteria (blue),” but the figure itself uses “insect symbiotic bacteria” and “other\nbacteria.”\n>>> Our response: Thank you for pointing this out. We have revised the terminology in the\ncaption to ensure consistency. The figure and the caption now use the same terms:\n“Fulgoromorpha symbionts”, “insect-symbiotic bacteria”, “other bacteria”. The legend now\nreads:\n“In terms of genome size and gene content, Fulgoromorpha symbiont genomes (red) fall\namong the very smallest known for insect-symbiotic bacteria (pink), which themselves are\namong the smallest bacterial genomes overall (other, non–insect-symbiotic bacteria shown in\nblue). Cellular organelles—mitochondria (orange) and chloroplasts (green)—are shown for\nreference.” (lines 141-144)\nLine 124: The authors state the they found no cases of gene gain in any lineage. Please clarify\nthe data that supports this statement. Does this mean that no lineage-specific orphan genes were\nfound?\n>>> Our response: We thank the reviewer for this valuable comment. By stating that we found\nno cases of gene gain, we meant that no lineage-specific genes of apparent external origin were\ndetected—that is, no evidence of horizontal gene transfer based on sequence similarity, GC\ncontent, or phylogenetic signal.\nIn this context, we would consider “orphan genes” as lineage-specific open reading frames\n(ORFs) lacking detectable homologs in any other known organism and not traceable to\nancestral genes within the same symbiont lineage. To evaluate this, we used a custom\nannotation pipeline combining hmmer searches with manually curated reference alignments to\ndetect highly diverged homologs and performed phmmer searches of all unannotated ORFs\n>50 amino acids in any of the genomes against the UniProt database. We included these ORFs\nas references in subsequent rounds of annotation, allowing us to understand their level of\nconservation.\nAcross all genomes, we identified 40 Sulcia and 86 Vidania ORFs with no significant similarity\nto known proteins - as explained in Results (lines 161-164). These ORFs are typically short,\nAT-rich, and often conserved among related species, often occupying syntenic positions\ncorresponding to degraded ancestral loci. These characteristics suggest that they represent\nancestral genes, or their remnants, rather than newly evolved, lineage-specific orphan genes.\nLines 169-170: To avoid misinterpretation, please use a more precise expression than a range.\nFor the two tiniest genomes, VFSACSP1 and VFMALBOS, it would be clearer to state the\nnumber of ribosomal proteins as “35 or 38, respectively.” The same applies to the “3-4\nribosomal DNA and RNA polymerase subunits.”\n>>> Our response: Thank you, the change has been made (lines 212-216)\nLines 186-192: The imaging results for the tiniest Vidania are highly interesting, but the\nderived observations are open to interpretation. Please clarify the number of samples on which\nthe statements regarding mitochondrial density and vacuolization are based. Additionally,\nproviding context on the host insect’s condition (e.g., larva vs. adult) would be valuable.\nIdeally, a comparison with a closely related insect hosting a non-tiniest Vidania would make\nthese findings more conclusive.\n>>> Our response: We thank the reviewer for this helpful comment. Using TEM, we analyzed\nten adult individuals of Malenia bosnica and consistently observed a high mitochondrial\ndensity in the cytoplasm of Vidania bacteriocytes, together with signs of cell degeneration,\nincluding vacuolization of the bacteriocyte cytoplasm. Similar features were observed in\nVidania bacteriocytes of other adult species lacking Sulcia and harboring only Vidania with\nreduced gene sets, including other derbids and Cixidia pilatoi (Achilidae).\nTo provide additional context and comparison, we have added Supplementary Fig. 7, which\nshows Vidania bacteriocytes from other planthoppers with highly reduced genomes alongside\nthose from species hosting Vidania with more complete genomes. The legend of Figure 4 has\nbeen updated to highlight that M. bosnica represents more general patterns:\n“The morphology and ultrastructure of bacteriome tissue inhabited by Vidania with the\nsmallest genomes differ from previously studied planthoppers by particularly high\nmitochondrial density—as exemplified by Malenia bosnica bacteriome and its tiny-genome\nVidania VFMALBOS using fluorescence (C1), light (C2), and transmission electron\nmicroscopy (C3)” (lines 245-249). We also revised the Results to note that: “These features\nwere also observed in the Vidania bacteriomes of other adult planthoppers where this symbiont\nhas highly reduced genome and Sulcia is absent, including the derbids Proutista sp. and\nPanmendanga sp., as well as the achilid Cixidia pilatoi (Supplementary Fig. 7)”. (lines 234-\n236)\nLine 188, 190: Fig. 4C?\n>>> Our response: Thank you for catching this. Of course, we meant to refer to figure 4C\nrather than 5C, and have corrected this.\nLine 203: mitochondrium -> mitochondrion\nLine 215: Please remove the comma after “Pseudomonas.”\nLine 355-356: Please state the specific software used for read mapping. This detail is essential\nfor the reproducibility of your analysis.\nLine 386: Please add details for the “Processing” and “Inkscape” software, as their specific\nversions should be specified for reproducibility.\n>>> Our response: Thank you for pointing these out. We have incorporated all of your\nsuggestions into the text.\nComments to SI\nLines SI 197-201: We acknowledge the difficulty of accurately annotating highly divergent\ngenes. However, we are not fully convinced by the conclusion that highly truncated genes are\nfunctional simply based on their conservation across lineages. Please provide a clearer rationale\nor discuss other evidence that supports their functionality.\n>>> Our response: We thank the reviewer for this helpful comment. As explained above, we\nhave expanded the Supplementary Materials (Supplementary Text, lines 228-242) to clarify\nthe rationale for considering some of the truncated genes as likely functional. Using rplI gene\nas an example, we explain how the rplI2 gene variant encodes a shortened form of the\nribosomal protein RplI that retains the essential N-terminal RNA-binding domain, responsible\nfor ribosome association and stability. The conservation of this truncated variant across nine\nplanthopper families supports its functional relevance rather than random sequence retention.\nThis clarification has also been noted in the main text (lines 452-458).\nLines SI 192, 208, 26, 246: We noticed distracting red lines under scientific names in the\nfigures. Please remove them, as they appear to be from an active spell-check.\n>>> Our response: Thank you for pointing these out. We have removed all those red lines.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\n>>> Our response: We thank Reviewer #4 for contributing to the review process and\nappreciate their participation in the Nature Communications Early Career Reviewer initiative."}, {"round": 2, "reviewer_comments": {"reviewer_1": "This is the second time I've reviewed this work. The authors have responded satisfactorily to all my suggestions and comments on the first version, so I have nothing further to add. Congratulations on an excellent job.", "reviewer_2": "From my perspective, the authors have successfully addressed the questions and concerns raised by reviewers, where possible, and the manuscript is greatly improved. I support the publication of this paper in Nature Communication and believes that it will be of broad value to the symbiosis community and beyond.", "reviewer_3": "We have read the revised version of the manuscript and the authors' responses to the comments. In conclusion, the authors have properly addressed our previous comments. We are satisfied with the revision and have no further major concerns regarding the work. The manuscript is now considered to have reached a standard suitable for publication in this journal. Specifically, the Introduction section has been substantially revised and significantly improved, clarifying the context and rationale of the study as we suggested. We also appreciate the addition of the TEM data. The inclusion of nice comparative controls is valuable for robust interpretation, even though these may not be the primary focus of the study. Furthermore, all minor points, including corrections to figure captions, have been thoroughly addressed, and I think that the additional discussion is appropriate and strengthens the manuscript's conclusions.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": ""}]

Decision letter