Genomic and metabolic divergence of the primary symbiont Candidatus Portiera between Bemisia tabaci and other whitefly species

Abstract

Background

The primary symbiont Candidatus Portiera is essential for nutrient provisioning in whiteflies. Genomic instability is a hallmark of Bemisia tabaci-associated Portiera, but the specific molecular evolutions and their metabolic consequences compared to Portiera from other whiteflies remain unclear.

Results

To overcome the limited sampling of previous studies, we assembled novel Portiera genomes from seven additional B. tabaci cryptic species. Comparative genomic, phylogenetic, and species delimitation analyses were conducted with other publicly available Portiera genomes. Branch-model selection analysis identified differentially evolved genes in the B. tabaci-associated Portiera, which were significantly enriched in amino acid biosynthetic pathways. The composition of essential amino acids biosynthetic pathways was systematically analyzed across Portiera, host nuclear, and secondary symbiont genomes. B. tabaci-associated Portiera formed a monophyletic lineage with larger genomes, lower coding density, and accelerated evolutionary rates, classified as a single species distinct from Portiera in other whiteflies. Twenty-two genes showed significantly different evolutionary rates with enrichment in amino acid biosynthetic pathways. Key genes for lysine and arginine biosynthesis were lost or pseudogenized in B. tabaci-associated Portiera but remained intact in other whiteflies like Trialeurodes vaporariorum. The synthesis of most other essential amino acids was similarly incomplete across all Portiera, relying on host or secondary symbiont genes for pathway completion.

Conclusions

The B. tabaci-associated Portiera represents a unique symbiotic metabolic architecture where host horizontally transferred genes may potentially compensate for Portiera’s genomic erosion, contrasting with the more autonomous Portiera in other whiteflies. This study reveals divergent evolutionary trajectories and metabolic integration strategies in whitefly symbiotic systems.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12864-026-13143-6.

Background

Whiteflies (Hemiptera: Aleyrodidae) are a globally distributed group of plant phloem-sap-feeding insects, many of which are significant agricultural pests due to virus transmission and direct feeding damage. Currently, the family comprises 1,556 extant species in 161 genera, classified mainly into two subfamilies, Aleyrodinae and Aleurodicinae, with a third, smaller subfamily Udamoselinae remaining contentious [1]. The sweetpotato whitefly Bemisia tabaci from Aleyrodinae represents a cryptic species complex of more than 44 putative species, and recent integrative approaches have established a robust phylogenetic framework of this complex [2, 3].

Sap-feeding insects feed on a nutritionally unbalanced diet deficient in essential amino acids (EAAs) and certain vitamins. To compensate for these dietary limitations, these insects harbor bacterial symbionts that synthesize and supply critical nutrients to the host [4, 5]. For instance, in aphids, Buchnera aphidicola supplies EAAs, enabling hosts to exploit nitrogen-poor plant sap [6, 7]. Comparative genomic studies of Buchnera from different aphid lineages reveal both conserved and divergent metabolic capabilities, underscoring the role of symbiont genome evolution in host adaptation [8]. Notably, in the aphid–Buchnera symbiosis, metabolic pathways for EAA biosynthesis are shared between host and symbiont, with host enzymes compensating for missing bacterial reactions [9]. Dual symbionts like Candidatus Sulcia muelleri and Ca. Baumannia cicadellinicola in the glassy-winged sharpshooter collaboratively synthesize the full complement of EAAs required by the host [10]. The primary symbiont of B. tabaci, Ca. Portiera aleyrodidarum, also produces a substantial proportion of EAAs required for host growth [11]. B. tabaci also harbors various secondary symbionts [12]. Although some secondary symbionts retain genes for EAA biosynthesis in their genomes [13], they may not necessarily provide nutritional benefits to the host [11]. Furthermore, host horizontally acquired genes can complement missing pathways in the primary symbiont Portiera to synthesize certain EAAs, such as lysine [14].

The primary symbiont Ca. Portiera aleyrodidarum, initially described as a single species in whiteflies [15], is now recognized to comprise distinct genomic lineages. Early comparisons revealed that Portiera from B. tabaci exhibits genome instability, including rearrangements, repeat proliferation, and loss of DNA repair genes such as dnaQ, unlike the stable genomes in Trialeurodes vaporariorum [16]. Subsequent sequencing of Portiera from Aleurodicinae (Aleurodicus dispersus, A. floccissimus) confirmed long-term structural stasis across whitefly subfamilies, except in the B. tabaci lineage [17]. Phylogenetic screening showed that genome instability and dnaQ loss are shared across the monophyletic Aleurolobini tribe including Bemisia, Aleurolobus, and Singhiella, dating back over 70 million years [18]. Comparative genomics of nine Portiera genomes identified three genetically distinct groups, one in Aleurodicinae and two in Aleyrodinae, rather than a single species [19].

In symbiotic systems, host-associated bacterial lineages often exhibit signatures of differential gene evolution as they adapt to specific host environments. For instance, comparative transcriptomic studies of deep-sea chemosymbiotic organisms have revealed that genes involved in nutrient transport, lysosomal activity, and immune modulation are frequently under positive selection or show differential expression, reflecting adaptive evolution to the symbiotic lifestyle [20, 21]. Similarly, pan-genome analyses of gut symbionts such as Bacteroides cellulosilyticus have shown that mixed symbioses foster genomic plasticity, with differentially evolved genes often enriched in metabolic and transport functions [22]. In the whitefly system, however, the identification and functional characterization of genes that have undergone differential evolution specifically in B. tabaci-associated Portiera remain largely unexplored.

Previous genomic comparisons of Portiera have been based on only four B. tabaci cryptic species, leaving the representativeness of reported traits unclear. Here, we assembled Portiera genomes from seven additional cryptic species, enabling a systematic test of whether the distinct genomic and metabolic features of B. tabaci-associated Portiera are consistently present across this species complex. Using this expanded dataset, we delineate the genomic diversity and species boundaries of Portiera, identify genes with differential evolutionary rates in the B. tabaci lineage, and systematically assess the completeness and complementation of essential amino acid biosynthetic pathways. Our findings reveal key genomic and metabolic divergences that underpin the co-evolutionary relationship between B. tabaci and its primary symbiont, providing new insights into the mechanisms of nutritional symbiosis in sap-feeding insects.

Methods

Sequencing data

To maximize the acquisition of Portiera genomes from B. tabaci, we downloaded nearly all available next-generation and third-generation sequencing data related to B. tabaci genomic DNA from National Center for Biotechnology Information (NCBI) by 1 March 2025. The sequencing data statistics are summarized in Table S1. Reads originating from Portiera were then extracted from the next-generation sequencing data using bowtie 2 v2.5.4 [23] with Portiera from B. tabaci MEAM1 (NCBI accession number CP003708) as the reference genome. For third-generation sequencing data, minimap2 v2.28 [24] were employed for read extraction. The obtained reads were subsequently filtered with fastp v0.20.0 [25] using default parameters. Based on the reference genome size, datasets achieving a minimum sequencing depth of 100 × for the Portiera genome were selected (Table S2). To delineate cryptic species within the B. tabaci complex, the mitochondrial genomes were assembled from the selected sequencing datasets using NOVOPlasty v4.3.3 [26] using default parameters. Subsequently, the following datasets were utilized to assemble novel Portiera genomes, with their respective cryptic species designations confirmed by the mitochondrial assemblies: SRR8142474 (New World 1), SRR11692175 (AsiaI), SRR11613040 (AsiaII1), SRR11609252 (AsiaII6), SRR11609259 (AsiaII7), SRR8142475 (SSA1-SG1), and ERR4153733 (SSA3).

Genome assembly and comparative genomic analysis

The Portiera genomes were assembled into circular sequences using Unicycler v0.4.8 [27] with default parameters. Then, initial annotation was performed with Bakta v1.11.3 [28]. Each sequence was then re-oriented to initiate from the dnaK gene, followed by a final round of annotation to update genomic coordinates. The final genomes were submitted to NCBI. The newly sequenced and previously published Portiera genomes downloaded from NCBI and the National Genomics Data Center, China National Center for Bioinformation (CNCB-NGDC) [29] were assessed for completeness using Benchmarking Universal Single-Copy Orthologs (BUSCO) v5.5.0 [30] against the bacteria_odb10 dataset in “genome” mode. Further, the orthologues were identified based on the amino acid sequences from all examined Portiera genomes using OrthoFinder v2.5.5 [31].

Phylogenetic analysis and species delimitation

To clarify the phylogenetic relationships of Portiera spp., we constructed a phylogenetic tree based on their identified orthologues. The nucleotide sequences of each single-copy orthologue group were aligned using MAFFT v7.471 [32]. The resulting multiple sequence alignments were then trimmed to remove poorly aligned regions using trimAl v1.5.0 [33] with the “automated1” option. The refined alignments were concatenated, and a partition file was generated using R package phylotools v0.2.2 [34]. The final concatenated supermatrix was used to reconstruct a maximum-likelihood (ML) phylogenetic tree with IQ-TREE v3.0.1 [35], employing automatic model selection and branch support assessed with 1,000 ultrafast bootstrap replicates [36]. Portiera genomes from the subfamily Aleurodicinae, specifically from A. dispersus and A. floccissimus, were designated as the outgroup. All other Portiera genomes, which are derived from the subfamily Aleyrodinae, constituted the ingroup.

Genomic synteny analysis was performed using JCVI v1.5.7 [37]. The annotation files in GFF format were first converted into BED format using jcvi.formats.gff module with “bed” action. Redundant entries were then removed with “uniq” action of jcvi.formats.bed module. Orthologous relationships between pairwise Portiera genomes were identified via “orthology” action in jcvi.compara.catalog module. Subsequently, synteny blocks were detected and filtered using “screen” action from jcvi.compara.synteny module with the parameters “–minspan = 5 –simple” to generate anchor files. Finally, synteny plots were generated using jcvi.graphics.karyotype module to visualize the genomic alignments.

To delineate species boundaries among these Portiera spp., the Average Nucleotide Identity (ANI) and digital DNA-DNA hybridization (dDDH) values among their genomes were calculated. The ANI values between all pairwise combinations of the Portiera genomes were calculated using fastANI v1.31 [38] with a fragment length of 1,000 bp. Genomic pairs with ANI values ≥ 95% were considered to belong to the same species. The dDDH values were computed using Genome-to-Genome Distance Calculator (GGDC) web server v3.0 [39].The analysis was run using the “BLAST + ” alignment method. As per the recommended threshold, a dDDH value ≥ 70% was used as the criterion for assigning genomes to the same species [40].

Identification of differentially evolved genes across B. tabaci-associated Portiera

To identify genes that have undergone differential evolution in B. tabaci-associated Portiera, we conducted branch-model analyses using CODEML program in PAML package v4.9 [41]. First, codon alignments were generated from the amino acid sequences of single-copy orthologues, which had been aligned using MAFFT. A fixed species tree topology, reconstructed previously from concatenated orthologues, was used as the input phylogeny. For each gene, branch lengths were re-optimized on the codon alignment in IQ-TREE under the best-fit substitution model selected by the “-m MFP” option, with the “-keep-ident” flag to retain identical sites in the alignment. In the branch-model test, the B. tabaci-associated Portiera were specified as the foreground branch, while all other Portiera lineages served as the background. Two models were compared: a one-ratio null model (model = 0, NSsites = 0), which assumes a uniform ω (dN/dS) ratio across all branches, and a two-ratio alternative model (model = 2, NSsites = 0), which allows a distinct ω ratio for the foreground branch. Likelihood ratio tests (LRTs) were performed for each gene, and the resulting p-values were adjusted for multiple testing using the Linear Discrete Rejection (LDR) correction method. Genes with an adjusted p-value < 0.05 were considered to be differentially evolved in the B. tabaci-associated Portiera. The gene symbols were submitted to the KOBAS-i web server [42] for KEGG pathway enrichment analysis using “Candidatus Portiera aleyrodidarum BT-B” as the reference genome.

Identification of genes involved in essential amino acid biosynthetic pathways

Given that differentially evolved genes are enriched in essential amino acid biosynthetic pathways, we focused on analyzing all genes related to these pathways in Portiera. Initially, we identified complete genes, pseudogenes, and lost genes within these pathways from the examined genomes. The analysis revealed that most pathways are incomplete, leading us to hypothesize that these pathways may be complemented by genes from the host or secondary symbionts. Therefore, we further identified genes associated with these pathways from the genomes of the whitefly symbiotic systems. The genomes of B. tabaci and T. vaporariorum, along with their secondary symbionts (Arsenophonus, Cardinium, Hamiltonella, Rickettsia, and Wolbachia), were downloaded from NCBI (Table S3). Using protein sequences from Portiera spp. and Escherichia coli (UniProt Taxon ID 83333) as queries, tblastn [43] was employed to identify homologous genes from these genomes, with a similarity threshold at > 30%. For the candidate genes obtained, multiple sequence alignment was performed to verify their integrity. Genes with high similarity in the NCBI database were selected, aligned using MAFFT, and subjected to phylogenetic tree construction with IQ-TREE to confirm their taxonomic classification. Additionally, manual verification was conducted by integrating the original genome annotation information.

Results

Genomic features of Portiera

We newly assembled the circular genomes of Portiera from seven cryptic species of B. tabaci (Fig. 1A). Divergence was observed in the Portiera genomes among B. tabaci cryptic species and other whitefly species. The genomes associated with B. tabaci were consistently larger, ranging from 352,228 to 359,359 bp, compared to those from other whiteflies which ranged from 277,700 to 290,376 bp. Correspondingly, the B. tabaci-associated genomes exhibited a lower coding density (66.99–68.18%) and a higher GC content (26.08–26.21%) with positive GC-skew. In contrast, the Portiera genomes from other whiteflies showed a higher coding density (90.72–92.78%), a lower GC content (23.76–24.69%), and a negative GC-skew (Table 1). Additionally, the Portiera symbionts of B. tabaci lacked the gene for tRNA-Ala with the anticodon GGC, which was present in the genomes of Portiera from other whitefly species. Based on whole-genome BUSCO assessment, all examined Portiera genomes exhibited notable gene content incompleteness, a common feature in many obligate symbionts. Portiera genomes from B. tabaci exhibited a lower proportion of complete single-copy genes and a higher proportion of both fragmented and missing genes than those from other whitefly species. Duplicated genes were not observed in any of the genomes (Fig. 1B). A core set of 223 orthologues was shared among all genomes, of which 210 were single-copy orthologues. Notably, the B. tabaci-associated Portiera had experienced significant gene loss, lacking 19 orthologues that were conserved in the symbionts of other whitefly species, while uniquely retaining five specific single-copy orthologues (Fig. 1C).

Fig. 1.

Signatures of Portiera genomes. A Genome circular map of Portiera from Bemisia tabaci New World 1. This panel presents one representative genome from the seven newly sequenced Portiera genomes of B. tabaci cryptic species. B BUSCO assessment of the newly sequenced and publicly available Portiera genomes. The newly sequenced genomes are highlighted in red. C Identification of orthologues across Portiera genomes. The total number of genes analyzed per genome is indicated at the Venn diagram. The numbers of shared orthologues among different genome sets are indicated at the UpSet plot

Table 1.

Genomic features of Portiera analyzed in this study

Portiera	Host	Genome size (bp)	CDS	Coding density (%)	GC content (%)	GC-skew	Accession number	Reference
P. BT AsiaI	Bemisia tabaci AsiaI	355,928	259	67.73	26.09	8.74	JBUYCK000000000	This study
P. BT AsiaII1	B. tabaci AsiaII1	353,769	254	67.76	26.12	9.03	JBUYCJ000000000	This study
P. BT AsiaII3	B. tabaci AsiaII3	354,523	259	67.94	26.10	9.09	CP016327	[44]
P. BT AsiaII6	B. tabaci AsiaII6	355,863	258	67.57	26.11	9.06	JBUYCI000000000	This study
P. BT AsiaII7	B. tabaci AsiaII7	355,459	259	67.78	26.08	9.02	JBUYCH000000000	This study
P. BT China1	B. tabaci China1	359,359	279	66.99	26.10	9.05	CP016343	[44]
P. BT MEAM1	B. tabaci MEAM1	358,242	259	67.18	26.17	8.55	CP003708	[5]
P. BT MED	B. tabaci MED	357,472	258	67.23	26.12	8.77	CP003835	[4]
P. BT New World 1	B. tabaci New World 1	352,228	258	68.07	26.21	8.23	JBUYCG000000000	This study
P. BT SSA1-SG1	B. tabaci SSA1-SG1	353,523	268	68.18	26.11	8.56	JBUYCF000000000	This study
P. BT SSA3	B. tabaci SSA3	353,900	262	67.19	26.12	8.77	JBUYCE000000000	This study
P. AdSh	Aleyrodes shizuokensis	283,014	268	91.68	23.76	− 15.49	GWHBOVO00000000a	[19]
P. PeMo	Pealius mori	277,700	266	92.30	24.10	− 14.66	LR744089	[18]
P. TrVa	Trialeurodes vaporariorum	280,663	264	92.78	24.69	− 14.25	CP004358	[16]
P. AlDi	Aleurodicus dispersus	290,195	270	91.59	23.94	− 12.18	LN649255	[17]
P. AlFl	Aleurodicus floccissimus	290,376	269	90.72	23.77	− 12.19	LN734649	[17]

^aThis genome is validated at the National Genomics Data Center, China National Center for Bioinformation (CNCB-NGDC, https://ngdc.cncb.ac.cn)

Phylogeny and species delimitation

In the phylogenetic tree rooted with P. AlDi and P. AlFl as outgroups, P. AdSh, P. PeMo, and P. TrVa were placed in basal positions, indicating an earlier divergence relative to B. tabaci-associated Portiera. B. tabaci-associated Portiera formed distinct clades characterized by remarkably long branch lengths implying accelerated evolutionary rates (Fig. 2A). The gene order within Portiera genomes was highly conserved across all cryptic species of B. tabaci. Similarly, a consistent gene arrangement was observed in Portiera from other whitefly species, including P. AlDi, P. AlFl, P. AdSh, P. PeMo, and P. TrVa. However, substantial differences were detected between the B. tabaci-associated Portiera and the Portiera from other whitefly hosts (Fig. 2B). This structural divergence suggests that extensive genomic rearrangement occurred in the ancestral Portiera following its association with B. tabaci. The pairwise ANI values among all B. tabaci-associated Portiera exceeded 98%, above the 95% species delineation threshold. Similarly, their dDDH values were all greater than 74%, above the 70% threshold. These results suggest that all Portiera from B. tabaci belong to the same species, corresponding to the proposed type species Candidatus Portiera aleyrodidarum as described by Thao and Baumann [15]. In contrast, ANI and dDDH values between them and Portiera from other whiteflies were below 87% and 31%, respectively, confirming distinct species boundaries. Among the latter group, P. AdSh, P. PeMo, and P. TrVa showed high pairwise ANI values over 94.8%, suggesting they may be closely related at the species level. However, their dDDH values fell below the 70% species threshold, indicating they likely represent separate species despite some genomic similarity. P. AlDi and P. AlFl exhibited both high ANI (98.1%) and dDDH (80.4%) values, strongly supporting their classification as the same species (Fig. 2C–D).

Fig. 2.

Phylogeny and species delimitation of Portiera spp. A Maximum-likelihood phylogenetic tree constructed from single-copy orthologues of Portiera spp. P. AlFl and P. AlDi were designated as outgroups. Bootstrap values less than 100 are indicated at the nodes. B Genomic synteny analysis of Portiera. Each horizontal bar represents one individual lineage, arranged in the identical taxonomic order as displayed in panel A. Interspersed grey segments denote conserved syntenic genomic regions shared across these lineages. C Matrix diagram of average nucleotide identity (ANI) across Portiera. D Matrix diagram of digital DNA–DNA hybridization (dDDH) across Portiera. An ANI value below 95% and a dDDH value below 70% are widely used thresholds for delineating bacterial species

Differentially evolved genes across B. tabaci-associated Portiera

Using CODEML program in PAML package with B. tabaci-associated Portiera as the foreground, we identified 22 differentially evolved genes (Table 2). The ω values for all these genes were below 1, indicating the purifying selection. Among them, 18 genes showed significantly lower ω values in the foreground, suggesting intensified purifying selection pressure in the B. tabaci-associated Portiera lineage (Fig. 3A). Functional enrichment analysis revealed significant enrichment in the KEGG pathways Biosynthesis of amino acids, with all enriched genes involved in amino acid biosynthesis (Fig. 3B).

Table 2.

Differentially evolved Portiera genes in Bemisia tabaci relative to other whiteflies

Gene	Product	Background ω	Foreground ω	Adjusted p
pal	Peptidoglycan-associated lipoprotein	0.369	0.025	0.001
leuD	3-isopropylmalate dehydratase small subunit	0.294	0.057	0.004
dnaB	Replicative DNA helicase	0.044	0.127	0.004
der	Ribosome biogenesis GTPase Der	0.174	0.062	0.004
trpB	Tryptophan synthase subunit beta	0.082	0.034	0.004
lysC	Aspartokinase	0.147	0.095	0.004
rplY	50S ribosomal protein L25	0.273	0.055	0.004
trhC	Threonine synthase	0.110	0.093	0.006
metE	5-methyltetrahydropteroyltriglutamate–homocysteine methyltransferase	0.161	0.073	0.008
mnmE	tRNA modification GTPase MnmE	0.144	0.057	0.009
hisA	1-(5-phosphoribosyl)−5-[(5-phosphoribosylamino)methylideneamino] imidazole-4-carboxamide isomerase	0.155	0.159	0.012
thrB	Homoserine kinase	0.238	0.052	0.012
leuA	2-isopropylmalate synthase	0.166	0.077	0.012
carA	Carbamoyl phosphate synthase small subunit	0.136	0.055	0.022
rbfA	30S ribosome-binding factor RbfA	0.429	0.070	0.025
nuoA	NADH-quinone oxidoreductase subunit	0.127	0.010	0.029
yadG	ABC transporter ATP-binding protein YadG	0.069	0.193	0.029
trpG	Anthranilate synthase component 2	0.163	0.053	0.029
gltP	Proton/glutamate-aspartate symporter	0.069	0.030	0.029
leuS	Leucine–tRNA ligase	0.162	0.103	0.037
mutS	DNA mismatch repair protein MutS	0.083	0.143	0.039
smpB	SsrA-binding protein SmpB	0.151	0.040	0.047

Fig. 3.

Differentially evolved genes in Portiera of B. tabaci. A Branch-model analysis was performed with Portiera from B. tabaci cryptic species as the foreground and those from other whiteflies as the background. The scatter plot displays genes with a significant difference in evolutionary rate (adjusted p < 0.05). B KEGG pathway enrichment analysis of the differentially evolved genes with the reference genome of B. tabaci MEAM1 (NCBI accession number CP003708)

Essential amino acid biosynthetic pathways in whitefly symbiotic systems

Notable differences in the gene content associated with essential amino acid biosynthesis were observed between Portiera from B. tabaci cryptic species and others whitefly species. In all examined B. tabaci-associated Portiera, dapF and lysA were consistently absent, and argH and dapB were all pseudogenized, which remained present and functional in other whiteflies (Figure S1). This divergence leads us to consider the B. tabaci–Portiera symbiosis and the Portiera associations in other whiteflies as two distinct whitefly symbiotic systems. It should be noted that in both systems, most biosynthetic pathways for essential amino acids are incomplete. All Portiera lack most genes required for the synthesis of methionine and arginine, as well as the terminal step enzymes for the synthesis of isoleucine, valine, leucine, phenylalanine, and histidine. Additionally, B. tabaci-associated Portiera specifically lacks certain genes in the lysine and arginine biosynthetic pathways (Fig. 4). Given that the synthesis of EAAs may be complemented by the host or secondary symbionts [13, 14, 45], we further analyzed the genes involved in essential amino acid synthesis in their genomes.

Fig. 4.

Essential amino acid biosynthetic pathways in Portiera spp. A Lysine, Threonine, and Methionine synthesis. B Arginine synthesis. C Isoleucine, Valine, and Leucine synthesis. D Tryptophan and Phenylalanine synthesis. E Histidine synthesis. Gene names are color-coded: genes present in all Portiera are in black; genes missing specifically in B. tabaci-associated Portiera are in blue; genes absent in all examined Portiera are in red

In the lysine biosynthetic pathway, the two symbiotic systems exhibited distinct patterns. In the B. tabaci system, the absence of dapB, dapF, and lysA genes in its Portiera corresponds with the presence of horizontally transferred genes (HTGs) in the host genome, suggesting a possible compensatory relationship (Fig. 5A; Figures S2, S3), consistent with the findings of Bao et al. [14]. Conversely, in other whiteflies like T. vaporariorum, no host genes for lysine synthesis were detected, while their Portiera maintained an intact lysine biosynthetic pathway (Fig. 5B). Secondary symbionts such as Arsenophonus, Hamiltonella, Rickettsia, and Wolbachia encode complete or partial pathways, implying a capacity for independent or collaborative lysine synthesis, despite studies indicating that Hamiltonella does not notably influence Portiera’s lysine production [14].

Fig. 5.

Lysine biosynthetic genes in divergent whitefly symbiotic systems. A Symbiotic system of B. tabaci. Diagram showing the presence and absence of lysine biosynthetic genes across B. tabaci-associated primary and secondary symbionts, and the whitefly host B. tabaci MEAM1 nuclear genome. B Symbiotic system of other whiteflies. Diagram showing the presence and absence of lysine biosynthetic genes across their symbionts and the whitefly host Trialeurodes vaporariorum nuclear genome

In the methionine biosynthetic pathway, all examined Portiera lacked multiple genes, including metA, metB, and metC, retaining only metE. No corresponding genes were detected in the host genome, and only certain strains of secondary symbionts were found to harbor metC (Fig. 6A; Figures S4, S5). Interestingly, insects possess a methionine cycle in which the host enzyme Homocysteine S-methyltransferase (MmuM) catalyzes the conversion of homocysteine to methionine. The Portiera-encoded MetE also performs this function, potentially enhancing the efficiency of the methionine cycle. Additionally, homocysteine can be converted by the host enzyme Cystathionine beta-synthase (Cbs) into cystathionine in an irreversible reaction. The bacterial enzyme MetC from some secondary symbionts catalyzes the reverse reaction from cystathionine back to homocysteine, thereby allowing homocysteine to re-enter the cycle (Fig. 6B).

Fig. 6.

Methionine metabolism in whitefly symbiotic systems. A Genes involved in the methionine biosynthesis pathway across genomes of two whitefly species and their symbionts. B Hypothetical pathway for methionine recycling, involving enzymatic steps potentially contributed by symbionts to recover methionine within the host

Analysis of the threonine biosynthetic pathway revealed that a complete gene set was present in all examined Portiera, Arsenophonus, and Hamiltonella. Conversely, no homologs of these biosynthetic genes were detected in either the host genomes or in the genomes of Rickettsia and Wolbachia (Figure S6).

In the arginine biosynthetic pathway, although Portiera has lost a substantial number of genes including argA, argB, argC, argE, and argH, their functions can be complemented by corresponding genes present in the B. tabaci host genome, as previously reported by Luan et al. Horizontally transferred argG and argH genes were identified in the B. tabaci MEAM1 host genome, which is consistent with a potential compensatory role for the absence of argH in its Portiera (Figures S7–S10). In contrast, in other whiteflies such as T. vaporariorum, neither argG nor argH was detected in the host genome, while their Portiera retained intact argG and argH genes (Fig. 7). Secondary symbionts also lacked a complete arginine biosynthesis pathway and uniformly did not possess argG or argH.

Fig. 7.

Arginine biosynthetic genes in divergent whitefly symbiotic systems. A Symbiotic system of B. tabaci. Diagram showing the presence and absence of arginine biosynthetic genes across B. tabaci-associated primary and secondary symbionts, and the whitefly host B. tabaci MEAM1 nuclear genome. B Symbiotic system of other whiteflies. Diagram showing the presence and absence of arginine biosynthetic genes across their symbionts and the whitefly host T. vaporariorum nuclear genome

In the biosynthetic pathways for isoleucine, valine, and leucine, all examined Portiera lacked only the terminal enzyme Branched-chain-amino-acid aminotransferase encoded by ilvE. This reaction can be catalyzed by eukaryotic-origin branched-chain amino acid aminotransferases (BCATs) present in the genomes of both B. tabaci MEAM1 and T. vaporariorum (Figure S11). Among secondary symbionts, B. tabaci-associated Arsenophonus spp. had lost numerous genes in these pathways. In contrast, Arsenophonus spp. from Aleurodicus floccissimus and T. vaporariorum retained nearly complete pathways, including ilvE gene. Furthermore, two distantly related Rickettsia spp. from B. tabaci, despite possessing highly incomplete biosynthesis pathways, both retain the ilvE gene (Figures S12, S13).

All examined Portiera retained a complete tryptophan biosynthetic pathway but lacked the terminal enzyme Aromatic-amino-acid aminotransferase encoded by tyrB required for phenylalanine synthesis (Figure S14). In both B. tabaci MEAM1 and T. vaporariorum, the eukaryotic‑origin Aspartate aminotransferase encoded by GOT2 exhibited the same catalytic activity. Although the phenylalanine biosynthetic pathway in Hamiltonella from B. tabaci was incomplete, it harbored the tyrB gene missing in Portiera (Figures S15, S16). The T. vaporariorum-associated Arsenophonus also carried tyrB, whereas Arsenophonus from other hosts lacks this gene. Other secondary symbionts possessed almost no genes involved in the biosynthesis of these two amino acids.

All examined Portiera possessed all genes of the histidine biosynthetic pathway except for hisD, which encodes the terminal enzyme Histidinol dehydrogenase. Additionally, no genes related to this pathway were detected in either the host genomes or the genomes of secondary symbionts (Figure S17).

Discussion

Portiera genomic differentiation and species delimitation

Our comparative genomic analyses confirm that Portiera comprises distinct genomic lineages co‑diverging with their whitefly hosts. The 11 B. tabaci-associated Portiera genomes showed consistent genomic features and gene loss patterns across phylogenetically divergent cryptic species. This indicates that the distinct symbiont architecture is a stable, lineage-wide trait of the B. tabaci complex, extending earlier observations based on limited sampling [16–18]. The B. tabaci‑associated Portiera exhibits larger genomes, lower coding density, altered GC‑skew, and extensive structural rearrangements relative to those from other whitefly genera, consistent with earlier reports of genomic instability in this lineage [16–18]. This pattern of genomic degeneration and structural plasticity mirrors observations in Portiera from the closely related species B. afer [46], suggesting a shared evolutionary trajectory toward genomic instability within the Bemisia clade. Gene order is highly conserved within each major structural group, underscoring a deep evolutionary split. Phylogenetic and species‑delimitation metrics (ANI > 98%, dDDH > 74% within the B. tabaci clade) support the classification of all B. tabaci‑associated Portiera as a single species, corresponding to the type description, yet genetically isolated from other Portiera lineages. This aligns with the proposal that Portiera from the Aleurolobini tribe (including Bemisia) share a unique evolutionary trajectory marked by genomic instability [18]. It should be noted that whitefly divergence is not a simple bifurcation, and the genomic and metabolic features of Portiera may vary in a continuous or graded manner across host lineages. For instance, the recently sequenced Portiera from B. afer shows a mixed pattern of gene retention and loss [46]. Therefore, whitefly–Portiera associations may span a spectrum of symbiotic integration, ranging from relatively autonomous to highly host-dependent. The B. tabaci lineage represents an extreme point on this continuum, where host genetic compensation has become particularly pronounced in response to extensive symbiont genome erosion.

The results extend our previous work, which proposed three distinct genetic groups within Portiera: one in Aleurodicinae, one in non‑Bemisia Aleyrodinae, and one exclusive to Bemisia [19]. The divergence in genomic features and evolutionary rates among these groups suggests that long‑term co‑evolution within different host lineages has driven bacterial differentiation. Similar patterns of cryptic diversification are observed in other obligate endosymbionts, such as Buchnera aphidicola in aphids [47], highlighting the general importance of host lineage history in shaping symbiont evolution.

The complex patterns of partner differentiation seen in Portiera, where strict vertical transmission ensures co‑speciation yet results in genomic and functional diversification, challenge a single‑species classification and illustrate the dynamic nature of obligate endosymbiont evolution. This mirrors phenomena in other intimate symbiotic systems, such as the splitting of Ca. Hodgkinia cicadicola in cicadas [48] and the fine‑scale genomic divergence in Photobacterium symbionts of marine fish [49]. Thus, the B. tabaci‑Portiera association represents a distinct evolutionary trajectory characterized by genomic instability, metabolic reorganization, and intensified selection, providing a model for understanding how long‑term symbioses balance functional interdependence and genomic degradation.

Differentially evolved genes across B. tabaci-associated Portiera

The accelerated evolutionary rates and intensified purifying selection detected in B. tabaci‑associated Portiera genes, particularly those involved in EAA biosynthesis, likely reflect increased functional constraint resulting from tighter co‑evolutionary metabolic integration with the B. tabaci host. In obligate nutritional symbioses, stronger purifying selection on symbiont metabolic genes is often a signature of host‑symbiont interdependence, where destabilizing mutations are deleterious and therefore purged more efficiently. Thus, rather than indicating adaptive innovation, these patterns point to stabilizing selection that maintains the finely tuned metabolic complementation between host and symbiont [50].

In the B. tabaci system, genomic and metabolic divergence of Portiera is accompanied by complementary gene losses and the genomic recruitment of host-encoded genes, which may serve to complete EAA pathways (e.g., lysine and arginine biosynthesis). This metabolic repartitioning suggests a co‑evolutionary integration where symbiotic stability is achieved through genomic streamlining of the symbiont and functional compensation by the host. Similar patterns of functional differentiation and metabolic complementarity are observed in other obligate symbiotic systems, such as the divergent energy‑conservation strategies in Ca. Nanohaloarchaeota lineages that adapt to distinct environmental niches [51].

While we found evidence of purifying selection in B. tabaci-associated Portiera, this pattern does not indicate adaptive innovation. Instead, stronger purifying selection typically reflects increased functional constraint arising from tighter host–symbiont metabolic interdependence. In obligate nutritional symbioses, deleterious mutations in genes governing core metabolic pathways are more efficiently purged when the host depends critically on symbiont-derived nutrients. The genomic instability in B. tabaci-associated Portiera may also create a landscape for non‑adaptive changes, potentially setting the stage for future diversification, a process analogous to the neutral splitting of Ca. Hodgkinia cicadicola in cicadas, which arose through constructive neutral evolution rather than adaptive selection [48]. Furthermore, the concept of cascading speciation [52] provides a broader ecological context: host‑driven selection and co‑evolution can shape symbiont genomic and functional divergence across multiple symbiotic partners, as seen in tsetse fly‑Wigglesworthia systems [53]. The deep genomic and metabolic schism between B. tabaci‑associated and other Portiera lineages likely represents a similar cascade of differentiation, initiated by the evolutionary divergence of the B. tabaci host complex and reinforced by the unique metabolic challenges posed by this host lineage.

Essential amino acid biosynthetic pathways in whitefly symbiotic systems

Our comparative genomic analysis reveals that B. tabaci-associated Portiera has lost or pseudogenized key genes in lysine and arginine biosynthesis, pathways that remain intact in Portiera from other whiteflies such as T. vaporariorum. This divergence has led to distinct metabolic integration strategies. The B. tabaci system may compensate for these losses through HTGs [14], whereas T. vaporariorum relies on autonomous synthesis by Portiera. Similar host–symbiont metabolic complementarity has been documented in other symbiotic systems, such as in trypanosomatids where horizontally acquired bacterial genes complete EAA pathways [54], and in the brown planthopper Nilaparvata lugens, where yeast-like symbionts contribute EAA biosynthesis [55]. These findings highlight host genetic compensation as an evolutionary response to symbiont genome erosion in the B. tabaci complex.

While all Portiera lineages retain complete pathways for tryptophan and threonine, they universally lack most genes for methionine synthesis, a function potentially linked to a host-mediated methionine cycle [56]. Terminal steps for branched-chain amino acids and phenylalanine are complemented by conserved eukaryotic host enzymes such as BCATs and GOT2 [13, 57]. Transcriptomic studies further support the complementarity between whitefly hosts and Portiera in amino acid biosynthesis [58]. Importantly, in aphid–Buchnera symbiosis, the production of EAAs by the bacterial symbiont is regulated by host demand through the supply of metabolic precursors, rather than changes in symbiont gene expression [59]. Although secondary symbionts (e.g., Arsenophonus, Hamiltonella) retain genes like ilvE and tyrB, their nutritional role in vivo appears limited [11], and their contributions may be modulated by host genetic background, as observed in the expression of symbiont metabolic genes in aphids [60]. This indicates that the metabolic network is shaped by long-term functional negotiation among host, primary, and secondary symbionts, rather than stochastic gene loss.

The host genetic compensation observed in B. tabaci reflects intensified metabolic dependency driven by co-evolution with its unstable Portiera lineage. This pattern contrasts with other sap-feeding insects. Aphids regulate Buchnera’s amino acid output via host transporters like ApGLNT1 [61, 62] and exhibit genotype-dependent coordination of host and symbiont gene expression [60], while in younger symbioses (e.g., Burkholderia in scale insects), symbionts retain near-complete EAA pathways [63]. Even within the ancient symbiotic system, Evansia in moss bugs retains relatively complete amino acid synthetic pathways, highlighting the role of host ecology in shaping symbiont metabolism [64]. Our findings align with the view that “symbiont genome erosion–host compensation” represents a common trajectory in long-term nutritional symbioses [65], and posit that in B. tabaci, this process may be particularly pronounced due to the accelerated gene loss observed in its Portiera.

Implications in insecticide resistance

The gene loss in B. tabaci–associated Portiera and the resulting host metabolic compensation may indirectly influence insecticide resistance. In other insect–symbiont systems, gut bacteria directly degrade insecticides or upregulate host detoxification genes [66, 67], whereas some symbionts increase susceptibility by downregulating xenobiotic metabolism [68]. In B. tabaci, the loss of Portiera genes for lysine and arginine biosynthesis, compensated by horizontally acquired host genes [14], could alter nitrogen availability for detoxification enzymes. This metabolic constraint may limit the host’s capacity to develop rapid resistance unless secondary symbionts which express diverse detoxification-related genes provide alternative pathways [69]. Consequently, targeting metabolic weaknesses in symbionts could synergize with insecticides. Integrating symbiont genotyping into resistance monitoring may thus offer a novel strategy for sustainable pest management.

Conclusions

Our comparative genomics and phylogenetic analyses demonstrate that B. tabaci-associated Portiera represents a monophyletic lineage characterized by larger genome sizes, lower coding density, accelerated evolutionary rates, and extensive genomic rearrangements. These features collectively contrast with the more stable Portiera genomes found in other whitefly species such as T. vaporariorum, representing opposite ends of an evolutionary continuum. The distinct genomic trajectory of B. tabaci-associated Portiera, marked by significant gene loss and pseudogenization, particularly in amino acid biosynthetic pathways, reflects a deep co-evolutionary history with its host. These genomic changes have reshaped the metabolic architecture of the symbiosis. The architecture of EAA biosynthetic pathways in whitefly symbiotic systems represents an evolutionary continuum from symbiont autonomy to host-dependent supplementation. The B. tabaci system exemplifies a highly derived state in which horizontally acquired host genes represent a candidate mechanism for potential metabolic complementation, reflecting deep metabolic integration and plasticity. It should be noted that beyond the whitefly species examined in this study, other whitefly lineages may harbor Portiera with intermediate genomic and metabolic states that are not captured by a simple binary framework. Future sampling of additional whitefly species will be required to fully resolve this continuum. These insights clarify how nutritional symbioses evolve under genomic instability and provide a framework for understanding the evolutionary diversity of host–symbiont metabolic networks in sap-feeding insects.

Abbreviations

ANI

Average Nucleotide Identity

BCAT

Branched-Chain Amino Acid Aminotransferase

BUSCO

Benchmarking Universal Single-Copy Orthologs

Cbs

Cystathionine beta-synthase

CNCB-NGDC

National Genomics Data Center, China National Center for Bioinformation

dDDH

Digital DNA-DNA Hybridization

EAA

Essential Amino Acid

GGDC

Genome-to-Genome Distance Calculator

LDR

Linear Discrete Rejection

MEAM1

Middle East-Asia Minor 1

ML

Maximum-Likelihood

MumM

Homocysteine S-methyltransferase

NCBI

National Center for Biotechnology Information

Authors’ contributions

YQL and XQ designed the research. TL, YJF, and CS extracted raw data. TL, YJF, HDS, XJZ and HLW analyzed the data. TL drafted the manuscript. All authors have read and approved the final manuscript.

Funding

This study was funded by the Zhejiang Provincial Natural Science Foundation of China (No. LMS26C040001), the Science & Technology Project of Taizhou (Nos. 25nya21 and 25sfa05), and the Earmarked Fund for China Agriculture Research System (No. CARS-23-C05).

Data availability

The datasets generated and analysed during the current study are available in the NCBI repository (https://www.ncbi.nlm.nih.gov) under accession numbers JBUYCE000000000, JBUYCF000000000, JBUYCG000000000, JBUYCH000000000, JBUYCI000000000, JBUYCJ000000000, and JBUYCK000000000.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.