Tracking the biogeography of the Asian Citrus Psyllid Diaphorina citri spread in China using mitogenomes and endosymbionts

Tracking the biogeography of the Asian Citrus Psyllid Diaphorina citri spread in China using mitogenomes and endosymbionts | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Tracking the biogeography of the Asian Citrus Psyllid Diaphorina citri spread in China using mitogenomes and endosymbionts Fengnian Wu, Zehan Dai, Min Shi, Jianjian Huang, Hui Zhu, Yuzhong Zheng, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3734048/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Sep, 2024 Read the published version in Journal of Pest Science → Version 1 posted 5 You are reading this latest preprint version Abstract Diaphorina citri is the vector of “ Candidatus Liberibacter asiaticus” (CLas), a bacterium associated with the citrus disease as Huanglongbing (HLB). Previous mitochondrial genome (i.e. mitogenome) analyses revealed the prevalence of two major mitochondrial groups (MGs) of D. citri in China, geographically distributed by elevation gradients. We estimated the population diversity of D. citri from sampling 54 major citrus-producing areas within 11 provinces/regions of China, through comparing assembled de novo mitogenomes. Additionally, endosymbiont genomes were recovered for “ Ca. Carsonella ruddii” ( Ca Cr) and “ Ca. Profftella armature” ( Ca Pa) from full-DNA next generation sequencing of 31 samples from the same pool. Most of quantified D. citri diversity came from single nucleotide polymorphisms (SNPs) in five genes: nad3 , cox2 , rrnL , cob , and atp6 . Nine SNPs clustered the analysed D. citri mitogenomes into two major mitochondrial groups (MG) comprising seven subgroups. Independent phylogenetic trees were generated for the endosymbionts Ca Cr and Ca Pa, and a Ca Pa plasmid, supporting patterns obtained for D. citri mitogenomes while adding complexity layers. Additional information from 64 Ca Cr, 58 Ca Pa, and 6 Ca Pa plasmids provided 38 SNPs and two gaps that converged with the independent results of mitogenomes analyses. Furthermore, subgroups within the original MG clusters were revealed by 87 SNPs and one gap. Thus, combined analyses of D. citri mitogenomes and associated endosymbionts proved useful in uncovering layers of population diversity, pointing to patterns in the natural history of an invasive species. Further understanding of D. citri and endosymbionts can aid D. citri HLB management protocols and forecast territorial expansion events. Plant disease vectors greening mitochondrial genes population genomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUTION The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae) is considered the most important pest of citrus worldwide. This psyllid insect transmits the phytopathogen “ Candidatus Liberibacter asiaticus” (CLas), an uncultured alpha-proteobacterium associated with the incurable citrus disease Huanglongbing (HLB), being considered a major threat to citrus production (Bové 2006 ). Locally, the main strategy for containing the spread of HLB is through quarantine, nurturing pathogen-free nursery stocks, extermination of CLas-infected trees from orchards, and local decimation of D. citri (Bové 2006 ). The psyllid D. citri was first recorded in China in 1934 at the Chaoshan area of Guangdong Province (Jiang et al., 1935 ; Hoffmann, 1936 ), and later invaded further 11 provinces/regions in China, gradually advancing northwards (Wang et al. 2016 ; Zhang et al. 2021 ; Zhang et al. 2022 ). The recorded geographical incidence of HLB disease almost perfectly overlaps with that of D. citri (Fan et al. 2009 ), illustrating how understanding the biogeographical dynamics of the disease vector is paramount for managing the spread of its vectored disease. Morphological characterization of various D. citri lineages has been the object of study of different research groups, using new generation sequencing (NGS) analysis of nuclear DNA for delimiting the psyllid lineages (De León et al. 2011 ; Boykin et al. 2012 ; Guidolin et al. 2013, 2014 ; Wang et al. 2017 ; Wu et al. 2016 , 2017 , 2018 ; Meng et al. 2018 ). The latest years have seen increased interest in the use of insect mitochondrial genomes (mitogenomes) for evolutionary and population analyses (Cameron 2014 ) given that insect genomes are relatively small and display low frequency of recombination (i.e. providing limited traits for population analysis after complex bioinformatics assembly). Typically, a single or few mitochondrial genes have been analyzed in D. citri populations from different locations, mainly cytochrome oxidase 1 ( cox1 ) (Boykin et al. 2012 ; Lashkari et al. 2014 ; Guidolin and Consoli 2013 ; Guidolin et al. 2014 ; De León et al. 2011 ). Through NGS, sampled D. citri mitogenomes can be directly recovered from total DNA raw data, enabling further traits useful in population structure (Wu et al. 2016 , 2017 , 2018 ). Currently, a total of 50 single nucleotide polymorphisms (SNPs) have been described from 23 sampled D. citri mitogenomes sampled from Guangdong (China), California (USA), and Florida (USA) (Wu et al. 2017 ), divided into two major mitochondrial groups (MGs): a cluster occurring from southwestern China at elevations above 1,000 meters, and another cluster from southeastern China at elevations below 180 meters (Wu et al. 2018 ). Endosymbionts – especially those intracellular endosymbionts with strict vertical transmission – accumulate convergent genetic structure with their hosts (Bennett and Moran 2015 ). It is thus reasonable to expect that endosymbionts genes can provide further useful information regarding their hosts’ evolutionary history. Total DNA NGS raw data can be mined further to recover the endosymbionts’ genetic structure from sampled populations, in the attempt to provide additional information enriching evolutionary patterns observed from nuclear genomes and mitogenomes. Therefore, NGS analyses of endosymbionts genomes can be a promising inexpensive strategy to analyze evolutionary history of host-parasite relationships. Genomes for D. citri endosymbionts other than CLas have already been sequenced, including “ Ca. Carsonella ruddii” ( Ca Cr) and “ Ca. Profftella armature” ( Ca Pa) (Nakabachi et al. 2006 , 2013; Tamames et al. 2007 ). Biologically, the primary D. citri endosymbiont Ca Cr is believed to provide essential amino acids that complement the insect host’s diet (Nakabachi et al. 2006 ; Tamames et al. 2007 ), while the secondary endosymbiont Ca Pa is known to produce a cytotoxic polyketide toxin (diaphorin) that shields infected D. citri against competitor microbes, including many pathogens (Nakabachi et al. 2013). Both endosymbionts exhibit traits indicating a long evolutionary history with their host: while Ca Cr shows strong evidence of tight cospeciation (Thao et al. 2000 , 2001 ; Hall et al. 2016 ), Ca Pa has the typical, drastically reduced genome of an obligatory endosymbiont (Nakabachi et al. 2013; Arp et al. 2017 ). There is phylogenetic evidence for the intimate cospeciation between Ca Cr and their host psyllids as a whole, stemming from higher taxonomic levels and spanning hosts from different genera and families within Psylloidea (Thao et al. 2000 ; Hall et al. 2016 ). Evidencing the coevolution of CaCr and D. citri , three housekeeping genes surveyed among populations from East and Southeast Asia illustrated how the endosymbiont accumulated greater genetic diversity between divergent D. citri lineages, mainly in cox1 (Nakabachi et al. 2006 ; Wang et al. 2017 ). Still, few published studies have analyzed the co-diversification of endosymbionts within their insect hosts down to the intraspecific level using genome structure, and none have examined D. citri endosymbionts and their plasmids. Given the continued advance of NGS technology, assessing mitogenomes and associated endosymbionts from whole-body extracts has become increasingly straightforward, often revealing loci which prove more insightful into evolutionary history and population structure than the traditionally used nuclear genome resources. The present study revisits the biogeographical patterns of D. citri populations in China using mitogenomes sampled from different regions, hypothesizing whether incorporating structural patterns recovered from the genomes of associated endosymbionts Ca Cr and Ca Pa would impact what was known about population genetics. We believe the approach adds relevant complexity layers to the body of knowledge of D. citri spread driving the HLB epidemic, notwithstanding the natural history of D. citri endosymbiotic microbiota. MATERIALS AND METHODS D. citri collection and DNA preparation Fifty-six samples of D. citri psyllids were obtained (listed as A1–A56 in Table S1 ) from which a single adult specimen representing each locality was extracted for sequencing and mitogenome analysis. The 54 samples comprised 23 samples from Wu et al. ( 2016 , 2018 ) plus 31 newly-collected samples, spanning 11 provinces in China. Two samples from the USA were included, to be used as outgroups. Insect samples were preserved in 75% ethanol and stored at 4°C until DNA extraction. Prior to NGS, total DNA was extracted from individual adult psyllids using the DNeasy blood and tissue kit (Qiagen, Valencia, CA). Genome sequence acquisition using NGS To reach sufficient DNA for sequencing, the obtained whole-genome DNA was amplified using the Illustra GenomiPhi V2 DNA amplification kit (GE Healthcare, Waukesha, WI, USA), towards amassing roughly 20 µg of total DNA. From this amount, 10 µg were sequenced with Illumina Hiseq-PE150, generating raw data that was analyzed with CLC Genomics Workbench v.20 (CLC Bio, Denmark), for mapping and de novo assembly of individual genomes. The raw data produced generated genomes for the insect mitochondria and associated endosymbionts, for each sample. Endosymbiont genomes came from a subgroup of 34 samples (listed as B1–B34 in Table S2 ), comprising 32 from China and the two collected from the USA (as outgroups). Draft genomes of D. citri mitochondria and endosymbionts were assembled by mapping the raw reads to reference GenBank deposits: mitogenomes (NC_030214); Ca Cr (CP012411); Ca Pa (CP012591); Ca Pa plasmid (CP012592). After mapping, all draft mitogenomes and endosymbiont genomes were double-checked with the results of independent de novo assemblies. Multiple bands were consistently generated from conventional PCR amplification of the mitogenome non-coding control region (CR) of each D. citri sample, so we opted to isolate and sequence the strongest amplicon band as representative of the region to complete the final mitogenome assembly. Gaps in the assembly were bridged using specifically-designed region-specific primers to generate amplicons matching the assembly gaps. Overall coverage of mitogenomes and endosymbiont genomes were estimated from mapping statistics by CLC Genomics Workbench software (length fraction = 0.98, similarity fraction = 0.95). Assembled genes were annotated with MITOS v.806.0. After identification of gene boundaries, they were re-examined and curated using published mitogenomes as references (listed in Table S1 ), sampled from other regions (Wu et al. 2016 , 2018 ). The typical cloverleaf secondary structure of tRNAs was predicted using ARWEN v 1.2.41.c. Three additional D. citri individuals from each region were selected for assessing tRNA poymorphisms. Nucleotide diversity analyses of D. citri mitogenomes from China Nucleotide variation in the D. citri mitogenomes was detected through sliding window analyses with DnaSP v.5.0 (Librado and Rozas 2009 ) based on the mitogenomes from the 54 Chinese samples (listed in Table S1 ), aligned using CLC software as above described. The nucleotide diversity (measured in Pi) of Protein-Coding Genes (PCGs), 22 tRNA genes, and two ribosomal RNA (rRNA) genes were estimated from 5-bp windows across 1-bp overlapping steps. Phylogenetic analysis using D. citri mitogenomes and endosymbiotic genomes Prior to phylogenetic analyses, mitochondrial cox1 and endosymbionts 16SrDNA were specifically compared among all samples. Mitogenomes non-coding CRs were excluded from such analyses as they are highly polymorphic (i.e. unstable and generating multiple PCR amplicons, see Wu et al., 2017 ). Phylogenetic trees were produced for the 56 D. citri mitogenomes and the 34 endosymbiont genomes (i.e. Ca Cr, Ca Pa, and Ca Pa plasmid), independently. Phylogenies were estimated by two different approaches, from which consensual phylogenetic trees were obtained: (i) maximum likelihood (ML) using "neighbor joining" in CLC software, and (ii) Bayesian inference (BI) using MrBayes v.3.2.7 software. The optimal substitution model was obtained with jModelTest v.2.1.1, where ML analysis nodal support among branches was established by bootstrapping 100 replicates, and for BI analyses two sets of four chains were run simultaneously for 1,000,000 generations, sampling at every 100 generations. After discarding the first 25% of samples as burn-in, a posteriori bayesian probability values were recalculated to generate consensus trees. We opted to combine two construction methods of Maximum Likelihood (Neighbor Joining and UPGMA), mainly to compensate for the fact that the length of Ca Pa genomes exceeded the running capacity of MrBayes software. Final consensus trees were produced using FigTree v.1.4.4. Identification of SNPs and gaps The amounts of identified SNPs and nucleotide gaps among 54 analyzed mitogenomes and 32 endosymbiont genomes from the Chinese D. citri samples were calculated using published correspondent genomes as references (i.e. mitogenome NC_030214; Ca Cr, CP012411; Ca Pa CP012591; Ca Pa plasmid CP012592) with CLC software tool “Create Pairwise Comparison”. To confirm the polymorphic site identifications of each sampled region, three additional D. citri individuals from each location were inspected by conventional PCR. Consistent polymorphic loci were recorded. RESULTS Description of genomes In total, 54 mitogenomes and 31 associated endosymbiont genomes and plasmids of Chinese D. citri were used in this study. Table S2 presents general information about the size and recorded coverage of the 34 D. citri samples that originated mitogenomes and genomes of Ca Cr, Ca Pa endosymbionts, and Ca Pa plasmid sequences. Psyllid mitogenome sizes ranged from 14,940 to 14,999 bp; endosymbionts Ca Cr ranged from 174,013 to 174,025 bp, and Ca Pa ranged from 457,563 to 457,579 bp; the Ca Pa plasmid ranged from 5,457 to 5,462 bp. The average coverages ranged from 56.13 to 97,058.36x (mitogenomes); 1.32 to 3,889.82x ( Ca Cr); 1.88 to 1,468.34x ( Ca Pa); 9.83 to 4,867.09x ( Ca Pa plasmid). Accession numbers for the reference annotated genomes at GenBank database are listed in Table S1 . Voucher specimens of D. citri pooled from each location are deposited at the Entomological Specimens Room of Hanshan Normal University in Chaozhou, China. Nucleotide diversity of mitogenomes Structurally, the assembled D. citri mitogenomes comprised 13 PCGs, 22 tRNA genes, two rRNA genes, and one A + T-rich CR, consistently displaying same gene order, read frame directions, and start/stop codons of PCGs, as the reference mt-GDpsy (NC_030214). The majority strand (J-strand) contained twenty-three genes and the minority strand (N-strand) contained 14 genes (Fig. 1 ). The nucleotide diversity of the 54 D. citri mitogenomes from China is summarized in Fig. 1 . The highest obtained average Pi value (0.00486) demarcated the non-coding control region (CR), and the five coding genes scoring the highest Pi values were: nad3 (Pi = 0.00093), cox2 (Pi = 0.00088), rrnL (Pi = 0.00079), cob (Pi = 0.00055), and atp6 (Pi = 0.00033). The gene cox1 had the relatively low Pi value of 0.00026. Like reported for other D. citri mitogenomes (Wu et al. 2016 , 2017 ), the tRNA genes (Fig. 2 ) presented standard cloverleaf structures in 20 out of the 22 tRNAs, as is typical of metazoan mitogenomes (Wolstenholme 1992 ). Of interest, we observed one polymorphic site inside the amino acid acceptor arm of trnTrp from samples collected from Mangshi and Ruili (Yunnan Province); furthermore, most samples from southeastern China presented the length of the TΨC arm of trnAsn measuring 7 bp, which is 1 bp longer than in samples from southwestern China. Phylogenetic analyses using mitogenome Figure 3 shows the topography of the consensus tree produced by combining ML and BI phylogenetic analyses of the 56 available mitogenomes. Phylogenies nodes are supported by strong bootstrap values (≥ 75%) in ML analyses and a posteriori Bayesian probabilities (≥ 0.96) in BI analyses, so that the presented topology is stable. Convergent information from Table S1 and Fig. 3 points to the existence of three major MGs, where two MGs are from China: (i) MG1 from southeastern China, at relatively low elevations under 550 m, and (ii) MG2 from southwestern China, at elevations above 970 m. The group MG1 was further subdivided into five subgroups: MG1-1 including most samples from Guangdong (18/23), Jiangxi (5/5), Guangxi (3/4), Hunan (2/2) and Hainan (2/2); MG1-2 including Fuzhou (Fujian) and Zhejiang; MG1-3 including Foshan (Guangdong) and Wuzhou (Guangxi); MG1-4 including Qinzhou (Gongxi) and Ceheng (Guizhou); and MG1-5 comprising the remaining samples regions from eastern Guangdong (4/23), Fujian (2/3), and Taiwan (2/2). Three samples from southwestern China did not match their geographic MGs as shown on a map in Fig. 4 : Ceheng (Guizhou Province), Jiangmen (Guangdong Province) and Chengmai (Hainan Province). For instance, sample no. A50 from Ceheng, Guizhou Province, unexpectedly grouped within MG2 (characterized by high elevations), while it is a region of more moderate elevations (about 1,002 m). Also unexpectedly, sample no. A52 from Leibo, in Sichuan Province at the highest latitude of southwestern China, was singled out as considerably dissimilar to MG1 or MG2. We herein opted to incorporate this sample no. A52 as a subgroup of MG2 (MG2-1) in order to conform with subsequent results from endosymbiont genomes data, see further below. Geographically, MG1 and MG2 intersected around southern Guizhou (Fig. 4 ), within which the two greater subgroups MG1-1 and MG1-5 were sympatric at Chaozhou (namely, samples no. A08 and A09) municipality of eastern Guangdong. Samples show clear regionalization, excepting subgroups MG1-3 and MG1-4, which showed as two independent clusters in Guangxi, Guizhou, and Guangdong Provinces (see Figs. 3 and 4 ). On the other hand, clearly D. citri populations are geographically discontinuous relative to their host plants species (Fig. 3 ). A phylogenetic tree constructed using cox1 polymorphisms (Figure S1 ) shows that this mitochondrial gene could only distinguish the most distantly related cluster MG3 from other Chinese samples, thus clustering MG1 with MG2. Notably, cox1 polymorphism separated samples from Jiangmen (Guangdong) from Chengmai (Hainan), similar to the pattern obtained using trnAsn (Fig. 2 ). Phylogenetic analyses using Ca Cr and Ca Pa genome A phylogenetic tree based on D. citri Ca Cr 16S rDNA (Figure S2 ) distinguished clearly between samples from the USA and China; however, it had insufficient resolution to separate among samples from China. Sequences of Ca Pa 16S rDNA proved identical overall, including USA samples. A consensus phylogenetic tree of Ca Cr genomes is presented in Fig. 5 , supported by bootstrap values (≥ 60%) in ML analyses and Bayesian a posterior probabilities (≥ 0.78) in BI analyses. The Ca Cr genome phylogeny separated Chinese D. citri samples from the USA outgroup, pointing to five subgroups in China clustered as follows: Group 1 containing MG1-1 and MG2-2, which are relatively genetically distant; Group 2 comprising MG1-2 and MG1-5; and monotypical Group 3 containing only MG1-3, Group 4 containing only MG1-4 and Group 5 containing only MG2-1. It is interesting to note how the phylogeny of this endosymbiont lineages seems unrelated to the distribution pattern of their host D. citri populations. The consensus phylogenetic tree for Ca Pa genomes converging Neighbor Joining and UPGMA results is shown in Fig. 6 A. The result was similar to the Ca Cr genome analysis (Fig. 5 ) in that all Chinese D. citri samples clustered within the same MG, subdividing into five subgroups. Unlike Ca Cr genomes, however, MG2-2 separated from MG1-1 and all Chinese D. citri samples split into four groups: Group 1 represented only by MG1-1, Group 2 represented only by MG1-3, Group 3 represented by MG1-2, MG1-4, and MG1-5; and Group 4 represented only by MG2. Sequences of Ca Pa plasmid were also analyzed (Fig. 6 B). Interestingly, although the plasmid genome is much shorter than Ca Pa, thus providing fewer mutation sites, its phylogeny returns the same three major MG groups revealed by the Ca Pa genomes analysis. It is worth noting that host plant strains showed no clear correlation with Ca Pa lineages – nor with their plasmid. Analyses of SNPs, gaps and polymorphic loci As shown in Figure S3, by comparing the assembled results with published references for (i) D. citri mitogenome (NC_030214), (ii) Ca Cr (CP012411), (iii) Ca Pa (CP012591), and (iv) the Ca Pa plasmid (CP012592) with the sequences sampled from Guangzhou (Tianhe District) city in Guangdong, China, it can be observed that the number of SNPs throughout other 53 Chinese mitogenomes varied from 0 (A15: Meixian, Meizhou, Guangdong) to 13 (A52: Leibo, Sichuan). The number of gaps ranged from 0 (in 26 samples) to 4 (A52: Leibo, Sichuan) (for details, see Table S1 and Figure S3). Similarly, the numbers of SNPs observed in the other 31 Chinese Ca Cr genomes ranged from 46 (B10: Yunan, Guangdong) to 95 (B29: Wenzhou, Zhejiang) and numbers of gaps ranged from 7 (B6: Xiangqiao, Chaozhou, Guangdong; B17: Maoming, Guangdong) to 24 (B12: Fengshun, Meizhou, Guangdong). Regarding Ca Pa, the numbers of SNPs ranged from 33 (B18: Zhanjiang, Guangdong) to 190 (B22: Guilin, Guangxi) and numbers of gaps from 7 (B10: Yunan, Guangdong) to 26 (B5: Jiedong, Jieyang, Guangdong). In the Ca Pa plasmids the number of SNPs ranged from 1 (seven samples) to 7 (B30: Mangshi, Yunnan) and the number of gaps ranged from 0 (18 samples) to 4 (B24: Wuzhou, Guangxi; B29: Wenzhou, Zhejiang). From organizing SNPs polymorphisms and gaps (through conventional PCR confirmation), we ended up with 23 D. citri mitogenomes, 108 Ca Cr genomes, 125 Ca Pa genomes, and 12 Ca Pa-associated plasmids (see Table S3, Table S4) supporting the population analyses. Regarding mitogenomes, all mapped SNPs were determinant to the consensual phylogenetic tree topology, among which nine base loci variations generated the major MGs separation (listed with details in Table 1 ). These were distributed as follows: the genes cox1 , atp6 , nad3 , cob , and nad1 had one SNP each, while cox2 and rrnL had two. Focusing on MG1-1 group, these nine loci presented their base positions relative to other MG groups as follows: MG1-2: C (11,729) and A (13,067); MG1-3: G (3,373); MG1-4: T (2,727) and G (4,192); MG1-5: A (13,067); MG2-1: T (5,426), A (13,067), and T (13,211); MG2-2: T (3,265), T (5,426), A (10,911), A (13,067), and T (13,211). Table 1 Polymorphic loci of the Diaphorina citri psyllid mitogenomes (minus the non-coding control region) for mitochondrial groups (MG) determination among representative collected samples from different locations in China and the USA. No. of MG Base loci position relative to the start of D. citri reference mitogenome (GenBank accession: NC_030214) 2,727 3,265 3,373 4,192 5,426 10,911 11,729 13,067 13,211 MG1-1 A C A A C C T G C MG1-2 A C A A C C C A C MG1-3 A C G A C C T A C MG1-4 T C A G C C T A C MG1-5 A C A A C C T A C MG2-1 A C A A T C T A T MG2-2 A T A A T A T A T MG3 A T A A T C T A T Gene cox1 cox2 cox2 atp6 nad3 cob nad1 rrnL rrnL Note: The MG subgroups on the left column were determined by the phylogenetic analyses of Fig. 3 . Samples from the USA are MG3 (underlined). Additional polymorphic mitochondrial loci are given in Table S3. Finally, the analyses of endosymbiont genomes and the associated plasmid reproduced the major groups also revealed by mitogenomes analysis, based on 38 confirmed SNPs (namely 18 from Ca Cr, 19 from Ca Pa, and one from Ca Pa plasmid) and two gaps (from Ca Pa) (Table S4), confirming the population patterns obtained. For instance, the SNP loci 17,005; 25,306; 64,605; 87,098; 88,946; 93,808; 141,570 in Ca Cr genomes will separate all MG1-1 samples from others. Likewise, 87 SNPs (namely 46 from Ca Cr, 37 from Ca Pa, and four from Ca Pa plasmid) and one gap ( Ca Pa plasmid) further refined patterns from the original populations. For example, 31 SNPs (e.g. SNP loci 2,390) and three gaps in Ca Cr split MG1-1 into smaller groups (Table S4). DISCUSSION Phylogenetic and population diversity studies based on the mitochondrial gene cox1 have been recurrent with D. citri psillids (de León et al. 2011 ; Boykin et al. 2012 ; Guidolin et al. 2014 ; Wang et al. 2017 ), as with many other organisms. Nonetheless, comprehensive mitogenome analyses indicate isolated cox1 sequences lack the resolution power to resolve psyllid population structure in China. Some previous studies did attempt to improve on population structure analyses of D. citri by including further mitochondrial genes. Meng et al. ( 2018 ) employed a 2,398 bp-long sequence concatenating cox1 , cob , and nad5 from 225 D. citri sampled from 7 distant locations in China, but reported no significant improvement. On the other hand, Wu et al. ( 2018 ) suggested cox2 , atp8 , nad3 , nad1 and rrnL provided more detailed information on D. citri populations diversity. The present study encompassed the high variability of the proposed cox2 , nad3 , and rrnL genes, and further reveals that cob and atp6 can provide more informative traits than the previously studied genes (e.g. atp8 and nad1 ; Fig. 1 ). Understanding invasive insect population distributions and their natural history can be useful to forecast further expansion events (Lachaise et al. 1988 ; Wu et al. 2018 ). The present study improved resolution on the D. citri haplotypes phylogenies and their patterns of distribution in China relative to previous investigations (mainly Wu et al. 2018 ). The added layers of structural complexity revealed unique population subgroups such as the six sub-major groups stemming from MG1, or the MG2 from Leibo (Guizhou sample), which might very well spawn another mitochondrial group later in evolutionary time. Furthermore, also the predicted structures of the trnTrp amino acid acceptor arm and of the trnAsn TΨC arm proved useful for further understanding D. citri population diversity (Fig. 2 ) by supporting the mitogenome analysis results. The increased regional sampling of the present study evidenced that a D. citri MG from southeastern China occurring at elevations below 180 m (Wu et al. 2018 ) can also be found in southern Guizhou among elevations of about 1,000 m. This observation illustrates that, although elevation can evidently impact D. citri prevalence, the psyllids seem able to eventually adapt to new environment conditions. This phenomenon seems inherent of invasive insect species, where a separate lineage will gradually adapt to new climate and environmental conditions, proliferating their haplotype (e.g. Dlugosch et al. 2008 ; Asplen et al. 2015 ). The boundaries of different haplotype groups of D. citri become gradually fuzzier as we observed locations harboring multiple haplotypes, such as southern Guizhou (the main intersection of major groups) and eastern Guangdong -- coincidentally or not, the latter being the original region where the psyllid-HLB association was first recorded (Hoffmann 1936 ; Chen 1943 ; Lin 1956). The identity of dominant haplotypes where HLB is particularly damaging remains an important aspect to be revealed by future investigations. The identification of distinct D. citri lineage groups and subgroups (as provided in Fig. 4 ) is essential for comprehending the epidemiology of disease insect vectors. Our previous study (Wu et al., 2018 ) inferred the surge of two MGs in Southeast China based on the distribution of D. citri populations and pertaining literature records on the region (Kuwayama 1908 ; Crawford 1919 ; Martin and Lau 1943), further suggesting that the southern subgroup MG1 likely originated around the Pearl River delta before migrating northwards, and that, on the other hand, an eastern subgroup of MG1 would have originated from between the eastern coastal area and Southeast Asia, potentially deriving of eastern Guangdong and Taiwan haplotypes, also prior to moving northwards. Nevertheless, previous studies were still unable to definitively appoint the geographical sources of MGs and issuing spreading patterns among their subgroups. The findings of this study (mainly as summarized by Figs. 3 and 4 ), indicate both lineage subgroups from Southeast China coexist at the eastern part of Guangdong (Chaozhou), unlike the isolated psyllid population at southern Guizhou (which represents a main intersection of the major MGs). It seems plausible to infer that the Southeastern China lineage emerged from Chaozhou, wherein MG1-5 represents the original lineage of MG1, which subsequently expanded eastwards into Southern China (including the Pearl River Delta) and then northwards into Eastern China and other affected regions. As for the Southwestern China MG2, its origin was postulated to the western part of Yunnan Province, with gradual dispersion to other areas in Yunnan, Guizhou, and Sichuan. Additionally, it has been observed that the mitogenome of psyllids from Leibo in Sichuan (no. A52) exhibit unique traits substantially distinct from MG1 and MG2, which would be suggestive of the origin of yet a third major lineage. This could only be confirmed by more extensive sampling from around this location. In short, the natural history of the spread of D. citri into China could be partially reconstructed from the obtained results. The main drivers of the spread of such pests like seedling transportation and climate events (e.g. typhoons) are known to often accelerate diversification in D. citri lineages (Deng et al. 2009; EFSA PLH et al. 2021). Remarkably, the fact that the distribution patterns of D. citri and their host plants proved poorly correlated is suggestive that the introduction of D. citri into new areas has been achieved by climate events. Although the spread among transported plants cannot be completely ruled out, the fact that D. citri host plant species present decoupled distribution suggests seedling transportation is of secondary importance. The observed distribution patterns of MG1-3 and MG1-4 was unexpected and merits future investigation. Compatible with previous reports of a strong association and co-evolution between D. citri and its endosymbiont Ca Cr (Nakabachi et al. 2013; Wang et al. 2017 ), the converging results with the present genomes point to strict, parallel evolution at a lower taxonomic scale. The fact that further useful traits were retrieved from D. citri endosymbionts, resolving further relationships, provided support to the obtained mitogenome phylogenies, while revealing more recent lineages at a lower scale. Most remarkably, endosymbiont genome analysis of CaCr indicate that the MG2 lineage samples from Mangshi in Yunnan Province (sample no. B30) grouped within the MG1 population of the mitogenome pattern (Fig. 5 ). This particular result is revealing of the original relationships between the two major MGs of D. citri in China, and will serve as the foundation for further experiments into the root of this diversification, and, potentially, into the identity of the original invasive lineage. In short, endosymbiont genomics can facilitate and improve studies of the evolution of large populations. CONCLUSION The spread of D. citri is a growing concern for citrus-producing areas in China. Overall, sequencing of citrus psyllid mitogenomes and associated endosymbionts followed by phylogenetic analyses revealed two major lineage groups (MG1 and MG2) and eight sub-groups in China. Particularly Southeastern China, wherein HLB remained endemic for > 100 years, presents diverse D. citri lineages which are evidently spreading and intermixing. It is plausible to infer that the Southeastern China lineage originated from eastern Guangdong, Southern Fujian and Taiwan, while the Southwestern China lineage likely originated from the western part of Yunnan Province. Additionally, a third major lineage may have originated from the Leibo region in Sichuan. These findings provide insights into the origins and dispersal patterns of the psyllid population in China. This information can be applied for current D. citri control and HLB management programs, as well as support evolutionary analysis between D. citri and their endosymbionts. From a technical point of view, the mapping of specific polymorphic sites in Chinese psyllid populations is bound to support practical applications, such as the design of more specific PCR primers and probes designed for rapid population identification. It would be relevant to further understand how seemingly trivial population genetics events support the spread of successful invasive pests. Declarations A CKNOWLEDGMENTS This research was supported by the projects of Guangdong Provincial Key Laboratory of Functional Substances in Medicinal Edible Resources and Healthcare Products (2021B1212040015), and from school-level project grants of Hanshan Normal University (QD202123, QD202122). EGPF was supported by a grant from FAPEG/CNPq (317847/2021-0). Funding This research was supported by the projects of Guangdong Provincial Key Laboratory of Functional Substances in Medicinal Edible Resources and Healthcare Products (2021B1212040015), and from school-level project grants of Hanshan Normal University (QD202123, QD202122). EGPF was supported by a grant from FAPEG/CNPq (317847/2021-0). Competing Interests The authors have no relevant financial or non-financial interests to disclose. AUTHOR CONTRIBUTIONS All authors contributed significantly to the study and revised the final version of the manuscript. Fengnian Wu, Zehan Dai, Jianjian Huang, Zikai Chen participated in methods design. Fengnian Wu, Zehan Dai, Min Shi, Xiuhong Li, Xiaoling Deng, and Eduardo G. P. Fox participated in the writing. Fengnian Wu, Zehan Dai, Min Shi, Jianjian Huang, Yuzhong Zheng, Zikai Chen and Eduardo G. P. Fox participated in data analysis and presentation. Hui Zhu was secured funding for the project and led project administration activities. 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SCI REP-UK 7: 10154. https://doi.org/10.1038/s41598-017-10713-3 Zhang Y, Dai S, Long G, Zhou M, Sun T, Li D (2021) Occurrence and control status of major citrus diseases and pests in Hunan Province [J]. Hunan Agricultural Science 12: 61–64 Zhang Y, Guo L, Yu X, Li W, Gu Q, Xiang M, Li X (2022) Occurrence, characteristics and control measures of citrus psyllid in Jiangxi. Biological Disaster Science 45: 311–317 Additional Declarations No competing interests reported. Supplementary Files Supplementaryfile1.2023.10.28fox.docx SUPPORTING INFORMATION Additional supporting information can be found online in the Supporting Information section at the end of this article. Fig. S1 Phylogenetic analyses of Diaphorina citri mitogenome sequences based on cox1 gene. Fig. S2 Phylogenetic analyses of Diaphorina citri “ Candidatus Carsonella ruddii” sequences based on the 16SrDNA gene. Fig. S3Heatmap table of numbers of single nucleotide polymorphisms (SNPs) and gaps among the sampled genomes from China belonging to (i) Diaphorina citri mitogenome, (ii) “ Candidatus Carsonella ruddii” ( Ca Cr), (iii) “ Ca . Profftella armature” ( Ca Pa) and (iv) Ca Pa associated plasmids. Table S1. Samples of Diaphorina citri used for mitogenome and endosymbiont analysis in this study. Table S2.Mitochondrial and endosymbiont genome information of 34 samples sequenced by Next Generation Sequencing (NGS) Illumina. Supplementaryfile2.TableS3andS4fox.xlsx Table S3.Polymorphic loci of the Diaphorina citri mitogenome sequences, assembled de novo from samples obtained in China. Table S4.Polymorphic loci of the Diaphorina citri endosymbiont genome separating mitochondrial groups of haplotypes among representative collected samples from different locations, in China and the USA. Cite Share Download PDF Status: Published Journal Publication published 11 Sep, 2024 Read the published version in Journal of Pest Science → Version 1 posted Reviewers agreed at journal 28 Dec, 2023 Reviewers invited by journal 22 Dec, 2023 Submission checks completed at journal 11 Dec, 2023 Editor assigned by journal 11 Dec, 2023 First submitted to journal 10 Dec, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3734048","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":258453792,"identity":"1416c2b7-33f1-4cfa-b4a5-96e721f3706a","order_by":0,"name":"Fengnian Wu","email":"","orcid":"","institution":"Hanshan Normal University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fengnian","middleName":"","lastName":"Wu","suffix":""},{"id":258453793,"identity":"44383931-4566-41e4-a085-3d559053a567","order_by":1,"name":"Zehan Dai","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zehan","middleName":"","lastName":"Dai","suffix":""},{"id":258453794,"identity":"e7f6e055-5d0b-4e0c-9591-96b9677e090d","order_by":2,"name":"Min Shi","email":"","orcid":"","institution":"Hanshan Normal University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Min","middleName":"","lastName":"Shi","suffix":""},{"id":258453795,"identity":"d56f5d06-7089-4519-968a-464c786be135","order_by":3,"name":"Jianjian Huang","email":"","orcid":"","institution":"Hanshan Normal University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jianjian","middleName":"","lastName":"Huang","suffix":""},{"id":258453796,"identity":"73c8f933-6013-45f5-9caf-11fa5203f08d","order_by":4,"name":"Hui Zhu","email":"","orcid":"","institution":"Hanshan Normal University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Zhu","suffix":""},{"id":258453797,"identity":"875a6118-2ea2-4aad-801f-a3226c68eed2","order_by":5,"name":"Yuzhong Zheng","email":"","orcid":"","institution":"Hanshan Normal University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuzhong","middleName":"","lastName":"Zheng","suffix":""},{"id":258453798,"identity":"d9c1c871-61fb-4312-ba70-97683e77c3d5","order_by":6,"name":"Zikai Chen","email":"","orcid":"","institution":"Hanshan Normal University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zikai","middleName":"","lastName":"Chen","suffix":""},{"id":258453799,"identity":"05c488ed-69cf-4f21-b342-4a7017791d0f","order_by":7,"name":"Xiuhong Li","email":"","orcid":"","institution":"Hanshan Normal University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiuhong","middleName":"","lastName":"Li","suffix":""},{"id":258453800,"identity":"fd02df57-cf1e-4ad9-b739-687a2af7d346","order_by":8,"name":"Xiaoling Deng","email":"","orcid":"","institution":"South China Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaoling","middleName":"","lastName":"Deng","suffix":""},{"id":258453801,"identity":"7ffeb6b2-23e6-4b41-be14-4591df4ea2e7","order_by":9,"name":"Eduardo G. P. Fox","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxklEQVRIiWNgGAWjYDAC5gNAwoaBgR/ESSggRgtbApBIY2CQbABpMSBFiwHINgZitPCzMR+T+JBgk7j5/OrEDw8MGOT5xQ7g1yLZxpZsOCMhzdjsxtvNEkCHGc6cnYBfi8H9HsPHvD8Oy5ndOLsBpCXB4DYhLcd4DA7zJBzmMZ5xdvMPYrUYPgZqkTPg791GnC1wv0jc4N1mkWAgQdgv8BDr7z+7+eaPCht5fmkCWhBAAqxSgljlYPsOkKJ6FIyCUTAKRhIAAJ2VQQPVXqvLAAAAAElFTkSuQmCC","orcid":"","institution":"Universidade Estadual de Goiás (UEG)","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Eduardo","middleName":"G. P.","lastName":"Fox","suffix":""}],"badges":[],"createdAt":"2023-12-10 12:29:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3734048/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3734048/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10340-024-01834-6","type":"published","date":"2024-09-11T15:57:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":48091116,"identity":"775ad19d-7b1f-4ee8-8174-edb51310165f","added_by":"auto","created_at":"2023-12-12 21:39:50","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":27882,"visible":true,"origin":"","legend":"\u003cp\u003eNucleotide diversity (Pi) among 54 Chinese \u003cem\u003eDiaphorina citri\u003c/em\u003e mitogenomes through a sliding window of 5 bp, with a step size of 1 bp. Gene names are given within orange arrows pointing to their reading frame direction; numbers under each gene name give their average Pi value as calculated using DnaSP v.5. Values in the five most variable genes shown in red. The majority strand (J-strand) coding is indicated with forward arrows, and minority strand (N-strand) in backward arrows.Gene abbreviations: \u003cem\u003eatp \u003c/em\u003e= ATP synthase, \u003cem\u003ecob \u003c/em\u003e= cytochrome oxidase b, \u003cem\u003ecox \u003c/em\u003e= cytochrome oxidase c, \u003cem\u003enad \u003c/em\u003e= NADH dehydrogenase subunits, \u003cem\u003errnL \u003c/em\u003e= large ribosomal RNA subunit, and \u003cem\u003errnS \u003c/em\u003e= small ribosomal RNA subunit. Other color codesof the arrows: blue arrows represent the short tRNA genes and the red arrows indicate rRNA genes\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3734048/v1/6ca32e65ef9802e43c0c57f1.jpg"},{"id":48091119,"identity":"4301beee-1ad4-41e2-bce0-ec2463d4b3dc","added_by":"auto","created_at":"2023-12-12 21:39:50","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":51497,"visible":true,"origin":"","legend":"\u003cp\u003ePredicted secondary structure of tRNAs \u003cem\u003etrnTrp\u003c/em\u003e and \u003cem\u003etrnAsn\u003c/em\u003e obtained from the mitogenomes of \u003cem\u003eDiaphorina citri\u003c/em\u003e psyllids. Watson-Crick base pairs are represented by “-”. Others are canonical base parings in tRNA: Plus sign “+”, a paring between G and U; Dot “•”, a paring between G and A. Detected differences among samples are indicated in red\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3734048/v1/67abad287dff74c775f2fd69.jpg"},{"id":48091121,"identity":"ceb06a8e-e67a-42a8-8b3b-7cbd7665e2b4","added_by":"auto","created_at":"2023-12-12 21:39:50","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":76451,"visible":true,"origin":"","legend":"\u003cp\u003eConsensus phylogenetic tree of \u003cem\u003eDiaphorina citri\u003c/em\u003e psyllid mitogenomes(excluding non-coding control regions) based on combined Maximum Likelihood (ML) and Bayesian Inference (BI). Numbers at the nodes are bootstrap values obtained for ML and BI analyses. Samples from China are shown in red. The red dots indicate samples obtained for this study. Blue stars indicate the samples selected endosymbionts analysis by NGS. MG stands for mitochondrial clustering groups. Samples from the USA and Pakistan were used as outgroups. Host plants are indicated by numbers: ① \u003cem\u003eCitrus reticulata\u003c/em\u003e, ② \u003cem\u003eMurraya paniculata\u003c/em\u003e, ③ \u003cem\u003eC. maxima\u003c/em\u003e, ④ \u003cem\u003eC. sinensis\u003c/em\u003e, ⑤ \u003cem\u003eC. medica\u003c/em\u003e, ⑥ \u003cem\u003eC\u003c/em\u003e. × \u003cem\u003eaurantium\u003c/em\u003e ‘sweet orange’, ⑦ \u003cem\u003eC. limon\u003c/em\u003e, ⑧ \u003cem\u003eC. japonica\u003c/em\u003e. Subgroups within MGs are given in different colors: MG1-1: deep red, MG1-2: blue, MG1-3: orange, MG1-4: green, MG1-5: light red, MG2-1: purple, MG2-2: magenta, MG3: black\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3734048/v1/1d742c53a1ecdba882ca3721.jpg"},{"id":48091117,"identity":"1389e4fe-a68c-4a6f-b393-140fadf20394","added_by":"auto","created_at":"2023-12-12 21:39:50","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":103604,"visible":true,"origin":"","legend":"\u003cp\u003eGeographical distribution of \u003cem\u003eDiaphorina citri \u003c/em\u003epopulations in China, grouped by mitochondrial groups (MGs) based on mitogenome analyses. The distribution of the major mitochondrial clusters MG1 and MG2 are tentatively demarked by dotted lines on the map. Colored circles represent 54 locations from where \u003cem\u003eD. citri\u003c/em\u003e adults were collected, coding for MGs subgroups (matching Figure 3) as follows: MG1-1: deep red, MG1-2: blue, MG1-3: orange, MG1-4: green, MG1-5: light red, MG2-1: purple, MG2-2: magenta. Respective sample numbers and details are provided in Table S1\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3734048/v1/a44fa95eb953b12580d0ae3f.jpg"},{"id":48091559,"identity":"665688b2-be0d-49a2-be34-f07e351f2b50","added_by":"auto","created_at":"2023-12-12 21:47:50","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":71879,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analyses of \u003cem\u003eDiaphorina citri\u003c/em\u003e “\u003cem\u003eCandidatus\u003c/em\u003e Carsonella ruddii” genome sequences converging two Maximum Likelihood strategies. Numbers at the nodes are bootstrap values of a posterior probabilities, which were analyzed by maximum likelihood and Bayesian inference respectively. Samples were identified by Mitochondrial Group (MG) subgroups matching Figure 3: MG1-1: deep red, MG1-2: blue, MG1-3: orange, MG1-4: green, MG1-5: light red, MG2-1: purple, MG2-2: magenta, MG3: black. Samples from the USA were used as outgroups. Host citrus cultivars are indicated by numbers: ① \u003cem\u003eCitrus reticulata\u003c/em\u003e, ② \u003cem\u003eMurraya paniculata\u003c/em\u003e, ③ \u003cem\u003eC. maxima\u003c/em\u003e, ④ \u003cem\u003eC. sinensis\u003c/em\u003e, ⑤ \u003cem\u003eC. medica\u003c/em\u003e, ⑥ \u003cem\u003eC\u003c/em\u003e. × \u003cem\u003eaurantium\u003c/em\u003e ‘sweet orange’, ⑦ \u003cem\u003eC. limon\u003c/em\u003e, ⑧ \u003cem\u003eC. japonica\u003c/em\u003e\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3734048/v1/d859b75c20889b6f773d0c0a.jpg"},{"id":48091558,"identity":"d4945c14-576b-4e42-93ae-aa8da07f9226","added_by":"auto","created_at":"2023-12-12 21:47:50","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":127753,"visible":true,"origin":"","legend":"\u003cp\u003eConsensus phylogenetic trees obtained from \u003cem\u003eDiaphorina citri\u003c/em\u003e “\u003cem\u003eCandidatus\u003c/em\u003e Profftella armature” genomes (A) and their plasmid sequences (B). Numbers at the nodes are bootstrap values from: (A) maximum likelihood analysis using Neighbor Joining/ UPGMA, or (B) maximum likelihood using posteriori probabilities from Bayesian inference analysis. Samples were identified by Mitochondrial Group (MG) subgroups matching Figure 3: MG1-1: deep red. MG1-2: blue, MG1-3: orange, MG1-4: green, MG1-5: light red, MG2-1: purple, MG2-2: magenta, MG3: black. Samples from the USA were used as outgroups. Host plant cultivars are indicated by symbols: ① \u003cem\u003eCitrus reticulata\u003c/em\u003e, ② \u003cem\u003eMurraya paniculata\u003c/em\u003e, ③ \u003cem\u003eC. maxima\u003c/em\u003e, ④ \u003cem\u003eC. sinensis\u003c/em\u003e, ⑤ \u003cem\u003eC. medica\u003c/em\u003e, ⑥ \u003cem\u003eC\u003c/em\u003e. × \u003cem\u003eaurantium\u003c/em\u003e ‘sweet orange’, ⑦ \u003cem\u003eC. limon\u003c/em\u003e, ⑧ \u003cem\u003eC. japonica\u003c/em\u003e\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3734048/v1/929f9053e32e022e46228b9a.jpg"},{"id":64618952,"identity":"adf48329-b156-4d92-951a-85d904953a78","added_by":"auto","created_at":"2024-09-16 16:08:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1199412,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3734048/v1/b730b728-e7cd-41c8-aee7-b4feff4452dc.pdf"},{"id":48091123,"identity":"5bf508c2-7566-4e0d-b674-123ee686ce35","added_by":"auto","created_at":"2023-12-12 21:39:50","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":723432,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSUPPORTING INFORMATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdditional supporting information can be found online in the Supporting Information section at the end of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. S1\u003c/strong\u003e Phylogenetic analyses of \u003cem\u003eDiaphorina citri\u003c/em\u003e mitogenome sequences based on \u003cem\u003ecox1 \u003c/em\u003egene.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. S2\u003c/strong\u003e Phylogenetic analyses of \u003cem\u003eDiaphorina citri\u003c/em\u003e “\u003cem\u003eCandidatus \u003c/em\u003eCarsonella ruddii” sequences based on the 16SrDNA gene.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. S3\u003c/strong\u003eHeatmap table of numbers of single nucleotide polymorphisms (SNPs) and gaps among the sampled genomes from China belonging to (i) \u003cem\u003eDiaphorina citri\u003c/em\u003emitogenome, (ii) “\u003cem\u003eCandidatus \u003c/em\u003eCarsonella ruddii” (\u003cem\u003eCa\u003c/em\u003eCr), (iii) “\u003cem\u003eCa\u003c/em\u003e. Profftella armature” (\u003cem\u003eCa\u003c/em\u003ePa) and (iv) \u003cem\u003eCa\u003c/em\u003ePa associated plasmids.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S1.\u003c/strong\u003e Samples of \u003cem\u003eDiaphorina citri\u003c/em\u003e used for mitogenome and endosymbiont analysis in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S2.\u003c/strong\u003eMitochondrial and endosymbiont genome information of 34 samples sequenced by Next Generation Sequencing (NGS) Illumina.\u003c/p\u003e","description":"","filename":"Supplementaryfile1.2023.10.28fox.docx","url":"https://assets-eu.researchsquare.com/files/rs-3734048/v1/2285ebacbab52d9ff24f5996.docx"},{"id":48091120,"identity":"7cfd1021-3f6e-4fc1-9966-77c20af44ef7","added_by":"auto","created_at":"2023-12-12 21:39:50","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":33587,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S3.\u003c/strong\u003ePolymorphic loci of the \u003cem\u003eDiaphorina citri\u003c/em\u003e mitogenome sequences, assembled de novo from samples obtained in China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S4.\u003c/strong\u003ePolymorphic loci of the \u003cem\u003eDiaphorina citri\u003c/em\u003e endosymbiont genome separating mitochondrial groups of haplotypes among representative collected samples from different locations, in China and the USA.\u003c/p\u003e","description":"","filename":"Supplementaryfile2.TableS3andS4fox.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3734048/v1/237b9a6c96cb0d5c7979b0bf.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Tracking the biogeography of the Asian Citrus Psyllid Diaphorina citri spread in China using mitogenomes and endosymbionts","fulltext":[{"header":"INTRODUTION","content":"\u003cp\u003eThe Asian citrus psyllid, \u003cem\u003eDiaphorina citri\u003c/em\u003e Kuwayama (Hemiptera: Liviidae) is considered the most important pest of citrus worldwide. This psyllid insect transmits the phytopathogen \u0026ldquo;\u003cem\u003eCandidatus\u003c/em\u003e Liberibacter asiaticus\u0026rdquo; (CLas), an uncultured alpha-proteobacterium associated with the incurable citrus disease Huanglongbing (HLB), being considered a major threat to citrus production (Bov\u0026eacute; \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Locally, the main strategy for containing the spread of HLB is through quarantine, nurturing pathogen-free nursery stocks, extermination of CLas-infected trees from orchards, and local decimation of \u003cem\u003eD. citri\u003c/em\u003e (Bov\u0026eacute; \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The psyllid \u003cem\u003eD. citri\u003c/em\u003e was first recorded in China in 1934 at the Chaoshan area of Guangdong Province (Jiang et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1935\u003c/span\u003e; Hoffmann, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1936\u003c/span\u003e), and later invaded further 11 provinces/regions in China, gradually advancing northwards (Wang et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The recorded geographical incidence of HLB disease almost perfectly overlaps with that of \u003cem\u003eD. citri\u003c/em\u003e (Fan et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), illustrating how understanding the biogeographical dynamics of the disease vector is paramount for managing the spread of its vectored disease.\u003c/p\u003e \u003cp\u003eMorphological characterization of various \u003cem\u003eD. citri\u003c/em\u003e lineages has been the object of study of different research groups, using new generation sequencing (NGS) analysis of nuclear DNA for delimiting the psyllid lineages (De Le\u0026oacute;n et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Boykin et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Guidolin et al. 2013, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Meng et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The latest years have seen increased interest in the use of insect mitochondrial genomes (mitogenomes) for evolutionary and population analyses (Cameron \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) given that insect genomes are relatively small and display low frequency of recombination (i.e. providing limited traits for population analysis after complex bioinformatics assembly). Typically, a single or few mitochondrial genes have been analyzed in \u003cem\u003eD. citri\u003c/em\u003e populations from different locations, mainly cytochrome oxidase 1 (\u003cem\u003ecox1\u003c/em\u003e) (Boykin et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Lashkari et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Guidolin and Consoli \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Guidolin et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; De Le\u0026oacute;n et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Through NGS, sampled \u003cem\u003eD. citri\u003c/em\u003e mitogenomes can be directly recovered from total DNA raw data, enabling further traits useful in population structure (Wu et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Currently, a total of 50 single nucleotide polymorphisms (SNPs) have been described from 23 sampled \u003cem\u003eD. citri\u003c/em\u003e mitogenomes sampled from Guangdong (China), California (USA), and Florida (USA) (Wu et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), divided into two major mitochondrial groups (MGs): a cluster occurring from southwestern China at elevations above 1,000 meters, and another cluster from southeastern China at elevations below 180 meters (Wu et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEndosymbionts \u0026ndash; especially those intracellular endosymbionts with strict vertical transmission \u0026ndash; accumulate convergent genetic structure with their hosts (Bennett and Moran \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). It is thus reasonable to expect that endosymbionts genes can provide further useful information regarding their hosts\u0026rsquo; evolutionary history. Total DNA NGS raw data can be mined further to recover the endosymbionts\u0026rsquo; genetic structure from sampled populations, in the attempt to provide additional information enriching evolutionary patterns observed from nuclear genomes and mitogenomes. Therefore, NGS analyses of endosymbionts genomes can be a promising inexpensive strategy to analyze evolutionary history of host-parasite relationships.\u003c/p\u003e \u003cp\u003eGenomes for \u003cem\u003eD. citri\u003c/em\u003e endosymbionts other than CLas have already been sequenced, including \u0026ldquo;\u003cem\u003eCa.\u003c/em\u003e Carsonella ruddii\u0026rdquo; (\u003cem\u003eCa\u003c/em\u003eCr) and \u0026ldquo;\u003cem\u003eCa.\u003c/em\u003e Profftella armature\u0026rdquo; (\u003cem\u003eCa\u003c/em\u003ePa) (Nakabachi et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2006\u003c/span\u003e, 2013; Tamames et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Biologically, the primary \u003cem\u003eD. citri\u003c/em\u003e endosymbiont \u003cem\u003eCa\u003c/em\u003eCr is believed to provide essential amino acids that complement the insect host\u0026rsquo;s diet (Nakabachi et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Tamames et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), while the secondary endosymbiont \u003cem\u003eCa\u003c/em\u003ePa is known to produce a cytotoxic polyketide toxin (diaphorin) that shields infected \u003cem\u003eD. citri\u003c/em\u003e against competitor microbes, including many pathogens (Nakabachi et al. 2013). Both endosymbionts exhibit traits indicating a long evolutionary history with their host: while \u003cem\u003eCa\u003c/em\u003eCr shows strong evidence of tight cospeciation (Thao et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2000\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Hall et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), \u003cem\u003eCa\u003c/em\u003ePa has the typical, drastically reduced genome of an obligatory endosymbiont (Nakabachi et al. 2013; Arp et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThere is phylogenetic evidence for the intimate cospeciation between \u003cem\u003eCa\u003c/em\u003eCr and their host psyllids as a whole, stemming from higher taxonomic levels and spanning hosts from different genera and families within Psylloidea (Thao et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Hall et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Evidencing the coevolution of \u003cem\u003eCaCr\u003c/em\u003e and \u003cem\u003eD. citri\u003c/em\u003e, three housekeeping genes surveyed among populations from East and Southeast Asia illustrated how the endosymbiont accumulated greater genetic diversity between divergent \u003cem\u003eD. citri\u003c/em\u003e lineages, mainly in \u003cem\u003ecox1\u003c/em\u003e (Nakabachi et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Still, few published studies have analyzed the co-diversification of endosymbionts within their insect hosts down to the intraspecific level using genome structure, and none have examined \u003cem\u003eD. citri\u003c/em\u003e endosymbionts and their plasmids. Given the continued advance of NGS technology, assessing mitogenomes and associated endosymbionts from whole-body extracts has become increasingly straightforward, often revealing loci which prove more insightful into evolutionary history and population structure than the traditionally used nuclear genome resources. The present study revisits the biogeographical patterns of \u003cem\u003eD. citri\u003c/em\u003e populations in China using mitogenomes sampled from different regions, hypothesizing whether incorporating structural patterns recovered from the genomes of associated endosymbionts \u003cem\u003eCa\u003c/em\u003eCr and \u003cem\u003eCa\u003c/em\u003ePa would impact what was known about population genetics. We believe the approach adds relevant complexity layers to the body of knowledge of \u003cem\u003eD. citri\u003c/em\u003e spread driving the HLB epidemic, notwithstanding the natural history of \u003cem\u003eD. citri\u003c/em\u003e endosymbiotic microbiota.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e \u003cb\u003eD. citri\u003c/b\u003e \u003cb\u003ecollection and DNA preparation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFifty-six samples of \u003cem\u003eD. citri\u003c/em\u003e psyllids were obtained (listed as A1\u0026ndash;A56 in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) from which a single adult specimen representing each locality was extracted for sequencing and mitogenome analysis. The 54 samples comprised 23 samples from Wu et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) plus 31 newly-collected samples, spanning 11 provinces in China. Two samples from the USA were included, to be used as outgroups. Insect samples were preserved in 75% ethanol and stored at 4\u0026deg;C until DNA extraction. Prior to NGS, total DNA was extracted from individual adult psyllids using the DNeasy blood and tissue kit (Qiagen, Valencia, CA).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGenome sequence acquisition using NGS\u003c/h2\u003e \u003cp\u003eTo reach sufficient DNA for sequencing, the obtained whole-genome DNA was amplified using the Illustra GenomiPhi V2 DNA amplification kit (GE Healthcare, Waukesha, WI, USA), towards amassing roughly 20 \u0026micro;g of total DNA. From this amount, 10 \u0026micro;g were sequenced with Illumina Hiseq-PE150, generating raw data that was analyzed with CLC Genomics Workbench v.20 (CLC Bio, Denmark), for mapping and \u003cem\u003ede novo\u003c/em\u003e assembly of individual genomes. The raw data produced generated genomes for the insect mitochondria and associated endosymbionts, for each sample. Endosymbiont genomes came from a subgroup of 34 samples (listed as B1\u0026ndash;B34 in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), comprising 32 from China and the two collected from the USA (as outgroups). Draft genomes of \u003cem\u003eD. citri\u003c/em\u003e mitochondria and endosymbionts were assembled by mapping the raw reads to reference GenBank deposits: mitogenomes (NC_030214); \u003cem\u003eCa\u003c/em\u003eCr (CP012411); \u003cem\u003eCa\u003c/em\u003ePa (CP012591); \u003cem\u003eCa\u003c/em\u003ePa plasmid (CP012592). After mapping, all draft mitogenomes and endosymbiont genomes were double-checked with the results of independent \u003cem\u003ede novo\u003c/em\u003e assemblies.\u003c/p\u003e \u003cp\u003eMultiple bands were consistently generated from conventional PCR amplification of the mitogenome non-coding control region (CR) of each \u003cem\u003eD. citri\u003c/em\u003e sample, so we opted to isolate and sequence the strongest amplicon band as representative of the region to complete the final mitogenome assembly. Gaps in the assembly were bridged using specifically-designed region-specific primers to generate amplicons matching the assembly gaps. Overall coverage of mitogenomes and endosymbiont genomes were estimated from mapping statistics by CLC Genomics Workbench software (length fraction\u0026thinsp;=\u0026thinsp;0.98, similarity fraction\u0026thinsp;=\u0026thinsp;0.95).\u003c/p\u003e \u003cp\u003eAssembled genes were annotated with MITOS v.806.0. After identification of gene boundaries, they were re-examined and curated using published mitogenomes as references (listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), sampled from other regions (Wu et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The typical cloverleaf secondary structure of tRNAs was predicted using ARWEN v 1.2.41.c. Three additional \u003cem\u003eD. citri\u003c/em\u003e individuals from each region were selected for assessing tRNA poymorphisms.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNucleotide diversity analyses of\u003c/b\u003e \u003cb\u003eD. citri\u003c/b\u003e \u003cb\u003emitogenomes from China\u003c/b\u003e\u003c/p\u003e \u003cp\u003eNucleotide variation in the \u003cem\u003eD. citri\u003c/em\u003e mitogenomes was detected through sliding window analyses with DnaSP v.5.0 (Librado and Rozas \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) based on the mitogenomes from the 54 Chinese samples (listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), aligned using CLC software as above described. The nucleotide diversity (measured in Pi) of Protein-Coding Genes (PCGs), 22 tRNA genes, and two ribosomal RNA (rRNA) genes were estimated from 5-bp windows across 1-bp overlapping steps.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhylogenetic analysis using\u003c/b\u003e \u003cb\u003eD. citri\u003c/b\u003e \u003cb\u003emitogenomes and endosymbiotic genomes\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePrior to phylogenetic analyses, mitochondrial \u003cem\u003ecox1\u003c/em\u003e and endosymbionts 16SrDNA were specifically compared among all samples. Mitogenomes non-coding CRs were excluded from such analyses as they are highly polymorphic (i.e. unstable and generating multiple PCR amplicons, see Wu et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Phylogenetic trees were produced for the 56 \u003cem\u003eD. citri\u003c/em\u003e mitogenomes and the 34 endosymbiont genomes (i.e. \u003cem\u003eCa\u003c/em\u003eCr, \u003cem\u003eCa\u003c/em\u003ePa, and \u003cem\u003eCa\u003c/em\u003ePa plasmid), independently. Phylogenies were estimated by two different approaches, from which consensual phylogenetic trees were obtained: (i) maximum likelihood (ML) using \"neighbor joining\" in CLC software, and (ii) Bayesian inference (BI) using MrBayes v.3.2.7 software. The optimal substitution model was obtained with jModelTest v.2.1.1, where ML analysis nodal support among branches was established by bootstrapping 100 replicates, and for BI analyses two sets of four chains were run simultaneously for 1,000,000 generations, sampling at every 100 generations. After discarding the first 25% of samples as burn-in, a posteriori bayesian probability values were recalculated to generate consensus trees. We opted to combine two construction methods of Maximum Likelihood (Neighbor Joining and UPGMA), mainly to compensate for the fact that the length of \u003cem\u003eCa\u003c/em\u003ePa genomes exceeded the running capacity of MrBayes software. Final consensus trees were produced using FigTree v.1.4.4.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of SNPs and gaps\u003c/h2\u003e \u003cp\u003eThe amounts of identified SNPs and nucleotide gaps among 54 analyzed mitogenomes and 32 endosymbiont genomes from the Chinese \u003cem\u003eD. citri\u003c/em\u003e samples were calculated using published correspondent genomes as references (i.e. mitogenome NC_030214; \u003cem\u003eCa\u003c/em\u003eCr, CP012411; \u003cem\u003eCa\u003c/em\u003ePa CP012591; \u003cem\u003eCa\u003c/em\u003ePa plasmid CP012592) with CLC software tool \u0026ldquo;Create Pairwise Comparison\u0026rdquo;. To confirm the polymorphic site identifications of each sampled region, three additional \u003cem\u003eD. citri\u003c/em\u003e individuals from each location were inspected by conventional PCR. Consistent polymorphic loci were recorded.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eDescription of genomes\u003c/h2\u003e \u003cp\u003eIn total, 54 mitogenomes and 31 associated endosymbiont genomes and plasmids of Chinese \u003cem\u003eD. citri\u003c/em\u003e were used in this study. Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e presents general information about the size and recorded coverage of the 34 \u003cem\u003eD. citri\u003c/em\u003e samples that originated mitogenomes and genomes of \u003cem\u003eCa\u003c/em\u003eCr, \u003cem\u003eCa\u003c/em\u003ePa endosymbionts, and \u003cem\u003eCa\u003c/em\u003ePa plasmid sequences. Psyllid mitogenome sizes ranged from 14,940 to 14,999 bp; endosymbionts \u003cem\u003eCa\u003c/em\u003eCr ranged from 174,013 to 174,025 bp, and \u003cem\u003eCa\u003c/em\u003ePa ranged from 457,563 to 457,579 bp; the \u003cem\u003eCa\u003c/em\u003ePa plasmid ranged from 5,457 to 5,462 bp. The average coverages ranged from 56.13 to 97,058.36x (mitogenomes); 1.32 to 3,889.82x (\u003cem\u003eCa\u003c/em\u003eCr); 1.88 to 1,468.34x (\u003cem\u003eCa\u003c/em\u003ePa); 9.83 to 4,867.09x (\u003cem\u003eCa\u003c/em\u003ePa plasmid). Accession numbers for the reference annotated genomes at GenBank database are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Voucher specimens of \u003cem\u003eD. citri\u003c/em\u003e pooled from each location are deposited at the Entomological Specimens Room of Hanshan Normal University in Chaozhou, China.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eNucleotide diversity of mitogenomes\u003c/h2\u003e \u003cp\u003eStructurally, the assembled \u003cem\u003eD. citri\u003c/em\u003e mitogenomes comprised 13 PCGs, 22 tRNA genes, two rRNA genes, and one A\u0026thinsp;+\u0026thinsp;T-rich CR, consistently displaying same gene order, read frame directions, and start/stop codons of PCGs, as the reference mt-GDpsy (NC_030214). The majority strand (J-strand) contained twenty-three genes and the minority strand (N-strand) contained 14 genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe nucleotide diversity of the 54 \u003cem\u003eD. citri\u003c/em\u003e mitogenomes from China is summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The highest obtained average Pi value (0.00486) demarcated the non-coding control region (CR), and the five coding genes scoring the highest Pi values were: \u003cem\u003enad3\u003c/em\u003e (Pi\u0026thinsp;=\u0026thinsp;0.00093), \u003cem\u003ecox2\u003c/em\u003e (Pi\u0026thinsp;=\u0026thinsp;0.00088), \u003cem\u003errnL\u003c/em\u003e (Pi\u0026thinsp;=\u0026thinsp;0.00079), \u003cem\u003ecob\u003c/em\u003e (Pi\u0026thinsp;=\u0026thinsp;0.00055), and \u003cem\u003eatp6\u003c/em\u003e (Pi\u0026thinsp;=\u0026thinsp;0.00033). The gene \u003cem\u003ecox1\u003c/em\u003e had the relatively low Pi value of 0.00026.\u003c/p\u003e \u003cp\u003eLike reported for other \u003cem\u003eD. citri\u003c/em\u003e mitogenomes (Wu et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), the tRNA genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) presented standard cloverleaf structures in 20 out of the 22 tRNAs, as is typical of metazoan mitogenomes (Wolstenholme \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). Of interest, we observed one polymorphic site inside the amino acid acceptor arm of \u003cem\u003etrnTrp\u003c/em\u003e from samples collected from Mangshi and Ruili (Yunnan Province); furthermore, most samples from southeastern China presented the length of the TΨC arm of \u003cem\u003etrnAsn\u003c/em\u003e measuring 7 bp, which is 1 bp longer than in samples from southwestern China.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analyses using mitogenome\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the topography of the consensus tree produced by combining ML and BI phylogenetic analyses of the 56 available mitogenomes. Phylogenies nodes are supported by strong bootstrap values (\u0026ge;\u0026thinsp;75%) in ML analyses and a posteriori Bayesian probabilities (\u0026ge;\u0026thinsp;0.96) in BI analyses, so that the presented topology is stable. Convergent information from Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e points to the existence of three major MGs, where two MGs are from China: (i) MG1 from southeastern China, at relatively low elevations under 550 m, and (ii) MG2 from southwestern China, at elevations above 970 m. The group MG1 was further subdivided into five subgroups: MG1-1 including most samples from Guangdong (18/23), Jiangxi (5/5), Guangxi (3/4), Hunan (2/2) and Hainan (2/2); MG1-2 including Fuzhou (Fujian) and Zhejiang; MG1-3 including Foshan (Guangdong) and Wuzhou (Guangxi); MG1-4 including Qinzhou (Gongxi) and Ceheng (Guizhou); and MG1-5 comprising the remaining samples regions from eastern Guangdong (4/23), Fujian (2/3), and Taiwan (2/2). Three samples from southwestern China did not match their geographic MGs as shown on a map in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e: Ceheng (Guizhou Province), Jiangmen (Guangdong Province) and Chengmai (Hainan Province). For instance, sample no. A50 from Ceheng, Guizhou Province, unexpectedly grouped within MG2 (characterized by high elevations), while it is a region of more moderate elevations (about 1,002 m). Also unexpectedly, sample no. A52 from Leibo, in Sichuan Province at the highest latitude of southwestern China, was singled out as considerably dissimilar to MG1 or MG2. We herein opted to incorporate this sample no. A52 as a subgroup of MG2 (MG2-1) in order to conform with subsequent results from endosymbiont genomes data, see further below.\u003c/p\u003e \u003cp\u003eGeographically, MG1 and MG2 intersected around southern Guizhou (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), within which the two greater subgroups MG1-1 and MG1-5 were sympatric at Chaozhou (namely, samples no. A08 and A09) municipality of eastern Guangdong. Samples show clear regionalization, excepting subgroups MG1-3 and MG1-4, which showed as two independent clusters in Guangxi, Guizhou, and Guangdong Provinces (see Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). On the other hand, clearly \u003cem\u003eD. citri\u003c/em\u003e populations are geographically discontinuous relative to their host plants species (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA phylogenetic tree constructed using \u003cem\u003ecox1\u003c/em\u003e polymorphisms (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) shows that this mitochondrial gene could only distinguish the most distantly related cluster MG3 from other Chinese samples, thus clustering MG1 with MG2. Notably, \u003cem\u003ecox1\u003c/em\u003e polymorphism separated samples from Jiangmen (Guangdong) from Chengmai (Hainan), similar to the pattern obtained using \u003cem\u003etrnAsn\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhylogenetic analyses using\u003c/b\u003e \u003cb\u003eCa\u003c/b\u003e\u003cb\u003eCr and\u003c/b\u003e \u003cb\u003eCa\u003c/b\u003e\u003cb\u003ePa genome\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA phylogenetic tree based on \u003cem\u003eD. citri Ca\u003c/em\u003eCr 16S rDNA (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e) distinguished clearly between samples from the USA and China; however, it had insufficient resolution to separate among samples from China. Sequences of \u003cem\u003eCa\u003c/em\u003ePa 16S rDNA proved identical overall, including USA samples.\u003c/p\u003e \u003cp\u003eA consensus phylogenetic tree of \u003cem\u003eCa\u003c/em\u003eCr genomes is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, supported by bootstrap values (\u0026ge;\u0026thinsp;60%) in ML analyses and Bayesian a posterior probabilities (\u0026ge;\u0026thinsp;0.78) in BI analyses. The \u003cem\u003eCa\u003c/em\u003eCr genome phylogeny separated Chinese \u003cem\u003eD. citri\u003c/em\u003e samples from the USA outgroup, pointing to five subgroups in China clustered as follows: Group 1 containing MG1-1 and MG2-2, which are relatively genetically distant; Group 2 comprising MG1-2 and MG1-5; and monotypical Group 3 containing only MG1-3, Group 4 containing only MG1-4 and Group 5 containing only MG2-1. It is interesting to note how the phylogeny of this endosymbiont lineages seems unrelated to the distribution pattern of their host \u003cem\u003eD. citri\u003c/em\u003e populations.\u003c/p\u003e \u003cp\u003eThe consensus phylogenetic tree for \u003cem\u003eCa\u003c/em\u003ePa genomes converging Neighbor Joining and UPGMA results is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA. The result was similar to the \u003cem\u003eCa\u003c/em\u003eCr genome analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) in that all Chinese \u003cem\u003eD. citri\u003c/em\u003e samples clustered within the same MG, subdividing into five subgroups. Unlike \u003cem\u003eCa\u003c/em\u003eCr genomes, however, MG2-2 separated from MG1-1 and all Chinese \u003cem\u003eD. citri\u003c/em\u003e samples split into four groups: Group 1 represented only by MG1-1, Group 2 represented only by MG1-3, Group 3 represented by MG1-2, MG1-4, and MG1-5; and Group 4 represented only by MG2. Sequences of \u003cem\u003eCa\u003c/em\u003ePa plasmid were also analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Interestingly, although the plasmid genome is much shorter than \u003cem\u003eCa\u003c/em\u003ePa, thus providing fewer mutation sites, its phylogeny returns the same three major MG groups revealed by the \u003cem\u003eCa\u003c/em\u003ePa genomes analysis. It is worth noting that host plant strains showed no clear correlation with \u003cem\u003eCa\u003c/em\u003ePa lineages \u0026ndash; nor with their plasmid.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAnalyses of SNPs, gaps and polymorphic loci\u003c/h2\u003e \u003cp\u003eAs shown in Figure S3, by comparing the assembled results with published references for (i) \u003cem\u003eD. citri\u003c/em\u003e mitogenome (NC_030214), (ii) \u003cem\u003eCa\u003c/em\u003eCr (CP012411), (iii) \u003cem\u003eCa\u003c/em\u003ePa (CP012591), and (iv) the \u003cem\u003eCa\u003c/em\u003ePa plasmid (CP012592) with the sequences sampled from Guangzhou (Tianhe District) city in Guangdong, China, it can be observed that the number of SNPs throughout other 53 Chinese mitogenomes varied from 0 (A15: Meixian, Meizhou, Guangdong) to 13 (A52: Leibo, Sichuan). The number of gaps ranged from 0 (in 26 samples) to 4 (A52: Leibo, Sichuan) (for details, see Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and Figure S3). Similarly, the numbers of SNPs observed in the other 31 Chinese \u003cem\u003eCa\u003c/em\u003eCr genomes ranged from 46 (B10: Yunan, Guangdong) to 95 (B29: Wenzhou, Zhejiang) and numbers of gaps ranged from 7 (B6: Xiangqiao, Chaozhou, Guangdong; B17: Maoming, Guangdong) to 24 (B12: Fengshun, Meizhou, Guangdong). Regarding \u003cem\u003eCa\u003c/em\u003ePa, the numbers of SNPs ranged from 33 (B18: Zhanjiang, Guangdong) to 190 (B22: Guilin, Guangxi) and numbers of gaps from 7 (B10: Yunan, Guangdong) to 26 (B5: Jiedong, Jieyang, Guangdong). In the \u003cem\u003eCa\u003c/em\u003ePa plasmids the number of SNPs ranged from 1 (seven samples) to 7 (B30: Mangshi, Yunnan) and the number of gaps ranged from 0 (18 samples) to 4 (B24: Wuzhou, Guangxi; B29: Wenzhou, Zhejiang).\u003c/p\u003e \u003cp\u003eFrom organizing SNPs polymorphisms and gaps (through conventional PCR confirmation), we ended up with 23 \u003cem\u003eD. citri\u003c/em\u003e mitogenomes, 108 \u003cem\u003eCa\u003c/em\u003eCr genomes, 125 \u003cem\u003eCa\u003c/em\u003ePa genomes, and 12 \u003cem\u003eCa\u003c/em\u003ePa-associated plasmids (see Table S3, Table S4) supporting the population analyses. Regarding mitogenomes, all mapped SNPs were determinant to the consensual phylogenetic tree topology, among which nine base loci variations generated the major MGs separation (listed with details in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These were distributed as follows: the genes \u003cem\u003ecox1\u003c/em\u003e, \u003cem\u003eatp6\u003c/em\u003e, \u003cem\u003enad3\u003c/em\u003e, \u003cem\u003ecob\u003c/em\u003e, and \u003cem\u003enad1\u003c/em\u003e had one SNP each, while \u003cem\u003ecox2\u003c/em\u003e and \u003cem\u003errnL\u003c/em\u003e had two. Focusing on MG1-1 group, these nine loci presented their base positions relative to other MG groups as follows: MG1-2: C (11,729) and A (13,067); MG1-3: G (3,373); MG1-4: T (2,727) and G (4,192); MG1-5: A (13,067); MG2-1: T (5,426), A (13,067), and T (13,211); MG2-2: T (3,265), T (5,426), A (10,911), A (13,067), and T (13,211).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePolymorphic loci of the \u003cem\u003eDiaphorina citri\u003c/em\u003e psyllid mitogenomes (minus the non-coding control region) for mitochondrial groups (MG) determination among representative collected samples from different locations in China and the USA.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eNo. of MG\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"10\" nameend=\"c11\" namest=\"c2\"\u003e \u003cp\u003eBase loci position relative to the start of \u003cem\u003eD. citri\u003c/em\u003e reference mitogenome (GenBank accession: NC_030214)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,727\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3,265\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3,373\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4,192\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5,426\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10,911\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e11,729\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e13,067\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003e13,211\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"1\" nameend=\"c11\" namest=\"c11\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMG1-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c11\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMG1-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eC\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eA\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c11\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMG1-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eG\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eA\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c11\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMG1-4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eT\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eG\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eA\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c11\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMG1-5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eA\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c11\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMG2-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eT\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eA\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eT\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c11\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMG2-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eT\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eT\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eA\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eA\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eT\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c11\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eMG3\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eT\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eT\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eA\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eT\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c11\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ecox1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ecox2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ecox2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eatp6\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003enad3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003ecob\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003enad1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cem\u003errnL\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cem\u003errnL\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c11\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"11\"\u003eNote: The MG subgroups on the left column were determined by the phylogenetic analyses of Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Samples from the USA are MG3 (underlined). Additional polymorphic mitochondrial loci are given in Table S3.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFinally, the analyses of endosymbiont genomes and the associated plasmid reproduced the major groups also revealed by mitogenomes analysis, based on 38 confirmed SNPs (namely 18 from \u003cem\u003eCa\u003c/em\u003eCr, 19 from \u003cem\u003eCa\u003c/em\u003ePa, and one from \u003cem\u003eCa\u003c/em\u003ePa plasmid) and two gaps (from \u003cem\u003eCa\u003c/em\u003ePa) (Table S4), confirming the population patterns obtained. For instance, the SNP loci 17,005; 25,306; 64,605; 87,098; 88,946; 93,808; 141,570 in \u003cem\u003eCa\u003c/em\u003eCr genomes will separate all MG1-1 samples from others. Likewise, 87 SNPs (namely 46 from \u003cem\u003eCa\u003c/em\u003eCr, 37 from \u003cem\u003eCa\u003c/em\u003ePa, and four from \u003cem\u003eCa\u003c/em\u003ePa plasmid) and one gap (\u003cem\u003eCa\u003c/em\u003ePa plasmid) further refined patterns from the original populations. For example, 31 SNPs (e.g. SNP loci 2,390) and three gaps in \u003cem\u003eCa\u003c/em\u003eCr split MG1-1 into smaller groups (Table S4).\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003ePhylogenetic and population diversity studies based on the mitochondrial gene \u003cem\u003ecox1\u003c/em\u003e have been recurrent with \u003cem\u003eD. citri\u003c/em\u003e psillids (de Le\u0026oacute;n et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Boykin et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Guidolin et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), as with many other organisms. Nonetheless, comprehensive mitogenome analyses indicate isolated \u003cem\u003ecox1\u003c/em\u003e sequences lack the resolution power to resolve psyllid population structure in China. Some previous studies did attempt to improve on population structure analyses of \u003cem\u003eD. citri\u003c/em\u003e by including further mitochondrial genes. Meng et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) employed a 2,398 bp-long sequence concatenating \u003cem\u003ecox1\u003c/em\u003e, \u003cem\u003ecob\u003c/em\u003e, and \u003cem\u003enad5\u003c/em\u003e from 225 \u003cem\u003eD. citri\u003c/em\u003e sampled from 7 distant locations in China, but reported no significant improvement. On the other hand, Wu et al. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) suggested \u003cem\u003ecox2\u003c/em\u003e, \u003cem\u003eatp8\u003c/em\u003e, \u003cem\u003enad3\u003c/em\u003e, \u003cem\u003enad1\u003c/em\u003e and \u003cem\u003errnL\u003c/em\u003e provided more detailed information on \u003cem\u003eD. citri\u003c/em\u003e populations diversity. The present study encompassed the high variability of the proposed \u003cem\u003ecox2\u003c/em\u003e, \u003cem\u003enad3\u003c/em\u003e, and \u003cem\u003errnL\u003c/em\u003e genes, and further reveals that \u003cem\u003ecob\u003c/em\u003e and \u003cem\u003eatp6\u003c/em\u003e can provide more informative traits than the previously studied genes (e.g. \u003cem\u003eatp8\u003c/em\u003e and \u003cem\u003enad1\u003c/em\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUnderstanding invasive insect population distributions and their natural history can be useful to forecast further expansion events (Lachaise et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The present study improved resolution on the \u003cem\u003eD. citri\u003c/em\u003e haplotypes phylogenies and their patterns of distribution in China relative to previous investigations (mainly Wu et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The added layers of structural complexity revealed unique population subgroups such as the six sub-major groups stemming from MG1, or the MG2 from Leibo (Guizhou sample), which might very well spawn another mitochondrial group later in evolutionary time. Furthermore, also the predicted structures of the \u003cem\u003etrnTrp\u003c/em\u003e amino acid acceptor arm and of the \u003cem\u003etrnAsn\u003c/em\u003e TΨC arm proved useful for further understanding \u003cem\u003eD. citri\u003c/em\u003e population diversity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) by supporting the mitogenome analysis results.\u003c/p\u003e \u003cp\u003eThe increased regional sampling of the present study evidenced that a \u003cem\u003eD. citri\u003c/em\u003e MG from southeastern China occurring at elevations below 180 m (Wu et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) can also be found in southern Guizhou among elevations of about 1,000 m. This observation illustrates that, although elevation can evidently impact \u003cem\u003eD. citri\u003c/em\u003e prevalence, the psyllids seem able to eventually adapt to new environment conditions. This phenomenon seems inherent of invasive insect species, where a separate lineage will gradually adapt to new climate and environmental conditions, proliferating their haplotype (e.g. Dlugosch et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Asplen et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The boundaries of different haplotype groups of \u003cem\u003eD. citri\u003c/em\u003e become gradually fuzzier as we observed locations harboring multiple haplotypes, such as southern Guizhou (the main intersection of major groups) and eastern Guangdong -- coincidentally or not, the latter being the original region where the psyllid-HLB association was first recorded (Hoffmann \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1936\u003c/span\u003e; Chen \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1943\u003c/span\u003e; Lin 1956). The identity of dominant haplotypes where HLB is particularly damaging remains an important aspect to be revealed by future investigations.\u003c/p\u003e \u003cp\u003eThe identification of distinct \u003cem\u003eD. citri\u003c/em\u003e lineage groups and subgroups (as provided in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) is essential for comprehending the epidemiology of disease insect vectors. Our previous study (Wu et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) inferred the surge of two MGs in Southeast China based on the distribution of \u003cem\u003eD. citri\u003c/em\u003e populations and pertaining literature records on the region (Kuwayama \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1908\u003c/span\u003e; Crawford \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1919\u003c/span\u003e; Martin and Lau 1943), further suggesting that the southern subgroup MG1 likely originated around the Pearl River delta before migrating northwards, and that, on the other hand, an eastern subgroup of MG1 would have originated from between the eastern coastal area and Southeast Asia, potentially deriving of eastern Guangdong and Taiwan haplotypes, also prior to moving northwards. Nevertheless, previous studies were still unable to definitively appoint the geographical sources of MGs and issuing spreading patterns among their subgroups. The findings of this study (mainly as summarized by Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), indicate both lineage subgroups from Southeast China coexist at the eastern part of Guangdong (Chaozhou), unlike the isolated psyllid population at southern Guizhou (which represents a main intersection of the major MGs). It seems plausible to infer that the Southeastern China lineage emerged from Chaozhou, wherein MG1-5 represents the original lineage of MG1, which subsequently expanded eastwards into Southern China (including the Pearl River Delta) and then northwards into Eastern China and other affected regions. As for the Southwestern China MG2, its origin was postulated to the western part of Yunnan Province, with gradual dispersion to other areas in Yunnan, Guizhou, and Sichuan. Additionally, it has been observed that the mitogenome of psyllids from Leibo in Sichuan (no. A52) exhibit unique traits substantially distinct from MG1 and MG2, which would be suggestive of the origin of yet a third major lineage. This could only be confirmed by more extensive sampling from around this location.\u003c/p\u003e \u003cp\u003eIn short, the natural history of the spread of \u003cem\u003eD. citri\u003c/em\u003e into China could be partially reconstructed from the obtained results. The main drivers of the spread of such pests like seedling transportation and climate events (e.g. typhoons) are known to often accelerate diversification in \u003cem\u003eD. citri\u003c/em\u003e lineages (Deng et al. 2009; EFSA PLH et al. 2021). Remarkably, the fact that the distribution patterns of \u003cem\u003eD. citri\u003c/em\u003e and their host plants proved poorly correlated is suggestive that the introduction of \u003cem\u003eD. citri\u003c/em\u003e into new areas has been achieved by climate events. Although the spread among transported plants cannot be completely ruled out, the fact that \u003cem\u003eD. citri\u003c/em\u003e host plant species present decoupled distribution suggests seedling transportation is of secondary importance. The observed distribution patterns of MG1-3 and MG1-4 was unexpected and merits future investigation.\u003c/p\u003e \u003cp\u003eCompatible with previous reports of a strong association and co-evolution between \u003cem\u003eD. citri\u003c/em\u003e and its endosymbiont \u003cem\u003eCa\u003c/em\u003eCr (Nakabachi et al. 2013; Wang et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), the converging results with the present genomes point to strict, parallel evolution at a lower taxonomic scale. The fact that further useful traits were retrieved from \u003cem\u003eD. citri\u003c/em\u003e endosymbionts, resolving further relationships, provided support to the obtained mitogenome phylogenies, while revealing more recent lineages at a lower scale. Most remarkably, endosymbiont genome analysis of \u003cem\u003eCaCr\u003c/em\u003e indicate that the MG2 lineage samples from Mangshi in Yunnan Province (sample no. B30) grouped within the MG1 population of the mitogenome pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This particular result is revealing of the original relationships between the two major MGs of \u003cem\u003eD. citri\u003c/em\u003e in China, and will serve as the foundation for further experiments into the root of this diversification, and, potentially, into the identity of the original invasive lineage. In short, endosymbiont genomics can facilitate and improve studies of the evolution of large populations.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThe spread of \u003cem\u003eD. citri\u003c/em\u003e is a growing concern for citrus-producing areas in China. Overall, sequencing of citrus psyllid mitogenomes and associated endosymbionts followed by phylogenetic analyses revealed two major lineage groups (MG1 and MG2) and eight sub-groups in China. Particularly Southeastern China, wherein HLB remained endemic for \u0026gt;\u0026thinsp;100 years, presents diverse \u003cem\u003eD. citri\u003c/em\u003e lineages which are evidently spreading and intermixing. It is plausible to infer that the Southeastern China lineage originated from eastern Guangdong, Southern Fujian and Taiwan, while the Southwestern China lineage likely originated from the western part of Yunnan Province. Additionally, a third major lineage may have originated from the Leibo region in Sichuan. These findings provide insights into the origins and dispersal patterns of the psyllid population in China. This information can be applied for current \u003cem\u003eD. citri\u003c/em\u003e control and HLB management programs, as well as support evolutionary analysis between \u003cem\u003eD. citri\u003c/em\u003e and their endosymbionts. From a technical point of view, the mapping of specific polymorphic sites in Chinese psyllid populations is bound to support practical applications, such as the design of more specific PCR primers and probes designed for rapid population identification. It would be relevant to further understand how seemingly trivial population genetics events support the spread of successful invasive pests.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cstrong\u003eCKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the projects of Guangdong Provincial Key Laboratory of Functional Substances in Medicinal Edible Resources and Healthcare Products (2021B1212040015), and from school-level project grants of Hanshan Normal University (QD202123, QD202122). EGPF was supported by a grant from FAPEG/CNPq (317847/2021-0).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the projects of Guangdong Provincial Key Laboratory of Functional Substances in Medicinal Edible Resources and Healthcare Products (2021B1212040015), and from school-level project grants of Hanshan Normal University (QD202123, QD202122). EGPF was supported by a grant from FAPEG/CNPq (317847/2021-0).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed significantly to the study and revised the final version of the manuscript. Fengnian Wu, Zehan Dai, Jianjian Huang, Zikai Chen participated in methods design. Fengnian Wu, Zehan Dai, Min Shi, Xiuhong Li, Xiaoling Deng, and Eduardo G. P. Fox participated in the writing. Fengnian Wu, Zehan Dai, Min Shi, Jianjian Huang, Yuzhong Zheng, Zikai Chen and Eduardo G. P. Fox participated in data analysis and presentation. Hui Zhu was secured funding for the project and led project administration activities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sequences data supporting the findings of this study are available from GenBank. For more details, see Tables S1 and S2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThis is an observational study dealing with insects. The local university Committee confirms no ethical approval is required.\u003c/em\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAsplen MK, Anfora G, Biondi A, Choi, DS, Chu, D, Daane, KM et al (2015) Invasion biology of spotted wing Drosophila (\u003cem\u003eDrosophila suzukii\u003c/em\u003e): a global perspective and future priorities. Journal of Pest Science 88(3): 469\u0026ndash;494. https://link.springer.com/article/10.1007/s10340-015-0681-z\u003c/li\u003e\n\u003cli\u003eArp AP, Martini, X, Pelz-Stelinski KS (2017) Innate immune system capabilities of the Asian citrus psyllid, \u003cem\u003eDiaphorina citri\u003c/em\u003e. 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Pest Management Science 11: 74. https://doi.org/10.1002/ps.5044\u003c/li\u003e\n\u003cli\u003eWu F, Kumagai L, Cen Y, Chen J, Wallis CM, Polek M, Jiang H, Liang G, Deng X (2017) Analyses of mitogenome sequences revealed that Asian citrus psyllids (\u003cem\u003eDiaphorina citri\u003c/em\u003e) from California were related to those from Florida. SCI REP-UK 7: 10154. https://doi.org/10.1038/s41598-017-10713-3\u003c/li\u003e\n\u003cli\u003eZhang Y, Dai S, Long G, Zhou M, Sun T, Li D (2021) Occurrence and control status of major citrus diseases and pests in Hunan Province [J]. Hunan Agricultural Science 12: 61\u0026ndash;64\u003c/li\u003e\n\u003cli\u003eZhang Y, Guo L, Yu X, Li W, Gu Q, Xiang M, Li X (2022) Occurrence, characteristics and control measures of citrus psyllid in Jiangxi. Biological Disaster Science 45: 311\u0026ndash;317\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-pest-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pest","sideBox":"Learn more about [Journal of Pest Science](https://www.springer.com/journal/10340)","snPcode":"10340","submissionUrl":"https://submission.nature.com/new-submission/10340/3","title":"Journal of Pest Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Plant disease vectors, greening, mitochondrial genes, population genomics","lastPublishedDoi":"10.21203/rs.3.rs-3734048/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3734048/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eDiaphorina citri \u003c/em\u003eis the vector of “\u003cem\u003eCandidatus \u003c/em\u003eLiberibacter asiaticus” (CLas), a bacterium associated with the citrus disease as Huanglongbing (HLB). Previous mitochondrial genome (i.e. mitogenome) analyses revealed the prevalence of two major mitochondrial groups (MGs) of \u003cem\u003eD. citri\u003c/em\u003e in China, geographically distributed by elevation gradients.\u003cstrong\u003e \u003c/strong\u003eWe estimated the population diversity of \u003cem\u003eD. citri\u003c/em\u003efrom sampling 54 major citrus-producing areas within 11 provinces/regions of China, through comparing assembled de novo mitogenomes. Additionally, endosymbiont genomes were recovered for “\u003cem\u003eCa. \u003c/em\u003eCarsonella ruddii” (\u003cem\u003eCa\u003c/em\u003eCr) and “\u003cem\u003eCa. \u003c/em\u003eProfftella armature” (\u003cem\u003eCa\u003c/em\u003ePa) from full-DNA next generation sequencing of 31 samples from the same pool. Most of quantified \u003cem\u003eD. citri\u003c/em\u003e diversity came from single nucleotide polymorphisms (SNPs) in five genes: \u003cem\u003enad3\u003c/em\u003e, \u003cem\u003ecox2\u003c/em\u003e, \u003cem\u003errnL\u003c/em\u003e, \u003cem\u003ecob\u003c/em\u003e, and \u003cem\u003eatp6\u003c/em\u003e. Nine SNPs clustered the analysed \u003cem\u003eD. citri\u003c/em\u003e mitogenomes into two major mitochondrial groups (MG) comprising seven subgroups. Independent phylogenetic trees were generated for the endosymbionts \u003cem\u003eCa\u003c/em\u003eCr and \u003cem\u003eCa\u003c/em\u003ePa, and a \u003cem\u003eCa\u003c/em\u003ePa plasmid, supporting patterns obtained for \u003cem\u003eD. citri \u003c/em\u003emitogenomes while adding complexity layers. Additional information from 64 \u003cem\u003eCa\u003c/em\u003eCr, 58 \u003cem\u003eCa\u003c/em\u003ePa, and 6\u003cem\u003e Ca\u003c/em\u003ePa plasmids provided 38 SNPs and two gaps that converged with the independent results of mitogenomes analyses. Furthermore, subgroups within the original MG clusters were revealed by 87 SNPs and one gap. Thus, combined analyses of \u003cem\u003eD. citri\u003c/em\u003e mitogenomes and associated endosymbionts proved useful in uncovering layers of population diversity, pointing to patterns in the natural history of an invasive species. Further understanding of \u003cem\u003eD. citri\u003c/em\u003e and endosymbionts can aid \u003cem\u003eD. citri\u003c/em\u003e HLB management protocols and forecast territorial expansion events.\u003c/p\u003e","manuscriptTitle":"Tracking the biogeography of the Asian Citrus Psyllid Diaphorina citri spread in China using mitogenomes and endosymbionts","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-12-12 21:39:45","doi":"10.21203/rs.3.rs-3734048/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"f3eecb95-78aa-4147-987f-db820902080b","date":"2023-12-28T11:26:41+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-12-22T10:01:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-12-11T15:00:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-12-11T15:00:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Pest Science","date":"2023-12-10T12:26:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-pest-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pest","sideBox":"Learn more about [Journal of Pest Science](https://www.springer.com/journal/10340)","snPcode":"10340","submissionUrl":"https://submission.nature.com/new-submission/10340/3","title":"Journal of Pest Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4b04fa06-74cb-4cad-b405-0c282c4ce8ea","owner":[],"postedDate":"December 12th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-16T15:59:24+00:00","versionOfRecord":{"articleIdentity":"rs-3734048","link":"https://doi.org/10.1007/s10340-024-01834-6","journal":{"identity":"journal-of-pest-science","isVorOnly":false,"title":"Journal of Pest Science"},"publishedOn":"2024-09-11 15:57:00","publishedOnDateReadable":"September 11th, 2024"},"versionCreatedAt":"2023-12-12 21:39:45","video":"","vorDoi":"10.1007/s10340-024-01834-6","vorDoiUrl":"https://doi.org/10.1007/s10340-024-01834-6","workflowStages":[]},"version":"v1","identity":"rs-3734048","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3734048","identity":"rs-3734048","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}