Background

Ticks (Acari: Ixodidae) are hematophagous arthropods and act as vectors for various infectious pathogens. Ticks are classified into three families: Argasidae (soft ticks), Ixodidae (hard ticks) and Nuttalliellidae. While there is only one extant species in the Nuttalliellidae, considered the closest extant relative to the ancestral tick lineage, > 700 and 200 recognized species have been identified within the Ixodidae and Argasidae, respectively [1,2,3].

China covers a large geographic area and possesses a variety of ecosystems. To date, at least 125 tick species from nine genera have been reported across 34 provinces in China, representing 13.9% of tick species identified globally [4]. The most frequently reported species in China are Haemaphysalis longicornis, Dermacentor silvarum, Ixodes persulcatus, Haemaphysalis conicinna, Rhipicephalus microplus and Rhipicephalus sanguineus sensu lato [5, 6]. Dermacentor sinicus, Ixodes sinensis, Haemaphysalis tibetensis and Haemaphysalis qinghaiensis have been only reported in China, although there is a lack of genomic data for these species [7].

Mitochondrial (mt) genomes are widely used in molecular systematics because their relatively high rate of evolutionary change provides greater phylogenetic resolution at the genus or family levels [8]. For ticks, complete mt genomes representing 66 species from 18 genera have been sequenced to date [9, 10]. In most arthropods, tick mt genomes are circular, 14–16 kb in length, and contain 37 genes, including 13 protein coding genes, 22 tRNA genes and two rRNA genes. Phylogenetic analyses based on protein coding and rRNA gene sequences show great consistency in genus level classification [9]. A single genome re-arrangement event has been identified in the Ixodidae, including the genera Rhipicephalus, Dermacentor, Amblyomma and Haemaphysalis, which possess a translocation of tRNA genes (trnL1, trnL2, trnC) and an inversion of the trnC gene [11, 12].

Ticks have long been regarded as disease vectors, with an increasing number of human disease associations described in recent years [13,14,15,31] and generating consensus sequences from the mapped reads. The clean reads were subsequently mapped to groEL gene sequences to estimated gene abundance as the number of mapped reads per million total reads (RPM, RPM of a gene = Number of reads mapped to a gene × 106/Total number of mapped reads from given library). Based on these genes, we then compared the bacterial pathogens identified in this study with those previously described by estimating phylogenetic relationships using the ML and Bayesian methods described above.

The co-phylogenetic relationship between ticks and their bacterial pathogens

We used the BaTS (Bayesian tip-association significance testing) program [41] to test whether bacteria pathogens (Rickettsia and Coxiella) form close co-phylogenetic relationship with their tick hosts. This analysis considered tick host and pathogen phylogenies at the genus level: that is, Rhipicephalus, Hyalomma, Dermacentor, Amblyomma, Haemaphysalis, Ixodes, Argas and Carios. Specifically, we estimated the Association Index [42] and compared it with a null distribution generated using 1000 replicates of state randomization (i.e. tick genera) across a credible set of pathogen trees generated by MrBayes version 3.2 [37] as described above.

To examine the extent of bacteria-tick co-divergence, we performed event-based co-phylogenetic reconstructions using the Jane program, version 4.0 [43]. The ‘cost’ scheme for analyses in Jane was set as follows: co-divergence = 0, duplication = 1, host switch = 1, loss = 1, failure to diverge = 1. The number of generations and the population size were both set to 100. The significance of co-divergence was derived by comparing the estimated costs to null distributions calculated from 100 randomizations of host tip map**. In addition, we performed a distance-based analysis to test the hypothesis of bacterial-tick co-divergence using ParaFit as implemented in the COPYCAT software package version 2.0 [44], comparing distance matrices derived from the bacteria and tick host phylogenies. Significance testing was based on 9999 randomizations of the association matrices. Additionally, to visualize the association between bacteria and their tick hosts, a tanglegram was generated by matching each bacterial species to their associated ticks using TreeMap version 3.0beta [45].

The influence of geographic and tick genetic distance on pathogen genetic diversity

Bacterial and tick genetic distance matrices were derived from pairwise genetic comparisons using MEGA version 5.2 [33]. Geographic distances (Euclidean distance) were calculated using spatial coordinates of the samples derived from information on their geographic location. We used Mantel correlation analysis [46] to test the extent of the correlation between these matrices. Both a simple Mantel’s test and partial Mantel’s test were performed, and the correlation was evaluated using 10,000 permutations. To access which of the two factors—geographic or tick genetic distances—best explained total variation in the bacteria genetic distance matrices, we performed a multiple linear regression analysis [47] on these distance matrices. The statistical significance of each regression was evaluated by performing 1000 permutations. All statistical analyses were performed using the Ecodist package implemented in R 3.4.4 [48], and all statistical results were considered significant at a P-value of 0.05.

Results

Morphological identification and characterization of ticks

Between 1959 and 2019, more than 20,000 ticks were collected from a wide range of hosts (e.g. cattle, goats, camels, hedgehogs) across China (Fig. 1, Table 1, Additional file 1: Table S1). Species identification of hard ticks was carried out based on morphological characters, such as palps, basis capituli, cornuae, auriculae, dentition formula, punctuations, coxal spurs, amongst others [49,50,51]. Identification of soft ticks was conducted using taxonomic keys proposed by Hoogstraal [54], Teng [50] and Sun et al. [52,53,54]. This revealed a total of at least 46 species comprising two families (Ixodidae and Argasidae) and eight genera: Haemaphysalis (19), Ixodes (10), Dermacentor (six), Rhipicephalus (four), Hyalomma (two), Amblyomma (three), Argas (one) and Carios (one). Among these genera are the six most common tick species in China [6]: R. microplus, R. sanguineus s.l., I. persulcatus, Hae. longicornis, D. silvarum and Hyalomma asiaticum. In addition, other common species were collected and analyzed, including I. sinensis (vector of B. burgdorferi [55]), Ixodes ovatus, D. steini, Haemaphysalis yeni, Hae. concinna, Hyalomma scupense, Rhipicephalus turanicus, Rhipicephalus haemaphysaloides and Argas persicus from at least two provinces. In comparison, other species were more locally defined, such as Hae. tibetensis from Tibet, Hae. qinghaiensis and Haemaphysalis danieli from Qinghai, Hy. asiaticum, Haemaphysalis punctata, Dermacentor marginatus and Dermacentor niveus from **njiang, Ixodes acutitarsus from Hubei and Haemaphysalis lagrangei and Haemaphysalis mageshimaensis from Hainan. In addition, we obtained a number of very rare species, particularly Amblyomma javanense, Ixodes simplex, Ixodes nuttallianus, Ixodes crenulatus, Ixodes kuntzi, Haemaphysali kitaokai and Carios vespertilionis, some of which were collected from wildlife animals including bats, pangolins and flying squirrels (Pteromyini). Other samples were collected by drag-flagging methods or were historical samples preserved in ethanol for > 60 years. For example, the oldest sample from our data set (A. javanense) was collected in the 1960s from a wild Chinese pangolin (Manis pentadactyla). In the case of two samples (i.e. C20, A29), identification could only take place to the genus level, in Amblyomma and Ixodes, respectively. Since we were unable to identify them at the species level using morphological characteristics, they were tentatively assigned as potential new species.

Table 1 Sampling locations, animal hosts and bacterial pathogens of tick species in China at the genus level
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Mitochondrial genomes of 74 ticks of 46 species

We sequenced the total DNA of 96 individual or mixed tick samples, which generated an average of 7.87 Gb of clean reads for de novo assembly and annotation. Complete or near-complete mt genome sequences were successfully obtained from 74 of the 96 libraries, including 23 species whose mt genomes were reported for the first time. The length of the newly identified mt genomes ranged from 14,428 bp to 15,307 bp, and the AT content varied from 72.29% (Ixodes sp. A29) to 81.06% (Hae. danieli Z14), similar to previously identified mt genomes of ticks [9]. Furthermore, the structure, composition and arrangement of genes largely followed their closest relatives within the same genus [9, 56]. The only differences were observed in the length and composition of non-coding regions, some of which contain more than one tandem repeat region (Additional file 1: Fig. S1–S2, Table S2). For example, the mt genome of D. marginatus E48 has an extra copy of the non-coding region so that its length (i.e. 15,307 bp) has surpassed that of Ixodes tasmani (NC 041,086.1, 15,227 bp) [9] to become the longest tick mt genome identified to date (Additional file 1: Fig. S1). Furthermore, we found inconsistencies in the control region within some individual samples. For example, cloning of sequencing of PCR products spanning the control region between trnQ and trnF reveals various copy numbers of short repeat sequences within the same (single tick, D. marginatus E1) sample (Additional file 1: Fig. S2C).

Molecular identification and genetic diversity of ticks

Both maximum likelihood (ML) and Bayesian phylogenetic trees were estimated based on sequences of 13 protein coding genes and two rRNA genes derived from 136 mt genomes of tick, including 74 generated in this study and 62 reference mt genomes from GenBank. The ML and Bayesian methods resulted in highly similar tree topologies that placed the diversity of Chinese ticks within a global context with high resolution (Fig. 2, Additional file 1: Fig. S3–S6). Importantly, the newly added genomes greatly expanded the diversity of many groups, particularly the genera Haemaphysalis, Ixodes and Dermacentor (Fig. 2). In addition to the new species identified in this study, 23 tick species previously only known through morphological characteristics or incomplete mt genomes (e.g. I. kuntzi, I. acutitarsus, Hae. mageshimaensis and Haemaphysalis colasbelcouri) were also included (Fig. 2, Additional file 1: Fig. S3–S6). Furthermore, at least five potential cryptic species were identified—R. sanguineus s.l., D. steini, D. marginatus and I. ovatus—adding to the previously reported cryptic species identified in R. microplus [6, 57]. Each contained at least two divergent (70.93–94.21% identity) phylogenetic clusters while sharing the same morphological characteristics based on palps, basis capituli, shape and ornamentation on scutum, spurs on coxae I–IV, syncoxae and ala, etc., although more morphological features need to be examined to confirm this observation (Fig. 2). Conversely, D. sinicus, Dermacentor nuttalli and D. silvarum shared a very close relationship (> 98.46% identity) even though these were separate species based on morphological characteristics. Interestingly, D. nuttalli and D. silvarum cannot be distinguished based on mt genome phylogeny, although they had quite distinctive trochanter I dorsal spur (Additional file 1: Fig. S7).

Fig. 2
figure 2

ML phylogenies of ticks based on all 13 protein coding genes and two rRNA genes. Two mite species act as the outgroup and the scale bar represents the number of nucleotide substitutions per site. For clarity, bootstrap values only shown for major nodes. The core phylogenetic tree is shown on the left, and sequences generated in this study are marked by a circle and colored according to different tick genera. The detailed subtrees of each group are shown on the right. Within each subtree, the sequences newly identified here are marked are colored accordingly along with number of sequencing libraries

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Discovery and characterization of bacterial endosymbionts and pathogens in ticks

We first used metaphlan2 [40] for bacterial taxonomic profiling, which revealed the presence of > 32 genera including Acinetobacter, Pseudomonas, Helicobacter and Escherichia (Additional file 1: Fig. S8). Among these, we identified of tick endosymbiotic bacteria or bacteria that are known to harbor human pathogens: namely, the order Rickettsiales, genus Coxiella and genus Borrelia. These discoveries were further confirmed and characterized with analyzing the marker genes (Fig. 3A). Overall, 56% (54/96) of tick libraries were positive for these bacterial groups, among which Coxiella had the highest prevalence (40/96, 42%), followed by Rickettsia (26/96, 27%), Wolbachia (1/96, 1%) and Borrelia (1/96, 1%) (Fig. 3A, Additional file 1: Table S3).

Fig. 3
figure 3

The abundance of tick-associated bacterial groups based on the groEL gene and the proportion of positive libraries of each group; Rickettsiales (NA) represented bacteria identified could not classified in a specific genus (A). Phylogenetic trees for bacteria from the order Rickettsiales based on groEL gene (B), genera Coxiella based on groEL gene (C) and Borrelia based on flaB gene (D). The trees were midpoint-rooted, and the scale bar represents the number of nucleotide substitutions per site. Sequences generated in this study marked by a circle and colored according to different tick genera. Bootstrap values only shown for major nodes. Within the Rickettsiales phylogeny, different genera are denoted by vertical lines. Within the Coxiella phylogeny, the position of C. bernetii is highlighted by a black arrow. Within the Borrelia phylogeny, “RF” denotes the Relapsing fever group, “REB” denotes the Reptile and echnida-associated Borrelia, while “LB” indicates the Lyme borreliosis group [69]

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Most of the species in the order Rickettsiales belonged to the genus Rickettsia, within which 14 bacterial species were identified from all tick genera included in this study (with the exception of Hyalomma; Fig. 3B), including a number of human pathogens. For example, Rickettsia raoultii, which causes human tick-borne lymphadenitis [58, 59], was identified from D. marginatus and D. niveus in **ghe, **njiang province, a region where R. raoultii have previously been reported [60,61,62]. Within **njiang (**ghe and Yining), we identified Rickettsia sibirica and Rickettsia africae circulating in D. sinicus and Ixodes vespertilionis, which are responsible for a range of tick-borne diseases, including Siberian tick typhus (STT) in Asia and African tick bite fever (ATBF) in Africa [20]. In addition, we discovered Rickettsia heilongjiangensis, the newly reported agent of Far-Eastern spotted fever (FESF) [63]. This bacterium was previously found in D. silvarum ticks from Heilongjiang, and herein it was associated with the tick species Haemaphysalis campanulata and Haemaphysalis cornigera in Hubei and Jiangxi provinces located in central China. Other pathogenic Rickettsia species included Rickettsia tamurae, Rickettsia monacensis and Rickettsia helvetica identified from Amblyomma testudinarium, I. sinensis and I. kuntzi.

We found a potentially novel species within Spotted Fever Group that was relatively divergent (< 99.46% genetic identity in six genes) from the other bacteria in this group (Additional file 1: Table S4). Since the species were identified from Haemaphysalis megaspinosa, we tentatively named it Rickettsia endosymbiont of Haemaphysalis megaspinosa. Furthermore, we identified four genetically divergent Rickettsia species that occupied basal phylogenetic positions. Among these, Rickettsia endosymbiont of Ixodes persulcatus H5 and N2 clustered with Rickettsia canadensis (97.78% identity), Rickettsia endosymbiont of Argas persicus H1 fell with Rickettsia bellii and Rickettsia sp. MEAM1 (Bemisia tabaci) (92.16% and 90.96% identity), and Rickettsia endosymbiont of Ixodes vespertilionis A54b, Rickettsia endosymbiont of Carios vespertilionis X1 formed a monophyletic group with Rickettsia endosymbiont of Culicoides newsteadi despite a high level of divergence (86.08% and 85.97% identity, respectively) (Additional file 1: Table S4, Fig. S9–S11).

In addition to Rickettsia, we identified two potential new species within the order Rickettsiales. One, Wolbachia endosymbiont of Ixodes vespertilionis A54, clustered with Wolbachia pipientis strain FL2016 (95.37%) and Wolbahcia endosymbiont of Drosophila melanogaster (95.16%) within the genus Wolbachia. The identification of Wolbachia in ticks has been reported in recent years [64, 65]. The other—Rickettsiales endosymbiont of Dermacentor—clustered with an unclassified Rickettsiales bacterium Ac37b identified from Amblyomma cajennense in Brazil (86.62% identity). Together they may represent a new genus or even family within the Rickettsiales (Additional file 1: Table S4, Fig. S9–S10).

Bacteria of the genus Coxiella had the highest prevalence among the tick species examined (40/96, 42%). The newly discovered Coxiella species in the present study are highly diverse and greatly expand the genetic diversity within this group (Fig. 3C). Indeed, new genetic lineages were defined based on our phylogenetic analysis, including the Coxiella endosymbiont of Dermacentor marginatus, the Coxiella endosymbiont of Haemaphysalis concinna and the Coxiella endosymbiont of Ixodes ovatus, most of which were divergent from existing members of Coxiella and generally associated with specific tick genera (Fig. 3C, Additional file 1: Fig. S12). Coxiella burnetii, the causative agent of Q fever [66], has been reported from D. sinicus tick sampled from **njiang province and was closely related with the “Dugway 5J108-111” strain sampled from the US [67] (Fig. 3C, Additional file 1: Fig. S11). In addition to C. burnetii, we identified a single species of Borrelia from a soft tick Carios vesperitilionis in Henan province. Based on the phylogenetic analyses, the newly identified bacteria, named Borrelia henanensis X1, fell within a clade “RF” that contains pathogens causing tick-borne relapsing fever (Fig. 3D, Additional file 1: Fig. S13) [68, 69].

Ecological and evolutionary patterns in ticks and their associated bacterial symbionts

We used Mantel tests to examine whether the tick host and/or geographic factors shape the genetic diversity of the bacteria they carry. For both Rickettsia and Coxiella, our results revealed positive and significant (P < 0.0005) correlations between tick and bacteria genetic distance matrices. However, no such significant correlation was found between bacterial genetic distance and geographic distance. Similar results were obtained using (i) partial Mantel analyses, in which we tested the effect between two factors while controlling for the third, and (ii) multiple linear regression analyses in which we tested the effect between three matrices (Table 2, Additional file 1: Table S5). These results suggested that bacterial genetic diversity was primary shaped by tick genetic distance, with geographic distribution having little or no impact. The strong impact of tick on bacterial genetic diversity was also reflected in the phylogenetic analysis in which we observed a significant clustering of bacterial genetic diversity at the tick general level [Rickettsia: association index (AI) = 2.760, P < 0.001; Coxiella: AI = 0.969, P < 0.001].

Table 2 Results of the Mantel test and partial Mantel test comparing two factors (tick genetic distance and geographic distance) that predict the structure of genetic diversity in bacterial pathogens
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We next examined whether the phylogeny of the ticks and their bacterial symbionts exhibited a pattern of bacterial-tick co-divergence over evolutionary time. We first tested hypothesis of co-divergence using an event-based framework, based on which we reconciled the phylogenies of ticks and their associated bacteria (i.e. Rickettsia and Coxiella, respectively) by accounting for four processes: co-divergence, duplication, host switching and loss [43]. This revealed significantly fewer non-co-divergence events (i.e. duplication, host switching and loss) than expected by chance alone. We similarly examined the co-divergence hypothesis using a distance method, in which we evaluated the overall phylogenetic congruence by comparing the tick and bacterial symbionts patristic distance [44]. This confirmed the significant overall similarity (ParafitGlobal, P = 0.0021 and 0.0003, respectively, for Rickettsia and Coxiella, at 9999 permutations) between the tick and bacterial symbionts phylogenies (Fig. 4). Collectively, these results suggest that the symbiotic bacteria from genera Rickettsia and Coxiella have co-diverged with their tick hosts for at least 264 million years.

Fig. 4
figure 4

Co-phylogenetic comparisons of Rickettsia and Coxiella bacteria phylogenies and their corresponding tick hosts. The table shows the results of the co-phylogeny analysis using Parafit and Jane4. The tanglegram shows the match between the phylogenies of the bacteria and tick hosts. The relationship between the two phylogenies is displayed to maximize topological congruence. Dotted line colors correspond to different tick groups as shown by figure legend at the right bottom

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Discussion

We collected > 20,000 ticks and determined the mitochondrial genomes of at least 46 species representing the diversity of both common and rare ticks in China. Our sampling mostly covered human residential areas as well as some biodiversity hotspots, namely Shennongjia forest (Hubei province), the Tibet plateau (Tibet and Qinghai provinces) and Hulun Beir prairie (Inner Mongolia). While most ticks were sampled from domestic animals and were commonplace [6], those identified from wildlife or directly from the environment yielded more unique diversity. Hence, there may be many more species of ticks in China that have not yet been identified by current disease or human-centered sampling schemes. Indeed, a number of tick species, such as I. kuntzi and Hae. colasbelcouri identified from Taiwan and Laos [70, 71], have not been sequenced and characterized previously. Furthermore, there is a general lack of genomic surveillance of ticks in reptiles and amphibians, such that substantial evolutionary gaps remain in studying the long-term tick-bacteria relationship.

Our study inferred the evolutionary history of ticks based on the entire sets of mitochondrial genes, which revealed a well-supported phylogenetic tree that resolves the inter-species relationships of ticks with high resolution (Fig. 2). Mitochondrial genes are an important tool in tick molecular systematics because they evolve faster than most nuclear genes and are therefore better suited to address evolutionary questions at lower taxonomic levels [8, 53, 54], and their small size and the abundance of mitochondria in cells make them easy to analyze. Indeed, the sequencing depth in this study (0.2–30.1 Gbp per library) is sufficient for mitochondrial genes but inadequate for nuclear genes.

The results of the morphological and molecular species identification performed here were largely similar, suggesting that sequencing can reliably identify tick species. However, there were several inconsistencies, mainly reflected in the presence of five cryptic species complexes, within which morphologically identical ticks can be separated by high levels of intra-specific genetic diversity (e.g. 73.40%, 70.93%) or had paraphyletic phylogenetic structure (e.g. D. marginatus and R. sanguineus s.l.) (Fig. 2). Although we treat all these as cryptic species, we could not completely exclude that there may be some additional morphological characteristics that can distinguish these ticks and were unrecognized during our species identification process. In addition to cryptic species, inconsistencies between morphological and mitochondrial taxonomy were also reflected in the observation that several morphologically distinct ticks (D. nuttali, D. silvarum, D. sinicus) had very similar mt sequences (i.e. > 98.46% genetic identity). This suggests a relatively recent speciation event, although this needs to be further confirmed with nuclear genes.

Our study also revealed the high prevalence and large diversity of bacterial endosymbionts within the ticks examined, many of which fell with groups/species that contain human pathogens, including members of Spotted Fever Group (nine species, genus Rickettsia), C. burnetii as well as B. henanensis. Given that our study only covers 219 individuals from 46 species of ticks, the prevalence level for pathogenetic bacteria within these ticks is relatively high. This broadly agrees with previous studies that revealed a 41.2%—68.5% prevalence for pathogenetic bacteria [72, 73]. Furthermore, the abundance of some of the bacterial pathogens was high (1.42—162.57 RPM), further facilitating inter-tick transmission. Interestingly, we did not detect other pathogenic genera within order Rickettsiales that are frequently carried by ticks, such as Anaplasma and Ehrlichia, although this may simply reflect a limited sample size.

Our results greatly enrich the diversity of both ticks and their associated bacteria, revealing that both endosymbiotic bacteria groups, namely those of the Rickettsia and Coxiella genera, had close association with ticks. This is also reflected in both the strong tick structure on the bacteria phylogeny and the high resemblance between bacteria and tick phylogenies indicative of long-term co-divergence. Previous studies have suggested a lack of co-divergence between Rickettsia [74] and Coxiella-like endosymbionts [27]. However, these were mainly based on bacteria detected from PCR assays that are highly sensitive and may easily include both endosymbionts as well as bacteria transmitted through co-feeding. In addition, some previous studies of co-divergence are based on the rRNA gene, which is too conserved to distinguish closely related species [75]. In contrast, the unbiased metagenomic sequencing performed here only detects those bacteria at relatively high abundance, such that these data are more reflective of the presence of endosymbiotic bacteria. Clearly, more data are required to fully resolve the co-evolutionary history between ticks and their endosymbiont bacteria.

Conclusions

In sum, our analysis of > 20,000 ticks collected over broad geographic range across China provides insights into diversity and evolutionary history of ticks and their associated bacteria symbionts/pathogens. Our data reveal that the genetic diversity of ticks in China goes beyond a few common species and includes rare and under-explored species, for which more diversity in wildlife hosts that remains to be discovered. Importantly, despite their low occurrence, these uncommon tick species can harbor diverse pathogens, some of which could pose a potential threat to human health.