The tick samples in this study were collected in different forested areas throughout Greater Hinggan in Hulun Buir City of Inner Mongolia, northeastern China (Fig. 1). The Greater Hinggan forest region of Inner Mongolia is in the northernmost area of the Greater Hinggan Mountains, accounting for 46% of the total area, with geographical coordinates ranging from 119°36′30″ to 125°24′00″ E and 47°03′40″ to 53°20′00″ N. The main habitat is primeval forest at an altitude of 250-1,745 meters, an average annual temperature of − 3.5℃, and an annual precipitation of 300–450 mm. In these areas, no specific permissions were required for the collection of ticks, and this study did not involve endangered or protected species.
Tick collection, DNA extraction, and borrelial cultivation
During 2016–2019, from May to June each year, host-seeking adult ticks were collected by flagging from vegetation. The collected tick samples were placed in a collection tube, which were classified and numbered according to the sampling time and place. I. persulcatus ticks were identified on the basis of the morphological characteristics. Ticks were washed with 0.1% sodium hypochlorite and 75% ethanol containing povidone iodine for 5 min, washed again with 3% hydrogen peroxide for 5 min, and then rinsed with sterile water. Genomic DNA PCR template was prepared from half of each tick body according to Yamazaki-Matsune et al. . The remaining half was used to cultivate B. miyamotoi in modified Barbour-Stoenner-Kelly medium (BSK-M: using minimal essential medium alpha [Bio West, Germany] as a substitute for CMRL-1066) under microaerophilic conditions [17, 22]. The tick samples that were positive for RF borreliae and negative for LD borreliae on qPCR were cultivated at 30℃ for 4 weeks, and the growth of spirochetes was examined by dark-field microscopy every 2 weeks.
Detection of borrelial DNA from ticks
Tick lysates were subjected to qPCR assay to detect borrelial infection. The assay was designed to specifically detect RF borreliae, including B. miyamotoi, and LD-related spirochetes. The system was originally established by Barbour et al. , and the borrelial DNA in the tick lysates was detected by multiplex qPCR targeting the 16S rRNA gene (16S rDNA). To allow detection of most Borrelia spp., common primers were designed to conserved sequences, and specific DNA probes conjugated to non-fluorescent quencher (NFQ) and minor groove-binder architectural protein (MGB) were designed. The two probes were labeled with either the fluorescence reporter group FAM or VIC, and the multi-qPCR reaction system was able to simultaneously detect RF and LD borreliae. The forward and reverse primers were 5′-GCTGTAAACGATGCACACTTGGT-3′ and 5′-GGCGGCACACTTAACACGTTAG-3’, respectively. The corresponding dye-labeled probes, FAM-TTCGGTACTAACTTTTAGTTAA-NFQ-MGB and VIC-CGGTACTAACCTTTCGATTA-NFQ-MGB, were purchased from Applied Biosystems (Foster City, CA). The qPCR was performed using Premix Ex Taq (Probe qPCR, Takara Bio Inc., Shiga, Japan) according to the manufacturer’s instructions and run on a Bio-Rad CFX96 system with 42 PCR cycles.
Conventional PCR and phylogeny reconstruction using glpQ sequences
To confirm the qPCR results, we performed conventional PCR on the tick-derived isolates. Ticks that were found to be RF-DNA-positive by qPCR were subjected to glycerophosphodiester diester phosphodiesterase gene (glpQ) analysis with PCR-based DNA sequencing  using primers purchased from Nanjing GenScript Biological Technology Company: forward primer (glpQ-F), 5′-CATACGCTTATGCYTTRGGMGCTGA-3′, and reverse primer (glpQ-R), 5′-GCAACCTCTGYCATACCTTCTTSTG-3′. The amplicon was approximately 610 bp in length. The reaction conditions of the first PCR were 3 min 94℃, then 30 cycles of 30 s at 94℃, 30 s annealing at 53℃, 30 s at 72℃, and finishing with 5 min at 72℃. In the second PCR, the annealing temperature was changed to 55℃. We employed the Blend Tag-Plus enzyme (TOYOBO, Osaka, Japan) in the PCR reactions, and the operation was conducted in accordance with the instructions. A negative control was used in each PCR amplification. After amplification, 5 µL of PCR product was separated on 1% agarose gel electrophoresis and visualized by ethidium bromide staining. PCR products containing the target fragment were sent to the Nanjing GenScript Biological Technology Company for bidirectional sequencing. We conducted phylogenetic analyses based on the nucleotide sequences of glpQ (555 bp) using the maximum likelihood method  in MEGA 6.0 . Homologous sequences were searched for with BLAST in NCBI and downloaded. ClustalW software was used for sequence alignment analysis, and its reliability was tested with bootstrap analysis with 1000 replicates.
De novo sequencing and multi-loci sequencing analysis based on draft genome data of cultured isolate
Genomic DNA was extracted from the B. miyamotoi strain Hetao-1 according to Lim et al. . For genomic library construction, 1 µg of DNA was used for DNA sample preparation, and sequencing libraries were generated using the Next Ultra DNA Library Prep Kit for Illumina (NEB, USA) following the manufacturer’s instructions. Briefly, the DNA sample was fragmented by sonication to approximately 350 bp, then DNA fragments were end-polished, A-tailed, and ligated with the full-length adaptor for Illumina sequencing with further PCR amplification. The PCR products were purified (AMPure XP system), and libraries were analyzed for size distribution on the Agilent 2100 Bioanalyzer and quantified using real-time PCR. The whole genome of B. miyamotoi strain Hetao-1 was sequenced using Illumina NovaSeq PE150. For genome assembly, the raw data were independently assembled using SOAP denovo v.1.0 , SPAdes , and ABySS v.2.0 . The assembly results for the three software packages were integrated with CISA software , and the assembly result with the fewest scaffolds was selected. De novo sequencing and assembling were performed at the Beijing Novogene Bioinformatics Technology.
Multi-loci sequencing analysis (MLSA) was performed using the MLSA dataset proposed by Margos et al. extracted from the draft genome sequence of strain Hetao-1. The loci of eight genes (clpA, clpX, nifS, pepX, pyrG, recG, rplB, and uvrA) were concatenated and used in this analysis according to Margos et al. .