*2.5. Phylogenetic Analysis of NEAU-wh3-1*

Extraction of genomic DNA, PCR amplification of the 16S rRNA gene sequence and sequencing of PCR products were carried out using a standard procedure [62]. The PCR product was purified and cloned into the vector pMD19-T (Takara Bio Inc., Dalian, China) and sequenced using an Applied Biosystems DNA sequencer (model 3730XL, Applied Biosystems Inc., Foster City, California, USA). The almost complete 16S rRNA gene sequence of strain NEAU-wh3-1, comprising 1487 bp, was obtained and compared with type strains available in the EzBioCloud server [63] and retrieved using NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi;), and then submitted to the GenBank database. The phylogenetic tree was constructed based on the 16S rRNA gene sequences of strain NEAU-wh3-1 and related reference species. Sequences were multiply aligned in Molecular Evolutionary Genetics Analysis (MEGA) using the Clustal W algorithm and trimmed manually where necessary. Phylogenetic trees were generated with the neighbor-joining [64] and maximum-likelihood [65] algorithms using MEGA software version MEGA 7.0 [66]. The stability of the topology of the phylogenetic tree was assessed using the bootstrap method with 1000 replicates [67]. A distance matrix was generated using Kimura's two-parameter model [68]. All positions containing gaps and missing data were eliminated from the dataset (complete deletion option). The *gyr*B gene was amplified with primers PF-1 and PR-2 [69] under the PCR program for 16S rRNA gene. PCR of the *atp*D, *rec*A, *rpo*B, and *trp*B genes were performed using primers and amplification conditions described by Guo et al. [70]. The sequence data were exported as single gene alignments or a concatenated five-gene alignment for subsequent analysis as described above. Trimmed sequences of the five housekeeping genes were concatenated head-to-tail in-frame in the order *atp*D (430 bp)-*gyr*B (354 bp)-*rec*A (431 bp)-*rpo*B (208 bp)-*trp*B (556 bp). Phylogenetic analysis was performed as described above.
