TAXONOMIC DESCRIPTION Wang et al., Int. J. Syst. Evol. Microbiol. DOI 10.1099/ijsem.0.004489 Aliikangiella coralliicola sp. nov., a bacterium isolated from coral Porites lutea, and proposal of Pleioneaceae fam. nov. to accommodate Pleionea and Aliikangiella Guanghua Wang1,2,3, Ge Dang1,2,3, Shuailiang Xu1,2,3, Jianfeng Liu1,2,3, Hongfei Su1,2,3, Jiayuan Liang1,2,3, Wen Huang1,2,3, Yinghui Wang1,2,3 and Kefu Yu1,2,3,* Abstract A novel Gram- stain- negative, non- endospore- forming, motile, and aerobic bacterial strain, M105T, was isolated from coral Porites lutea, and was subjected to a polyphasic taxonomic study. Global alignment based on 16S rRNA gene sequences indi- cated that M105T shares the highest sequence identity of 94.5 % with Aliikangiella marina GYP-15T. The average nucleotide identity (ANI) and average amino acid identity (AAI) between M105T and A. marina GYP-15T was 69.8 and 71.6 %, respectively. On the basis of the results of phenotypic, chemotaxonomic, phylogenetic, phylogenomic, and comparative genomic analyses, it is concluded that M105T should represent a novel species in the genus Aliikangiella, for which the name Aliikangiella coralliicola sp. nov. is proposed. The type strain is M105T (=MCCC 1K03773T= KCTC 72442T). Furthermore, the family Kangiellaceae was clas- sified into two families on the basis of phylogenetic, phylogenomic, polar lipid profile and motility variations. The novel family Pleioneaceae fam. nov. is proposed to accommodate the genera Aliikangiella and Pleionea. The family Kangiellaceae was established to accommodate (designated strain M105T) was picked and purified by some species of the family Alcanivoracaceae in the order streaking on one- tenth strength marine agar 2216. M105T was Oceanospirillales, on the basis of phylogenetic, chemotaxo- stored at −70 °C in 25 % (v/v) glycerol. This strain has also nomic and physiological characteristics, which included the been deposited in the Marine Culture Collection of China and genera Kangiella, Pleionea and Aliikangiella [1]. Cells from the Korean Collection for Type Cultures under the accession the members of the genus Kangiella are usually described as numbers MCCC 1K03773T and KCTC 72442T, respectively. non- motile, rod shaped and genome reduced [2–11]. Spe- The reference strain Aliikangiella marina GYP-15T (=MCCC cies from the genera Pleionea [12, 13] and Aliikangiella [1] 1K01163T) was obtained from the Marine Culture Collection have also been described as non-motile rods. However, both of China. Pleionea mediterranea DSM 25350T was purchased genome analysis and microscopic examination indicated that from the German Collection of Microorganisms and Cell cells of Pleionea and Aliikangiella are motile by means of a Cultures, and Kangiella koreensis KCTC 12182T was obtained flagellum. So the exact taxonomy of the genera Pleionea and from the Korean Collection for Type Cultures. The novel Aliikangiella needs further study. isolate and the reference strains grew well on marine agar Strain M105T was isolated from the hermatypic coral Porites 2216 (BD). lutea [sampled from Weizhou Island (21°03ʹ42ʹʹ N, 109°08ʹ35ʹʹ The 16S rRNA gene sequence was obtained by polymerase E), PR China)] by spread dilutions of coral tissue and skel- chain reaction (PCR) amplification with the universal primers eton homogenate on one-tenth strength marine agar 2216 27F and 1492R [15]. Alignment of 16S rRNA gene sequences (BD) plates [14]. Inoculated plates were incubated at 25 °C was performed using the sina software package [16] in the for about 2 weeks, following which a pale- yellow colony silva rRNA database. Phylogenetic trees were reconstructed Author affiliations: 1Guangxi Key Laboratory on the Study of Coral Reefs in the South China Sea, Nanning 530004, PR China; 2Coral Reef Research Center of China, Guangxi University, Nanning 530004, PR China; 3School of Marine Sciences, Guangxi University, Nanning 530004, PR China. *Correspondence: Kefu Yu, kefuyu@ scsio. ac. cn Keywords: Aliikangiella coralliicola; Pleioneaceae; Kangiellaceae. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and the whole-genome sequence of strain M105T are MN094887 and VIKS00000000, respectively. The GenBank/EMBL/DDBJ accession number for the whole-genome sequence of strain Aliikangiella marina GYP-15T is VIKR00000000. Eight supplementary figures and three supplementary tables are available with the online version of this article. 004489 © 2020 The Authors 1 Wang et al., Int. J. Syst. Evol. Microbiol. 2020 97 83 Halomonadaceae Marinospirillum minutulum ATCC 19193T (AB006769) Oceanospirillaceae II 59 Balneatrix alpica 4-87T (Y17112) 0.05 Oceanospirillaceae I Litoricola lipolytica IMCC1097T (EF176580) 99 Endozoicomonadaceae Saccharospirillaceae 95 Oleiphilus messinensis ME102T (AJ295154) Halospina denitrificans HGD 1-3T (DQ072719) Hahellaceae T 82 Aliikangiella marina GYP-15 (KR078283) 92 Kangiella sp. K17-13 (FJ449700) T 100 Aliikangiella coralliicola M105 (MN094887) Kangiella sp. KLE1212 (GU812863) M105 clade / Pleioneaceae fam. nov 69 100 S26-25 (EU287325) 99 Gammaproteobacterium JAM-GA16 (AB526339) Pleionea mediterranea MOLA115T (AM990889) 100 Pleionea sediminis S1-5-21T (MK791303) T 94 Kangiella spongicola A79 (GU339304) 53 Kangiella japonica KMM 3899T (AB505051) 68 Kangiella marina KM1T (JN559388) 77 Kangiella geojedonensis YCS-5T (CP010975) 93 Kangiella sediminilitoris KCTC 23892T (CP012418) Kangiellaceae 99 Kangiella taiwanensis KT1T (JN559389) Kangiella koreensis DSM 16069T (CP001707) 100 T 95 Kangiella profundi FT102 (KM209197) 98 Kangiella aquimarina DSM 16071T (ARFE01000004) ‘Kangiella chungangensis’ CAU 1040 (KP064189) 100 Arenicellaceae Marinicella litoralis KMM 3900T (NR112913) Alcanivoracaceae 93 Streptomyces albus subsp. albus DSM 40313T (AJ621602) Fig. 1. Maximum likelihood phylogenetic tree (expanded) based on the 16S rRNA gene sequences of M105T and related taxa. The sequence from Streptomyces albus subsp. albus was used as an outgroup. Numbers at nodes indicate percentages of 1000 bootstrap resamplings; only values above 50% are shown. Bar, 0.05 substitutions per nucleotide position. using the maximum likelihood [17], neighbor- joining [18], members of the genus Kangiella form another branch (Figs and maximum parsimony [19] methods using mega software S2 and S3). v7.0 [20]. Phylogenetic distance matrices were estimated Genome sequencing was performed by Beijing Novogene by the Kimura two-parameter model [21]. Topology of the Bioinformatics Technology (Beijing, China) under its phylogenetic tree was evaluated using Felsenstein’s bootstrap resampling method [22] with 1000 replicates. standard procedure. Genomic DNA was extracted using the sodium dodecyl sulphate (SDS) method [24]. Harvested DNA About 1 410 bp of 16S rRNA gene sequence was obtained and was detected by agarose gel electrophoresis and quantified deposited in GenBank under the accession number MN094887. with a Qubit 2.0 Fluorometer (Thermo Fisher Scientific). Global alignment based on the 16S rRNA gene sequences The whole genomes of M105T and A. marina GYP-15T were in the EzBioCloud database (https://www. ezbiocloud. net/) sequenced using a NovaSeq PE150 platform (Illumina). The T indicated that M105 shares 94.5 % sequence similarity with Illumina PCR adapter reads and low- quality reads were T A. marina GYP-15 , 89.8–90.6 % sequence similarities with filtered during quality control using Readfq v10. All good- members of the genus Kangiella and 88.9–91.5 % sequence quality paired reads were assembled using the SOAPdenovo similarities with members of the genus Pleionea. This indi- [25, 26], SPAdes [27], and ABySS [28] methods and integrated T cated that M105 may represent a novel species of the genus into a number of scaffolds by CISA [29]. Genes were predicted Aliikangiella [23]. The results of phylogenetic analysis based using GeneMarkS programme [30]. Genome information was T on the maximum likelihood algorithm indicated that M105 extracted according to the method of Chun et al. [31]. The forms a distinct branch in a large clade composed of members average nucleotide identity (ANI) was calculated using the of the genera Aliikangiella and Pleionea (M105 clade) (Figs 1 ChunLab’s online calculator [32]. Average amino acid identity and S1). Meanwhile, the members of the genera Kangiella, (AAI) was calculated using the online AAI- profiler [33].The Arenicella and Marinicella formed another large clade (Figs 1 phylogenomic tree was reconstructed using the up- to- date and S1). Trees reconstructed using the using the neighbor- bacterial core gene set (UBCG v.3) [34]. joining and maximum parsimony algorithms indicated that M105T and members of the genera Aliikangiella, Pleionea and Genome sequences for Kangiella koreensis KCTC 12182T Kangiella form a large clade, where M105T and members of (CP001707), Kangiella profundi FT102T (CP025120), the genera Aliikangiella and Pleionea form one branch, and Kangiella sediminilitoris KCTC 23892T (CP012418), 2 Wang et al., Int. J. Syst. Evol. Microbiol. 2020 Streptomyces albus subsp. albus NRRLF-4971 (NZ_JOID01000001) 92 Halomonas elongata HEK1 (NZ_MAJD01000001) Halomonadaceae Halotalea alkalilenta IHB B 13600 (CP015243) Alcanivorax pacificus W11-5T (CP004387) 91 92 Alcanivorax dieselolei B-5T (NC_018691) Alcanivorax xenomutans P40 (CP012331) 92 Alcanivorax hongdengensis A-11-3T (AMRJ01000001) Alcanivoraceae T 88 Alcanivorax nanhaiticus 19-m-6 (ARXV01000001) Alcanivorax borkumensis SK2T (NC_008260) 92 90 92 Alcanivorax jadensis T9T (ARXU01000001) Saccharospirillum