Leptodesmis xinxiangensis Y. Zhang et Y. Liu, 2024

Liu, Yang, Zhang, Yang, Wang, Xue, Geng, Ruozhen & Zhang, Bangjun, 2024, Description of a novel filamentous cyanobacterial species of Leptodesmis (Leptolyngbyaceae, Cyanobacteria) in China based on the polyphasic approach, Phytotaxa 678 (2), pp. 96-108 : 100-104

Leptodesmis xinxiangensis Y. Zhang et Y. Liu sp. nov. ( Fig. 1 )

Description: The strain was observed to exhibit filamentous characteristics under an optical microscope. The filaments appeared long, straight, or slightly curved, and varied in color from blue–green to dark green. Electron microscopy revealed that the ultrastructure consisted of cylindrical cells, measuring approximately 1.1–1.7 μm in width and 1.2– 2.0 μm in length. The cells were generally longer than they were wide, although some were occasionally shorter than their width. The cell wall was notably thick, featuring a solid, transparent sheath with multiple filaments arranged within the thick sheath ( Fig. 2 ). The transverse walls between the cyanobacterial cells were slightly constricted, and necrotic cells were observed. Some cell centers contained granules. The thylakoids were organized in a ring formation, with each cell containing 6–8 layers of thylakoids that ran parallel to the intracellular periphery. The growth pattern in BG11 medium was a blue–green cyanobacterial mat.

Holotype (here designated): Dry material of strain Leptodesmis xinxiangensis HNU2023 was preserved in the Herbarium of Wenzhou University ( WZUH), Wenzhou, Zhejiang Province, China, as specimen No. WZUH- ZLXX202301 .

Type locality: In Xinxiang, Henan Province, China. (April 2023, 113°30′– 115°01′ E, 34°55′– 35°50′N).

Reference strain: The living culture was preserved in the Laboratory No. 404 of Life of Sciences, Henan Normal University, Henan Province, China. The strain number is HNU 2023.

Etymology: This strain was isolated from Xinxiang city where xinxiangensis refered to and transliterated into Latin.

Habitat: Adhesion grows in the freshwater area.

Diagnosis: It differs from Leptodesmis paradoxa in its narrower cells and from L. sichuanensis and L. alaskaensis in its shorter cells. A broader range of cell lengths was observed in L. alaskaensis and L. lacustris , whereas L. xinxiangensis exhibited medium cell length. Meanwhile, L. xinxiangensis showed moderate cell width and length compared to other representatives of Leptodesmis . L.sichuanensis A121 and L. alaskaensis are motile, whereas L. xinxiangensis , L. paradoxa and L. lacustris are immotile. Leptodesmis xinxiangensis was also distinguished based on its phylogenetic position.

Molecular and Phylogenetic Analysis

The target gene fragment of the filamentous cyanobacteria HNU2023 was amplified using the above primers, and the 16S rRNA, ITS and partial 23S rRNA sequences with a full length of 2177 bp were obtained. The 16S rRNA sequence of HNU2023 was compared with that of the online alignment tool BLAST at NCBI. The similarity between Leptodesmis xinxiangensis HNU2023 and the four species of Leptodesmis in this study was 95.67–96.81% (Supplementary Table S1), which was higher than the 95% threshold defining bacterial genus classification and lower than the 97.5% threshold defining bacterial species classification ( Hoffmann et al., 2005). Sequences of the HNU2023 and Cf. Leptolyngbya sp. CCNUM2 (isolated from humid moss on forest limestone in Wuhan, China) ( Zhang et al., 2019) and Cf. Leptolyngbya sp. Greenland 9 (isolated from Arctic hot springs in Greenland) ( Roeselers et al., 2007) were found to have similarities of between 99.63% and 100% (Supplementary Table S1), thereby indicating a high probability that the uncultured cyanobacterium and Leptodesmis xinxiangensis HNU2023 were strains of the same species.

A phylogenetic tree of the 16S rRNA gene was constructed based on the neighbor-joining method (NJ), maximum likelihood method (ML) and Bayesian methods (BI). As shown in Fig. 3 , the sequences of HNU2023 and the four species of the genus Leptodesmis were clustered into a phylogenetic branch, with a high bootstrap support value (100/73/1.00), indicating that HNU2023 belongs to the genus Leptodesmis and may represent a new species of the filamentous cyanobacteria Leptodesmis .

Analyses of ITS between 16S and 23S rRNA Gene and Secondary Structures

In this study, the partial 16S-23S ITS sequence of HNU 2023 was obtained with a total length of 524 bp ( Table 1), and they were used, together with seven species of the five genera Leptodesmis , Kovacikia , Alkalinema , Chroakolemma and Stenomitos downloaded from NCBI, to construct the ITS secondary structures. The ITS region can be used as a tool to distinguish closely related organisms ( Caires et al., 2018; Pietrasiak et al., 2014). In general, all the sequences contained both tRNAIle and tRNAAla ( Table 1).

Analyses of D1-D1′ helix ( Fig.4 ) of the studied strains showed unique structure and different nucleotides in comparison with other Leptodesmis species. The eight related species were classified into eight types. The D1-D1′ helix structure of the strain HNU2023 showed that the basal stem consisted of a 5-bp helix, followed by a 1:8-bp bilateral bulge, two small unidirectional bulges, and then further followed by two bilateral bulges of 4:5- and 3:2-bases, the terminal loop contained 5-bp bases.

The Box-B helix ( Fig.5 ) of strain HNU2023 was similar to that of several species of similar species, such as Leptodesmis alaskaensis BACA 0748, Leptodesmis sichuanensis A121 and Chroakolemma opaca 701 clone 2 in the basal and apical stem-loop. There was a 5-bp helix, followed by a 1:2 bp-base bilateral bulge, in the basal stem of strain HNU2023, and the apical structures contained a 5-bp helix with a 4-bp loop.

No regular patterns were found for the V3 helices between HNU2023 and the other seven filamentous species ( Fig.6 ). The studied strain HNU2023 had a 3-bp helical basal stem, followed by four bilateral bulges (4:4-, 1:1- and two 1:2-bases), and the apical structures contained a 9-bp helix with a 3-bp loop, which significantly differed from that of species.

Figures

FIGURE 1. Light microscopy of Leptodesmis xinxiangensis strains. (a) Randomly entangled trichomes. (b,c) Filaments without sheaths. (d-f) Filaments with colorless sheaths. Trichome fragmentation and formation of necridia. Scale bars: 10 μm.

FIGURE 2. Ultrastructure of Leptodesmis xinxiangensis strains. (a) Transverse section. (b–d) Longitudinal sections. Cw: cell wall, Th: thylakoids, Sh: sheath. Scale bars: 500 nm.

FIGURE 3. Maximum Likelihood (ML) phylogenetic tree of Leptodesmis xinxiangensis HNU2023 based on 16S rRNA gene sequences. Bootstrap values greater than 50% are showed on the ML tree for NJ/ML methods and Bayesian posterior probabilities. “*” indicates bootstrap values of 100 in ML and NJ and BI posterior probabilities of 1.00. The novel filamentous strains of this study indicate in bold. Bar, 0.04.

FIGURE 4. D1-D1' helix in Leptodesmis xinxiangensis and other seven related species. (a). Leptodesmis xinxiangensis HNU2023. (b). Leptodesmis lacustris BACA0204. (c) Leptodesmis alaskaensis BACA0748. (d). Leptodesmis sichuanensis A121. (e). Kovacikia muscicola HA7619-LM3. (f). Alkalinema pantanalense CENA528. (g). Chroakolemma opaca 701 clone 2. (h). Stenomitos rutilans HA7619-LM2.

FIGURE 5. Box-B helix in Leptodesmis xinxiangensis and other seven related species. (a). Leptodesmis xinxiangensis HNU2023. (b). Leptodesmis lacustris BACA0204. (c). Leptodesmis alaskaensis BACA0748. (d). Leptodesmis sichuanensis A121. (e). Kovacikia muscicola HA7619-LM3. (f). Alkalinema pantanalense CENA528. (g). Chroakolemma opaca 701 clone 2. (h). Stenomitos rutilans HA7619-LM2.

FIGURE 6. V3 helix in Leptodesmis xinxiangensis and other seven related species. (a). Leptodesmis xinxiangensis HNU2023. (b). Leptodesmis lacustris BACA0204. (c). Leptodesmis alaskaensis BACA0748. (d). Leptodesmis sichuanensis A121. (e). Kovacikia muscicola HA7619-LM3. (f). Alkalinema pantanalense CENA528. (g). Chroakolemma opaca 701 clone 2. (h). Stenomitos rutilans HA7619-LM2.