Two new non-chlorophyll f-producing species in the Kovacikia genus (Leptolyngbyaceae, Leptolyngbyales) from the Poyang Lake Basin, China

For the filamentous cyanobacteria, the order Leptolyngbyales is the larger and obviously polyphyletic group, although its members are morphologically similar. In this study, three Leptolyngbya-like strains were isolated from the soil of three different habitats in the Poyang Lake Basin, China—dark microhabitats similar to those found in far-red light (FRL) habitats. The three strains were phylogenetically identified as Kovacikia diezihuensis sp. nov. and Kovacikia jiangxiensis sp. nov. (Leptolyngbyaceae, Leptolyngbyales) using a polyphasic approach, respectively. The phylogenetic tree showed that they clustered within the Kovacikia genus (species type: Kovacikia muscicola) with five other Kovacikia species. The 16S rRNA gene sequences of K. diezihuensis and K. jiangxiensis shared 97.2% similarity with each other (data not shown) and 94.9–96.8% similarity with five other Kovacikia species. Furthermore, the secondary structures of the internal transcribed spacer (ITS) regions and ITS sequences exhibited uniqueness. The two species were similar to the other five Kovacikia species in morphology, but K. diezihuensis was bright blue-green, and K. jiangxiensis was gray-green to blue-green in color, and its length was usually greater than its width in cells under white light, respectively. Pigment analysis showed that the two strains did not produce phycoerythrin. FRL adaptation experiments further showed that they could neither grow nor produce chlorophyll (Chl) f under FRL. In summary, K. diezihuensis and K. jiangxiensis were new non-Chl f-producing species in the Kovacikia genus. This is the first report of both Chl f-producing and non-Chl f-producing species in the same genus within the Leptolyngbyales, shedding light on the diversity and the evolutionary divergence of Chl f-producing cyanobacteria.

1 Introduction

Cyanobacteria, as a highly diverse group, have long faced challenges in phylogenetic studies at the genus and subgenus levels (Hoffmann et al., 2005). In the past, classifying cyanobacteria solely based on their morphological characteristics led to a serious underestimation of cyanobacterial diversity (Taton et al., 2003; Komárek, 2006; Engene et al., 2011). Among them, filamentous cyanobacteria are particularly difficult to classify due to their subtle morphological differences and diverse species. The introduction of molecular genetics methods and electron microscopy has fundamentally driven the transformation of cyanobacteria classification (Komárek et al., 2014; Komárek, 2016). For example, Komárek et al. (2014) revised the classification of cyanobacteria, dividing Oscillatoriales into Oscillatoriales and Synechococcales, based on the preliminary results of phylogenetic analyses and the ultrastructural patterns of thylakoids. The filamentous cyanobacteria, Leptolyngbya (Anagnostidis and Komárek, 1988), are therefore classified into the new family Leptolyngbyaceae, Synechococcales. In recent years, with the increasing number of reference strains of currently identified cyanobacteria and well-described genome sequences, Strunecký et al. (2023) reconstructed a robust phylogenomic tree to classify the entire cyanobacteria phylum, including 10 new orders and 15 new families. Therefore, many Leptolyngbya-like cyanobacteria no longer belong to the Synechococcales but to Leptolyngbyales (including Leptolyngbyaceae, Trichocoleusaceae, and Neosynechococaceae), Oculatellales, and Nodosilineales.

Leptolyngbya is a larger and obviously polyphyletic group, but it is almost impossible to separate it based on morphological characteristics (Komárek, 2016). A polyphasic approach combining ecological, molecular, and morphological data has been more recently applied, providing more evidence for the genera and species levels classification (Iii et al., 2011; Vaz et al., 2015; Engene et al., 2018). The traditional definition of Leptolyngbya actually includes several distinct and not closely related phylogenetic clusters, some of which have been formally identified as new genera separated from Leptolyngbya by polyphasic approach, such as Phormidesmis (Turicchia et al., 2009; Raabová et al., 2019), Tapinothrix (Bohunická et al., 2011), Alkalinema (Vaz et al., 2015), Pantanalinema (Vaz et al., 2015), Scytolyngbya (Song et al., 2015), Limnolyngbya (Li and Li, 2016), Kovacikia (Miscoe et al., 2016; Shen et al., 2022; Kaštovský et al., 2023), Stenomitos (Miscoe et al., 2016; Shalygin et al., 2020), Chamaethrix (Dvořák et al., 2017), Onodrimia (Jahodářová et al., 2017), Chroakolemma (Becerra-Absalón et al., 2018), Myxacorys (Pietrasiak et al., 2019; Soares et al., 2019), Leptodesmis (Raabová et al., 2019; Strunecky et al., 2020; Tang et al., 2022; Luz et al., 2023; Kaštovský et al., 2023; Shen et al., 2024), Apatinema (Davydov and Vilnet, 2022), Romeriopsis (Hentschke et al., 2022), Khargia (Rasouli-Dogaheh et al., 2023), Pseudoleptolyngbya (Hentschke et al., 2024), and Leptolyngbyopsis (Hentschke et al., 2024). It is worth noting that there are relatively few chlorophyll (Chl) f-producing studies in the aforementioned cyanobacteria.

Chl f was the newest member of the Chl family, and was a far-red light (FRL) induced Chl (Chen et al., 2010, 2012). Cyanobacteria restructured their photosynthetic apparatus and produced Chl f through a complex and extensive light adaptation reaction called FRL photoadaptation (FaRLiP), greatly improving their photosynthetic performance under FRL and playing an important ecological role as primary producers under FRL environment (Gan et al., 2014; 2015; Li et al., 2016; Kato et al., 2020; Gisriel et al., 2020, 2022a, 2022b). At present, it is known that Chl f only exists in some cyanobacteria, and these cyanobacteria that can produce Chl f including Halomicronema (Li et al., 2014), Aphanocapsa (Akutsu et al., 2011; Miyashita et al., 2014; Zhang et al., 2019), Chlorogloeopsis (Airs et al., 2014; Gan et al., 2014), Leptolyngbya (Gan et al., 2014; Ohkubo and Miyashita, 2017; Zhang et al., 2019), Calothrix (Gan et al., 2014), Fischerella (Gan et al., 2015), Synechococcus (Gan et al., 2015), Chroococcidiopsis (Gan et al., 2015; Zhang et al., 2019), Altericista (Averina et al., 2021), “Leptothermofonsia” (Tang et al., 2022), Kovacikia (Shen et al., 2022), Elainella (Shen et al., 2023), Pegethrix (Shen et al., 2023), and Leptodesmis (Shen et al., 2024). This indicates that Chl f-producing cyanobacteria possess significant diversity and warrant further taxonomic research. Recently, a polyphasic approach has also been gradually applied to the classification of Chl f-producing cyanobacteria, and some new species have been successfully identified, such as Altericista variichlora (Averina et al., 2021), Kovacikia minuta (Shen et al., 2022), Pegethrix sichuanica, and Elainella chongqingensis (Shen et al., 2023), and four new species of the Leptodesmis genus (Shen et al., 2024).

In this study, three Leptolyngbya-like cyanobacterial strains were isolated from the Poyang Lake Basin, China. Their preliminary molecular data showed they are genetically diverse, forming a new clade in Kovacikia that deserves further investigation regarding their taxonomic status. Therefore, a polyphasic approach was adopted to classify the three strains based on comprehensive morphological characters, 16S rRNA gene sequences phylogeny and sequence similarity, internal transcribed space (ITS) secondary structure, and ecological characters. Finally, these three strains were classified as Kovacikia diezihuensis sp. nov. (reference strain, ACCP0342) and Kovacikia jiangxiensis sp. nov. (reference strain, ACCP0444) by a polyphasic approach. Since the three strains were isolated under white light (WL), it is speculated that they might lack the ability to produce Chl f under FRL. Therefore, FRL adaptation experiments were designed, revealing that K. diezihuensis and K. jiangxiensis could neither produce Chl f nor grow under the induction of FRL. Up to now, five species have been reported in the Kovacikia genus, including Kovacikia muscicola (type species) (Miscoe et al., 2016), K. minuta (Shen et al., 2022), Kovacikia anagnostidisii (Kaštovský et al., 2023), Kovacikia brockii (Kaštovský et al., 2023), and Kovacikia atmophytica (Luz et al., 2023). Following the discovery of Chl f-producing species K. minuta in 2022, this study found that K. diezihuensis and K. jiangxiensis were the only two new species in the Kovacikia genus that do not produce Chl f. This discovery indicates that Chl f-producing and non-Chl f-producing cyanobacteria can belong to different species at the same genus level in the Lepolyngbylales. This provides significant clues for further exploring the evolutionary divergence of Chl f-producing cyanobacteria and their adaptability to the environment.

2 Materials and methods

2.1 Strain isolation, purification, and maintenance

The samples used in this study were isolated from the soil surface under a bamboo forest in the corner of a wall at Diezihu Avenue, Nanchang City, Jiangxi Province (28°41′12.13″N, 115°50′06.57″E), moss-like soil in the corner of a wall at Diezihu Avenue, Nanchang City, Jiangxi Province (28°41′12.32″N, 115°50′05.56″E), and dried fluffy soil in the corner of Jiangxi Normal University, Nanchang City, Jiangxi Province (28°41′0.786″N, 116°1′50.467″E) (Figure 1, Table 1). These were shadowy environments similar to an FRL habitat.

Figure 1

Figure 1. Strain habitats. (A) (28°41′12.13″N, 115°50′06.57″E), the soil under the bamboo forest in the corner at Diezihu avenue, Nanchang City, Jiangxi Province; (B) (28°41′12.32″N, 115°50′05.56″E), moss-like soil in the corner of the wall at Diezihu Avenue, Nanchang City, Jiangxi Province; (C) (28°41′0.786″N, 116°1′50.467″E), dried fluffy soil in the corner of Jiangxi Normal University, Nanchang City, Jiangxi Province.

Table 1

Table 1. Basic information on sample collection and pure algae strains.

The appropriate samples were added to 100 mL sterile BG11 culture medium (Ichimura, 1979) in the 250 mL conical flask and incubated at 25 °C and 5–10 μmol photons m⁻² s⁻¹ under WL until the culture appeared. Then the cultures were homogenized to disperse the cells as evenly as possible and purified using the dilution spread plate method on a 0.8% solid BG11 plate. Finally, unialgal strains were successfully isolated and expanded in BG11 culture medium. The purity of the isolated strains was determined using an inverted microscope (Olympus, Tokyo, Japan). The pure strains were maintained in the 250 mL conical flask containing 120 mL BG11 medium under WL, respectively, at the Algae Culture Collection of Poyang Lake (ACCP), Jiangxi Key Laboratory of Flood and Drought Disaster Defense, Jiangxi Academy of Water Science and Engineering (JAWSE).

2.2 PCR amplification and phylogenetic analysis

Cyanobacterial genomic DNA was extracted using the modified version of the CTAB method (Neilan et al., 1995). The oligonucleotide primers of PA (5′-AGAGTTTGATCCTGGCTCAG-3′) and B23S (5′-CTTCGCCTCTGTGTGCCTAGGT-3′) were used to amplify the 16S rRNA gene and the complete 16S–23S rRNA internal transcribed spacer (ITS) region (Edwards et al., 1989; Lepère et al., 2000). The polymerase chain reaction (PCR) reaction system comprised 25 μL of PCR Mix 2 × Taq Plus Master Mix II (Dye Plus, P213-01/02/03, Vazyme, Nanjing, China), 2 μL of each forward and reverse primer, 2 μL of DNA, and sterile water to a final volume of 50 μL. The thermal cycling conditions were set to an initial denaturation step at 95 °C for 5 min; 35 cycles of DNA denaturation at 95 °C for 30s, primer annealing at 58 °C for 30 s, and strand extension at 72 °C for 2 min 30 s; and a final extension at 72 °C for 10 min. The PCR products were detected by electrophoresis and were then recovered. The recovered products were ligated with the pMD™ 18-T vector (Takara, Japan) and then transfected into competent Escherichia coli cells. Cloning experiments were performed according to the manufacturer’s protocol. Finally, positive single clones were sequenced by Sangon Biotech (Shanghai, China). The sequences were processed using BioEdit 7.2.5 (Hall, 1999) software and uploaded to NCBI GenBank, with accession numbers PQ881585, PQ881586, and PQ895252, respectively.

The 16S rRNA gene sequences were aligned using BioEdit 7.2.5 (Hall, 1999). The best-fit models under the Akaike Information Criterion (AIC), estimated by ModelFinder (Kalyaanamoorthy et al., 2017), were adopted for both Bayesian inference (BI) and maximum likelihood (ML) analyses, respectively. The particular parameters of the substitution model for BI and ML were individually estimated using MrBayes version 3.2.6 (Ronquist et al., 2012) and IQ-TREE version 1.5.6 (Nguyen et al., 2015), respectively. For the BI analysis, two independent runs of four Markov chains were executed for 10 million generations with sampling every 100 generations. A total of 1,000 bootstrap replicates were conducted to evaluate the relative support of branches. Bootstrap analysis was performed with 1,000 replicates for the ML phylogenetic tree to estimate the degree of confidence for each branch node. Phylogenetic relationships of the 16S rRNA genes were also analyzed by neighbor-joining (NJ) phylogenetic trees (Shen et al., 2023). Gloeobacter violaceus PCC 7421 was used as the outgroup. The three different methods yielded the same phylograms on the branches that gave the bootstrap values. FigTree version 1.5.6 was used to visualize the phylogenetic tree (Nguyen et al., 2015). Bootstrap values greater than 70% with BI/ML/NJ methods are shown in the Bayesian phylogenetic tree. The sequence similarity of the 16S rRNA gene was calculated in MEGA.

2.3 Analysis of ITS secondary structure

The 16S–23S rRNA sequences were aligned using BioEdit 7.2.5 (Hall, 1999) to determine similarity and dissimilarity percentages. The sequences of 16S–23S rRNA ITS regions were also utilized for taxonomic resolution of the strains under investigation at the species level. The complete ITS sequences for these strains were aligned with the corresponding sequences, and different conserved and variable regions were identified in accordance with the method described by Iteman et al. (2000). The secondary structure of the ITS regions, such as D1–D1’, Box-B, and V3 helices, were predicted with the m-Fold web server (Zuker, 2003), and each fragment was folded individually. Default parameters were used in this study.

2.4 Morphological and ultrastructural examination

An appropriate amount of homogenate of algal filaments was added to the fresh liquid BG11 medium and grown for approximately two weeks at 25 °C and 5–10 μmol photons m⁻² s⁻¹ under WL. The cultures were examined using a Nikon Eclipse 80i light microscope with an external digital camera of Nikon DS-Ri1 (Tokyo, Japan). At least 100 measurements were obtained for cell length and width from at least 10 different algal filaments. Colony morphology was recorded using a Nikon D5600 digital camera. The subcellular ultrastructure was examined using transmission electron microscopy (TEM) (Hitachi HT-7700, Tokyo, Japan) according to a previously described method (Shen et al., 2022).

2.5 Pigment compositions of the new isolates

The absorption spectra of cultured algal filaments in good condition, as described above, including the cyanobacterial culture and lipid-soluble and water-soluble pigments, were analyzed in the range of 400–800 nm using an Integrating Sphere Ultraviolet (UV) Spectrophotometer (Specord 210 Plus; Analytik Jena, Jena, Germany). All operations were conducted under dim or dark conditions. Water-soluble pigments were extracted by phosphate buffer with appropriate modifications (Bennett and Bogorad, 1973; Shen et al., 2024). Lipid-soluble pigments were extracted by methanol with appropriate modifications (Zhang et al., 2019). The algae were centrifuged at 4 °C, 13,200g for 2 min, and the resulting supernatant was removed, and the algal pellet was retained. Fifty percent methanol was added, the samples were mixed, and the supernatant was removed after centrifugation. Pre-cooled (at −20 °C) 100% methanol was added, and the algae were extracted overnight at −20 °C. After centrifugation at 4 °C, 13,200g for 2 min, the supernatant was collected for absorption spectrum analysis of lipid-soluble pigments.

3 Results

3.1 Phylogenetic evaluation

Partial sequences of the 16S rRNA gene of K. diezihuensis ACCP0342 and K. jiangxiensis ACCP0444 were obtained. The phylogenetic tree based on 16S rRNA gene sequences contained 84 nucleotide sequences with a total of 1,185 nt positions in the final dataset, further revealing the phylogenetic relationship between these two strains and their sister taxa (Figure 2). From the phylogenetic tree based on 16S rRNA gene sequences, ACCP0342, ACCP0444, K. muscicola, K. minuta, “Leptothermofonsia sichuanensis,” K. anagnostidisii, K. brockii, and K. atmophytica were clustered into a group, and assigned to the Kovacikia genus, while other sister taxa Chroakolemma, Pantanalinema, and Stenomitos were significantly separated into individual lineages at the genus level (Figure 2) (Becerra-Absalón et al., 2018; Vaz et al., 2015; Miscoe et al., 2016; Shalygin et al., 2020). Leptothermofonsia Daroch, Tang and Shah 2022 was merge to Kovacikia Miscoe, Pietrasiak and Johansen 2016, while Leptothermofonsia is not considered a valid name, due to improper designation of the holotype (Miscoe et al., 2016; Tang et al., 2022; Kaštovský et al., 2023). Therefore, these sequences of “L. sichuanensis” are only used as background references for the Kovacikia genus.

Figure 2

Figure 2. Bayesian phylogenetic tree based on the 16S rRNA gene sequences showing the relationships of Kovacikia diezihuensis ACCP0342 and Kovacikia jiangxiensis ACCP0444 (bold part) with their similar taxa. The nodes marked by black dots were genus horizontal, and the black square nodes were horizontal nodes of the Leplyngbyaceae family. Bootstrap values greater than 70% were given in front of the corresponding nodes for BI/ML/NJ phylogenetic analysis. The scale bar represents the rate of nucleotide substitutions per site.

Comparing the 16S rRNA gene sequences of K. diezihuensis (1,481 bp) with K. jiangxiensis (1,482 bp) separately, the similarity was 97.2% (data not shown). Cyanobacteria with 98.7% or less 16S rRNA gene sequence similarity may be considered different species (Yarza et al., 2014). Compared with K. minuta, the 16S rRNA gene sequences of ACCP0342 and ACCP0444 shared 96.8 and 95.7% similarities, respectively (Table 2). The 16S rRNA gene sequences (Table 2) of K. diezihuensis and K. jiangxiensis showed 94.9%–96.8% similarity with those of the other five Kovacikia species, indicating that they are distinct species within the same genus (Stackebrandt and Goebel, 1994; Sciuto et al., 2012; Kim et al., 2014; Komárek et al., 2014; Yarza et al., 2014; Sciuto and Moro, 2016; Eckert et al., 2015).

Table 2

Table 2. Similarities of the 16S rRNA gene sequences between the representative strains in this study (black bold part) and related sister taxa in the Kovacikia genus.

3.2 16S-23S ITS analysis

The lengths of 16S–23S rRNA ITS sequences of ACCP0342 and ACCP0444 strains were 493 bp and 488 bp, with two types of tRNA: tRNA^Ile (74 bp) and tRNA^Ala (73 bp), respectively. The 16S–23S rRNA ITS sequences of the five representative strains, including the K. muscicola HA7619-LM3 clone 41A (KU161669.1), K. minuta CCNU0001 (OK110207.1), K. anagnostidisii YS86-RH1 (OR236708.1), K. brockii YNP74-RH1 clone 1 (OR220391.1), and K. atmophytica BACA0619 (OM732256.1), were complete with 617, 610, 550, 519, and 602 bp, respectively. The percent dissimilarity among 16S–23S rRNA ITS region has been shown to be very effective at establishing cyanobacterial species (Boyer et al., 2001; Komárek et al., 2014; Osorio-Santos et al., 2014; Kaštovský et al., 2023). Compared with K. minuta, the 16S-23S ITS sequences of ACCP0342 and ACCP0444 shared 38.9 and 38.2% similarities, respectively (data not shown). The 16S–23S rRNA ITS sequences of ACCP0342 and ACCP0444 strains showed 10.6% divergences with each other and 33.9 to 38.9% divergences with the other five Kovacikia species (data not shown), which matched the thresholds for different species (Becerra-Absalón et al., 2018; González-Reséndiz et al., 2018; Mai et al., 2018; Vázquez-Martínez et al., 2018; Mareš et al., 2019; Pietrasiak et al., 2019; Becerra-Absalón et al., 2020).

The secondary structures of representative regions of 16S–23S rRNA ITS, namely, D1–D1’, Box-B, and V3 helices, are shown in Figure 3. ACCP0342 and ACCP0444 strains have similar lengths of D1–D1’ helix with 62 nucleotides. All strains had a basal stem (GACCU-AGGUC), with little difference in the middle and lower parts of the D1–D1’ helix. The main difference was concentrated at the top, where only three nucleotides differed between ACCP0342 and ACCP0444.

Figure 3

Figure 3. The representative regions of 16S-23S ITS secondary structure of D1-D1’, Box-B, and V3 helices about Kovacikia diezihuensis ACCP0342, Kovacikia jiangxiensis ACCP0444 strains, and other strains of Kovacikia species.

The length of the Box-B helix was 37–47 nucleotides. About the length of the Box-B helix, ACCP0342 was the shortest with 37 nucleotides, and ACCP0444 was 41 nucleotides, which was the same as K. muscicola HA7619-LM3 clone 41A (KU161669.1) and K. atmophytica BACA0619 (OM732256.1). The Box-B helix was partially identical at the bottom part (AGCA-UGCU), and the difference was greater in the upper part, especially at the top.

The V3 helix varied greatly overall in length (range 40–110 nucleotides) and secondary structure conformation. The V3 helix was absent in K. anagnostidisii YS86-RH1 (OR236708.1) (Figure 3). The V3 region of K. anagnostidisii YS86-RH1 (OR236708.1) was incomplete and contained 88 nucleotides, and its tail portion was missing, resulting in the failure to form a stable helical structure (Figure 3). The V3 helix of ACCP0342 was most similar to ACCP0444; both of them were short, 40 and 41 nucleotides in length, respectively, with only five base differences, and concentrated at the top. All V3 helices had the same basal stem structure (GUCAGGU-ACAGAC), and the V3 helices of ACCP0342 and ACCP0444 were generally very different from the other strains on the whole.

In summary, based on the differences in the 16S–23S rRNA ITS sequences and their secondary structures, ACCP0342 and ACCP0444 exhibited uniqueness to be distinguished as different species.

3.3 Morphological description

Kovacikia diezihuensis L.-Q. Shen & R. Li sp. nov. (Figures 4A–I).

Figure 4

Figure 4. Morphological characteristics of Kovacikia diezihuensis ACCP0342 and Kovacikia jiangxiensis ACCP0444 strains on the white light (WL). (A,J) Morphological characteristics in a conical flask containing BG11 medium. (B–G) and (K–P) Differential interference contrast microscope images. (B) and (K) scale bar, 50 μm. (C–G) and (L–P) scale bar, 5 μm. (H,I,Q,R) Transmission electron micrographs. (H,I) Scale bar, 1 μm; (Q,R) Scale bar, 500 nm.

Diagnosis: Its color was bright blue-green under WL, which was different from other Kovacikia species. Its 16S rRNA gene sequence had a similarity of 97.2% with K. jiangxiensis ACCP0444, and a similarity of 95.5%–96.8% with five other species in the Kovacikia genus. It had unique secondary structures in the ITS region.

Description: In liquid culture, the filaments extended and entangled to form thin to slightly thick mats, most of which were attached to the bottom or small parts, and were clustered on the liquid culture surface and in contact with the bottle body. The color was bright blue-green under WL. The filaments were elongated, occasionally slightly curved, without false branches, heterocysts, and akinetes. The sheath was colorless, slightly thin, and occasionally visible. The trichomes were not attenuated to the ends, slightly constricted at the crosswalls, and occasionally had necridia. The cells were cylindrical, with rounded apical cells, without calyptras; sometimes small circular particles were observed in the center of the cells. The cells were isodiametrical or had a length slightly greater than or slightly less than their width, with 0.90–2.92 μm (mean 1.67 μm) length and 1.46–2.21 μm (average 1.86 μm) width. The cells had 4–6 layers of parietal thylakoid membrane. Reproduction occurred by hormogonia, trichome breakage via necridia, and subsequent disintegration, then releasing hormogonia.

Etymology: The specific epithet “diezihuensis” refers to the collection location of K. diezihuensis from Diezihu Avenue, Nanchang City, Jiangxi Province, China.

Holotype: ACCP-ZLJX20210342, a packet consisting of culture material of the ACCP0342 strain preserved at 4% formaldehyde in a 10 mL centrifuge tube and dried biomass of the same strain preserved in 2 mL frozen storage tubes, deposited at the ACCP, Jiangxi Key Laboratory of Flood and Drought Disaster Defense, JAWSE.

Isotype: WZUH-ZLJX20210342, a packet consisting of culture material of the ACCP0001 strain preserved at 4% formaldehyde in a 10 mL centrifuge tube and dried biomass of the same strain preserved in 2 mL frozen storage tubes, deposited at the College of Life and Environmental Sciences, Wenzhou University.

Reference strain: ACCP0342. Culture deposited at ACCP, Jiangxi Key Laboratory of Flood and Drought Disaster Defense, JAWSE.

Habitat and type locality: The strain was isolated from soil under the bamboo forest in the corner of a wall at Diezihu Avenue, Nanchang City, Jiangxi Province (28°41′12.13″N, 115°50′06.57″E).

Other strain, habitat, and locality: Strain ACCP0340 was isolated from moss-like soil in the corner of a wall at Diezihu Avenue, Nanchang City, Jiangxi Province (28°41′12.32″N, 115°50′05.56″E).

Kovacikia jiangxiensis L.-Q. Shen & R. Li sp. nov. (Figures 4J–R).

Diagnosis: Its color was gray-green to blue-green in color, and the cell length was usually longer than the width under the WL, which was different from other Kovacikia species. Its 16S rRNA gene sequence had a similarity of 97.2% with K. diezihuensis ACCP0342, and a similarity of 94.9%–96.2% with five other species in the Kovacikia genus. It had unique secondary structures in the ITS region.

Description: In liquid culture, the filaments extended and entangled to form thin to slightly thick mats. Small parts were attached to the bottom, whereas large clusters of filaments floated on the liquid culture surface. The color was gray-green to blue-green under WL. The filaments were elongated, occasionally slightly curved, without false branches, heterocystes, and akinetes. The sheath was colorless, slightly thin, and occasionally visible. The trichomes were not attenuated to the ends, slightly constricted at the crosswalls, and occasionally had necridia. The cells were cylindrical, with rounded apical cells, without calyptras. The cells, usually with a length greater than their width, measure 1.11–2.92 μm (mean 1.92 μm) long and 0.96–1.49 μm (average 1.17 μm) wide. The cells had 3–5 layers of parietal thylakoid membrane. Reproduction occurred by hormogonia, trichome breakage via necridia and subsequent disintegration, then releasing hormogonia.

Etymology: The specific epithet “jiangxiensis” refers to the collection location of K. jiangxiensis from Jiangxi Normal University, Nanchang City, Jiangxi Province, China.

Holotype: ACCP-ZLJX20210444, a packet consisting of culture material of the ACCP0444 strain preserved at 4% formaldehyde in a 10 mL centrifuge tube and dried biomass of the same strain preserved in 2 mL frozen storage tubes, deposited at the ACCP, Jiangxi Key Laboratory of Flood and Drought Disaster Defense, JAWSE.

Isotype: WZUH-ZLJX20210444, a packet consisting of culture material of the ACCP0444 strain preserved at 4% formaldehyde in a 10 mL centrifuge tube and dried biomass of the same strain preserved in 2 mL frozen storage tubes, deposited at the College of Life and Environmental Sciences, Wenzhou University.

Reference strain: ACCP0444. Culture deposited at ACCP, Jiangxi Key Laboratory of Flood and Drought Disaster Defense, JAWSE.

Habitat and type locality: The strain was isolated from dried fluffy soil in the corner of Jiangxi Normal University, Nanchang City, Jiangxi Province, China (28°41′0.786″N, 116°1′50.467″E).

3.4 Pigment analysis

The absorption spectra for ACCP0342 and ACCP0444 strains under WL are shown in Figure 5. The pigments mainly included Chl a, phycocyanin (PC), and carotenoids by the cell absorption spectrum (Figures 5A,D). The water-soluble pigment analysis (Figures 5B,E) showed that ACCP0342 and ACCP0444 strains produced a large amount of PC (absorption peak around 618 nm under WL), but did not produce phycoerythrin (PE). Based on the absorption spectrum of lipid-soluble pigments (Figures 5C,F), ACCP0342 and ACCP0444 strains produced Chl a (absorption peak around 667 nm in methanol under WL). The two strains could not produce red-shifted complexes and Chl f induced by FRL and could not grow under FRL (Figure 5).

Figure 5

Figure 5. Pigment absorption spectra of Kovacikia diezihuensis ACCP0342 (A–C) and Kovacikia jiangxiensis ACCP0444 (D–F) strains. The solid line shows white light (WL). (A,D) Cell absorption spectrum was homogenized according to the absorption peak of Chl a (680 nm); (B,E) Absorption spectra of water-soluble pigments were homogenized with the absorption peak of PC (618 nm); (C,F) Fat-soluble absorption spectrum, homogenized with the absorption peak of Chl a in methanol (667 nm).

4 Discussion

According to previous reports, the species of Kovacikia were widely distributed, being found in Jiangxi Province, Hubei Province and Sichuan Province in China; across the Eurasian continent to the Azores Archipelago (Portugal) in the Atlantic Ocean, and across Yellowstone National Park (USA) in North America, as well as the Hawaiian Islands in the Pacific Ocean (Miscoe et al., 2016; Shen et al., 2022; Kaštovský et al., 2023), which across through the whole Northern Hemisphere, and are all in the mid-latitude zone (Table 1). This also illustrates the geographical universality of species in the Kovacikia genus. Despite their geographically distant distribution, their ability to reproduce in a diverse range of habitats, including freshwater, atmophytic environment attachment beneath plants, cave walls, soil surfaces, and hot springs, reflects their strong adaptability to different conditions. These species were filaments that formed mat-like groups, microbial mats, mosses, or crusts, which may help them adapt to environmental changes. In addition, the Kovacikia species were widely distributed and had diverse habitats, most likely a ubiquitous class of cyanobacteria present in the environment.

Kovacikia was first identified as a new genus by a polyphasic approach, which is mostly defined by its unique phylogenetic position and the secondary structure of the ITS region (Miscoe et al., 2016). Molecular genetics methods are commonly used in a polyphasic approach and are crucial in the modern cyanobacterial classification. In this study, the phylogenetic analysis showed that they were clustered in the Kovacikia genus with the other five Kovacikia species. Comparing the 16S rRNA gene sequences (Table 2), K. diezihuensis, K. jiangxiensis, and the other five Kovacikia species shared 94.9%–97.2% similarity, which belongs to the appropriate threshold for different species in the same genus (Stackebrandt and Goebel, 1994; Sciuto et al., 2012; Kim et al., 2014; Komárek et al., 2014; Yarza et al., 2014; Eckert et al., 2015; Sciuto and Moro, 2016). Compared to other Kovacikia species, the secondary structures of the ITS regions of K. diezihuensis and K. jiangxiensis exhibit significant differences, demonstrating their uniqueness. All these molecular genetic data support the separation of K. diezihuensis and K. jiangxiensis from the other five Kovacikia species.

Actually, Kovacikia is considered to be similar to Leptolyngbya (Anagnostidis and Komárek, 1988) in morphology, making it difficult to distinguish (Miscoe et al., 2016). In this study, the most obvious morphological difference between K. diezihuensis and other Kovacikia species is that its color is bright blue-green. In contrast, other species are either purple-brown or gray-green to blue-green under WL. Secondly, its cell length is slightly smaller than the average width, which is similar to that of K. anagnostidisii (Kaštovský et al., 2023). K. Jiangxiensis is the most similar to K. atmophytica (Luz et al., 2023) in morphological characteristics, but there is still a slight difference in that its cell width is slightly smaller. K. Jiangxiensis is different from other Kovacikia species in color or cell length and width (Supplementary Table S2). Therefore, the two new species, K. diezihuensis and K. jiangxiensis, were similar to the other five Kovacikia species in morphology. At the same time, they have fine distinctions in color and length-to-width ratio in the cell.

Among the five officially reported and effectively named Kovacikia species, only K. minuta was reported to produce Chl f. “Leptothermofonsia” was merged with Kovacikia, while “Leptothermofonsia” is not considered a valid name, due to improper designation of the holotype (Kaštovský et al., 2023). However, “L. sichuanensis” E412, a strain of Kovacikia, can also grow, and the color was green under FRL. Combined with genome data, it is speculated that the strain also has the ability to produce Chl f. In terms of habitat, K. minuta was isolated from the colony underneath macrophytes in a shaded pond, “L. sichuanensis” E412 was isolated from the colony in the pond of Lotus Lake Hot Spring, while K. diezihuensis and K. jiangxiensis in this study were isolated from moss-like soils in the corner. These habitats are common habitats for Chl f-producing cyanobacteria (Antonaru et al., 2020), suggesting that there may be niche overlap between Chl f-producing cyanobacteria and non-Chl f-producing cyanobacteria. The results of Ohkubo and Miyashita suggest that the deeper layer of the microbial mat was a habitat for Chl f-producing cyanobacteria, and Chl f enabled them to survive in a habitat with little PAR (Ohkubo and Miyashita, 2017). Therefore, in microbial mats or colonies, the non-Chl f-producing cyanobacteria may be closer to WL, while the Chl f-producing cyanobacteria are closer to FRL.

In terms of molecular genetics, the phylogenetic tree showed that they clustered in the Kovacikia genus (Figure 2). The non-Chl f-producing K. diezihuensis and K. jiangxiensis in this study diverged at the species level from K. minuta and “L. sichuanensis” E412, and had 95.0%–96.8% similarity with 16S rRNA gene sequences (Table 2). Furthermore, the secondary structures of the ITS regions and ITS sequences exhibit significant differences, demonstrating their uniqueness (Figure 3). All these show that both Chl f-producing and non-Chl f-producing species at the same genus in Kovacikia. This provides significant impetus for further analysis of the evolutionary divergence of Chl f-producing cyanobacteria and their adaptability to the different environments.

Compared to K. diezihuensis, in morphology, K. jiangxiensis is more similar to K. minuta and “L. sichuanensis” E412, both of which have slender cells. In Kovacikia, both K. minuta and “L. sichuanensis” E412, which produce Chl f and contain PE, are brown or purplish-brown under WL and grass-green or green under FRL(Shen et al., 2022; Tang et al., 2022). However, K. diezihuensis and K. jiangxiensis, which do not produce Chl f and do not contain PE, are bright blue-green or blue-green to gray-green under WL. It is speculated that K. muscicola containing PE may also have the ability to produce Chl f. Among the reported Leptolyngbya-like cyanobacteria producing Chl f, it was found that all other cyanobacteria contain phycoerythrin except Leptodesmis undulata. Therefore, Chl f may be more easily found in Leptolyngbya-like cyanobacteria containing phycoerythrin.

5 Conclusion

In summary, three Leptolyngbya-like cyanobacteria were phylogenetically identified as K. diezihuensis sp. nov. (reference strain, ACCP0342) and K. jiangxiensis sp. nov. (reference strain, ACCP0444) (Leptolyngbyaceae, Leptolyngbyales) using a polyphasic approach combining ecological, molecular, and morphological data, respectively. These two new species of Kovacikia did not produce phycoerythrin under WL and Chl f under the induction of FRL. This is the first report of both Chl f-producing and non-Chl f-producing species within the same genus in the Leptolyngbyales, shedding light on the diversity and the evolutionary divergence of Chl f-producing cyanobacteria.

In addition, their habitats are common habitats for Chl f-producing cyanobacteria, suggesting that there may be niche overlap between Chl f-producing cyanobacteria and non-Chl f-producing cyanobacteria. Among the reported Leptolyngbya-like cyanobacteria producing Chl f, it was found that all other cyanobacteria contain phycoerythrin except Leptodesmis undulata. Therefore, it is assumed that Chl f may be more easily found in Leptolyngbya-like cyanobacteria containing phycoerythrin. These discoveries can help elucidate the evolution of FaRLiP in cyanobacteria and provide a new perspective for the ecological application of Chl f cyanobacteria with its FaRLiP gene cluster in the future.

Data availability statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: https://www.ncbi.nlm.nih.gov/genbank/, PQ881585; PQ895252; PQ881586.

Author contributions

L-QS: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. GD: Funding acquisition, Resources, Writing – review & editing. YL: Writing – review & editing. SL: Writing – review & editing. XL: Writing – review & editing. RL: Methodology, Writing – review & editing. JL: Funding acquisition, Project administration, Resources, Writing – review & editing.

Funding

The author(s) declare that financial support was received for the research and/or publication of this article. This study was supported by the Project of Science and Technology Department of Jiangxi Province (20213AAG01012 and 2022KSG01004), the National Natural Science Foundation of China (No. 42161016; No. 32160305), and the Jiangxi Provincial Natural Science Foundation (20242BAB20242).

Acknowledgments

We thank Xing-Yuan Wu for helping with sample collection. Thanks to Bao-Sheng Qiu and Zhong-Chun Zhang of the School of Life Sciences, Central China Normal University, for their support and help in the separation and purification of algal strains. The authors thank Zhen-Fei Xing of the Analysis and Testing Center, Institute of Hydrobiology, Chinese Academy of Sciences, for the technical assistance in TEM images.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The authors declare that no Gen AI was used in the creation of this manuscript.

Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2025.1578689/full#supplementary-material

Abbreviations

ACCP, Algae Culture Collection of Poyang Lake; BI, Bayesian inference; Chl f, chlorophyll f; CTAB, cetyltrimethylammonium bromide; FaRLiP, far-red light photoacclimation; FRL, far-red light; ITS, internal transcribed spacer; ML, maximum likelihood; NJ, neighbor-joining; PC, phycocyanin; PE, phycoerythrin; TEM, transmission electron microscopy; WL, white light; JAWSE, Jiangxi Academy of Water Science and Engineering.

References

Airs, R. L., Temperton, B., Sambles, C., Farnham, G., Skill, S. C., and Llewellyn, C. A. (2014). Chlorophyll f and chlorophyll d are produced in the cyanobacterium Chlorogloeopsis fritschii when cultured under natural light and near-infrared radiation. FEBS Lett. 588, 3770–3777. doi: 10.1016/j.febslet.2014.08.026

PubMed Abstract | Crossref Full Text | Google Scholar

Akutsu, S., Fujinuma, D., Furukawa, H., Watanabe, T., Ohnishi-Kameyama, M., Ono, H., et al. (2011). Pigment analysis of a chlorophyll f-containing cyanobacterium strain KC1 isolated from Lake Biwa. Photomed. Photobiol. 33, 35–40.

Google Scholar

Anagnostidis, K., and Komárek, J. (1988). Modern approach to the classification system of cyanophytes. 3. Oscillatoriales. Algol. Stud. / Arch. für Hydrobiol 50–53, 327–472.

Google Scholar

Antonaru, L. A., Cardona, T., Larkum, A. W. D., and Nürnberg, D. J. (2020). Global distribution of a chlorophyll f cyanobacterial marker. ISME J. 14, 2275–2287. doi: 10.1038/s41396-020-0670-y

PubMed Abstract | Crossref Full Text | Google Scholar

Averina, S., Polyakova, E., Senatskaya, E., and Pinevivich, A. (2021). A new cyanobacterial genus Altericista and three species, A. Lacusladogae sp. nov., A. violacea sp. nov., and A. Variichlora sp. nov., described using a polyphasic approach. J. Phycol. 57, 1517–1529. doi: 10.1111/jpy.13188

PubMed Abstract | Crossref Full Text | Google Scholar

Becerra-Absalón, I., Johansen, J. R., Muñoz-Martín, M. A., and Montejano, G. (2018). Chroakolemma gen. Nov. (Leptolyngbyaceae, Cyanobacteria) from soil biocrusts in the semi-desert central region of México. Phytotaxa 367, 201–218. doi: 10.11646/phytotaxa.367.3.1

Crossref Full Text | Google Scholar

Becerra-Absalón, I., Johansen, J. R., Osorio-Santos, K., and Montejano, G. (2020). Two new Oculatella (Oculatellaceae, Cyanobacteria) species in soil crusts from tropical semi-arid uplands of México. Fottea 20, 160–170. doi: 10.5507/fot.2020.010

PubMed Abstract | Crossref Full Text | Google Scholar

Bennett, A., and Bogorad, L. (1973). Complementary chromatic adaptation in a filamentous blue-green alga. J. Cell Biol. 58, 419–435. doi: 10.1083/jcb.58.2.419

PubMed Abstract | Crossref Full Text | Google Scholar

Bohunická, M., Johansen, J. R., and Fucíková, K. (2011). Tapinothrix clintonii sp. nov. (Pseudanabaenaceae, Cyanobacteria), a new species at the nexus of five genera. Fottea 11, 127–140. doi: 10.5507/fot.2011.013

PubMed Abstract | Crossref Full Text | Google Scholar

Boyer, S. L., Flechtner, V. R., and Johansen, J. R. (2001). Is the 16S-23S rRNA internal transcribed spacer region a good tool for use in molecular systematics and population genetics? A case study in cyanobacteria. Mol. Biol. Evol. 18, 1057–1069. doi: 10.1093/oxfordjournals.molbev.a003877

PubMed Abstract | Crossref Full Text | Google Scholar

Chen, M., Li, Y., Birch, D., and Willows, R. D. (2012). A cyanobacterium that contains chlorophyll f - a red-absorbing photopigment. FEBS Lett. 586, 3249–3254. doi: 10.1016/j.febslet.2012.06.045

PubMed Abstract | Crossref Full Text | Google Scholar

Chen, M., Schliep, M., Willows, R. D., Cai, Z. L., Neilan, B. A., and Scheer, H. (2010). A red-shifted chlorophyll. Science. 329, 1318–1319. doi: 10.1126/science.1191127

Crossref Full Text | Google Scholar

Davydov, D., and Vilnet, A. (2022). Review of the cyanobacterial genus Phormidesmis (Leptolyngbyaceae) with the description of Apatinema gen. Nov. Diversity-Basel 14:731. doi: 10.3390/d14090731

Crossref Full Text | Google Scholar

Dvořák, P., Hašler, P., Pitelková, P., Tabáková, P., Casamatta, D. A., and Poulíčková, A. (2017). A new cyanobacterium from the Everglades, Florida – Chamaethrix gen. nov. Fottea 17, 269–276. doi: 10.5507/fot.2017.017

PubMed Abstract | Crossref Full Text | Google Scholar

Eckert, E. M., Fontaneto, D., Coci, M., and Callieri, C. (2015). Does a barcoding gap exist in prokaryotes? Evidences from species delimitation in cyanobacteria. Life – Basel 5, 50–64. doi: 10.3390/life5010050

PubMed Abstract | Crossref Full Text | Google Scholar

Edwards, U., Rogall, T., Blöcker, H., Emde, M., and Böttger, E. C. (1989). Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Res. 17, 7843–7853. doi: 10.1093/nar/17.19.7843

PubMed Abstract | Crossref Full Text | Google Scholar

Engene, N., Choi, H., Esquenazi, E., Rottacker, E. C., Ellisman, M. H., Dorrestein, P. C., et al. (2011). Underestimated biodiversity as a major explanation for the perceived prolific secondary metabolite capacity of the cyanobacterial genus Lyngbya. Environ. Microbiol. 13, 1601–1610. doi: 10.1111/j.1462-2920.2011.02472.x

PubMed Abstract | Crossref Full Text | Google Scholar

Engene, N., Tronholm, A., and Paul, V. J. (2018). Uncovering cryptic diversity of Lyngbya: the new tropical marine cyanobacterial genus Dapis (Oscillatoriales). J. Phycol. 54, 435–446. doi: 10.1111/jpy.12752

PubMed Abstract | Crossref Full Text | Google Scholar

Gan, F., Shen, G., and Bryant, D. A. (2015). Occurrence of far-red light photoacclimation (FaRLiP) in diverse cyanobacteria. Life. 5, 4–24. doi: 10.3390/life5010004

Crossref Full Text | Google Scholar

Gan, F., Zhang, S., Rockwell, N. C., Martin, S. S., Lagarias, J. C., and Bryant, D. A. (2014). Extensive remodeling of a cyanobacterial photosynthetic apparatus in far-red light. Science 345, 1312–1317. doi: 10.1126/science.1256963

PubMed Abstract | Crossref Full Text | Google Scholar

Gisriel, C. J., Flesher, D. A., Shen, G., Wang, J., Ho, M. Y., Brudvig, G. W., et al. (2022a). Structure of a photosystem I-ferredoxin complex from a marine cyanobacterium provides insights into far-red light photoacclimation. J. Biol. Chem. 298:101408. doi: 10.1016/j.jbc.2021.101408

Crossref Full Text | Google Scholar

Gisriel, C. J., Shen, G., Ho, M. Y., Kurashov, V., Flesher, D. A., Wang, J., et al. (2022b). Structure of a monomeric photosystem II core complex from a cyanobacterium acclimated to far-red light reveals the functions of chlorophylls d and f. J. Biol. Chem., 298:101424. doi: 10.1016/j.jbc.2021.101424

Crossref Full Text | Google Scholar

Gisriel, C., Shen, G., Kurashov, V., Ho, M. Y., Zhang, S., Williams, D., et al. (2020). The structure of photosystem І acclimated to far-red light illuminates an ecologically important acclimation process in photosynthesis. Sci. Adv., 6:eaay6415. doi: 10.1126/sciadv.aay6415

Crossref Full Text | Google Scholar

González-Reséndiz, L., Johansen, J. R., Alba-Lois, L., Segal-Kischinevzky, C., Escobar-Sánchez, V., Jimenez-García, L. F., et al. (2018). Nunduva, a new marine genus of Rivulariaceae (Nostocales, Cyanobacteria) from marine rocky shores. Fottea 18, 86–105. doi: 10.5507/fot.2017.018

PubMed Abstract | Crossref Full Text | Google Scholar

Hall, T. A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucl. Acids Symp. Ser. 734, 95–98. doi: 10.1021/bk-1999-0734.ch008

PubMed Abstract | Crossref Full Text | Google Scholar

Hentschke, G. S., Morais, J., de Oliveira, F. L., Silva, R., Cruz, P., and Vasconcelos, V. (2024). Taxonomic updates in the family Leptolyngbyaceae (Leptolyngbyales, Cyanobacteria): the description of Pseudoleptolyngbya gen. Nov, Leptolyngbyopsis gen. Nov., and the replacement of Arthronema. Eur. J. Phycol. 0, 1–14. doi: 10.1080/09670262.2024.2404887

Crossref Full Text | Google Scholar

Hentschke, G. S., Ramos, V., Pinheiro, Â., Barreiro, A., Costa, M. S., Rego, A., et al. (2022). Zarconia navalis gen. Nov., sp. nov., Romeriopsis navalis gen. Nov., sp. nov. and Romeriopsis marina sp. nov., isolated from inter- and subtidal environments from northern Portugal. Int. J. Syst. Evol. Microbiol. 72, 1–12. doi: 10.1099/ijsem.0.005552

PubMed Abstract | Crossref Full Text | Google Scholar

Hoffmann, L., Komárek, J., and Kaštovský, J. (2005). System of cyanoprokaryotes (cyanobacteria) –state in 2004. Arch. Hydrobiol. Suppl. Algol. Stud. 117, 95–115. doi: 10.1127/1864-1318/2005/0117-0095

Crossref Full Text | Google Scholar

Ichimura, T. (1979). “Media for the freshwater cyanobacteria” in Methods in Phycological studies. ed. K. Nishizawa (Tokyo, Japan: Kyouritsu Shuppan), 295–296.

Google Scholar

Iii, R. B. P., Johansen, J. R., Kovácik, L., Brand, J., Kaštovský, J., and Casamatta, D. A. (2011). A unique Pseudanabaenalean (Cyanobacteria) genus Nodosilinea gen. Nov based on morphological and molecular data. J. Phycol. 47, 1397–1412. doi: 10.1111/j.1529-8817.2011.01077.x

PubMed Abstract | Crossref Full Text | Google Scholar

Iteman, I., Rippka, R., Marsac, N. T. D., and Herdman, M. (2000). Comparison of conserved structural and regulatory domains within divergent 16S rRNA-23S rRNA spacer sequences of cyanobacteria. Microbiology 146, 1275–1286. doi: 10.1099/00221287-146-6-1275

PubMed Abstract | Crossref Full Text | Google Scholar

Jahodářová, E., Dvořák, P., Hašler, P., and Poulíčková, A. (2017). Revealing hidden diversity among tropical cyanobacteria: the new genus Onodrimia (Synechococcales, Cyanobacteria) described using the polyphasic approach. Phytotaxa 326, 28–40. doi: 10.11646/phytotaxa.326.1.2

Crossref Full Text | Google Scholar

Kalyaanamoorthy, S., Minh, B. Q., Wong, T. K., von Haeseler, A., and Jermiin, L. S. (2017). ModelFinder: fast model selection for accurate phylogenetic estimates. Nat. Methods 14, 587–589. doi: 10.1038/nmeth.4285

PubMed Abstract | Crossref Full Text | Google Scholar

Kaštovský, J., Johansen, J. R., Hauerová, R., and Akagha, M. U. (2023). Hot is rich - an enormous diversity of simple trichal cyanobacteria from Yellowstone hot springs. Diversity-Basel 15, 1–29. doi: 10.3390/d15090975

PubMed Abstract | Crossref Full Text | Google Scholar

Kato, K., Shinoda, T., Nagao, R., Akimoto, S., Suzuki, T., Dohmae, N., et al. (2020). Structural basis for the adaptation and function of chlorophyll f in photosystem І. Nat. Commun. 11, 238. doi: 10.1038/s41467-019-13898-5

Crossref Full Text | Google Scholar

Kim, M., Oh, H. S., Park, S. C., and Chun, J. (2014). Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int. J. Syst. Evol. Microbiol. 64, 346–351. doi: 10.1099/ijs.0.059774-0

PubMed Abstract | Crossref Full Text | Google Scholar

Komárek, J. (2006). Cyanobacterial taxonomy: current problems and prospects for the integration of traditional and molecular approaches. Algae 21, 349–375. doi: 10.4490/ALGAE.2006.21.4.349

Crossref Full Text | Google Scholar

Komárek, J. (2016). A polyphasic approach for the taxonomy of cyanobacteria: principles and applications. Eur. J. Phycol. 51, 346–353. doi: 10.1080/09670262.2016.1163738

Crossref Full Text | Google Scholar

Komárek, J., Kaštovský, J., Mareš, J., and Johansen, J. R. (2014). Taxonomic classification of cyanoprokaryotes (cyanobacterial genera) 2014, using a polyphasic approach. Preslia 86, 295–335.

Google Scholar

Lepère, C., Wilmotte, A., and Meyer, B. (2000). Molecular diversity of Microcystis strains (Cyanophyceae, Chroococcales) based on 16S rDNA sequences. Syst. Geogr. Plants 70, 275–283. doi: 10.2307/3668646

Crossref Full Text | Google Scholar

Li, X., and Li, R. (2016). Limnolyngbya circumcreta gen. & comb. nov. (Synechococcales, Cyanobacteria) with three geographical (provincial) genotypes in China. Phycologia 55, 478–491. doi: 10.2216/15-149.1

Crossref Full Text | Google Scholar

Li, Y., Lin, Y., Garvey, C. J., Birch, D., Corkery, R. W., Loughlin, P. C., et al. (2016). Characterization of red-shifted phycobilisomes isolated from the chlorophyll f-containing cyanobacterium Halomicronema hongdechloris. BBA-Bioenergetics. 1857, 107–114. doi: 10.1016/j.bbabio.2015.10.009

Crossref Full Text | Google Scholar

Li, Y., Lin, Y., Loughlin, P. C., and Chen, M. (2014). Optimization and effects of different culture conditions on growth of Halomicronema hongdechloris - a filamentous cyanobacterium containing chlorophyll f. Front. Plant Sci. 5:67. doi: 10.3389/fpls.2014.00067

Crossref Full Text | Google Scholar

Luz, R., Cordeira, R., Kaštovský, J., Johansen, J. R., Dias, E., Fonseca, A., et al. (2023). Description of four new filamentous cyanobacterial taxa from freshwater habitats in the Azores archipelago. J. Phycol. 59, 1323–1338. doi: 10.1111/jpy.13396

PubMed Abstract | Crossref Full Text | Google Scholar

Mai, T., Johansen, J. R., Pietrasiak, N., Bohunická, M., and Martin, M. P. (2018). Revision of the Synechococcales (Cyanobacteria) through recognition of four families including Oculatellaceae fam. Nov. and Trichocoleaceae fam. Nov. and six new genera containing 14 species. Phytotaxa 365, 1–59. doi: 10.11646/phytotaxa.365.1.1

Crossref Full Text | Google Scholar

Mareš, J., Strunecký, O., Bučinská, L., and Wiedermannová, J. (2019). Evolutionary patterns of thylakoid architecture in cyanobacteria. Front. Microbiol. 10:277. doi: 10.3389/fmicb.2019.00277

PubMed Abstract | Crossref Full Text | Google Scholar

Miscoe, L. H., Johansen, J. R., Vaccarino, M. A., Pietrasiak, N., and Sherwood, A. R. (2016). The diatom flora and cyobacteria from caves on Kauai, Hawaii. II. Novel cyanobacteria from caves on Kauai, Hawaii. Bibl. Phycol. 120, 75–152.

Google Scholar

Miyashita, H., Ohkubo, S., Komatsu, H., Sorimachi, Y., Fukayama, D., Fujinuma, D., et al. (2014). Discovery of chlorophyll d in Acaryochloris marina and chlorophyll f in a unicellular cyanobacterium, strain KC1, isolated from Lake Biwa. J. Phys. Chem. B 4, 149–158. doi: 10.4172/2161-0398.1000149

PubMed Abstract | Crossref Full Text | Google Scholar

Neilan, B. A., Jacobs, D., and Goodman, A. E. (1995). Genetic diversity and phylogeny of toxic cyanobacteria determined by DNA polymorphisms within the phycocyanin locus. Appl. Environ. Microb. 61, 3875–3883. doi: 10.1128/aem.61.11.3875-3883.1995

PubMed Abstract | Crossref Full Text | Google Scholar

Nguyen, L. T., Schmidt, H. A., von Haeseler, A., and Minh, B. Q. (2015). IQ–TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 32, 268–274. doi: 10.1093/molbev/msu300

PubMed Abstract | Crossref Full Text | Google Scholar

Ohkubo, S., and Miyashita, H. (2017). A niche for cyanobacteria producing chlorophyll f within a microbial mat. ISME J. 11, 2368–2378. doi: 10.1038/ismej.2017.98

PubMed Abstract | Crossref Full Text | Google Scholar

Osorio-Santos, K., Pietrasiak, N., Bohunická, M., Miscoe, L. H., Kováčik, L., Martin, M. P., et al. (2014). Seven new species of Oculatella (Pseudanabaenales, Cyanobacteria): taxonomically recognizing cryptic diversification. Eur. J. Phycol. 49, 450–470. doi: 10.1080/09670262.2014.976843

Crossref Full Text | Google Scholar

Pietrasiak, N., Osorio-Santos, K., Shalygin, S., Martin, M. P., and Johansen, J. R. (2019). When is a lineage a species? A case study in Myxacorys gen. Nov. (Synechococcales: Cyanobacteria) with the description of two new species from the Americas. J. Phycol. 55, 976–996. doi: 10.1111/jpy.12897

PubMed Abstract | Crossref Full Text | Google Scholar

Raabová, L., Kovacik, L., Elster, J., and Strunecký, O. (2019). Review of the genus Phormidesmis (Cyanobacteria) based on environmental, morphological, and molecular data with description of a new genus Leptodesmis. Phytotaxa 395, 1–016. doi: 10.11646/phytotaxa.395.1.1

Crossref Full Text | Google Scholar

Rasouli-Dogaheh, S., Noroozi, M., Khansha, J., Amoozegar, M. A., Rahimi, R., Nikou, M. M., et al. (2023). Khargia gen. Nov., a new genus of simple trichal Cyanobacteria from the Persian Gulf. Fottea 23, 49–61. doi: 10.5507/fot.2022.012

PubMed Abstract | Crossref Full Text | Google Scholar

Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D. L., Darling, A., Höhna, S., et al. (2012). MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 61, 539–542. doi: 10.1093/sysbio/sys029

PubMed Abstract | Crossref Full Text | Google Scholar

Sciuto, K., Andreoli, C., Rascio, N., Rocca, N. L., and Moro, I. (2012). Polyphasic approach and typification of selected Phormidium strains (Cyanobacteria). Cladistics 28, 357–374. doi: 10.1111/j.1096-0031.2011.00386.x

PubMed Abstract | Crossref Full Text | Google Scholar

Sciuto, K., and Moro, I. (2016). Detection of the new cosmopolitan genus Thermoleptolyngbya (Cyanobacteria, Leptolyngbyaceae) using the 16S rRNA gene and 16S-23S ITS region. Mol. Phylogenet. Evol. 105, 15–35. doi: 10.1016/j.ympev.2016.08.010

PubMed Abstract | Crossref Full Text | Google Scholar

Shalygin, S., Shalygina, R. R., Redkina, V. R., Gargas, C. B., and Johansen, J. R. (2020). Description of Stenomitos kolaenensis and S. Hiloensis sp. nov. (Leptolyngbyaceae, Cyanobacteria) with an emendation of the genus. Phytotaxa 440, 108–128. doi: 10.11646/phytotaxa.440.2.3

Crossref Full Text | Google Scholar

Shen, L.-Q., Zhang, Z.-C., Huang, L., Zhang, L.-D., Yu, G., Chen, M., et al. (2023). Chlorophyll f production in two new subaerial two cyanobacteria of the family Oculatellaceae. J. Phycol. 59, 370–382. doi: 10.1111/jpy.13314

PubMed Abstract | Crossref Full Text | Google Scholar

Shen, L.-Q., Zhang, Z.-C., Shang, J.-L., Li, Z.-K., Chen, M., Li, R., et al. (2022). Kovacikia minuta sp. nov. (Leptolyngbyaceae, Cyanobacteria), a new freshwater chlorophyll f-producing cyanobacterium. J. Phycol. 58, 424–435. doi: 10.1111/jpy.13248

PubMed Abstract | Crossref Full Text | Google Scholar

Shen, L.-Q., Zhang, Z.-C., Zhang, L.-D., Huang, D., Yu, G., Chen, M., et al. (2024). Widespread distribution of chlorophyll f-producing Leptodesmis cyanobacteria. J. Phycol. 61, 1–17. doi: 10.1111/jpy.13538

Crossref Full Text | Google Scholar

Soares, F., Tiago, I., Trovão, J., Coelho, C., Mesquita, N., Gil, F., et al. (2019). Description of Myxacorys almedinensis sp. nov. (Synechococcales, Cyanobacteria) isolated from the limestone walls of the old Cathedral of Coimbra, Portugal (UNESCO world heritage site). Phytotaxa 419, 77–90. doi: 10.11646/phytotaxa.419.1.5

Crossref Full Text | Google Scholar

Song, G., Jiang, Y., and Li, R. (2015). Scytolyngbya timoleontis, gen. Et sp. nov. (Leptolyngbyaceae, Cyanobacteria): a novel false branching Cyanobacteria from China. Phytotaxa 224, 72–84. doi: 10.11646/phytotaxa.224.1.5

Crossref Full Text | Google Scholar

Stackebrandt, E., and Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int. J. Syst. Evol. Micr. 44, 846–849. doi: 10.1099/00207713-44-4-846

Crossref Full Text | Google Scholar

Strunecký, O., Ivanova, A. P., and Mareš, J. (2023). An updated classification of cyanobacterial orders and families based on phylogenomic and polyphasic analysis. J. Phycol. 59, 12–51. doi: 10.1111/jpy.13304

PubMed Abstract | Crossref Full Text | Google Scholar

Strunecky, O., Raabova, L., Bernardova, A., Ivanova, A. P., Semanova, A., Crossley, J., et al. (2020). Diversity of cyanobacteria at the Alaska north slope with description of two new genera: Gibliniella and Shackletoniella. FEMS Microbiol. Ecol. 96:fiz189. doi: 10.1093/femsec/fiz189

PubMed Abstract | Crossref Full Text | Google Scholar

Tang, J., Du, L. M., Li, M., Yao, D., Jiang, Y., Waleron, M., et al. (2022). Characterization of a novel hot-spring cyanobacterium Leptodesmis sichuanensis sp. nov. and genomic insights of molecular adaptations into its habitat. Front. Microbiol. 12:739625. doi: 10.3389/fmicb.2021.739625

PubMed Abstract | Crossref Full Text | Google Scholar

Taton, A., Grubisic, S., Brambilla, E., Wit, R. D., and Wilmotte, A. (2003). Cyanobacterial diversity in natural and artificial microbial mats of Lake Fryxell (McMurdo dry valleys, Antarctica): a morphological and molecular approach. Appl. Environ. Microb. 69, 5157–5169. doi: 10.1128/AEM.69.9.5157-5169.2003

PubMed Abstract | Crossref Full Text | Google Scholar

Turicchia, S., Ventura, S., Komárková, J., and Komárek, J. (2009). Taxonomic evaluation of cyanobacterial microflora from alkaline marshes of northern Belize. 2. Diversity of oscillatorialean genera. Nova Hedwigia 89, 165–200. doi: 10.1127/0029-5035/2009/0089-0165

Crossref Full Text | Google Scholar

Vaz, M. G. M. V., Genuário, D. B., Andreote, A. P. D., Malone, C. F. S., Sant'Anna, C. L., Barbiero, L., et al. (2015). Pantanalinema gen. Nov. and Alkalinema gen. Nov.: novel pseudanabaenacean genera (Cyanobacteria) isolated from saline-alkaline lakes. Int. J. Syst. Evol. Micr. 65, 298–308. doi: 10.1099/ijs.0.070110-0

PubMed Abstract | Crossref Full Text | Google Scholar

Vázquez-Martínez, J., Gutierrez-Villagomez, J. M., Fonseca-García, C., Ramírez-Chávez, E., Mondragón-Sánchez, M. L., Partida-Martínez, L., et al. (2018). Nodosilinea chupicuarensis sp. nov. (Leptolyngbyaceae, Synechococcales) a subaerial cyanobacterium isolated from a stone monument in Central Mexico. Phytotaxa 334, 167–182. doi: 10.11646/phytotaxa.334.2.6

Crossref Full Text | Google Scholar

Yarza, P., Yilmaz, P., Pruesse, E., Glöckner, F. O., Ludwig, W., Schleifer, K. H., et al. (2014). Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences. Nat. Rev. Microbiol. 12, 635–645. doi: 10.1038/nrmicro3330

PubMed Abstract | Crossref Full Text | Google Scholar

Zhang, Z.-C., Li, Z.-K., Yin, Y.-C., Li, Y., Jia, Y., Chen, M., et al. (2019). Widespread occurrence and unexpected diversity of red-shifted chlorophyll producing cyanobacteria in humid subtropical forest ecosystems. Environ. Microbiol. 21, 1497–1510. doi: 10.1111/1462-2920.14582

PubMed Abstract | Crossref Full Text | Google Scholar

Zuker, M. (2003). Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406–3415. doi: 10.1093/nar/gkg595

PubMed Abstract | Crossref Full Text | Google Scholar