Polysaccharide biosynthesis-related genes explain phenotype-genotype correlation of Microcystis colonies in Meiliang Bay of Lake Taihu, China

The 16S rDNA, 16S-23S rDNA-ITS, cpcBA-IGS, mcy gene and several polysaccharide biosynthesis-related genes (epsL and TagH) were analyzed along with the identification of the morphology of Microcystis colonies collected in Lake Taihu in 2014. M. wesenbergii colonies could be distinguished directly from other colonies using espL. TagH divided all of the samples into two clusters but failed to distinguish different phenotypes. Our results indicated that neither morphology nor molecular tools including 16S rDNA, 16S-23S ITS and cpcBA-IGS could distinguish toxic and non-toxic species among the identified Microcystis species. No obvious relationship was detected between the phenotypes of Microcystis and their genotypes using 16S, 16S-23S and cpcBA-IGS, but polysaccharide biosynthesis-related genes may distinguish the Microcystis phenotypes. Furthermore, the sequences of the polysaccharide biosynthesis-related genes (espL and TagH) extracted from Microcystis scums collected throughout 2015 was analyzed. Samples dominated by M. ichthyoblabe (60–100%) and M. wesenbergii (60–100%) were divided into different clade by both espL and TagH, respectively. Therefore, it was confirmed that M. wesenbergii and M. ichthyoblabe could be distinguished by the polysaccharide biosynthesis-related genes (espL and TagH). This study is of great significance in filling the gap between classification of molecular biology and the morphological taxonomy of Microcystis.


Primer
For sequence (5′-3′) Rev sequence (5′-3′) Reference   morphology to that of a typical M. novacekii. Li et al. 18 illustrated that solubilization of mucilage could induce changes in colonial morphology and the authors suggested that seasonal succession of Microcystis species was due to morphological changes. Therefore, the taxonomy of this genus should be re-evaluated via molecular genetic analyses.
The phenotype-genotype correlation of Microcystis is helpful in filling the gap between classification of molecular biology and the morphological taxonomy of Microcystis. The phylogenetic analysis based on 16S rDNA was considered as one of the most reliable criteria for determining relationships among organisms with close relation 19 . However, the similarity of colonies in different morphology was high as measured by 16S rDNA sequencing 20,21 , and thus the unification of five species of Microcystis has been proposed 22 . In addition, the events of horizontal gene transfer would cause flexibility of several informative genes including 16S rDNA of Microcystis 23 . A more reliable gene sequence should be explored to analyze the phenotype-genotype correlation of Microcystis. Otten and Paerl 24 indicated that M. wesenbergii could be identified from four different Microcystis morphospecies using 16S-23S rDNA-ITS sequences, but the other four morphospecies could not. Tan et al. 25 indicated cpcBA-IGS could be used as an effective tool to identify M. wesenbergii. Several polysaccharide biosynthesis-related genes were also found to identify morphospecies of Microcystis 26 . Thus, these genes were hypothesized to be significantly related to Microcystis colonial morphology, and this hypothesis has been preliminarily verified by Xu et al. 27 .
In addition, microcystin-producing genes were also postulated to divide Microcystis into toxic species and non-toxic species 28 . The morphospecies was considered to relate to the toxicity of Microcystis. Generally, M. ichthyoblabe was considered as non-toxic species 29 , while M. aeruginosa and M. wesenbergii as toxic species [30][31][32] . The microcystin synthetase (mcy) gene cluster in different Microcystis morphospecies was thus analyzed to reveal the phenotype-genotype correlation of Microcystis colonies 33 . However, it was still poorly understood whether there was a relationship between the phenotype and microcystin-producing genes.
The current study aimed to gain insight into the phenotype-genotype correlation of Microcystis. The 16S rDNA, 16S-23S rDNA-ITS, cpcBA-IGS, mcy gene (mcyB) 34 and several polysaccharide biosynthesis-related genes were analyzed along with the identification of the morphology of Microcystis colonies collected in the field. This study also attempted to resolve that polysaccharide biosynthesis-related genes might distinguish the Microcystis morphospecies as EPS played great roles in colony formation and morphological changes of Microcystis 18,35 .

Materials and Methods
Experimental design. This study has two parts. (I) Seeking novel functional gene which may distinguish the Microcystis morphospecies. Individual Microcystis colonies were isolated from natural samples and then axenically cultured for PCR amplification and sequencing. Afterwards, phenotype-genotype correlation of Microcystis colonies was investigated and the function gene was identified. (II) Confirming the functional gene. Microcystis "scum" at different seasons were collected and divided into varying classes consisting of various Microcystis Sample collections. Algal samples for colony isolation and culture in part I were collected during a Microcystis bloom in Meiliang Bay in northern Lake Taihu (China) on 15 August and 1 November 2014. Lake Taihu was selected in the current study because Microcystis spp. is the dominant species at most of the time and heavy Microcystis blooms occurs frequently 10 . In addition, the colony morphology and phylogenetic inference of Microcystis species has been well investigated in this lake 8,24,36 , which could be referred to. The water samples containing abundant Microcystis colonies were collected directly from the lake surface (30 cm depth) and were transferred into plastic bottles with a capacity of 5 L. The samples were then stored in a cold closet and transported    DNA extraction. The DNA extraction method was referred to Sun et al. 17 . Microcystis pellets were dispersed into 0.8 mL extraction buffer (1.5 M NaCl, 1% CTAB, 100 mM Tris-HCl, 100 mM Na 2 EDTA, 100 mM Na 3 PO 3 , pH 0.8) and 20 μ L of proteinase K (30 mg mL −1 ). Afterwards, they were incubated at 37 °C for 30 min and then, 0.48 mL of 20% SDS was added to each sample, incubating at 65 °C for 1 h. The samples were extracted using phenol-chloroform-isoamyl (25:24:1) and chloroform-isoamyl (24:1) successively. Centrifuged at 8000 × g for 5 min, the supernatant was transferred to new tubes. Thereafter, 0.6 mL pure isopropyl alcohol was injected to purify the DNA sample. After 20-min centrifugation at 16000 × g, 70% ethanol was used to rinse the DNA sample. Each DNA sample was dried and dissolved in 100 μ L of Tris-EDTA (10 mM Tris and l mM EDTA, pH 8.0). Finally, the DNA sample was analyzed using a Nanodrop-2000.
PCR amplification and sequencing. Seven pairs of primers targeting the 16S rRNA, 16S-23S ITS(A)/(S), cpcBA-IGS, mcyB, TagH and epsL genes were used for the amplification and sequencing of all of the samples (see Table 1

Treatment of samples for part II.
The sample for part II was poured gently through sieves (divided into four classes: > 500 μ m, 300-500 μ m, 150-300 μ m and 75-150 μ m). Each class was re-suspended in BG-11 medium. For each subsample from sieving, the photomicrographs were taken using an Olympus C-5050 digital camera coupled with an optical microscope (Olympus CX31). The length and width of Microcystis colonies was analyzed using the UTHSCSA ImageTool (v3.00, University of Texas Health Science Center, San Antonio, TX, USA). The biovolume of Microcystis colony was calculated as volume = π /6 (length × width) 3/2 as it is hard to measure the thickness of colonies. A total of 300 colonies were analyzed in each sample. Afterwards, the percentage of different Microcystis morphospecies in the total Microcystis biovolume of each subsample was calculated. Microcystis morphospecies was identified according to Yu et al. 7 . In the current study, M. ichthyoblabe, M. aeruginosa and M. wesenbergii was identified as in Fig. 1 and other Microcystis colonies were defined as unidentified Microcystis. For each subsample, DNA for PCR templates was extracted. Only epsL and TagH were used for amplification and sequencing according to the results of part I. All the procedure and method was as same as those described for part I.

Data analysis. Alignment for all of the sequences was determined by Muscle and edited by software
Bioedit 37 . Some related sequences in the NCBI database were also used for alignment. MEGA5 was used to construct neighbor-joining tree of phylogeny analysis 38 , with bootstrap for 1000 replications, Maximum Composite Likelihood, and d: Transitions + Transversions.

Results and Discussion
Relationship between species and toxicity. Figure 2 shows an electropherogram of the PCR products with the primer of mcyB. Our results showed that one M. aeruginosa colony contained mcyB but the other did not. Two out of five M. ichthyoblabe colonies contained mcyB in this study. Mazur-Marzec et al. 39 showed similar results in the Vistula Lagoon (southern Baltic Sea). However, M. aeruginosa colonies are generally considered as toxic species 30,40 . M. ichthyoblabe has never been reported to produce microcystins 29,41,42 . M. wesenbergii was classified as a non-toxic species 31 , but our results showed that both two M. wesenbergii colonies contained mcyB. Nevertheless, some investigations 32,42 also illustrated that M. wesenbergii is toxic. All of the conflicting conclusions above indicated that there is not an exact relationship between the phenotype and microcystin-producing genes.
Yoshida et al. 32 divided 47 strains of Microcystis into three clusters based on the sequences of 16S-23S rDNA-ITS. Their results showed that the first cluster contained both non-toxic and toxic strains, the second only had toxic ones, and the last only had non-toxic strains. This result implied that the 16S-23S gene may distinguish the toxic and non-toxic Microcystis species, which was also reported by Janse et al. 43 . On the contrary, our results demonstrated that the 16S-23S gene sequences failed to distinguish nine strains with different phenotypes, four of which possessed the mcyB gene. This result suggested that 16S-23S rDNA-ITS gene failed to distinguish toxic and non-toxic strains. Yoshida et al. 44 suggested that 16S rDNA could used to identify toxic and non-toxic Microcystis species in some bloom stages. However, our results did not reach a similar conclusion. Therefore, the Microcystis species identified by morphology or molecular tools (16S rDNA, 16S-23S ITS and cpcBA-IGS) could not be used to distinguish toxic and non-toxic species. All the above studies considered that M. wesenbergii could be distinguished using the 16-23S rDNA ITS and the cpcBA-IGS sequences. Conversely, in the current study, the sequences displayed high homozygosity for each 16S-23S and cpcBA-IGS in all of the samples except for the M. aeruginosa colony, TH32 (Figs 4 and 5). Similarly, the phylogenic tree for the 63 Microcystis strains in China based on the cpcBA-IGS gene sequences showed that this gene did not always succeed in identifying different morphospecies 47 . These occasional failures may be resulted from genetic variations among the strains of Microcystis 48 . One Microcystis genotype was reported to have more than one phenotype 29,49 . In East Africa, 24 isolated strains of M. aeruginosa could be separated into 10 genotypes based on the DNA sequences of the PC-IGS and ITS1 rDNA regions 50 . Thus, there was no obvious relationship between these phenotypes and the phenotypes of Microcystis based on 16S, 16S-23S and cpcBA-IGS because of the significant genetic variations among the strains of Microcystis.
Polysaccharide biosynthesis-related genes. Figure 6 shows a phylogenetic tree based on the analysis of the sequences of the polysaccharide biosynthesis-related genes (espL and TagH). The results demonstrate that the M. wesenbergii colonies could be divided directly from other colonies using espL. Xu et al. 27 suggested that the polysaccharide biosynthesis-related gene TagH may explain the diversity of the Microcystis morphospecies. In the current study, TagH divided all of the samples into two clusters but failed to distinguish the different phenotypes.
Since very small amount of colonies were tested and cultured, there would be a risk that the final Microcystis morphotype would change compared with the initially identified Microcystis due to intraspecific competition. Therefore, part II was carried out to confirm as the polysaccharide biosynthesis-related genes could distinguish the Microcystis phenotypes. The phylogenetic tree based on the analysis of the sequences of the polysaccharide biosynthesis-related genes (espL and TagH) extracted from Microcystis "scum" collected from June and Scientific RepoRts | 6:35551 | DOI: 10.1038/srep35551 November 2015, was shown in Figs 7 and 8, respectively. The gene espL divided all of the samples into two clusters and the first cluster was divided into three subclades (Fig. 7). The samples in clade 2 was dominated by M. wesenbergii (60-100%). The samples in subclade 1 of clade 1 was dominated by M. ichthyoblabe (60-100%). As shown in Fig. 8, the gene TagH divided all of the samples into two clusters. All the samples collected in June and November were brought into subclade 1 in clade 1 and samples in August were brought into subclade 2 in clade 1. The former samples was dominated by M. ichthyoblabe (60-100%) and the latter samples was dominated by M. wesenbergii (60-100%). In consequence, it was confirmed that M. wesenbergii and M. ichthyoblabe could be distinguished by the polysaccharide biosynthesis-related genes espL and TagH. However, the two polysaccharide biosynthesis-related genes (epsL and TagH) may not be qualified for identifying all the species of Microcystis. These two genes combined with some other functional genes may succeed in identifying all the Microcystis species based on further researches. Extracellular polysaccharide (EPS) was considered to be the material basis of Microcystis colony formation. A positive relationship between colony size and EPS content has been reported during recent years 51,35 . Li et al. 18 illustrated that solubilization of mucilage, which consists of EPS, induced changes in Microcystis colonial morphology. Forni et al. 52 indicated that the composition of EPS in different Microcystis species varied. The EPS content of various Microcystis morphospecies was also different 53 . Therefore, the content and composition of EPS has been postulated to be related to Microcystis colony morphology. In conclusion, the polysaccharide biosynthesis-related genes could distinguish the Microcystis phenotypes.

Conclusions
(1) Microcystis species identified by morphology or molecular tools (16S rDNA, 16S-23S ITS and cpcBA-IGS) could not be distinguished as toxic and non-toxic species. (2) There was no obvious relationship between the phenotypes of Microcystis species based on 16S, 16S-23S and cpcBA-IGS because of the significant genetic variations among the strains of Microcystis. (3) It was confirmed that polysaccharide biosynthesis-related genes could distinguish the Microcystis phenotypes.