## 3 Results and Discussion
### Analysis of Geosmin-Associated Genes: Origin and Evolution  
#### 3.4.1 Inconsistent Topologies between Geosmin Gene and 16S rDNA Phylogenetic Trees  
To investigate the evolutionary history in the distribution of the geosmin/germacradienol synthase gene among cyanobacteria, the reported sequences of this gene were collected and phylogenetically analyzed with the housekeeping 16S rRNA gene from the same taxa. As shown in Figure 5, the NJ/MP topologies of these phylogenetic trees generated from the geosmin/germacradienol synthase gene and 16S rRNA gene were not congruent. In the geosmin/germacradienol gene tree, _A.__ucrainica_ strains were separated from other heterocystous cyanobacterial species, but clustered with non-heterocystous _Phormidium_ sp. (Geo A) and _Oscillatoria_ sp. PCC 6506. These relationships differed from the recognized relationships revealed by the 16S rRNA gene-based tree. The inconsistent phylogenetic topologies of the geosmin gene and 16S rRNA gene (Figure 5) implied that the geosmin synthesis genes did not co-evolve with the corresponding genomes during the evolutionary history of cyanobacteria, suggesting that these genes were acquired by horizontal gene transfer (HGT) among different groups. HGT is an important evolutionary force in bacteria, and the  
\begin{table}
\begin{tabular}{c c c c c} \hline
**Species** & **Taxonomy** & \begin{tabular}{c} **Primers** \\ **GTC1F/R** \\ \end{tabular}  & \begin{tabular}{c} **Primers** \\ **GTC2F/R** \\ \end{tabular}  & \ \begin{tabular}{c} **Homology** \\ **genes** \\ \end{tabular}  & \begin{tabular}{c} **Organization of** \\ **genes** \\ \end{tabular}  \\ \hline _Anabaena_ sp. 3585 & Nostocales & +/–  
genes responsible for cyanobacteria and saxitoxin synthesis in cyanobacteria have a history of HGT [27, 28]. Sequences from various cyanobacterial taxa are necessary to ascertain the occurrence of HGT in geosmin evolution and the role of HGT in the distribution of the geosmin synthase gene in cyanobacteria. However, geosmin-associated genes were only elucidated in a few strains, including _N. punctiforme_ PCC 73102, _Phormidium_ sp., and _Lyngbya kuetzingii_ UTEX 1547 [11, 13, 29]. 4.2 Comparison of _geo_, Single-Domain Sesquiterpene Gene, MIB Synthesis Gene and Their Related Gene Clusters in Cyanobacteria  
Another typical sesquiterpene synthase gene with a single domain in cyanobacteria was revealed through the BLAST search and analysis on the homologous genes of _geo_ (Figure 6). The single-domain genes are highly homologous with _geo_ in functional sites, such as Mg\({}^{2+}\)-binding sites, but different in several other sites (Figure S3), and could catalyze the FPP to sesquiterpenes, such as germacrene [12]. As shown in Figure 6, the single-domain gene clustered with a cytochrome P450 gene and a hybrid two-component protein gene, and the gene array and constitution were universal in the published genomes of cyanobacteria, including _N. punctiforme_ PCC 73102, _Nostoc_ sp. PCC 7120 and _Anabaena variabilis_ ATCC 29413. As elucidated in the present study, the two-domain _geo_ gene and two _cnb_ genes formed an operon structure, and this operon structure was proven to be consistent in cyanobacteria. Interestingly, [PERSON] _et al_. [25] revealed that the _cnb_ genes are also closely linked to MIB synthesis genes and form an operon structure in cyanobacteria (Figure 6). The _cnb_ genes of the geosmin operon are highly homologous with those of the MIB gene cluster in cyanobacteria. Table 2 shows the similarities in DNA sequences among these _cnb_ genes from geosmin and MIB gene clusters. The similarities among six _cnb_ genes from representative cyanobacterial species, including _A. urcanica_ CHAB 1432 (geosmin), _Pseudanabaena_ sp. dqh15 (MIB) and _Planktothricoides raciborskii_ CHAB 3331 (MIB), are between 60% and 88.5%. These _cnb_ genes associated with MIB synthesis have higher similarities (77.1% to 88.5%) to those between geosmin and MIB operon (60% to 62.8%).