The diversity, origin, and evolutionary analysis of geosmin synthase gene in cyanobacteria

Science of the Total Environment 689 (2019) 789–796

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

The diversity, origin, and evolutionary analysis of geosmin synthase gene in cyanobacteria Zhongjie Wang a, Gaofei Song b, Yeguang Li a, Gongliang Yu b, Xiaoyu Hou a,c, Zixuan Gan d, Renhui Li b,⁎ a

Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, The Innovative Academy of Seed Design, Chinese Academy of Sciences, Wuhan 430074, PR China Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, PR China University of Chinese Academy of Sciences, Beijing 100049, PR China d Wuhan Foreign Language School Meiga Academy, Wuhan 430200, PR China b c

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• 16 geosmin-producing cyanobacteria species representing 11 genera were isolated. • Geosmin synthase gene (geo) of 16 cyanobacteria species was cloned and characterized. • Purifying selection and highly conserved in the evolution of cyanobacterial geo • Horizontal gene transfer of geo in cyanobacteria evolution history was confirmed. • Results shed light on the molecular detection and monitoring of geosmin.

a r t i c l e

i n f o

Article history: Received 26 March 2019 Received in revised form 27 June 2019 Accepted 27 June 2019 Available online 28 June 2019 Editor: Ewa Korzeniewska Keywords: Odor Phylogeny Purifying selection Horizontal gene transfer Evolution Sesquiterpene

a b s t r a c t The sesquiterpene geosmin, mainly originating from cyanobacteria, is considered one of the problematic odor compounds responsible for unpleasant-tasting and -smelling water episodes in freshwater supplies. The biochemistry and genetics of geosmin synthesis in cyanobacteria is well-elucidated and the geosmin synthase gene (geo) has been cloned and characterized in recent years. However, understanding the diversity, origin, and evolution of geo has been hindered by the limited availability of geo sequences to date. On the basis of the cloned geo sequences from16 filamentous geosmin-producing cyanobacterial species, representing 11 genera in Nostocales and Oscillatoriales, the diversity and evolution of geo in cyanobacteria was systematically analyzed in this study. Homologous alignment revealed that geo is highly conserved among the examined cyanobacterial species, with DNA sequence identities N0.72. Phylogenetic reconstruction and codon bias analysis based on geo suggest that cyanobacterial geo form a monophyletic branch with a common origin and ancestor for cyanobacteria, actinomycetes, and myxobacteria. The global ratio of nonsynonymous/synonymous nucleotide substitutions (dN/dS) was 0.125, which is substantially b1 and indicates strong purifying selection in the evolution of cyanobacterial geo. To add to further interest, horizontal gene transfer of cyanobacterial geo in evolutionary history was confirmed by the discovery of an incongruent coevolutionary relationship between geo and housekeeping genes 16S rDNA and rpoC. The present study enhances the fundamental understanding of cyanobacterial geo in diversity and evolution, and sheds light on the development of molecular assays for detection and molecular ecology research of geosmin-producing cyanobacteria. © 2019 Elsevier B.V. All rights reserved.

⁎ Corresponding author at: No. 7 Donghu South Road, Wuchang District, Wuhan 430072, PR China. E-mail address: [email protected] (R. Li).

https://doi.org/10.1016/j.scitotenv.2019.06.468 0048-9697/© 2019 Elsevier B.V. All rights reserved.

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1. Introduction The aquatic ecosystem of drinking water is threatened by secondary compounds produced by cyanobacteria, including various toxins and volatile organic compounds, accompanied by increasing cyanobacterial blooms and eutrophication (Lee et al., 2017; Scholz et al., 2017). Among the numerous volatile organic compounds released by cyanobacteria, the earthy-musty smelling geosmin (trans-1, 10-dimethyl-trans-9decalol) is perhaps the most problematic odorant and has become a focus of biological studies (Jüttner and Watson, 2007; Watson et al., 2016). Geosmin was first identified and characterized in Streptomyces as a sesquiterpene-derived alcohol (Gerber and Lecheval, 1965), and subsequently found in various strains of distantly related filamentous cyanobacterial taxa, with or without heterocyst differentiation (Izaguirre and Taylor, 2004; Jüttner and Watson, 2007; Watson et al., 2016). This odorant has a very low sensory threshold (4–20 ng L−1 for humans) and is considered a major cause of most taste and odor episodes in drinking water (Cook et al., 2001; Jüttner and Watson, 2007; Smith et al., 2008). The biochemistry and genetics of geosmin synthesis have been elucidated in Streptomyces and cyanobacteria successively for the last 30 years. Initially, geosmin was assumed to be synthesized through two alternative biosynthesis pathways: the mevalonate (MV) isoprenoid pathway mainly used by Streptomyces (Hamano et al., 2002) and myxobacteria (Dickschat et al., 2005); and the 2-methylerythritol-4phosphate (MEP) pathway mainly used by cyanobacteria (Kuzuyama, 2002). The elucidation of the synthesis from the direct precursor, farnesyl diphosphate (FPP), to geosmin, was aided by the genome sequencing of several geosmin-producing actinomycetes. Gust et al. (2003) and Cane and Watt (2003) independently revealed that SCO6073, a putative 726-amino acid sesquiterpene cyclase encoded by sco6073 in Streptomyces coelicolor A3(2), is required for geosmin synthesis. Further studies by Jiang et al. (2006, 2007) demonstrated that SCO6073 is a bifunctional enzyme composed of two domains, the Nand C-terminal domains, and is defined as geosmin synthase (Geo). This enzyme first catalyzes the conversion of FPP to germacradienol by its N-terminal domain, and finally to geosmin by its C-terminal domain, solely in the presence of Mg2+. Subsequently, several geosmin synthase genes (geo) sharing high sequence similarity with sco6073 have been identified in some genome-sequenced actinomycetes (Streptomyces, Frankia, Saccharopolyspora) and myxobacteria (Cane and Ikeda, 2012). The synthesis of geosmin in cyanobacteria has similar biochemical mechanisms and homologous geo with Streptomyces. Ludwig et al. (2007) cloned two putative geo genes (geoA1 and geoA2) from a geosmin-producing Phormidium sp. for the first time, using degenerated primer PCR from the conserved regions of geo in Streptomyces. Later, a hypothetical geo (npun2003620) with incomplete C-terminal was identified in the sequenced genome Nostoc punctiforme PCC 73102, however, it was unable to catalyze FPP to geosmin (Agger et al., 2008). Giglio et al. (2008) identified the sequencing error in the genome data of npun2003620 and demonstrated that this gene is a completed geo with the ability of convert FPP to geosmin. To date, geo has also been identified from the genome-sequences of Oscillatoriasp. PCC 6506, Calothrix sp. PCC 7507 and Cylindrospermum stagnale PCC 7417, and characterized in Anabaena ucrainica, Lyngbya kuetzingii, and Leptolyngbya bijugata (Zhang et al., 2014; Wang et al., 2015a, 2015b). In addition, Giglio et al. (2013) and Wang et al. (2015b) revealed that geo is closely associated with two putative cyclic nucleotide-binding protein genes (cnb) and furthermore forms an operon in cyanobacteria. Though several articles on cyanobacteria-related geosmin have been published in recent years, they have mostly focused on the aquatic environment or the characterization of geo in a specific cyanobacterium (Zhang et al., 2014; Wang et al., 2015a, 2015b; Watson et al., 2016). Nearly 70 geosmin-producing cyanobacterial species have been documented, including those from the genera Anabaena, Nostoc, Lyngbya,

Oscillatoria, Phormidium, Planktothrix, and Aphanizomenon (Izaguirre and Taylor, 2004; Watson et al., 2016), however, there are very rarely cyanobacterial geo sequences released. Interestingly, our previous research revealed a possible horizontal gene transfer (HGT) of geo in cyanobacteria (Wang et al., 2015b). Therefore, the exploration of the origin, diversity, and evolution of geo in cyanobacteria is not only significantly helpful to understand the function and regulation of geosmin, but also conducive to the development of geo-based molecular assays. In the process of studying odorous cyanobacteria, we isolated and purified 16 geosmin-producing cyanobacterial species, representing 11 genera in Nostocales and Oscillatoriales. The purposes of the present study are to reveal the origin of geo and analyze its diversity and evolutionary lineage in cyanobacteria thoroughly, on the basis of geo sequences amplified from these isolates and previously reported sequences. 2. Materials and methods 2.1. Isolation and identification of geosmin-producing cyanobacteria Water/soil samples with earthy-musty smell were collected from various habitats of six provinces in China, including Hubei, Jiangsu, Zhejiang, Sichuan, Yunnan, and Inner Mongolia in the last ten years for the isolation of geosmin-producing cyanobacteria by our lab. Pasteur capillary pipette method was used for the separation and purification of cyanobacterial strains from the samples, as previously described by Wang et al. (2015a). The strains obtained were further cultured in CT medium and maintained in 10 mL screw-capped tubes under an illumination of 30 μmol photons m−2 s−1 at 25 °C and were deposited in the Collection of Harmful Algae Biology (CHAB), Institute of Hydrobiology, Chinese Academy of Sciences (CAS). Taxonomic identifications were accorded following the descriptions of Komárek and Anagnostidis (2005) and AlgaeBase (http://www.algaebase.org). In addition to these isolated strains, three geosmin-producing cyanobacteria from the Freshwater Algae Culture Collection at the Institute of Hydrobiology (FACHB), CAS, namely Anabaena minutissima FACHB 250, Nostoc commune FACHB 261 and Lyngbya kuetzingii FACHB 388, were confirmed as geosmin-producing organisms by our group for the first time and were used in the present study. Detailed information of the isolated strains is listed in Table 1. All the strains used were confirmed as geosmin-producers using solid phase microextraction (SPME) and Polydimethylsiloxane/ Divinylbenzene fiber (Supelco, Bellefonte, USA) coupled with gas chromatography-mass spectrometer (GC–MS) (Hewlett Packard, Palo Alto, USA), as described by our previous studies (Wang et al., 2015a). 2.2. DNA extraction, cloning and sequencing of geo, 16S rDNA and rpoC Cyanobacterial cells were collected by centrifugation (12,000g, 10 min) from 2 mL cultures during the exponential growth phase, and the total genomic DNA was extracted using a DNA Mini Spin kit (TIANGEN BioTech, Beijing, China) according to the manufacturer's instructions. After determining purity and concentration using microvolume spectrophotometers (NanoDrop 8000, Thermo Scientific, Waltham, USA), the isolated DNA was stored at −20 °C. Based on the available cyanobacterial geo sequences, i.e. in Anabaena ucrainica (Wang et al., 2015b), Nostoc punctiforme PCC 73102 (Agger et al., 2008; Giglio et al., 2008), Phormidium sp. (Ludwig et al., 2007), Lyngbya kuetzingii UTEX 1547 (Zhang et al., 2014), and Leptolyngbya bijugata (Wang et al., 2015a), we designed a pair of universal primers, GSMIf (5′-TGGTATGTNTGGGTRTTCTT-3′) and GSMIr (5′-ATGTATTC RATGGGGTTRGC-3′) for cyanobacterial geo. The PCR reaction was carried out in a GeneAmp PCR System 9700 (Applied Biosystems, Waltham, USA) using the following program: preliminary denaturation at 94 °C for 3 min, followed by 40 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 20 s. The target PCR products (311 bp) were cloned into PMD-18 T vector (Takara

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Table 1 Geosmin-producing cyanobacteria strains isolated in this study and collected species with geo in genome. Species and strains

Taxonomy

Origin

Habitats

VOCs

Anabaena ucrainica CHAB 1434 Anabaena planctonica SDZ-1 Anabaena circinalis CHAB 3585 Anabaena minutissima FACHB 250 Calothrix sp. CHAB 2384 Cylindrospermum sp. CHAB 2115 Nostoc commune FACHB 261 Nodularia sp. Su-A Aphanizomenon sp. CHAB 1684 Aphanizomenon gracile CHAB 2417 Nostoc flagelliforme CHAB 2816 Scytonema sp. CHAB 3651 Tychonema bourrellyi CHAB 663 Lyngbya kuetzingii FACHB 388 Phormidium sp. D6 Leptolyngbya bijugata A4 Oscillatoria sp. PCC 6506 Nostoc punctiforme PCC 73102 Cylindrospermum stagnale PCC 7417 Calothrix sp. PCC 7507 Calothrix sp. NIES-2100 Nostoc linckia NIES-25 Nostoc sp. NIES-2111

Nostocales Nostocales Nostocales Nostocales Nostocales Nostocales Nostocales Nostocales Nostocales Nostocales Nostocales Nostocales Oscillatoriales Oscillatoriales Oscillatoriales Oscillatoriales Oscillatoriales Nostocales Nostocales Nostocales Nostocales Nostocales Nostocales

Dianchi Lake, Yunnan Sidouzhu Reservoir, Hubei Qiandao Lake, Zhejiang FACHB Dianchi Lake, Yunnan Dali, Yunnan FACHB A pond in Japan Taihu Lake, Jiangsu Xinghu Lake, Hubei Inner Mongolia Ganzhi, Sichuan Erhai Lake, Yunnan FACHB A fish pond, Hubei Xiaogan, Hubei PCC PCC PCC PCC NIES NIES NIES

Planktonic, freshwater Planktonic, freshwater Planktonic, freshwater Benthic, freshwater Periphytic, freshwater Benthic, freshwater Periphytic, soil Planktonic, brackish water Planktonic, freshwater Planktonic, freshwater Periphytic, soil Periphytic, rock Periphytic, freshwater Periphytic, soil Periphytic, freshwater Periphytic, soil – – – – – – –

geosmin geosmin geosmin geosmin geosmin geosmin geosmin geosmin geosmin geosmin geosmin geosmin geosmin geosmin geosmin geosmin & MIB geosmin geosmin geosmin geosmin Unknown Unknown Unknown

geo length (bp) 2256 (W) 2256 (W) 2256 (W) 2253 (W) 2289 (W) 1552 (P) 2235 (W) 2235 (W) 2259 (W) 2262 (W) 953 ((P)) 2259 (W) 2268 (W) 2313 (W) 2298 (W) 2142 ((P)) 2240 (W) 2262 (W) 2292 (W) 2256 (W) 2292 (W) 2131 ((P)) 2131 ((P))

Note: FACHB, Freshwater Algae Culture Collection at the Institute of Hydrobiology, CAS; CHAB, Collection of Harmful Algae Biology (CHAB), Institute of Hydrobiology, CAS; PCC, Pasteur Culture Collection; NIES, Microbial Culture Collection at the National Institute for Environmental Studies (Tsukuba, JAPAN); Su-A, D6 and A4 are personal codes of strain collector; W, whole length, P, partial length.

Bio, Kusatsu, Japan) and sequenced. For the whole geo length of each geosmin-producing species, genome walking technique using a kit (Takara Bio, Dalian, China) was used as described by our previous studies (Wang et al., 2015b) and the instrument, on the basis of the corresponding sequenced geo fragment. Open reading frames (ORFs) of geo were identified using ORF finder tool of NCBI (https://www.ncbi.nlm. nih.gov/orffinder/). The housekeeping genes 16S rDNA and rpoC were selected as molecular markers for phylogenetic analysis. Two primer sets, Cya106F (5′CGGACGGGTGAGTAACGCGTGA-3′) and 1492R (5′-GGTTACCTTGTTAC GACTT-3′); rpoCf (5′-GATATGCCNYTGCGGGATGT-3′) and rpoCr (5′TGGTCBCCRTCAAARTCRGC-3′), were used for the amplification of these two genes, respectively, using the aforementioned PCR programs with the exception of a longer extension time (60 s). All target products were cloned and sequenced as described above. The obtained geo, 16S rDNA and rpoC sequences of isolated geosminproducing cyanobacteria have been submitted to GenBank. The accession numbers are MK213943-MK213958 for geo, MK209088MK209103 for 16S rDNA and MK204447-MK204462 for rpoC; detailed information is provided in Table S1.

2.3. Diversity, codon bias, and phylogenetic analysis of the geo sequences Other than cyanobacteria, the two microbial groupsactinomycetes and myxobacteria also produce geosmin in the natural environment, and geo shared high similarity among these groups (Giglio et al., 2008; Zhang et al., 2014; Wang et al., 2015a). Thus we collected 7 geo sequences of cyanobacteria, 30 geo sequences of actinomycetes and 10 geo sequences of myxobacteria, from GenBank using the BLAST algorithm (www.blast.ncbi.nlm.nih.gov/Blast.cgi) (Table 1, Table S1). The obtained geo sequences in the present study and other collected sequences were edited and aligned using BioEdit (http://www.mbio. ncsu.edu/bioedit/bioedit.html), and the identities of DNA and amino acids sequences were calculated by MEGA-X (Kumar et al., 2018). After a best-fit nucleotide substitution models search, geo's phylogenetic tree was constructed using the neighbor-joining (NJ) method and the maximum likelihood (ML) method in MEGA-X, using the GTR substitution model, with a bootstrap value of 1000.

The relative synonymous codon usages of geo in different geosminproducing groups, including 23 cyanobacterial species, 30 actinomycetes species and 10 myxobacteria species, and of geo and rpoC within cyanobacteria, were calculated using the Compute Codon Usage Bias module equipped in MEGA-X. 2.4. Recombination detection and natural selection analyses of cyanobacterial geo The global ratio of nonsynonymous nucleotide substitutions (dN) to synonymous nucleotide substitutions (dS), known as ω (ω = dN/dS), was considered as the indicator of selection pressure, where N1, =1, and b1 indicated positive selection, neutral evolution, and purifying selection, respectively. In the present study, we calculated the ω value of cyanobacterial geo on the basis of 23 obtained sequences to assess the selection pressure on this gene during evolution. In order to avoid possible recombination, which often leads to a significant increase in false positives, the genetic algorithm for recombination detection (GARD) method in the Datamonkey web-server (www.datamonkey.org) (Weaver et al., 2018) was used to identify potential recombination breakpoints before ω calculation. Furthermore, five different sitespecific methods in the Datamonkey web-server were used to calculate the ω value of geo: the single likelihood ancestor counting (SLAC) method; the fixed effects likelihood (FEL) method; the random effects likelihood (REL) method; the mixed effects model of evolution (MEME); and Fast Unconstrained Bayesian AppRoximation (FUBAR). In addition, a web server for the identification of site-specific selection, ‘The Selecton Server’ (http://selecton.tau.ac.il/index.html), was also used to examine the natural selection of geo in cyanobacteria, using an evolutionary model with positive selection (M8, beta + ω ≥ 1). 2.5. Phylogenetic reconstruction of cyanobacterial geo, 16S rDNA and rpoC Phylogenetic relationships of geo and corresponding 16S rDNA and rpoC were reconstructed to test the coevolutionary features of geo in cyanobacteria. The cyanobacterial geo, 16S rDNA and rpoC sequences, including 16 from the present study and 7 from GenBank, were edited and aligned using BioEdit, and were later used to construct phylogenetic

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(AAs), and Nodularia sp. Su-A has the shortest 2235-bp geo, encoding 744 AAs. In terms of homology, geo of 23 cyanobacterial strains shared high sequence identities. As shown in Table 2, identity matrix revealed high conservation of geo in DNA, with identities of 0.68–1.00, and in AAs, with identities of 0.75–0.00, among tested cyanobacterial groups. The geo sequences of 16 isolated cyanobacterial strains are homologous to the elucidated reference geo in Nostoc punctiforme PCC 73102 with DNA identities ranging from 0.78 to 0.91 and AAs identities ranging from 0.78 to 0.96. In contrast, cyanobacterial geo sequences showed a certain degree of diversity according to the taxonomic positions. The three planktonic Anabaena species had geo genes with nearly identical sequences (identities over 0.99), however these were considerably different from Anabaena minutissima FACHB 250 geo gene sequence (identities b0.78). Scytonema sp. CHAB 3651 had the lowest homology (0.72–0.74) with other heterocystous taxa. In addition, homology of cyanobacterial geo with geo genes from other two geosmin-producing groups, actinomycetes and myxobacteria, was also explored. Cyanobacterial geo shared DNA identities of 0.49–0.72 with homologous genes in actinomycetes and DNA identities of 0.51–0.73 with myxobacteria.

trees using NJ and ML methods as described above. The myxobacteria Myxococcus xanthus DK 1622 was used as outgroup. 3. Results 3.1. Geosmin-producing cyanobacteria isolated in this study A total of 16 pure filamentous cyanobacteria strains from different habitats with unique morphological characteristics and earthy-musty odors were isolated or collected, and were confirmed for their ability to synthesize geosmin (Table 1). These geosmin-producers were taxonomically divided into different species on the basis of morphological characteristics and 16S rDNA sequences, representing 11 genera in filamentous Nostocales and Oscillatoriales of cyanobacteria: Anabaena, Nostoc, Cylindrospermum, Nodularia, Aphanizomenon, Calothrix, Scytonema, Tychonema, Lyngbya, Leptolyngbya, and Phormidium. Except for Nodularia sp. Su-A, Anabaena minutissima FACHB 250, and Nostoc commune FACHB 261, which were isolated in Japan or obtained from the FACHB collection, most cultures were isolated from various habitats in six Chinese provinces (shown in Table 1), including lakes, reservoirs, fish ponds, soil, and rock surfaces. From a habitat perspective, six geosmin-related species were planktonic with gas vesicles and were categorized as typical bloom-forming cyanobacteria (such as Anabaena planktonica, Anabaena circinalis, and Aphanizomenon gracile), while the other 10 cultures were categorized as benthic/periphytic in most periods of life.

3.3. Phylogenetic and codon bias analysis of geo in cyanobacteria, actinomycetes, and myxobacteria Geosmin exists widely in cyanobacteria, actinomycetes, and myxobacteria, and similar mechanisms of geosmin synthesis and homology of geo genes have been elucidated (Giglio et al., 2008; Zhang et al., 2014; Wang et al., 2015a). To explore the origin and evolutionary development of geo in cyanobacteria and other groups of microorganisms, phylogenetic analysis of geo was performed based on 23 sequences of cyanobacteria, 30 sequences of actinomycetes, and 10 sequences of myxobacteria using NJ/ML algorithms. As shown in Fig. 1, three monophyletic branches were observed. All cyanobacterial species clustered and formed a monophyletic branch with bootstrap values of 100/100. Leptolyngbya bijugata A4 represents an independent sub-branch within the cyanobacterial branch but removed from other cyanobacterial strains. Actinomycetes and myxobacteria also formed independent branches. For further understanding of the evolutionary relationship of geo among cyanobacteria, actinomycetes, and myxobacteria, the codon bias of geo was analyzed by relative synonymous codon usage (RSCU) after excluding three stop codons. Fig. 2 shows that the cyanobacterial geo has significant differences in RSCU at most sites of the 61 informative codons from homologous genes of actinomycetes and myxobacteria. In contrast,

3.2. The diversity of geo in cyanobacteria Using geo universal primers GSMIf/r and genome walking technique, the whole gene sequence of geo was cloned and identified in 13 isolated geosmin-producing strains (species). Only partial geo sequences ranging from 953-bp to 2142-bp length were obtained for the other strains, Cylindrospermum sp. CHAB 2115, Nostoc flagelliforme CHAB 2816, and Leptolyngbya bijugata A4 (Table 1). In order to explore the diversity of geo in cyanobacteria, 7 representative corresponding geo sequences were downloaded from GenBank through homologous BLAST including Nostoc punctiforme PCC 73102 and six other strains belonged to the genera Oscillatoria, Cylindrospermum, Calothrix, and Nostoc. Detailed information is provided in Table 1. The cyanobacterial geo varies in whole length. Lyngbya kuetzingii FACHB 388 has the longest 2313-bp geo, encoding 770 amino acids Table 2 Identity matrix of cyanobacterial geosmin synthase gene (geo). Strains

A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

S

T

U

V

W

CHAB 1434 (A) SDZ-1 (B) CHAB 3585 (C) FACHB 250 (D) CHAB 2384 (E) CHAB 2115 (F) FACHB 261 (G) Su-A (H) CHAB 1684 (I) CHAB 2417(J) CHAB 2816 (K) CHAB 3651 (L) CHAB 663 (M) CHAB 388 (N) D6 (O) A4 (P) PCC 6506 (Q) PCC 73102 (R) PCC 7417 (S) PCC 7507 (T) NIES 2100 (U) NIES 25 (V) NIES 2111 (W)

/ 1.00 1.00 0.78 0.81 0.78 0.79 0.79 0.88 0.89 0.78 0.72 0.82 0.80 0.80 0.69 0.81 0.79 0.77 0.80 0.77 0.71 0.71

1.00 / 1.00 0.78 0.81 0.78 0.79 0.79 0.88 0.89 0.78 0.72 0.82 0.81 0.80 0.69 0.81 0.79 0.78 0.80 0.77 0.71 0.71

1.00 1.00 / 0.77 0.81 0.78 0.79 0.79 0.88 0.89 0.78 0.72 0.82 0.80 0.79 0.69 0.80 0.78 0.77 0.80 0.77 0.71 0.71

0.86 0.86 0.85 / 0.80 0.85 0.83 0.84 0.78 0.79 0.81 0.74 0.76 0.80 0.81 0.73 0.80 0.84 0.83 0.83 0.81 0.73 0.72

0.88 0.88 0.88 0.87 / 0.84 0.83 0.82 0.83 0.83 0.81 0.72 0.79 0.83 0.85 0.73 0.83 0.84 0.82 0.87 0.82 0.70 0.70

0.88 0.88 0.87 0.90 0.90 / 0.88 0.87 0.82 0.81 0.87 0.73 0.78 0.86 0.87 0.74 0.87 0.91 0.89 0.88 0.85 0.71 0.71

0.86 0.86 0.86 0.89 0.88 0.93 / 0.88 0.81 0.82 0.98 0.74 0.77 0.86 0.84 0.74 0.86 0.91 0.87 0.87 0.83 0.72 0.72

0.88 0.88 0.87 0.87 0.88 0.92 0.93 / 0.81 0.81 0.86 0.74 0.77 0.84 0.84 0.75 0.85 0.90 0.87 0.86 0.85 0.73 0.72

0.96 0.96 0.95 0.86 0.89 0.88 0.87 0.88 / 0.99 0.80 0.72 0.84 0.81 0.81 0.73 0.82 0.81 0.80 0.84 0.78 0.71 0.72

0.96 0.96 0.95 0.86 0.89 0.88 0.87 0.88 0.99 / 0.80 0.72 0.84 0.81 0.81 0.72 0.82 0.81 0.80 0.84 0.78 0.71 0.72

0.83 0.83 0.83 0.86 0.85 0.90 0.97 0.90 0.84 0.84 / 0.73 0.76 0.84 0.82 0.73 0.84 0.89 0.85 0.85 0.81 0.71 0.70

0.78 0.78 0.78 0.79 0.78 0.78 0.78 0.80 0.77 0.77 0.75 / 0.70 0.73 0.72 0.71 0.73 0.74 0.72 0.72 0.73 0.92 0.90

0.91 0.91 0.91 0.84 0.88 0.86 0.85 0.85 0.91 0.91 0.82 0.76 / 0.77 0.76 0.68 0.77 0.78 0.75 0.79 0.76 0.70 0.69

0.88 0.88 0.88 0.86 0.90 0.92 0.91 0.91 0.88 0.89 0.88 0.79 0.86 / 0.87 0.72 1.00 0.85 0.84 0.86 0.81 0.71 0.72

0.87 0.87 0.87 0.87 0.89 0.91 0.88 0.89 0.87 0.87 0.85 0.77 0.85 0.92 / 0.72 0.87 0.85 0.85 0.85 0.80 0.70 0.70

0.76 0.76 0.76 0.79 0.79 0.79 0.79 0.79 0.77 0.77 0.77 0.76 0.76 0.78 0.77 / 0.73 0.75 0.75 0.74 0.74 0.70 0.70

0.89 0.89 0.88 0.87 0.90 0.92 0.92 0.91 0.89 0.89 0.89 0.79 0.86 1.00 0.92 0.79 / 0.85 0.85 0.86 0.81 0.71 0.72

0.87 0.87 0.87 0.89 0.90 0.94 0.94 0.96 0.88 0.88 0.91 0.79 0.86 0.91 0.89 0.78 0.91 / 0.88 0.89 0.85 0.72 0.72

0.86 0.86 0.86 0.90 0.88 0.93 0.91 0.91 0.87 0.88 0.88 0.78 0.85 0.90 0.88 0.79 0.90 0.92 / 0.85 0.85 0.71 0.70

0.91 0.91 0.91 0.87 0.91 0.92 0.92 0.92 0.92 0.92 0.89 0.79 0.89 0.92 0.91 0.78 0.93 0.93 0.91 / 0.84 0.71 0.72

0.86 0.86 0.86 0.89 0.89 0.91 0.90 0.92 0.87 0.87 0.87 0.78 0.86 0.89 0.87 0.78 0.89 0.92 0.92 0.91 / 0.71 0.71

0.77 0.77 0.76 0.77 0.77 0.77 0.78 0.79 0.76 0.76 0.75 0.95 0.75 0.78 0.76 0.76 0.79 0.78 0.77 0.79 0.78 / 0.92

0.77 0.77 0.77 0.78 0.78 0.78 0.79 0.81 0.76 0.76 0.76 0.95 0.75 0.79 0.76 0.76 0.79 0.79 0.78 0.79 0.79 0.98 /

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Fig. 1. Unrooted NJ/ML phylogenetic tree based on geosmin synthase gene (geo) (2148 bp) of geosmin-producing microorganisms. The blue, red and green lines are cyanobacteria, actinomycetes and myxobacteria species respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

geo has similar RSCU in most codons in actinomycetes and myxobacteria. Within the cyanobacterial group, no significant differences in RSCU were observed at most codons between geo and rpoC. In addition, most RSCUs of cyanobacterial geo fluctuated around 1.0% (between 0.5 and 1.5%), indicating average usage of synonymous codons, whereas RSCUs with great differences in actinomycetes and myxobacteria imply obvious usage bias of synonymous codons. 3.4. The selection pressures of cyanobacterial geo in evolution No evidence of recombination was identified in the 23 cyanobacterial geo sequences by the GARD test. Thus, these sequences were used directly for the evolutionary analysis. SLAC and FUBAR methods were used to calculate the dN-dS and ω, respectively, of each codon site. Fig. 3 shows that dN-dS b 0, which means dN b dS, in most sites of cyanobacterial geo. Though dN-dS N 0 was found in several sites in SLAC analysis, the ω of most sites was under 0.4 and no site with ω N 1 was observed. The global ω value of cyanobacterial geo was calculated as 0.125, which is far b1.0 and implies strong purifying selection using SLAC, FEL, MEME, and FUBAR methods (Table 3). In site-specific selection analysis, no positively selected site was detected by SLAC and FUBAR analysis, although 365 and 553 negatively selected sites were confirmed, respectively. Analysis results of ‘The Selecton Server’ did not reveal any positively selected sites either (Fig. S1). The other methods, FEL, REL, and MEME, identified 2, 3, and 11 positively selected sites, respectively, including only one common site (733). No positively selected functional domain sites were located after thorough comparison with Geo from N. punctiforme PCC 73102.

3.5. Comparison of geo- and housekeeping genes-based phylogenetic trees A phylogenetic topology comparison with a housekeeping gene could provide coevolution information of the target gene, such as horizontal gene transfer (HGT) in the genome during evolutionary history. Using the myxobacteria Myxococcus xanthus DK 1622 as the outgroup, NJ/ML phylogenetic trees of cyanobacterial geo and housekeeping 16S rDNA and rpoC genes were reconstructed and compared. As shown in Fig. 4, the phylogenetic trees based on geo, 16S rDNA and rpoC revealed incongruent topologies. Five species of Oscillatoriales are clustered in the 16S rDNA and rpoC tree, which reflects true taxonomic relationships, however, the geo tree indicates different branch positions of these species. Tychonema bourrellyi CHAB 663 grouped with Anabaena and Aphanizomenon with bootstrap values of 100/100. Phormidium sp. D6, Oscillatoria sp. PCC 6506 and Lyngbya kuetzingii FACHB 388 formed a mini-cluster in Nostocales group far from Leptolyngbya bijugata A4. Topology incongruencies were also observed at the genera level in Nostocales. In the 16S rDNA and rpoC tree, species belonging to same genus clustered perfectly, however, they were distributed in different clades in the geo tree, mainly including the six Nostoc-Nodularia species, which clustered into two separate clades, and the species of one genus (Calothrix and Cylindrospermum) belonged to two clades which are far removed.

4. Discussion To better understand geo in cyanobacteria, we cloned the geo sequences of 16 isolated cyanobacterial species. We systematically

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Fig. 2. The relative synonymous codon usages of geo in cyanobacteria, actinomycetes and myxobacteria (A), and of geo and rpoC within cyanobacteria (B).

explored characteristics of geo including sequence diversities, phylogenetic relationships among geosmin-producing microorganisms, natural selection, and determination of evolutionary lineage. Increasing numbers of geosmin-related cyanobacteria have been identified and recorded worldwide for decades due to the great concern over off-flavor events. The summaries by Izaguirre et al. (1982); Izaguirre and Taylor (2004), Jüttner and Watson (2007), and Smith et al. (2008) list over 40 geosmin-producing cyanobacterial species taxonomically belonging to unicellular/colonial Chroococcales and filamentous Nostocales, Oscillatoriales, and Stigonematales, and the list

Fig. 3. The dN-dS and dN/dS (ω) values of each geo codon sites calculated by SLAC and FUBAR methods. dN: nonsynonymous nucleotide substitutions; dS: synonymous nucleotide substitutions.

has rapidly increased to nearly 70 species in recent years (Watson et al., 2016). The geosmin-producing strains used in the present study, Nostoc commune, Nostoc flagelliforme, Scytonema sp., Nodularia sp., and Anabaena minutissima were identified as geosmin-producing taxa for the first time, which not only expanded the known diversity of odorproducing cyanobacteria, but also implied an underestimation of the quantity of these species. Thus, further work on the species diversity of geosmin-producing cyanobacteria should be pursued. The present study highlighted the genetic diversity and conservation of geo in cyanobacteria. Since the first illustration of geo in cyanobacteria Phormidium sp. (Ludwig et al., 2007) and sequencing of genome of N. punctiforme PCC 73102 (Agger et al., 2008; Giglio et al., 2008), only whole cyanobacterial geo sequences had been identified and sequenced, such as those of Anabaena ucrainica (Wang et al., 2015b) and Lyngbya kuetzingii (Zhang et al., 2014). Thus, the extent of the genetic diversity of geo in cyanobacteria has been hindered by a lack of sufficient sequencing data. The alignment and homology comparison of obtained geo sequences revealed that although there are slight differences in length, geo is highly conserved among the cyanobacteria surveyed, with identities over 0.72. Cyanobacteria is considered the main biological factor responsible for most geosmin episodes (Izaguirre and Taylor, 2004; Jüttner and Watson, 2007; Watson et al., 2016; Zhang et al., 2016). The sequence conservation of geo in cyanobacteria provides the possible basis for development of molecular strategies for geosmin detection. In fact, several detection techniques for specific geosminproducing cyanobacteria have been reported, which mainly include an assay incorporating melting curve analysis developed by Giglio et al. (2008), quantitative PCR (qPCR) targeting geosmin-producing Anabaena developed by Su et al. (2013) and Tsao et al. (2014), a molecular assay for geosmin-producing cyanobacteria and actinomycetes developed by Kutovaya and Watson (2014), and a geosmin PCR tool for quantitative assay recently reported by John et al. (2018). Procedures for qualitative and quantitative detection using primers or probes for most of the geosmin-producing cyanobacteria have not been developed largely due to the lack of reference geo sequences. However, the present study now provides diverse cyanobacterial geo sequence data for the development and establishment of new protocols. Geosmin synthesis ability is widely found in the three microorganism groups, cyanobacteria, actinomycetes and myxobacteria (Dickschat et al., 2005; Jüttner and Watson, 2007; Watson et al., 2016). The direct precursor of geosmin, FPP, is used in different pathways, such as the MV isoprenoid pathway in Streptomyces and myxobacteria (Hamano et al., 2002; Dickschat et al., 2005), and the MEP pathway in cyanobacteria Synechocystis (Kuzuyama, 2002). The Geo of cyanobacteria, which catalytically converts FPP to geosmin, shares a similar catalytic mechanism and high homology of corresponding coding geo with the other groups (Jiang et al., 2007; Giglio et al., 2008; Zhang et al., 2014; Wang et al., 2015b). Thus, the origin of cyanobacterial geo and its relationships with the other two geosminproducing groups in evolution is of major interest. Phylogenetic reconstruction based on the geo sequences of the representative groups showed that cyanobacteria formed a monophyletic group independent of actinomycetes and myxobacteria (Fig. 1). Considering the homology and phylogenetic relationships of geo described above, it is reasonable to conclude that geo of cyanobacteria has a common origin and ancestor with homologous genes of actinomycetes and myxobacteria. Such a conclusion was further supported by the observed substantial difference in the geo RSCU of cyanobacteria with other microorganisms (Fig. 2). All reported geosmin-producing cyanobacteria are widely distributed in different genera, with most belonging to filamentous Oscillatoriales and Nostocales (Jüttner and Watson, 2007; Watson et al., 2016), and a few belonging to Chroococcales, e.g. Coelosphaerium and Synechococcus (Watson et al., 2016; Godo et al., 2017), and Stigonematales, e.g. Fischerella muscicola (Wu and Jüttner, 1988). Therefore, it is likely that geo was inherited from the common ancestor of cyanobacteria, and is retained in many species in evolution.

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Table 3 Selection test of cyanobacterial geo using different methods. Method

Global dN/dS

SLAC FEL REL MEME FUBAR

0.125 0.125 – 0.125 0.125

Positively selected sites

No. of negatively selected sites

– 41, 733 370, 382, 733 41, 207, 345, 422, 456, 535, 603, 725, 726, 733, 743 –

The analysis of natural selection pressure on geo in cyanobacteria revealed that the global ω values derived from SLAC, FEL, MEME, and FUBAR were significantly b1, and more than half of the codon sites were confirmed as negatively selected sites (Table 3). These results suggest strong purifying selection in the evolution of geo. Based on the aforementioned findings, it can be theorized that geo and its product geosmin must be very important for cyanobacteria. Terpenoid compounds such as geosmin mostly function as allelochemicals in plants (Kiran et al., 2007). The results of Ikawa et al. (2001) revealed that geosmin derived from cyanobacteria plays a significant function in suppressing the growth of green alga Chlorella pyrenoidosa. Ozaki et al. (2008) confirmed that geosmin has lytic activity on Microcystis cells. Increased synthesis and extracellular proportion of geosmin were also observed under unsuitable environmental conditions in many cyanobacteria, e.g. Anabaena ucrainica, Fischerella muscicola, and Lyngbya kuetzingii (Wu and Jüttner, 1988; Zhang et al., 2009; Wang and Li, 2015). Research by Zimba et al. (1999), however, proposed that geosmin is only a byproduct of increased metabolism and is not necessary for cyanobacteria. Thus, elucidating the exact functions of geosmin in cyanobacteria demands further in-depth and mechanistic studies. It is noteworthy that geo has a close structural relationship with putative cyclic nucleotide-binding protein genes in both cyanobacteria and actinomycetes (Wang et al., 2015b), and a functional connection between these two genes deserves more attention and future research.

365 431 381 – 553

Significance p-Values b 0.05 p-Values b 0.05 Bayes factor N 50 p-Values b 0.05 Posterior probability ≥0.95

Our previous study suggested possibly inconsistent topologies between geo and 16S rDNA in six cyanobacterial species (Wang et al., 2015b). In this study, a topological comparison of phylogenetic trees for geo and housekeeping genes 16S rDNA and rpoC confirmed the incongruent between these genes (Fig. 4), and clearly indicated geo was acquired by cyanobacteria and then a mixture of co-evolution and HGT. HGT is an important form of gene exchange among microorganisms and is generally believed to confer selective advantage to the host (Gogarten and Townsend, 2005). There are some secondary metabolite-associated genes, e.g. cyanobactin synthetase gene (Leikoski et al., 2009) and saxitoxin synthesis genes (Kellmann et al., 2008), which spread by HGT in cyanobacteria. Unfortunately, HGT will cause difficulties in the development of monitoring techniques for geosmin-producing cyanobacteria. The expected techniques mainly focus on two aspects: quantification of geosmin-producing cyanobacteria using geo primers, such as the qPCR assays reported by Su et al. (2013) and Tsao et al. (2014) and analysis of the species composition of geosmin-related cyanobacteria in various environments, such as the method developed by Kutovaya and Watson (2014). Considering that there have been multiple instances of HGT of geo, it is very likely that researchers will be unable to obtain reliable results for the identification of geosmin-producing taxa only through geo sequences, and increased the difficult on quantification of specific geosminproducing taxa, in environmental monitoring. Therefore, further indepth exploration of the diversity and evolution of cyanobacterial geo, and associated monitoring techniques, are still needed.

Fig. 4. NJ/ML phylogenetic trees based on cyanobacterial geo (2154 bp), 16S rDNA (1233 bp) and rpo C (905 bp). Myxococcus xanthus DK 1622 was used as outgroup. The blue and red lines are Nostocales and Oscillatoriales species respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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5. Conclusions The present study enhances the understanding of geo genetic diversity, phylogeny, and evolution in cyanobacteria. On the basis of 16 filamentous geosmin-producing cyanobacterial species belonging to 11 genera, we demonstrated that geo is highly conserved with identities over 0.72. The phylogenetic analysis of geosmin-producing microorganisms revealed that geo of cyanobacteria formed a monophyletic branch and shared a common ancestor. Further studies confirmed strong purifying selection pressure of cyanobacterial geo in evolution. Taken together, these findings not only illustrate the conservation of geo, which provides the possible basis for development of general molecular probes targeting geosmin-producing cyanobacteria, but they also highlight the importance of geo and its product geosmin in cyanobacteria. Unfortunately, the function of geosmin is poorly understood to date, and thus demands more focused studies with special emphasis on horizontal gene transfer (HGT) of geo in evolutionary history. The difficulty in detecting the specific geosmin-producing cyanobacteria with respect to HGT of geo sequences poses complications for future molecular ecology research. Acknowledgement The authors would like to sincerely acknowledge Dr. Yang Liu (Henan Normal University), Dr. Jie Wang (Taiyuan Normal University), Dr. Xiaochuang Li and Yilu Gu (Institute of Hydrobiology, Chinese Academy of Sciences) for their kindly help in sample collection and strains isolation. Funding This research was supported by National Science Foundation of China (NSFC 51779247), National Key Research and Development Program of China (2017YFA0605201), and National Science Foundation of China (NSFC 31502192). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2019.06.468.

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