Microbiology (2009), 155, 642–656 DOI 10.1099/mic.0.022848-0

Molecular identification, typing and traceability of from freshwater reservoirs

Elisabete Vale´rio,1,2 Le´lia Chambel,1 Se´rgio Paulino,2 Nata´lia Faria,2 Paulo Pereira2 and Roge´rio Tenreiro1

Correspondence 1Universidade de Lisboa, Faculdade de Cieˆncias, Centro de Biodiversidade, Geno´mica Integrativa e Roge´rio Tenreiro Funcional (BioFIG), Edifı´cio ICAT, Campus da FCUL, Campo Grande, 1749-016 Lisboa, Portugal [email protected] 2Laborato´rio de Microbiologia e Ecotoxicologia, Instituto Nacional de Sau´de Dr Ricardo Jorge, Avenida Padre Cruz, 1649-016 Lisboa, Portugal

In order to assess the potential of several molecular targets for the identification, typing and traceability of cyanobacteria in freshwater reservoirs, molecular techniques were applied to 118 cyanobacterial isolates mostly sourced from Portuguese freshwater reservoirs and representative of three orders of cyanobacteria: Chroococcales (54), Oscillatoriales (15) and (49). The isolates were previously identified by morphological methods and subsequently characterized by composite hierarchical cluster analysis of STRR and LTRR (short and long tandemly repeated repetitive sequences) PCR fingerprinting profiles. Representative isolates were selected from each cluster and their molecular identification, at the species level, was obtained or confirmed by phylogenetic positioning using 16S rRNA gene and rpoC1 phylogenies. A highly congruent association was observed between STTR- and LTRR-based clusters and taxonomic affiliation, revealing the usefulness of such PCR fingerprinting profiles for the identification of cyanobacteria. Composite analysis of hierarchical clustering of M13 and ERIC PCR fingerprints also appeared suitable for strain typing and traceability within a reservoir, indicating its potential for use in cyanobacterial monitoring, as a quality management control. Based on Simpson (D) and Shannon–Wiener (J9) indices a high diversity was observed within all species, with Planktothrix Received 1 August 2008 agardhii showing the lowest diversity values (D50.83; J950.88) and Aphanizomenon flos-aquae Revised 13 October 2008 the highest ones (D5J950.99). A diagnostic key based on 16S-ARDRA, ITS amplification and Accepted 14 October 2008 ITS-ARDRA for identification purposes is also presented.

INTRODUCTION Bergey’s Subsection IV) or branching filaments (Order Stigonematales, or Bergey’s Subsection V) (Castenholz, Cyanobacteria are a morphologically diverse group of 2001). A sixth cyanobacterial order, Gloeobacterales, was ranging from unicellular to colonial and filament- proposed by Cavalier-Smith (2002) to accommodate the ous forms. Taxonomically, cyanobacteria are grouped into genus Gloeobacter, formerly included in the Chroococcales. unicellular forms that divide by binary fission (Order Chroococcales, or Bergey’s Subsection I) or multiple fission Traditionally, the classification of cyanobacteria has been (Order Pleurocapsales, or Bergey’s Subsection II); and based on morphological characters such as trichome width, filamentous forms that are non-heterocystous (Order cell size, division planes, shape and arrangement, pig- Oscillatoriales, or Bergey’s Subsection III) or differentiate mentation and the presence of characters such as gas heterocysts in non-branching (Order Nostocales, or vacuoles and a sheath (Baker, 1991, 1992; Bourrelly, 1970; Koma´rek & Anagnostidis, 1986, 1989). Beyond the considerable expertise required to identify species by such Abbreviations: ARDRA, amplified rDNA restriction analysis; ERIC, enterobacterial repetitive intergenic consensus; ITS, intergenic tran- characters, subjective judgment by operators can lead to scribed spacer region; LTRR, long tandemly repeated repetitive errors, resulting in incorrect assignment of isolates. sequences; STRR, short tandemly repeated repetitive sequences; Koma´rek & Anagnostidis (1989) estimated that more than UPGMA, unweighted pair group method with arithmetic average. 50 % of the strains in culture collections are misidentified. The GenBank/EMBL/DDBJ accession numbers for the rpoC1 and 16S Moreover, some diagnostic features, such as gas vacuoles or rDNA sequences are EU078443 to EU078480 (rpoC1) and akinetes, can show variations with different environmental AY699989, DQ497635 and EU078482 to EU078548 (16S rDNA). or growth conditions and even be lost during cultivation Two supplementary tables and seven supplementary figures are (Rudi et al., 1997; Lyra et al., 2001). Such limitations of available with the online version of this paper. phenotypic characters have highlighted the requirement for

642 022848 G 2009 SGM Printed in Great Britain Molecular diagnostics for cyanobacteria more reliable methods and promoted molecular A sequential polyphasic approach was used in this study. approaches in cyanobacterial , including DNA Isolates were identified by observation of their morpho- base composition (Kaneko et al., 1996, 2001), DNA logical features. A hierarchical analysis with STRR and hybridizations (Kondo et al., 2000), gene sequencing LTRR PCR fingerprinting patterns was performed and (Nu¨bel et al., 1997) and PCR fingerprinting (Rasmussen representatives of the clusters obtained were identified by a & Svenning, 1998; Versalovic et al., 1991). Cyanobacterial- phylogenetic analysis using two genes, one coding for the specific methods not requiring axenic cultures are of small-subunit rDNA (16S rRNA gene) and the other for utmost importance since such cultures are difficult to the DNA-dependent RNA polymerase subunit (rpoC1). obtain (Choi et al., 2008). Subsequent characterization of all isolates by M13 and ERIC fingerprints allowed the discrimination of strains, Repetitive sequences constitute an important part of the revealing also the traceability potential of these last prokaryotic genome (van Belkum et al., 1998). Despite methods for routine freshwater monitoring. Furthermore, their unknown function, and lack of knowledge about how a diagnostic key was constructed for the identification of they are maintained and dispersed, the presence, wide- cyanobacterial species, based on the use of 16S-ARDRA, spread distribution and high conservation of these ITS length and ITS-ARDRA. sequences make them methodologically important for DNA fingerprinting and allow their use as an alternative for the identification of species or strains and in diversity studies. In the particular case of cyanobacteria, a family of METHODS repetitive sequences, the short tandemly repeated repetitive Cyanobacterial strains. A total of 118 unicyanobacterial non-axenic sequences (STRRs), has been described (Mazel et al., 1990). cultures, belonging to the orders Chroococcales (54), Oscillatoriales These heptanucleotide sequences have been identified in (15) and Nostocales (49), mainly isolated from Portuguese freshwater several cyanobacterial genera and species, so far mostly in blooms and maintained in the LMECYA culture collection heterocystous cyanobacteria (Rasmussen & Svenning, 1998; (Cyanobacteria Culture Collection Estela Sousa e Silva – INSA) using Zheng et al., 1999; Nilsson et al., 2000; Wilson et al., 2000; Z8 medium (Skulberg & Skulberg, 1990), were used in this study Teaumroong et al., 2002; Lyra et al., 2005; Prasanna et al., (Supplementary Table S1, available with the online version of this paper). Isolates were kept under a 14 : 10 h light : dark cycle 2006), but also in some non-heterocystous ones 22 21 (20±4 mmol m s )at20±1 uC. Lyophilized samples of the (Rasmussen & Svenning, 1998). Furthermore, a 37 bp isolates were obtained as described by Saker et al. (2003). long tandemly repeated repetitive sequence (LTRR) has also been identified in some cyanobacterial species Morphological identification. The morphology of cells and (Masepohl et al., 1996; Rasmussen & Svenning, 1998; filaments was studied using an Olympus BX60 light microscope with Prasanna et al., 2006). Analysis of STRRs and LTRRs has a digital camera. The following parameters were analysed: length and been described as a powerful tool for taxonomic studies width of vegetative cells; morphology of the terminal cell; the presence or absence of heterocysts, akinetes and gas vesicles; the distance (Mazel et al., 1990). Moreover, the specificity of these between heterocysts and the distance between a heterocyst and the sequences has made STRRs useful even for non-axenic nearest akinete; and finally, the shape of filaments and their potential cyanobacterial cultures (Nilsson et al., 2000). aggregation into colonies.

A universal marker for DNA fingerprinting is the DNA extraction from cyanobacteria and heterotrophic bacteria. oligonucleotide csM13. It has already been tested in a Genomic DNA of cyanobacterial isolates was extracted according to a small number of cyanobacteria (Vale´rio et al., 2005), and previously described method (Vale´rio et al., 2005). has a demonstrated ability even to discriminate strains of For isolation of heterotrophic (non-obligately oligotrophic) bacteria, the same species. Techniques based on the enterobacterial associated with some cyanobacterial cultures (randomly selected), an repetitive intergenic consensus (ERIC) have also been used aliquot of the supernatant of isolates LMECYA 64, AQS, LMECYA 79, for identification and discrimination purposes in some LMECYA 13 and LMECYA 173 was grown on LB agar (tryptone 1 % cyanobacteria (Rasmussen & Svenning, 1998; Lyra et al., w/v; yeast extract 0.05 %, w/v; NaCl 0.05 %, w/v; agar 2 %, w/v) at 2001; Vale´rio et al., 2005; Bruno et al., 2006). 22 uC for 3 days. The strains obtained were subsequently purified and grown in LB medium (tryptone 1 %, w/v; yeast extract 0.05 %, w/v; The restriction fragment length polymorphisms (RFLPs) of NaCl 0.05 %, w/v) overnight at 22 uC for DNA extraction. Cells were particular PCR products can provide signature profiles recovered by centrifugation and total DNA extracted using the specific to the genus, species, or even strain. Genetic guanidium thiocyanate method, according to Pitcher et al. (1989). characterization of cyanobacterial strains has been under- PCR fingerprinting and data analysis. STRR PCR fingerprinting taken using RFLPs of the 16S rRNA gene (16S-ARDRA) was performed using both STRR1F (59-CCCCARTCCCCART-39) and (Lyra et al., 1997) and of the intergenic transcribed spacer STRR3F (59-CAACAGTCAACAGT-39) primers (Wilson et al., 2000). region (ITS-ARDRA) (Lu et al., 1997; West & Adams, 1997). For LTRR PCR fingerprinting both LTRR1 (59-GGATTTTTGTT- Furthermore, amplification of the 16S–23S rRNA ITS, which AGTTAAAAC-39) and LTRR2 (59-CTATCAGGGATTGAAAG-39) has been shown to be variable in length (Rocap et al., 2002; primers (Rasmussen & Svenning, 1998) were used. Iteman et al., 2002; Neilan, 2002, Laloui et al., 2002) and M13 PCR fingerprinting was performed using oligonucleotide csM13 number (West & Adams, 1997; Iteman et al., 2002) in (59-GAGGGTGGCGGTTCT-39) (Huey & Hall, 1989) as single cyanobacteria, can also be used as an identification tool. primer. For ERIC PCR fingerprinting both ERICIR (59-ATGTAA- http://mic.sgmjournals.org 643 E. Vale´rio and others

GCTCCTGGGGATTCAC-39) and ERIC2 (59-AAGTAAGTGACT- GGGGTGAGCG-39) primers (Versalovic et al., 1991) were used. PCRs were performed in 50 ml containing 16 PCR buffer (Invitrogen), 0.4 mM of each of the four dNTPs (Invitrogen), 0.5 mM (for STRR and LTRR fingerprinting) or 1 mM (for M13 and Microcystis Microcystis

ERIC fingerprinting) of each primer, 10–15 ng genomic DNA, 3 mM Microcystis Microcystis Microcystis (for STRR, LTRR and M13 fingerprinting) or 2 mM (for ERIC 21 fingerprinting) MgCl2, 1 mg BSA ml and 1 U Taq DNA polymerase (Invitrogen). Amplification was performed in a T Gradient thermo- cycler (Biometra), consisting of an initial denaturation step at 95 uC for 10 min, followed by 35 cycles of 90 s at 95 uC, 2 min at 56 uC (for M13 fingerprinting), 40 uC (for ERIC fingerprinting) or 38 uC (for STRR and LTRR fingerprinting) and 2 min at 72 uC, and a final extension step of 5 min at 72 uC. (2005) Universal for cyanobacteria (2005) Universal for cyanobacteria The PCR products were electrophoresed in 1.4 % (w/v) agarose gel in (1997) Universal for cyanobacteria 6 et al. 0.5 TBE buffer at 90 V for 3 h 30 min, using the 1 kb plus DNA et al. Ladder (Gibco-BRL) as molecular size marker. After staining with et al. rio rio ´ ´ ethidium bromide, the image obtained under UV transillumination bel ¨ This study Universal for cyanobacteria This study Universal for cyanobacteria This study Specific for was digitized using the Kodak 1D 2.0 system. This study Specific for To assess reproducibility of the fingerprinting methods according to Sneath & Johnson (1972), at least 10 % of isolates were randomly D d

selected and analysed in duplicate in independent experiments. The d similarity between each pair of duplicates was obtained from the analysis based on a dendrogram computed with Pearson’s correlation sequencing coefficient and the unweighted pair group method with arithmetic average (UPGMA) as the agglomerative clustering, using

BioNumerics software v4.0 (Applied Maths) (data not shown). The rpoC1 reproducibility value was determined as the average similarity for all pairs of duplicates. The hierarchical clustering of isolates, based on their genomic fingerprinting patterns, was performed with Pearson’s correlation coefficient and UPGMA using BioNumerics software v4.0. The accuracy of each dendrogram to represent the similarity matrix was assessed by the cophenetic correlation coefficient (r), which ideally should be between 0.6 and 0.9 (Priest & Austin, 1993). Composite analyses were performed by constructing dendrograms based on the patterns obtained with two different methods. Clusters of isolates were correlated with taxonomic positioning, geographical origin and available toxicity data. ) Amplicon size (nt) Target site Reference or source Observation

The diversity within each cyanobacterial species comprising more § –3 than five isolates was evaluated with the indices of Simpson (Hunter § & Gaston, 1988) and Shannon–Wiener (Zar, 1996). The Simpson index (D) is based on the number of types (fingerprinting profiles) and isolates for each type and measures the probability of two non- related strains, taken from the tested population, belonging to two

different genomic types. The Shannon–Wiener index (J9) also and ITS region amplification and for 16S rRNA and provides an evenness measure, expressing the observed diversity as the proportion of the possible maximum diversity and reflecting the rpoC1

homogeneity/heterogeneity of the distribution of isolates among the sp. WH7803 (CT971583). sp. WH7803 (CT971583). genomic types.

PCR amplification and sequencing of 16S rRNA and rpoC1 PCC 7806 (AY425000). sp. PCC 6803 (BA000022). genes. Since universal primers for direct sequencing of 16S rRNA Synechococcus PCC 6803 (NC000911). genes are usually designed to be used with axenic cultures, and Synechococcus available primers for rpoC1 amplification and sequencing are highly degenerate, specific primers were selected or designed in order to CYAN738R GCTAGGACTACWGGGGTAT 630 738–765 CYAN738F ATACCCCWGTAGTCCTAGC 738–765 Vale CYAN1281R GCAATTACTAGCGATTCCTCC 1260 1281–1302 Vale RPOCM61F GGAAATGGATGGGTTATTCTGC 580 61–82 RPOCM624R TAAAACCATCCATTCTGCCTC 624–643 ITSCYA225R TGCAGTTKTCAAGGTTCT Variable 225 RPOC145FRPOC683RRPOC1006R ACTCTGAARCCAGAAATGGA AARTTRTCAATTACCCGCA TGCTTACCTTCAATAATGTC 555 880 145–164§ 1006–1025§ 683–701§ This This study study This study Except Except for for Except for M. aeruginosa obtain clean sequences for both genes without the need for a cloning Synechocystis step. Primers used in the amplification and sequencing of the 16S rRNA and rpoC1 genes are listed in Table 1. The PCRs were Synechocystis

performed in 50 ml containing 16 PCR buffer (Invitrogen), 0.4 mM Primers used for 16S rRNA, of each of the four dNTPs (Invitrogen), 0.25 mM of each of the two sequence of sequence of end of 23S rRNA of end of 16S rRNA of 9 primers, 10–15 ng genomic DNA, 2 mM MgCl2 (Invitrogen), 0.5 mg 9 BSA ml21 (Invitrogen) and 1 U Taq DNA polymerase (Invitrogen). Gene Primer* Sequence (5 16S rRNA CYA106F CGGACGGGTGAGTAACGCGTGA 106–127 Nu rpoC1 ITS region ITSCYA236F CTGGTTCRAGTCCAGGAT 236|| This study Universal for cyanobacteria At 5 16S rRNA of rpoC1 rpoC1 *F, Forward primer; R,D reverse primer. Table 1. d § Amplification was performed in a T Gradient thermocycler ||At 5

644 Microbiology 155 Molecular diagnostics for cyanobacteria

(Biometra) using the following conditions: 10 min of initial For 16S-ARDRA, approximately 1 mg of the amplicon of 1260 bp, denaturation at 95 uC, followed by 35 cycles at 94 uC for 45 s, obtained with CYAN106F and CYAN1281R primers (Table 1), was 55 uC for 45 s and 72 uC for 1 min. The amplification was completed digested without further purification with AvaII (BioLabs) and BanII by holding for 5 min at 72 uC to allow the complete extension of the (Takara) restriction endonucleases in separate reactions, according to PCR product. PCR products were visualized as described above but the manufacturer’s instructions. using 1 % (w/v) agarose gel. Amplification reaction products were purified with a Jet Quick-PCR Purification kit (Genomed) as The ITS region was amplified using DNA of non-axenic cultures and described by the manufacturer. The purified PCR products were two cyanobacterial-specific primers designed in this study, sequenced in both directions using an automated ABI PRISM 3130xl ITSCYA236F and ITSCYA225R (Table 1). The PCRs were performed Genetic Analyzer (Applied Biosystems) with dye terminators, using in 50 ml containing 16 PCR buffer (Invitrogen), 0.4 mM of each of the four dNTPs (Invitrogen), 0.5 mM of each primer, 10–15 ng standard protocols and the cyanobacterial-specific primers used for 21 amplification, as well as primers CYAN738F and CYAN738R (Table 1) genomic DNA, 2.5 mM MgCl2, 0.5 mg BSA ml and 1 U Taq DNA for the 16S rRNA gene sequencing. polymerase (Invitrogen). Amplification was performed in a T Gradient thermocycler (Biometra), consisting of an initial denatura- Phylogenetic analysis and identification at species level. A tion step at 95 uC for 6 min, followed by 35 cycles of 45 s at 95 uC, two-step strategy was used to obtain a 16S rRNA gene phylogeny for 45sat52uC and 1 min at 72 uC, and a final extension step of 5 min the cyanobacterial strains under analysis. Firstly, a separate phylogeny at 72 uC. was constructed for each order (Chroococcales, Oscillatoriales and Also in this case, a theoretical study was performed with NEBcutter Nostocales), including the sequences from 71 LMECYA strains and v2.0 software (Vincze et al., 2003), using cyanobacterial ITS sequences reference sequences from well-characterized and reliably identified available in databases, resulting in the selection of TaqI enzyme. strains of validated species: sequences from completely sequenced genomes; sequences from the strains used by Suda et al. (2002) for the For ITS-ARDRA, amplified DNA (10 ml, approximately 1 mg) was taxonomic revision of the genus Planktothrix; Limnothrix redekei digested without further purification with TaqI restriction endonu- sequences determined by Gkelis et al. (2005); and Microcystis spp. clease (Fermentas) according to the manufacturer’s instructions. sequences determined by Otsuka et al. (2001). ITS amplicons and restriction products for 16S-ARDRA and ITS- In the next step, a subgroup of LMECYA isolates and reference ARDRA were visualized as described above, using 1.2 % (w/v) and organisms was selected from the phylogeny of each order as 2 % (w/v) agarose gels, respectively. representatives of each tree structure and phylogenetic distances and a global 16S rRNA gene phylogeny was constructed to include the six cyanobacterial orders. As indicated in the figure legends, the RESULTS alignments used encompassed 1182 or 1203 nucleotides of the 16S rRNA gene. Morphological identification Since rpoC1 sequences were determined for only 38 LMECYA isolates and fewer reference sequences are available, a global phylogeny Identification of isolates at the species or genus level was including the sequences of all these isolates and reference sequences previously assessed by their morphology (Supplementary was constructed using an alignment of 452 nucleotides. Table S2). Out of the 118 isolates, 99 could be identified at All alignments were made with CLUSTAL_X version 1.83 and visually the species level and the remaining 19 only at the genus corrected. Phylogenetic trees were constructed based on a Bayesian level. These results suggest that the cyanobacterial species approach using MrBayes software version 3.0b4 (Huelsenbeck & present in Portuguese freshwaters, most of which are Ronquist, 2001). Each analysis consisted of 2 000 000 generations responsible for bloom formation, are diverse, and belong to from a random starting tree and four Markov chains (with default three of the six cyanobacterial orders: Chroococcales, heating values) sampled every 100 generations. The first 8000 sampled trees were discarded, resulting in a set of 12 000 analysed trees Oscillatoriales and Nostocales. Of the 118 isolates under sampled after the chains became stationary. The eubacterial study, 54 (45.8 %) belong to Chroococcales, 15 (12.7 %) to Escherichia coli sequence was used as an outgroup. Neighbour-joining Oscillatoriales and 49 (41.5 %) to Nostocales. The species and maximum-parsimony trees were also constructed using the same predominantly found were Microcystis aeruginosa (52 alignments and PAUP 4.0b10 software (Swofford, 2003). isolates; 44 %) and Aphanizomenon flos-aquae (13 isolates; The BLAST tool of the National Center for Biotechnology Information 11 %). (http://www.ncbi.nlm.nih.gov) was used to find homologous and other close sequences (97–100 % identity and E-values ¡10220)tobe included in the phylogenies as reference sequences. Assessment of PCR fingerprinting reliability To achieve the final identification of the isolates, the following criteria The average reproducibility of the tested fingerprinting were used. When the phylogenetic positioning of an isolate retrieved techniques, estimated by the similarity average value for all identification at the species level, this prevailed; if the phylogenetic pairs of duplicates, was 92 %, representing a good positioning allowed only genus determination or no molecular reproducibility (Sneath & Johnson, 1972), since the identification was possible, then the identification at the species level Pearson’s correlation coefficient used is very sensitive to obtained by morphological analysis was retained. the band intensity, leading to lower final similarity values. 16S rRNA gene and ITS analysis. To select the most suitable In all dendrograms the cophenetic value (r) is above 0.85, restriction endonucleases to provide an easily identifiable 16S rRNA which corresponds to a good representation of the original gene restriction profile, a theoretical study was performed with data . NEBcutter v2.0 software (Vincze et al., 2003), using cyanobacterial 16S rRNA gene sequences available in databases, resulting in the In order to assess the effect of the non-axenic nature of selection of AvaII and BanII enzymes. the uni-cyanobacterial cultures, the PCR fingerprinting http://mic.sgmjournals.org 645 E. Vale´rio and others methods to be tested (STRR, LTRR, M13 and ERIC) were applied to a set of cyanobacterial cultures and their co- cultured heterotrophic (non-obligately oligotrophic) bac- teria. From the same culture (Fig. 1 underlined group of lanes), cyanobacteria (in bold) and cultivated hetero- trophic bacteria displayed M13 (Fig. 1c) and ERIC (Fig. 1d) PCR fingerprints with very few co-migrating fragments (white boxes). However, isolates LMECYA 79 and LMECYA 173 displayed identical fingerprinting profiles with each technique, despite the fact that their corres- ponding cultivated bacteria showed M13 and ERIC profiles different from each other but with some co-migrating fragments with the cyanobacteria (Fig. 1, lanes 8–11). On the other hand, STRR and LTRR PCR fingerprinting (Fig. 1a, b) showed specificity for cyanobacteria, since no amplification was obtained for the cultivated bacteria.

STRR and LTRR PCR fingerprinting A composite analysis of hierarchical clustering of STRR and LTRR patterns was performed with all isolates (data not shown) and subsequently for each order as presented in Fig. 2. It can be observed that the isolates grouped according to their genomic similarity. To confirm the species identification, a set of representative isolates (marked with bold italics in Fig. 2), including members of different clusters and members of the same cluster, was selected and their identification was obtained or confirmed by phylogenetic positioning using 16S rRNA gene and rpoC1 phylogenies. The identification presented in Fig. 2 is in agreement with the final identification of the isolates obtained by 16S rRNA gene sequencing (which in some cases was also confirmed by the rpoC1 sequencing, as presented below). It can be observed that the majority of the clusters correspond to different species, showing the taxonomic potential of STRR and LTRR fingerprints. The three species (two of them represented by only one isolate) belonging to the order Chroococcales (Fig. 2a) are intermixed and could not be separated according to their STRR and LTRR fingerprinting patterns. In fact, the isolates of Microcystis aeruginosa show very diverse profiles (grouping in three distinct clusters) and the isolate of Synechocystis sp. is more similar (52.5 %) to cluster II of M. aeruginosa than the M. aeruginosa clusters are to each other (15–27 %). Synechococcus nidulans, together with one Fig. 1. Fingerprinting profiles of cyanobacteria and cultivable isolate of M. aeruginosa, has the lowest similarity (10 %) bacteria from each non-axenic culture. (a) STRR PCR fingerprint- ing; (b) LTRR PCR fingerprinting; (c) M13 PCR fingerprinting; (d) with all the other isolates. Fingerprinting patterns of ERIC PCR fingerprinting. (1) LMECYA 64; (2) LMECYA 64 isolates belonging to the order Oscillatoriales (Fig. 2b) led heterotrophic bacteria #1; (3) LMECYA 64 heterotrophic bacteria to the formation of two clusters and four isolated strains at #2; (4) LMECYA 64 heterotrophic bacteria #3; (5) LMECYA 64 40 % similarity, while nine major clusters and four isolated heterotrophic bacteria #4; (6) AQS; (7) AQS heterotrophic strains were observed at 50 % similarity for isolates of the bacteria #1; (8) LMECYA 79; (9) LMECYA 79 heterotrophic order Nostocales (Fig. 2c). In both cases almost all the bacteria #1; (10) LMECYA 173; (11) LMECYA 173 hetero- clusters correspond to different species, except for trophic bacteria #1; (12) LMECYA 13; (13) LMECYA 13 circinalis and Aphanizomenon gracile, which heterotrophic bacteria #1. The white boxes mark the common formed two distinct clusters (genotypes). fragments.

646 Microbiology 155 http://mic.sgmjournals.org oeua igotc o cyanobacteria for diagnostics Molecular

Fig. 2. Dendrograms obtained by composite hierarchical analysis of STRR and LTRR fingerprinting profiles using Pearson’s correlation coefficient and the UPGMA clustering method for the orders used in the study: (a) Chroococcales, (b) Oscillatoriales and (c) Nostocales. The cophenetic correlations (r) of the dendrograms are shown. The scale 647 corresponds to global percentage similarity. gen, genotype. A ‘weight of 2’ was attributed to the STRR patterns, and a ‘weight of 1’ to the LTRR patterns. E. Vale´rio and others

Phylogenetic analysis using the 16S rRNA gene Phylogenetic analysis using rpoC1 The phylogenetic tree presented in Fig. S1 corresponds to The rpoC1-based phylogeny presented in Fig. 3(b) allowed the partial 16S rRNA gene phylogeny of the order the identification of six of the 38 sequenced isolates. Two of Chroococcales. This phylogeny confirmed the identifica- them (LMECYA 17 and 126) belong to Anabaena circinalis, tion of the 27 sequenced isolates only at the genus level. In one (LMECYA 134) to Cylindrospermopsis raciborskii, one fact, although the genus Microcystis forms a monophyletic (LMECYA 153E) to Planktothrix agardhii and one cluster, all five species are highly homologous and thus (LMECYA 68) to Synechocystis sp. All these identifications cannot be distinguished at the 16S rRNA gene level. were congruent with those obtained by 16S rRNA gene sequencing. The partial 16S rRNA gene phylogeny of the order Oscillatoriales is depicted in Fig. S2; it allowed the Isolates LMECYA 9, 40, 64 and 148, identified both identification at species level of seven sequenced isolates morphologically and by 16S rRNA gene sequencing as (out of 10), four of these (LMECYA 152, 153, 153E and Aphanizomenon gracile, clustered into two distinct groups, 153F) as Planktothrix agardhii, one (LMECYA 203) as neither of them closely related to the available Planktothrix rubescens, one (LMECYA 162) as Planktothrix Aphanizomenon gracile rpoC1 sequence. pseudoagardhii and one (LMECYA 145) as Limnothrix Phormidium sp. isolates (LMECYA 79 and 173) grouped redekei. The three remaining isolates (LMECYA 79, 173 into a cluster containing another Phormidium sequence but and 214) were grouped in a cluster containing sequences of also Leptolyngbya and Synechococcus sequences. Phormidium and Leptolyngbya isolates. However, taking into account their morphological characteristics, these Some isolates did not group with a particular species, due isolates were identified as Phormidium sp. The distribution to the low number of rpoC1 sequences available. of Oscillatoria and Lyngbya isolates in several distinct clades In the rpoC1 phylogeny, the Nostocales split into two also reveals the polyphyletic nature of these genera. clusters, a major one containing almost all species of the The partial 16S rRNA gene phylogeny of the order clade obtained with the 16S rRNA gene and a second Nostocales (Fig. S3) allowed the identification of 21 out grouping the remaining species, including Scytonema sp., of 34 sequenced isolates. The isolate LMECYA 177 groups with species from Stigonematales and Pleurocapsales. in a cluster where several Anabaena species are present. Oscillatoriales were separated in distinct clades, mostly This phylogenetic positioning does not allow an identifica- equivalent to the 16S rRNA ones. Chroococcales presented tion of this isolate; however, its morphological features are a monophyletic cluster, except for the Prochloron and two similar to those of Anabaena planctonica, which is also Synechococcus spp. sequences, which were distributed in present in the cluster. This is also the case with isolates 77A, other clusters. When this rpoC1 Bayesian tree was 77B, 88, 99, 125, 129 and 141, grouped in a cluster with compared with neighbour-joining and maximum-par- mainly Aphanizomenon flos-aquae but also Aphanizomenon simony trees (see Supplementary Fig. S5), similar phylo- gracile. However, morphological features of these isolates genetic relationships were observed. are typical of Aphanizomenon flos-aquae. Isolates LMECYA 9 and 33 grouped in a cluster with Anabaena flos-aquae, Strain discrimination and traceability Aphanizomenon flos-aquae and Aphanizomenon gracile sequences, but morphological features of these isolates Representative M13 and ERIC PCR fingerprinting profiles are typical of Aphanizomenon gracile. The isolates LMECYA of the genomic types of the species comprising more than 178, 182 and 185 did not group with any species or genus. five isolates are presented in Supplementary Fig. S6. As this In fact, their morphology is congruent with Anabaena, and figure shows, the richness and diversity of the DNA LMECYA 178 is identical to Anabaena flos-aquae. This banding patterns allowed the discrimination of all distinct phylogeny also showed that Anabaena and Aphanizomenon isolates and the detection of identical ones. do not form monophyletic clusters, with isolates of both Individual dendrograms were constructed for each of the genera intermixed along the tree. seven species (data not shown). Based on the M13 and The global 16S rRNA gene phylogeny of representative ERIC PCR fingerprints of all the Microcystis aeruginosa species of the six cyanobacterial orders, using a Bayesian isolates evaluated by a composite analysis of hierarchical approach, is illustrated in Fig. 3(a). It shows that the clustering, a cut-off level of 75 % similarity was established Nostocales form a monophyletic cluster, except for the sole for definition of a genomic type. Table 2 presents the Scytonema sequence presented. Stigonematales are also percentage of types and values of Simpson and Shannon– monophyletic, containing the genera Chlorogloeopsis, Wiener diversity indices (for the species with more than Fischerella and Nostochopsis, except the unique Stigonema five isolates). The lowest diversity values were obtained for sequence here presented. The order Pleurocapsales forms a Planktothrix agardhii, where four of the nine isolates have monophyletic cluster, whereas the orders Chroococcales and the same genomic type. The other four species whose Oscillatoriales are both divided into two distinct clades. diversity indices were determined revealed higher diversity Identical results were obtained using neighbour-joining and values, with Aphanizomenon flos-aquae showing the highest maximum-parsimony trees (Supplementary Fig. S4). one.

648 Microbiology 155 http://mic.sgmjournals.org oeua igotc o cyanobacteria for diagnostics Molecular

Fig. 3. (a) Global MrBayes tree of representative species using 1182 nt of the 16S rRNA gene sequences in the alignment. (b) MrBayes tree of the rpoC1 sequences using 452 nt in the alignment. Posterior probabilities values above 0.5 are indicated to provide branch support. Bars: 10 % sequence divergence. GenBank accession numbers are

649 indicated after the species designation (names in bold correspond to sequences determined in this study). E. Vale´rio and others

Table 2. Genomic diversity of cyanobacterial species In the dendrogram of all Microcystis aeruginosa isolates (Supplementary Fig. S7), most of those sourced from the Species % types* DD J§d same reservoir seem to group together and a general pattern of association also emerges in terms of microcystin- Microcystis aeruginosa 59.6 (28/47) 0.96 0.93 Planktothrix agardhii 66.7 (6/9) 0.83 0.88 producing ability. In fact, two major clusters were formed Anabaena circinalis 85.7 (6/7) 0.95 0.98 at the 20 % similarity level: cluster I with 22 toxic and five Aphanizomenon flos-aquae 92.3 (12/13) 0.99 0.99 non-toxic isolates, and cluster II with 18 non-toxic and 2 Aphanizomenon gracile 75.0 (6/8) 0.93 0.97 toxic isolates. Furthermore, only for one type defined at Aphanizomenon issatschenkoi 80.0 (4/5) - - 75 % similarity did a toxic isolate (from Magos reservoir) Cylindrospermopsis raciborskii 80.0 (4/5) - - group with a non-toxic one (from Roxo reservoir). To test the traceability potential of these fingerprints, a *% types: (number of types/number of isolates)6100. dendrogram comprising a group of Microcystis aeruginosa D D, Simpson diversity index. isolates, recovered from the same reservoir (Montargil) on d J9, Shannon–Wiener diversity index. different dates, was constructed (Fig. 4a) and 10 genomic types were defined (at the 75 % similarity level). Although the non-toxin-producing isolates (NT) were distributed into distinct clusters, all the toxin-producing ones (T)

Fig. 4. Composite hierarchical analysis of M13 and ERIC fingerprints (a) and genomic and temporal relationships amongst isolates (b) of Microcystis aeruginosa sourced from Montargil reservoir on different dates. NT, non-toxic; T, toxic. The dotted lines indicate the cut-off levels of reproducibility (92 %) and genomic type definition (75 %). Black and white stars refer to toxic and non-toxic genomic types (numbered from 1 to 10), respectively. Circles indicate genomic types introduced de novo.

650 Microbiology 155 Molecular diagnostics for cyanobacteria grouped together at a similarity level above 55 %, AvaII four out of the 18 species are identified: Limnothrix indicating the ability of M13 and ERIC fingerprints to redekei (630 bp, double band), Cylindrospermopsis raci- discriminate toxic from non-toxic isolates. Two isolates, borskii (1100 and 160 bp bands), Phormidium sp. (900 and LMECYA 92C and LMECYA 110, recovered in different 360 bp bands) and Synechocystis sp. (960 and 300 bp years, showed 92 % similarity (reproducibility level; dotted bands). For the remaining species, seven show a 1260 bp line in Fig. 4a), indicating that they should be assumed to band (no restriction) and the others show a profile of 810 represent the same strain. and 450 bp bands. Further restriction with BanII enabled In spite of the reduced number of isolates under analysis, the identification of seven out of the 14 species not the cross-analysis of temporal occurrence and genomic previously identified. However, Microcystis aeruginosa, relationships (Fig. 4b) points to the probable independent Anabaena circinalis, Anabaena flos-aquae, Anabaena plac- introduction of seven genomic types (two of them toxic), tonica, Anabaena spiroides, Aphanizomenon flos-aquae and as well as the existence of resident and evolving genomic Aphanizomenon gracile cannot be distinguished, since they types (types 4-3, 6-7 and 8-9), revealing traceability all show a profile with two bands of 815 and 445 bp. potential for these fingerprints. The ITS amplicons obtained with the cyanobacterial- specific primers designed in this study retrieved the Diagnostic key for cyanobacterial identification amplification fragments also summarized in Table 3. The number of amplified fragments varied between one and With the aim of achieving fast and reliable identification of three, and their size ranged from 180 to 930 bp. Most of the isolates, PCR amplification and subsequent restriction the species included in the Nostocales showed a 300 bp of 16S rRNA gene and ITS region were tested. fragment. The range of ITS sizes for members of the genus The sizes of the fragments obtained after 16S rRNA gene Microcystis overlapped in some cases with Anabaena and digestion are summarized in Table 3. After restriction with Aphanizomenon. Some amplification profiles can also

Table 3. 16S-ARDRA fragments generated with AvaII and BanII endonucleases, ITS amplicon dimensions and ITS-ARDRA fragments generated with TaqI

16S-ARDRA fragments (bp) ITS amplicons (bp) ITS-ARDRA fragments (bp)* AvaII BanII

Chroococcales Microcystis aeruginosa 1260 815; 445 Type 1: 930; 300; 180 ND Type 2: 470; 300 ND Type 3: 300; 180 ND Type 4: 300 190; 110 Type 5: 180 180 Synechococcus nidulans 1260 715; 430; 115 300 190; 110 Synechocystis sp. 960; 300 715; 350; 195 170 170 Oscillatoriales Limnothrix redekei 630; 630 715; 445; 100 350; 250 ND Planktothrix agardhii 1260 665; 420; 175 220 120; 100 Planktothrix pseudoagardhii 1260 815; 320; 125 220 100; 90; 30 Planktothrix rubescens 1260 640; 490; 130 220 120; 100 Phormidium sp. 900; 360 670; 420; 170 220 115; 105 Nostocales Anabaena circinalis 810; 450 815; 445 300 180; 100; 20 Anabaena flos-aquae 1260 815; 445 420; 300 ND Anabaena planctonica 810; 450 815; 445 300 160; 95; 45 Anabaena spiroides 810; 450 815; 445 300 160; 95; 45 Anabaenopsis circularis 810; 450 1260 380 190; 110; 80 Aphanizomenon flos-aquae 810; 450 815; 445 300 160; 95; 45 Aphanizomenon gracile 810; 450 815; 445 300 170; 110; 20 Aphanizomenon issatschenkoi 810; 450 815; 360; 85 300 160; 80; 60 Aphanizomenon ovalisporum 1260 715; 445; 100 315 225; 90 Cylindrospermopsis raciborskii 1100; 160 715; 430; 115 300; 195 ND

*ND, not determined. http://mic.sgmjournals.org 651 E. Vale´rio and others provide an identification, as is the case for Synechocystis sp. 16S rRNA gene sequencing, and thus can be used as an (170 bp band), Limnothrix redekei (350 and 250 bp bands), identification tool, decreasing the need for 16S rRNA gene Anabaena flos-aquae (420 and 300 bp bands), Anabaenopsis sequencing, often used in association with the morpho- circularis (380 bp band), Aphanizomenon ovalisporum logical identification of a cyanobacterial strain (Nu¨bel et (315 bp band) and Cylindrospermopsis raciborskii (300 al., 1997). and 195 bp). Moreover, a 220 bp fragment was always The genus Microcystis forms a monophyletic group distant obtained for the Planktothrix spp. isolates. from the other genera in both 16S rRNA and rpoC1 The ITS-ARDRA fragments obtained after restriction of the phylogenies. All five Microcystis species showed a high ITS with TaqI are summarized in Table 3. Some of the homology (.98.5 % with both genes), preventing their isolates show a profile that can be used for identification. discrimination. This has already been shown by Otsuka et The genotypic pattern produced using this restriction al. (2001), who proposed the unification of all five species, enzyme can provide a species identification marker: for with Microcystis aeruginosa having nomenclatural priority. instance Planktothrix pseudoagardhii, which showed a However, despite the high sequence homology and also the profile distinct from the other two Planktothrix species, high DNA–DNA reassociation levels found among these Planktothrix agardhii, difficult to distinguish from the species, Kondo et al. (2000) suggested their maintenance as former by morphological characteristics, and Planktothrix distinct taxa based on their distinct morphological features. rubescens, which can be identified by its cell dimensions Both 16S rRNA and rpoC1 gene phylogenies showed that and brownish colour. the genera Synechococcus and Synechocystis form distinct Based on the 16S-ARDRA, ITS amplification and ITS- clades. Nevertheless, our results recommend a revision of ARDRA a diagnostic key was constructed (Fig. 5) allowing the genus Synechococcus as already suggested by Robertson the identification of 15 out of the 18 species analysed. Only et al. (2001). Anabaena planctonica, Anabaena spiroides and Regarding the order Oscillatoriales, we also found that the Aphanizomenon flos-aquae could not be distinguished. genus Planktothrix forms a monophyletic cluster closer to Nostocales than to other Oscillatoriales, as already DISCUSSION described by Suda et al. (2002). It is also evident that the genus Oscillatoria still needs major revision, its species In this study, several molecular techniques were used to being widely distributed in 16S rRNA gene trees. In fact, examine the level of genetic diversity of 118 cyanobacterial this genus has recently undergone revisions (Suda et al., isolates for taxonomic purposes. 2002) and its polyphyly was also inferred by others (Ishida et al., 2001; Marquardt & Palinska, 2007; Suda et al., 2002). All the PCR fingerprinting methods showed good repro- Similarly to Joyner et al. (2008), a polyphyletic origin of the ducibility, and the inter- and intra-specific variability, genus Lyngbya was also inferred from the 16S rRNA gene observed in all species analysed, was revealed to be tree, justifying its taxonomic revision. This might be related appropriate for assessment of genomic relationships, to the fact that traditional criteria used for classification of isolate discrimination and traceability, in spite of the the Oscillatoriales at the genus level predominantly rely on non-axenic character of the cyanobacterial cultures. STRR the characteristics of external sheaths and colony forma- fingerprints were obtained for a larger number of non- tion, rather than on cellular features (Marquardt & heterocystous cyanobacteria than described so far Palinska, 2007). Other 16S rRNA analyses have already (Rasmussen & Svenning, 1998) and the LTRR PCR demonstrated the polyphyletic origin of members of the fingerprinting was also tested in a large number of species. orders Chroococcales (Litvaitis, 2002; Seo & Yokota, 2003) Beyond some discrimination ability, the specificity of the and Oscillatoriales (Ishida et al., 2001; Litvaitis, 2002). STRR and LTRR fingerprinting patterns revealed iden- tification potential. In fact, all species from the Nostocales We also can verify from the rpoC1 phylogeny that the order and Oscillatoriales could be discriminated by the compos- Nostocales is not monophyletic and that its members are ite hierarchical analysis of the STRR and LTRR fingerprints closely related to those of the Stigonematales, giving when only species/genera of the same order were analysed. additional support to the statement already made by For most species, a good correlation was found between Gugger & Hoffmann (2004) based on 16S rRNA gene morphological identification and genomic clustering. In phylogeny. Anabaena and Aphanizomenon isolates were the case of Anabaena circinalis two clusters were formed, genetically heterogeneous and intermixed in both gene corresponding to two genotypes whose 16S rRNA gene was trees, confirming previous results obtained with other 96.5 % similar, whereas the isolates of the same genotype strains (Lyra et al., 2001; Gugger et al., 2002; Rajaniemi et displayed 99.9 % similarity. A similar finding was observed al., 2005). Furthermore, the possible presence of incorrectly for Aphanizomenon gracile isolates, which were also divided identified sequences in databases may also distort the into two clusters, showing 98 % 16S rRNA gene similarity inferred conclusions. Although the results obtained with between them and 99–100 % within the clusters. The rpoC1 phylogeny seem to indicate possible lateral gene composite hierarchical analysis of the STRR and LTRR transfer events in Anabaena and Aphanizomenon species, fingerprints reflects the identification results obtained by the hypothesis that the morphological features are

652 Microbiology 155 http://mic.sgmjournals.org oeua igotc o cyanobacteria for diagnostics Molecular

Fig. 5. Diagnostic key based on 16S-ARDRA with AvaII and BanII, ITS amplification and ITS-ARDRA with TaqI, for cyanobacterial identification purposes. C, Chroococcales; O, Oscillatoriales; N, Nostocales. 653 E. Vale´rio and others insufficient to delineate the true taxonomic structure of ACKNOWLEDGEMENTS these genera cannot be ruled out. Cylindrospermopsis raciborskii forms a monophyletic group, more distant from The authors are deeply indebted to the anonymous journal referees for their thorough review and helpful comments on both scientific the other species of the Nostocales with both markers. issues and terminology. Elisabete Vale´rio is recipient of Fundac¸a˜o The identification of genomic types by hierarchical para a Cieˆncia e a Tecnologia grant SFRH/BD/8272/2002. clustering analysis of M13 and ERIC fingerprints allowed the assessment of intra-specific diversity for the represent- REFERENCES ative species of this study and also revealed this approach to be a useful tool for traceability purposes within a Baker, P. (1991). Identification of Common Noxious Cyanobacteria freshwater reservoir. Part I – Nostocales: Urban Water Research Association of Australia. Research Report no. 29. Although a rather low number of isolates was analysed for Baker, P. (1992). the majority of the species, except for Microcystis Identification of Common Noxious Cyanobacteria Part II – Chroococales Oscillatoriales: Urban Water Research aeruginosa, a high diversity was observed within all species, Association of Australia. Research Report no. 46. with Planktothrix agardhii showing the lowest diversity Bourrelly, P. 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