w a t e r r e s e a r c h 5 9 ( 2 0 1 4 ) 2 4 8 e2 6 1
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/watres
Polyphasic identification of cyanobacterial isolates from Australia Elvina Lee a, Una M. Ryan a, Paul Monis b, Glenn B. McGregor c, Andrew Bath d, Cameron Gordon d, Andrea Paparini a,* a
School of Veterinary and Life Sciences, Murdoch University, 90 South Street, Murdoch, Western Australia 6150, Australia b Australian Water Quality Centre, South Australian Water Corporation, 250 Victoria Square, Adelaide 5000, Australia c Department of Science, Information Technology, Innovation and the Arts, GPO Box 5078, Brisbane, Queensland 4001, Australia d Drinking Water Quality Branch, Water Corporation, 629 Newcastle Street, Leederville, Western Australia 6007, Australia
article info
abstract
Article history:
Reliable identification of cyanobacterial isolates has significant socio-economic implica-
Received 15 November 2013
tions as many bloom-forming species affect the aesthetics and safety of drinking water,
Received in revised form
through the production of taste and odour compounds or toxic metabolites. The limitations
10 April 2014
of morphological identification have promoted the application of molecular tools, and
Accepted 12 April 2014
encouraged the adoption of combined (polyphasic) approaches that include both micro-
Available online 24 April 2014
scopy- and DNA-based analyses. In this context, the rapid expansion of available sequence data is expected to allow increasingly reliable identification of cyanobacteria, and ulti-
Keywords: Cyanobacteria identification Morphology
mately resolve current discrepancies between the two approaches. In the present study morphological and molecular characterisations of cyanobacterial isolates (n ¼ 39), collected from various freshwater sites in Australia, were compared. Se-
Molecular phylogeny
quences were obtained for the small ribosomal subunit RNA gene (16S rDNA) (n ¼ 36), the
16S rDNA
DNA-dependent RNA polymerase gene (rpoC1) (n ¼ 22), and the phycocyanin operon, with
rpoC1
its intergenic spacer region (cpcBA-IGS) (n ¼ 19). Phylogenetic analyses identified three
Phycocyanin operon
cyanobacterial orders: the Chroococcales (n ¼ 8), Oscillatoriales (n ¼ 6), and Nostocales (n ¼ 25). Interestingly, multiple novel genotypes were identified, with 22% of the strains (17/ 77) having 50%), while Baldwin Park type 2, although on an isolated branch, was found well within the Nostocales. This is in contrast to the 16S locus, where GS2-1 and Baldwin Park type 2, respectively grouped with Nostoc commune (bootstrap > 70%; well within the Nostocales), or formed a clearly distinct branch, basal to the order (Fig. 3). As with the 16S tree (Fig. 3), multiple clusters of the Chroococcales were also evident from the rpoC1 tree (Fig. 4), with four sequences grouped within this order: MIC058-B, Vasse River types 9, and 13 and GS6-1. Apart from GS2-1, the remaining 17 sequences clustered within the
Nostocales, which was characterized by the distinct positions of Vasse River type 2, Buayanup drain type 2 and GS5-2. Analysis of the cpcBA-IGS locus (423 characters; 396 parsimony informative sites) showed that, apart from GS2-1, which grouped strongly (bootstrap value >80%) with Pseudanabaena sp. (Oscillatoriales) on an isolated branch, the overall topology was similar to that obtained for the 16S rDNA (Fig. 5). As with the 16S and rpoC1 loci, the Nostocales (10 sequences) and the Chroococcales (2 sequences) formed monophyletic groups, while the Oscillatoriales (7 sequences) were paraphyletic (Fig. 5). As the majority of cpcBA-IGS sequences available from GenBank to date mainly belong to relatively few genera (e.g., Arthrospira, Synechococcus, Phormidium etc.), large distance values and the presence of isolated branches were observed for the tree based on this locus (Fig. 5). Baldwin Park type 2 could not be successfully amplified at this locus, and only GS41, GS4-2, Baldwin Park type 1 and Vasse River type 3 exhibited almost complete (99%) homology with available sequences (Fig. 5, Table 2).
3.3. Comparison between morphological and molecular identifications Discrepancies between morphological and molecular identifications were observed for several isolates (Table 2). For example, Hyde Park type 1 was identified, at the cpcBA-IGS locus and morphologically, as An. elenkinii. However, at the 16S rDNA it was closest to Anabaenopsis circularis. Further molecular identification of Hyde Park type 1 was hampered by the paucity of An. circularis and An. elenkinii sequences at both the rpoC1 and cpcBA-IGS loci which prevented confident identification at these loci. The isolate Buayanup drain type 2, which was identified morphologically as A. torulosa was most similar to A. oscillaroides at the 16S rDNA locus, and to Anabaena sphaerica at the cpcBA-IGS locus (no A. oscillaroides sequences were available at this locus). Isolates GS6-1 and Vasse River types 9, 12 and 13 were identified as Aphanothece sp. based on morphology. However, using molecular methods, they were phylogenetically more similar to Synechococcus sp. (HE975005) than Aphanothece minutissima (FM177488) (Fig. 3). Morphologically, ANA196-A was identified as Dolichospermum circinale, but was phylogenetically placed with Aphanizomenon gracile, using the 16S rDNA and rpoC1 sequence data (Table 2). This was also observed for GS4-2, which was identified morphologically as a Nostoc sp. or Sp. aphanizomenoides, but was found to be most closely related to Anabaena bergii (100% similarity) at both the 16S rDNA and cpcBA-IGS loci. Similarly, although Baldwin Park type 1 and GS4-1 were identified morphologically as Planktolyngbya and Oscillatoriales/Geitlerinema sp., they showed 100% similarity to various Limnothrix spp. and Planktothrix spp., at the 16S rDNA locus, and to Geitlerinema amphibium (FJ545644), at the cpcBA-IGS locus. Overall, for the nine isolates that amplified at all loci studied, microscopic and molecular data from at least one locus, were in agreement at genus level for all isolates, except Vasse River type 13 (Table 2). However, agreement between morphological and molecular identifications, from all three loci, was obtained for only one isolate (Vasse River type 6)
Table 2 e Cyanobacteria identification based on 16S, rpoC1, Phycocyanin (Cpc) DNA sequences and isolate morphology. Percentage similarity at each locus was calculated in MEGA 5 (Tamura et al., 2011), as the pairwise evolutionary divergence, with a p-distance model (Kimura, 1980). Sub-group
Isolate
Molecular identification
Morphological identification
Related sequence (percentage similarity) 16S rDNA (n ¼ 36) Nostocales (n ¼ 25)
D. affinejFN691906 (100%) e e e (D. circinale AWQC150-AjAF247573) Ap. gracilejHQ157688 (100%) D. flos-aquaejAB551438 (100%) D. circinalejAF247588 (100%) D. circinalejAF247581 (100%) A. oscillaroidesjAJ630428 (94%)
Chelodina wetlands type 1 GS1-2 GS2-1
cpcBA-IGS (n ¼ 19)
Taxonomist 1
Taxonomist 2
e D. circinalejAF199423 (100%) D. circinalejAF199423 (100%) D. circinalejAF199423 (100%) D. circinalejAF199423 (100%) Ap. gracilejEU078450 (97%) e e D. circinalejAF199425 (100%) Anabaena sp.jAF199432 (86%) Anabaena sp.jAF199433 (86%) Nostoc sp.jAY424997 (88%)
e e e e e e e e e e
D. circinale D. circinale D. circinale D. circinale D. circinale D. circinale D. circinale D. circinale D. circinale N.A.
N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. Anabaena sp. 1
e
Sp. aphanizomenoides
A. oscillaroides
A. sphaericajDQ439645 (92%)
A. torulosa
A. torulosa
Ap. gracilejEU078532 (99%)
Sp. aphanizomenoidesjFJ234841 (86%) Sp. aphanzomenoidesjFJ234848 (86%) e
Anabaena sp.
Anabaena sp.
A. variabilisjCP000117 (89%) Pseudanabaena sp. PCC7367j CP003592 (76%) e C. stagnale PCC7417jCP003642 (87%) A. cylindrica PCC7122jCP003659 (84%) e
Anabaena sp. Nostocales
Anabaena sp. Nostoc sp.
Sp. aphanizomenoides Cylindrospermum sp.
Nostoc sp. Anabaena sp.
Anabaena sp.jGU935369 (97%)
N.A.
Anabaena sp.
GS5-3
N. punctiformejGQ287652 (97%) N. communejDQ185223 (99%) N. communejAB251863 (99%) A.bergiijFR822617 (100%) Nostocaceae cyanobacteriumj GQ389643 (100%) T. variabilisjAJ630456 (100%) T. variabilisjJQ390607 (100%) Nostoc sp.jFJ948088 (99%)
Ap. gracilejFN552318 (97%) D. compactajAY702239 (97%) e Pseudanabaena sp.jEF680776 (80%) A. bergiijFJ234863 (100%) e
Anabaena sp.
Anabaena sp.
Hyde Park type 1 Vasse River type 1
An. circularisjGQ859629 (100%) D. flos-aquaejAY701573 (100%)
An. circularisjEU078479 (89%) Anabaena sp.jAF199432 (98%)
An. elenkinii Anabaena sp.
An. elenkinii D. flos-aquae
Vasse River type 2
Anabaena sp. PCC9109jAY768408 (99%)
Anabaena sp.jEU078475 (87%)
N.A.
Anabaena sp.
Vasse River type 3 Vasse River type 6
A. sphaericajGQ466513 (100%) Sp. aphanizomenoidesjFM177473 (100%)
N.A. Sp. aphanizomenoides
Anabaena sp. Sp. aphanizomenoides
Vasse River type 8
Calothrix sp.jGQ859627 (98%)
A. variabilisjAB074795 (100%) Sp. aphanizomenoidesjFJ830555 (96%) e
Nostoc sp. PCC6720j JF740673 (94%) An. elenkiniijFN552383 (96%) Sp. aphanizomenoidesj GU197719 (99%) Anabaena sp. PCC9109j AY768473 (96%) N. linckiajAY466120 (99%) Sp. aphanizomenoidesj GU197719 (97%) e
Gloeotrichia sp.
Rivularia sp.
GS4-2 GS5-1 GS5-2
Nostoc sp. PCC8976jAM711525 (100%) Nostoc sp.jAB087403 (100%) A. oscillaroidesjAJ630428 (99%)
w a t e r r e s e a r c h 5 9 ( 2 0 1 4 ) 2 4 8 e2 6 1
ANA019-BR ANA118-AR ANA131-CR ANA148-CR ANA150-Aa ANA196-A ANA278-FR ANA335-C AWQC318 Baldwin Park type 2 Baldwin Park type 3 Buayanup drain type 2
rpoC1 (n ¼ 22)
(continued on next page)
253
254
Table 2 e (continued ) Sub-group
Isolate
Molecular identification
Morphological identification
Related sequence (percentage similarity) 16S rDNA (n ¼ 36) Oscillatoriales (n ¼ 6)
Taxonomist 1
Taxonomist 2
G. amphibiumjFJ545644 (100%)
Oscillatoriales
Planktolyngbya sp. 1
e
N.A.
Trichocoleus sp
Leptolyngbya sp.jHM217044 (100%)
e
N.A.
Planktolyngbya sp.
GS3-2
Pseudanabaena sp.jGU935355 (100%)
e
Pseudanabaena sp.
Ps. galeata
GS4-1
e
Geitlerinema sp.
Planktolyngbya sp. 1
GS6-2
Planktothrix sp.jAF212922 (100%) L. redekeijEU078512 (100%) Limnothrix sp.jEF088338 (100%) Ps. mucicolajGQ859642 (99%)
Cyanobacterium txid129981j AJ401183 (75%) Spirulina laxissimaj DQ393286 (84%) Pseudanabaena sp. PCC7409jM99426 (82%) G. amphibiumjFJ545644 (100%)
e
Pseudanabaena sp. PCC7409jM99426 (83%)
Pseudanabaena sp.
Ps. galeata
GS3-1 MIC058-B GS6-1
M. flos-aquaejAF139328 (98%) M. flos-aquaejAF139328 (100%) Synechococcus sp.jHE975005 (100%)
e e e
N.A. M. flos-aquae N.A.
Microcystis sp. N.A. Aphanothece sp.
Vasse River type 9 Vasse River type 12 Vasse River type 13 Vasse River type 14 Vasse River type 15
Synechococcus sp.jHE975005 (100%)
e M. aeruginosajAP009552 (98%) Synechococcus sp. PCC7920j AF245158 (96%) Synechococcus sp. PCC7920j AF245158 (96%) e
e
Aphanothece sp.
Aphanothece sp.
N.A.
Aphanothece sp. 1
N.A.
Aphanothece sp. 1
Synechococcus sp.jHE975005 (100%)
Synechococcus sp. PCC7920jAF245158 (96%) e
Synechococcus sp. PCC7918j AF223462 (97%) Synechococcus sp. PCC7918jAF223462 (96%) e
N.A.
Aphanothece sp.
Synechococcus sp.jHE975005 (100%)
e
e
N.A.
Aphanothece sp. 1
Synechococcus sp.jHE975005 (100%) Synechococcus sp.jHE975005 (100%)
16S rDNA sequence previously available (GenBank Acc. No AF247573); e ¼ No sequence data obtained; N.A. ¼ Not examined; A. ¼ Anabaena; An. ¼ Anabaenopsis; Ap. ¼ Aphanizomenon; C. ¼ Cylindrospermum; D. ¼ Dolichospermum; G. ¼ Geitlerinema; L. ¼ Limnothrix; M. ¼ Microcystis; N. ¼ Nostoc; O. ¼ Oscillatoria; P. ¼ Planktothrix; Ps. ¼ Pseudanabaena; Sp. ¼ Sphaerospermopsis; T. ¼ Trichormus. Sequences with percentage molecular similarity above the threshold used for identification are in bold. For species identification, the threshold was 98% for the 16S rDNA, and 95% for the rpoC1 and cpcBA-IGS loci. For genus, the threshold was 95% for the 16S rDNA, and 90% for rpoC1 and cpcBA-IGS.
w a t e r r e s e a r c h 5 9 ( 2 0 1 4 ) 2 4 8 e2 6 1
a
cpcBA-IGS (n ¼ 19)
e
Buayanup drain type 4 GS1-1
Chroococcales (n ¼ 8)
rpoC1 (n ¼ 22)
Planktothrix sp.jAF212922 (100%) L. redekeijEU078512 (100%) Limnothrix sp.jEF088338 (100%) Leptolyngbya sp.jEU729062 (98%)
Baldwin Park type 1
w a t e r r e s e a r c h 5 9 ( 2 0 1 4 ) 2 4 8 e2 6 1
255
Fig. 3 e Maximum likelihood tree based on the 16S rDNA sequences (313 bp) showing the clustering of isolates obtained. Branch support values greater than 50% for Maximum Likelihood, Maximum Parsimony, and Distance analyses respectively are indicated left of the nodes. Bar, 0.05 substitutions per site. The outgroup was removed to facilitate the visualisation of the isolates. Sequence previously submitted to GenBank with accession number AF247573.
256
w a t e r r e s e a r c h 5 9 ( 2 0 1 4 ) 2 4 8 e2 6 1
Fig. 4 e Maximum likelihood tree based on the rpoC1 sequences (409 bp) showing the clustering of isolates obtained. Branch support values greater than 50% for Maximum Likelihood, Maximum Parsimony, and Distance analyses respectively are indicated left of the nodes. Bar, 0.1 substitutions per site. The Synechococcus cluster containing GS6-1, Vasse River types 9 and 13 were removed to facilitate visualisation of the other isolates.
(Table 2). When morphology was compared with two loci for all isolates (16S rDNA plus, either rpoC1, or cpcBA-IGS), species identities for ANA150-A (D. circinale) and AWQC318 (D. circinale) were in agreement. When morphological data was combined with molecular identification from any one locus, a further seven isolates could be identified to the species level. These included ANA118-AR, ANA131-CR, ANA148-CR, ANA335-C (D. circinale), Hyde Park type 1 (An. elenkinii), MIC058-B (Microcystis flos-aquae), and Vasse River type 1 (Dolichospermum flos-aquae). Of the remaining isolates, 31% (12/39) were in agreement at the genus level, 28% (11/39) to the order level, while no agreement was obtained for the remaining 15% (6/39) (Table 2).
3.4.
Identification of novel isolates
Based on their unique/variable phylogenetic positions and large genetic distances from available sequences, two potentially new members of the Nostocales (Baldwin Park type 2 e morphologically Anabaena sp. 1, and GS2-1 e morphologically Nostoc sp.) were identified. These new strains had no particularly atypical morphology, and could only be morphologically identified to genus level (Anabaena sp. 1, and Nostoc sp., respectively). Noteworthy, 17 novel sequences with
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/watres
Polyphasic identification of cyanobacterial isolates from Australia Elvina Lee a, Una M. Ryan a, Paul Monis b, Glenn B. McGregor c, Andrew Bath d, Cameron Gordon d, Andrea Paparini a,* a
School of Veterinary and Life Sciences, Murdoch University, 90 South Street, Murdoch, Western Australia 6150, Australia b Australian Water Quality Centre, South Australian Water Corporation, 250 Victoria Square, Adelaide 5000, Australia c Department of Science, Information Technology, Innovation and the Arts, GPO Box 5078, Brisbane, Queensland 4001, Australia d Drinking Water Quality Branch, Water Corporation, 629 Newcastle Street, Leederville, Western Australia 6007, Australia
article info
abstract
Article history:
Reliable identification of cyanobacterial isolates has significant socio-economic implica-
Received 15 November 2013
tions as many bloom-forming species affect the aesthetics and safety of drinking water,
Received in revised form
through the production of taste and odour compounds or toxic metabolites. The limitations
10 April 2014
of morphological identification have promoted the application of molecular tools, and
Accepted 12 April 2014
encouraged the adoption of combined (polyphasic) approaches that include both micro-
Available online 24 April 2014
scopy- and DNA-based analyses. In this context, the rapid expansion of available sequence data is expected to allow increasingly reliable identification of cyanobacteria, and ulti-
Keywords: Cyanobacteria identification Morphology
mately resolve current discrepancies between the two approaches. In the present study morphological and molecular characterisations of cyanobacterial isolates (n ¼ 39), collected from various freshwater sites in Australia, were compared. Se-
Molecular phylogeny
quences were obtained for the small ribosomal subunit RNA gene (16S rDNA) (n ¼ 36), the
16S rDNA
DNA-dependent RNA polymerase gene (rpoC1) (n ¼ 22), and the phycocyanin operon, with
rpoC1
its intergenic spacer region (cpcBA-IGS) (n ¼ 19). Phylogenetic analyses identified three
Phycocyanin operon
cyanobacterial orders: the Chroococcales (n ¼ 8), Oscillatoriales (n ¼ 6), and Nostocales (n ¼ 25). Interestingly, multiple novel genotypes were identified, with 22% of the strains (17/ 77) having 50%), while Baldwin Park type 2, although on an isolated branch, was found well within the Nostocales. This is in contrast to the 16S locus, where GS2-1 and Baldwin Park type 2, respectively grouped with Nostoc commune (bootstrap > 70%; well within the Nostocales), or formed a clearly distinct branch, basal to the order (Fig. 3). As with the 16S tree (Fig. 3), multiple clusters of the Chroococcales were also evident from the rpoC1 tree (Fig. 4), with four sequences grouped within this order: MIC058-B, Vasse River types 9, and 13 and GS6-1. Apart from GS2-1, the remaining 17 sequences clustered within the
Nostocales, which was characterized by the distinct positions of Vasse River type 2, Buayanup drain type 2 and GS5-2. Analysis of the cpcBA-IGS locus (423 characters; 396 parsimony informative sites) showed that, apart from GS2-1, which grouped strongly (bootstrap value >80%) with Pseudanabaena sp. (Oscillatoriales) on an isolated branch, the overall topology was similar to that obtained for the 16S rDNA (Fig. 5). As with the 16S and rpoC1 loci, the Nostocales (10 sequences) and the Chroococcales (2 sequences) formed monophyletic groups, while the Oscillatoriales (7 sequences) were paraphyletic (Fig. 5). As the majority of cpcBA-IGS sequences available from GenBank to date mainly belong to relatively few genera (e.g., Arthrospira, Synechococcus, Phormidium etc.), large distance values and the presence of isolated branches were observed for the tree based on this locus (Fig. 5). Baldwin Park type 2 could not be successfully amplified at this locus, and only GS41, GS4-2, Baldwin Park type 1 and Vasse River type 3 exhibited almost complete (99%) homology with available sequences (Fig. 5, Table 2).
3.3. Comparison between morphological and molecular identifications Discrepancies between morphological and molecular identifications were observed for several isolates (Table 2). For example, Hyde Park type 1 was identified, at the cpcBA-IGS locus and morphologically, as An. elenkinii. However, at the 16S rDNA it was closest to Anabaenopsis circularis. Further molecular identification of Hyde Park type 1 was hampered by the paucity of An. circularis and An. elenkinii sequences at both the rpoC1 and cpcBA-IGS loci which prevented confident identification at these loci. The isolate Buayanup drain type 2, which was identified morphologically as A. torulosa was most similar to A. oscillaroides at the 16S rDNA locus, and to Anabaena sphaerica at the cpcBA-IGS locus (no A. oscillaroides sequences were available at this locus). Isolates GS6-1 and Vasse River types 9, 12 and 13 were identified as Aphanothece sp. based on morphology. However, using molecular methods, they were phylogenetically more similar to Synechococcus sp. (HE975005) than Aphanothece minutissima (FM177488) (Fig. 3). Morphologically, ANA196-A was identified as Dolichospermum circinale, but was phylogenetically placed with Aphanizomenon gracile, using the 16S rDNA and rpoC1 sequence data (Table 2). This was also observed for GS4-2, which was identified morphologically as a Nostoc sp. or Sp. aphanizomenoides, but was found to be most closely related to Anabaena bergii (100% similarity) at both the 16S rDNA and cpcBA-IGS loci. Similarly, although Baldwin Park type 1 and GS4-1 were identified morphologically as Planktolyngbya and Oscillatoriales/Geitlerinema sp., they showed 100% similarity to various Limnothrix spp. and Planktothrix spp., at the 16S rDNA locus, and to Geitlerinema amphibium (FJ545644), at the cpcBA-IGS locus. Overall, for the nine isolates that amplified at all loci studied, microscopic and molecular data from at least one locus, were in agreement at genus level for all isolates, except Vasse River type 13 (Table 2). However, agreement between morphological and molecular identifications, from all three loci, was obtained for only one isolate (Vasse River type 6)
Table 2 e Cyanobacteria identification based on 16S, rpoC1, Phycocyanin (Cpc) DNA sequences and isolate morphology. Percentage similarity at each locus was calculated in MEGA 5 (Tamura et al., 2011), as the pairwise evolutionary divergence, with a p-distance model (Kimura, 1980). Sub-group
Isolate
Molecular identification
Morphological identification
Related sequence (percentage similarity) 16S rDNA (n ¼ 36) Nostocales (n ¼ 25)
D. affinejFN691906 (100%) e e e (D. circinale AWQC150-AjAF247573) Ap. gracilejHQ157688 (100%) D. flos-aquaejAB551438 (100%) D. circinalejAF247588 (100%) D. circinalejAF247581 (100%) A. oscillaroidesjAJ630428 (94%)
Chelodina wetlands type 1 GS1-2 GS2-1
cpcBA-IGS (n ¼ 19)
Taxonomist 1
Taxonomist 2
e D. circinalejAF199423 (100%) D. circinalejAF199423 (100%) D. circinalejAF199423 (100%) D. circinalejAF199423 (100%) Ap. gracilejEU078450 (97%) e e D. circinalejAF199425 (100%) Anabaena sp.jAF199432 (86%) Anabaena sp.jAF199433 (86%) Nostoc sp.jAY424997 (88%)
e e e e e e e e e e
D. circinale D. circinale D. circinale D. circinale D. circinale D. circinale D. circinale D. circinale D. circinale N.A.
N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. Anabaena sp. 1
e
Sp. aphanizomenoides
A. oscillaroides
A. sphaericajDQ439645 (92%)
A. torulosa
A. torulosa
Ap. gracilejEU078532 (99%)
Sp. aphanizomenoidesjFJ234841 (86%) Sp. aphanzomenoidesjFJ234848 (86%) e
Anabaena sp.
Anabaena sp.
A. variabilisjCP000117 (89%) Pseudanabaena sp. PCC7367j CP003592 (76%) e C. stagnale PCC7417jCP003642 (87%) A. cylindrica PCC7122jCP003659 (84%) e
Anabaena sp. Nostocales
Anabaena sp. Nostoc sp.
Sp. aphanizomenoides Cylindrospermum sp.
Nostoc sp. Anabaena sp.
Anabaena sp.jGU935369 (97%)
N.A.
Anabaena sp.
GS5-3
N. punctiformejGQ287652 (97%) N. communejDQ185223 (99%) N. communejAB251863 (99%) A.bergiijFR822617 (100%) Nostocaceae cyanobacteriumj GQ389643 (100%) T. variabilisjAJ630456 (100%) T. variabilisjJQ390607 (100%) Nostoc sp.jFJ948088 (99%)
Ap. gracilejFN552318 (97%) D. compactajAY702239 (97%) e Pseudanabaena sp.jEF680776 (80%) A. bergiijFJ234863 (100%) e
Anabaena sp.
Anabaena sp.
Hyde Park type 1 Vasse River type 1
An. circularisjGQ859629 (100%) D. flos-aquaejAY701573 (100%)
An. circularisjEU078479 (89%) Anabaena sp.jAF199432 (98%)
An. elenkinii Anabaena sp.
An. elenkinii D. flos-aquae
Vasse River type 2
Anabaena sp. PCC9109jAY768408 (99%)
Anabaena sp.jEU078475 (87%)
N.A.
Anabaena sp.
Vasse River type 3 Vasse River type 6
A. sphaericajGQ466513 (100%) Sp. aphanizomenoidesjFM177473 (100%)
N.A. Sp. aphanizomenoides
Anabaena sp. Sp. aphanizomenoides
Vasse River type 8
Calothrix sp.jGQ859627 (98%)
A. variabilisjAB074795 (100%) Sp. aphanizomenoidesjFJ830555 (96%) e
Nostoc sp. PCC6720j JF740673 (94%) An. elenkiniijFN552383 (96%) Sp. aphanizomenoidesj GU197719 (99%) Anabaena sp. PCC9109j AY768473 (96%) N. linckiajAY466120 (99%) Sp. aphanizomenoidesj GU197719 (97%) e
Gloeotrichia sp.
Rivularia sp.
GS4-2 GS5-1 GS5-2
Nostoc sp. PCC8976jAM711525 (100%) Nostoc sp.jAB087403 (100%) A. oscillaroidesjAJ630428 (99%)
w a t e r r e s e a r c h 5 9 ( 2 0 1 4 ) 2 4 8 e2 6 1
ANA019-BR ANA118-AR ANA131-CR ANA148-CR ANA150-Aa ANA196-A ANA278-FR ANA335-C AWQC318 Baldwin Park type 2 Baldwin Park type 3 Buayanup drain type 2
rpoC1 (n ¼ 22)
(continued on next page)
253
254
Table 2 e (continued ) Sub-group
Isolate
Molecular identification
Morphological identification
Related sequence (percentage similarity) 16S rDNA (n ¼ 36) Oscillatoriales (n ¼ 6)
Taxonomist 1
Taxonomist 2
G. amphibiumjFJ545644 (100%)
Oscillatoriales
Planktolyngbya sp. 1
e
N.A.
Trichocoleus sp
Leptolyngbya sp.jHM217044 (100%)
e
N.A.
Planktolyngbya sp.
GS3-2
Pseudanabaena sp.jGU935355 (100%)
e
Pseudanabaena sp.
Ps. galeata
GS4-1
e
Geitlerinema sp.
Planktolyngbya sp. 1
GS6-2
Planktothrix sp.jAF212922 (100%) L. redekeijEU078512 (100%) Limnothrix sp.jEF088338 (100%) Ps. mucicolajGQ859642 (99%)
Cyanobacterium txid129981j AJ401183 (75%) Spirulina laxissimaj DQ393286 (84%) Pseudanabaena sp. PCC7409jM99426 (82%) G. amphibiumjFJ545644 (100%)
e
Pseudanabaena sp. PCC7409jM99426 (83%)
Pseudanabaena sp.
Ps. galeata
GS3-1 MIC058-B GS6-1
M. flos-aquaejAF139328 (98%) M. flos-aquaejAF139328 (100%) Synechococcus sp.jHE975005 (100%)
e e e
N.A. M. flos-aquae N.A.
Microcystis sp. N.A. Aphanothece sp.
Vasse River type 9 Vasse River type 12 Vasse River type 13 Vasse River type 14 Vasse River type 15
Synechococcus sp.jHE975005 (100%)
e M. aeruginosajAP009552 (98%) Synechococcus sp. PCC7920j AF245158 (96%) Synechococcus sp. PCC7920j AF245158 (96%) e
e
Aphanothece sp.
Aphanothece sp.
N.A.
Aphanothece sp. 1
N.A.
Aphanothece sp. 1
Synechococcus sp.jHE975005 (100%)
Synechococcus sp. PCC7920jAF245158 (96%) e
Synechococcus sp. PCC7918j AF223462 (97%) Synechococcus sp. PCC7918jAF223462 (96%) e
N.A.
Aphanothece sp.
Synechococcus sp.jHE975005 (100%)
e
e
N.A.
Aphanothece sp. 1
Synechococcus sp.jHE975005 (100%) Synechococcus sp.jHE975005 (100%)
16S rDNA sequence previously available (GenBank Acc. No AF247573); e ¼ No sequence data obtained; N.A. ¼ Not examined; A. ¼ Anabaena; An. ¼ Anabaenopsis; Ap. ¼ Aphanizomenon; C. ¼ Cylindrospermum; D. ¼ Dolichospermum; G. ¼ Geitlerinema; L. ¼ Limnothrix; M. ¼ Microcystis; N. ¼ Nostoc; O. ¼ Oscillatoria; P. ¼ Planktothrix; Ps. ¼ Pseudanabaena; Sp. ¼ Sphaerospermopsis; T. ¼ Trichormus. Sequences with percentage molecular similarity above the threshold used for identification are in bold. For species identification, the threshold was 98% for the 16S rDNA, and 95% for the rpoC1 and cpcBA-IGS loci. For genus, the threshold was 95% for the 16S rDNA, and 90% for rpoC1 and cpcBA-IGS.
w a t e r r e s e a r c h 5 9 ( 2 0 1 4 ) 2 4 8 e2 6 1
a
cpcBA-IGS (n ¼ 19)
e
Buayanup drain type 4 GS1-1
Chroococcales (n ¼ 8)
rpoC1 (n ¼ 22)
Planktothrix sp.jAF212922 (100%) L. redekeijEU078512 (100%) Limnothrix sp.jEF088338 (100%) Leptolyngbya sp.jEU729062 (98%)
Baldwin Park type 1
w a t e r r e s e a r c h 5 9 ( 2 0 1 4 ) 2 4 8 e2 6 1
255
Fig. 3 e Maximum likelihood tree based on the 16S rDNA sequences (313 bp) showing the clustering of isolates obtained. Branch support values greater than 50% for Maximum Likelihood, Maximum Parsimony, and Distance analyses respectively are indicated left of the nodes. Bar, 0.05 substitutions per site. The outgroup was removed to facilitate the visualisation of the isolates. Sequence previously submitted to GenBank with accession number AF247573.
256
w a t e r r e s e a r c h 5 9 ( 2 0 1 4 ) 2 4 8 e2 6 1
Fig. 4 e Maximum likelihood tree based on the rpoC1 sequences (409 bp) showing the clustering of isolates obtained. Branch support values greater than 50% for Maximum Likelihood, Maximum Parsimony, and Distance analyses respectively are indicated left of the nodes. Bar, 0.1 substitutions per site. The Synechococcus cluster containing GS6-1, Vasse River types 9 and 13 were removed to facilitate visualisation of the other isolates.
(Table 2). When morphology was compared with two loci for all isolates (16S rDNA plus, either rpoC1, or cpcBA-IGS), species identities for ANA150-A (D. circinale) and AWQC318 (D. circinale) were in agreement. When morphological data was combined with molecular identification from any one locus, a further seven isolates could be identified to the species level. These included ANA118-AR, ANA131-CR, ANA148-CR, ANA335-C (D. circinale), Hyde Park type 1 (An. elenkinii), MIC058-B (Microcystis flos-aquae), and Vasse River type 1 (Dolichospermum flos-aquae). Of the remaining isolates, 31% (12/39) were in agreement at the genus level, 28% (11/39) to the order level, while no agreement was obtained for the remaining 15% (6/39) (Table 2).
3.4.
Identification of novel isolates
Based on their unique/variable phylogenetic positions and large genetic distances from available sequences, two potentially new members of the Nostocales (Baldwin Park type 2 e morphologically Anabaena sp. 1, and GS2-1 e morphologically Nostoc sp.) were identified. These new strains had no particularly atypical morphology, and could only be morphologically identified to genus level (Anabaena sp. 1, and Nostoc sp., respectively). Noteworthy, 17 novel sequences with
