Marine cyanobacteria from Bahia
TAXINOMY AND PHYLOGENY
MATERIALS AND METHODS
The field work was done in Bahia Concepcion, Gulf of Califomia, BCS, Mexico, from October 1995 to October 1997. Three sampling stations were selected on the west side of the bay.
1.Thermal springs in coastal evaporitic system at Santispac Beach. In this locality there are two thermal springs on an evaporite fiat ("mini sabkha"), bordered towards the sea by mangrove trees, a smalllagoon, and a beach. The first pond is a shallow hot spring with three hydrothermal vents spurting hot fresh water, which mixes with seawater. The water is clear and colorless, with temperatures ranging from 41 to 52°C, 2.5% to 2.8% salinity, and pH 6.8.
Macroscopic prokaryotic phototrophic masses composed of filaments were found in the vents, with short columns, microbial mats, and scums around it.
The second pond had the ranged oftemperature from 28 to 45°C, 2.9% salinity, and pH 7.1. Benthic microbial communities were attached to the surface and walls of the pond. Others detached from the bottom and formed scums fioat-ing on the water surface. Of particular interest were the nodules (oncoids) from the walls and edges of this second thermal spring.
2. Calcareous algae, shells, rock, sand, and slime mud from the bottom (3.5 to 18 m) in front of El Coyote Beach were used as sampIes. The tempera-ture was 19 and 20°C, salinity was 3.5%, and the pH varied from 6.5 to 7.6.
3. Hypersaline fiat, Los Pinos. Laminated microbial mats rich in evaporitic mineraIs occurred in a shallow pond. The temperature was from 21 to 37°C, salinity was 2.4 to 4.0% , and pH was 6.9 to 7.2.
Two media were used for the enrichment and isolation of prokaryotic pho-totrophs, Pfennig medium (Trüper, 1970) and ASNIII medium (Rippka et al., 1981). BottIes filled with liquid Pfennig medium were incubated at tempera-ture of 25°C and light intensity of 25 f.lE m-2 S-1 for six weeks. Most of the cyanobacteria were isolated by pour-plate technique in ASNIII, but, sorne also from liquid Pfennig medium. The ASNIII plates with subsamples from bot-tom, oncoids, and microbial mats were incubated at light intensity of 50 f.lE m-2 S-1 at 25°C, and those from hot springs at light intensity of 25~E m-2s-l and temperature of 50°C.
Microscopic analyses of subsamples, previously fixed in a buffered (MgC03) 3% solution of formaldehyde in sea water of each site, were made.
Preparation for light microscopy of calcareous algae was done by dissolving the substrate using 3% HCl as a carbonate solvent (LeCampion-Alsumard &
Golubic, 1985). Isolated strains of cyanobacteria were identified to the species level by phenotypic traits including morphological and, in sorne cases, bio-chemical, physiological and ecological characteristics (Geitler, 1925; Desika-chary, 1959; Tilden, 1910; Golubic, 1976; Komârek& Anagnostidis, 1986;
Anagnostidis& Komarek, 1988).
Eight different species of monocyanobacterial high-density cell cultures from the stationary phase were chosen for preservation in liquid nitrogen (Rippka et al., 1981). The relational database is made with eight entries;
TAXONOMY, SYNONOMY, CURATOR, PHYSIOLOGY, GEOGRAPHY, BIBLIOGRAPHY, INSTITUTION, andPERSON.
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RESULTS AND DISCUSSION
Microorganisms in thermal springs, at Santispac, grow and develop to macroscopically visible filamentous, cylindrical, domal masses, and scums (Fig. lA). At the end of the wann period in October of 1995, a green, colorful, filamentous microbial community occurred in a hydrothermal vent (52°C).
The filament structure was formed in the direction of flowing hot water from the vent, reaching the surface (35 cm), and adopting a flat shape. The struc-ture was mainly composed of filamentous cyanobacteriaLeptolyngbya ther-malis(Fig. 1C),Limnothrix amphigranulata(Fig. ID), and anoxygenic photo-trophic bacteriumChlorofiexus, but also contained unicellular cyanobacteria Aphanothece bullosa(Fig. IF),Chroococcus minutus,andCh. turgidus(Fig. lB).
A few types of specialized microorganisms proliferate in an unusual environ-ment characterized by high temperature, flow ofwater, high salinity and pres-ence of sulfur. Similar environments and microbial communities dominated byLeptolyngbya thermalis have been reported for the Blue Lagoon geother-mal area in Iceland (Pétursd6ttir& Kristjansson, 1996), and others from the Tengchong hot sea geotherrnal region in China, dominated by Chlorofiexus and Synechococcus (Zhang Yun, 1986). Spherical to subspherical oncoids were found in hot springs. The average diameter of the oncoids seen are about 2 to 4 cm. The oncoids show a well-laminated cortex composed of alternating layers, light, white evaporitic-rich laminae, and porous, darker, organic-rich material. From the oncoids,Synechococcus elongatus(Fig. 1E),Synechocystis pevalekii(Fig. IN lower right), andGleocapsasp. have been identified.
From the bottom at Isla El Coyote, epilithic cyanobacteria were common at 3.5 to 15 m depth, growing on the surface of calcareous red algae (Rhodo-phora). The main species wereCalothrix contarenii (Fig. 11), Phormidium rubrum (Fig. 1G), Phormidium spp, Lyngbya aestuarii (Fig. lH), Oscilla-toria vizagapatensis, and Spirulina meneghiniana (Fig. 11). P. rubrum is a scarlet filamentous material rich in phycoerythrin, suggesting complementary chromatic adaptation in environments with low light intensity. AIso, epipelic cyanobacteria were found at depths of 15 m, where only slime mud exists.
The cyanobacteria identified wereSpirulina sp. (Fig. lM), Phormidium pur-purascens(Fig. IL),P.molle,andLimnothrix amphigranulata,which coexist withBeggiatoasp. (Fig. 1K), a chemolithotrophic bacterium.
Laminated microbial mats found in the hypersaline flat at Los Pinos, show a pattern of layers colored orange-yellow, green, red-purple, and black. The mat was dominated byLynbgya aestuarii, Microcoleus chthonoplastes(Fig. IN), Spirulina subtilissima (Fig. IN, left), S. subsalsa (Fig. 1N), Phormidium tenue, Oscillatoria limnetica, Limnothrix amphigranulata, Synechocystis pevalekii, Synechoccoccus aeruginosa, and Chromatium sp .. Similar mats have been reported for the Pacifie Ocean coast of Baja California (Cohen&
Rosenberg, 1989; Lopez-Cortes, 1998). The mats from Los Pinos, Bahia Concepcion, Gulf of California are different in texture and are the first described for this area. A summary of the distribution, environments, micro-bial structures, cyanobacteria and related organisms, from Bahia Concepcion are shown in Table1.
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Figure 1. Macroscopic phototropruc structures and prokaryotic cells from Bahia Concep-cion, B.CS., Mexico. (A) Macroscopic phototrophic prokaryotic masses in thennal spring at Santispac. (B)Chroococcus minutus,associated with pholotrophic filaments.
Inset showChroococcus /lIrgidus. (C) Leptolyngbya thernuzlis.(D)Limnothrix amphi-granulata. (E)Synechococcus elongatus. (F)Aphanothece buLlosa. (G)Phormidium rubrum.(H)Lyngbya aes/ltarii. (1)Calothrix contarenii.(1)Spirulina meneghiniana.
(K)Beggiatoasp. (L)Phormidium purpurascens. (M)Spirulinasp. (N)Microcoleus chthonoplastes.(N)Spirulina submlsa.Upper left inselSpirulina subtilissirna.Lower right insetSynechocystis pevalekii.See color plates at the end of the volume.
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Table1. Distribution and diversity of cyanobacteria in different environments of Bahia Concepcion, Baja Califomia Sur, Mexico. The species names appear
in order of relative abundance.
Geoposition Locality Environment Structure Cyanobacteria species 26°45'46", Santispac Hot spring Filaments Leptolyngbya thermalis*
111 °53'38" mangroves in Sabkha in vent Limnothrix amphigranulata * microbial Chroococcus minutus mats Chroococcus turgidus Aphanothece bullosa*
Oncoids Gleocapsasp.
Synechococcus elongatus*
Synechocystis pevalekii 26°43'32", El Coyote Sea bottom Epilithic Calothrix sp.
1W54'06" Punta Cola (3.5 m) on red algae Phormidium rubrum *
De Ballena Rhodophora
26°44'29", El Coyote Sea bottom Epilithic on Lyngbya aestuarii 111°52'30" Isla (12.0 m) caIcareous Calothrix contarenii
La Cueva and other Phormidium rubrum
surfaces Spirulina meneghiniana Oscillatoria vizagapatensis Sea bottom Slime mud Spirulinasp.
(18 m) Phormidium purpurascens
Phormidiumspp.
26°43'08", El Coyote Sea bottom Epilithic on Calothrix contarenii 1W52'04" Isla Blanca (15 m) Rhodopora Phormidium tenue
... ...
Seabottom Slîme mud Spirulinasp.
(18 m) Phormidium purpurascens
Phormidium molle Limnothrix amphigranulata 26°43'19", El Coyote Sea bottom Epilîthic on Hydrocoleumsp.
1W53'20" Isla Coyote (6.0 m) shell and rocks Phormidium okenii*
26°32'10", Los Pinos Hypersaline Laminated Microcoleus chthonoplastes 111 °44'20" fiat microbial Spirulina subtilissima
mats Spirulina subsalsa Phormidium tenue Oscillatoria limnetica * Limnothrix amphigranulata Synechocystis pevalekii * Synechococcus aeruginosa
*
Isolates preserved in liquid nitrogen as monocyanobacterial strains.Bulletin de l'Institut océanographique, Monaco,n° spécial 19(1999) 91
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Macroscopic structures, filamentous mass, oncoids and mats grow under different combined and unusual environmental conditions; relatively high temperature (28-52°C), salinity (2.4-4.0%), high light intensity (1500 ilE m-2
S-I),or low light intensity (10 ilE m-2 S-I),and presence of elemental sulfur.
The use of classification systems built for cyanobacteria from terrestrial freshwater environments, had limitations for the identification of the strains isolated from these singular biotopes.
Besides the taxonomie importance of the extremophiles, the prokaryotic phototrophs of extreme environments have been considered relicts of primor-dial forms of life. The microbial ecology of thermal springs associated with hypersaline environments from Santispac could help explain sorne paleonto-logical and geopaleonto-logical features of the rocks from Warrawoona Group, Aus-tralia (3.5 x 109years old). The sedimentary evidence shows that Warrawoona rocks were deposited in a shallow hypersaline water body, associated with volcanic activity (Barley et al., 1979). From the Precambrian fossil record, morphological similarities and ecological setting of hot salt sulfur waters of recent counterparts suggest that cyanobacteria and other prokaryotic photo-trophs (Chloroflexus) could be related with hot salt water environments and may have been dominant in early Precambrian.
Strains of eight different species have been preserved in liquid nitrogen and are recorded in a data base available for consulting in SEMARNAP and CIBNOR. These strains are part of the CIBNOR microalgae collection and their description appears on the internet (http://www.cibnor.mx/malgas/
ealgas.html).
ACKNOWLEDGEMENTS
This work was financially supported by grant G035 from La Comision Nacional para el Conocimiento y Uso de la Biodiversidad, CONABIO, and projects BIT4 and EDA4 from CIBNOR.
REFERENCES
ANAGNOSTIDIS K., KOMÂREK J., 1988. - Modern approach to the classification system of cyanophytes. 3. Oscillatoriales. - Arch. Hydrobiol., Suppl 80, 327-472.
BARLEY M.E.. DUNLOP J.S., GLOVER J.E., GROVES D.I., 1979. - Sedimentary evidence for an Archean shallow-water volcanic sedimentary facies, eastern of Pilbara Block, Western Australia. - Earth and Planetary Science Letters,43(1), 74-84.
COHENY.,ROSENBERG E., 1989. - Microbial Mats.-American Society for Micro-biology. Washington D.C., 494 p.
DESlKACHARY T.V., 1959. - Cyanophyta. - Indian Council of Agricultural Research, New Delhi, 686 p.
GEtTLER L., 1925. - Cyanophyceae. - Pascher's Süsswasserflora, G. Fischer-Verlag, Jena, 12, 1-450.
GOLUBIC S., 1976. - Taxonomy of extant stromatolite-building cyanophytes. - In:
Walter M.R. (ed),Stromatolites.Elsevier Scientific Pub. Co., New York, 127-140.
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KOMÀREK l, ANAGNOSTlDIS K, 1986. - Modem approach to the classification system of cyanophytes. 2. Chroococcales. - Arch. Hydrobiol., Suppl. 73, 157-226.
LECAMPION-ALSUMARD T., GOLUBIC S., 1985. - Hyella caespitosa Bornet et Flahault andHyella balaniLehman (Pleurocapsales, Cyanophyta): a comparartive study. - Arch. Hydrobiol., Suppl.71, 119-148.
LOPEZ-CORTESA., 1998. - Ecologfa y biotecnologfa de las comunidades microbia-nas. Ciencia y Desarrollo. - SEP-CONACYT-Mexico,24 (138),10-17.
PÉTURSD6ITIR S.K, KRrSTJÀNssON lK,1996. - The relationship between physical and chemical conditions and ow microbial diversity in the Blue Lagoon geother-mallake in leeland. - FEMS Microbiol. Ecol., 19,39-45.
RIPPKA R., WATERBURY lB., STANIER R.Y., 1981. - Isolation and purification of cyanobacteria: Sorne general principles. - In:Starr M.P.et al. (eds),The Proka-ryotes.Springer-Verlag. New York.,l, 212-223.
TRÜPER RG., 1970. - Culture and isolation of phototrophic sulfur bacteria from the marine environment. - Helgoliinder Wiss Meeresunters,20,6-16.
TrLDEN l, 1910. - The myxophyceae of North America. - Verlag Von J. Cramer, New York,328p.
ZHANG YUN,1986. - Therrnophilic microorganisms in the hot springs of Tengchong geothermal area, west Yunnan, China. - Geothermies, 15,347-358.
Bulletin de l'Institut océanographique. Monaco, n° spécial 19(1999) 93
Molecular biological characterizatîon of unicellular marine Cyanobacteria
from the Baltic Sea
Andrea MIKûLAJCZAK(l) *, Rainer SOLLER(2),
Dietmar BLûHM(2),Ulrich FISCHER(2)
University of Bremen, FB2, Zentrumfür Umweltforschung und Technologie,
(1)Abt. Marine Mikrobiologie, (2)Abt. Biotechnologie und Molekulare Genetik, Leobener Str., 28359Bremen, Germany
*
Corresponding author (miko@biotec.uni-bremen.de)ABSTRACT
Six unicellular marine cyanobacteria strains of the order Chroococcales isolated from the Baltic Sea were examined by DNA sequence analysis and RAPD-PCR. The results were correlated with the classical determination relying on morphological features. After sequencing the complete 16S rDNA of one strain, a highly variable 305 bp fragment was further investigated from aIl six strains. RAPD-PCR was used to reveal DNA sequence polymorphisms on a level beyond the taxonomic range approached by the 16S rDNA. Fur-therrnore, a unique band obtained from the RA PD-pattern was cloned and finally used to compare the strains by Dot-Blot hybridization using this sequence as probe.
Three of the six cyanobacterial strains showed almost 100% sequence identity of the 16S rDNA investigated. No polymorphism was detected in the RAPD band pattern of these strains. In addition, positive Dot-Blot signaIs were obtained using the cloned RAPD-probe with these three strains.
These results suggest that three of the six unicellular marine cyanobacteria strains examined should be considered as a single species. The investigation shows that the molecular techniques applied are useful tools to complement cyanobacterial taxonomy and to infer interspecies relationships.
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INTRODUCTION
Cyanobacteria are a large group of phototrophic microarganisms with highly variable morphological featmes. For a long time, mainly morphologi-cal characteristics were taken into account for a taxonomimorphologi-cal classification of cyanobacteria, following the assignments of Rippka et al. (1979). Recent reports of phenotypic variations without correlation of genetic traits underline the need for new approaches in cyanobacterial taxonomy (Lu, 1997; for review see Wilmotte, 1994). Consequently, this work addresses the question of morphological versus genetic diversity focussing on six unicellular marine cyanobacteria strains of the arder Chroococcales isolated from shallow waters of the southem Baltic Sea (Germany). DNA sequence analysis and RAPD-PCR were performed and the results were correlated with the classical deterrnination. A highly variable 305 bp 16S rDNA fragment (base position 545-850) was chosen from the complete 16S rDNA fragment of strain Bü 79 for further investigations of all six strains.Inaddition, RAPD-PCR was used to reveal DNA sequence polymorphisms of those strains which have shown similarities in their 16S rDNA sequence. Finally, one 654 bp RAPD-band common to three of the RAPD-pattems was cloned and used to compare the strains by Dot-Blot hybridization using this sequence as a probe.
Itwas the aim of this study to use modem molecular techniques far a better classification of unicellular marine cyanobacteria which do not differ signifi-candy in their morphology.
MATERIALS AND METHODS
The unicellular marine cyanobacteria strains examined are part of the cul-ture collection of the "Abteilung für Marine Mikrobiologie, Universitat Bremen, Germany" and are listed in Table1.Taxonomical classification was
Table1.Examined unicellular marine cyanobacterial strains collected at Hiddensee (HI) and Boiensdorf station (BO), 165 rDNA bases sequenced and cornmon RAPD bands (classification was performed by Rethmeier, 1995;
n=no data; nt=nucleotides).
Organisms Strain 16SrDNA cornmon
sequenced RAPDbands
Synechocystissp. HI 19 305 nt 5
Chroococcus turgidus BO 1 305 nt 0
Synechococcussp. B045 305 nt n
Synechococcus elongatus B052 305 nt 0
Synechocystissp. B079 1479 nt 5
Microcystissp. BO 83 305 nt 5
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do ne by lightmicroscopy only (Rethmeier 1995) according to the assignments of Rippkaet al. (1979).
After DNA preparation, 16S rDNA-PCR, RAPD-PCR, sequence analysis, and Dot-Blot hybridization were perforrned. For further details see Schenk (1996). A 1479 bp 16S rDNA-fragment from strain BO 79 was amplified, sequenced, and compared ta known cyanobacterial 16S rDNA-sequences found in the EMBL-database (http//www.ebi.ac.ukl) in order ta choose a highly variable region to discriminate the examined strains.
Finally, the DNA-sequence of the cloned RAPD-band was determined and compared to the complete EMBL-database using the BLAST N- and FAST A-aJgorithms available via the Husar-programm (DKFZ, Heidelberg) in order to find out a possible function or a related species.
RESULTS AND DISCUSSION
Sequence alignment of the investigated 305 bp 16S rDNA fragment has shown complete identity within the cyanobacteria strains BO 79, Ba 83 and HI 19. Strain Ba 45 revealed only one base difference whereas strains BO 1 and BO 52 differed in more base positions (28 and 70 bp respectively). The genetic distances determined from clustal analysis are shown in Table II.
These results indicate a high genetic sirnilarity of four of the six strains.
RAPD analysis using primers CRA22 and CRA 23 (Neilan, 1995) confirmed the homogeneity of strains Ba 79, BO 83, and HI 19 by cornmon RAPD pat-tern (Fig. 1). In contrast, the RAPD patpat-terns of Ba 1 and Ba 52 revealed no similarity (Table1). Unfortunately, no RAPD-pattern of strain Ba 45 could be obtained.
For further evidence of genetic homogeneity, a 654 bp RAPD band from Ba 79 (Fig. 1)was cloned and used as a probe in a Dot-Blot experiment with
TableII.Differences in base pairs (under diagonal) and genetic distances in percent divergence (above diagonal) of a 305 nt 16S rDNA-fragment
from the unicellular marine cyanobacterial strains examined.
Sequence pair divergence were calculated by the program MEGALIGN (Lasergene, DNASTAR) using the default options of the ClustalVmethod.
Strain B045 B052 B079 B083 HI 19
BO 1 18.7 9.3 9.3 9.3
B045 0.3 0.3 0.0
B052 58 22.2 21.9
B079 28 0,0
BO 83 28 70
HII9 28 0 69 0
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2 3 4 5 6 bp
<Il 4072
<Il 1636
<Il 1018
<Il 506
<Il----C
Figure 1. RAPD-pattern of the unicellular marine cyanobacteria examined. Lane 1 - 5:
strainsBa l,Bü52, Ba79,Bü83, HI 19. Lane 6: 1-kb-Iadder. (bp= base pairs, c=cloned RAPD band from strainBa 79).
genomic DNA from the other cyanobacteria examined. Positive Dot-Blot signais from the strainsBa 83 and HI 19 confirm the presence of this sequence in these strains. The sequence analysis data of the probe have revealed the highest similarity with Synechocystis sp. PCC 6803 (EMBL-database entry AC D9û916).
A possible function is still unknown.
These data show that at least three of the six unicellular cyanobacteria strains examined form a coherent phylogenetic group. Therefore, we suggest to combine the strains BO 79,Ba83, andHI 19 to one Synechocystis species.
ACKNOWLEDGEMENT
This work was partly supported by the Bundesminister für Bildung, Wissenschaft, Forschung und Technologie under the project "DY SMaN"
(03Fû123D).
98 Bullelin de l'Institut océanographique, Monaco, nOspécial 19(1999)
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REFERENCES
Lu W., EVANs E.R., MCCOLL S.M., SAUNDERS VA., 1997. - Identification of cyanobacteria by polymorphisms ofPCR-amplified ribosomal DNA spacer region.
- FEMS Microbiol. Leu., 153, 141-149.
NEILAN B.A., 1995. - Identification and phylogenetic analysis of toxigenic cyano-bacteria by multiplex randomly amplified polymorphie DNA PCR. -Appl. environ.
Microbiol.,61,2286-2297.
RETHMEIER J., 1995. - Untersuchungen zur Okalogie und zum Mechanismus der Sulfidadaptation mariner Cyanobakterien der Ostsee. - Ph. D. thesis, University of Bremen.
RIPPKA R., DERUELLES1.,WATERBURY J.B., HERDMAN M., STANIER R.Y., 1979. -Generic assignment, strain histories and properties of pure cultures of cyano-bacteria. - J.Gen. Microbiol., Ill,1-61.
SCHENK A., 1996. - Untersuchungen zur Identifizierung unizel!uliirer mariner Cyanobakterien der Ostsee aufmolekularbiologischer Ebene unter besonderer Berücksichtigung der PCR-Technik. - Master thesis, University of Oldenburg.
WILMOTTE A., 1994. - Molecular evolution and taxonomy of the cyanobacteria. -In: Bryant DA. (ed), The molecular biology of cyanobacteria. Kluver Academie Publishers, Dordrecht, 1-25.
Bulletin de l'Institut océanographique, Monaco, n° spécial 19 (1999) 99
Cyanobacterial diversity in marine ecosystems as seen by RNA polymerase (rpoCl)
gene sequences
Brian PALENIK *, Gerardo roLEDO, Mike FERRIS
Scripps /nst. ofOceanography, University ofCalifornia, San Diego, La Jolla, CA 92093-0202, USA
*
Corresponding author (bpalenik@ucsd.edu)Cyanobacteria comprise one of the major eubacteriallineages. The diver-sity within this lineage, including both that of morphology (singles ceUs, branching filaments, akinetes, etc.) and physiology (nitrogen fixation, hetero-trophy, motility, etc.) has fascinated microbiologists. Cyanobacteria have also provided model systems for understanding those processes that occur more broadly than the lineage itself such as photosynthesis, nitrogen fixation, pat-tern development, and circadian rhythms (Bryant, 1994).
Cyanobacteriologists have been greatly interested in the relationships between morphology or unique physiological processes and evolution. What characteristics are related to the evolution of specifie groups within the cyanobacterial lineage? For example true branching seems to have evolved once and is characteristic of only one lineage, the Group V cyanobacteria (Rippka et al., 1979; Giovannoni et al., 1988) although this idea has been more recently questioned based on its low bootstrap values in 16S rRNA data (Wilmotte, 1994). In contrast, light harvesting by chlorophyU b (in cyanobac-teria referred to as prochlorophytes) seems to have arisen multiple times within the cyanobacteria (Urbach et al., 1992; Palenik& Haselkom, 1992).
These relationships between morphology and physiological characteristics and the evolution of various groups has been investigated using a number of phylogenetic tools, sorne based on protein sequences, sorne on 16S rRNA.
Reviews of these approaches can be found in Packer & Glazer (1988);
Wilmotte (1994). We have been pursuing these questions using as another
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tool, DNA sequences of a fragment (rpoCl) of the protein DNA-dependent RNA polymerase (Palenik & Haselkorn, 1992; Palenik, 1992; Palenik &
Swift, 1996). Cyanobacterial specifie primers allow cyanobacterial rpoCl sequences to be pcr amplified from DNA obtained from water samples, cyanobacterial symbiont (tunicate) hosts, and nonaxenic cultures.
Environmental variables such as light, temperature, and nutrients seem to be influencing cyanobacterial genetic diversity in marine ecosystems, but to an extent that is still poorly characterized. This influence is feh at different levels. For example, the presence of nitrogen fixing groups of cyanobacteria is likely to be related to the concentration of dissolved nitrogen sources or N:P nutrient ratios (Karlet al., 1997). More subtle are the changes in the pres-ence of different groups within what we think of as the dominant abundant lineages. We are just beginning to see that there are "ecotypes" or species within the genera "Synechococcus" and "Prochlorococcus" and these "ecotypes"
are associated with particular ecological niches.
In the oligotrophic ocean environment covering much of the globe, large changes in sorne environmental parameters occur with depth. For example in the oligotrophic regions of the Califomia CUITent (Eppley, 1986), light flux drops by 3 orders of magnitude from the surface to the bottom of the cyano-bacterial growth range (0-150 meters), and large shifts in Iight quality occur.
Nutrient gradients are also large with undetectable nitrate through much of the upper water column up to a few micromolar concentration at depth. Tem-perature gradients are smaller with the range in the California CUITent from around 20°C at the surface to about l3°C at the bottom of the euphotic zone.
This picture is then complicated by a seasonal mixing cycle in many environ-ments including the California CUITent. During the winter, the water column is mixed through the euphotic zone, while during summer and fall the water column is generally stratified. During stratification, different parts of the cyanobacterial population are presumably under very different selection pres-sures with high light, low nutrients at the surface and lower, blue light and higher nitrate conditions at depth. We would expect the most genetic differen-tiation in the cyanobacterial population thus between surface and deep sam-pIes at this time. However, since seasonal mixing changes these selective pressures so drastically it is possible that they do not exist long enough for specifie "high" or "low" light cyanobacterial species or "ecotypes" to have developed. Sequence data from cyanobacterial DNA-dependent RNA polymerase (rpoCl) genes obtained from marine isolates and from bulk sea-water DNA samples can be used to describe and understand cyanobacterial diversity as a function of depth and geographical region in order to answer such questions.
Recently, a total of 15 cyanobacterial strains were isolated from the oligo-trophic edge of the California CUITent from two depths (5 and 95 m) (Toledo
& Palenik, 1997). RNA polymerase (rpoCl) gene sequences of the strains revealed two major genetic lineages, distinct from other marine or freshwater cyanobacterial isolates, or groups seen in shotgun c10ned sequences from the oligotrophic Atlantic Ocean. One group, the California CUITent low phy-courobilin group represented by 6 isolates in a single lineage (CC9311) was less diverse than the Califomia CUITent high phycourobilin group with 9 iso-102 Bulletin de l'Institut océanographique, Monaco, na spécial 19 (1999)
CYANOBACTERIAL DIVERSITY AS SEEN BYRNAPOLYMERASE GENE SEQUENCES
lates in three relatively divergent lineages (represented by CC9317, CC9318, andCC930S-3).The former group was found to be the closest known genetic group toProchlorococcus, the chlorophyll b containing marine cyanobacte-rial group. Both groups included strains obtained from surface(S m) and deep (9S m) samples, thus there was no clear correlation between sampling depth and isolation of genetic groups.
Further work onrpoCl gene sequences PCR amplified from DNA samples from the same region clearly show the presence of the "low phycourobilin group", but less clearly the other ("high phycourobilin") group in surface sea-water samples (Ferris & Palenik, manuscript in prep.). There is a cluster of DNA library strains that appear related to the high PUB group (clones G Il, 12, 23, 40, 96, lOS in Fig. 1), but we are still unclear on what constitutes a definable clade in these organisms. These results may be because the two approaches of isolation and environmental library preparation are providing different information about these groups, with the later likely to be more related to abundance than the former. Synechococcus clades may have sorne
Cluster B clones T5,T9,T13,T14 clones A13&.14
clone A2
ProchlorococcU& MED (Mediterranean Bea isolate)
Cluster A
clones G 11.12.23.
40.96,105
.. •
~'lim
mo n...
~
PCC79&2
Figure 1.An rpoCl DNA fragment library from a sample from the oligotrophic Califomia CUITent (station 77.100, 1996, 3 meters). Aiso included are Califomia Current isolates (CC), Woods Hole Culture Collection(WH) and other isolates and Sargasso Sea DNA clones (IAR, IAB, ICNC, 2CNA, lCNH, ICNI). The sequence data (270 bp) were analyzed using distance matrix (Jukes-Cantor) and the neighbor-joining methods using the program PHYLIP (Felsenstein, U. Washington). The data show at least twoProchlorococcus(cluster A and B) and fiveSynechococcusgroups.
Subsequent libraries have shown that the IAB cluster is typically surface associated while the1AR group is more typically found deeper in the water column.
Bulletin de l'Institut océanographique. Monaco. naspécial 19(1999) 103