Cyanobacterial–Bacterial Complexes in Plant Syncyanoses

The morphology and ultrastructure of associative microsymbiont complexes (AMC) isolated from the ferns Azolla pinnata and Azolla sp. and the apogeotropic roots of the cycad Cycas revoluta were studied. The composition of the AMC obtained includes the cyanobionts (symbiotic cyanobacteria) and satellite bacteria (SB). It was found that two types of cyanobacteria that substantially differ in their morphological organization are likely present as cyanobionts in the coralloids of C. revoluta. The isolated cyanobiont strains exhibited the morphological traits and regularities of development typical of the genus Nostoc; they were characterized by the ability of their cells to divide in mutually perpendicular planes. When isolating AMC from different morphological zones of C. revoluta apogeotropic roots, SB growth was revealed only around the pieces corresponding to the coralloid apical zone. No AMC components were revealed around the segments of the basal growth zone. Pure cyanobiont cultures were obtained from the AMC of C. revoluta coralloids. The AMC isolated from the ferns A. pinnata and Azolla sp. are characterized by obligate mutual dependence of the partners (the cyanobiont and SB).     

PHYLOGENETIC PATTERNS AMONG NOSTOC CYANOBIONTS WITHIN BI‐ AND TRIPARTITE LICHENS OF THE GENUS PANNARIA1

Phylogenetic relationships between Nostoc cyanobionts in the lichen genus Pannaria were studied to evaluate their correlation to geography, habitat ecology, and other patterns previously reported. The 16S rRNA gene sequences of a total of 37 samples of 21 Pannaria species from seven countries from the Northern and Southern hemispheres were analyzed and compared with 69 free‐living and symbiotic cyanobacterial strains. The sequences from Pannaria were distributed throughout a branch of Nostoc sequences previously called “the Nephroma guild,” and within two subgroups from another branch, referred to as the “Peltigera guild,” although there was a gradual transition between the two major groups. There is a more diverse pattern of relationships between Nostoc sequences from bipartite versus tripartite lichen species in Pannaria, compared with other well‐studied genera, such as Nephroma and Peltigera. Cyanobionts from several tripartite Pannaria species from the Southern Hemisphere and corticolous bipartite species from both hemispheres were grouped together. Four sequences of Pannaria and Pseudocyphellaria cyanobionts from rocks in the Chilean Juan Fernández Islands were nested within corticolous cyanobionts, whereas the terricolous “Pannaria sphinctrina clade” was placed with other terricolous strains. The cluster patterns derived from phylogenetic analysis were partly reflecting lichen taxonomy, in two groups of lichen species, possibly indicating coevolution. The phylogram partly also reflected lichen ecology. Three Pannaria species have very different cyanobiont strains when they grow in different habitats.     

Comparative analysis of cyanobacteria in the rhizosphere and as endosymbionts of cycads in drought-affected soils

Does the diversity of cyanobacteria in the cycad rhizosphere relate to the cyanobiont species found in the coralloid roots of these ancient plants? The aim of this study was to identify the diversity of soil cyanobacteria occurring in the immediate vicinity of 22 colonized coralloid roots belonging to members of the cycad genera: Macrozamia, Lepidozamia, Bowenia and Cycas. The majority of coralloid roots were sampled at depths >?10?cm below the soil surface. A total of 32 cyanobacterial isolates were cultured and their 16S rRNA gene partially sequenced. Phylogenetic analysis revealed nine operational taxonomic units of soil cyanobacteria comprising 30 Nostoc spp., a Tolypothrix sp. and a Leptolyngbya sp. Microscopy indicated that all isolates were unialgal and confirmed their genus identity. Rhizospheric diversity was compared to existing data on cyanobionts isolated at the same time from the cycad coralloid root. The same isolate was present in both the cycad coralloid root and rhizosphere at only six sites. Phylogenetic evidence indicates that most rhizosphere isolates were distinct from root cyanobionts. This weak relationship between the soil cyanobacteria and cycad cyanobionts might indicate that changes in the soil community composition are due to environmental factors.     

Response of 19 Azolla strains to a high concentration of ammonium ions

The response of 19 Azolla strains from five species to 20 mM ammonium ions was examined. The response varied even among strains of the same species. The tolerance indexes of both relative growth rate and nitrogen fixation [(values for plants grown in the presence of ammonium ions)/(values for plants grown in the absence of ammonium ions)] showed that ammonium ions did not always simultaneously inhibit growth and nitrogen fixation of individual strains. The tolerance of each Azolla–Anabaena association to ammonium ions is assumed to be determined separately by both the host fern and the symbiotic Anabaena. The inhibitory effects of ammonium appeared predominantly in the mature region of Azolla–Anabaena associations. In the sensitive strains, both chlorophyll content and the number of cyanobionts were reduced only in the mature region when ammonium ions were present. Possible strains for treatment of wastewater, which contains a high concentration of ammonium ions, are discussed.     

Does the Reproductive Strategy Affect the Transmission and Genetic Diversity of Bionts in Cyanolichens? A Case Study Using Two Closely Related Species

Observed levels of population genetic diversity are often associated with differences in species dispersal and reproductive strategies. In symbiotic organisms, the genetic diversity level of each biont should also be highly influenced by biont transmission. In this study, we evaluated the influence of the reproductive strategies of cyanolichen species on the current levels of population genetic diversity of bionts. To eliminate any phylogenetic noise, we selected two closely related species within the genus Degelia, which only differ in their reproductive systems. We sampled all known populations of both species in central Spain and genotyped the fungal and cyanobacterial components of lichen samples using DNA sequences as molecular markers. We applied population genetics approaches to evaluate the genetic diversity and population genetic structure of the symbiotic components of both lichen species. Our results indicate that fungal and cyanobiont genetic diversity is highly influenced by the reproductive systems of lichen fungus. We detected higher bionts genetic diversity values in the sexual species Degelia plumbea. By contrast, the levels of fungal and cyanobiont genetic diversity in the asexual species Degelia atlantica were extremely low (almost clonal), and the species shows a high specificity towards its cyanobiont. Our results indicate that reproduction by vegetative propagules, in species of the genus Degelia, favors vertical transmission and clonality, which affects the species’ capacity for resources and competition, thereby limiting the species to restricted niches.     

Pigment distribution and nitrogen fixation inAnabaena azollae

Summary The symbiotic heterocystous cyanobacteriumAnabaena azollae present in the leaf cavities of the water fernAzolla spp. was studied. The cyanobacteria extracted from the leaf cavities showed differences in pigment composition in three species ofAzolla, i.e A.pinnata var.pinnata, A.caroliniana and A.filiculoides, as observed by pigment absorption and epifluorescence tests. These differences suggest that of these species the cyanobiont ofA. pinnata is the most actively nitrogenfixing form. This has been confirmed by nitrogen fixation (acetylene reduction) tests. Heterocysts of the symbiont ofA. pinnata were characterized by high chlorophylla and low phycocyanin content, a low fluorescence yield of chlorophyll in the heterocysts compared to vegetative cells and a gradient of phycocyanin concentration in the vegetative cells adjacent to heterocysts. This indicates that only photosystem I is present in the heterocyst. In the two otherAzolla species quantitative shifts in the pigment composition occurred suggesting a lower nitrogen fixation activity.In the cyanobiontAnabaena azollae the heterocyst frequency could reach a value of 44–45%. It is argued that there are two generations of heterocysts in a matureAzolla plant, which are concomitant with two peaks of nitrogen fixation activity correlated with leaf age,i.e. leaf number along the main axis of the plant. At both peaks of maximal N₂-ase activity, only 20–25% of the heterocysts present are metabolically active as demonstrated by the reduction of Neotetrazolium chloride (NTC) in the heterocysts and darkening of nuclear emulsions by silver salt reduction. Vegetative cells of the cyanobiont reduce Neotetrazolium chloride (NTC) to formazan more rapidly than has been observed in the free-living heterocystous cyanobacteriumAnabaena cylindrica tested in parallel experiments. This feature may be due to a more permeable cell wall of the vegetative cells of the cyanobiont compared to the free-living form, since the vegetative cells of the symbiont play a role in cross-feeding of the host (Azolla).Evidence is obtained that only the heterocysts of the cyanobiont ofAzolla are involved in the nitrogen fixation process as in free-living heterocystous cyanobacterium species. This situation is different from other cyanobacterial symbioses such as inGunnera, Blasia andAnthoceros, where physiological modifications are reported in the symbiosis with another photosynthetic partner such as the absence of O₂ evolution and the absence of photo-fixation of CO₂ in the cyanobionts.Pigment composition and N₂-ase activity in the symbiotic cyanobacteria of three Azolla species have indicated the superiority of theA. pinnata symbiont.A. pinnata var.pinnata is a semidomesticated form used in S.E. Asia for agricultural purposes (irrigated rice culture) to increase soil fertility.It is suggested that by selection (domestication) more efficient strains (clones) can be obtained, and further that with more advanced techniques such as gene mutation and genetic manipulation even more efficient and for agriculture more beneficial clones can be obtained.     

Pseudocyphellaria crocata, P. neglecta and P. perpetua from the Northern and Southern Hemispheres are a phylogenetic species and share cyanobionts

Pseudocyphellaria crocata, P. neglecta and P. perpetua specimens were examined to investigate links between genetic variation and morphology, geographical distribution and cyanobiont specificity. Fungal internal transcribed spacer (ITS), beta-tubulin and cyanobacterial tRNA(Leu) (UAA) intron sequences were used to investigate symbiont diversity in these lichens. Specimens were morphologically distinct but could not be distinguished by ITS sequences. Phylogenetic analyses split the P. crocata specimens into two clades, the larger of which contained P. neglecta and P. perpetua. Five cyanobionts were identified; two of these were in a number of specimens, while three were each restricted to a single lichen thallus. Fungus-specific molecular markers indicated that all specimens belonged to a single phylogenetic species. However, this may contain a cryptic species. Geography was linked to genetic diversity with Canadian specimens forming a monophyletic group, and most Southern Hemisphere specimens grouping together, although Chile represented a hot spot of genetic diversity. There was no connection between fungal genetic diversity and cyanobiont choice, consistent with the presence of a common pool of cyanobionts.     

Brasilonema lichenoides sp. nov. and Chroococcidiopsis lichenoides sp. nov. (Cyanobacteria): two novel cyanobacterial constituents isolated from a tripartite lichen of headstones

Cyanolichens are an assemblage of fungi and cyanobacteria from diverse, cosmopolitan habitats. Typically composed of a single species of cyanobacterium, with or without another eukaryotic alga, here we present two novel cyanobionts isolated from an undescribed tripartite lichen. This endolithic lichen was isolated from a granite cemetery tombstone from Jacksonville, FL, and contains two potentially nitrogen‐fixing cyanobionts. Employing a total evidence approach, we characterized the cyanobionts using molecular (the 16S rDNA and ITS gene region), morphological, and ecological data. Phylogenetic analyses revealed two novel taxa: Brasilonema lichenoides and Chroococcidiopsis lichenoides, both of which fell within well‐supported clades. To our knowledge, this represents the first instance of a tripartite lichen with two cyanobacterial and no eukaryotic members. These types of lichens may well represent an unexplored reservoir of cyanobacterial diversity. The specific epithets are proposed under the provisions of the International Code of Nomenclature for algae, fungi, and plants.     

Biology and host specificity of the genusPsoroptes Gervais (Acarina: Psoroptidae), with reference to its occurrence in Australia

Psoroptes mites are obligate ectoparasites of mammals that cause psoroptic mange in a number of domestic and wildlife species world-wide. Correct identification of species in this genus is important for eradication and quarantine programs. However, conflicting reports about species identity and host specificity within this morphologically conservative genus have raised questions regarding the usefulness of the taxonomic characters previously used to distinguish species. Records that rely only on host species do not provide accurate identification ofPsoroptes mites at the species level. This paper reviews the information available on life history, geographic distribution, and host specificity for this genus, with special reference to problems associated with identifying species in Australia.     

Azolla fern lectins that specifically recognize endosymbiotic cyanobacteria

Lectins were extracted from whole fern grindings ofAzolla pinnata (AP) andAzolla filiculoides (AF) by precipitation with ammonium sulfate to 20% of saturation. At high pH both lectins dissociate into inactive subunits (5000 mol wt) which reassociate into active aggregates (>500,000 mol wt) following concentration by ammonium sulfate precipitation or freezing and thawing. Although amino sugars inhibited hemagglutinating activity of both AP and AF lectins,d-fructose was inhibitory only to the AP lectin hemagglutinating activity, andd-galactose was slightly inhibitory to the AP lectin but not to the AF lectin. Both lectins exhibited specificity for freshly extracted cyanobionts from homologous fern species: AP lectin agglutinated cyanobiont filaments from AP, but not from AF; AF lectin agglutinated cyanobiont filaments from AF, but not AP. Neither lectin reacted with cultured cyanobionts from either fern species. Hemagglutinating titers were likewise reduced by adsorption of these lectins to homologous cyanobiont cells. This report provides strong suggestive evidence for specificity in this N-fixing symbiosis between aquatic fern and cyanobacterium.     

Taxonomic status ofAnabaena azollae: An overview

Despite the long-standing and widespread use of the symbiotic association between the aquatic fern Azolla and its cyanobacterial symbiontAnabaena azollae to augment nitrogen supplies in rice paddy soils, very little is known about taxonomic aspects of the symbiosis. The two partners normally remain associated throughout vegetative and reproductive development, limiting the opportunities for interchanges. We have used monoclonal antibodies and DNA/DNA hybridization techniques to show that the cyanobacterial partner is not uniform throughout the genus Azolla, and that substantial diversification has occurred. With these procedures it will be possible to characterize genotypes of the cyanobacterium and to monitor experiments aimed at synthesizing new combinations ofAzolla species andAnabaena azollae strains.     

Fingerprinting of freshly separated and cultured cyanobionts from different Azolla species using morphological and molecular markers

Differentiation of freshly separated (uncultured) and cultured cyanobionts of Azolla species was carried out employing morphological markers and profiles generated using SDS-PAGE and PCR–RFLP of 16S rRNA. The cell dimensions of vegetative cells, heterocysts and heterocyst frequency (25–28%) of the freshly separated cyanobionts were distinctly higher than that those recorded for the cultured cyanobionts (7–10%). The SDS-PAGE profiles of whole cell proteins of cultured cyanobionts (comprising 28–30 bands) and those of freshly separated cyanobionts (consisting of 6–10 bands) exhibited distinct differences and unique bands. AluI was able to discriminate freshly separated cyanobionts from cultured cyanobionts of same species of Azolla. The profiles of cyanobionts (freshly separated and cultured) of A. rubra (RU6503) generated using the restriction enzyme AluI were distinctly different from other cyanobionts and unique bands were observed in both cyanobionts. The cultured cyanobionts from A. microphylla (MI4018), A. filiculoides (FI1001) and A. pinnata (PP7001) showed the presence of a distinct band of 450, 622 and 307 bp, respectively. Three common bands of 500, 400 and 275 bp were recorded in the AluI restriction profiles of all the freshly separated cyanobionts. 16S rRNA PCR–RFLP analyses confirmed the existence of primary and secondary cyanobionts in the leaf cavities of different Azolla species. These techniques can be utilized for discriminating between freshly separated and cultured cyanobionts of Azolla and provide reliable fingerprints for these strains.     

Are classification and phytopathological diversity compatible in Xanthomonas?

The genus Xanthomonas is characterized by its phytopathogenic diversity and the host specificity of its members. In the past, the classification of the members of this genus has been based primarily on the criterion of host specificity. This has led to a classification system which focused only on naming phytopathogenic variants on different hosts. Extensive taxonomic examination of Xanthomonas has shown that the phytopathogenic specialization of the bacteria is not correlated with the actual relationships within the genus. Based upon total genomic DNA homology, the genus has been reclassified into 20 species. At present, non-pathogenic xanthomonads are frequently isolated from plant material. As these strains often cannot be classified to existing species, it becomes clear that the diversity of the genus is much greater than expected from the phytopathogenic subpopulation, which has been the primary subject in the past. The example of Xanthomonas also illustrates that attempts to divide bacterial populations into discrete taxa conflict with the actual continuous nature of biodiversity. Received 16 April 1996/ Accepted in revised form 27 September 1996     

The luggage hypothesis: Comparisons of two phototrophic hosts with nitrogen-fixing cyanobacteria and implications for analogous life strategies for kleptoplastids/secondary symbiosis in dinoflagellates

Nostoc and Richelia belong to a group of heterocystous cyanobacteria and are unique within this group in forming intracellular symbioses with phototrophic hosts, the angiosperm Gunnera and the diatoms (algae) Rhizosolenia and Hemiaulus, respectively. The function of the cyanobiont is similar in the symbioses, namely providing fixed atmospheric nitrogen to their hosts; also the cyanobionts are contained in a host compartment, the symbiosome. The evolutionary timescale for the cyanobiont-endosymbiosis formation is in both instances about ≈90 Ma. However, the potentials for further co-evolution of host and microsymbiont, are different. Nostoc is regarded as preyed upon by its host, while in the Richelia-Rhizosolenia symbiosis example the evolution towards a new type of permanent organelle is possible. It is proposed that symbiosis is ruled by divergent host strategies. In the case of Richelia-Rhizosolenia the evolution of a permanent symbiosis is linked to diatom hosts needing to carry the cyanobiont permanently, as it is not available free-living in the oceans. However, in the case of Nostoc/Gunnera, the host exploits an abundant cyanobacterial species. A model where the relative abundance of microsymbionts determines the nature of the symbiosis comes into view: If environmental ratios of host/microsymbiont are so that hosts are the dominating party, then the host has to carry the microsymbiont as luggage (vertical transmission). Likewise, if the ratio of microsymbiont is higher than host, than the host will prey on the microsymbiont (horizontal transmission). The article also discusses the retention of secondary plastids in dinoflagellates. We show that dinoflagellates are organisms that exemplify both types of strategies that is either preying or harbouring a permanent organelle. The difference from the cyanobacterial example is that only parts of the eukaryotic microsymbionts are kept, usually only the plastid. We emphasize that the dinoflagellates can obtain their plastids from various different organisms. The luggage theory offers an explanation to why some dinoflagellate species contain kleptoplastids, while others have permanent, secondary plastids and some have tertiary plastids.     

Sub-cellular Localization of Calcium in Azolla-Anabaena Symbiosis by Chlortetracycline,ESI and EELS

The subcellular localization of calcium in cells of symbiotic partners located within leaf cavities of Azolla was investigated by using chlorotetracycline, ESI and EELS analysis. Loosely membrane-bound calcium was evidenced by using CTC or EGTA and CTC, in cytoplasmic regions of Azolla hair cells and in cytoplasm of the cyanobiont. Tightly membrane-bound calcium revealed by CTC, and ESI and EELS analysis, was observed in cyanophycin granules and carboxysomes of the cyanobiont. A third calcium type, revealed by ESI and EELS analysis, was localized at the level of cell walls of simple and branched Azolla hairs, in the envelope of heterocysts, and in the cell walls of the cyanobiont.     

Patterns of Symbiodinium (Dinophyceae) diversity and assemblages among diverse hosts and the coral reef environment of Lizard Island,Australia

Large‐scale environmental disturbances may impact both partners in coral host–Symbiodinium systems. Elucidation of the assembly patterns in such complex and interdependent communities may enable better prediction of environmental impacts across coral reef ecosystems. In this study, we investigated how the community composition and diversity of dinoflagellate symbionts in the genus Symbiodinium were distributed among 12 host species from six taxonomic orders (Actinaria, Alcyonacea, Miliolida, Porifera, Rhizostoma, Scleractinia) and in the reef water and sediments at Lizard Island, Great Barrier Reef before the 3rd Global Coral Bleaching Event. 454 pyrosequencing of the ITS2 region of Symbiodinium yielded 83 operational taxonomic units (OTUs) at a 97% similarity cut‐off. Approximately half of the Symbiodinium OTUs from reef water or sediments were also present in symbio. OTUs belonged to six clades (A‐D, F‐G), but community structure was uneven. The two most abundant OTUs (100% matches to types C1 and A3) comprised 91% of reads and OTU C1 was shared by all species. However, sequence‐based analysis of these dominant OTUs revealed host species specificity, suggesting that genetic similarity cut‐offs of Symbiodinium ITS2 data sets need careful evaluation. Of the less abundant OTUs, roughly half occurred at only one site or in one species and the background Symbiodinium communities were distinct between individual samples. We conclude that sampling multiple host taxa with differing life history traits will be critical to fully understand the symbiont diversity of a given system and to predict coral ecosystem responses to environmental change and disturbance considering the differential stress response of the taxa within.     

Diverse avian malaria and other haemosporidian parasites in Andean house wrens: evidence for regional co‐diversification by host‐switching

Recent research has revealed well over 1000 mtDNA lineages of avian haemosporidian parasites, but the extent to which this diversity is caused by host–parasite coevolutionary history or environmental heterogeneity is unclear. We surveyed haemosporidian and host mtDNA in a geographically structured, ecological generalist species, the house wren Troglodytes aedon, across the complex landscape of the Peruvian Andes. We detected deep genetic structure within the house wren across its range, represented by seven clades that were between 3.4–5.7% divergent. From muscle and liver tissue of 140 sampled house wrens we found 23 divergent evolutionary lineages of haemosporidian mtDNA, of which ten were novel and apparently specific to the house wren based on searches of haemosporidian databases. Combined and genus‐specific haemosporidian abundance differed significantly across environments and elevation, with Leucocytozoon parasites strongly associated with montane habitats. We observed spatial stratification of haemosporidians along the west slope of the Andes where five lineages were restricted to non‐overlapping elevational bands. Individual haemosporidian lineages varied widely with respect to host specificity, prevalence, and geographic distribution, with the most host‐generalist lineages also being the most prevalent and widely distributed. Despite the deep divergences within the house wren, we found no evidence for host‐specific co‐diversification with haemosporidians. Instead, host‐specific haemosporidian lineages in the genus Haemoproteus were polyphyletic with respect to the New World parasite fauna and appeared to have diversified by periodic host‐switches involving distantly related avian species within the same region. These host‐specific lineages appeared to have diversified contemporaneously with Andean house wrens. Taken together, these findings suggest a model of diffuse co‐diversification in which host and parasite clades have diversified over the same time period and in the same geographic area, but with parasites having limited or ephemeral host specificity.     

Geographic Distribution and Genetic Diversity of Ceanothus-Infective Frankia Strains

Little is known about Ceanothus-infective Frankia strains because no Frankia strains that can reinfect the host plants have been isolated from Ceonothus spp. Therefore, we studied the diversity of the Ceonothus-infective Frankia strains by using molecular techniques. Frankia strains inhabiting root nodules of nine Ceanothus species were characterized. The Ceanothus species used represent the taxonomic diversity and geographic range of the genus; therefore, the breadth of the diversity of Frankia strains that infect Ceanothus spp. was studied. DNA was amplified directly from nodular material by using the PCR. The amplified region included the 3′ end of the 16S rRNA gene, the intergenic spacer, and a large portion of the 23S rRNA gene. A series of restriction enzyme digestions of the PCR product allowed us to identify PCR-restriction fragment length polymorphism (RFLP) groups among the Ceanothus-infective Frankia strains tested. Twelve different enzymes were used, which resulted in four different PCR-RFLP groups. The groups did not follow the taxonomic lines of the Ceanothus host species. Instead, the Frankia strains present were related to the sample collection locales.     

Genetic diversity among and within cultured cyanobionts of diverse species of <Emphasis Type="Italic">Azolla</Emphasis>

The cyanobionts isolated from 10 Azolla accessions belonging to 6 species (Azolla mexicana, A. microphylla, A. rubra, A. caroliniana, A. filiculoides, A. pinnata) were cultured under laboratory conditions and analyzed on the basis of whole cell protein profiles and molecular marker dataset generated using repeat sequence primers (STRR(mod) and HipTG). The biochemical and molecular marker profiles of the cyanobionts were compared with those of the free-living cyanobacteria and symbiotic Nostoc strains from Anthoceros sp., Cycas sp. and Gunnera monoika. Cluster analysis revealed the genetic diversity among the selected strains, and identified 3 distinct clusters. Group 1 included cyanobionts from all the 10 accessions of Azolla, group 2 comprised all the symbiotic Nostoc strains, while group 3 included the free-living cyanobacteria belonging to the genera Nostoc and Anabaena. The interrelationships among the Azolla cyanobionts were further revealed by principal component analysis. Cyanobionts from A. caroliniana-A. microphylla grouped together while cyanobionts associated with A. mexicana-A. filiculoides along with A. pinnata formed another group. A. rubra cyanobionts had intermediate relationship with both the subgroups. This is the first study analyzing the diversity existing among the cultured cyanobionts of diverse Azolla species through the use of biochemical and molecular profiles and also the genetic distinctness of these free-living cyanobionts as compared to cyanobacterial strains of the genera Anabaena and Nostoc.     

Genetic diversity of symbiotic cyanobacteria in Cycas revoluta (Cycadaceae)

The diversity of cyanobacterial species within the coralloid roots of an individual and populations of Cycas revoluta was investigated based on 16S rRNA gene sequences. Sixty-six coralloid roots were collected from nine natural populations of cycads on Kyushu and the Ryukyu Islands, covering the entire distribution range of the species. Approximately 400?bp of the 5'-end of 16S rRNA genes was amplified, and each was identified by denaturing gradient gel electrophoresis. Most coralloid roots harbored only one cyanobiont, Nostoc, whereas some contained two or three, representing cyanobiont diversity within a single coralloid root isolated from a natural habitat. Genotypes of Nostoc within a natural population were occasionally highly diverged and lacked DNA sequence similarity, implying genetic divergence of Nostoc. On the other hand, Nostoc genotypes showed no phylogeographic structure across the distribution range, while host cycads exhibited distinct north-south differentiation. Cycads may exist in symbiosis with either single or multiple Nostoc strains in natural soil habitats.