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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.
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- Materials and methods
Many land plants can form symbiotic associations with nitrogen-fixing cyanobacteria. These cyanobacteria have symbiotic associations with fungi (Geosiphon and lichens), bryophytes (liverworts and hornworts), pteridophytes (Azolla), gymnosperms (Cycadaceae), and angiosperms (Gunnera) (Bergman et al., 1996; Rai et al., 2000).
Cycads are an ancient group of seed plants that have existed for approximately 300 million years. In general, cycads develop three types of roots: a tap root, which is equivalent to the primary root system found in most types of plants, lateral roots, and ‘coralloid roots’, which are highly specialized lateral roots containing cyanobionts (Costa & Lindblad, 2002). Mature coralloid roots are thick, finger-like structures usually embedded in soil at a shallow depth. Cyanobionts are restricted to the intercellular region of the cortex where they grow in a microaerobic and dark environment. Although not actively photosynthesizing, they maintain their clear green color in this environment (Costa & Lindblad, 2002). Nostoc spp. are the most common cyanobionts in Cycadaceae, whereas Anabaena and Calothrix species form symbiotic associations less frequently (Obukowicz et al., 1981; Zhu, 1982; Grobbelaar et al., 1986). All known cycad cyanobionts are of the botanical order Nostocales (Komarek & Anagnostidis, 1989). Several hypotheses of the mechanism of cyanobiont infection have been suggested, but the details are yet to be determined.
Costa et al. (1999) collected 11 coralloid roots from cycads cultivated in a botanical garden, including the genera Cycas, Encephalartos and Zamia. Only a single Nostoc strain was detected in each coralloid root, based on DNA sequence analyses of the tRNALeu (UAA) intron. They also determined Nostoc diversity within single or multiple coralloid root(s) of individual plants. Zheng et al. (2002) collected coralloid roots from indigenous cycad plants in a Chinese national park and found a large variety of cyanobacteria in a single coralloid root using polymerase chain reaction (PCR) fingerprinting. They also suggested that different strains of cyanobacteria could exist in the apical, middle and basal parts of a single coralloid root. However, Costa et al. (2004) suggested that the finding of multiple strains of cyanobacteria by Zheng et al. (2002) was likely due to a methodological limitation of fingerprinting: fingerprinting patterns may differ according to the DNA extraction method used, even with identical DNA samples. Costa et al. (2004) also pointed out that the results of Zheng et al. (2002) would be hard to corroborate, as no plant or bacterial controls were used. Thus, the number of cyanobiont strains that exist in a single coralloid root remains uncertain. Costa et al. (2004) also identified only a single strain of Nostoc within individual coralloid roots and in individual plants of indigenous cycads by sequencing of the tRNALeu (UAA) intron. They also found that plants growing in close proximity (female plants and their offspring) shared the same cyanobionts in their coralloid roots, suggesting a selective mechanism between the host plant and its cyanobionts. However, most previous studies used coralloid roots collected from botanical gardens and/or greenhouses. Zheng et al. (2002) used coralloid roots collected from cultivation in China and greenhouse specimens from Stockholm. The latter source exhibited lower cyanobiont species diversity. Gehringer et al. (2010) were the first to report the use of samples collected from their natural habitats. They collected coralloid roots from 31 Macrozamia species throughout their distribution range in Australia and sequenced the 16S rRNA genes. The data confirmed that coralloid roots harbored a single Nostoc strain, and the same strain was sometimes symbiotic with cycads in several locations. Host specialization of cyanobionts within the cycad genus Macrozamia was not observed in the wild.
In general, host specialization of the genus Nostoc is low (O'Brien et al., 2005). However, the water fern Azolla harbors unique cyanobionts: Papaefthimiou et al. (2008) analyzed the phylogeny of plural strains of symbiotic cyanobacteria and free-living cyanobacteria and found a robust cluster of Azolla symbionts, corroborating that the association of a unique cyanobacterial type with a host plant is strict and selective. They assumed that this may have been caused by coevolution and that Azolla symbiosis might have evolved in an aquatic environment.
Cycas revoluta Thunb. grows on rocky and sandy coasts of southern Japan, ranging from southern Kyushu to the most southern edge of the Ryukyu Islands (Fig. 1). Thus, distribution of C. revoluta is mostly isolated and scattered on islands and its range is wide, approximately 900 km in a northeast–southwest direction. This plant is a 1.5–5-m-tall dioecious tree that produces large seeds approximately 4 cm in length and 3 cm in width (Yamazaki, 1995). Because fertile seeds sink in seawater, seed dispersal of C. revoluta is thought to be limited, and dispersal over the sea is likely problematic. Phylogeography of this cycad is highly geographically structured: chloroplast and mitochondrial DNA haplotype analysis indicated two major areas, comprising northerly and southerly populations that were demarcated north and south of Okinoerabujima (Kyoda & Setoguchi, 2010). In addition, leaflet margin morphology also exhibited north–south differentiation (Setoguchi et al., 2009). The phylogeographic structure of C. revoluta is assumed to be determined by the formation and division of a landbridge between mainland Asia and Kyushu via the Ryukyu Islands during the Quaternary climatic oscillations (Kizaki & Oshiro, 1977, 1980; Ujiie, 1990; Kimura, 1996, 2000). However, no phylogeographic study of cyanobionts in Cycas coralloid roots has been conducted. Nostoc can be common in the environment, but the cycad cyanobionts might also exhibit a phylogeographic structure across the distribution range if they share the host plant's migration history. In contrast, multiple Nostoc strains can occur in the environment, and so cycad coralloid roots may use indigenous strain(s). In the latter case, no phylogeographic structure would be observed among cycad cyanobionts in the habitats of the Ryukyu Islands and Kyushu.
Figure 1. Coralloid root sampling locations. Locations of the nine sampling sites on the Ryukyu Islands and Kyushu. Population name abbreviations correspond to those in Table 1. (Inset) Location of the Ryukyu Islands and Kyushu.
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We aimed to investigate the genetic diversity of cyanobionts within single or multiple coralloid root(s) of individual plants, among the coralloid roots of individual plants and within populations of C. revoluta. All materials were collected from natural populations. In addition, we studied the phylogeography of the coralloid root cyanobionts in relation to their host cycads across their distribution range in the Ryukyu Islands and Kyushu. We amplified cyanobacterial 16S rRNA genes, and genotyping was conducted by a combination of denaturing gradient gel electrophoresis (DGGE) and sequencing.