Cryptic species in cyanobacterial systematics: a case study of Phormidium retzii (Oscillatoriales) using RAPD molecular markers and 16S rDNA sequence data
Introduction
Cyanobacteria are among the most widespread, morphologically distinct, and abundant prokaryotes known (Whitton, 1992). However, phylogenetic relationships among the cyanobacteria are relatively poorly understood. Traditionally, they have been classified using morphological and ecological characters (Geitler, 1932, Desikachary, 1959). Stanier et al. (1978) and Rippka et al. (1979) argued that the systematic treatment of cyanobacteria should not be based on traditional botanical methods, but rather on type cultures deposited in permanent culture collections as with other bacteria. However, morphological changes induced by culture conditions and environmental plasticity can be problematic for cyanobacterial taxonomy based solely on morphology (Stanier et al., 1971, Palinska et al., 1996).
In total, very few cyanobacteria have been analyzed using new molecular techniques such as DNA sequencing, random amplified polymorphic DNA (RAPD), and DNA polymorphisms (Mullins et al., 1995, Neilan, 1995, Neilan et al., 1997, Otsuka et al., 1999). For broad phylogenetic studies, sequence data from the 16S rRNA gene are most commonly employed due to its utility for distinguishing higher level taxonomic groups as well as traditional species (Neilan, 1995, Nelissen et al., 1995). Of the sequenced taxa, most have been those responsible for potable water problems (Neilan et al., 1997, Otsuka et al., 1999) or taxa primarily from marine environments (e.g., Moore et al., 1998, Urbach et al., 1998). In terms of molecular data, one particularly understudied section of cyanobacteria is the Oscillatoriales, which ostensibly falls within Section 3 of the cyanobacteria (filamentous members that never form heterocysts, Rippka, 1988). This grouping has traditionally included the well known genera Oscillatoria, Phormidium, and Lyngbya, which are primarily differentiated by sheath properties (Geitler, 1932). However, sheath production is only one of the typical diagnostic features subject to the direct effects of both environmental and culture conditions and as such may not be phylogenetically informative (Rippka et al., 1979, Whitton, 1992). This section has been shown to be polyphyletic, and some authors have suggested that any species designations at this point would be premature (e.g., Castenholz, 2001), although others would disagree (i.e., Anagnostidis and Komárek, 1988).
Phormidium retzii (C.A. Agardh) Gomont is a cosmopolitan cyanobacterium (Geitler, 1932). Sheath and Cole (1992) reported that it is the most widespread macroalgal (forming macroscopic mats) species in North America and collected in all biomes sampled. This cyanobacterium also has been recorded in Europe and Asia (e.g., Desikachary, 1959, Drouet, 1968). In North America alone, it has been recorded from a wide range of habitats, including epiphytic on mosses in Newfoundland, springs in Wisconsin, ponds in Minnesota, as well as collections from Florida, Alabama and Texas, warm mud in North Carolina and streams in south-central Alaska (Tilden, 1910, Whitford and Schumacher, 1973, Sheath et al., 1986). Further, P. retzii was reported as the most widespread taxon encountered from lotic ecosystems of São Paulo State, Brazil (Branco et al., 1999). This taxon was also found in a survey of cyanobacterial diversity of King George Island, Antarctica, which as the author points out, had a similar ecology with populations outside of Antarctica (Komárek, 1999). Thus, this species is truly a widespread, ecologically amorphous taxon. In addition, this taxon has an unusually broad species description encompassing samples with occasionally slightly constricted cell walls, straight cells 4.5–12 μm wide by 4–9 μm long with a thin sheath (Geitler, 1932). Due to the potentially great degree of environmentally induced morphological variability, molecular markers may be invaluable for resolving questions pertaining to the systematics and biogeography of this organism. Therefore, the present study was initiated investigating 12 strains of P. retzii using RAPD analysis and 16S rDNA sequence data to examine genetic variation across North America in this cosmopolitan cyanobacterium.
Section snippets
Field and tissue collection
A total of 12 P. retzii strains were collected from across North America (Fig. 1). All strains morphologically fit the species concept for P. retzii, even after recent systematic revision (sensu Anagnostidis and Komárek, 1988). Motile trichomes were 4.5–12 μm in diameter, more or less isodiametric and granular, with thin, diffluent sheaths and truncated apical cells. After collection, some strains (Table 1) were grown on 1.5% agar plates containing BG-11 medium (Stanier et al., 1971). Unialgal
Results
A total of 133 RAPD bands were generated using 9 primers. The total number of bands per strain ranged from 36 to 55 (mean=47.25,S.D.=±7.4). Seven of the nine primers generated bands in all strains, although OPAA-06 and OPAA-09 did not. The removal of the bands generated by these primers did not greatly alter the patterns of grouping (data not shown). Bands generated from all primers were utilized, as the Jaccard coefficient is suitable for data which may include double-negative values. Since
Discussion
The species circumscription of P. retzii is one of the most broadly delimited in the cyanobacteria. Geitler (1932) describes the species as: filaments±straight, mostly not or sometimes constricted at the cross-walls, end cells not capitate nor tapering, 4.5–12 μm wide with a thin sheath, cells shorter than wide, 4–9 μm long, non-granular cross-walls, in flowing or standing water on rocks and cosmopolitan. Given the wide range of morphology attributed to this taxon, Drouet (1968) combined this
Conclusion
This analysis of P. retzii strains from across North America was initially undertaken to be an assessment of the biogeography of this taxon. Microscopic examination of the strains indicated that they all correspond to the species description sensu stricto Geitler (1932). However, 16S rDNA sequencing to confirm taxonomic assignment revealed 16S rDNA similarity scores often <97%, which has typically been used to infer different species. At this time, it is premature to assign new species to this
Acknowledgments
The authors extend their thanks to Melissa Hall, Mary Koske, Kirsten Müller, G. Lemon, Tara Rintoul and Alison Sherwood for assistance in collection of sample materials. We thank Nanda Filkin for translation of German texts. Jeff Johansen provided valuable discussion on species concepts. In addition, this work was partially supported by a Sigma-Xi grant in aid of Research to DAC. Portions of this manuscript were written while DAC was supported by an Ohio University Graduate Fellowship.
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