Cryptic species in cyanobacterial systematics: a case study of Phormidium retzii (Oscillatoriales) using RAPD molecular markers and 16S rDNA sequence data

Abstract

The genetic variability of the cosmopolitan, ubiquitous freshwater cyanobacterium Phormidium retzii was assessed using random amplified polymorphic DNA (RAPD) markers and nearly complete (ca. 95%) 16S rDNA sequences. Strains consistent with the morphological species circumscription were utilized from geographically distant locations ranging from British Columbia, Canada, in the north to Rio Claro, Costa Rica in the south, and from Rhode Island, USA in the east coast to Washington State, USA on the west coast. In addition, some strains were from geographically close (<35 km apart) locales. To assess biogeographic trends, the nine RAPD primers utilized yielded 133 distinct bands in total from the 12 strains. Non-metric multidimensional scaling (NMDS) analyses revealed that the strains isolated from Ohio, Connecticut, and Rhode Island were closely related with the Costa Rica strain and one Washington State strain associated with this group. The two strains from Canada, British Columbia and Ontario, were closely related and clustered with the two other strains from Washington State. The remaining two strains from Mexico and Missouri were loosely associated with this cluster and genetically distinct. Geographic proximity did not correlate with genetic similarity (Mantel test, r=0.235,P>0.05). A 1340 bp region of the 16S rRNA gene was sequenced for 8 of the 12 strains used in the RAPD analysis. Sequence homology among strains ranged from 88.4 to 98.4%, implying the presence of cryptic species within this group of strains. Given the lack of sequence similarity, P. retzii as presently circumscribed most likely represents several cryptic species not clearly distinguishable with light microscopy of morphological features. This conclusion may explain the lack of correlation between geographic proximity and genetic similarity. This research provides further strength for the case of species of cyanobacteria being more localized rather than having global distributions.

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.