Effects of inorganic nitrogen on taxa-specific cyanobacterial growth and nifH expression in a subtropical estuary

The potentially toxic, diazotrophic filamentous cyanobacterium Cylindrospermopsis raciborskii has recently become a common component in the summer phytoplankton in the St. Johns River (SJR) estuary, Florida, where Anabaena spp. historically dominated. Using a microcosm nutrient enrichment experiment, we investigated the ability of C. raciborskii and Anabaena spp. to compete under a range of available NO 3 and NH z 4 concentrations, to test the hypothesis that C. raciborskii benefits from increased dissolved inorganic nitrogen (DIN) availability. TaqMan quantitative polymerase chain reaction (PCR) probes were designed, tested, and applied to target the nifH gene in one C. raciborskii and two Anabaena spp. strains from the SJR. N limitation prevailed, as shown by increased N2-fixation rates if no N was added, increased chlorophyll a concentrations when DIN was added, and depletion of added DIN. Anabaena spp. and C. raciborskii showed rapid growth with no DIN additions and were the main taxa responsible for N2 fixation. Abundances of C. raciborskii increased if NH z4 was added, but nifH was expressed at low levels, suggesting growth was relying on NH z 4 . Anabaena spp. and C. raciborskii expressed nifH genes when NO 3 or NH z 4 were present, but expression was higher with NO { 3 . The narB gene sequence was amplified from Anabaena spp. and C. raciborskii from the SJR, suggesting these taxa are capable of assimilating NO 3 . However, even small NO { 3 additions blocked the growth of Anabaena spp. in the mixed phytoplankton community but not that of C. raciborskii. The results suggest that C. raciborskii in the SJR is a stronger competitor than Anabaena spp. when DIN is present. The ability to compete and use different nitrogen (N) sources for growth is an important factor in determining the competitive success of individual taxonomic groups in mixed phytoplankton assemblages. Differences in nutrient uptake affinities or storage capabilities can lead to competitive exclusion in controlled laboratory conditions (Tilman 1982), and these models can be applied in nature (Sommer 1993). However, in complex natural phytoplankton assemblages, species composition is difficult to predict and model based on laboratory data, since many different taxa coexist and numerous environmental conditions simultaneously affect growth rates. Based on laboratory experiments and field observations, under N-replete conditions competition between diazotrophic (N2-fixing) and nondiazotrophic cyanobacteria is generally thought to lead to competitive exclusion by nondiazotrophs since they tend to have higher affinities for dissolved inorganic nitrogen (DIN) (Flores and Herrero 1994) and faster growth rates than diazotrophs. However, some cyanobacterial diazotrophs may be adapted for growth on NH z4 or NO { 3 and can effectively compete with nondiazotrophs during time periods when DIN is available (Agawin et al. 2007). In the eutrophic, subtropical St. Johns River (SJR) estuary in Florida, phytoplankton growth tends to be N limited in the summer, manifested as high abundances of diazotrophic cyanobacteria, but shortterm and localized inputs of DIN occur (Phlips et al. 2007). Owing to the pulsed nature of the nutrient inputs, the system may locally shift between N-limited and non–Nlimited conditions several times during the cyanobacterial growth season. Cyanobacteria that have the ability to fix atmospheric N2 and effectively compete for combined N sources may have a competitive advantage under these conditions. Although it is known that diazotrophic cyanobacteria can use NH z4 and the ability to use NO { 3 is widespread (Flores et al. 2005), little is known about the roles these functions play in an ecological context. In general, NH z4 inhibits the function of nitrogenase, the enzyme mediating N2 fixation, and the magnitude of this inhibition appears taxa specific (Stewart 1973). In the SJR, Anabaena spp., and Aphanizomenon flosaquae have historically dominated diazotroph communities, while Microcystis spp. and Aphanocapsa spp. are Acknowledgments S. Gordon, A. E. Morrison, J. Braddy, A. Joyner, J. Joyner, L. Kelly, K. Rossignol, M. Leonard, and L. A. Cheshire are acknowledged for technical assistance. B. Jenkins, R. Foster, and M. Piehler are acknowledged for discussions. This work was supported by the National Science Foundation (grant DEB0452324) to P.M. and H.P., and the Gordon and Betty Moore Foundation (J.Z). Limnol. Oceanogr., 53(6), 2008, 2519–2532 E 2008, by the American Society of Limnology and Oceanography, Inc.