Blackwell Science, LtdOxford, UKEMIEnvironmental Microbiology 1462-2912Society for Applied Microbiology and Blackwell Publishing Ltd, 200474519529Original ArticleCyanobacterial diversity in variable salinity pondsA.-D. Jungblut et al. Environmental Microbiology (2005) 7(4), 519–529 doi:10.1111/j.1462-2920.2004.00717.x Diversity within cyanobacterial mat communities in variable salinity meltwater ponds of McMurdo Ice Shelf, Antarctica Anne-Dorothee Jungblut,1 Ian Hawes,2 first oxygenically active organisms with fossil findings con- Doug Mountfort,3 Bettina Hitzfeld,4 Daniel R. Dietrich,5 firming the antiquity (Castenholz and Waterbury, 1989). Brendan P. Burns1 and Brett A. Neilan1* Cyanobacteria are of key interest for a better understand- 1University of New South Wales, Australia. ing of the evolution of their present diversity and mecha- 2National Institute of Water and Atmospheric Research, nisms of adaptation, which are essential for their survival New Zealand. in cold environments. 3Cawthron Institute, New Zealand. The McMurdo Ice Shelf comprises an area of about 4Swiss Agency for the Environment, Forests and 1500 km2 with a thickness from 10 to 50 m (Swithnbank, Landscape, Switzerland. 1970; Howard-Williams et al., 1990). During the summer, 5University of Konstanz, Germany. the area is covered with a network of meltwater ponds that vary in size, shape and physicochemical conditions, even though some of them are only several meters apart (De Summary Mora et al., 1991). Three of these ponds, Fresh, Orange This study investigated the diversity of cyanobacterial and Salt Ponds, have been intensively studied with regard mat communities of three meltwater ponds – Fresh, to their physicochemical parameters as well as the mor- Orange and Salt Ponds, south of Bratina Island, phological diversity of cyanobacteria, protozoa, rotifers, McMurdo Ice Shelf, Antarctica. A combined morpho- tardigrades and nematodes present (Howard-Williams logical and genetic approach using clone libraries was et al., 1990). High salinities in these ponds originated used to investigate the influence of salinity on cyano- from diluted seawater or chemical weathering of sedi- bacterial diversity within these ecosystems without mentary material such as the dissolution of mirabilite prior cultivation or isolation of cyanobacteria. We were (Na2NO3SO4H2) and thernadite (Na2SO4), in combination able to identify 22 phylotypes belonging to Phormid- with small amounts of gypsum (CaSO4H2O) (Keys and ium sp., Oscillatoria sp. and Lyngbya sp. In addition, Williams, 1981; De Mora et al., 1994). we identified Antarctic Nostoc sp., Nodularia sp. and In these hypersaline environments, cyanobacteria need Anabaena sp. from the clone libraries. Fresh (17 phy- to maintain their intracellular osmotic equilibrium and cell lotypes) and Orange (nine phylotypes) Ponds showed turgor to ensure survival through mechanisms such as the a similar diversity in contrast to that of the hypersaline production of organic osmolytes (Oren, 2000). High UV Salt Pond (five phylotypes), where the diversity within radiation is another major stress factor for cyanobacteria cyanobacterial mats was reduced. Using the compar- in Antarctic aquatic ecosystems on McMurdo Ice Shelf ison of identified phylotypes with existing Antarctic (Ross and Vincent, 1998), which can lead to the photoin- sequence data, it was possible to gain further insight hibition of photosynthesis, mutagenesis, or chlorophyll- into the different levels of distribution of phylotypes bleaching (Castenholz, 1992; Ehling-Schulz and Scherer, identified in the investigated cyanobacterial mat com- 1999). munities of McMurdo Ice Shelf. Cyanobacteria are often found in the form of thick microbial benthic mats, having a characteristic vertical stratification of different functional microbial groups Introduction (Howard-Williams et al., 1989). Cyanobacteria are most Cyanobacteria are the dominant primary producers in dominant in the top layer of these mats (Stal et al., 1985) extreme permafrost environments of the aquatic systems and motile species such as Oscillatoriales, as well as the on McMurdo Ice Shelf, Antarctica. They are also one of nitrogen-fixing Nostocales, are very prominent in these the oldest groups of organisms and are thought to be the communities (Howard-Williams et al., 1990). Previous attempts to classify species in these ponds Received 1 March, 2004; revised 6 July, 2004; accepted 8 July, 2004. *For correspondence. E-mail [email protected]; Tel. (+61) have relied on traditional microscopic studies (Howard- 2 9385 3235; Fax (+61) 2 9385 1591. Williams et al., 1990; Broady and Weinstein, 1998) and © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd 520 A.-D. Jungblut et al. Table 1. Physicochemical conditions of the meltwater ponds – Fresh, Orange and Salt Ponds, McMurdo Ice Shelf, Antarctica. a 3 b 3 c 3 d 3 e 3 Site Conductivity (mS/cm) DRP (mg/m )NH4-N (mg/m )NO3-N (mg/m ) DOP (mg/m ) DON (mg/m ) Fresh Pond 158 5.5 18 2 34 1 131.5 Orange Pond 937 39 18.5 2.5 99 2 080.5 Salt Pond 52 900 29 50.5 4 565.5 13 356.5 a. Dissolved reactive phosphorous. b. Ammonium. c. Nitrate. d. Dissolved organic phosphorous. e. Dissolved organic nitrogen. culturing of morphotypes (Nadeau et al., 2001). Because tation and distribution, using a polyphasic characteriza- of the variable morphological appearance of cyanobacte- tion. Finally, this study will also address the question of ria depending on the prevailing environmental conditions, the role of the McMurdo meltwater ponds as biotic pools this traditional approach of characterizing cyanobacteria for the aquatic systems in the ice-free regions of Victoria is very difficult. Additionally, culturing only provides limited Land during interglacial periods (Howard-Williams et al., information on the composition of communities because 1990; Vincent, 2000a). of selectivity. In the present study, benthic cyanobacterial mat communities of three meltwater ponds on McMurdo Results Ice Shelf were investigated for the first time via a polypha- Conductivity sic morphological and genotypic approach. These melt- water ponds are Fresh, Orange and Salt Ponds (unofficial The investigated McMurdo meltwater ponds Fresh, names), and they are situated south of Bratina Island on Orange and Salt Ponds had very different conductivities McMurdo Ice Shelf. The ponds were chosen because of (Table 1). The highest conductivity was measured in Salt their significant differences in salinities and physicochem- Pond with 52 900 mS/cm. In Orange Pond the conductivity ical parameters described above. was 937 mS/cm and the lowest conductivity was found in Cyanobacterial mats are not only a dominant feature of Fresh Pond with only 158 mS/cm. Antarctic ecosystems, but also in Arctic as well as other Morphological diversity extreme environments such as hot springs and desert crusts (Rudi et al., 1997; Ward et al., 1998; Vincent, The cyanobacterial mat communities in the three meltwa- 2000a; Wynn-Williams, 2000). Therefore, it is of interest ter ponds investigated were dominated by cyanobacteria to compare the diversity of these mats on McMurdo Ice belonging to the filamentous orders Oscillatoriales and Shelf in the context of speciation and physiological adap- Nostocales (Table 2). Nostoc sp. and Nodularia sp. were Table 2. Identified morphogroups and species of the cyanobacterial mat communities in the meltwater ponds – Fresh, Orange and Salt Ponds, McMurdo Ice Shelf, Antarctica. Morphogroups Order Genus (tichome-width) Species Fresh pond Orange pond Salt pond Oscillatoriales Oscillatoria Phormidium Lyngbya A: <3 mm + + + P. cf. deflexum + + + (Anagnostidis and Komarek) O. cf. fragile (Gom) + P. cf. Priestleyi (Fritsch) + Lyngbya cf. limnetica + B: 3–5 mm ++ + C: 5–9 mm + + + Osc cf. priestleyii + + (West and West) P. cf. autumnale + + (Anagnostidis and Komarek) D: 9–12 mm + Oscillatoria cf. limosa + (Anagnostidis and Komarek) Nostocales Nostoc ++ Nodularia + + + N. cf. harveyana (Thuret) + + + © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 519–529 Cyanobacterial diversity in variable salinity ponds 521 observed from the order Nostocales. The morphotypes restriction enzymes, AluI and ScrFI (data not shown). Two belonging to Oscillatoriales were identified as Phormid- clones were chosen from each novel restriction enzyme ium sp., Oscillatoria sp. and Lyngbya sp. Four morpho- pattern for sequencing to evaluate the levels of discrimi- groups were defined for morphotypes belonging to nation of the amplified restriction digest rDNA analysis Oscillatoriales but did not agree clearly with the oscillato- (ARDRA), and to determine the 16S rDNA sequence of rian identification keys. These morphogroups were distin- each restriction pattern type. guished on the basis of the width of the trichome such as Phylogenetic analysis was performed using reference trichome width <3 mm (morphogroup A), 3–5 mm (mor- sequence data from the orders Oscillatoriales and Nosto- phogroup B), 5–9 mm (morphogroup C) and 9–12 mm cales. Sequence data of the orders Pleurocapsales, (morphogroup D). Following this approach, Orange Pond Chroococcales and Prochloron were excluded, because exhibited the largest diversity of morphotypes. The the clones did not show a high similarity to these orders number of identified morphotypes in Fresh Pond was in the GenBank (NCBI) BLAST searches. Sequence data slightly lower and the lowest morphological diversity was from other Antarctic environments and various Arctic observed