Microbial mat communities along an oxygen gradient in a perennially ice-covered Antarctic lake ...

45 Pages·2015·1.11 MB·English

AEM Accepted Manuscript Posted Online 13 November 2015
Appl. Environ. Microbiol. doi:10.1128/AEM.02699-15
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1 Microbial mat communities along an oxygen gradient in a perennially ice-covered
2 Antarctic lake
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4 Anne D. Jungblut,a# Ian Hawes, b Tyler J. Mackey,c Megan Krusor, d Peter T. Doran, e Dawn
5 Y. Sumner, c Jonathan A. Eisen, d,e Colin Hillman,b Alexander K. Goroncyb
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7 Life Sciences Department, The Natural History Museum, London, United Kingdom a; lo
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8 University of Canterbury, Christchurch, New Zealand b; Department of Earth and Planetary e
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9 Sciences, University of California, Davis, California, USA c; Microbiology Graduate Group, ro
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10 University of California, Davis, California, USA d; Department of Geology and Geophysics, h
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11 Louisiana State University, Baton Rouge, Louisiana, USA e; Genome Center, University of ://
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12 California, Davis, Davis, California, USA f. m
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16 Running head: Oxygen-stratified mat communities in a polar lake a
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18 # Address correspondence to Anne D. Jungblut, [email protected] ,
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19 Abstract
20 Lake Fryxell is a perennially ice-covered lake in the McMurdo Dry Valleys, Antarctica, with
21 a sharp oxycline in a water column that is density-stabilized by a gradient in salt
22 concentration. Dissolved oxygen falls from 20 mg L-1 to undetectable over one vertical meter
23 from 8.9 to 9.9 m depth. We provide the first description of the benthic mat community that
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24 falls within this oxygen gradient on the sloping floor of the lake using a combination of w
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25 micro- and macroscopic morphological descriptions, pigment analysis, and 16S rRNA gene lo
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26 bacterial community analysis. Our work focused on three macroscopic mat morphologies that e
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27 were associated with different parts of the oxygen gradient: (I) “cuspate pinnacles” in the o
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28 upper hyperoxic zone, which displayed complex topography and were dominated by h
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29 phycoerythrin-rich cyanobacteria attributable to the genus Leptolyngbya and a diverse but ://
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30 sparse assemblage of pennate diatoms; (II) a less topographically complex “ridge-pit” mat m
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31 located immediately above the oxic-anoxic transition containing Leptolyngbya and an m
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32 increasing abundance of diatoms; and (III) flat prostrate mats in the upper anoxic zone, r
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33 dominated by a green cyanobacterium phylogenetically identified as Phormidium n

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34 pseudopriestleyi and a single diatom, Diadesmis contenta. Zonation of bacteria was by lake a
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35 depth and by depth into individual mats. Deeper mats had higher abundances of
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36 bacteriochlorophylls and anoxygenic phototrophs, including Chlorobi and Chloroflexi. This ,
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37 suggests that microbial communities form assemblages specific to niche-like locations. Mat 9

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38 morphologies, underpinned by cyanobacterial and diatom composition are the result of local y

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39 habitat conditions likely defined by irradiance and oxygen and sulphide concentrations. e
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40 Introduction
41 The McMurdo Dry Valleys (MDV) region of Southern Victoria Land, Antarctica, contains a
42 number of perennially ice-covered lakes, each home to complex and diverse microbial
43 communities (1). Unusual properties of these lakes that are sustained by the year-round ice
44 cover include the absence of wind-induced turbulence and persistent, salinity-dependent
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45 density gradients, which often encompass a significant proportion of the lake water columns w
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46 (2). These properties make the MDV lake systems amongst the most physically stable lo
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47 lacustrine habitats on Earth, and research over the past decades has shown that microbial e
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48 communities and processes are consistently embedded at specific positions in environmental o
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49 gradients within their stable water columns (e.g., 3). There is a strong connection between h
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50 dominant microbial physiologies and redox-related biogeochemistry through the water ://
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51 column (4, 5, 6, 7, 8, 9), and microbial distributions along stable redox gradients have m
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52 provided useful test cases for understanding links between environment and metabolism. To m
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53 date, such observations have largely been confined to planktonic communities (3, 6, 10), r
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54 despite the fact that in all MDV lakes studied, thick microbial mats cover the floors of the n

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55 lakes to the base of the photic zone. In lakes Hoare and Vanda, these benthic mats have been a
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56 estimated to contribute at least as much biomass and biological productivity, on a whole-lake
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57 basis, as plankton (11, 12, 13, 14, 15). ,
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58
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59 Previous work on microbial mat community composition in MDV lakes has focused almost u
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60 exclusively on the oxygenic communities, dominated by cyanobacteria, in the upper parts of
61 the lakes. Two distinct mats zones are recognized. Below the ice cover, where liquid water
62 persists year-round, mats are rich in light-harvesting pigments (12) and extracellular
63 polysaccharides, are laminated (11) and often take the form of complex, three-dimensional
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64 structures (13, 16, 17). During summer, solar heating melts the shallow margins of most
65 MDV lakes to some extent, creating shallow “moats” that seasonally alternate between ice
66 free and frozen. Here, mat morphological complexity is suppressed and communities are
67 acclimated to much higher irradiance (e.g. 11, 18). Approaches to community assemblage
68 analysis in both zones have been primarily based on microscopy, and have shown that mats
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69 provide habitats for cyanobacteria, microbial eukaryotes, microalgae, and heterotrophic w
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70 bacteria (19, 20). Though molecular methods applied to cyanobacterial diversity have lo
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71 confirmed an assemblage dominated by Oscillatoriales and Nostocales in mats from moats e
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72 (21), mats growing on the sloping lake floors from the underside of the ice to greater depths o
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73 were dominated by Oscillatoriales, with most variation in diversity being related to to h
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74 irradiance and conductivity (22). ://
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75 .a
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76 To date, while some effort has been made to understand changes within the cyanobacterial .o
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77 community at different lake depths in the MDV lakes, this has not extended into the anoxic or o
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78 micro-oxic parts of the water column (12, 23, 24). In traversing the oxycline, we expect a M
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79 change in microbial community, both functionally and compositionally, as described for h

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80 planktonic communities (25). Furthermore, it is not known how the composition and 8
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81 dominant metabolisms of bacteria other than cyanobacteria shift with lake depth or redox 1
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82 gradients, and how these changes might relate to observed shifts in macroscopic mat b
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83 morphology with lake depth (18). The goal of this study is to address this gap by describing u
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84 the benthic community across the oxycline in Lake Fryxell, a MDV lake.
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86 Lake Fryxell is among the better studied MDV lakes, particularly from the perspective of
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87 anoxic microbiology (7). It has a stable geochemical and salinity stratification (26), within
88 which dissolved oxygen falls from 20 mg L-1 close to the ice cover to undetectable at a
89 readily accessible 9-10 m depth (27). This transition coincides with increasing sulphide and a
90 shift from oxygen to sulphur-based water column metabolism (7). Lake Fryxell also has a
91 gently sloping lake floor colonized by microbial mats, and therefore vertically steep
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92 geochemical gradients translate to relatively long and readily sampled horizontal distances. w
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94 In the present study, our aim was therefore to describe the mat composition, including fr
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95 cyanobacteria, other bacteria, and Archaea, and the macroscopic mat morphology along a
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96 transect from oxic to anoxic conditions in Lake Fryxell using a combination of morphological :
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97 descriptions, pigment analysis, and next generation sequencing of 16S rRNA genes for e
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98 community analysis. As part of the description of the mat communities in Lake Fryxell, we s
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99 identified a highly abundant cyanobacterium that occupies a unique niche below the oxycline .o
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100 where it produces an oxygenated “oasis” (28), and we therefore undertook a comprehensive o
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101 phylogenetic analysis of this organism. M
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103 Materials and methods 2
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104 Study site 9

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105 Lake Fryxell (77° 36´S 162° 6´ E) (Fig. 1) is located near the eastern end of Taylor Valley. It
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106 is a meromictic lake where water layers do not intermix. It is 5 x 1.5 km in extent and es
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107 permanently covered by 4.5 meters of ice (7, 27). The maximum depth is ~20 m, including
108 the ice cover. Water is supplied to Lake Fryxell by 13 glacial melt-water streams, with most
109 water coming from the Canada and Commonwealth glaciers (30). Water balance is achieved
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110 by evaporation and ablation from the surface, and there are no out-flowing streams (31).
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112 Like most lakes of the MDVs, the level of Lake Fryxell has fluctuated over time, which is
113 reflected in its current physical structure. It has been suggested that 24,000 years ago, Lake
114 Fryxell formed part of a large water body (Lake Washburn). Prior to ca. 1,000 years ago,
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115 Lake Fryxell may have evaporated to a small playa before inflowing melt-water flooded over w
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116 this brine (34). Though recent research has questioned the full extent of this dry-down (35), it lo
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117 seems certain that the lake has had a series of relatively low level events, with evaporation to e
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118 at least 5 m below present lake level (36). The legacy of these evaporation-refilling events is o
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119 a salinity gradient from the floor of the lake to the underside of the ice and an inherently h
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120 stable water column (2). Physical mixing is weak at best in large sections of lake water ://
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121 column (10) and vertical flux of solutes occurs largely by diffusion rather than turbulence m
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122 (37). m
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123 r
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124 The ice cover of Lake Fryxell has been shown to transmit a few percent of incident irradiance n

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125 (38), which is sufficient to support a deep chlorophyll maximum reaching >20 mg m-3 (39) a
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126 just above the oxygen limit/nutricline, which is at 9-11 meters depth (10, 40). Based on
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127 studies to date, benthic phototrophic microbial mats appear to be present to depths of at least ,
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128 10.5 m all around the lake (18). 9

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130 Sample collection e
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131 Sampling was undertaken in November 2012 by divers operating through a single hole
132 melted in the ice cover on the northern side of the lake, at 77° 36.4´S, 163° 09.1´ E (Fig. 1).
133 In November 2006, a transect was established and marked by a rope from 8.9 to 11.0 m depth
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134 (all depths are based on hydrostatic water level in November 2012). Nine stations along the
135 transect were marked with pegs between 8.9-11 m depth (Table 1). The dive hole in 2012 was
136 over 8 m of water near the transect, and all sampling was undertaken at transect stations
137 unless stated otherwise. The gentle slope of the lake bottom at the transect site meant that the
138 vertical drop of 3 m corresponded to a horizontal distance of ~50 m.
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140 Physical properties lo
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141 Irradiance at each transect station was determined by a diver equipped with surface e
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142 communications carrying a LiCor Li 1400 meter in a waterproof housing, to which a Li 192 o
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143 photosynthetically active radiation (PAR) sensor was connected. As underwater h
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144 measurements were made, simultaneous measurement of irradiance incident to the lake ://
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145 surface (Li 190 PAR sensor) allowed the percent surface irradiance to be calculated. The m
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146 attenuation coefficient for down-welling radiation was determined from these observations m
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147 and transect-station depth (see below) by log-linear regression analysis (41). The r
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148 transmission of the ice cover was measured similarly, but with the underwater sensor held up n

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149 to the underside of the ice cover while the diver swam a series of radial patterns from the dive a
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150 hole, taking care to avoid the area of unnaturally bright ice close to the dive hole.
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151 ,
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152 Conductivity-temperature-depth-oxygen (CTDO) profiles were obtained in November 2012 9

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153 using a recently calibrated Richard Branker “Concerto” CTD, recording at 6 Hz, to which y

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154 was attached a Unisense UWM picoammeter recording signals at 1 Hz from a Unisense e
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155 oxygen microelectrode (42). The microelectrode had a 50 µm diameter tip and a 90%
156 response time of <2 s. It was calibrated at ambient temperature against air-saturated distilled
157 water and water deoxygenated using sodium dithionite. The combined instrument was first
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158 lowered though a hole in the lake ice close to the deepest part of the lake to obtain a
159 continuous CTDO cast. Post-processing aligned data from the two instruments by time, using
160 a 6-point running mean for the CTD data to accommodate the higher sampling rate. To
161 determine the conditions along the benthic transect, the combined instrument was later
162 carried along the transect by a diver, and lowered to within 50 mm of the lake floor at each
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163 station while recording continuously, as described above, with the time stamp of each w
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164 instrument record used to reconcile location and data. These data were used to confirm the lo
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165 depths of the transect sites. e
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166 o
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167 Water chemistry h
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168 Water samples were taken at each station using acid-washed, 60 ml syringes. Divers drew ://
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169 water into the syringes while the syringes were held just above the lake bottom, rinsing them m
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170 thrice prior to collecting water samples. Once returned to the lake surface, one syringe sample m
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171 from each depth was analyzed immediately for pH using a calibrated portable meter. Three r
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172 replicate samples of 2 ml of lake water from this syringe were also injected into 12-ml serum n

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173 tubes, pre-loaded with 0.2 ml concentrated phosphoric acid, for subsequent determination of a
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174 dissolved inorganic carbon (DIC). Prior to sample injection, a suitable volume of air was
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175 withdrawn from the tubes to ensure approximate pressure equilibrium after sample injection. ,
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176 A second syringe of water was filtered (Whatman GF/F) directly into an acid-washed HDPE 9

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177 bottle and frozen in a portable freezer at -20 °C. Bottles were transported frozen to New y

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178 Zealand and stored at -20°C until nutrients were analyzed. e
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180 Upon return to New Zealand, DIC was measured as CO2 by injecting a subsample of the
181 head-space directly into a stream of nitrogen gas, which then passed through the measuring
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182 channel of a Li-Cor Infra Red Gas Analyzer (IRGA). DIC concentration was estimated from
183 peak height using suitable blanks and bicarbonate calibration standards. Frozen water
184 samples were melted and NH4+-N, NO2-+NO3--N, and dissolved reactive phosphorus (DRP)
185 were measured using an Astoria autoanalyzer.
186
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188 Classification and quantification of macroscopic mat morphology lo
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189 The macroscopic characteristics of the microbial mats on the bottom of Lake Fryxell were e
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190 recorded by a diver who operated a downward-facing high resolution digital video camera o
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191 (Go-Pro HD Hero 2), an LED light source, the Brancker CTD for depth recording, and two h
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192 parallel lasers spaced 3 cm apart for scale. The CTD measured the depth of the camera at 1 s ://
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193 intervals, which allowed subsequent depth referencing of time-stamped video frames. Laser m
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194 pointers were offset at an angle to the video camera to allow calculation of camera height m
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195 above the mat surface from the location of the laser points in the camera field of view. r
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196 Distance from the lake floor was calibrated using images obtained at 11 defined distances n

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197 from a gridded calibration target. The absolute depth of the lake floor at a given point was a
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198 calculated based on the CTD depth and distance above the lake floor, which was calculated
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199 from the laser positions in the image. Calculated depths were cross-checked at three staked ,
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200 sites along the transect, and the error was <100 mm. 9

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202 The mat morphology survey involved a diver swimming a series of meandering tracks e
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203 through a 25 m wide swath to one side of the transect. Recorded images were reviewed and
204 used to produce a mat typology based on dominant features including presence or absence of
205 pinnacles or ridges, color, and texture. A total of 197 frames along the video transects were
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206 then scored for lake depth and mat type at the point where the laser scale impinged on the mat
207 surface, and mat type frequencies were compiled for 0.1 m depth bins.
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209 Representative mat samples were collected by divers and vertically sectioned to document
210 lamination of the main mat morphologies present along the transect. The dominant
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211 cyanobacterial morphotypes in different microbial mat sections were identified on site using w
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212 an Olympus light microscope (BX51, Olympus) at 400–1000x magnification, and assigned to lo
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213 genera based on Komarek & Anagnostidis (43-45). e
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214 o
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215 Pigment and diatom analysis h
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216 Samples for quantitative pigment and diatom analysis were taken with a 38 mm diameter ://
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217 corer, with four replicates at each transect station. Individual cores were placed into a 60 ml, m
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218 wide-mouthed bottle underwater, sealed and returned to the surface. On site processing m
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219 involved draining excess water and freezing (-20 °C). Upon return to New Zealand, samples r
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220 were freeze-dried and ground to a fine powder. Weighed aliquots were then taken for analysis n

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221 of phycoerythrin, chlorophyll, pigment HPLC and diatom relative abundance. a
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223 For the phycoerythrin analysis, aliquots were extracted into 5 ml 0.1 M Tris Buffer (pH 7.6) ,
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224 using ultrasonication (15 W for 30 seconds) in an ice-water bath and left to extract at 4 °C for 9

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225 12-16 h. After clarification by filtration (Whatman GFF) extracts were analyzed y

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226 fluorometrically according to Downes and Hall (46). Subsamples for chlorophyll were e
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227 extracted by sonication in ice-cold 95 % acetone and left at 4 °C in the dark for 12-16 h to
228 complete extraction. After clarification by centrifugation, chlorophyll concentration was
229 initially determined by spectrophotometry at 665 nm, without acidification using a method
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