Vertical models of phototrophic bacterial distribution in the metalimnetic microbial communities of several freshwater North-American kettle lakes

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Vertical models of phototrophic bacterial distribution in the metalimnetic microbial communities of several freshwater North-American kettle lakes X. Vila a; *, C.A. Abella a , J.B. Figueras a , J.P. Hurley a

b;c

Institute of Aquatic Ecology, Microbiology Laboratory, University of Girona, Campus Montilivi, E-17071 Girona, Spain b Wisconsin Department of Natural Resources, Monona, WI, USA c University of Wisconsin, Water Chemistry Program, Madison, WI, USA Received 25 September 1997; revised 19 November 1997; accepted 20 November 1997

Abstract The composition and the structure of the metalimnetic communities of phototrophic microorganisms were studied in 24 lakes of Wisconsin and Michigan (USA), during the period of summer stratification, and related to environmental parameters. The presence of phototrophic bacteria was reported for the first time in some lakes. Different types of phototrophic microorganisms were separated in three different clusters, by decreasing values of photosynthetic available radiation (PAR) and redox potential (Eh): (1) cyanobacteria and eukaryotic phytoplankton, (2) Chromatiaceae and multicellular filamentous green bacteria, (3) green sulfur bacteria. Each cluster conformed one different layer of the vertical distribution of the community. At each layer, usually one type of microorganisms outcompeted the others, but at times they were found together in competition. The three layers were mixed at the bottom of the lake at the beginning of the stratification period, before widening and colonising the water column. At the end of summer, the contraction of the metalimnion resulted in overlapping or suppressing some layers. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. Keywords : Phototrophic bacterium ; Metalimnion; Community structure; Kettle lake ; PAR ; Eh

1. Introduction The water column of lakes could present a high diversity of ecological niches for the phototrophic microorganisms, since several physical and chemical gradients could establish along depth through the whole water column, but mixing processes usually tend to homogenise the epilimnia [1]. However, if * Corresponding author. Tel.: +34 (72) 418176; Fax: +34 (72) 418748; E-mail: [email protected]

the metalimnion is stable enough, the gradients can develop there during the seasonal strati¢cation. Then, the microbial community consists of populations at di¡erent vertical positions in a complex multilayered structure. Each species develops at its respective optimal situation in these metalimnetic zone of gradients (gradient zone), where the di¡erent combinations of environmental parameters depending on depth select for di¡erent species in the competitive exclusion process. When the hypolimnion or monimolimnion is an-

0168-6496 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 1 6 8 - 6 4 9 6 ( 9 8 ) 0 0 0 1 0 - 5

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oxic, the gradient goes from the upper warm, illuminated and oxic layers to the deeper cold, dark, anoxic, and usually sul¢de-rich layers. Anoxygenic phototrophic bacteria appear when sul¢de and adequate light are present [2,3]. According to their particular taxonomic and ecophysiological attributes, several microbial groups can be di¡erentiated in these metalimnetic communities: green sulfur bacteria (GSB) with di¡erent (brown or green) pigmentations, Chromatiaceae, multicellular ¢lamentous green bacteria (MFGB), eukaryotic phytoplankton and cyanobacteria with phycocyanin or phycoerythrin as dominant pigments. Some groups have been previously found to separate within the vertical gradient. Two di¡erent communities of phototrophic bacteria (A and B) were distinguished by Caldwell and Tiedje [4] in lakes Wintergreen and Burke, above a bottom community (C) of colourless bacteria. Community A was red in colour, contained Chromatiaceae and was positioned above community B, which was green and composed by GSB. Subsequently, Chromatiaceae were found above GSB in di¡erent lakes [5^14]. The layering of phytoplankton and cyanobacteria above the populations of anoxygenic phototrophic bacteria has also been reported in several lakes [11,14^20]. For the present work, the metalimnion of 24 lakes from Wisconsin and Michigan (USA) was studied in detail to investigate the composition and structure of phototrophic microbial communities. The presence of phototrophic bacteria was reported for the ¢rst time in some of these lakes. Main physical and chemical parameters were analysed in relation to the distribution of microorganisms, in order to understand the conditions for the dominance of particular microbial groups. Finally, the community structure was examined and summarised in ¢ve models of vertical distribution. The models could be related to the sequence of metalimnetic succession along the period of strati¢cation.

2. Material and methods 2.1. Studied lakes The 24 studied lakes were situated in the NorthAmerican Great Lakes Region, in the states of Mich-

igan and Wisconsin (Table 1), and were sampled during the summer of 1994. They were small freshwater lakes of glacial origin (kettle lakes), dimictic or biogenically meromictic, and contained anoxic hypolimnetic waters. Physical and chemical parameters determined stable strati¢cation during the study period, but gradients were always soft. The previously best known lake was the eutrophic Wintergreen Lake, in which complex microbial communities of Chromatiaceae and green species of GSB were identi¢ed by Caldwell and Tiedje [4]. In the same work, lakes Duck and Cassidy were also found to contain anoxygenic phototrophic bacteria. The bacterial communities of lakes Fish, Mirror, Mary, Peter and Paul were studied in relation to light intensity and quality by Parkin and Brock [21,22], who found GSB populations in all lakes and Chromatiaceae in lakes Peter, Fish and Mirror. Dystrophic Mary Lake was also well studied because of its high concentrations of dissolved humic substances (gilvin) [23], that determined special light quality conditions in the water column [24]. The deep communities of phytoplankton and phototrophic bacteria in lakes Wood, Crystal, Minocqua, Sparkling, Fish and Mirror were studied during the period of the present work by Hurley and collaborators [25,26]. The presence of phototrophic bacterial populations in the other lakes (Baker, Jones, Lefevre, Little Mill, Mud, Palmetier, Round, Warner, Little Long, Trout Bog and basins I and II of Silver) had not been previously reported. 2.2. Sampling methods and physical and chemical measurements Water samples were collected from these lakes using a special device designed for the study of thin (20^25 cm) water layers [27], and were stored in dark bottles until analyses were performed. Relevant chemical parameters for lake characterisation and habitat de¢nition (conductivity, temperature, oxygen concentration, pH, Eh) were measured in situ by selective electrodes. Sul¢de concentrations were quanti¢ed in the laboratory following the Pachmayr method [28], from water samples ¢xed with Zn acetate and NaOH 1 M. Integrated irradiance values for photosynthetic available radiation (PAR) were calculated from downward spectral irradiance measure-

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Table 1 Sample dates (year-month-day), geographical situation and some general limnological features of the studied lakes Lake

Date

Latitude

Longitude

M. depth

Eh depth

Epilim. Kd

940725 940722 940720 940723 940721 940722 940723 940724 940818 940818 940725 940724 940718

42³40P 42³28P 42³25P 42³31P 42³13P 42³28P 42³31P 42³34P 46³15P 46³15P 42³40P 42³28P 42³24P

N N N N N N N N N N N N N

85³30P 85³10P 85³23P 85³25P 85³23P 85³16P 85³25P 85³26P 89³30P 89³30P 85³30P 85³32P 85³23P

W W W W W W W W W W W W W

8.00 10.50 4.00 13.00 15.00 10.50 8.00 11.50 11.00 17.50 6.50 16.50 6.00

7.00 7.00 3.00 4.75 14.00 9.00 4.00 9.25 7.00 8.50 5.00 13.50 4.75

1.20 0.67 0.71 1.61 0.67 0.60 1.50 0.46 0.78 0.65 1.20 0.58 1.06

940811 940804 940809 940819 940817 940809 940811 940811 940816 940817 940802

43³48P 43³17P 44³38P 46³15P 45³51P 44³21P 43³23P 43³23P 46³01P 46³03P 43³59P

N N N N N N N N N N N

88³01P 89³39P 88³56P 89³54P 89³43P 89³05P 88³13P 88³13P 89³42P 89³41P 89³30P

W W W W W W W W W W W

18.00 17.00 8.00 18.00 12.50 13.00 14.00 8.00 18.00 8.00 15.00

12.75 11.25 4.50 3.25 10.50 8.00 12.75 6.75 17.50 3.50 13.50

0.41 0.92 0.99 2.60 0.60 0.62 0.45 0.58 0.37 2.70 0.35

Michigan Baker Cassidy Duck Jones Lefevre Little Mill Mud Palmetier Paul Peter Round Warner Wintergreen Wisconsin Crystal Fish Little Long Mary Minocqua Mirror Silver I Silver II Sparkling Trout Bog Wood

Maximum depth (M. depth, m), depth of the redoxcline, measured as the depth where an Eh value of 0 mV was found (Eh depth, m), and vertical attenuation coe¤cient for downward quantum irradiance of PAR in the epilimnion (epilim. Kd , m31 ).

ments performed with a Li-Cor underwater spectroradiometer (LI-1800 UW). 2.3. Identi¢cation of phototrophic microorganisms Photosynthetic microbial groups and species were identi¢ed from pigment composition and microscopic observation of the samples. Pigment concentration, as an estimation of population density, was spectrophotometrically determined [29]. Water samples were ¢ltered on cellulose nitrate ¢lters of 0.45 Wm pore diameter and photosynthetic pigments extracted in acetone and stored 24 h at dark in the freezer (320³C). The extinction coe¤cient for Chl b was used for the calculation of BChl e concentrations, because of the lack of a true BChl e coe¤cient and the similarity between both pigments [30]. These extracts were also analysed by HPLC [31], using a Waters instrument (Waters 510 Pumps and Waters

996 Diode Array Detector) provided with a Spherisorb C-18 column. HPLC data were used to better discriminate among similar pigments and calculate their proportions, in order to improve the spectrophotometric quantitative measurements. 2.4. Statistical analyses Analyses of variance on physical and chemical data were performed with the ONEWAY procedure of the SPSSx statistical pack for Apple Macintosh.

3. Results and discussion 3.1. Composition of the metalimnetic phototrophic communities and environmental factors The densest populations of photosynthetic pig-

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Table 2 Concentrations of the photosynthetic pigments and species composition Lake

Lefevre Warner Crystal Silver I Wood Wintergreen Baker Duck Jones Mud Round Mary Troutbog Cassidy Minocqua Mirror Paul Peter Little Mill Little Long Sparkling Palmetier Fish Silver II

[Chl a]

[BChl a]

[BChl d]

[BChl e]

Max. Depth Int.

Species Max. Depth Int.

Species

Max. Depth Int.

Species

101.8 25.6 42.4 28.7 32.4 276.8 21.0 101.0 202.6 81.4 105.5 109.8 20.6 55.7 43.1 73.1 224.1 69.3 159.8 283.9 8.5 85.5 22.3 168.6

eu eu eu eu eu eu eu eu eu eu eu eu eu cb cb cb eu eu cb eu eu cb eu eu

Tv C Tv Tv C Tv Lr Tr Tv Tr Co ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ Tr C Tv Tv C Tv C Tr C Tv Lr

^ ^ ^ ^ ^ 570.9 179.3 29.6 132.4 81.8 889.0 140.1 83.7 109.5 50.5 823.7 252.5 366.4 87.5 403.0 7.5 24.8 2.5 42.2

^ ^ ^ ^ ^ Ap Cn Ca Ca Cn Ca Cn Ca Cn Ca Cn Ca Cl Cn Ca Pc Cv Cn Ca Cn Ca Cn Ca Ca Cn Ca Cn Cn Ca Cn Cn

5.00 12.00 10.50 11.50 13.25 3.75 4.50 3.00 3.50 3.75 4.50 2.25 2.25 5.50 9.50 7.00 6.25 8.00 8.50 4.00 15.50 9.00 4.00 6.50

315.5 163.0 172.9 198.3 110.6 535.2 56.6 81.9 303.3 241.5 274.3 128.1 53.2 133.4 183.6 202.9 188.5 241.8 261.9 404.4 94.7 180.6 140.7 221.6

cb cb cb cb cb cb cb cb cb cb cb cb

cb cb cb

cb

20.2 12.0 78.1 107.1 17.6 426.7 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ 36.5 157.5 12.6 83.2 2.1 197.8

13.25 12.25 12.00 12.00 13.25 4.25 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ 9.00 4.20 16.25 9.25 10.00 6.50

25.1 20.4 90.0 164.9 18.5 309.9 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ 36.5 108.9 14.4 37.8 5.3 181.8

^ ^ ^ ^ ^ 5.00 6.00 3.50 4.75 4.00 5.00 3.75 3.00 6.50 11.00 7.25 7.00 9.00 9.00 4.20 16.50 9.25 10.50 6.75

^ ^ ^ ^ ^ 902.1 376.8 20.2 392.8 171.8 1394.4 306.0 130.3 228.2 82.4 829.3 628.5 1088.7 87.3 471.5 22.3 37.6 2.6 33.8

Max. Depth Int. 266.8 61.7 265.7 158.2 48.1 ^ ^ ^ Pc ^ Pc Ap ^ Pc Ap ^ ^ ^ 23.6 7.2 101.6 Cl 20.2 Cl 16.5 168.1 695.1 51.4 31.8 0.4 46.6

13.75 12.75 12.00 12.50 13.25 ^ ^ ^ ^ ^ ^ ^ ^ 9.00 11.00 7.50 7.00 10.00 10.00 4.60 18.00 9.25 11.00 7.50

284.1 46.8 285.0 243.7 75.5 ^ ^ ^ ^ ^ ^ ^ ^ 62.8 7.9 194.1 46.4 93.5 137.8 736.6 61.7 47.5 0.6 37.2

Species Pr Pr Pr Pr Pr ^ ^ ^ ^ ^ ^ ^ ^ Pr Pr Pr Pr Pr Pr Pr Pr Pr Pr Pr

The di¡erent columns correspond to the maximum metalimnetic values (max., Wg l31 ), their depths (m), the integrated values for the whole water column (int., Wg l31 ) and the dominant species of metalimnetic phototrophic microorganisms, for each photosynthetic pigment : chlorophyll a (Chl a), bacteriochlorophyll a (BChl a), bacteriochlorophyll d (BChl d) and bacteriochlorophyll e (BChl e). Abbreviations for the microbial species: eu (eukaryotic phytoplankton), cb (cyanobacteria), Tv (Thiocystis violacea), C (Chromatium spp. ), Lr (Lamprocystis roseopersicina), Tr (Thiopedia rosea), Co (Chromatium okenii), Ap (Ancalochloris per¢lievii), Cn (Chloronema spp. ), Ca (`Chlorochromatium aggregatum'), Pc (Pelodictyon clathratiforme), Cl (Chlorobium limicola), Cv (`Chloroplana vacuolata'), Pr (`Pelochromatium roseum').

ments in these lakes were always found at the metalimnion, where the deep communities of phototrophic microorganisms could account for the highest biomass values of the whole water column ^ however, they are usually not correlated with the highest productivity values [16,32]. The composition of deep communities (Table 2) also indicated a high diversity, either by the di¡erences among lakes and the internal diversity of some communities. Almost all combinations of the di¡erent microbial groups were found. Cyanobacteria or eukaryotic phytoplankton species (Chl a) were represented in all 24 lakes and both types of microorganisms were found in most lakes. Only lakes Lefevre and Fish lacked metalimnetic Chl a maxima. For deep communities, maximum densities ( s 200 Wg Chl a l31 ) were

found in lakes Jones, Wintergreen, Paul and Little Long. Chromatiaceae (BChl a) were identi¢ed by their photosynthetic pigments in 12 lakes. However, these bacteria were observed with the microscope in almost all the other lakes, although their pigment concentrations were too low to be detected by spectrophotometry or HPLC. The densest populations were found in Wintergreen Lake ( s 400 Wg Bchl a l31 ), but high pigment concentrations ( s 100 Wg Bchl a l31 ) were also found in Little Long Lake and both basins of Silver Lake. The most widespread species were Thiocystis violacea, Thiopedia rosea and some species of Chromatium sp. (Fig. 1). Green-coloured species of green sulfur bacteria (GSB) or multicellular ¢lamentous green bacteria (MFGB), both containing BChl d as main photosyn-

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Fig. 1. Common phototrophic bacteria from studied lakes. Microphotographs (U600) at the optical microscope of phase contrast, showing some of the most common phototrophic bacteria in metalimnetic communities: A: Wintergreen Lake (4.75 m): Chromatium okenii, Thiopedia rosea, Ancalochloris per¢lievii. B: Cassidy Lake (6 m): Thiocystis gelatinosa, Chloronema spp. C: Baker Lake (5.5 m): Lamprocystis roseopersicina, Chloronema spp. D: Round Lake (5 m): Thiopedia rosea, Pelodictyon spp., Ancalochloris per¢lievii, Chloronema spp., `Chlorochromatium aggregatum', `Pelochromatium roseum', `Chloroplana vacuolata'.

thetic pigment, were found in most of the lakes. They were absent only in the deepest communities (Lefevre, Warner, Crystal, Silver I and Wood), probably due to their severe light limitations [3,29,33]. The highest concentrations of BChl d were measured in lakes Round and Mirror ( s 800 Wg BChl d l31 ),

and also lakes Wintergreen, Paul, Peter and Little Long ( s 200 Wg BChl d l31 ). These pigments were contained by a high diversity of species, which often coexist at high densities in the same community. The most abundant pigments were from the MFGB of genus Chloronema, the consortium `Chlorochromati-

Table 3 Mean values of the main physical and chemical parameters for the di¡erent microbial groups PAR (%)

Eh (mV)

[H2 S] (WM)

[O2 ] (mg31 )

Euk. phytoplankton PC cyanobacteria PE cyanobacteria Chromatiaceae Multicellular ¢lamentous green bacteria (MFGB) Green-pigmented GSB Brown-pigmented GSB

0.71 0.45 0.43 0.14 0.14 0.05 0.05

197 223 182 105 110 48 320

0.00 2.91 4.71 14.50 8.30 15.47 35.57

0.25 1.06 1.63 0.29 0.27 0.57 0.18

F probability

0.0000

0.0302

0.0011

0.1469

Means have been calculated from the values corresponding to the maximum density depth at the metalimnion of each group of phototrophic microorganisms: eukaryotic phytoplankton, phycocyanin- (PC-) or phycoerythrin- (PE-) containing cyanobacteria, Chromatiaceae, multicellular ¢lamentous green bacteria (MFGB), green-coloured green sulfur bacteria (GSB) and brown-coloured GSB. F probabilities arising from the analysis of variance are indicated at the bottom of the table.

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um aggregatum' and the vacuolated GSB of genera Pelodictyon and Ancalochloris. The pigments of brown-coloured species of GSB were found in all the lakes lacking the BChl d phototrophic bacterial species. These brown bacteria were also observed in most lakes that contained greencoloured GSB, although in some of them the BChl e concentration was too low to be detected. The highest concentrations of BChl e were measured in lakes Little Long ( s 600 Wg BChl e l31 ), Lefevre and Crystal ( s 200 Wg BChl e l31 ). The dominant browncoloured GSB was always the consortium `Pelochromatium roseum'. The values of several physical and chemical parameters have been compared to the composition of the microbial communities at di¡erent depths, in order to ascertain which environmental factors determine the vertical distribution of these microorganisms. Seven types of phototrophic microorganisms were considered, according to their ecophysiological characteristics: eukaryotic phytoplankton, phycocyanin-containing cyanobacteria, phycoerythrin-containing cyanobacteria, Chromatiaceae, MFGB, green- and brown-coloured GSB. Values of the main parameters (light intensity as % PAR, Eh, [O2 ] and [H2 S]) for the positions of the highest density of their populations have been treated by variance analysis (Table 3). Light intensity and [H2 S] were determined to be the main factors related to the microbial distribution, with F probability values smaller than 0.01. Eh values, that are integrative of the di¡erent chemical compounds contained by the water, had also a signi¢cative F probability (lower than 0.05) and distributed inversely to [H2 S] values ^ in fact, H2 S gives rise to negative Eh when it accumulates [34]. Both PAR and Eh have been used to separate the di¡erent groups of microorganisms (Fig. 2). Algae and both types of cyanobacteria developed at the highest PAR and Eh values, Chromatiaceae and MFGB at intermediate values and both types of GSB at the lowest light intensities and Eh values. However, there are some Eh di¡erences among green and brown GSB, that can be also related to di¡erences on [H2 S]. Oxygen did not have a signi¢cant relationship with the distribution of these microbial groups, as revealed by an F probability value higher than 0.05. The oxygen concentrations were always low, because all the pop-

Fig. 2. Distribution of the microbial groups in relation to environmental parameters. Mean values and standard errors of PAR (as a percentage of light at each depth relative to the surface value) and Eh (measured in mV) corresponding to the maximal density depth at the metalimnion of each group of phototrophic microorganisms are represented. The three clusters (1^3) are de¢ned according to these values. A: Eukaryotic phytoplankton, B: phycocyanin-containing cyanobacteria, C : phycoerythrin-containing cyanobacteria, D: Chromatiaceae, E: multicellular ¢lamentous green bacteria (MFGB), F : green-coloured green sulfur bacteria (GSB), G: brown-coloured GSB.

ulations developed at the deepest part of the oxycline or even below it. 3.2. Structure of the community: three-layered models of vertical distribution The metalimnetic community of phototrophic microorganisms was usually structured in three distinct layers (1^3), which vertical distribution can be summarised in ¢ve models (I^V). The combination of light and Eh (Fig. 2) clearly separated them in three clusters: (1) phytoplankton and both types of cyanobacteria, (2) Chromatiaceae and MFGB, and (3) both types of GSB. In the latter, the green-coloured GSB appeared in an Eh intermediate situation between the brown-coloured GSB and the microorganisms of group 2. As both light and Eh usually decrease with depth, the three microbial clusters must be placed at di¡erent positions in the water column, thus resulting in three di¡erent layers in the vertical distribution of the metalimnetic microbial commun-

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ity, from the shallowest to the deepest part of the community. This separation occurred at depths with low light intensities (less than 1% of surface PAR) and must be related to the redoxcline, where at least the populations of layer 2 must be positioned. Layer 3 was often found at the redoxcline too, because hypolimnetic Eh usually reached lower values than those measured where these populations developed. Layers 2 and 3 agree with the communities A and B previously described in lakes Wintergreen and Burke by Caldwell and Tiedje [4]. The occurrence of phytoplankton and cyanobacteria (layer 1) at the oxycline, just above the populations of anoxygenic phototrophic bacteria, has been extensively reported [11,14^20] and usually was related to an increase in oxygen concentration at the uppest part of the thermocline [35^38]. Oxygen production by the photosynthetic activity in this layer precludes the development of the anoxygenic bacterial populations, which develop lower in the water column. The distribution of Chromatiaceae (layer 2) over the GSB (layer 3), also reported for several lakes [5^14], has been attributed to di¡erent requirements of light and sul¢de [2,39] or to a higher ecological £exibility of Chromatiaceae [3]. Both explanations are consistent with these results. The ¢rst one can be related to the di¡erences in % PAR, [H2 S] and Eh mean values found among these groups. The £exibility attributed to Chromatiaceae by the second explanation is necessary to thrive in layer 2, because of the changing conditions that must tolerate them in this position. It is not surprising that Chloronema populations were found at shallower depths than GSB. Although traditionally considered to be closely related taxonomic groups [40], marked physiological di¡erences are thought to separate them, as well as the evident morphological di¡erences. From previous knowledge of the Chloronema ecology in several lakes of central Europe [41,42], they were considered to have special adaptive features to the environmental characteristics of the transition zone. This zone was de¢ned by Abella and Garcia-Gil [42] as an intermediate anaerobic fraction of the vertical gradient, between the aerobic shallower layers and the anaerobic deeper layers with high sul¢de concentrations. The conditions de¢ned for the transition zone agree better with the values of the main parameters at the depths

293

where Chloronema populations (layer 2) developed, than where the GSB (layer 3) were found. Thus, an interesting ecological situation that has not been examined before, arises from this consideration, since also Chromatiaceae species can be found at the same depths. The competition between the microorganisms of these groups for the resources available at the transition zone must be minutely examined in further studies of their seasonal population dynamics. Di¡erent microbial populations can coexist in the same layer in competition for light and nutrients. In most cases, however, one microbial group is dominant. This frequent situation, represented among others by lakes Wintergreen, Jones and Silver I (Fig. 3), has been considered as the model I of distribution. The selection of the dominant microorganisms within each layer must be related to environmental factors other than light intensity and Eh, because these parameters can only separate the microorganisms belonging to di¡erent layers, except for the selection of green- or brown-coloured GSB, that can be in£uenced by [H2 S] and thus also by Eh. Light quality has been found to discriminate between both pigmentary types of GSB [29,33] and the analysis of the spectral distribution of light reaching the populations of these and several European lakes indicates that this factor also operates on the selection within the other layers (X. Vila and C.A. Abella, unpublished data). It is also well known that the N:P ratio controls the competition between cyanobacteria and eukaryotic phytoplankton in many lakes [43]. The presence of more than one microbial group in the same layer characterises the model II and was found, for example, in Little Long Lake (Fig. 3), where Chromatiaceae and MFGB were mixed in layer 2 and also both types of GSB reached high densities in layer 3. In these situations, the competition is still intense and will lead to the selection of one microbial group or to the coexistence of di¡erent microorganisms by vertical separation in microlayers. Evidence of such microstrati¢cation appeared in Mirror Lake (Fig. 4), where both groups of GSB were found in layer 3 and their maxima were vertically separated by 25 cm depth. However, this lake has not been considered as representing model II because of the lack of layer 2. These mechanisms

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Fig. 3. Examples of metalimnetic vertical distribution of the phototrophic microorganisms for models I and II. Pigment concentrations (Wg l31 ) at di¡erent depths are represented and the di¡erent layers are indicated by numbers 1^3. Chl a (¢lled squares) indicates cyanobacteria and eukaryotic phytoplankton (layer 1), BChl a (empty squares) indicates Chromatiaceae populations (layer 2), BChl d (empty circles) indicates MFGB (layer 2) and green-coloured GSB (layer 3), BChl e (¢lled circles) indicates brown-pigmented GSB (layer 3). Chl a concentrations are magni¢ed in Silver I (U3). They are also magni¢ed in Little Long (U2), as well as BChl a (U4) and BChl d (U2). The vertical ranges for the oxycline (oxy) and the redoxcline (red) are also indicated at the right of each panel.

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Fig. 4. Examples of metalimnetic vertical distribution of the phototrophic microorganisms for models III, IV and V. Pigment concentrations (Wg l31 ) at di¡erent depths are represented and the di¡erent layers are indicated by numbers 1^3. Chl a (¢lled squares) indicates cyanobacteria and eukaryotic phytoplankton (layer 1), BChl a (empty squares) indicates Chromatiaceae populations (layer 2), BChl d (empty circles) indicates MFGB (layer 2) and green-coloured GSB (layer 3), BChl e (¢lled circles) indicates brown-pigmented GSB (layer 3). Several pigment concentrations are magni¢ed : Chl a in Round (U5) and Mirror (U10), BChl d in Palmetier (U8), BChl e in Mirror (U7) and Palmetier (U8). The vertical ranges for the oxycline (oxy) and the redoxcline (red) are also indicated at the right of each panel.

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Fig. 5. Theoretical temporal evolution of the vertical distribution of the metalimnetic populations. The succession of the ¢ve di¡erent models (I^V) for the vertical distribution of the three microbial layers (1^3) is represented in relation to the evolution of the transition zone, delimited by the oxycline and the redoxcline, during the strati¢cation period. The arrows indicate the shifts of the oxycline and the redoxcline as O2 and H2 S spread (upper panel) and the movement trends of the metalimnetic community (lower panel).

can also produce the species diversity within the same microbial group in some layers, as in the green-coloured GSB layer of lakes Round and Mud (Table 2). In some communities, however, a lower number of layers could be di¡erentiated (Fig. 4), because some of them overlapped (model III, as lake Palmetier) or were suppressed, usually the layer 2 (model IV, as lakes Round and Mirror). Finally, in a few lakes all of the di¡erent populations were mixed in the same layer (model V, as lake Wood). Caldwell and Tiedje [4] suggested the possibility of temporal expansion or contraction of the bacterial layers, while their relative position remained constant. These ¢ve models can be considered as di¡erent stages of the metalimnetic community evolution

and thus all of them could be found in each lake, but at di¡erent times of the strati¢cation period. The model V can be imputed to a microbial community in its initial stages of development ^ at the beginning of the strati¢cation ^ where sul¢de is available only near the sediment (Fig. 5). The increase of sul¢de concentration and its di¡usion to shallower parts of the water column generate the wide gradients that allow the microbial populations to segregate in the three-layer distribution characteristic of models I and II, found in most of the studied communities. If microstrati¢cation is not developed and is stable for a long period of time, the distributions of model II will tend to evolve toward model I when some species will be selected at the detriment of the others.

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Finally, at the end of summer, the metalimnion is reduced [18], as both epilimnion and hypolimnion expand due to heating of the surface and sul¢de releasing from the bottom, respectively. Therefore, the microbial populations can be overlapped or outcompeted in some layers, resulting in the distributions of models III (overlapping) and IV (suppression). The model V can also be related to the pattern I (bottom plates) of vertical distribution of the microbial biomass described by Montesinos and Van Gemerden [32], while the models I^IV are di¡erent possibilities of their pattern II, in which the maximum biomass occurs in a narrow layer at the boundary between the aerobic and the anaerobic zones. There are many lakes containing anoxic hypolimnia, in which phototrophic bacterial populations are still unknown or only dense metalimnetic populations of cyanobacteria have been reported [35^37]. The presence of anoxygenic phototrophic bacteria can be expected in most of these lakes, because they have limnological features quite similar to the kettle lakes of Michigan and Wisconsin in which these bacteria were previously unknown. Since this type of lake is abundant in regions a¡ected by glacial processes, their populations must be considered more widespread and common than was previously thought. More accurate studies focused on the hypotheses suggested by this work will provide more relevant information about the structure of the metalimnetic community and their microstrati¢cation and dynamics.

Acknowledgments We are indebted to Dr. J. Tiedje and Dr. M. Klug, from the Michigan State University, and to the sta¡ at the Kellogg Biological Station (Kalamazoo, Michigan) for facilities provided for the study of the lakes of southern Michigan. We also acknowledge the sta¡ of Water Chemistry Program and Center for Limnology at the University of Wisconsin (Madison and Woodru¡, Wisconsin) for their collaboration during the ¢eld work in Wisconsin and northern Michigan, with a special mention to G. Lauster. The HPLC analyses of photosynthetic pigments were possible by the signi¢cant contribution of Dr. C. Borrego and X. Cristina, from the Institute of Aquatic Ecol-

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ogy (University of Girona, Spain). During the work in the USA, Dr. X.V. and J.B.F. were the recipients of doctoral scholarships from the Spanish Ministry of Education and Science (FP90-77910108) and the Generalitat of Catalunya (FI/93-155), respectively. Dr. C.A.A. was supported by the research project NAT91-0708, from the Spanish Ministry of Education and Science. This is the publication number 152 of the Institute of Aquatic Ecology (University of Girona).

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