PRTMARY PRODWTIVITY, CHEMO- ORGANOTROPHY, AND NUTRITFONAJ. ENTERACTIONS OF EPIPHYHC ALGAE ' AND BACTERIA 0N MMRGPHYTES IN ' THE UTTORAL OF A LAKE Thesis for the Degree of Ph. D. MECHI’GAN STATE UNIVERSITY HAROLD LeROY ALLEN 1969 L1 13 1‘? AR Y Michigan State University T'HtStS g 1 This is to certify that the thesis entitled PRIMARY PRODUCTIVITY, CHEMO-ORGANOTROPHY, AND NUTRITIONAL INTERACTIONS 0F EPIPHYTIC ALGAE AND BACTERIA ON MACROPHYTES IN THE LITTORAL OF A LAKE presented by Harold LeRoy Allen has been accepted towards fulfillment of the requirements for Ph .D. degree in Botanz (BL/Ajlb/ Major pro%or Date 19 November 1969 0-169 amoma av HMS & SUNS' BUC“ MDERY "W. Lln' 'IV am IERQF ’— «1:! “fl ll—Aaaggfigg ...n‘|\l...\|‘llllal'll\wtllsvln r ABSTRACT .PRIMARY PRODUCTIVITY, CHEMO-ORGANOTROPHY, AND NUTRITIONAL INTERACTIONS OF EPIPHYTIC ALGAE AND BACTERIA ON MACROPHYTES IN THE LITTORAL OF A LAKE By Harold LeRoy Allen Assessment of epiphytic algal and bacterial in E122 community metabolism, and physiological-nutritional inter— relationships of macrOphyte—epiphyte systems, were investi— gated in the littoral zone of a small temperate lake from April 1968 through May 1969. Annual primary productivity, chemo—organotrophic utilization of dissolved organic com- pounds, and field and laboratory studies of macrOphyte- epiphyte interactions were monitored by carbon—14 techniques. Qualitative and quantitative photosynthetic pigment compo- sition, and a brief taxonomic examination of the sessile complex, accompanied measurement of field parameters. Productivity measurements of epiphytic algae on artificial substrata colonized in emergent (Scirpus acutus Muhl.) and submergent (NaJas flexilis L. and Chara spp.) macrophytic vegetation sites were compared over an annual period with pigment (chlorOphyll a and total plant carote— noids) estimates of biomass. The results indicate biomass Harold LeRoy Allen estimates are not indicative of photosynthetic activity,- except during periods of intense productivity. The annual mean productivity of epiphytic algae was higher per unit surface area of the submerged portions of emergent plants 2 (336 mg C m- day-l) than on that of submergent forms (258 mg C m-2 day—l); annual mean productivity per unit area of the littoral zone, for all of the macrOphytic sur- face area colonized, was 195 and 1807 mg C m-2 day-1 in each of the respective sites. The results indicate that algal epiphytes on submerged vascular vegetation may be the dominant primary producers in shallow—water ecosystems, and may easily exceed the phytoplankton. Spatial and temporal rates of epiphytic productivity are discussed in relation to pigment composition and algal distribution, organic utilization, structural integrity of the matrix, and taxo- nomic composition of the epiphytic community. Deposition of lb'C-monocarbonates during in situ_measurements of pro— ductivity represented 38.5 to 71.7% of the total intra— cellular fixed carbon. Acidification of 1uC-productivity samples by rinsing with dilute hydrochloric acid (0.001 g) removed 2U% of previously incorporated carbon and is not recommended as a routine procedure. Potential physiological interactions in macrOphyte- epiphyte systems were investigated by bioassay procedure. Inorganic iron added at less than 10 pg 1’1, and at 100 ug l_1 in combination with organic compounds of Harold LeRoy Allen chelatory or complexatory ability, stimulated photosynthesis of epiphytic algae. Results of bioassay experiments with vitamins, trace metals, and inorganic phosphorus are dis- cussed. Chlorophyll a (corrected for pheophytin degradation products) and total plant carotenoid levels are among the highest standing crOps reported in the literature (annual maximum of chlorophyll a: 7.3 g m-2; plant carotenoids: no.7 SPU m-2 ). Maximum concentrations were found during winter under ice cover. Epiphytic bacterial chemo—organotrOphy with glucose and acetate substrates, measured at ecologically reasonable concentrations (11 to 160 ug 1-1), was evaluated through Michaelis-Menten enzyme kinetic analysis. First-order active transport kinetics dominated throughout the annual period with uptake of acetate (submergent substrata annual mean rate: 893 pg 1-1 hr-1 dm-2; emergent: 106 pg 1"1 hr"1 dm_2) being greater than that of glucose (submergent: 586 pg 1-1 hr-1 dm-Z; emergent: 5“ pg 1_1 hr.1 dm-2). Sources of dissolved organic matter at the epiphytic surface are discussed in relation to high rates of utilization and to in situ_metabolism of attached primary producers. Scirpus acutus was photosynthetically labelled in situ with natural concentrations of luco 2. Uptake of labelled materials of macrophytic origin by the epiphytic complex was determined. Extracellular release of 1”Cudissolved organic matter with respect to depth in the littoral water Harold LeRoy Allen column was followed in three plants over a 5 hour period. The nature of extracellular release, in comparison with 1“002 fixed by the hydrOphyte and incorporation by the epiphytic complex, suggests functional interactions that may be prevalent in other macrophyte-epiphyte systems. Najas flexilis, germinated and grown under axenic conditions in defined medium, was photosynthetically labelled and placed into Plexiglas chambers partitioned into compartments by organic matter-free membrane filters. Uptake of extracellularly released, labelled dissolved organic materials by cultured algal and bacterial epiphytes, separately and mixed in simulated natural communities, was followed under variously controlled conditions. Compari- sons of uptake of known concentrations of glucose and acetate at 5, 11-12 and 21—230 by cultured epiphytes per— mitted evaluation of both laboratory macrOphyte—epiphyte systems and field studies. Nutritional and physiological interactions of simulated communities of algae and bacteria are discussed. Functional aspects of macrOphyte—epiphyte metabolism in littoral ecology and lake trOphic dynamics are described in the form of a model. Major metabolic and nutritional interactions in the littoral zone of a representative freshwater ecosystem are discussed. Macrophyte-epiphyte metabolism is stressed as a source of dissolved organic materials and extracellular metabolites, potentially capable of regulatory effects on autotrophic productivity Harold LeRoy Allen in the pelagial environment and, as a dynamic system, nutritionally and physiologically interacting to sustain high levels of primary productivity and chemo-organotrOphy. PRIMARY PRODUCTIVITY, CHEMO-ORGANOTROPHY, AND NUTRITIONAL INTERACTIONS OF EPIPHYTIC ALGAE AND BACTERIA ON MACROPHYTES IN THE LITTORAL OF A LAKE By Harold LeRoy Allen A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1969 "T N; i i) \ VT 2'» T n 1 I \& ACKNOWLEDGMENTS The author would like to express his sincere appreci- ation to Dr. Robert G. Wetzel, w. K. Kellogg Biological Station and the Department of Botany and Plant Pathology, Michigan State University, for advice and much valuable criticism during the course of the investigation. Appreci- ation is also due Dr. Brian Moss and Dr. Stephen N. Stephenson, both of the Department of Botany and Plant Pathology, Michigan State University, and to Dr. James T. Staley of the Department of Environmental Sciences and Engineering, University of North Carolina, for critical reading of the manuscript. Dr. Peter Hirsch, Department of Microbiology and Public Health, Michigan State University, kindly provided a Zeiss Photomicroscope for examination of epiphytic samples. Dr. Eugene Stoermer, Great Lakes Research Division, University of Michigan, identified certain of the sessile diatoms. Dr. Robert R. L. Guillard, Woods Hole Oceanographic Institution, offered assistance with the selection of an apprOpriate inorganic medium for growth of isolated epiphytic algae, and provided several cultures of axenic algae, including the Cyclotella taxon employed in portions of this study. 11 These investigations were supported, in part, by the U. S. Atomic Energy Commission, Contract AT(ll-l)—1599 (C00-l599—24), and by the National Science Foundation Grant GB-6538 to R. G. Wetzel. The study was further assisted by the National Science Foundation Grant B0—15665 to G. H. Lauff, gt a1. (Coherent Areas Program for Investi- gation of Freshwater Ecosystems). iii TABLE OF CONTENTS Page ACKNOWLEDGMENTS . . . . . . . . . . . . . 11 LIST OF TABLES . . . . . . . . . . . . . Vi LIST OF FIGURES . . . . . . . . . . . . . ix I. INTRODUCTION. . . . . . . . . . . . 1 II. METHODS . . . . . . . . . . . . . 9 A. Lawrence Lake, Michigan. . . . . . 9 B. In situ assessment of epiphytic community metabolism. . . . . . 1A 1. Preliminary studies. . . . . l6 2. Measurement of annual cycle parameters 17 a) Primary productivity . . . . . 17 b) Chemo-organotrophy . . . . . . 22 c) Pigment composition. . . . . . 2H d) Taxonomic treatment. . . . . . 26 3. Determination of naturally colonized surface areas of macrOphytes. . . . 26 C. Field experimental studies. . . . . . 29 l. Precipitation of luC—monocarbonates and decontamination procedure . . . 30 2. Application of bioassays to the detection of physiological—nutritional interactions . . . . . . . . . 32 D. In situ macrOphyte— epiphyte interactions . 33 E. Axenic macrophyte- epiphyte interaction studies . . . . . . . . . . 37 iv Page III. RESULTS AND DISCUSSION . . . . . . . . A6 A. In situ methodological studies . . . 46 B. Epiphytic algal and bacterial community metabolism. . . . . . . . . . . 55 1. Productivity of epiphytic algal primary producers . . . 55 2. Physiological interactions through bioassay . . 89 3. Pigment composition and distribution in epiphytic standing crops. . . . 9A A. Chemo-organotrOphy of dissolved organic compounds . . . . . . . 108 C. In situ 1uC- -labelling of macrophyte- epiphyte systems. . . . 1A2 D. Nutritional and physiological inter- actions of axenic Najas flexilis L. and cultured epiphytes . . . . . . . 150 E. Functional aspects of macrophyte— epiphytic metabolism in littoral ecology and lake trOphic dynamics. . . . . 168 IV. LITERATURE CITED . . . . . . . . . . 177 Table LIST OF TABLES Composition of Artificial Lakewater Patterned After That of Lawrence Lake, Barry County, Michigan . . . . . . . . . . . Rates of Primary Productivity by Heterogeneous Epiphytic Algal Communities on Several Natural Macrophytic Substrata. Lawrence Lake, Barry County, Michigan; 19-20 September 1967 . . . . . . . . . A Comparison of Mean Annual Productivity Rates for Epiphytes on Artificial Substrata at the Scirpus Site (Station I) and at the Najas—Chara Site (Station II), Lawrence Lake. Carbon Fixation Data are Expressed on the Basis of Equivalent Square Meters of MacrOphytic Surface Mean Annual Primary Productivity by Attached Algae on Artificial Substrata at the Scir us Site (Station I) and Najas-Chara Site (Station II), Lawrence Lake, per m2 of the Littoral Zone . . . . .. . . . Mean Annual Primary Productivity for Attached Algal Organisms on Artificial Substrata at the Scirpus Site (Station I) and Najas- Chara Site (Station II) Substrata, Lawrence Lake, Integrated for the Entire Littoral Water Column. . . . . . . . . . A Comparison of Mean Annual Production by Algal Epiphytes in Lawrence Lake, Michigan, to Yearly Community Production Values from Various Aquatic Ecosystems Total Mean Annual Production by Attached Algae on Artificial Substrata at the Scirpus Site and NaJas-Chara Site, Lawrence Lake, Michigan . . vi Page 39 A7 63 85 86 88 9O Table 10. ll. l2. 13. 1“. Rates of Chemo-organotrophic Utilization of Glucose and Acetate by Heterogeneous Epiphytic Communities on Several Natural Macrophytic Substrata, Lawrence Lake, Michigan, 21-23 September 1967 . Comparison of Chemo-organotrOphic Utilization of Dissolved Organic Compounds by Epiphytic Communities Removed from the Emergent HydrOphyte, Scirpus acutus, Lawrence Lake, Michigan; 2“ September 1968 . . Mean Annual Rates of Chemo-organotrophic Utilization of Dissolved Organic Compounds by Epiphytic Communities, Lawrence Lake, Michigan. Data Were Collected from Artificial Substrata . . . Release of Dissolved Organic Matter (IAC_ labelled) by the Emergent Hydrophyte, Scirpus acutus Muhl., With and Without a Natural Epiphytic Community, Simultaneous to In Situ Photosynthesis in the Presence of THC02. Station I, Lawrence Lake, Michigan; 30 June 1969. . Release of Dissolved Organic Matter (IAC_ labelled) by the Emergent Hydrophyte, Scirpus acutus Muhl., Simultaneous to I Situ Photosynthesis in the Presence of 002. Station I, Lawrence Lake, Michigan; 30 June 1969 . . . . . . . . . . . Intracellular Fixed Carbon-1A in the Epiphytic Community of Scirpus acutus Muhl., Following In Situ Photosynthetic lEVIL-labelling of the Emergent Vascular Hydrophyte. (5 Hour Incubation; Station I, Lawrence Lake, Michigan; 30 June 1969) . . . . . Intracellular Fixed Carbon-1A in Scirpus acutus Muhl., Followin In Situ Photosynthetic Labelling With “C02. (5 Hour Incubation; Station I, Lawrence Lake, Michigan; 30 June 1969) . . . . . . . . . vii Page 115 117 1A1 IAN INS 1U8 1A9 Table Page 15. Comparison of Rates of Chemo-organotrophic Utilization of Glucose and Acetate by Individual Axenic Cultures of Algal and Bacterial Epiphytes Under Different Thermal Conditions. (G = Glucose; A = Acetate) . . . . . . . . . . . . 153 16. Comparison of Rates of Chemo—organotrophic Utilization of Glucose and Acetate by Mixed Axenic Cultures of Algal and Bacterial Epiphytes Under Different Thermal Conditions. (G = Glucose; A = Acetate) . . . . . . . . . . . . ISA 17. Uptake of INC—labelled Extracellular Products of Axenic Najas flexilis by Cultured Algal and Bacterial Epiphytes. (20C; Medium II; See Text for Experimental Procedure). (L = Light; D = Dark Incubation) . . . . 163 18. Uptake of luC-labelled Extracellular Products of Axenic NaJas Flexilis by Mixed Cultures of Algal and Bacterial Epiphytes. (20C; Medium II) . . . . . . . . . . . 165 l9. Utilization of Extracellular Products of Axenic Najas flexilis by Cultured Algal Epiphytes Subsequent to Microbial Metabo- lism of the Released Material. See Text for Explanation. (20C; Medium II; L = Light; D = Dark) . . . . . . . . . 167 viii LIST OF FIGURES Figure Page 1. Morphometric Map of Lawrence Lake, Barry County, Michigan, Showing Stations I (Dominant Macrophyte: Scirpus acutus) and II (Dominant Macrophytes: Najas flexilis and Chara spp ). This Map was Constructed with the Aid of Sonar (200kc sec- -1, Model F- 850- A, Furuno Electric Co., Ltd., Japan) Measurements Along Predetermined Transects; at Depths of Less Than 2 m Direct Measures Were Employed (cf. Wetzel, et a1. , in Preparation, for Further Details) . . . . ll 2. Systematic Treatment of Samples for Determi- nation of Routinely Monitored Community Metabolic Parameters of Primary Produc- tivity, Chemo- -organotrophy, and Pigment Composition Within the Epiphytic Complex (see Text). . . . . . . . . . 19 3. Experimental Plexiglas Chamber Used for Partitioning Uptake Kinetics of Extra— cellular Products from MacrOphytes by Algal and Bacterial Epiphytes . . . . . . A3 A. Mean Percentage Loss of 1”C Activity (as Precipitated Monocarbonates) from Filtered Epiphytic Algae Upon Exposure to Fumes of Concentrated HCl for Varying Periods of Time (Upper), and Loss of Activity (as Intracellular Fixed Carbon) by Rinsing Filters with Various Concentrations of Dilute Acid (Lower). Natural Epiphytic Algae Removed from Scirpus acutus Following In Situ Photosynthetic 180 Fixation in Lawrence Lake (Station I), Michigan, 1A March 1969. . . . . . . . . . . . 52 ix Figure 50 7. 8. In Situ Primary Productivity (mg C m-2 Macro- ) phytic Surface Area Day by Attached Algae at 5, 15, and 25 cm Above the Sedi- ments at the Scirpus Site (Station I), Lawrence Lake, Michigan. Data were Col- lected from Artificial Substrata. The Area Between the Upper and Lower Lines for Each Depth Represents Quantitative Precipitation as Inorganic Carbonates by Attached Microflora During Measurements of Photosynthesis . . . . . . . . . Situ Primary Productivity (mg C m 2 Macro— phytic Surface Area Day- 1) by Attached Algae at 5 and 10 cm Above the Sediments at the Najas—Chara Site (Station II), Lawrence Lake, Michigan. Data were Col- lected from Artificial Substrata . . . Isopleths of In Situ Primary Productivity (mg C m-2 MacrOphytic Surface Area Day 1) by Attached Algae at 5 to 25 cm Above the Sediments at the Scirpus Site (Station I), Lawrence Lake, Michigan. Data were Cor- rected for Radiocontamination as 1 C In- organic Carbonates Precipitated During Measurements of Photosynthesis. (Shaded Area = Ice Cover) . . . . . . Isopleths of In Situ Primary Productivity (mg C m-2 Macrophytic Surface Area Day 1) by Attached Algae at 5 to 10 cm Above the Sediments at the Najas—Chara Site (Station II), Lawrence Lake, Michigan. Data were collected from Artificial Substrata, and Are Corrected for Radiocontamination as 1”C Inorganic Carbonates Precipitated During Measurements of Photosynthesis . Situ Primary Productivity (mg C m 2 of Littoral Zone Day-1) by Attached Algae at 25 cm (A), 15 cm (B), and 5 cm (C) Above the Sediments at the Scirpus acutus Site Station I), Lawrence Lake, Michigan. Data were Collected from Artificial Substrata. The Area Between the Upper and Lower Lines for Each Depth Represents Productivity Error the Result of luc Precipitation as Inorganic Carbonates by Attached Micro- flora During Measurements of Photosynthesis X Page 58 6O 67 69 73 Figure 10. ll. 12. 13. Page 3; Situ Primary Productivity (mg c m"2 of Littoral Zone Day-1) by Attached Algae at 10 cm (A) and 5 cm (B) Above the Sediments at the Najas—Chara Site (Station II), Lawrence Lake, Michigan. Data were Col- lected from Artificial Substrata. The Area Between the Upper and Lower Lines for Each Depth Represents Productivity Error the Result of 1“C Precipitation as Inorganic Carbonates by Attached Micro- flora During Measurements of Photosynthesis . 75 Isopleths of In Situ Primary Productivity (mg C m"2 of Littoral Zone Day‘l) by Attached Algae from the Sediment-Water Interface to 30 cm Above the Sediments at the Scirpus acutus Site (Station I), Lawrence Lake, Michigan. Data were Collected from Artificial Substrata, and are Corrected for Radiocontamination as 1“C Inorganic Carbonates Precipitated During Measurements of Photosynthesis. (Shaded Area = Ice Cover) . . . . . . . . . . . . . 78 Isopleths of In Situ Primary Productivity (g C m"2 of—Littoral Zone Day‘l) by Attached Algae from the Sediment-water Interface to 20 cm Above the Sediments at the NaJas-Chara Site (Station II), Lawrence Lake, Michigan. Data were Collected from Artificial Sub- strata, and are Corrected for Radiocontami- nation as 1”C Inorganic Carbonates Precipi- tated During Measurements of Photosynthesis . 80 Situ Integrated Primary Productivity (g C m72 of Littoral Zone Day-l) by Attached Algae at the Scirpus acutus Site (A; Station I), and at the NaJas flexilis and Chara spp. Site (B; Station II), Lawrence Lake, Michigan. Data were Collected from Artificial Sub- strata. The Area Between the Upper and Lower Lines for Each Vegetative Site Repre- sents Productivity Errors the Result of luc Precipitation as Inorganic Carbonates by Attached Microflora During Measurements of Photosynthesis. . . . . . . . . . . 82 xi Figure Page I“. Effect of Iron and Chelating Agents (Ethylene- diaminetetraacetic Acid, Disodium Salt; 8—hydroxy-5-quinoline Sulfonic Acid; and Extracted Limnohumic Substances) on Growth of Natural Epiphytic Algae Removed from Scirpus acutus in Lawrence Lake (Station I), Michigan, 8 to 10 September 1968 . . . 93 15. Isopleths of Corrected Chlorophyll a Concen- trations (mg m- -2 of Littoral Zone) of Attached Algae at the Scirpus Site (Station I), Lawrence Lake, Michigan . . . . . . 96 16. Isopleths of Plant Carotenoid Concentrations (Millispecified Plant Pigment Units m*2 of Littoral Zone) of Attached Epiphytic Algae at the Scirpus Site (Station I), Lawrence Lake, Michigan . . . . . . . . 96 17. Is0pleths of Corrected ChlorOphyll a Concen— trations (g m 2 of Littoral Zone) of Attached Algae at the NaJas- -Chara Site (Station II), Lawrence Lake, Michigan. . . 98 l8. Isopleths of Plant Carotenoid Concentrations (Millispecified Plant Pigment Units - 103 m-2 of Littoral Zone) of Attached Algae at the Najas- -Chara Site (Station II), Lawrence Lake, Michigan . . . . . . . . . 98 19. Integrated Chlorophyll a Concentrations (g m-2 of Littoral Zone) of Attached Algae at (A) the Scirpus acutus Site (Station I), and (B) the Najas flexilis and Chara spp. Site (Station II), Lawrence Lake, Michigan. Data were Collected from Artificial Sub- strata and are Corrected for the Presence of Pheophytin Degradation Products. . . . 10A 20. Integrated Plant Carotenoid Concentrations 3 (M%llispecified Plant Pigment Units - 10 of Littoral Zone) of Attached Algae at (A) the Scirpus acutus Site (Station I), and (B) the Naias flexilis and Chara spp. Site (Station II), Lawrence Lake, Michigan. Data were Collected from Artificial Sub- strata . . . . . . . . . . . . . 106 xii Figure Page 21. Kinetics of Chemo-organotrOphic Utilization of Dissolved Organic Compounds by Fresh- water Microflora, Illustrating (A) Differentiation of Bacterial and Algal Uptake in Response to Increasing Sub- strate Concentrations, and (B) Graphical Representation of Uptake Kinetics of Dilute Concentrations of Glucose-luC by Attached Bacteria from 5, 15, and 25 cm Above the Sediments in Scirpus acutus Site (Station I), Lawrence Lake, Michigan, 28 July 1968 (Cut/c = l/% of Uptake; See Text) . . . . . . . . . 112 22. Chemo-organotrOphic Utilization of Glucose (VmaXS pg Glucose Removed l’1 hr-l) by Attached Bacteria from 1 dm2 of Colonized Surface Area at 5, 15, and 25 cm Above the Sediments at the Scirpus Site (Station I), Lawrence Lake, Michigan. Data were Collected from Artificial Substrata . . . 121 23. Chemo-organotrophic Utilization of Acetate (Vmax3 ug Acetate Removed 1"l hr-l) by Attached Bacteria from 1 dm2 of Colonized Surface Area at 5, 15, and 25 cm Above the Sediments at the Scirpus Site (Station I), Lawrence Lake, Michigan. Data were Col- lected from Artificial Substrata . . . . 123 24. Chemo-organotrophic Utilization of Glucose (VmaxS ug Glucose Removed 1"l hr'l) by Attached Bacteria from 1 dm2 of Colonized Surface Area at 5 and 10 cm Above the Sediments at the Najas-Chara Site (Station II), Lawrence Lake, Michigan. Data were Collected from Artificial Substrata . . . 125 25. Chemo-organotrophic Utilization of Acetate (Vmax; ug Acetate Removed 1-l hr'l) by Attached Bacteria from 1 dm2 of Colonized Surface Area at 5 and 10 cm Above the Sediments at the Naias-Chara Site (Station II), Lawrence Lake, Michigan. Data were Collected from Artificial Substrata . . . 127 xiii Figure Page 26. Is0pleths of Chemo-organotrOphic Utilization of Glucose (VmaxS ug Glucose Removed 1"l hr-l) by Attached Bacteria from 1 dm2 of Littoral Zone at the Scirpus Site (Station I), Lawrence Lake, Michigan. Data were Collected from Artificial Substrata . . . 131 27. ISOpleths of Chemo-organotrophic Utilization of Acetate (Vmax3 ug Acetate removed 1"1 hr-l) by Attached Bacteria from 1 dm2 of Littoral Zone at the Scirpus Site (Station I), Lawrence Lake, Michigan. Data were Collected from Artificial Substrata . . . 131 28. ISOpleths of Chemo-organotrOphic Utilization of Glucose (Vmax3 ug Glucose Removed 1'1 hr- ) by Attached Bacteria from 1 dm2 of Littoral Zone at the Najas-Chara Site (Station II), Lawrence Lake, Michigan. Data were Collected from Artificial Sub- strata. . . . . . . . . . . . . 133 29. Isopleths of Chemo-organotrophic Utilization of Acetate (Vmaxs ug Acetate Removed 1-l hr-l) by Attached Bacteria from 1 dm2 of Littoral Zone at the Naias-Chara Site (Station II), Lawrence Lake, Michigan. Data were Collected from Artificial Sub— strata. . . . . . . . . . . . . 133 30. Integrated Chemo-organotrOphic Utilization of Glucose (Vmax3 ug Glucose Removed l-l hr’l) by Attached Bacteria from 1 dm2 of Littoral Zone at (A) the Scirpus acutus Site ' (Station I), and (B) the Najas flexilis and Chara spp. site (Station II), Lawrence Lake, Michigan. Data were Collected from Artificial Substrata . . . . . . . . 137 31. Integrated Chemo-organotrophic Utilization of Acetate (Vmax3 pg Acetate Removed 1‘ hr'l) by Attached Bacteria from 1 dm2 of Littoral Zone at (A) the Scirpus acutus Site (Station I), and (B) the Naias flexilis and Chara spp. Site (Station II), Lawrence Lake, Michigan. Data were Collected from Artificial Substrata . . . . . . . . 139 xiv Figure Page 32. Rates of Photosynthetic Incorporation of Carbon-1H (uCi g' '1 Dry Weight hr 1) by Cultured Axenic Najas flexilis L. . . . 159 33. Percentage of Mean Excretion of Dissolved Organic Compounds to the Mean Rates of Photosynthetic Carbon Fixation by Axenic Najas flexilis L. . . . . . . . 161 3A. Diagrammatic Representation of Metabolic and Nutritional Interactions Existing in the Littoral Zone of a Representative Calcareous Aquatic Ecosystem. (See Text; PS = Photosynthesis; DOM = Dissolved Organic Matter) . . . . . . . . . 173 XV I. INTRODUCTION A substantial portion of temperate North America, inclusive of the Great Lakes region, is represented with a large number of lakes, ponds, marshes, and similar aquatic habitats of shallow to moderate depth. A large majority of these aquatic ecosystems characteristically possess well- defined littoral zones, with extensive deve10pment of sub- merged and emergent vascular hydrophytic vegetation. The communities of shallow habitats are commonly thought to contribute significantly to the total energy fixation, utilization, and transformation. Little investi- gative effort, however, has been directed to comprehensive studies of littoral metabolism and the functional inter- actions and dynamics of these communities (see discussion by Wetzel, 196”). Frequently, a quantitatively significant portion of the total biomass of primary producers and de- composers constitutes the sessile community (herein referred to as the periphytic or attached algal and bacterial flora) and strongly suggests biomass that is often quantitatively dominant over their pelagial counterpart. Thus, the littoral community may be capable of exerting a strong influence on observed annual cycles and dynamics of pelagic Dhyto- and zooplankton through direct and indirect interactions. Such influences, based on the function of dissolved organic compounds and metabolites in natural waters (see discussion of Wetzel, 1968), may have pronounced effects in regulation of eutrOphicational ontogeny within many of these fresh waters. Wetzel and Allen (1970) have suggested interactive mechanisms, functioning in littoral and pelagial environ- ments, which are potentially responsible for regulation of various aspects of trophic dynamics. Detailed theoretical and experimental evidence continues to reaffirm the close physiological interrelationships and nutritional inter— dependencies existent between the epiphytic, epibenthic, and pelagial floras. Exceedingly little information exists with regard to the quantitative contribution of epiphytes (hereafter con- sidered the sessile microflora colonizing only vascular macrOphytic vegetation) to the total primary producers pool within an aquatic ecosystem. There is, however, a volumi- nous literature generally applicable to the periphytic community, with stress being placed on the descriptive aspects of spatial and temporal population dynamics and community structure (Wetzel, 196“), but little consider- ation of nutrition, energetics, or physiological metabo- lism is evident. Few investigators have sought to elucidate the interactions existing between the host macrophytic vege- tation and the attached algal and bacterial flora in freshwater environments. Linskens (1963) has determined the direction of transport of inorganic phosphorus between several marine macro-algal species and certain of their dominant epiphytic algae and noted specifically in which cells (epiphytes) and morphological structures (host macro— forms) the translocated isotope became localized. Other physiologically related studies on marine algae and their attached algal flora include Funk (195“), v. d. Ende and Linskens (1962), v. d. Ende and v. Oorschot (1963), and Beth and Linskens (196“), but similar studies on freshwater systems are lacking. Studies contributing to our present understanding of the distribution of algal (or bacterial) epiphytes and causal mechanisms influencing attachment and subsequent growth are exiguous in the literature. Annual periodicity of the attached algal flora, especially with reference to changes in environmental and water-quality parameters (light, thermal conditions, inorganic chemistry, wave activity, etc.) has received cursory attention (see repre- sentative studies by Willer, 1923; Godward, 1934; Knudson, 1957; Cannon, 32 al., 1961; Kowalczewski, 1965; and others). Many of the studies in this area have not dealt with the epiphytic vegetation in its natural heterogeneity, but have isolated representative taxa from natural substrata to evaluate their individual growth and metabolic requirements under controlled laboratory conditions. Such studies would be of greater value in the interpretation of observed in §i§u_changes in population densities, species compo- sitional shifts, and community metabolism, if experimental conditions simulated natural conditions. Some studies (Godward, 193“; Prowse, 1959) have demonstrated a distinct specificity of certain epiphytic algae for select macrophytic hosts. If their observations are representative and extrapolatable to the dominant taxa and vascular hydrOphytes commonly colonizing littoral areas, then a host of potential interactive mechanisms will re- quire eventual investigation. Certain of these mechanisms are most likely to function and be detectable at very subtle levels and probably involve both commonly shared and more specific unidirectional metabolic interactions. Jorgensen (1957) suggested that the epiphytic diatom, Tabellaria flocculosa, is capable of utilizing silica directly from the stems of Phragmites, especially since Si is easily dissolved and decreases synchronously within the hydrOphyte during periods of peak population density of the diatom. His circumstantial evidence is further sup- ported by spring and fall epiphytic maxima which do not coincide with the maximum development of planktonic diatom communities. Such prOposed nutritional supplementation by the macrophyte may be beneficial in the development of seasonal growth patterns distinctly different from those of the epibenthic or planktonic communities, and further decrease the probability of direct competition for specific nutritional elements or essential metabolites. The epibenthic habitat (epilithic, epipelic, and epipsammic communities) has been little studied function- ally. Studies are beginning to emerge where estimates of their integrated ecological importance and various aspects of their productivity and overall metabolism are considered. Much of the exhaustive literature prior to 1963 has been reviewed by Wetzel, 196“; similarly, much literature specific to epipelic and epilithic habitats has been in- cluded by Round, 196“. Other lotic and lenitic studies of particular interest, primarily since 1963, include Grbntved, 1960, 1962; Eichelberger, 1963; Sladecek and Sladeckova, 1963, 196“; Wetzel, 1963, 1965a, 1969c; Pieczyfiska, 1965; McIntire and Phinney, 1965; Kevern and Ball, 1965; Kevern, gt al., 1966; King and Ball, 1966; Szczepanski and Szczepanska, 1966; Cushing, 1967; Moss and Round, 1967; Backhaus, 1967, 1968; Grzenda, gt_al., 1968; Peters, et al., 1968; Steele and Baird, 1968; Moss, 1968, 1969; Pamatmat, 1968; and Hargrave, 1969). As in previous studies of epiphytic algal metabolism, there have been no investi- gations which emphasize synthesis or integration of com— munity parameters that would, in turn, allow a direct estimate of their function or contribution to the lake as a whole. For at least “0 years the majority of these studies were concerned with assessment of biomass (dry weight or organic matter content), and species composition, and distributional changes. Recently, emphasis has shifted to in sitg measurements of primary productivity. Variations of the carbon-1“ technique have been employed in a few cases to measure photosynthetic rates of attached com- munities under natural conditions. .Unfortunately, much of the work accomplished with artificial substrata is of little relevance in determining the importance of ig_§itg metabolism by attached communi- ties. Where slides (or other substrata) have been sus- pended vertically in reservoirs and deeper waters, and subsequently allowed to undergo colonization, communities which are atypical in a planktonic environment may have developed. Attachment during prolonged stratification may reveal periphytic communities which are representative of natural populations and distributions of organisms in the plankton (especially applicable to the bacterial micro— flora). The real value in employing artificial substrata is to obtain attached community deve10pment where the natural substrata do not rapidly lend themselves to repro- ducible sampling procedures in littoral areas, and where attached communities are normally found, in littoral and shallow water zones. Sampling of the natural heterogeneous community com- plex presents many obvious problems and depends upon the question being asked and precision required. Undoubtedly Such problems are responsible in part for the lack of supporting information on littoral ecology that exists for the pelagial environment. The littoral is frequently deleted from even the most thorough and comprehensive investigations. Further, of those studies which have attempted to look at the ecological role of attached com- munities from their overall systems-importance, few have achieved the necessary partitioning of the littoral environ- ment into some of its basic structural components, i.g. into macrophytic vegetation, periphyton, etc., in order to make more direct and valid comparisons with the Open water communities (Wetzel, 196“; Hargrave, 1969). The present studies were designed to elucidate the relative importance of primary productivity and chemo— organotrOphy (with selected supporting descriptive param- eters) of epiphytic algae on submerged and emergent aquatic vegetation within a small lake to lake metabolism in general. Certain of the nutritional and metabolic inter- actions of host vascular hydrOphytes and their attached heterogeneous algae and bacteria were studied. Potential direct and indirect regulatory effects of the collective littoral community on the pelagial environment and subse- quent control over aspects of lake trophic ontogeny were considered. Estimates of in §i£u_primary productivity and chemo-organotrophic utilization of dissolved organic com- pounds, as employed in this study were based on modifi- cations of lL‘C-methodology. Metabolism of extracellularly released organic materials by axenic cultures of a fresh- water macrOphyte (Najas flexilis L.), and subsequent partitioning of uptake by cultured algal and bacterial epiphytes in specially constructed chambers, also employed lMC tracer techniques. A technique designed for the in situ labelling (with 1” CO2) of an emergent hydrOphyte (Scirpus acutus Muhl.), with potential applicability to other littoral metabolic studies, was develOped and used under field conditions. Resultant data, from both field and concomitant laboratory studies of epiphytic metabolism, strongly sug- gest that this community may easily be a dominant primary producer in many of our typical freshwater ecosystems, and through effective interaction in the cycling of dissolved organic compounds function at subtle levels to accelerate or retard eutrophicational processes and lake ontogeny. From the magnitude of measured annual rates of in gitu photosynthesis, it is becoming increasingly obvious that we can no longer Justify neglect of this part of the aquatic ecosystem, especially when evaluating trophic dynamics at the inter- and intrasystems level. II. METHODS A. Lawrence Lake, Michigan Lawrence Lake (85° 21' W, “2° 27' N) is located in the southwestern corner of lower Michigan (Barry County) which, and in consociation with a smaller lake and sur- rounding marsh area, forms a basin confluent with the southern outwash apron of the Kalamazoo morainic system (Rich, 1970). The surrounding terrain is characterized geologically by the presence of glacial till and undulating plains. The origin of the lake, as evidenced by the steep— ness of the basin (Wetzel, gt al., in preparation), proba- bly followed melting of a buried terminal ice block. The cultural history of Lawrence Lake, indicating post- settlement influences to the lake system proper, and sur- rounding watershed, are documented in considerable detail by Rich (1970). Lawrence Lake lies along a NE-SW axis at an elevation of 275.3 m above sealevel, and receives visible drainage from two very small inlets (Figure l). The single outflow eventually terminates at Augusta Creek. There is some evidence that vernal springs may also contribute to the lake. The watershed is approximately 10 times the surface area of the lake and consists primarily of fallow fields. 9 10 II II II .Amafimpov pmnppsm mom .coapMpmdmuo CH A.Hm um .Hmupmz .mov UmmOHQEm who: monummoe pommfip Em cmnp mmoa no mnpmmp pm mmpomwcmpp pmcHEmmumbmpm wcoam mpcmEmpdmmoE Acmamh ..ppq ..oo campomam ocspsm . 500 pg 1'1) has little relevance in interpretation of naturally observed bacterial—decomposer and chemo- organotrOphic activity, in that detectable responses may be induced or the result of site-inhibition phenomena. Short incubation periods are necessary to avoid possible recycling of metabolites through extracellular release (or as accumulated by-products) and subsequent re- utilization, in accordance with previously established rates of bacterial turnover of organic substrates (see discussion in Allen, 1969). For a more detailed discussion 2“ of the suitability, and applicability, of these measure- ments based on Michaelis-Menten enzyme kinetic analyses and active transport capabilities, to the study of fresh- water microbial metabolism, the reader is referred to the original work of Wright and Robbie (1965a, 1965b, 1966). Glucose and acetate were routinely employed because of their representative involvement in more gener- alized microbial metabolic schemes. On several occasions, other hexose sugars (fructose, galactose), amino-acids (glycine, serine, alanine), and Krebs-Cycle intermediates (succinate, glycolate) were tested. As with the photo- synthetic measurements, maximum rates of utilization of organic substrates by the microflora (pg substrate removed ‘1 hr'l) were equated to surface areas of natural or 1 artificial substrata. In strict interpretation, the new parameter of uptake would become: pg of substrate removed per liter per hour by the microflora colonizing 1 dm2 of plant or artificial substrata. Presentation of chemo- organotrophic data on a dm2 as opposed to per m2 basis emphasizes the dynamics of utilization from small, colonized surface areas where the bacterial component is very hetero- geneously distributed in the epiphytic community. 0) Pigment composition Small aliquots (20 ml; Figure 2) of resuspended epiphytic organisms were concentrated onto Millipore HA membrane filters under low vacuum (< 0.5 atm), placed 25 between absorbant pads, and immediately frozen under desiccation until extraction and analysis could be per— formed (1 to 2 weeks). Extraction was accomplished by cell homogenization (1 minute; compressed—air driven Teflon—glass homogenizer) in reagent grade, aqueous 90% basic acetone. After centrifugation, standard spectro- photometric (Hitachi-Perkin Elmer, Model UV-VIS 139, or Beckman DK-2A, ratio—recording spectrophotometer; 10 cm light path) techniques were applied (Strickland and Parsons, 1965). Computation of concentrations of chloro- phyll g and total plant carotenoids followed equations given by Strickland and Parsons. Subsequent to acidifi— cation of samples (1 drop of l N HCl) in spectrophotometer cuvettes, absorption peaks were redetermined to permit estimations of the concentration of pheo-degradation pig- ment products. Calculations of concentrations of chloro— phyll g corrected for the presence of pheophytin products (see discussion by Moss, 1967a, 1967b) followed equations of Lorenzen (1967). Although absorption data were deter- mined for chlorophylls g, t, and g, quantitative evaluation of g and g was not attempted in this study. As with pri- mary productivity field data, chlorophyll g, and total plant carotenoids are expressed as mg or g, and milli- specific pigment units (MSPU) or MSPU - 103, per m2 of artificial substrata or littoral zone. 26 d) Taxonomic treatment Samples of epiphytic communities (10 ml; Figure 2) were routinely preserved from each depth, at each of the two stations, throughout the annual period. Evaluation of species composition and distribution was limited to a cursory examination by the settling-chamber technique (UtermOhl, 1958) and an inverted microscope (Wild, Model M-“0). Dominance among the attached flora was usually determined, however, through the use of orthodox prepared slides and a compound light microscope. Both, living and preserved samples of intact, epiphytic community structure were examined with a Zeiss PhotomicrOSCOpe, after embedding thin-sections of natural macrOphytic cross-sections and surfaces in vaspar (1:1 mixture of vaseline and paraffin wax). Photographic documentation has revealed distinctly stratified patterns within natural communities of epiphytes. 3. Determination of naturally colonized surface areas of macrOphytes Quantitative and qualitative data of rates of photo— synthesis, chemo-organotrophy, and pigment composition from artificial substrata were expressed in two ways: 1) as a rate or concentration per dm2 or m2 of colonized surface area at a specific depth in either of the two stations or 2) by employing factors for the macrophytic surface area 2 present per dm2 or m of littoral zone, proportionally corrected for the macrophyte biomass present, per unit 27 area (dm2 or m2) of the littoral zone. Expression of these data on the basis of total macrophytic surface areas per unit area of littoral permits a direct comparison of moni- tored values with identical parameters in the pelagial. Several procedures were used to obtain estimates of macrophytic surface area supported per m2 of the littoral zone. Station II is dominated by Najas flexilis and several species of Qgggg, Owing to the high heterogeneity in natural distribution, representative plants were excised at the sediment-water interface by removal of replicated cores (diameter: l“.9 cm2) along predetermined transects parallel and perpendicular to the shoreline. The macro- phytic biomass was carefully rinsed in a floating sieve, blotted to remove excess water, and gently placed in a tared, shallow aluminum pan containing a known quantity (by weight) of surfactant (Teepol 610R, Shell Chem. Corp., N. Y.; and a 50% dilution of Tween 80R, E. H. Sargent & Co.; both were independently tested). Plants, after 10 to 15 seconds immersion, were drained for several seconds and removed. Loss in weight of the surfactant is equivalent to the area covered as determined by previous calibration with various objects of known surface area (technique closely followed that of Harrod and Hall, 1962). Differences in the weight of surfactants, used for conversion to surface area equivalents, were not corrected for evaporative losses during tg situ measurements. Transport of the 28 surfactant to and from the field in air-tight, screw- capped containers minimized such losses. Surface areas of individual Scirpus acutus plants (Station I) were determined by computation, using the formula for a subtended cone, with data of the leaf (stem) diameter at various heights above the sediment and the vertical distance to the surface. The mean number of Scirpus plants per m2 was determined in transects perpen- dicular and parallel to the shoreline, and multiplied by the mean colonizable surface area per plant for a measure- ment of the total colonizable area exposed per m2 of the littoral zone. Estimates of macrophytic surface area were determined during spring and fall; interpolated surface areas for sampling intervals throughout the annual period were applied to monitored parameters only to gain a better understanding of the magnitude of natural biomass change. The surface area conversions represent only approximate values. Compu- tation of all annual cycle data for Scirpus substrata includes correction in surface area per m2 of littoral zone, for fluctuation in surface water level. Data from the uppermost sample (25 cm above the sediments) are used for calculation of rates or concentrations for the interval from 30 cm to the surface. Sample data from 5, 15, and 25 cm above the sediments (Station I) were used for calcu- lation of parameters in iSOpleth figures (0-10 cm; 10-20 cm; and 20-30 cm); at Station II, data from 5 and 10 cm 29 samples are used to express the vertical intervals (0-10 cm; 10—20 cm). Mean height of the submergent plant story by transect measurement was 20 cm. Although expression of data in isopleth form may be criticized for potential over-resolution, such presentation is of definite value in visualizing dynamic changes which would be obscured, or lost entirely, in individual line— drawings and histograms. Annual cycle data are graphically presented in several forms: 1) as individual parameters at depth versus time, 2) as isopleths, integrative of several depth intervals against time, and 3) as integrated line- drawings of the summation of a parameter with time. Estimates of the contribution of epiphytic metabolism to the total lake were obtained by multiplication of annual mean rates (planimetrically Obtained) by the surface areas (means for vegetative season) of the littoral zone from 0 to 60 cm depth in which species of Chara, Najas flexilis, and Scirpus acutus are found. These latter estimates were determined by planimetry of species vegetational maps of Lawrence Lake, from 0 to 60 cm depth, for the above species. C. Field experimental studies Several experimental approaches were applied in an attempt to elucidate some of the physical, chemical, and biological interactions occurring within the epiphytic community complex under natural conditions and to aid in 30 interpretation of observed annual fluctuations in assessed metabolic parameters. 1. Precipitation ofluC-monocarbonates and decontamination procedure Measurements of rates of photosynthetic activity in aquatic environments employing inorganic carbon-l“ have been demonstrated to be erroneously high in certain in- stances, owing to extracellular precipitation of 1”C mono— carbonates (g.g. Paasche, 1963 cited by Wetzel, 1965c). Various procedures have been used for removal of deposited carbonates, including immediate rinsing of filtered algae with dilute acid (Strickland, 1960; Goldman, 1963), and by brief exposure to fumes of concentrated HCl (Steeman- Nielsen, 1952; Wetzel, 19650). McAllister (1961) and Paasche (1963) have demonstrated that even 1 to 2 minute exposure to fumes of concentrated hydrochloric acid is sufficient to remove the contaminant activity. A 10 minute exposure has been suggested by Wetzel (1965c) as a standard procedure in radioassay studies of photosynthetic activity. Such decontamination procedures are necessary in studies involving the epiphytic complex. Deposition of luC as monocarbonates by epiphytic algae and bacteria is rapid, even during 3 to “ hour 1g.gttg incubation periods. This precipitation was especially evident in the dark-bottle samples for photosynthesis during winter periods under ice cover and poor illumination. 31 Epiphytic samples were removed from natural Scirpus acutus substrata (15 cm above the sediments; 1“ March 1969) after tg_gttg_photosynthetic labelling and redistributed into 150 ml of ultrafiltered lakewater from the growth site. Thirty duplicate aliquots (5 ml each) were filtered onto Millipore HA filters. Triplicate samples were dried, desiccated, and exposed to fumes of concentrated HCl for varying periods of time. Identical triplicate samples were also rinsed with 5 ml aliquots of various concentrations of dilute acid. In both treatments l“CO2 evolved was precipitated with barium hydroxide, concentrated onto mem- brane filters, and radioassayed in a G.-M. prOportional counter. Loss of intracellularly fixed carbon from the algae by treatment with small aliquots of HCl solution was estimated by conversion of the removed dissolved organic 1“ material to CO by persulfate oxidation (Menzel and 2 Vaccaro, 196“). Radioassay of the evolved carbon dioxide was accomplished with a vibrating-reed ionization-chamber electrometer system. Data obtained from all samples analyzed by a conventional Geiger-Muller end-window, gas- flow system were similarly converted to absolute radio- activity by combustion of replicate control samples in gas-phase. A series of filtered samples was also treated by direct immersion in 0.10 N hydrochloric acid for 30 seconds. 32 2. Application of bioassays to the detection of physiological—nutritional interactions The tg'gttg_carbon—l“ bioassay of limiting factors for stimulatory or inhibitory response of actively photo- synthesizing algal organisms has been applied in both freshwater and marine environments (for example, see Ryther and Guillard, 1959; Menzel and Ryther, 1961; Goldman and Mason, 1962; Goldman, 1960a, 1960b, 1962, 1963, 196“, 1965, 1967; Goldman and Wetzel, 1963; Wetzel, 196“, 1965b; Gold- man and Carter, 1965; and others). Although this technique is most frequently applied to planktonic algal populations for detection of nutrient limiting factors to primary pro- ductivity, its sensitivity and rapidity is suitable for a general assay of nutritional and metabolic interactions between host macrOphytes and their epiphytic community complexes. Detection of the level of bacterial or algal stimulation or inhibition or causal mechanisms involved in perpetuating the response, was not the parameter sought here. Rather the composite community response was desired. Responses to added micro-nutrients by autotrOphic organisms within the epiphytic complex might reveal the direction and magnitude of movement of certain nutritionally important micro-nutrients and metabolites. The likelihood is that materials are translocated between the macrophyte and its epiphytes. Epiphytic samples were removed from natural Scirpus substrata (15 cm above the sediments; 8 September 1968) 33 and redistributed into ultrafiltered lakewater (2000 ml) collected from the growth site. Identical 100 m1 aliquots were dispensed into ground-glass stOppered, transparent Pyrex bottles (125 ml) and labelled. The bottles were incubated under controlled laboratory conditions (gg. 1000 lux; 25C; on a 3 rpm rotating table). Prior to incubation, micro-additions of the following were made: (1) inorganic iron (FeCl3) separately and in combination with low con- centrations of artificial and natural complexing agents, (2) inorganic phosphorus (K2HPOu), (3) vitamin mixture of B biotin, and thiamine hydrochloride, and (“) trace 12’ metal mixture of CuSOu, ZnSOu, CoCl MnCl2, Na2MoOu, and 2’ H3BO3. Algae were concentrated onto Millipore HA filters and treated as described for photosynthetic samples. Activity of prefiltered controls were subtracted from those of the experimental, to eliminate possible error by in- organic precipitation of 1“C where micro—additions of iron were used (Goldman and Mason, 1962). D. Ig_situ macrophyte-epiphyte interactions Although extracellular release of dissolved organic compounds has been demonstrated with axenic laboratory cultures of Najas flexilis (Wetzel, 1969a, 1969b), there have been no confirmatory studies which would support the occurrence of a similar release under completely natural conditions in the field (Sieburth, 1969; Sieburth and 3“ Jensen, 1969; and Khailov and Burlakova, 1969, have docu- mented lg gltg exudation by marine macroalgae). The pur- pose of this experiment was to demonstrate the presence of extracellular dissolved organic matter (DOM) production by an emergent hydrOphyte, Scirpus acutus, and chemo- organotrophic utilization by epiphytic organisms of these materials arising from the plant. Small, light (< “ g) chambers of transparent Handi- WrapR (Dow Chemical Co., Midland, Mich.) with reinforced polystyrene supports (8 x 8 x 16 cm; approximately 1 liter volume) were placed over the emergent tips of three Scirpus acutus plants (Station 1, Lawrence Lake). A small ampoule was affixed to the innerside of each chamber, containing BaluCO3 of known radioassay (52.0 mCi mM-l, 261 pCi mg-l; 8“% isotOpic abundance in the carbon atom). The exact amount of barium carbonate-luC was determined with gravi— metric precision (1.89579 mg BaluCO3 per ampoule; Cahn Electrobalance) such that when 0.20 ml of 1 N HCl was injected through the chamber wall, l”C02 would be evolved at near atmospheric concentrations (0.035% v/v) similar to the technique employed by Dahlman and Kucera (1968) in translocation studies of grassland vegetation. Diffusion of radioisotopes in liquid (glucose and acetate-1H0) and gaseous (1“002) phase through the membrane could not be demonstrated. Three types of experimental treatment were designed. On one hydrOphyte the epiphytic complex was removed by 35 gentle friction of glassfiber filter pads, after which a transparent Plexiglas tube (inside diameter: “.5 cm) was carefully inserted into the sediments surrounding the plant. The top of the Plexiglas tube remained above the water surface and effectively isolated the immediate en- vironment of the Scirpus plant. A second tube was posi— tioned around arother Scirpus plant with the epiphytic community intact. A third plant, without a tube enclosure and with its natural attached flora undisturbed, served as a control. 1“ Upon evolution of the CO in the field water samples 2 adjacent to the plants and samples of the attached com- munities were systematically collected at 0, 15, 30, 60 minutes, and 1 to 5 hours. At the termination of the experiment, the Scirpus plants were sectioned at 2 cm intervals from the emergent tip into the rhizome tissue to determine internal labelling patterns. All samples, except those of the attached com- plex, were assayed for the presence of radioactivity by liquid scintillation techniques (employing a Beckman LS-150 ambient temperature Scintillation counter). Epiphytic samples were assayed after concentration, by solid—counting procedures. Dissolved organic matter samples were collected in the field by a simple suction-tube device, filtered, and pipetted (1 m1 duplicate aliquots) directly into glass vials containing 10 ml of scintillation fluid (8.05 g 36 FluoralloyR mix (Beckman Instruments, Inc., Calif.) dis- solved in 1 liter of toluene; 2-aminoethanol, a carbon dioxide absorbant, was added at a ratio of 1:10 parts toluene in certain samples, but Triton-XR (Packard Inst. Co., 111.) at 6:7 (v/v) of toluene plus Fluoralloy mix was primarily used; the latter solvent:scintillator mixture is especially recommended where the sample to be assayed contains considerable water). Carbon dioxide-lac contained within the water, and within the plant tissue at the termi- nation of the experiment, was determined by immediate acidification (“ drops of l N HCl) of the counting fluid to a pH of less than “.0, and trapping the evolved 1“CO2 in an NCSR (0.6 N solubilizer solution in toluene; Amersham/Searle, Ill.)-based scintillator fluid; NCS functions as a quantitative carbon dioxide absorbant (Zimmerman, 1967). Activity remaining in purged samples was present as dissolved organic compounds. Digestion of Scirpus plant tissue was accomplished with NCS (1.“ ml added to 8.6 ml of toluene-Fluoralloy mixture) at “C for “8 hours in total darkness. Similar digestion of plant material was effected with Bio-Solv solubilizer (Beckman; Formula BBS-3) at the same concentrations. Severe color-quenching from chlorOphylls resulted from digested macrophytic tissue and required standardi- zation of the instrument with a color-quenching series prior to assay of plant samples. Standardization with liquid, organic isotOpes (in the above toluene-based 37 scintillator fluids) of known radioassay was necessary prior to each assay. Additional standards were prepared by addition of organic (glucose and acetate-luC) isotopes to various concentrations of extracted pigments from un- labelled Scirpus plants. All samples assayed by liquid- scintillation techniques were counted to a preset error of t 2.0% (equivalent to 105 disintegrations per minute). E. Axenic macrophyte-epiphyte interaction studies Studies of epiphytic algal and bacterial metabolism, with isolated and purified cultures under controlled laboratory conditions, have not previously been reported. Interactions of epiphytic organisms and their host macro- phytes under natural field conditions are also exiguous in the current literature. For correlative purposes and evaluation of lg gltg_monitored parameters and observable growth patterns, some knowledge of the interactive mechan- isms and interrelations of bacterial, algal, and macro- phytic metabolism under fixed conditions is desirable. For purposes of conducting experiments with direct interpretative value to field conditions, isolation and purification of several species of algae and bacteria from natural macrOphytes was initiated. Resuspended (into ultrafiltered lakewater) epiphytic samples from Scirpus acutus, Najas flexilis, and Chara sp. were repeatedly streaked onto plates of solidified (0.5% agar) Lawrence 38 Lake water from the growth site. Small aliquots were also introduced into 1) Rodhe's VIII medium (Rodhe, l9“8), 2) solidified and liquid WC medium (Dr. R. R. L. Guillard, personal communication), 3) solidified and liquid medium II of Forsberg (1965, as modified by Wetzel and McGregor, 1968), and “) artificial lakewater with an ionic compo- sition and total ionic content similar to that of the parent lakewater of Lawrence Lake (Table 1). The latter medium did not support growth of algal organisms beyond three routine transfers. Repeated streaking on plates and slants and serial dilution transfers in liquid media in— volved one treatment with dilute concentrations of anti- biotics (penicillin "G"-sodium, and streptomycin sulfate) at 0, 10, 50, 100, and “00 pg 1"1 of each. Procedures and concentrations of each bactericidal agent are similar to those used for algal organisms by Droop (1967). Cultures of epiphytic bacteria included Pseudomonas sp., and Caulobacter sp. (see Allen, 1968b for details of isolation of two similar species of freshwater Caulobacter). The isolated epiphytic algae were Gomphonema sp., Cyclotella sp. (cultures of Cyclotella nana were later obtained from Dr. R. R. L. Guillard, Woods Hole Oceanogr. Inst., which were morphologically identical to the epiphytic Cyclotella), and Chlorella sp. Occasional sterility tests with a broad spectra of media (Wetzel and McGregor, 1968) demonstrated axenic conditions throughout the period of laboratory experimentation. 39 TABLE l.--Composition of artificial lakewater patterned after that of Lawrence Lake, Barry County, Michigan. 1 Concentration Constituents -1 (mgl ) Ca 80.96 Mg 17.50 Na 7.50 K 3.01 HCO3 116.68 SO“ 69.23 Cl 10.0“ Fe 0.020 PO“ 0.010 NO3 » 0.300 Chelator2 “0.0 Buffer3 1000.0 1Vitamins and trace metal mix are identical to those recommended in WC medium (see text). 2NTA (Nitrilotriacetic acid); Eastman Organic Chemicals, Rochester, N. Y.; No. 5“l7. 3TRIS-(hydroxymethyl)-aminoethane; ultrapure; supplied by Mann Research Laboratories, N. Y. “0 Upon isolation, minimal nutritional requirements were established for the algae and bacteria. Constant growth conditions were employed for the cultured organisms at 16 to 200 with800 to 1000 lux intensities. Growth curves were established for the bacteria in liquid media (WC and medium II) with glucose or acetate added. Growth was recorded as optical density of the culture at 650 nm. Algal growth was also monitored in liquid media using similar methods, but was recorded as optical density at 750 nm. Cultures, grown for rate of pOpulation increase studies, were incubated in 100 ml of media in side-arm flasks. Cell numbers were determined in a Petroff—Hausser Bacterial Counter with a standard compound light micro— sc0pe at 1000x magnification and oil immersion optics. Prior to experimental treatment, all cultures were grown until log phase of growth pOpulation densities were ob- tained (optical density monitored). Najas flexilis, a common taxon of submerged macro- phytic species inhabiting freshwater littoral zones, was grown and maintained under axenic conditions from surface- sterilized seeds, until seedlings reached 2 to 3 cm in length (medium 11; 750 lux; 20C; see Wetzel and McGregor, 1968, for details of growth conditions). Initially, rates of photosynthetic incorporation of inorganic carbon (Inc) by Nglgg were determined in essentially the same manner as Wetzel and McGregor (1968) with the exception of the radioassay procedure of labelled plant biomass. A “1 homogenization technique and planchette-plating and solid- counting procedure was used (O'brien and Wardlaw, 1961). Extracellular release of luC-labelled organic compounds by Nglgg, subsequent to photosynthetic labelling, was deter- mined by combustion of DOM to 1“C02 and radioassay in gas- phase. Studies on the transfer of labelled dissolved organic compounds and 1“CO2 from axenic Nglgg_into cultured epi- phytic algae and bacteria were accomplished with specially constructed chambers (Figure 3). The chambers were fabri- cated from highly transparent Plexiglas tubing (inside diameter: 3.75 cm; length of each section: 5.0 cm) in three sections, where each section was separated from the adja- cent one by pre—eluted (with 60 ml of 0.1 g HCl, following recommended procedures of Parker, 1967) Millipore GS (porosity: 0.22 i 0.02 p) membrane filters. Tests were made to determine the time interval required for small concentrations of 1LlC-labelled organic compounds to move and equilibrate from the center chamber section into the two outer sections under exact conditions of normal trans- fer experiments. Rapid movement Of labelled organic com- pounds resulted in equilibrium activities within 3 to 11 minutes. Adsorption of the isotopes onto filter surfaces was found to be negligible over several hours. Najas flexilis plants (5 to 6 per flask) were pulse- labelled with inorganic carbon in 200 m1 of medium II for a period of “ hours and carefully transferred to the central “2 .mmumnafiom Hmfipmpomn cum Hmem an nonzndonoms Eopm mpodcopa pmasaamomppxm mo moapmcfix mmeQs MCHCOHpHuLmQ pom pom: pmoemzo mmawfixmam HmpcmEfiLOQxMIl.m mpdwfim “3 .m Onswfim ““ section of the experimental chamber. After brief accli- mation, aliquots of algae or bacteria at known pOpulation densities in log phase of growth were introduced into the adjoining chamber sections containing 50 m1 of medium. Uptake by the epiphytes of materials extracellularly re- leased by the Ngjgg were monitored by periodically removing small duplicate aliquots of these cultures and filtering onto GS and HA filters. A serial dilution of the extra- cellular materials was made for application of Michaelis— Menten enzymatic assays with separate cultures of epiphytes. Similar culture experiments were performed on macrOphytes, algae, and bacteria present in a single 250 ml flask to gain further insight into interaction phenomena. Studies were further performed to determine the rates of chemo-organotrophic utilization of glucose and acetate- 124C (at the same concentrations and incubation intervals as routinely used in the field) at 5, 11 to 12, and 21 to 23C, by cultured algae and bacteria, to aid in evaluation of previously described studies with partitioned chambers in the laboratory. Data from separate and mixed cultures are expressed as maximum velocities of utilization (bacterial Vmax of Michaelis-Menten enzyme kinetics) and rates of diffusion (algal Kd; gt. discussion in Allen, 1969). Resultant data on utilization of organic compounds are further expressed per unit Optical density (cell numbers in log phase of “5 growth) to allow direct comparisons of rates under different experimental conditions. III. RESULTS AND DISCUSSION A. lg situ methodological studies A brief survey of rates of primary productivity of epiphytic algae removed from identical surface areas of several natural macrophytic substrata revealed similar rates among communities on different hydrophytic species (Table 2). In that measurements were conducted during two consecutive days at approximately the same time of day, diel photosynthetic patterns for autotrophic metabolism are largely negated. Differences shown are likely to be representative for the season, depth, and specific macro- phyte sampled. Although species composition was observed to be different on each of the supporting macrOphytes and between sample replicates, rates of inorganic carbon fix- ation were similar. It is noteworthy that rates of carbon fixation by epiphytes on the stem of Potamogeton sp. were lower than rates from other macrOphytes tested. Replicate measurements of rates of carbon fixation by epiphytic algae were made on 11 samples from Scirpus acutus to determine variance associated with epiphytic communities from the same macrOphytic species within the same vegetational stand. At a depth of 15 cm above the sediments a mean rate of photosynthetic productivity of “6 “7 TABLE 2.—-Rates of primary productivity by heterogeneous epiphytic algal communities on several natural macrOphytic substrata. Lawrence Lake, Barry County, Michigan; 19-20 September 1967. Productivity Plant and Depth (mg c m“2 day‘l)a Above Sediments Mean Range Scirpus acutus Muhl. 10 cm 821 i 75 20 cm 1300 i 26“ Potamogeton sp. 50 cm “03 i “9 75 cm “65 i 31 Nuphar sp. 25 cm 1515 i 110 60 cm 1297 i 218 Nymphaea sp. 25 cm 1136 i 230 “0 cm 809 i 137 aRates (N = 2) expressed as net mg C assimilated per m of macrophytic surface area at depth indicated; samples acidified to remove radiocontaminated carbonates. “8 1162 (range = i “09) mg C was assimilated per m2 of plant substrata per day by attached samples on spatially separ- ated hydrophytes. Plants selected for epiphyte removal were randomly chosen from within an area of approximately 80 m2 on the marl bench in Lawrence Lake (Figure l). Variance estimates of rates of photosynthesis by epiphytes from 15 cm above the sediments on the same emergent hydrOphyte were 892 (range = 1 116) (3 replicates of 0.785 cm2 each; 17 September 1967), 126“ (range = i 201) and 990 (range = i 83) mg C m-2 of macrophytic substrata day-1 for three spatially separated plants. These few results suggest that rates of photosynthesis are relatively uniform despite variation in species composition for epi- phytes on the same plant within a homogeneous stand at a specific height in the littoral water column. Additional support was demonstrated for similar rates from replicated measurements on the same plant equidistant above the sedi- ments. Uniformity of epiphytic response appears to be more closely related, at least for Scirpus acutus substrata, to constancy in light and thermal conditions at a particular vertical stratum than to a high degree of overall homo- geneity in species composition of attached communities. Photosynthetic rates were measured for epiphytes on both young and second year Scirpus acutus plants (Station I; 17 September 1967). The mean response for communities attached to over-wintered substrata dominated by diatoms was 10“2 (range = i 381) mg C m"2 of plant substrata day-1 “9 (for 5 replicates of two samples each). Epiphytes on plants produced during the same vegetative season had photosynthetic rates of 697 (range = i 103) mg C m-2 of plant substrata day-l (same number and type of replicates) which reflects a distinctly different community structure. Ranges of rates reported here only allow an intrasystem evaluation of the sampling procedure employed for the 1“C method for measuring primary productivity rates of algal epiphytes. NO attempt was made to place these photo- synthetic rates on a littoral zone basis, as variance in natural macrophytic surface area per m2 of the littoral zone would obscure the differences observed per unit sur- face on individual plants. Owing to problems in quantitative removal of the epiphyton from natural plant surfaces, artificial substrata were employed for studying the annual cycle of lg situ epiphytic metabolism. Prior to initiation of routine monitoring the Plexiglas substrata were allowed to undergo colonization for six weeks, before comparative productivity of epiphytes on natural and artificial substrata was mea- sured. These measurements were conducted on Scirpus sub- strata (natural and artificial at 15 cm above the sediments) at Station 1. Rates of photosynthesis measured on 12 repli- cates of equal surface area (1.6 cm2) from young Scirpus plants were 135 (range = i 51) mg C m"2 of plant substrata day-1; the mean rate for equivalent areas on artificial 2 substrata was 82 (range = i 26) mg C m’ day"1 (12 April 50 1968). Community structure in the attached algae on both substrata was poorly develOped with colonization of only 6 weeks subsequent to ice retreat. Comparisons of over- wintered Scirpus plants with artificial substrata were not undertaken during initial studies. The following spring (19 April 1969) an independent comparison of photosynthetic rates (luC method) of epiphytes from over-wintered surfaces of Scirpus (271 (range = i 59) mg C m"2 of plant day-l; N = 3; 15 cm above the sediments) showed favorable agree- ment with similarly placed artificial substrata (162 (range = t “8) mg C m"2 substrata day-1; N = 3). Resultant data from initial studies have shown the 1“C technique to be adequately suited to the measurement of primary productivity of algal epiphytes. Incubation periods were limited to a 3 to “ hour interval during mid- day. Rates were additive where carbon fixation was measured from (1) 0900 to 1200, (2) 1200 to 1500, and from 0900 to 1500. A larger portion of the inorganic carbon was fixed during the 0900 to 1200 interval than during the afternoon period (27%). A major source of inherent error in applying the 114C method of epiphytic complexes, especially true in calcareous environments, is the rate at which deposition of 1“C_ labelled monocarbonates occurs during lg gltg incubation. Data from photosynthetically labelled epiphytic samples sub- jected to two procedures of decontamination are summarized in Figure “. Filtered samples exposed to fumes of concentrated 51 Figure “.--Mean percentage loss of 1“C activity (as precipitated monocarbonates) from filtered epiphytic algae upon exposure to fumes of concentrated HCl for varying periods of time (upper), and loss of activity (as intra- cellular fixed carbon) by rinsing filters with various con- centrations of dilute acid (lower). Natural epiphytic algae removed from Scirpus acutus following in situ photo- synthetic l“0 fixation in Lawrence Lake (Station 1'), Michigan, 1“ March 1969. Figure “. ABSQUTE RADIOACTIVITY i COUNTS / MONUTE MOO l200 ' IOOO 800 600 400 l200 I000 600 400 200* 52 YVTTTj V V mm D 30 60 EXPOSURE TO C(NC. ch FLNES (MIN) 24% cmnmx (mm mm on NORMALITY HCI (5m! RINSE) 53 acid over 10 to 30 minute intervals lost from 5 to 6% of their absolute activity. Identical epiphytic algal samples treated by rinsing with small aliquots of dilute acid re— leased intracellular fixed carbon, equivalent to 22 to 30% of their total absolute activity in addition to 1“002 evolved from the dissolution of monocarbonates. A single set of triplicate samples immersed in 0.1 N HCl released 32% of the fixed organic carbon as dissolved organic com- pounds, a loss similar to that of rinsed samples. It cannot be over-emphasized that 0.001 N HCl, although very dilute, effectively removes incorporated carbon from 1“C_ labelled algae upon rinsing. From these findings it was concluded that all photosynthetic samples (both light and dark bottles following lg gltg incubation) should be de- contaminated by standard 10-minute exposure to fumes of concentrated hydrochloric acid prior to radioassay (Wetzel, 1965c). The quantitative significance of precipitated monocarbonates over the annual period is discussed below. To evaluate the contribution of epiphytic metabolism on a lake basis, it was necessary to recompute data ob- tained from known surface areas of substrata at depth on the basis of total macrophytic surface areas present per square meter of the littoral zone in each of the two sites. For Scirpus acutus during the vegetative season (spring to fall) the range for each 10 cm segment of the stem above the sediments was 0.07728 to 0.1595 m2 of colonizable surface area per m2 of the littoral zone. The total 5“ available surface from the sediments to a height of 30 cm in the water column was 0.2318 to 0.“785 m2 of plant sur- face m—2 of the littoral zone for the vegetative stand. Factors were determined to correct all annual cycle data at each depth (5, 15, and 25 cm above the sediments), on each sampling occasion, on the basis of a square meter of littoral zone for each 10 cm interval on the plant, by random counting estimates of changes in the quantity of Scirpus plants per m2 of the littoral zone over the growing season. Initially, in the spring (1968) a mean of 51.3 plants per m2 of the littoral was found. During the fall (1968) 6“.8 occurred per m2 and in the spring of 1969, 56.3. The assumption of these conversions is that single sample measurements (from 2 x 1.6 cm2 surface area) are representative of the natural vertical heterogeneity in epiphytic metabolism and thus permit extrapolation of data from each of these points (5, 15 and 25 cm vertical dis- tance) to each of the three 10 cm intervals. Adsorbed surfactant procedures for obtaining surface areas of Najas flexilis and Chara spp. depended upon core samples of natural macrOphytes along predetermined tran- sects both parallel and perpendicular to the shore line at Station II. A high variance in availability of Nglgg and Qggtg surfaces at station II was associated with lg gltg heterogeneity in distribution of the submerged plants (range = 9.0 to 21“.9 cm2 of macrOphytic surface area per 2 1“.90 cm of littoral zone; spring, 1969). An annual 55 range of 6.15 to 10.63 m2 plant surface/m2 littoral zone was determined for the vegetative season of 1968. Since the mean vertical height of the submerged plant story was 19.8 cm, the actual macrOphytic surface areas per m2 of littoral zone were divided by 2 to provide factors to correct point measurements of metabolic parameters at 5 and 10 cm intervals to a vertical height of 0 to 10 cm and 10 to 20 cm, respectively. Factors were extrapolated for all sampling intervals to proportionally correct point measurements to an aerial littoral zone basis. Approxi- mately 23 to 27 times more surface area exists on submerged macrophytic species per m2 of the littoral zone than on the emergent Scirpus acutus. Considering the morphological similarities in submerged and emergent freshwater vege- tation, these conversions are likely to be suitable to other shallow aquatic habitats where these taxa occur, provided distributional patterns are shown to be quanti— tatively and qualitatively similar and the length of the growing season is congruous. I B. Epiphytic algal and bacterial community metabolism 1. Productivity of epiphytic algal primary producers The current literature on annual cycles of primary productivity in lakes and marine environments contains no single study on epiphytic algae. As discussed in the introductory section, there have been several extensive 56 studies on primary productivity within the epibenthos, with considerable work having been accomplished with peri- phytic communities, and to some extent with epipelic com- munities. The importance of epiphytic carbon fixation and annual contribution to the total primary productivity of an aquatic ecosystem is frequently thought to be insignifi— cant. Observations of large standing crOps of attached algal biomass, however, suggest that macrOphytes may well support quantitatively dominant primary producers in many of our shallow and more productive natural systems. Rates of photosynthesis by algae attached to artifi- cial substrata, representative of epiphytes of emergent and submerged vegetation, were monitored in Lawrence Lake for one year (Figures 5 and 6). Data are expressed in each of these figures on the basis of a square meter of macro- phytic surface area, permitting a direct comparison of photosynthetic activity between attached communities in two spatially separated macrophytic stands. Rates of photosynthesis on substrata in a stand of Scirpus acutus located on a wind-swept calcareous bench were markedly different over the annual period at each of the three depths monitored, even though separated vertically only by 10 cm intervals. A significant feature is that rates near the surface showed considerable oscillation, with a progressively more stable and persistent response close to the sediment. Wave and mechanical activity throughout the ice-free season was likely responsible for this 57 Figure 5.—-lg situ prigiry productivity (mg C m-2 macrophytic surface area day ) by attached algae at 5, 15, and 25 cm above the sediments at the Scirgus site (Station 1), Lawrence Lake, Michigan. Data were collected from artificial substrata. The area between the upper and lower lines for each depth represents quantitative l“C precipitation as inorganic carbonates by attached micro- flora during measurements of photosynthesis. 58 Figure 5, 25 CM : \ I600» '-—--' ~01 Acorn) \ \ \ *—-——- WED "'0 C In" DAY" M. JIL. AUG. 55;: [968 09'— NOV. DEC. 59 .mpmpmeSm HmHOHMHpmm 80pm Uopooaaoo who: mums .cmmHQOHz .oqu mocopzmq .AHH coapmpmv mufim anaconmmndz on» an magma Ifipmm Ono o>oom 80 OH pcm m um Omwam bosomppm mo.hHlmmo mmbm oommp5m Ofipzcdopome NIB 0 mEv mpfi>fipospopa upmefiha Spam CHII.m omswfim 60 mwm 093 P03 olllllo .200. .m ogzwfim 61 stratified pattern. Community cohesiveness and the ability to maintain structural integrity (probably related to the matrix of deposited monocarbonates and biotic components), in a position essentially perpendicular to the macrOphytic substrata, probably would be decreased by such physical perturbations. Factors contributing to metabolic fluctu- ations near the surface may be related to surface dilution effects of nutrients through direct precipitation and run- off, oscillatory movements of the upper portion of the plant, and diel patterns of high light intensity and ther- mal heating at the surface. Rates of photosynthesis at the Scirpus site at all depths exhibited annual patterns similar to the general growth patterns of the macrOphytes themselves. Photo- synthesis at all depths after six weeks colonization in- creased steadily until the first annual maximum was reached in early June. Growth then decreased to 22 mg C m"2 of plant substrata day_1 5 cm above the sediments and coin— cided with a period of intense precipitation and possible nutrient dilution effects. The annual maxima for all depths occurred during July and August and rapidly de- creased to winter levels in mid-November. A trimodal photosynthetic response was evident in the Najas—Chara site (Figure 6). Although rates from both depths were similar temporally, maximum annual primary productivity occurred near the sediments. The similarity 62 of annual cycles from both depths probably reflects the small 5 cm vertical distance between the colonized samples. A comparison of annual mean productivity from equi- valent surface areas (Table 3) shows epiphytes on substrata of the Scirpus site were fixing 22.8% more carbon per unit time than attached algae from the submergent site. Pro- ductivity maxima, corrected for precipitation of l“c_ labelled monocarbonates, at the Scirpus site ranged from 115“ to 1517 mg C assimilated m-2 of plant substrata day—1 for all depths; samples from the Najas-Chara site ranged from 7“2 to 1055 mg C m-2 day-1 for both depths. Mean annual contamination by deposition of 14C carbonates amounted to 38.5 to 71.7% of the actual carbon fixed for Scirpus substrata. Over the annual period “0.3 to “5.7% of the determinations of mean annual productivity were due to errors of 1MC deposition as inorganic carbon within the submerged site. These high percentages stress the necessity for decontamination procedures and indicate the potential error if neglected. Rates of deposition of carbonates were commonly found to be highest during periods of maximum photosynthe— sis but were not directly proportional to carbon fixation rates. As much as 53% of the radioactivity was present in precipitated form during the vegetative season in both experimental sites but average prOportions varied from 10 t0 30%. Deposition of 1“C during incubation was greatest during winter conditions under ice cover in dark 63 TABLE 3.--A comparison of mean annual productivity rates for epiphytes on artificial substrata at the Scirpus (Station I) and Najas-Chara site (Station 11), Lawrence Lake. Carbon fixation data are expressed on the basis of equivalent square meters of macrOphytic surface. 32' Annual Productivity Depth above Sediments (mg C m-2 of substrata day-l) Scirpus Site 25 cm 330.1 15 cm 297.7 5 cm 281.9 Najas-Chara Site 10 cm 2“1.3 5 cm 278.8 6“ photosynthesis samples (where precipitation was likely due largely to bacterial activity, see Kusnezow, 1966). Loss of activity through acidification predominated in dark photosynthesis bottles and supports findings reported by Wetzel (1965c). High standing crops of attached organisms remained on the artificial (gt. discussion on pigments) and natural substrata during winter periods. Rates of net carbon fix- ation, however, decreased to almost indetectable levels. During the winter season several dark bottles from photo- synthesis measurements possessed more 1“C activity than did their complementing light bottles, prior to acidifi- cation treatment ("net" photosynthesis = 0.01 to 0.09 mg C m2 of plant substrata day—1). This finding is commonly observed in phytoplankton and has significant ecological implications. Two alternatives are available to the attached algal flora for maintenance of high standing crops under conditions seemingly adverse to photosynthesis. Either the cells are supporting themselves energetically through utilization of metabolic storage products at low respiratory rates, or they are supplementing photosynthesis with chemo-organotrophy by direct uptake of macrophyti- cally, bacterially, or allochthonously derived organic compounds. It is ecologically feasible that both alter- natives are functional to some extent under natural conditions. 65 Visualization of the more dynamic changes in photo- synthetic activity based on rates of carbon fixation per square meter of macrOphytic surface area at depth, is en- hanced by presentation of data in isopleth form for the two macrOphyte sites (Figures 7 and 8). Although single data measurements are not decipherable, overall patterns of annual change become accentuated and lend these data to a better interpretation of the dynamics involved. Epiphytic productivity in the Scirpus site remained less than 50 mg C m—2 plant substrata day"l throughout the first week in May and was followed by simultaneous in— creases near the surface and the sediments. Light and temperature would appear to be critical factors during initial colonization and may lead to "light" and "shade" adaptation during this period. A strong stratification occurred during the latter half of July and August (surface maximum = 1251 mg C m—2 plant substrata day-l; near sedi— ment maximum = 1517 mg C m"2 plant substrata day-l). Similar pronounced shifts occurred in pigment deve10pment (gt. Figures 15 and 16) and correlate with the establish— ment of stratified diatom communities (Gomphonema sp. and Eunotia sp.) which persisted to some extent into winter months. By early fall a gradual decrease in photosynthesis was found and by the end of December low winter rates pre- vailed. At Station I natural and artificial substrata were frozen to within “ cm of the sediments throughout 66 .Ahm>oo OOH u mono oopmnmv .mfimmnugmmOpona mo meoEOLSmmoE wcfigso popdpfinfiompd monocoopmo OficmprCH 02H Wm coapmcaampsOOOHpmp LOM bmpoompoo mums mama .cmenOHz .Oqu mocmpzmq .AH coapmpmv opfim msmpfiom on» no mummefibmm 03p m>onm Eu mm Op m pm mmwam Omnomppm zfllhalmmc mono mommpSm oaumndopome NIB 0 wev mpa>flpospopa mmeapa Spam CH mo mnpmHQOmHll.s mmswam 67 3f ' \ / I969 \ P / 1 1“ 000 000 . so 0m . < _ mo w “:1 .A A \ , , . . APR MA M JJL. “16.. SEP. OCT. NOV. DEC. JAN B68 68 .mfimmnpcszpocd mo mpcmEOLSmmoE wcfipso Umpwpfiofiomaa moumgonhmo Oficmwpocfi 02H 9w coapmcfiEOOQOOOHU E .mom Omuooppoo who: mama .qmeEOHz .Oxmq monopzmq .AHH coapmpmv Opfim assaulwmndz map pm mucoEHOmm 03p m>onm 80 OH O» m an mamas pogomppm mplhalmmb mops motmpdw OHumndomomE NIB 0 wEv zpfi>apospopm hmeHpa Spam CH mo mnpoadomHll.m opswfim Figure 8. 69 1 Y 1 Y Y i V 1' Ex AN .11.. AUG 'SEP. OCT. wov. DEC JAN. FEB MAR APR. MAY u APR. ( 8 a a a as lNBMESIDGVCWHldBO I969 968 70 most of the month of March. Subsequent to ice—retreat on 27 March 1969, rates increased in nearly identical patterns to those of spring, 1968. Rates of photosynthesis of algae in the Najas-Chara (Figure 8) showed a much stronger initial intensification during the spring months through May (500 mg C m-2 plant substrata day-l) than occurred on the Scirpus substrata. However, stratification in early summer was weak, possibly in response to changes in day length and photoperiod. Contrary to the pronounced stratification observed in mid- summer at Station 1, maximum rates of slightly over 1000 mg C m-2 plant substrata day-1 were observed at 5 cm above the sediments. It is likely that a 5 cm interval was not sufficient to detect a strong vertical difference, if it existed to any significant extent, in the attached algal metabolic rates. Pigment distribution, discussed below, was strongly correlated with rate increases towards the sediment in mid-year samples (see Figures 17 and 18). A gradual loss of vertical stratification in rates of pro- ductivity occurred from September through November. By mid-December winter values of 50 mg C m"2 plant substrata day-1 or less were found in all samples. Station II had a significant ice cover (19 cm), but did not freeze to the sediments. All samples between 22 January and 13 March, 1969, with one exception, had photosynthetic rates of less than 50 mg C m"2 plant substrata day-l, and confirm the independence of pigment concentration (g.g. biomass) 71 and intensities of photosynthesis. It may be deduced from data discussed thus far that monitoring productivity rates on the basis of changes in pigments for epiphytic communi- ties may lead to erroneous conclusions (Wetzel, 196“). lfl.§l§2 epiphytic primary productivity, expressed per square meter of the littoral zone for the two coloni- zation areas (Figures 9 and 10), immediately demonstrates that the attached algal flora on emergent hydrophytic vegetation contributed only slightly to the total littoral primary production on an annual basis. Much higher epi- phytic productivity per area of littoral zone is shown by the communities on submergent vegetation, as was antici- pated from the ratio of surface areas per m2 of benthic area for the two types of vegetation. Rates of 1”C fix- ation at Station I for the three depths monitored (Figure 9) reveal annual maxima of 196 to 232 mg C m—2 of littoral zone day"1 with winter minima of 0.3 to 5 mg C m—2 day-l. For epiphytic algae on submergent simulated substrata (Figure 10) annual maxima also occur during the middle of the vegetative period (July and August) with 3.8“ to 5.“6 g of carbon fixed per m2 of the littoral zone day-l. Minima observed from December through April were 57 to 2“8 mg C m-2 of the littoral zone day-l. It is noteworthy that winter rates for carbon fixation under ice cover by algae supported on simulated submergent macrOphytic substrata are equivalent to summer maxima Observed for sessile forms on simulated emergent vegetation. 72 .mfimonpcmmOponQ mo mu2080p5mmme wcfinsc whoam IOLOfiE Oonomppw an wepmcoppmo OHcmwhocH mm goapmpfidfiompa 03H mo pazmmn map Loppm zpfi>apozpopa masommpamp nuamp 30mm pom mocfia sozoa bum pond: on» COOZOOQ mmpm one .mOmmpmosm HmHOHmemm Eomm OOOOOHHOO who: spam .cmeSOHz “Oxmq mocopzmq .AH soapmpmv moan mapzom msqufiom on» um mpgmefipmm on» o>oom on 80 m can .Amv Eo ma .Apfl>fipozpopa mmeHmQ Spam cHll.m mpzwfim H' amp 73 6 Sat o .-AVO I‘m 3 M $2.3 o ”‘3‘ .m onsmfim 7“ Figure 10.--In situ primary productivity (mg C m-2 of littoral zone da-y-I) by attached algae at 10 cm (A) and 5 cm (B) above the sediments at the Najas-Chara site (Station 11), Lawrence Lake, Michigan. Data were col- lected from artificial substrata. The area between the upper and lower lines for each depth represents produc— tivity error the result of 1“C precipitation as inorganic carbonates by attached microflora during measurements of photosynthesis. 75 Figure 10. 3000- o———o NOY ACNE!) 0—4 KlUFlED 4000* 3000r 2000~ APR. MAY M. .11.. AUG. SEP OCT. NOV. DEC. I968 JAN FEB. MAR. APR. 969 MAY 76 Isopleths of rates of photosynthetic intensity for the two types of substrata simulated are presented in Figures 11 and 12. Extrapolated macrophytic substrata surface area per unit area of the littoral zone alter only slightly the patterns of these rates (compare Figures 11 and 12 to Figures 7 and 8). Annual maxima (acidified samples) for both substrata occurred during the late fall period adjacent to the sediments. Winter rates for the Najas-Chara substrata were 100 to 200 times those on Scirpus substrata. In general, similar stratificational patterns were found at both stations. An increase occurred near the surface and sediments during spring and was followed by an annual maximum associated with the sediments and rapid erosion from September into winter conditions under ice cover. The general pattern suggests a stringent regulatory effect by environmental and physical conditions common to both areas. Productivity rates for attached algae from both vegetative sites were integrated (Figure 13) by summation of rates per vertical increment in the littoral water column. The total productivity per square meter of the littoral zone for the Scirgus substrata include rates extrapolated from the uppermost point measurements (l.g. data from 25 cm above the sediments which were expanded to the 20 to 30 cm interval) to the surface area from 30 cm above the sediments to the air-water interface. Data 77 .Ahm>oo OOH u wont Oopmnmv .mfimmnucmmoponm mo mp208095mmms wcfipso umpmpadfiomma mmpmconpmo oacmwnocfi 03H mm coapmcaEmpcooloavmp pom pouompmoo mum paw mumpmeSm HOHOHMHOMO 80pm mOpOOHHOO who: mama .cmeQOHS .oxmq 00209384 0 AH coapmpmv mpfim mapsOm.mmmmmmm on» an mucosavmm map m>opm Eo om op Oommpmpcfi mmpmzupcmEHomm on» Eopm mmemImOnomppm an Aaummv meow ammOOOHH mo NIB 0 wev hpa>fiposOOAQ hamsfimm Spam CH mo mnpmaaomHII.HH opswam 78 9x: /4 s; I968 0 APR MAY .m. JUL. AUG. SEE OCT. NOVLDEC. llll 8 N 9 “GWIOBS amaVCun) Hid-30 79 .mfimmeCmmOpOCQ mo mpCmEmCSmmmE wCHCdO Ompmpfiafiomnm mmumCOoCmo OHmeCOCH oza mm COHmefiEmpCOo|OHOmC Com Cmpomppoo Ohm mCm mumppmosm Hmfioamfiupm EOCC Cmpomaaoo 0C03 mpmo .meHCo«z .mxmq moCmpzmq .AHH Coapmpmv mpfim mCmConmmwmz 0C» pm mpCosHOmm 0C» Osoom Eu cm on mommpmpCH COOmZIpCmEHOOm 0C» Eopm mmmamlmOComupm an Aalmmm 0Com HmCOppHH Co NIB 0 mv mpfi>fiposmopd zmmefina Spam CH mo memHQOmHTI.mH mmswfim 80 >42 .mdd mmwzm \ X/ : Nd no 0.. 0.. a L — I“ _ tag... _ . tQ/s 4,18... O i D Q Q 8 SlNBWIOBS 3A08v (m) Hld30 .NH ossmam 81 .mfimmnpcmmouosm mo mpcme Impzmmme wnapso whoamopofie Umnompum mp mmpwconpmo oacmwmocfi mm coapmp uHQHompa 03H no pHSwmu map mponnm mpfi>fiposvopn mpcmmmnamn mpfim m>aump Imwm> nomm pom mmCHH pmzoa cam pmaa: mg» cwmspmn mmmm mne .mpmppmpsm HmHOfiMHupm 60pm Umpomaaoo mum: Mums .smeQOHz .mxmq mocmpzmq .AHH soapMpm mmv mpflm .Qam mnmco ucm mfiaaxmam mmnmz map pm 6cm .AH coaumpm mfiposvopa mumsfipa cmpmmwmpnfi SPflm cHll.mH mmswfim 82 mwm. mwm. >43 Ed g .8... .246 Gun .62 FOO mm .g 35.. .5 i g \‘IWIIJIJ'II ..nl..n , - . . 4 * 1 1 q 4. . oflunug 'Illlo QUE P2 QII'IO .ma mmswfim 83 from all measurements at the Scirpus site were corrected for fluctuations in water level during the ice—free season. In summary, annual maximum rates of productivity for the attached algal microflora for the entire water column above 1 m2 of benthic area on the calcareous bench was 0.962 g C assimilated m"2 day-l. A minimum rate of 0.506 mg C m"2 day-1 was observed under ice cover in February for samples. An order of magnitude difference was found for colonized algal species in the zone dominated by sub- mergent macrOphytes. A maximum of 9.299 g of carbon m-2 day'-1 was fixed during August while winter values reached a low of 155 mg C m-2 day—l. The annual maximum rates noted probably reflect near maximum rates sustained by this system in relation to available light. It is notable that variations in annual patterns at individual depths give markedly similar annual patterns. Each exhibits a sharp increase in photosynthesis by attached communities during the first week in June, followed by a decrease, and the annual observed maximum in mid-August. A third peak, the smallest of the year, occurred in both sites during the latter half of October. Subsequent rates decreased pro— portionately from October into December with falling water temperatures to persistent winter ranges. By early spring, 1969, intensified rates were observed and agree closely with the previous spring pulse, subsequent to initial colonization. 8U Mean annual primary productivity rates for epiphytic algae per m2 of the littoral zone are summarized with respect to vertical increments monitored (Table 4). Sur- face area effects are readily apparent in the order of magnitude difference between the two mean annual rates for both of the vegetative sites. By summation of individual increments and extrapolation to the maximum height of each of the macrOphytic stands, planimetrically determined values for the entire littoral water column above 1 square meter for the two representative sites were estimated (Table 5). Although rates of algal carbon fixation for Scirpus sub- strata were low, they are probably collectively significant in a system such as Lawrence Lake where suppressed pelagic photosynthetic activity is in effect (93. earlier dis- cussion on planktonic photosynthesis in relation to chemical-physical and nutritional interactions in a marl lake). Westlake (1963, p. “0“) in a review of plant pro— ductivity, has stated that "freshwater benthic and epi- phytic algae usually account for only a small part of the production of communities where they are present." Without question, an annual mean rate of productivity of 1.807 grams of carbon assimilated per m2 per day represents one of the dominant producers within this system. To further emphasize the importance of total epi— phytic primary productivity in Lawrence Lake, and to place these data into proper perspective, comparison is made with annual production rates in various aquatic ecosystems 85 TABLE u.—-Mean annual primary productivity by attached algae on artificial substrata at the Scirpus site (Station2 I) and Najas—Chara site (Station II), Lawrence Lake, per m of the littoral zone. i Annual Productivity Depth above Sediments (mg C m—2 of littoral zone day-1) Scirpus Site 20—30 cm 37.2 10-20 cm 37.2 0-10 cm “3.3 Najas—Chara Site 10-20 cm 8U0.7 0—10 cm 966.5 86 TABLE 5. Mean annual primary productivity for attached algal organisms on artificial substrata at the Scirpus (Station I) and Najas—Chara site (Station II), Lawrence Lake, integrated for the entire littoral water column. E Annual Productivity Site 2 (mg C m- of littoral zone day-l) Scirpus acutus Muhl. l96 Najas flexilis and Chara spp. 1807 87 (Table 6). Such a comparison must be viewed as only' approximate as techniques of assessment, conversion factors, and units of expression differ considerably among the investigations cited. Annual mean planktonic algal productivity of Lawrence Lake (1968) was 73.6 g C m-2 year-l. It is apparent from this current study that epi- phytic algae represent the dominant form of primary producer in a shallow—water ecosystem. In aquatic systems where the littoral zone is well colonized by macrOphytes, particu- larly submerged species, attached algal production is likely to exceed that occurring in the pelagial water column. An exception might be polluted pond systems and shallow water habitats of advanced eutrOphic conditions. From these comparative data it can be seen that the epiphytic algal community ranks among the highest for both fresh water and marine environments and is likely to be more productive than most epibenthic habitats, especially if the submerged vascular flora is extensively develOped. Mean annual productivity rates of attached algae per square meter of littoral zone per day were used to estimate the approximate magnitude of epiphytic carbon fixation for the entire lake, within restricted depth ranges. Planimetric analyses of vegetational maps of Scirpus acutus, Najas flexilis, and Chara spp. from the shoreline to a depth of 60 cm in Lawrence Lake were used to calculate the total surface area colonized by these 88 wmsHm> >pH>Hposooaa OHQmeo Eopm UmpMHSOHmo who: AmmmHv meHpmm3 Song «poo .pcmpcoo cooan Roz wcHESmmm m .AmmmHV m>mhwhmm an cmpHoH AmmmHv UpHmm cam Hmmpm 02H ml: coceemeHam mchmE AmmmHv em>pcnpo 03H msaumHH mpcmefiomm mcfipme Hmcfippmch H HAmmmHv augumemm gm~35 mmmumzH ApmHmncmmv mmmHm oaspcmnfiam HAmmmHv mopofiom :mwzxo oom Anmpme uHmmv mmem OHQpcmoHam cam mmpAEQOhomE pmwhmEndm osm AmeH smpsHHoav cepxcmHQOpsnm omH AHmpmmoov coaxcmHQOBsnm om AocHnmSV coaxcwHQOpmnm mflmmev mmeummz om Apmpmznmmpmv COpxCMHQOpzsm mm mmpmzaonomz Hm coaxcmHQOpmsm AzmmHv Hmupmz o H pom GonznaHpmm : mxmq xmpom AmmmH mzv mH>mo o H mH mmpmnaopomz H AmmmHv whommm o: w coaxanQOpznm AmmmHv m>mpwsmm cmwwxo 0: mmem ofinpcmnfiam mqu COHpmz A.ompo cHV .AMIMM .Hmupoz o H as couxcmHQopmnm mBSpm pcmmmnm : owe mpmhmeSm pcmwmensm mUSpm pcmmmmm 0 HF mpmnmeSm pcmwnmem :H mmem oHpmnaHam mxmq mocmpzmq moCmmemm umwswmmMMmz pm NIB 0 wv maze mpHQSEEoo GQHposoopm . .msmpmmmOOm OHpmsum monpm> Eopm mmsHm> COHposuouQ szCSEEoo kflpm mme monopzmq CH mmpmcaHmm meHm no SOHpoSUOLQ Hmsccm cams Q m o «1‘ 1 .cwmfinoflz o comfiamasoo <...© ms mas 89 species (emergent: 1987 m2; submergent: 263“ m2). Multipli- cation of the mean annual epiphytic productivity per m2 of the littoral water column by these aerial estimates permits the calculation of the mean annual epiphytic production per lake (littoral zone: 0 to 60 cm depth only) per day (Table 7). Although these data represent a minor portion of the total littoral zone and do not include all taxa of hydro- phytic vegetation supporting epiphytic algal floras, they easily exceed the total mean annual productivity (as kg C 1 lake“ day-l) of macrophytes in Borax Lake, California (Wetzel, 1964, p. 30). 2. Physiological interactions through bioassay Potential regulation of epiphytic primary productiv- ity by availability of trace micronutrients and external metabolites has been investigated to a limited extent. Two hypotheses were considered: (1) that the high sus- tained rates of productivity observed are due to the move- ment of trace materials and micronutrients outward from the macrOphyte during the vegetative season, $.3. a uni- directional flow into the attached communities, and (2) that materials released by the macrophytes during normal photosynthesis as dissolved organic compounds may function- ally serve as complexing agents for required metabolites which are being supplied from the littoral water column (an example might be Fe). 9O .pzooo opr OHpanoppzn pompoEQSm pom pcmmpoao poems o>oom onp ohms: Eo om mo spoon m on ocHHoponm on» Eopmw amua 005.: .oom noono ooo maHaonm wohoz mzH mmm.o .Hndz mapzom mzmpHom upmom Hlocou HmLOppHH o mxv mAHImmU Hlmcou HmAOppHH o wxv COHpospopm Hmscc< M COHuospopm Hmscc¢ m A H maze Enumppmnzm .cmeSOHE .oxmq mocopzmq .opHm mpmnoamodmz cam opHm msmpHom mzp pm mumpmeSm HmHOHmepm co omem poQQMppm mo CQHpospopd Hmzccm some HmpOBII.~ mqm¢e 91 Quantitatively small and ecologically relevant con- centrations of added vitamins, trade metals, and inorganic phosphate failed to stimulate increased rates of carbon fixation by natural epiphytes from Scirpus acutus above controls. Presumably, concentrations of these materials from (1) bacterial metabolic activity, (2) extracellular macrophytic release, (3) entrappment in the muco-organo- carbonate complex of the macrophytic substrata, and (u) availability from the littoral free water area were suf- ficient that limiting effects to the community response were not evident at the time (8-10 September 1968) of analysis. Addition of inorganic iron and organic compounds stimulated rates of photosynthesis (Figure l“). Concen— trations of inorganic iron in the epilimnnetic waters of the pelagial of Lawrence Lake are frequently in the range of < l to 5 ug 1.1, and are often below the sensitivity of standard techniques (Wetzel, 33 al., in preparation). Increased productivity by epiphytes in response to added Fe below 10 pg 1"1 suggests a previous limitation. Iron, added at 10 to 100 ug 1'1, was inhibitory. Stimulatory effects upon provision of sodium-EDTA and quinolinesulfonic acid, an artificial chelator, at concentrations of 1 mg l-l, together with 100 pg 1'1 of FeCl3 (inhibitory at this level when added independent of the chelator) has several important ecological implications: (1) macrophytically released dissolved organic matter may 92 .momH aooEoooom OH on m .cmenon .AH COHpmpmv oxmq mocohzmq CH m5u50m msmpHom 80mm pm>oEom mmem oHpchHam HmQSpmc go gasoaw co AmmocmmeSm oHESLOCEHH popchpxo paw mpHom 0HcomH5m ocHHocHSUImumxoppmnlm mpHMm Eanome .UHom oHumommhpou locHemeoCmHmzpmv musomm mchmHoco was comH mo poommmll.:H onstm 93 mfifli ow on o¢ on an Hg 93 5588+ -.-.-- (88.0. .369 + --- \x v (Faun; (959+ .-....--....-.. \x . one 3 on (9:86 Illll- .\ \ \.\ ,\. . s \. a, \. s, .\ \.. \ A. 3s... , \.. .\ x.. 1 .\.\ x .\ .\ \ \ .. \ x .. x... X. .2 T l o g ONOOBS/SiNnOD l ‘2 ON mfix¥. 8 on 0? On ON Q noon 8 3:28! IIIII .I not 8 £36on --- .. 6.... I on. .3508 .5. m 2.560 E: omoas/smnoo L .zH ohdem 9“ be largely labile and serve only as a carbon and energy source to the attached microflora or largely escape the sessile community, and (2) that organic compounds of allochthonous or autochthonous origin in the littoral water column do not serve as efficient and satisfactory chelators. Limnohumic substances, known to be closely associated with inorganic iron in freshwater ecosystems (see for example Shapiro, 1957), was strongly inhibitory to the epiphytic complex even at 5 pg 1'1 levels. It should be emphasized that photosynthetic responses were evident in experimental samples within 1 to 2 hours of initiation, and were not reliant upon long term responses in which recycling of micronutrients or respiratory l“C02 may have occurred. 3. Pigment composition and distribution in epiphytic standing crops Pigment composition (chlorOphyll a and plant carote- noids) of the epiphytic complexes have been summarized in Figures 15, l6, l7, and 18. All estimates for chlorOphyll a is isopleth figures have been corrected for the presence of pheo—degradation products and theoretically show annual changes in the active chlorophyll pigments. Further, col- lection of samples at the same time of day has reduced errors due to possible diel changes in pigment content. Vertical stratification patterns are evident for both chlorophyll a and total plant carotenoids for 95 .cmmHQOHS .oxmq ooCoMZmH .AH QOHpMpmV muHm msmpHom on» pm ommHm ponomppm mo Aocon HmAOppHH mo m-E mpHcs pcoEwHa pcmHQ ooHMHooom IHHHHEV mQOHpmppcoocoo pHocopOAMo pcmHQ mo wnpoHQomHI-.mH opstm .cmenOHz .mxmq mocmpzwq .AH COprpmv mpHm mSQAMom on» pm omem oosomppw mo Hocon HopOppHH mo mle MEV mCOHpmppcmocoo m HHanOLOHno oopoouhoo mo mnpoHQOmHII.mH opdem O V 120;; (E ./"Q 9:2 \\\Sb/ com 2030 $9 so 20 n j UN. “allAUG. SEP OCT MVADEC. M9. 96 KE’JE \\ / APR. MAY JUN. JJL. AUG. SEE IS 97 .cmeEOHz .mxmq mononzmq .AHH COHpmpmv mpHm mpmzo-mmnmz o£p pm mmem posomppm mo Hocou HmpOpuHH mo m-E moH . mpHc: pcostQ pGMHQ pmHmHomam -HHHHEV mCOHumppcmocoo oHocmpOAMO pcmHQ mo mnpmHaomH-I.mH mmstm .cmenon .ome mocmpzmq .AHH COHpMQmV mpHm mango-mmwmz map pm omem ponomppm go Aocow HMLOppHH mo IE wv mQOHpmppcoocoo m HHhsmopOHno oouoopmoo mo mnpmHQomH-I.>H omstmm 98 mmm. >48 E4 £42 dun. .245 .omo 59,. H00 mwm .034. J? .35 >42 .E4 0 2111.11. . .6 1 L. Q 0 O C v s i n / n w 0 m N. 0L 9 Q o o Q Q m V . 10— . n r o. n. / ;/M V /l- r p- » ON mom. 8m. >43 .E4 £42 .mw... .24.. .Uwo $02 H00 mwm .024 .155 .22. 3 .054 O J A N‘ 1 — /<“<\Q “(N g d 1 g! \ d N._ N 10 1K1 K... 1 11 W11 NO 1 Q SLNMOBS 3A08V (wo) H1d3Cl 9 O N SiNBWIOES 3A08V (“13) H1d30 .wH opstm .KH oazwaa 99 epiphytes colonizing both the emergent and submerged type of simulated substrata. Initial studies with epiphytes from natural substrata showed considerable uniformity in vertical position of pigment concentration on the same macrophytic species, but significant disagreement among macrOphytic species and especially between emergent and submergent forms. Standing crops of both pigments occurred in identical concentrations per unit surface area of the macrophytes in the initial studies. This was especially true for artificial substrata colonized on opposing litto— ral zones, following six weeks incubation. Differences in summary figures are primarily related to surface area effects and differences in colonizable macrOphytic area per m2 of the littoral zone. Patterns of annual change in pigment concentration at both stations showed a number of spatial and temporal similarities in their deve10pment. Chlorophyll 2 increased rapidly at both Stations (Figure 15 and 17) during initial phases of colonization through the vernal months. A paucity of diatoms were present by early June (Synedra sp., Eunotia sp., and some Tabellaria sp.), with concomitant development of several cyanOphytes. Bacterial compactions were also present during this phase of colonization. Similarities in community development at both stations up to this period were marked. A spring peak in chlorophyll 9 adjacent to the surface and the sediments on Scirpus substrata correlated with an increase in Fragilaria and lOO Tabellaria respectively at the upper and lower depths. Early summer developmental activity of diatoms in the sub- merged stands consisted of Fragilaria, Tabellaria, and Cymbella. No pronounced vertical stratification was seen on emergent simulative substrata. By mid-summer at both sites vertical stratification was lost. Community structure from July through early fall was dominated on both substrata by Gomphonema sp., primarily perpendicular to the macro- phytic substrata on secreted mucilaginous stalks. A secondary community structure of epiphytes adherent to the Gomphonema developed, primarily consisting of Eunotia and small forms of Cymbella, Fragilaria, and Synedra. Dominant forms possessing hold-fasts or growing prostrate during much of the vegetative season were: Oedogonium, Bulbochaete, Zygnema, ChaetOphora, Navicula, Cyclotella, Synedra, and Chlorella. Many other taxa, in— cluding common planktonic and littoral forms, were occasion- ally identified, but their quantitative contributions were minor. Considerable numbers of epiphytic stalked Caulo- bacter and flyphomicrobium were observed attached to the secreted stalks of diatoms. Frequency of attachment of stalked bacteria and the presence of fungi correlated strongly with algal epiphytes undergoing decomposition. Compactions of blue—green algae (Gloeotrichia) and mucila— ginous communities of bacterial organisms were occasionally the dominant understory of the epiphyton. Deposition of calcium carbonate crystals and chlorotic diatoms interwoven 101 in a mucilaginous matrix believed to be cell wall residues of initial colonization by adsorbed bacteria, effectively cover the plant surface beneath the stratified and epi- phytic climax community. Annual maxima of both pigments at the two growth sites are temporally separated (Figures 15, 16, 17, and 18). Accumulations of both pigments from simulated sub- strata in the submerged zone (Station II) were very high under ice cover (greater than 3.6 g chlorophyll a and 20 SPU m—2 of the littoral zone). 0n the Opposite side of the lake (emergent substrata) samples were frozen over much of the January to April interval. Yet pigment con- centrations were increasing Just prior to freezing of all but A cm of the water column. Annual maximum and minimum pigment concentrations for both sides of the lake were weakly correlated with periods of intense primary productivity. For Scirpus substrata, maximum rates of carbon fixation in the Spring and fall coincide to within one week with peak pigment concentrations. No similar relationship was found from mid- summer through autumnal overturn. Following ice retreat in the spring (1969), pigments rapidly develOped a vertical stratification. Photosynthesis, on the other hand, re- mained at low pre-ice cover winter rates. For epiphytic algae on substrata in the submergent stand, very little proportionality and direct correspondence between rates <3f carbon fixation and standing crops of chlorophyll a or 102 plant carotenoids was found to occur. The maxima noted during the annual study (greater than 3.6 g a m-2, and greater than 20 SPU m-2), coincided with the lowest ob- served rate of photosynthesis (0.1 g m_2 of littoral zone day-l) for this site. Periods of intense precipitation of 1MC carbonates during photosynthesis measurements by epiphytes on arti- ficial Scirpus substrata (see Figure 9) temporally show some visual agreement with periods of maximum pigment deve10pment of chlorophyll a. Such a relationship may reflect deposition by algal rather than bacterial micro- organisms. Maximum intensities of monocarbonate formation in epiphyte samples from the submergent simulated site bear no correlation with changes observed in pigment con- centrations. Certainly, from a physical point of view, the structural integrity of the sessile communities would be enhanced by the deposited crystalline understory which resists decomposition and allows increased standing crOps. Patterns of integrated concentrations of chlorophyll a and plant carotenoids for the entire littoral water column (Figures 19 and 20) over an annual basis accentuate the differences between the two types of substrata. Maxi- mum annual concentrations of chlorophyll a for algal epiphytes at Station I (Scirpus substrata) were 0.195 and 2 0.383 g m— at the spring and early fall peaks. Annual maxima for submergent substrata (Station 11) were 4.24 103 .mp03p0ha COHme Immwop cHuchooca mo monomopa on» you Umuooppoo ohm Ucm waspprSm HmHOHMprm 80mm oopooHHoo ohms mama .cmenon .oxmq mocopzmq .AHH coaooomv moan .ooo oaooo ooo mHHonHo monoz on» Amv ocm .AH coaooomv opHm mapsow mzmnHom on» A42 .E4 mwg dds. .mmm .>Oz H00 mum www. .84 i5... .22. d >42 E4 4 4 H .1 J) H l md v.0 .mH opsmHm 105 .mpmpmeSm HmHOHMHpnw Bonn popomHHoo who: mpmo .cmmHBOHz .mxmq mononzmq .AHH QOHpmumv ouHm .Qom mango cum mHHonHN mmnmz on» Amv paw .AH COHpmpmv mpHm mandom mammHom map A45 #54 E42 .mmu .24.. .08 >02 mwm. Huomww.034.-_2..§>42.¢& BACTERIAL-ALGAL MNED UPTAKE RESPONSE (INS/TU) o I 2 3 4 5 ADDED SUBSTRATE (fr-oi") I / (% OF UPTAKE) 400 r B 25 CM 300 I0 CM 5 CM D 20 «3 en so no GLUCOSE¢ng5 113 responses likely reflect epiphytic bacterial and algal enzyme and diffusion systems that are highly specific for only certain concentration ranges of the solute. 0n addition of near natural concentrations of glucose and acetate, epiphytic microbial populations nearly always responded with first order kinetics. A typical kinetic response to the addition of glucose-luC to suspended epi- phytic communities from substrata in the Scirpus acutus site on 28 July 1968 is graphically represented in Figure 21, Part B. Velocity of organic uptake in routine measure- ments usually increased with depth and lowest velocities were observed near the surface or upper portion of the macrophytes. Although duplicate measurements were seldom made after the initial studies, some indication of measure- ment variance and reliability was obtained by calculation of standard deviations and correlation coefficients for each of the linear responses to the addition of labelled organic isotopes (standard deviation range = l to 10%; r = 0.950 or greater for over 90% of the field measurements). Loss of respiratory l“CO2 during incubation with organic isotopes was estimated by precipitation as barium carbonate and liquid scintillation procedures. In late fall and early spring respiratory losses were equivalent to 25 to “0% of the carbon remaining intracellularly (gf. discussion by Hobbie and Crawford, 1969). Annual cycle data were not corrected for such losses. 11A Experiments were designed initially to assess ranges of rates of utilization of glucose and acetate (0 to 100 pg 1-1) from several equal surface areas removed from natural macrophytic substrata. Summarized results (Table 8) indi- cate minor differences in rates between epiphytes on various types of aquatic vegetation. The small differences in rates may reflect variations in actual bacterial biomass or species within the adherent complexes. It is noteworthy that (1) rates of organic utilization for the same sub- strate are quantitatively very close to one another, and (2) that acetate is utilized much more rapidly than glucose (the ratio of acetate to glucose uptake = 1.5 to 3.2: l). The increased uptake of acetate over glucose has been ob- served for planktonic bacterial populations in a near polluted pond, also by 1“c methods (Allen, 1969). In other freshwater studies on utilization of dissolved organic compounds, their in situ concentrations, and turn— over times (Hobbie, 1967; Wetzel, 1967), this preference is not markedly shown. Assessment of rates of chemo—organotrophy of glucose was made on triplicate surface areas (each 0.785 cm2) from the same plant at depth (Scirpus acutus; 15 cm above the sediments; 1“ September 1967; Vma 1-1 hr'.’1 dm_2) and from six plants spatially separated x range: 11.7 to 19 pg within the same macrophytic stand (Scirpus acutus; V range: 8 to 21 pg 1-1 hr-1 dm—2). Statistical differences max could not be demonstrated between uptake responses on 115 TABLE 8.--Rates of chemo—organotrophic utilization of glucose and acetate by heterogeneous epiphytic communities on several natural macrophytic substrata, Lawrence Lake, Michigan, 21-23 September 1967. Velocity of Utilization Plant (pg 1'1 hr.1 per dm2)a Glucose Acetate Scirpus acutus Muhl. 10 cmb 11.3 37.9 20 cm 12.9 25.6 Chara sp. 5 cm 9.0 17.6 15 cm 8.7 16.“ Nuphar sp. 25 cm 13.“ 21.2 60 cm 17.8 30.9 Najas flexilis L. 5 cm 7.3 1N.5 15 cm 6.2 10.5 aUptake rates expressed as velocity of removal of organic compounds by epiphytic bacteria from 1 dm2 of macrophytic surface area at depth indicated. Standard deviations of response lines were within the range of l to 10%. bDepth above the sediments for stem, petiole, and internodal substrata collection. 116 artificial substrata and on the natural substrata subse- quent to six weeks in §i£g_colonization of the Plexiglas substrata (only glucose and acetate tested; ranges and standard deviations overlapped considerably). There appeared to be a more uniform response in microbial activ- ity with regard to spatial distribution of samples, but this may be an artifact of the small surface to which uptake velocities were extrapolated (1 dm2). Expressing organic uptake data per dm2 attempts to reduce the error involved as species heterogeneity and area covered probably have a very high variance. Secondly, the high rates per small area emphasize dynamics of i2_§itu removal of organic compounds. Chemo-organotrOphy of several organic compounds was tested in addition to glucose and acetate (Table 9). Utilization kinetics of glycolate, glycine, and serine showed unusually high variance and poor linearity at low substrate levels. It may be concluded from these limited data, and from other studies (see Allen, 1969, for example), that glucose and acetate are two of the most suitable organic substrates with which to monitor annual chemo- organotrophic metabolism. Certainly testing of annual cycles with a large array of organic compounds would be necessary to offer any statement of their quantitative importance to one another or to the total soluble organic carbon pool. Were in situ concentrations of fructose, galactose, glycolate, succinate, and the amino acids very 117 TABLE 9.--Comparison of chemo-organotrophic utilization of dissolved organic compounds by epiphytic communities re- moved from the emergent hydrophyte, Scir us acutus, Lawrence Lake, Michigan; 2“ September 1968.a Velocity of Utilization l -1 Organic Compound . - 2 b (Vmax’ pg 1 hr per dm ) Glucose 12.5 i 1.3 Fructose 1.5 i 0.25 Galactose 2.0 i 0.59 Acetate 31.0 i 3.5 Glycolate “.5 i 3.60 Succinate 3.9 t 1.8 Glycine 9.7 i 3.0c Alanine 7.7 t 1.6 Serine 2.0 i 1.030 aNatural epiphytic samples removed from 15 cm above the sediments; resuspended in “00 ml of ultrafiltered lakewater from the site prior to measurements. _ bConcentration range for added substrates: 0—160 pg 1 ; each mean velocity is based on duplicate measurements; : = range. 0Linearity of response kinetics poor. 118 low in comparison with those of glucose and acetate, the ecological importance of their utilization would assume much larger prOportions. Two further tests were conducted to establish the effect of experimental conditions upon the use of Michaelis- Menten enzyme kinetics for routine field studies of micro- bial populations. Replicate epiphytic samples from Scirpus acutus substrata, incubated in the dark in the presence of glucose-lac, were subjected to (1) a slow oscillatory movement (Eberbach shaker; approximately 60 oscillations per minute), (2) a fast oscillatory movement of approxi- mately 180 oscillations per minute, and (3) no movement. Rates of uptake under the respective conditions were 26, 29, and 18 pg 1‘1 hr-1 dm-2. The increase is probably due to dispersion of aggregated bacteria and provision of more bacterial cell wall surface area for isotopic uptake. Annual cycle samples were subjected to shaking (wrist— action Burrel shaker), primarily to destratify the isotDpe and to provide constant dispersion of nutrients naturally occurring in the sample. Variations in incubation periods of l to N hours showed rate of change of velocity decreased considerably during the 3rd and Nth hours, suggesting high bacterial mortality or a severe "bottle effect", although other ex- planations are possible. Incubation of l to 1.5 hours was routinely employed throughout the annual period. 119 On two occasions rates of organic utilization ob- served on substrata near the sediments (5 cm above the sediments in the Scirpus acutus site) were nearly identical for both substrates to rates from similar surface areas of epibenthic (upper 1 mm) samples. There is a possibility that microbial floras are migratory between the lower plant portions and the sediments but it is more likely that species composition within epiphytic and epibenthic habi- tats is similar. Annual cycles of glucose (Figures 22 and 2M) and ace- tate uptake (Figures 23 and 25) by epiphytic communities showed similar patterns of utilization. Highest rates of uptake of both substrates occurred in communities near the sediments. Acetate was preferentially utilized over glucose and had two annual peaks of utilization in both sites (July and September, although temporal agreement is not exact), ranging from 90 to 130 pg 1-1 hr-1 dm"2 for substrata in Station I to no to 70 pg 1’1 hr'1 mi2 in Station II. Minimal rates of acetate utilization (5 to 20 pg 1'1 hr"1 dm-2) generally occurred from October through the period of ice deposition with increases near the bottom under ice cover at both Sites. This latter phenomenon, pronounced near the sediments prior to freezing of substrata in mid- February at the Scirpus Site, appeared in the flélii and Ch§§§_site Just prior to loss of ice. Neither of these pulses coincided with increases in photosynthetic rates for the same periods. Development of both chlorophyll a, 120 Figure 22.-—Chemo—organotr0phic utilization of glucose (Vmax; pg §lucose removed 1"1 hr-l) by attached bacteria from 1 dm of colonized surface area at 5, 15, and 25 cm above the sediments at the Scirpus site (Station 1), Lawrence Lake, Michigan. Data were col— lected from artificial substrata. 121 Figure 22. anunirwwmfi 8§§§§§ 300 ~ zoo» E 8 O O 122 Figure 23.—-Chemo-organotr0phic utilization of acetate (Vmax3 pg acetate removed 1-1 hr-l) by attached bacteria from 1 dm2 of colonized surface area at 5, 15, and 25 cm above the sediments at the Scirpus site (Station I), Lawrence Lake, Michigan. Data were col— lected from artificial substrata. 123 Figure 23. T h P nmmmm nun-mum 1 . 1 by. D P hr + b I? m... m . . «ZS... tifoEEHaR l2“ .MuMSmeSm HmHoHMprm Eopm UopooHHoo who: puma .cwanon .oxmq mocmpzmq .AHH CDHpmpmv opHm mpmno-mmwdz on» up mpsoeHoom on» o>oom Eo 0H ncm m up mono mommpzm wouHmo 00 mo New H Song mempomn Umnomppm an AH-nn H-H po>oEom mmoous m: m m >1 omoous mo SOHpmNHHHps oHnaopuocmeo-oEono-I.:m opstm 125 O j A I L A 1 l O twp-JIM asoomobfl A L A A L .sm magmas 126 .mpmpmeSm HmHOHMHupm Song pmpooHHoo ohms mama .cmenon .oxmq mocopzmq .AHH coHumumv opHm mpmno-mmhmz opp pm mpsmEHpmm map o>oom 50 OH pew m pm mops mommpsm poNHcoHoo no man H .Eopm manopomo pmnomppw an AH-pn H-H pm>oth mumpmom w: mxme>v mumpmom mo SOHpmNHHHps OHonpuocmwpo-osono-I.mm mpstm 127 mmw. 8m. >4! 2&4 .24! dun .245 .08 :02 H8 mm .§.a§...§.>§.g 0 fl I 1. .8 v .1 T .QON . .8.» r i r 18'“ 1- .. . am 1. .1 V 1 so. gm. .1 .r .oosw. - - a .1. O .09 1 Y 1% f . . 12001 .8. r r L t 8? .mm oasmam 128 and total plant carotenoids correlated strongly with the increase in chemo-organotrophy at the Scirpus site. Rate changes in spatial and temporal utilization of glucose were similar to those seen for acetate. Perio- dicity of epiphytic utilization of glucose on the marl bench area was nearly identical to acetate near the sur- face but was reduced by 50%. Bimodal responses for glucose uptake were found at 5 and 15 cm above the sediments and agreed with acetate, but differed again by 50%. The upper- most substratum at the Scirpus site revealed a peak which occurred between those seen at the lower levels. A strong bimodal response was not evident for glucose uptake at the Nalas-Chara Site. At the submerged site an annual maximum spanning July and August was found. Rate increases ob- served for acetate during ice cover were similar for glu- cose. Glucose annual maxima (greater than 30 to 55 pg 1'1 hr-1 dm'2 for Station I; greater than 20 to U5 pg 1"1 hr-1 mm.2 for Station II) were considerably lower than those of acetate; minima values were in the range of 5 to 10 pg 1-1 hr-1 dm—2 for glucose uptake. Several summarizing remarks with respect to chemo- organotrophic utilization per unit of macrophytic surface area are pertinent here. Annual patterns showed gradual and continuous change without rapid oscillation (several short-term studies during initial investigations confirm this), contrary to changes seen in annual pelagic patterns of eutrophic waters (see Allen, 1969, for example). Peak 129 activities of utilization of the organic compounds seldom demonstrated strong agreement, Spatially or temporally, although increases towards the sediment were commonly present. This relationship suggests separate sources of lfl.§l§3 organic compounds, physiological differences in bacterial species, or, indeed, a community composition distinctly different at each of the sampled strata. Rates of uptake for both substrates during the vegetative season showed extremely close temporal correspondence with rates of primary productivity and may indicate interdependencies in nutritional metabolism via algal excretion, bacterial respiration, etc. Rates per unit area of substratum were higher on substrata colonized on the calcareous bench than in submerged vegetation. Annual rates determined in this study are among the highest presently known, and reflect high rates of metabolism in this portion of the epiphytic system. Isopleths of epiphytic utilization of glucose and acetate per square meter of the littoral zone for the Scirpus acutus site (Figure 26 and 27) and the Najas-Chara site Show dynamic changes over the annual period (Figures 28 and 29). The differences in available macrophytic surface area per m2 of the littoral zone now obscures differences seen previously on a unit growth substrata basis. Potential removal of organic carbon exceeds 2000 to 3000 pg 1"1 hr_1 mm2 by attached microflora colonizing 130 .wpmpquSm HmHOHMHpmm 80pm pmpooHHoo mum: some .cme30Hz .oxmq mocopzmq .AH COHpmpmv ouHm msmpHom on» up ocou proppHH mo Nev H Eopm mHmmpomo pogomppm mp AH-pn H-H pm>oEmp oompoom m: mxme>v opwpoom mo SOHpmNHHHp: oHcQoapocmwSo-DEDSO mo mcuoHaomH-I.Sm mpsmHm .mpmnmeSw HwHOHmHupm 80pm oopooHHoo who: mama .cmeEOHE .oxmq monopzmq .AH SOHumpmv mpHm msmuHom one up msoa HmLODDHH mo so H Scam meopomn oosomppm an AH-pn H-H po>oEop omoosz w: mxme>v omoozH mo SOHpmNHHHpS oHnQomuosmmpo-oEmno mo mnuquomH-I.mm opstm 131 1. g :9 \S. m /" g P“) 01: i \”\l: \1:: 19V§ ~11. ’3 W2 #N\ -g 132 .mpmpmeSm HmHonHupm Song oopooHHoo who; mama .cmegon .mxmq mocmnzmq .AHH COHpmpmV opHm amazo-mmnmz one am ocou HmSOppHH mo New H 80pm meopomn pmnomupm mp AH-pn H-H po>oEop oompoom w: mxms>v mumpmom mo SOHpmNHHde OHnaoppocmwpo-osono mo mzpoHQomH-I.mm mpsmHm .mpmpmeSm HmHoHMprm Song wouooHHoo ohms puma .cmmHson .mxmq monomzmq .AHH COHpmpmV opHm mpmnolmmnmz one up msom HmAOppHH mo men H Eopm meopomn convened an AH-Ln HIH po>oEop omoous w: mxwe>v mmoous mo SOHpmNHHHu: OHQQoppocmmno-oaozo mo mapmHaomH-I.wm opdem 133 .000 .>02 80. :4— 35H. ID 1 9 mwm. >4! dad £42 .00... mum .0 .5 oz/H1/\ 58.5“? 88 888\\11/ .11 1 .1 .08 § RN09 8 7 \1 8 1 1 .1. _ 18 1.18.1 . . mwm. . >42 .Ed .mSz .00... .23. .000 .>02 mum .0D<__\ JR. 7:. >42 Ed 1 . . M... - ”\WWM .12 . .1../\./ 8v8~ CON 8. 09 000 00.» SN 1 8. f81. 1 1 f: 8.1 Q 1 '2 O Q SLNBWIOBS BMW (“19) H1d30 O N O I!) .8 Si~3wuoas 3Aosv0m) H1d30 .mm oasmam .mm mhstm 134 'l (infz c>f ‘the littoral zone (for both strata = 4978 pg 1"1 hr_1 dm-Z). Assuming the water column above a littoral surface areaofldm2 contains an estimated 2 to 4 liters of water, each liter containing 2 to 10 mg dissolved organic carbon, epiphytic bacteria (together with epi— benthic forms) are probably responsible for removal of ‘mucrlcmf the labile and refractory organic compounds which occh?‘there. It is impossible to assume such a large 'portixnu of the labile soluble carbon pool exists as glu- cose and acetate, but high substrate approximations (as Kt + Sn values through graphical analyses of the enzyme kinetics; 100 to 300 pg 1"1 of each) may be indicative of extremely rapid regenerative mechanisms followed by efficient removal. Wave and mechanical activity in the littoral area probably contributes to replacement of litto- ral water and an increased organic nutrient availability against "dead water spaces" at the epiphyte—macrophyte surface. During normal thermal stratification, with cycl— inggnmmably limited to the epilimnetic waters, and normal stagunm water of macrOphyte beds, the size of the littoral andgxflagial dissolved organic matter pools may be strongly affected by epiphytic metabolism. Idttle evidence for significant vertical stratifi- cathxlwas found prior to or following the vegetative and icedrmaseason. Freezing of all samples for a minimum oftwmfiw days during the winter at the Scirpus site was prdmbhrpartially responsible for the low rates seen 135 subsequent to ice retreat, as pOpulations may have been severely damaged with respect to enzymatic activity. It seems apparent that incomplete colonization had occurred by tfiue conclusion of the first spring, 1.3. rates in May <1f 1969 were already equivalent to those observed during July of 1968. Annual glucose and acetate utilization rates were integrated for the total macrophytic surface area present for colonization per dm2 of the littoral zone, for each of the respective sites (Figures 30 and 31). Scant, non- reticulate emergent substrata (50 to 60 Scirpus acutus 2 plants m- of the littoral zone) for colonization on the marl bench explains the disparity in annual rates. Maximum summer rates of removal per unit of littoral zone for Scirpus site substrata did not equal winter rates for sub— mergent site substrata. In essence, if these data are representative, the only significant epiphytic removal of dissolved organic compounds occurs in littoral zones within submerged macrophytic vegetation, and is due only to pro- vision of considerable biomass on an expanded surface area. Integrated data accentuate the bimodal response of maximum acetate uptake and the single peaks for glucose. Secondary peaksikH'Station II epiphytic metabolism occur during the vfimmer months and are probably of species—specific signifi- cance. Annual ranges for chemo-organotrophic uptake of ghumme are 3.7 to 2““ pg 1‘1 hr-1 dm”2 for the Scirpus siteeum 119.8 to 3155.0 pg 1'1 hr—1 dm—2 for the submergent 136 .mumpquSm HMHOHMprm 80pm umpomHHoo who: mpmo .cmeQOHz .mxmq monopsmq .AHH soaumpmv mpfim .ddm mnmno one mfiafixmam wwnmz on» Amy cam .AH coapmpmv muam msuSom msmufiom on» A¢v um msos HmLOpuHH mo map H 80pm wfihmpomn ponompum an Aalhn HtH Um>oEmp mmoosaw w: mxms>v mmoosaw mo coapmNHHHps candoppocmwpouoEmso umpmpwmchlu.om madman 137 9X2 9X2 >42 dad M32 .mu... ZS. duo >02 Foo mum .034 :55 23... >42 in? o .80. .88M 88% F f b h » ooocx J. U. 4 o 1.. . D. m .8. r Sou P r L1 L1 F g .om mhswfim 138 .mpmpmeSm HmfiOHMprm 80mm Umpomaaoo who; sumo .cmMHQOfiz .mxmq mosoazmq .AHH soapMpmV mpam .adm wpmno ocm mfiaaxoam momma map Amv cam .AH coapmumv mpfim mSpSOm msahfiom on» AoEmp mumpmom w: mxme>v muwpoom mo soapmufiaaus candompocmwhouoEmno UmumpwmpsHln.Hm mpdwfim 139 me. 8m. >42 .E4 M2: .mmn. .24.. .0mo .>02 Foo mum fi .02 35.. .22. >42 4&4 J. 4 d u q A d d (I) O 0 § § § § § rum PM ,.I 31V133V 5r! . Hm 3ng 1H0 Annual acetate utilization ranges for 1 dm2 of the l hr-l, and 71.2 to These latter site. water column are 10.4 to 502.2 mg 1- 14977.8 ug 1-1 hr-1 at these respective sites. values suggest a paramount effect upon prevailing dissolved organic matter in shallow water littoral plant beds, re— gardless of its origin. Annual mean values of chemo—organotrophic utilization of glucose and acetate were determined by planimetric measurement of the respective integrated curves (Table 10). If these mean values are summed together for both sites and extrapolated to a square meter basis over a 2“ hour day, and converted to the equivalent percentage of carbon in glucose and acetate, a total of 610 mg of glucose and 958 mg of acetate are potentially used per m2 per day in the litto— ral zone by epiphytic communities. Assumptions are that these rates persist over a 24-hour period (which is likely to be an overestimate), and that a continuous regeneration of these substrates occurs. A comparison of these data to net mean annual primary productivity by algal epiphytes 2 of the litto- (1807.2 + 195.7 2002.8 mg C assimilated m- ral zone day-l) indicates 78% of the carbon produced on a mean daily basis could be removed if it were directly con- verted to free glucose and acetate. If autolysis of attached algal forms and excretion by them accounted for 5 to 10% of the dissolved organic matter at the plant this would not be enough to sustain the rates of surface, chemo—organotrophy observed, and further suggests the use 141 TMHE NL--Mean annual rates of chemo-organotrOphic uttUzatum of dissolved organic compounds by epiphytic cmmmnflfles, Lawrence Lake, Michigan. Data were collected from artificial sub strata . i Annual Velocity of Utilization 1 hr.1 per dm2)a vegetation Site (ug 1" Glucose Acetate 5A 106 Scirpus acutus Muhl. Najas flexilis and Chara spp. 586 893 aBased on total macrOphytic surface area present per unit area of littoral zone. 1142 of organic solutes from other sources, 3.5. macrOphytic If the release and surrounding littoral water column. asmmmtnn1that natural chemo-organotrOphic uptake is Lmuted‘mathe 6 to 8 hours of diel darkness is made, the annual rates assume even greater importance in littoral metabolism. C. In situ lLlC-labelling of macrophyte- epiphyte systems Extracellular release of dissolved organic compounds has been established and reconfirmed for axenic laboratory grown Najas flexilis (Wetzel, 1969a, 1969b; Allen, 1970). Similar measurements have not been attempted to demonstrate the occurrence of extracellular organic release under Further, no studies have natural conditions in the field. been conducted to establish if natural epiphytic communi- ties cmui'utilize such products. Circumstantial evidence derivemi:flrom laboratory cultures presents a very strong case»fxn?‘this, but field evidence for this interactive systmnn is 21 necessary prerequisite to application of laboratory data to in situ observations. {To chocument the release of extracellular materials under natural conditions the emergent hydrophyte, Scirpus xvais pulse-labelled above the water surface with acutus, evolved 114002, of high specific activity, at close to Liberation of dis- ambient concentrations (0.035% v/v). solved organic materials was followed with respect to 1143 depth and time to show patterns of release for plants with and without their epiphytic communities (Table 11). The difference in activities of dissolved organic matter escap- ing from the plant surface can be attributed to efficiency of removal through epiphytic metabolism. A third plant, serving as a control, was also labelled with carbon dioxide, but without a Plexiglas tube surrounding the plant and restricting its external aquatic milieu (Table 12). Verti- cal distribution of dissolved organic matter release is not a prominent feature for either of the plants surrounded by Plexiglas tubes. Loss of exchange ability and water — renewal may have had adverse effects upon the hydrOphytes themselves and disrupted any effects upon the epiphytic communities. Removal of the epiphytes showed little effect upon dissolved carbon loss from the macrOphytes during the first 2 hours of incubation. Activities increased where epiphytes were present following this period and after 5 hours in situ incubation values were approximately twice those where epiphytes were removed. These data suggest interactive mechanisms may exist between the epiphytic community and the host vascular hydrophyte, and further may indicate that sessile communities are fundamentally important in inducing the physiological release by the macrophyte. A second possibility is that the response is due to physiological damage sustained by the plant during physical removal of its epiphytic vegetation. A similar increase in extracellular release is seen in the 1U“ .oopooHHoo mama mmHoSmm momMLSm Loom: on» onoo gamma 0 .moQSp mmemeHm SH oomOHoSo who; mopzndHao pdoszz cam cpHs mucmHQ mandom mSQSHomo .mpH>HpoMOHomS opszHE Sod monsoom sz mmm as: mmm mam mHm mH: So om me mm: am: Hmm smm omm omm So om mos mHm sm: Hom mmm om: :zm So OH osm mmm mo: mmm mam mmm omm So 0 moossofioo pSOSpHB .Hnsz mSpSom msmpHom mHm sw: mm: mmm mmm ssm ozm So om :sm omm Hem sHm mHm mHm um: So om mmm mom omm mmm osm mmm mmm So OH mHm mom mmm Hsm mam mHm Hmm oSo o LbMopsnaHdo anz .anz minnow madeom SHE Sam Lam SEN mmnmm :mumm mHIHH muo mHm>moch wSHHdEmm owmsoo mEHB Eopmsm guano pm youmSome mo HIHS mASQoV mounds oHSmwho oo>HommHo «“5th a . m . IHSEHm .MMMSSH SOHuwpm oozH mo mosmmmpo on» CH mHmmnmmmH wand leMfimmH£0fiz .mxmq £PH3 USN 3P 3 we oHcmwmo wo>H0Hssz mapsom mmmmwmm .m .mSmOSOSQ Spam S I! H 0p msoocwp saga mmHv mo mmm SH mamas mHmman. 145 .oopooHHoo mums mdeSmm oomMSSm Looms onp SOHoo cocoon .mmbjp mmemeHm so ooUSSOLSSm mucde mmozp on Hopucoo m mm mm>pmmm sow mes ems :Hm 2mm mam SSMH So om Hos mmm mom mmm mo: 00: mmm So om Hm: mHo mom mmm ssm so: wow 50 OH mmm mHm mmm 2mm msm msm mmm oSo o mmpmdedo anz .Hcdz mandom mSQSHom .SHS pzm pzm Szm molmm :mlmm mHlHH mlo mHm>Smch wSHHQSmm mo mmpsoo oEHB Smpmmm andmo pm Somemme mo HIHS Sdov hoops: oHSmwho om>HommHQ .m mH we: . (ll wocmmmha asp CH mfimwnuwhmOponm om .fiWWHEOHZ .meA mocwhzmq «H COHQMpm . N lopozn pcmm Spam C 0 post on» so AooHHopmHnquv woowmwswmmmmHsSHm ..Hn:z.mmmmmw m: c oozH go no How oo>Homeo mo ommmHomau. .mpmsa 146 armrolpflant after 1 hour incubation, especially towards Whether the release is light dependent and the sediments. These a true stratification exists in nature is not known. expanhmnts were conducted at mid-day from 0950 to 1450, mkiremuts may be correlated with day length and photo- response of increase in early afternoon may period, 1.3. be rekued to diel photosynthesis patterns of the host plant. 4 CO2 In the above dissolved organic matter samples was purged from the scintillation fluid, to yield only carbon-14 in the dissolved organic phase. On several occasions this sparged “CO2 was trapped in an NCS solu— bilizing agent (see methods), or in a diamino-ethanol based fluor mixture for quantification. Estimates of 12 C of the total amount released as dissolved to 29% as 1“ organic compounds (for 6 samples only), indicates epiphytic autotrophs may be obtaining a certain amount of their in- organic carbon from the supporting plant. .Non-quantitative samples of attached communities wermelnmnoved by gentle friction on the plant surface, .folltwnai by suction into a glass vial containing solu— biLLizen? arui fluors, at time intervals of dissolved organic matter collection only to determine if they were labelled Scintillation counting and if a transfer had occurred. 1“0 was being utilized by the proc edure s c onfirmed that the label was detectable within a three—minute epxipfluytnes; Iaezricmi. 4‘ ..' ( til-Ill} "l llll I. ll! 147 FdUowing termination of the field experiments, all threelflants were removed from their growth site. Epi— gflwtesihbm known surface areas (0.785 cm2) were removed at 6 cm hwervals from the portion of the Scirpus plant norm~ allytumerwater (on 2 plants), resuspended into fresh lake- All three waterzhxmlthe site, filtered and radioassayed. plants were sectioned at 2 cm intervals and 1 mm thick discs of stem tissue were dissolved in NCS and Triton-X, plus fluors to establish localization of the isotOpe following photosynthetic labelling. Vertical distribution of carbon incorporation by the epiphytic complex shows little quantitative change (Table 13), although too few data were obtained to make any definitive statements. It is apparent, however, that under natural conditions carbon transfers do occur within Such data may this particular epiphyte—macrOphyte system. suggest that a host of physiological interactions are probably functional within epiphyte—macrophyte systems elsewhere. Attmnnpts were made to determine patterns of carbon incorporation by Scirpus via liquid scintillation techni- tuu: errors may have been introduced into the data ques, Precautions were taken to mini- due to pigment quenching. mize these effects. Internal translocation of the 1“C label (Table 14) showed little similarity between the three Activity with Scirpus plants after 5 hours incubation. 148 TAMfill3umIntracellular fixed carbon—14 in the epiphytic cmmmnimlof Scirpus acutus Muhl., following in situ photo- synumtnzluC-labelling of the emergent vascular hydrophyte. (5 hmn7incubation; Station I, Lawrence Lake, Michigan; 30 June 1969). Epiphytic Activity Emergent Substrata and 2 Depth Sampled (cpm per 0.785 cm area)a Scirpus acutus Muhl.b 0 cm = surface 669 6 cm 326 12 cm 464 18 cm 469 24 cm 302 30 cm 291 36 cm 311 42 cm 655 46 cm = sediments Scirpus acutus Muhl.c 0 cm = surface 463 6 cm 821 12 cm 325 18 cm 342 24 cm 329 30 cm 221 36 cm 275 42 cm 339 48 cm 271 51 cm = sediments aSanuiles processed by gas-flow counting techniques and were treated by acidification for removal of monocarbonate contaminant acthflty. bSunnnerged portion incubated with a Plexiglas tube enclosure. CConsidered "control" plant; no experimental treatment. E‘t fl - 149 TMHE NL--Intrace11u1ar fixed carbon-14 in Scirpus acutus Mrfl.,fbllowing in situ photosynthetic labelling with J-C02. (5 hour incubation; Station I, Lawrence Lake, Michigan; 30 June 1969).* Activity Incorporated DepmiBelow (cpm per mg dry weight) WaMfi°Surface Scirpus Scirpus Scirpus No. 1 No. 2 No. 3 0 cm = surface 4052 . 1870 1132 4 cm 6608 1431 1562 8 cm 4730 1542 3901 12 cm 2199 1307 2842 16 cm - 1986 3652 20 cm 4688 2146 2143 24 cm 6772 6342 3144 28 cm 3465 2879 1555 32 cm 1760 3161 2008 36 cm 2297 4107 7681 40 cm 4633 5517 7239 *Measurements of incorporated activity were made at 2 cm intervals from the emergent tip into the rhizoid- horizontal stem; Scirpus No. 1 = epiphytes present, Plexi- glas enclosure; Scirpus No. 2 = epiphytes absent, Plexiglas enclosure; Scirpus No. 3 = control plant, epiphytes present, .no surrounding enclosure. 150 depw1emnessed as counts per minute per mg dry weight wascmtahmd by extrapolation of dry weights from 1 mm Huck dkms adjacent to those sampled on the plant for radimfimay. (Activities above the water surface approached 100,mM)cpm per equivalent dry weights, in the vicinity of the 1“002 chambers). (knmiderable dissolved organic matter was present at distances of 1 to 3 meters from the plant after 2 hours in sflnlincubation (depth: 20 cm below the surface; 1 m distance: 381; 2 m: 31, and 3 m: 18 cpm ml—l). When the amount of water above a square meter of the littoral zone is taken into consideration, these activities assume greater importance, and indicate that products of macrophyte- epiphyte metabolism are detectable at some distance from Such documentation furthers the possibility their origin. of macrophytic—epiphyte interactions in littoral metabolism, particularly with epibenthic communities in close associ— ation. I). Nutritional and physiological interactions of axenic Naias flexilis and cultured epiphytes Ihmior to assessment of mixed community interactions between isolated algal and bacterial epiphytes, and the freshwater vascular angiospern, Najas flexilis studies were undertaken to establish rates of uptake of glucose and acetate-lac by the epiphytes. Initially, however, 151. somefmflliarity with growth kinetics and minimum nutri— tflonalrequirements of cultured epiphytes was necessary. hmmedures for determination of optical density and emflvahnm cell numbers have been described in detail in The period over which log phase of the methods section . grmMflxkinetics could be observed was established by spafinmmhotometric readings prior to each measurement of organhztudlization in media II and WC (for media, see Wetzel and McGregor, 1968; and Dr. R. R. L. Guillard, For bacterial personal communication, respectively). species (Caulobacter and Pseudomonas) log phase was ob- served within the Optical density (O.D.) range of 0.01 to 0.150, extending over a 1 to 3 day period, followed subse- 0.240 to 0.400 quently by a population plateau (0.D. Algal species (Gomphonema, Cyclotella, and maximum). Chlorella) generally reached maximum rates of cell increase within 4 days of inoculation and persisted in log growth Bacteria cultured fins 2 to 5 days and occasionally longer. iJIInediiun II required the presence of a vitamin mix (same as Lanai for WC), trace metal mix (as for WC), ammonium The presence of source (NHLIC1), and glucose (1 mg l- ). had no effect upon enzyme glucose at l to 100 mg 1— kinetic responses when determined by 1 apparently there was no adaptive response to higher exter- Epiphytic algal cultures required “0 uptake, 1.3. mal concentrmflons. vitamins and trace metals but no additional carbon or .‘ .II/\I..cl\ ( 152 enemy smntes. Further, they were not enriched with CO2; pHcfl‘aLlexperimental media was 7.5 to 8.2. Allspecies of epiphytic organisms were grown into log;flmse,wmshed twice, concentrated, and placed into 100 mlcfl‘fresh medium. Near in situ concentrations of high:nmmific—activity, uniformly labelled-lac glucose and acetate were added by micropipettes to cultures of Con— individual and mixed algal and bacterial epiphytes. centrations were extended to 5 mg 1-1 where algal species Uptake measurements monitored by Michaelis- were used. Menten and diffusion kinetic graphical and mathematical analyses, at temperature ranges simulating winter (5C), vernal and autumnal (11 to 12C), and summer (21 to 23C) conditions in shallow—water littoral areas are summarized in Tables 15 and 16. Numbers of organisms extrapolated from optical density measurements did not change appreci- ablgrchxring the course of incubation and were usually wittdxi‘the range of 10Ll to 105 ml-1 for bacteria and zilgae. thile cell concentrations are likely to be higher than in many freshwaters, they were lower than numbers commonly employed in microbiological and algological studies (frequently 106 to 109 ml'l). Raixes of organic uptake for individual algal and baxrtertial.<3ultures showed a strong general correlation In several instances low temperatures with temperamne. seemed to inactivate enzymatic and diffusion mechanisms nl‘ II III} 153 .mSOHpmSpsooSoo Sost: am ozonm mQOHm HmpsouHSon on» on SOHpHUom SH .moHpmSHx pmoso pmSHm mpmowwdm mSOHpmppcooSoo mumpmeSm 30H pm wHHopOHomu mo omSOQmmm** .szSmmSHH hood oouHoanm mmSOQmoS SOHpmHsooo mmpMoHoSH* OHm. OHN. 10m. emsH. I *mso. mHHoSOHno Hmm. cam. 3mm. OHS. *msH. omo. Sem.: «HHooOHosU sow. mms. mam. mom. . *mHo. oeocoemeoo H.s m.m *H.m :.m *m.o *H.o mMSoEoodomm m.: H.H N.H s.m :.m m.m nooomQOHsmo a o a o a o a o a o a o ommuHm omHnHH om ommuHm omHuHH om o xme moSSszo AHILS m xv AHISQ HIH m: m >V mofipmcfix Coamswmfio mafipmcfim pkommcmhe m>apo¢ .Ampmpoom u g momoous n ov Hpopomo cam Hmem mo mop: HHp: oHcooppoSmwponoSmgo hops: mmphcafimm Hm .mco ocm mmoode mo COHpmNfi Hpfiocoo HmELmnp pCme pHso oHcoxm Hose > no moods mo cemHH HUSH z ooHc m monsoonlm popmom V\’ I «l / ,ll\|| ( k 154 .mpHpmmSHH Loon omuHosto uncommon SOHpmHzooo mopmoHccHx oom.m 0mm. moH.m OHs. 00H. mmH. o.H m.m m.m m.= o.H m.o meococdeoo + mHHoSOHso + nooomDOHsmo Hoo.m omm.H smo.H 0mm. OOH. 30H. m.m m.: :.o m.m .m.o *:.o mwmmmmmmm + mSoS02QSoo + mmcoEoosomm o=.H com. moo.H sHm. a moo. =.m 3.5 m.m H.m - .=.o «Soconqeoo + mmeoeoosomm som. Hmz. moo. 0mm. u .Hmo. s.m S.m m.m H.m . .H.O mmmmmmmmm + mmcoeoosomm mms. 0mm. 0mm. .mOH. . .oHo. :.m m.m *m.o *o.H - *w.m mHHoSOHgo + noooNQOHsmo Ham. mmm. mms. smm. a memo. 3.0 m.m =.m m.m n m.m mSoconmSoo + pooomQOHsmo < o < o a o < o a o < o ommuHm omHuHH om ommuHm omHuHH om monsoHso AHILS moxv AHlpz HIH w: mxme>v mofioocfix counsoowo moHuoSHx upcomcmpe o>Hpo< .Hopmpoom u < momoous u ow .mSOHpHocoo HmESonu pcopmmoHv Sons: mouzndHao HmHSopomo ocm Hmem mo mopsuHSo oacmxm vmxHE an mamamow Ucw wmoode MO COHpmNHHHp: UfiSQOLpocmwhOIOEmno MO mmpMQ no COmHHMQEOOII.mH mqm<8 155 Rates of glucose uptake of Caulobacter increased at lower temperatures, indicative of highly efficient and specialized enzyme systems under cold conditions. Pseudomonas showed an increase in rates of uptake for both substrates at 11 to 12C, and at 21 to 23C. A common response by algal organisms to substrate concentrations greater than 100 ug 1—1 for all species except Cchlotella was an increase in rate of diffusion of both substrates. Rates of increase of both algal and bacterial cultures were not proportional to increases in temperature and likely reflect optimal enzymatic activity. Uptake by Cyclotella is of noteworthy importance in that first order kinetics were expressed at concentrations generally dominated by bacterial uptake. This observation may be indicative of direct competition with bacteria for organic substrates. Similar results were not observed with the remaining cultures. Uptake responses with mixed populations of algae and bacteria in medium II showed definite metabolic inter— actions (Table 16). Bacterial responses definitive at the lower concentration ranges as in monoculture experiments showed little change in rates of organic uptake in the presence of algae although, as noted, linear responses for glucose at 5C were characteristically poor. Algae, on the other hand, with very few exceptions showed increased accumulation of radioactivity over monoculture conditions. Additional uptake may have been due to uptake of 14 C02 previously respired by the bacteria. Increases in 156 diffusion rate of the algae were greatest for diatom- bacteria and bacteria-diatom-chlorophyte combinations. Nutritional interactions such as these may, in part, be responsible for high rates of organic uptake in natural epiphytes and further be indicative of C02 cycling within the attached community. Increases in the cycling speed within the solute phase and recovery of respiratory losses as carbon dioxide by autotrOphs and metabolic forms capable of using inorganic carbon probably represents an ecological advantage to the epiphytic complex in comparison with spatially separated communities, e_.g. pelagial. Although measurements employing Michaelis-Menten enzyme kinetics and diffusion mechanisms permit the deri- vation of turnover times of the organic substrate being tested, such parameters have little relevance in that regenerative rates (as T1; and Td; see Allen, 1969) cannot be linearily added in heterogeneous communities to explain effects of one taxon on another. Velocity and rate measure- ments are far more applicable to the interpretation of results where fixed or known pOpulation numbers are used. Extracellular production of dissolved organic materials by Najas flexilis in response to variously modi- fied inorganic nutrient conditions has been previously established (Wetzel, 1969a, 1969b). For purposes of this study, autotrophic assimilation of 1’4 C-labelled inorganic carbon and subsequent release of lac-labelled organic compounds by axenic cultures of Najas flexilis were 157 redetermined (Figures 32 and 33). Techniques of sterili- zation of seed surfaces, procedures for germination and general nutritional responses of this angiosperm have been presented elsewhere (Wetzel and McGregor, 1968). Constant labelling of macrOphytic tissue occurred subsequent to 3 hours incubation under constant laboratory conditions (750 lux; 200; medium II). The assumption was made that rates of excretion would be maximal only after the entire metabolic pool became labelled to a constant specific activity. To substantiate the pattern of extracellular release in medium II, plants (5 or 6 plants per flask; in triplicate) were pulse-labelled with inorganic carbon-l4 for four hours, followed by placement into label-free fresh media, after which changes in absolute activity in the dissolved organic carbon phase were followed (Figure 33). Over a 3.45 hour incubation interval, under conditions identical to those used for autotrophic assimilation, a mean excretion rate of 7% of the total intracellular fixed carbon was released. Utilization of macrophytic extracellular products by cultured epiphytes was determined in partitioned chambers of special design (see Figure 3). Following 4 .hours of photosynthesis on lL‘C-labelled carbon, the plants (5 or 6 per flask) were introduced into the center chamber section of each Plexiglas container. After brief acclimiti- zatioh by the macrophytes, aliquots of algal and bacterial cniltures in log phase of growth were added under aseptic 158 .A mHHonHm mmnmz oHcoxm ooSSpHSo so AHlmson panoz moo Hum Honv :choopwo mo SOHumSOQSooSH oHponucmmouond no mopmmll.mm opstm 159 . . Illill «Illa 1-1JI-‘I u d 4 d .mm madman 160 .q mHHonHm mmfimz OHSoxm so SOHpmme Sooamo oHponpSmwOpond mo mopmm cams on» o» monsooeoo oHcmwSo oo>HommHo mo SOHuoSoxo come no ommpcmopomll.mm opstm Figure 33. 161 Q ' So a ' é ' é; ‘ war-5 Sd 0” oo'x._1u..boxewoo orf HOURS 162 conditions to the two outer chamber sections. Aliquots of the algal and bacterial cultures were removed with time, dried on planchetes or filters, and radioassayed. A summary of intracellular uptake by cultured epi- phytes (Table 17), under light and dark conditions at 20C confirms that a significant transfer occurs from the macro- phytes into the adherent taxa during brief incubation periods. Data expressed were obtained by equating optical density measurements in aliquots removed from the chambers, immediately after inoculation, to dry weights (105C) of equal aliquots of organisms on tared filters using a Cahn Electrobalance. Activities from dried, filtered or plated, organisms were used to compute specific activities on a dry weight basis. Such data presentation readily permits a comparison of epiphytic utilization of macrophytic extra- cellular organic products to the total amount released (3a. 7% of the macrophyte photosynthate). Based on the maximum epiphytic removal rates (Table 17; 0.72 to 11.45 uCi - 10"3 mg dry weight-l), 0.00413 to 0.0545% of the total extracellular carbon can be removed if 17.4 to 21.0 uCi mg”l hr'l is produced by Najas flexilis under these conditions. Patterns of uptake of extracellular macro- phytic products for algae and bacteria were similar, with increased uptake occurring initially, followed by a de- crease and gradual plateau. Rates of uptake in total darkness are higher than in the light but these data fail to discriminate if this is due to (1) increased 163 HN.H om.H N3.H so.m om.H mm.H mm.m q «HHoooaoso ms.o mm.o mm.o 30.3 mm.H ms.3 s3.m 3 «HHoSOHzo mo.H s3.H om.H mm.H om.m mm.m s3.m o om.o 03.3 m3.H No.0 mH.m Hm.m 3m.H q meozoeoeoo m3.m om.m 33.5 Ho.m os.m mm.» m3.HH a m3.m om.3 m3.o mm.m Ho.s om.o mm.m q mmeoeoosomm NH.3 mm.m sm.o om.o 3H.m os.m om.HH a pooomQOHsmo 03m omH omH om om m3 0 H.2HEV owpsoo oEHB mmSSpHso AHuonwfioz see we m-OH Hoav so3>3oo< oaofiooom .ASOHmeSoSH sumo n o mpanH u Av .thsoooOLQ Hapcoe IHSodxo pom pxop mom MHH EsHooE moomv .mopzcdfido Hmeopomo cam Hmem ooSSpHSo mo mHHonHm mmwmz oHSoxw no mposoopd SmHzHHoompro ooHHoomHlozH mo oxmpdoll.sH mqmoe 164 extracellular release by Wales, (2) increased uptake efficiency by epiphytes, or use of different pathways, or more probably, (3) a combination of these. Rates of bacteria were approximately 10 times those of the algae during the first 15 minutes and gradually decreased to rates only twice those of the algae. A comparison of mixed algal and bacterial uptake was made by removal of culture aliquots of algae and bacteria, growing simultaneously in the presence of Najas flexilis in Pyrex flasks (Table 18). Optical density measurements were equated for mixed cultures to their dry weights to gain an acceptable expression of their specific activities per unit of time. Certain of these responses are difficult to interpret. Only in mixed cultures of one alga and one bacterium were uptake activities greater than those of individual cultures. Mixed communities of a single bacter- ium, a chlorophyte, and a diatom, showed poor uptake efficiencies with time. Such results suggest interspecific interactions where competition for specific external metabo- lites or organic solutes may have existed or where accumu- lation of extracellular products caused toxicity effects. The brief time interval of sampling in these studies needs to be stressed. There exists the inherent problem of re- cycling of 1”CO2 and external metabolites, confounding interpretation, if experiments under fixed conditions are extended over lengthy periods of time. 165 u o.m m.m m.m s.m s.3 m.3 oHHoSOHno mEoSonQEoo + mmsoEoosomm u s.s s.s m.m m.m m.m s.o mHHoSOHno wEoconQEoo + hopomnoHSMo u 3.mH s.mH 0.0m H.mm m.mH 0.33 meoeoemeoo + mmcoeoosomm u o.m s.m m.m m.m m.m 3.m oHHoSOHao + nooomQOHsoo w.mH o.mH m.mH s.MH H.mH m.mH m.mH «Soconaeoo + pooomQOHsmo 03m omH omH om om mH o A.SHSV ompsoo oEHB monopHso ootz AHuoemfioz see we m-OH . Aoav so3>3oo< oaofiooam .mousndeo HmHSopomn cam Hmem mo woodeSo ooxHS so me oxoooouu.m3 mamas meH Ssfiooe moomv mHHonHm mmnmz OHSoxm mo mposoopa mmHsHHoomnpxo ooHHmQMHIozH 166 Two additional experiments were performed to eluci- date existing nutritional interactions between the cultured epiphytes and Naias flexilis. Naias plants were photo- synthetically labelled, placed into fresh media, and allowed to produce extracellular products for 2 hours in the light. The plants were carefully removed and aliquots of medium were dried on planchetts to obtain control values of extracellular activity. Bacteria were then inoculated into this medium and permitted to grow there for 2 hours before they were concentrated and removed (membrane filter). Algal cultures were then allowed to grow on this and media to which no bacteria had been added. A significant increase in algal activity, in both light and dark incubation Condi— tions, following bacterial metabolism (Table 19), suggests materials (either 114CO2 or luC-labelled by-products) re- leased by the flalag were not wholly suitable as carbon and energy substrates without prior microbial degradation. Similar pathways are certainly functional in the natural environment. Aliquots of lLlC-extracellular products of Mala§_were diluted in series (1:0.5, 1:2, 1:4, 1:8, and 1:16) with fresh unlabelled medium II and used for esti- mation of uptake response kinetics by cultured algae and bacteria. No diffusion or zero-order kinetics could be demonstrated with any validity and may have been related to the low prOportions of dissolved organic carbon released or possibly to its unsuitability as a C or energy source. Velocities of substrate uptake did follow enzyme kinetics 167 TABLE 19.--Utilization of extracellular products of axenic Najas flexilis by cultured algal epiphytes subsequent to microbial metabolism of the released material. See text for explanation. (200; medium 11; L = light; D = dark). Specific Activity of Algae (uCi - 10'3 mg dry weight-l) Cultures Incubation Without With Bacteria Bacteria* Gomphonema L 1.36 1.89 D 1.92 2.45 Cyclotella L 2.45 2.93 Chlorella L 1.91 - *Medium II control before bacteria (Caulobacther + Pseudomonas) were added: 0.00236 uCi ml-l; medium II control after two hours of microbial activity in the light, with bacteria removed: 0.00205 uCi ml'l. 168 with a definite first order linear response (a = 0.994; significant at the 0.05% level). Maximum uptake ratefor Pseudomonas was 0.023 pg 1"1 hr-l, and is two orders of magnitude lower than respective rates of glucose and ace- tate at approximately the same temperatures (200; see Table 15) and optical densities. A final laboratory experiment to demonstrate the percentage of 1“CO2 release by Najas flexilis in pure culture was undertaken. Through precipitation as barium 14 carbonate-lac release of extracellular CO was 15 to 19% 2 of the total fixed carbon at 20C (incubation = 4 hours). Data herein discussed for the various laboratory studies were not corrected for these losses, or for respiratory losses in individual and mixed uptake experiments. E. Functional aspects of macrophyte—epiphytic metabolism in littoral ecology and lake trophic dynamics The magnitude of extracellular release of dissolved organic matter (DOM) under natural conditions in the lake, as well as the subsequent rapid incorporation into the epiphytic complex and loss to the surrounding littoral zone, suggests (1) the littoral vegetation and attached periphytic growth are capable of significant contributions to the total DOM "pool", and (2) that considerable utili- zation and transformation of the macrOphytically produced DOM is likely to occur prior to its availability in the littoral and pelagial areas. 169 Many of the potential ramifications of macrOphytically released DOM on profiles, annual patterns, and rates of primary productivity of autotrOphic microorganisms in the pelagial environment, have been derived through (1) bio- assay responses of pelagial plankton communities to varying inorganic and organic nutrient conditions, and (2) through further knowledge of succinct physical—chemical-nutritional interactions existing and Operating in specific freshwater habitats, g,g. in marl lakes (see relevant discussion in Wetzel and Allen, 1970). The relationships of dissolved organic matter, whether allochthonously or autochthonously produced, to sustained and persistent rates of autotrOphic metabolism, through direct and indirect interactions intra- primary producer trophic level, are becoming more defined (Wetzel, 1968). Indeed, the presence or absence of an abundance of organic materials functioning as physiological chelators, for example, by provision of inorganic iron to autotrOphic plankton metabolism, may be responsible for significant differences in daily rates of openwater photo- synthesis. Similar interactions, if persistent and charac- teristic for the system, may very well lead to advanced or retarded eutrOphicational development, regardless of lake morphometry, basin characteristics, depth and surrounding geological features, although the latter certainly play important roles in this process. A point to be considered in this discussion is that functional interactions of organic matter on pelagial 170 dynamics would likely be pronounced and accentuated in a system where annual mean primary productivity is low and where the ratio of the littoral to the pelagial zone is high. Prolifically developed submerged and emergent vegetation, with its epiphytic flora in close nutritional association, is probably capable of imposing severe demands on dissolved organic materials at the micro- and macro- levels. It may ultimately deprive plankton communities of the more labile carbon and energy rich compounds, and trace organic micronutrients. Certainly the intensity of rates of primary productivity and chemo-organotrophy reported in this study (among the highest in the litera- ture) for epiphytic algae and bacteria, must have an impact in a system where plankton photosynthesis is suppressed. Even in aquatic ecosystems where significant amounts of allochthonous and autochthonous dissolved and particulate organic matter are available to pelagial metabolism, and are reflected in increased carbon fixation rates, the epiphytic complex may represent the dominant producer and may still be capable of indirectly affecting the pOpulation dynamics and community metabolic patterns of the phyto- plankton (see relevant discussion by Khailov and Gorbenko, 1967). Quantification of various epiphyte metabolic param- eters, coupled with descriptive observations of communi- ties, under laboratory and field conditions have permitted 171 a more thorough understanding of epiphytic dynamics and host macrOphytic interactions (Figure 34). During initial colonization littoral bacteria adhere and probably form a mono-cellular layer on the new vege- tative substratum interwoven with time by deposition of calcium carbonate by the macrOphyte and attaching algal forms. Such a matrix formation may enhance the integrity of community structure and is probably fundamental in the adherence of large standing crops of organisms. Deposition of particulate monocarbonates is especially prevalent in a calcareous environment such as Lawrence Lake. As coloni- zation intensifies (late spring and early summer for natural substrata) a dissolved organic carbon "pool" is probably established within the matrix of deposited carbonates. Sources contributing to this pool would probably include (1) extracellular release from the macrophyte, (2) active excretion by attached algal and bacterial flora, (3) de- composition products following autolysis of epiphytes, and (4) allochthonously and autochthonously derived particulate and dissolved carbon present in the littoral zone. High rates of chemo-organotrOphy and primary productivity are probably sustained to a large extent by provision of trace elements, P, N, biotics, growth factors, etc., as well as labile and more refractile compounds from within the pool. The bioassay conducted, indicating stimulation of epiphytic primary productivity by addition of chelators and inorganic iron, may reflect deficiencies in the qualitative 172 .Hpopums oHSmwSo oo>HommHo n :09 mmHmonpcmmOpOSQ n mm mpxop oomv .Eopmamooo oHpmsom msooSMono o>HpmpSomonQop m mo ocou proppHH on» SH wcfipmeo mSOHpoMSopSH HMSOHuHSpss cam oHHonwpoS mo SOHpMpSomondop oHpmSSmSmeosl.:m madem 173 Vw///////////////////////eels. 7////i/_/7//, T. 33 fan. ’1 fl :8 6 "(Hi .11 II... 8.! E .966 a £553.. .. Shikoku i. W sensual 5 ea 2 ea .5... m SQ :8 0.2.86.5 g .3535 EU! qv‘xbvfimo. .5 :8 uvmxukk: .m m SQ 65: .0! M :5 IA _ _ .30 I. .(u: x. i E .393. 3 .............. Win... 835 g8 38.50 g g.” xgggfi L; g ..<_o<4wn_ 455:5 .nm onanm 174 composition of this pool or competition by attached bacteria for such organic substrates. In that adsorption of organic compounds on carbonates has been confirmed (Wetzel and Allen, 1970; see also Chave, 1965), this pro- cess may contribute to removal and retention of much DOM of epibenthic and eulittoral origin. Quantitatively insignificant, but detectable, chemo-organotrophy has been established for Najas flexilis grown in axenic culture under controlled conditions (Dr. R. G. Wetzel, personal communication), and may represent a nutritional feedback mechanism from the epiphytic flora to the host substratum. Although not yet documented, excretion of DOM by epiphytic algae is likely to occur (as has been shown for marine and freshwater phytoplankton, 93. for example Fogg, 1962, 1966; Lefevre, 1964; Fogg and Watt, 1965; Hellebust, 1965; Forsberg and Taube, 1967; and others), and probably contributes together with bacterial and macrOphytic extra- cellular products to the presence of a littoral dissolved organic matter pool. In that specific organic metabolites required for epiphytic metabolism are likely not to be continuously regenerated under natural conditions, com— pounds of littoral or even pelagial origin may function as feedback mechanisms through the supply of these neces- sary materials. Without prior knowledge the quality and quantity composition of each of the DOM pools are suspected to be different, in that communities contributing to each 175 of these pools maintain distinct species composition, which in turn upon autolysis or in situ excretion probably re- lease a specific array of compounds. As an example, intense epiphytic microbial activity may supply certain of the much needed water-soluble vitamins (B-complex) to littoral pro- ducers. The effect of release of CO by the macrOphyte, 2 algae and bacteria has not been investigated, but intricate mechanisms of combined respiratory cycles may lead to pro— vision of considerable localized CO2 accumulations for epiphytic algae and epibenthic communities for early morning photosynthetic requirements. Certainly the release of oxygen by the macrophyte regulates to some extent rates of decomposition processes within the muco-organo—carbonate complex by reducing the intensity to which anaerobic metabo— lism may proceed. Compactions of sulfur bacteria were observed micrOSCOpically on natural Scirpus substrata. Other nutritional advantages to epiphytes over pelagial communities can be speculated upon briefly. Autotrophic metabolism can probably more easily and readily be shifted on the attached surface to chemo-organotrophic supplementation during periods of poor light and adverse inorganic nutrient conditions. There is the possibility that exosmotic release by macrOphytes and maintenance of a surface adsorbed dissolved organic carbon source (the distinction is not herein made between the actual dis- solved phase and particulate-dissolved, in various stages 176 of decomposition; functionally, they may be the same) pro- vides organic compounds easily capable of serving in more than one functional capacity, as a chelator, complexing agent, or as a carbon or reductive substrate. In summary, the potential importance of the littoral zone as a functional unit within a lake has been emphasized from the standpoint of (l) a source of dissolved organic matter and (2) as a dynamic macrophyte-epiphyte system, nutritionally and physiologically interacting to sustain high levels of primary productivity and chemo-organotrophy. A detailed discussion of the epiphyte-macrophyte inter- actions alluded to in this study, coupled with functional aspects of epibenthic and pelagial metabolism, has been presented by Wetzel and Allen (1970). Such proposed causal mechanisms and nutritional interrelationships as suggested here (for macrophyte-epiphyte interacting systems), while derived from studies in a typical marl lake, are thought to be generally applicable to representative aquatic eco- systems of shallow to moderate depth. IV. LITERATURE CITED Allen, H. L. 1967. Acetate utilization by heterotrophic bacteria in a pond. Hidrolégiai Kozlony 41:295-297. . 1968a. 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