COMPARATIVE POPULATION DYNAMICS OF FRESHWATER DINOFLAGELLATES OF THE GENERA PERIDINIUM AND PERIDINIOPSIS

by

Andy Kyle Canion

A thesis submitted to the faculty of The University of Mississippi in partial fulfillment of the requirements of the Sally McDonnell Barksdale Honors College.

Oxford

May 2004

Approved by

___________________________________ Advisor: Dr. Clifford Ochs

___________________________________ Reader: Dr. Tamar Goulet

___________________________________

Reader: Dr. Michael Mossing

© 2004 Andy Kyle Canion

ALL RIGHTS RESERVED

ii

ACKNOWLEDGEMENTS

I would like to express my gratitude to everyone who provided assistance with this research. Dr. Susan Carty of Heidelberg College and Dr. Jack Holt of Susquehanna University provided much needed help with the identification of dinoflagellate species. Fortune Ogbebo and Jim Weston at the University of Mississippi and John Hart at the Center for Water and Wetland Resources assisted with sample processing. The Sally McDonnell Barksdale Honors College provided the financial support for this research. Lastly, I would like to thank Dr. Cliff Ochs, who served as the advisor for this project. The involvement of all these people was essential for the advent and completion of this project.

iii

ABSTRACT ANDY KYLE CANION: Comparative Population Dynamics of Freshwater

Dinoflagellates of the genera Peridinium and Peridiniopsis (Under the direction of Cliff Ochs)

Freshwater dinoflagellates, like their marine relatives, have the potential to

reach large population sizes known as blooms. Though they are not toxic like some

marine forms, freshwater blooms may have a large impact in their respective

ecosystem. This study investigated the temporal and spatial changes in population

density of four Peridinium species (P. deflandrei, P. volzii, P. wisconsinense, and P.

limbatum) and one Peridiniopsis species (Peridiniopsis polonicium) in Boondoggle

Lake, a shallow lake (4.5 m) in northern Mississippi (Lafayette Co.). On each

sampling date, measurements of the abiotic environment were made, including

dissolved oxygen, temperature, pH, and turbidity. Also, water samples were taken

from three depths: 0.25 m, 1 m, and 2 m. Estimates of total dinoflagellate density

were made by enumeration of concentrated samples on LM slides. Individual species

were identified using SEM, and estimates of the relative abundance of each species

were made at the SEM, which allowed estimates of the actual densities of each

species to be made. One species, P. deflandrei, was by far the most dominant in the

summer bloom (90% of the total dinoflagellate population) and reached a maximum

population density of 2.75 X 105 cells/ L. P. volzii was the most abundant species in

the spring and late fall, with its maximum density being 5.7 X 104 cells/ L. The other

iv

three species had temporal patterns similar to either P. deflandrei or P. volzii but

never comprised more than 25% of the total dinoflagellate population. P. limbatum

reached maximum densities in the spring and fall, similar to P. volzii. P.

wisconsinense and Peridiniopsis polonicium reached maximum densities in the

summer, similar to P. deflandrei. The species composition and population density of

dinoflagellates in Boondoggle Lake are determined by many environmental factors.

Those that are likely to be most important include low nutrient levels, an acidic pH

(5.5-6.5), warm temperatures, summer anoxia, wind protection, and interspecific

competition.

v

TABLE OF CONTENTS

LIST OF FIGURES………………………………………………………………….vii SECTION 1: INTRODUCTION……………………………………………………...1 SECTION 2: MATERIALS AND METHODS……………………………………...11 SECTION 3: PHYSICAL AND CHEMICAL CHARACTERISTICS OF BOONDOGGLE LAKE………………………………………………17 SECTION 4: INDENTIFICATION OF DINOFLAGELLATE SPECIES FROM BOONDOGGLE LAKE………………………………………………28 SECTION 5: SPATIAL AND TEMPORAL PATTERNS IN POPULATION FOR DINOOFLAGELLATES IN BOONDOGGLE LAKE……………….41 SECTION 6: DISCUSSION…………………………………………………………55 LIST OF REFERENCES…….………………………………………………………61

vi

LIST OF FIGURES

Figure 1.1: Key morphological features of freshwater armored dinoflagellates...........3 Figure 1.2: Basic life cycle of sexually reproducing freshwater armored dinoflagellates…………………………………………………………….7

Figure 3.1: Temperature and dissolved oxygen profiles for selected dates beginning March 2002 and ending July 2003………………………………..…18-20

Figure 3.2: Temporal patterns of pH for three depths: 0.25 m, 1 m, and 2 m……….23 Figure 3.3: Temporal patterns in Secchi depth………………………………………24 Figure 5.1: Total dinoflagellate population density………………………………….42

Figure 5.2: Population proportion at 0.25 m…………………………………………44 Figure 5.3: Population proportion at 1 m…………………………………………….45 Figure 5.4: Population proportion at 2 m…………………………………………….46 Figure 5.5: Individual population trends at 0.25 m…………………………………..48 Figure 5.6: Individual population trends at 1 m……………………………………...49 Figure 5.7: Individual population trends at 2 m……………………………………...50 Figure 5.8: Correlation of total dinoflagellate density to temperature……………….53 Figure 5.9: Correlation of P. deflandrei density to temperature……………………..54

vii

Section 1: General Introduction

1.1 Overview of Dinoflagellates

Dinoflagellates are a monophyletic group of single-celled eukaryotes that could

originally be found in the classification schemes of both zoologists and botanists.

Although they share cellular features with members of both the plant and animal

kingdom, they are now believed to be most closely related to either photosynthetic

protists or protozoa (Carty 2003). Molecular evidence suggests they branched from the

eukaryotic lineage early in the evolution of eukaryotes (Herzog 1986). At present,

approximately 2000 extant species of dinoflagellates have been identified (Pfiester and

Anderson 1987).

The most extensive studies of dinoflagellates have been in marine environments

due to their important ecological roles and potential detrimental effects on marine biota

as well as humans. Blooms of marine dinoflagellates known as red tides have the

potential to cause fish kills, and dinoflagellate toxins, which are produced only by marine

forms, can cause human illnesses such as toxic shellfish poisoning and ciguatera (Graham

and Wilcox 2000).

Freshwater dinoflagellates, although not as dangerous to humans, can have an

equally large role in their respective ecosystems. Freshwater blooms of the genera

Ceratium and Peridinium have been documented in numerous lakes (Moore 1981;

Pollingher and Hickel 1991; Stewart and Blinn 1976; Whiting et. al. 1978; Wu and Chou

1

1998; Yan and Stokes, 1978). Certain Peridinium blooms have been known to reach high

enough population densities to be labeled as freshwater “red tides.” One such red tide

was documented in Clear Lake, California, where Peridinium pernardii reached densities

of 5 X 106 cells/ L (Horne, et. al. 1971). Another freshwater bloom of Peridinium with

densities reaching 10-93 X 106 cells/L occurred in an oligotrophic reservoir near Tokyo

in 1975 (Nakomoto 1975). A bloom of Peridiniopsis polonicium was responsible for fish

kills in Lake Sagami near Tokyo, one of the few cases of a toxic freshwater dinoflagellate

bloom (Hashimoto, et. al. 1968).

Below, I review the basic biology of dinoflagellates, including morphology,

classification, nutrition, life history, and ecology. This review focuses on motile

freshwater armored dinoflagellates and the two genera that were investigated in this

study, Peridinium and Peridiniopsis.

1.2 Morphology and Classification

Freshwater dinoflagellate species show diverse patterns of morphology. They are

loosely grouped in the categories of thecate (armored) and athecate (naked), depending

on the presence or absence of sub-membranous cellulose plates. If present, the collection

of plates is known as the theca (Taylor 1987). The two genera of focus in this study,

Peridinium and Peridiniopsis, are thecate genera. A diagram of a freshwater armored

dinoflagellate is shown in Figure 1.1. The view is of the ventral side. Important features

to note are the cingulum (girdle), sulcus, flagella, epitheca, hypotheca, and sutures. One

flagellum lies in the transverse groove known as the cingulum (also girdle). The other is

found in the longitudinal groove, the sulcus.

2

Figure 1.1: Key morphological features of freshwater armored dinoflagellates (Modified after Lefevré 1932)

3

The epitheca and hypotheca refer to the collection of thecal plates above and below the

cingulum respectively. The ridges where thecal plates meet are known as sutures (Taylor

1987). Classification of freshwater armored dinoflagellates is based on the arrangement

and number of the thecal plates (Carty 1986).

1.3 Nutrition

Dinoflagellates are also diverse in their modes of nutrition. Autotrophic,

auxotrophic, mixotrophic, and heterotrophic forms have been documented (Carty 2003,

Holt and Pfiester 1981). Only six freshwater species have been documented as

completely autotrophic, meaning they require no organic substances for growth in the

light. Four of these are species from the genus Peridinium: P. cinctum f. ovoplanum, P.

inconspicuum, P. volzii, and P. willei (Gaines and Elbrächter 1987). Many

photosynthetic dinoflagellates require vitamins for growth (auxotrophy). There are only

three vitamins that are known to be required by auxotrophic algae: B12, thiamine, and

biotin (Graham and Wilcox 2000), and dinoflagellates have been shown to require one,

two, or all three of these vitamins for growth (Gaines and Elbrächter 1987). Pure

heterotrophs (living exclusively on organic compounds) comprise approximately half of

all dinoflagellates. In freshwater systems, photosynthetic species dominate, but

heterotrophic benthic species are not uncommon (Gaines and Elbrächter 1987).

1.4 Life History

An important characteristic of the motile freshwater dinoflagellate life cycle is

the alternation between a swimming, vegetative form and a resting stage known as a cyst

4

(Pollingher 1988). Cyst formation is associated with most sexually reproducing forms

and occurs after a zygote ceases to be motile and forms a thick cell wall. Encystment is

believed to be an adaptation for surviving environmental stress, and it usually occurs in

conjunction with specific environmental conditions (Pollingher 1988). Some of the

possible environmental factors that can lead to encystment include temperature, nutrient

depletion, light intensity, and changes in concentration of dissolved gasses. Of these,

nutrient depletion of nitrogen has been best demonstrated to induce encystment in

cultures (Pfiester and Anderson 1987). Sexual reproduction followed by encystment was

shown to be induced by nitrogen depletion in cultures of four Peridinium species:

Peridinium cinctum, Peridinium willei, Peridinium gatunense, and Peridinium volzii.

(Pfiester 1975; Pfiester 1976; Pfiester 1977; Pfiester and Skvarla 1979).

Dinoflagellates undergo asexual reproduction and sometimes sexual reproduction

depending on the species and environmental conditions. The vegetative (also called

assimilative) stage of freshwater armored dinoflagellates is haploid and divides asexually

through mitosis (Pfiester and Anderson 1987). Cells increase their size before dividing,

which is accomplished by expanding the theca at the sutures between thecal plates.

These expanded areas are referred to as intercalary bands (Taylor 1987).

Sexual reproduction begins when the haploid vegetative stage forms gametes

through mitosis. Gametes then fuse to form a planozygote. The planozygote is motile,

with two longitudinal flagella, and can remain motile to continue cell growth (Pfiester

and Anderson 1987). Planozygotes accomplish cell enlargement in the same manner as

vegetative cells by expansion at the sutures between thecal plates (Pfiester and Skvarla

1980). Formation of a hypnozygote occurs when the planozygote loses motility and

5

forms a thick cell wall. Hypnozygotes rest on the lake bottom until conditions are right

for excystment, at which time vegetative cells are returned to the water column. Meiosis

must occur between formation of the planozygote and excystment of the hypnozygote,

and when it occurs varies among taxa.

The causes and mechanisms of sexual reproduction have been studied in only 22

of all 2000 dinoflagellate species (Pfiester and Anderson 1987). As previously

mentioned, many environmental factors are thought to induce sexual reproduction and

subsequent encystment. In previous studies of freshwater dinoflagellates, environmental

cues have been shown to influence the emergence of vegetative cells from resting stages

(Pfiester and Anderson 1987; Kawabata and Ohto 1989; Anderson et al. 1987).

Life history traits are an important deterministic component of the population

dynamics and ecology of dinoflagellates because reproductive strategies influence

temporal and spatial patterns in dinoflagellate abundance. Figure 1.2 shows the basic

sexual and asexual life cycle of freshwater armored dinoflagellates.

1.5 Ecology

Dinoflagellates usually reach their maximum population size in the summer

months. Though they are usually found in low numbers, some species may form large

blooms given the correct environmental conditions. Members of the genera Ceratium

and Peridinium are well studied bloom formers (Carty 2003; Pollingher 1988). The

population ecology of freshwater dinoflagellates is widely thought to be influenced by

many bottom-up and top-down control factors. Bottom-up factors include pH,

temperature, organic matter, inorganic ions (Ca, Cl, N, P), light intensity, vitamins, and

6

Figure 1.2: Basic life cycle of sexually reproducing freshwater armored dinoflagellates (Modified after Carty 2003)

7

inoculum from cysts. Top- down factors include predation, disease, life history traits

(cyst formation), lake outflow, and turbulence (Carty 2003; Pollingher 1988; Popovsky

and Pfiester 1990). The effects of bottom-up and top-down factors on patterns of

freshwater dinoflagellate abundance have been investigated in many studies. A brief

review of the well-studied control factors is now presented.

Lake pH has been demonstrated to influence the growth rate of freshwater

dinoflagellates. However, the effect of pH on growth rate is variable across species. In

the genus Peridinium, studies of laboratory cultures have shown species with maximum

growth occurring at a pH ranging from 5.5 (P. limbatum) to 8 (P. cinctum), and one

species, Peridinium inconspicuum, showed little change in growth rate over a range of

5.5-8.5 (Holt 1981; Pollingher 1988).

The influence of temperature on freshwater dinoflagellate populations is only a

general trend. Most dinoflagellates show maximum growth during the summer, but there

is a wide tolerance range for temperature. That is, dinoflagellates are not strong

stenotherms (Carty 2003; Pollingher 1988). Though most freshwater dinoflagellates

form cysts during the winter, some have been found in the water column throughout an

entire year. These include Peridinium bipes and Peridinium willei (Pollingher 1988).

Freshwater dinoflagellates are not tolerant of oxygen stress. Low oxygen levels

can cause Peridinium cinctum fa. Westii to shed its flagella, shed its theca, and become

immobile. Insufficient oxygen has also been shown to induce encystment in Peridinium

species from Lake Kinneret (Pollingher 1987). In situ observations of Peridinium in

Lake Kinneret and Ceratium in Esthwaite Water have revealed that these freshwater

8

dinoflagellates are rarely, if ever, found in anoxic hypolimnic waters (Pollingher 1988;

Harris et. al. 1979).

The concentrations of inorganic nutrients, particularly nitrogen and phosphorus,

are important in determining the growth of dinoflagellates. Freshwater dinoflagellates

are generally more abundant relative to other algal taxa when nutrients are low. It is

thought that the resilience of dinoflagellates under low nutrient conditions along with

reduced competition from other algae is largely responsible for their summer success in

nutrient poor systems (Pollingher 1987; Pfiester 1971). Mechanisms for tolerance of low

nutrient levels have been studied. These include luxury consumption of phosphorus, long

generation times, and vertical migration to obtain nutrients (Heany and Eppley 1981;

Carty 2003; Pollingher 1988). As mentioned earlier, low nitrogen levels may contribute

to sexual reproduction and encystment of freshwater dinoflagellates (Pfiester and

Anderson 1987; Pfiester 1975, 1976, 1977; Pfiester and Skvarla 1979).

Usually, photosynthetic dinoflagellates prefer abundant light and are concentrated

in the upper layers of the water column (Pollingher 1987). Their motility makes them

able to move to an optimal depth for photosynthesis as well as nutrients. Dinoflagellates

have been shown to undergo vertical diel migrations, which are thought to be most

influenced by light, temperature, and nutrient availability. However, these migrations are

frequently variable for different species (Heany and Eppley 1981). One study of vertical

migration in Ceratium hirudinella suggests that a compromise between light availability

from the surface and nutrients concentrated at depth will determine the depth where

maximum densities of dinoflagellates will occur (Heany and Furnass 1980). Irradiation

9

is important not only in how productive dinoflagellates will be but also where they will

be found in the water column.

The role of top-down factors in the population ecology of freshwater

dinoflagellates also has been investigated, but to a lesser extent. Measurements of

physiological death of Peridinium in Lake Kinneret showed a decrease of 8-10% during

exponential growth and 40-80% at the end of the summer bloom (Serruya et. al. 1975).

Large freshwater dinoflagellates, such as some species of Peridinium and Ceratium, are

subject to grazing only by certain species of rotifers and in some cases, fish. Parasitism

by chytrid fungi has been documented in Peridinium (Pollingher 1988).

Other factors contributing to loss include turbulence, which was associated with a 62.5%

death rate in continuously shaken Peridinium cultures (Pollingher and Zemel 1981), and

lake outflow, which was observed to remove 7.65 X 1013 dinoflagellate cells/ day from a

lake population (Heany and Talling 1980).

1.6 This Study

The objective of this study was to describe the population dynamics of the

freshwater armored dinoflagellates in Boondoggle Lake (Oxford, Mississippi). This

objective was accomplished by collecting physical and chemical data from the lake,

identifying the species of freshwater armored dinoflagellates present, and estimating

population densities of dinoflagellates throughout the study period.

There is a paucity of information on the ecology and distribution of freshwater

dinoflagellates. This study will provide some of the much needed information on the

dinoflagellates in Mississippi.

10

Section 2: Materials and Methods 2.1 Sampling site and surrounding area

Boondoggle Lake is a man-made lake located approximately 10 miles east of

Oxford, MS on the edge of Holly Springs National Forest and is approximately 70 yrs

old. The lake has an elevation roughly 120 m above sea level and is surrounded by

mixed bottomland forest on all sides. It is about 1.5 hectares in surface area and has a

maximum depth of approximately 4.5 m. It undergoes one major period of

stratification in the summer and has been observed to go through a mixing and

restratification in the fall after intense rain. The lake experiences anoxia in the

summer that may begin as high as 1.5 m from the surface, and it exhibits a dark

reddish-brown color during summer months, which becomes clearer upon cooler

temperatures and mixing.

2.2 Water Sampling, Temperature and Oxygen Measurements

Water samples were collected on 44 dates between March 2002 and

September 2003. For each sampling date, water samples and measurements were

taken at a marked point near the middle of the lake (depth ~4 m). Sampling was

performed during mid-day (between 11:00 a.m. and 1:00 p.m.) each date and took

approximately 45 minutes to complete. Samples were returned to the lab and

processed within approximately one hour of sampling time.

11

Two replicates of water samples were taken at 0.25 m, 1 m, and 2 m using 2-

liter Nalgene® plastic bottles. A peristaltic pump was used to obtain samples from

1 m and 2 m. Water was run through the pump at each depth before filling the bottles

in order to prevent contamination of samples by water from a different depth.

Along with collection of water samples, basic physical and chemical data

from the lake were collected on each sampling date. Secchi depth measurements

were used to estimate light penetration. A YSI model 57 oxygen meter was used to

measure dissolved oxygen and temperature profiles with readings taken every 0.25 m

from the surface to a depth of 4 m.

2.3 Water Sample Measurements and Preservation

In the lab, 200 mL of each water sample were vacuum filtered through a

47 mm Whatman® GF/F-filter. The filters were wrapped in aluminum foil and saved

in a -800 C-freezer until needed for HPLC pigment analysis. The filtrate was saved in

Whirl pak® bags in a -200 C-freezer for nutrient measurements.

Turbidity was measured for all unfiltered water samples using a Hach model

2100A Turbidimeter. The turbidimeter was calibrated using a 9.9 NTU Gelex®

secondary turbidity standard.

The pH of unfiltered water samples was determined using a Fisher Scientific

Accumet® 1001 pH meter. The pH meter was standardized using a pH 4 potassium

biphthalate buffer solution and a pH 7 potassium phosphate monobasic-sodium

hydroxide buffer solution.

12

Water samples to be used for enumeration of dinoflagellates were

concentrated before being preserved. One liter of water from each of two replicates

from each depth was filtered through a 20 µm mesh filter and concentrated to a

volume of approximately 20 mL. These water samples were placed into plastic vials

and fixed with 200 µL of acid Lugol’s solution so that they could be used later for

enumeration and identification. Also, for most dates, 100 mL of unconcentrated

sample water from .25 m, 1 m, and 2 m was preserved with 1mL of Acid Lugol’s for

potential later use.

2.4 Population Counts

Slides were prepared for population counts of dinoflagellates at each depth

according to the following procedure. 2-3 mL of previously concentrated sample was

filtered onto an 8 µm-nitrocellulose filter. Filters were dried briefly on top of an

upside down petri dish that was placed on a hotplate set to the lowest heat setting. 2-

3 drops of immersion oil were placed on a microscope slide, and the filter was placed

on top of the immersion oil. The filters cleared upon contact with the immersion oil.

A cover slip was placed on each slide and all slides were refrigerated until counted.

Counting was performed using a Jena-Lumar© light microscope. All counts

were made at 125X magnification. Two different counting methods were employed

depending on the abundance of organisms. If few dinoflagellates were present, then

slides were counted by two transects across the diameter of the filter: one horizontal

and one vertical. The width of one transect was the width of the eyepiece grid at

125X magnification (790 µm). When many dinoflagellates were present, the slides

13

were counted by random fields. Counts were made in ten random fields giving a total

number of organisms per the area of 10 fields (each field was 0.624 mm2). Slide

counts and concentration factors for each sample were used to back-calculate the

population density of all dinoflagellates. Estimates of actual densities (given in

cells /L) were made using the following formula:

# of cells counted X area of filter (mm2) X Concentration Factor area counted (mm2) mL filtered

where the area counted was equal to 0.6241 mm2 per field x 10 fields = 6.241 mm2 or

when transects were used, the length x width of a transect. The area of the filter was

227 mm2. The concentration factor was given by:

volume of sample after concentrating (L)

volume of field sample concentrated (L)

2.5 Species Identification and Population Proportion Counts using SEM

In order to identify species of dinoflagellates, scanning electron microscopy

was used to visualize the morphological features used to classify freshwater

dinoflagellates. Dinoflagellates were identified by the number and pattern of thecal

plates and also by the presence of reticulation on thecal plates. Species were

identified using three taxonomical keys to the freshwater dinoflagellates (Popovsky

1990; Starmach 1974; Lefevre 1932).

Scanning electron microscope stubs were prepared by filtering between 2 and

5 mL of concentrated sample water (see section 2.3) onto a 0.8µm-membrane filter.

14

The filter was dried by the same method as section 2.5. After drying, filters were

affixed to the stubs using double-sided tape and gold coated using a sputter coater.

The specimens were examined using a JEOL® JSM-5600 scanning electron

microscope. The magnification was changed as needed to examine morphological

features.

In addition to identification of species present in the water samples, scanning

electron microscopy was employed to estimate the proportion of the total

dinoflagellate population contributed by each species throughout the 16 month

sampling period. SEM stubs were prepared by the same method used for species

identification for two sampling dates from each month. For each date, stubs were

prepared from all three sampled depths (.25 m, 1 m, 2 m). Relative abundances were

determined by counting a series of transects across the stub until 100 cells were

counted. All counts were made at 300X magnification. The percent of total

dinoflagellates represented by each species was multiplied by the total density

obtained from the light microscope counts (see section 2.4) in order to estimate

individual species densities on each sampling date.

2.6 Dissolved Inorganic Nutrient Measurements Ion chromatography was used to quantify dissolved inorganic nutrients. In

particular, concentrations of nitrate, orthophosphate, and ammonium were measured

to estimate trophic status of the lake. Measurements of sulfate were also made.

Filtrate from the GF/F-filters (see section 2.3) was analyzed on a Dionex® 600

system with an AS14 column and conductivity detector. Peaknet® 6 software was

15

used for analysis. The detection limits were 10 ppb for nitrate and phosphate. The

detection limits for ammonium and sulfate were 5 ppb and 3 ppb, respectively.

2.7 Pigment Measurements using HPLC

High performance liquid chromatography (HPLC) was used to quantify

pigments from the GF/F-filters (see section 2.3). Chlorophyll a was quantified to

estimate total phytoplankton biomass. Peridinin was quantified because it is a

pigment unique to dinoflagellates. Sample preparations were performed after the

method of Wright and Jeffrey (1997). The filters were extracted with 5 mL of 90%

acetone, sonicated, and the resulting pulp was filtered again using centrifugation.

Samples were run using a Dionex® reversed phase HPLC column with both UV-Vis

and photodiode array detectors. Chromeleon® software was used for data analysis.

16

Section 3: Physical and Chemical Characteristics of Boondoggle Lake

This section presents the physical and chemical data recorded over the study

period, including measurements of temperature, dissolved oxygen, pH, Secchi depth,

dissolved inorganic ions, and photosynthetic pigments.

3.1 Temperature

Throughout the study period, water surface temperatures ranged from 7.5 to

310 C, with an average of value of 210 C. Vertical temperature profiles show that the

lake has well defined temperature stratification that lasts throughout the summer

months. It mixes completely during the remainder of the year and rarely, if ever,

experiences ice cover. Therefore, according to the classification scheme of Lewis

(1983), Boondoggle Lake is a warm monomictic lake (Kalff 2002).

Figure 3.1 presents temperature profiles for selected dates from each month

sampled. For the profiles from 2002, stratification began in March, and the

thermocline remained poorly defined through April and May. In June, a well defined

thermocline was seen beginning at 1.5 m. By October, the lake was mixing again.

A similar trend was seen in the 2003 temperature profiles. The lake began to

stratify in March and showed weak stratification from April through May. A well

defined thermocline was seen in June and July.

17

Mar

ch 2

7, 2

002

O2

(mg/

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24

68

1012

Depth

0 1 2 3 4

Tem

p (C

)5

1015

2025

30

Apr

il 12

, 200

2

O2

(mg/

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24

68

1012

0 1 2 3 4

Tem

p (C

)5

1015

2025

30

May

10,

200

2

O2

(mg/

L)

24

68

1012

0 1 2 3 4

Tem

p (C

)5

1015

2025

30

June

14,

200

2

O2

(mg/

L)

24

68

1012

Depth

0 1 2 3 4

Tem

p (C

)

510

1520

2530

July

12,

200

2

O2

(mg/

L)

24

68

1012

0 1 2 3 4

Tem

p (C

)

510

1520

2530

Aug

ust 2

9, 2

002 O

2 (m

g/L)

24

68

1012

0 1 2 3 4

Tem

p (C

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510

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18

Sep

13, 2

002

O2

(mg/

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24

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1012

Depth

0 1 2 3 4

Tem

p (C

)5

1015

2025

30

Oct

18,

200

2

O2

(mg/

L)

24

68

1012

0 1 2 3 4

Tem

p (C

)5

1015

2025

30

Nov

22,

200

2

O2

(mg/

L)

24

68

1012

0 1 2 3 4

Tem

p (C

)5

1015

2025

30

Dec

6, 2

002 O

2 (m

g/L)

24

68

1012

Depth

0 1 2 3 4

Tem

p (C

)

510

1520

2530

Jan

9, 2

003 O2

(mg/

L)

24

68

1012

0 1 2 3 4

Tem

p (C

)

510

1520

2530

Feb

21, 2

003

O2

(mg/

L)

24

68

1012

0 1 2 3 4

Tem

p (C

)

510

1520

2530

O2

Tem

p

Figu

re 3

.1 (c

ontin

ued)

19

Mar

ch 1

5, 2

003

O2

(mg/

L)

24

68

1012

Depth

0 1 2 3 4

Tem

p (C

)5

1015

2025

30

Apr

il 17

, 200

3

O2

(mg/

L)

24

68

1012

0 1 2 3 4

Tem

p (C

)5

1015

2025

30

May

20,

200

3

O2

(mg/

L)

24

68

1012

0 1 2 3 4

Tem

p (C

)5

1015

2025

30

June

24,

200

3

O2

(mg/

L)

24

68

1012

Depth

0 1 2 3 4

Tem

p (C

)

510

1520

2530

July

10,

200

3

O2

(mg/

L)

24

68

1012

0 1 2 3 4

Tem

p (C

)

510

1520

2530

O2

Tem

p

Figu

re 3

.1 (c

ontin

ued)

20

3.2 Dissolved Oxygen

A stratification of dissolved oxygen levels accompanied temperature

stratification in the summer. Anoxic conditions were present during the summer

months, which began near the lake bottom and rose to depths just below the

epilimnion.

Dissolved oxygen in surface waters (0.25 m) ranged from 6 mg/L to

11.5 mg/L during the study period. When the lake was mixing, dissolved oxygen

was between 7-10 mg/L at all depths.

Figure 3.1 presents dissolved oxygen profiles for selected dates from each

month sampled. In 2002, stratification of dissolved oxygen began in mid April,

and by mid May, anoxia developed below 4 m. The depth at which anoxia began

became shallower during June and July and receded during August and

September. The shallowest depth at which anoxia began was recorded on August

22, 2002 and was 1.25 m. By October, the lake was mixing and the dissolved

oxygen concentration was approximately 6 mg/L. Dissolved oxygen increased

during the colder winter months, reaching a maximum of 10 mg/L in December.

Data from 2003 showed a similar trend in dissolved oxygen concentration.

Again, strong stratification and anoxia began in May. In the profiles from June

and July, oxygen concentration dropped quickly after a depth of 1 m, with

complete anoxia occurring between 1.25 m and 1.5 m.

21

3.3 pH

For all dates and depths, pH was always between 5.5 and 6.5. Figure 3.2

shows the temporal changes in pH values measured at three depths. The data

suggest a stratification of pH that corresponds to the temperature and oxygen

stratification mentioned in sections 3.1 and 3.2.

In Figure 3.2, from May 2002 through September 2002, a clear separation

was seen between the pH values of each depth. The pH measured at .25 m

remained the highest and the pH measured at 2 m remained the lowest through the

stratification period, with the pH at 1 m falling in between. This pattern of

decreasing pH with depth is likely due to buildup of carbon dioxide and carbonic

acid associated with anoxia at deeper depths. Measurements of pH remained

similar between depths during lake mixing (October 2002- April 2003).

3.4 Secchi Depth

Figure 3.3 shows temporal patterns in Secchi depth. Secchi depth ranged

from 2.1 m in the early spring to 0.72 m in late summer. Average Secchi depth

was 1.2 m over the study period.

The major pattern observed in Secchi depth was the sharp decrease in

early June to a depth of approximately 1 m. Secchi depth then gradually

decreased through the summer and remained shallow until November, when a

sharp increase in depth occurred.

22

Dat

e

27-M

ar-02 5-A

pr-02

12-A

pr-02

21-A

pr-02

26-A

pr-02 2-M

ay-02

10-M

ay-02 15

-May

-02 26-M

ay-02 2-J

un-02 7-J

un-02 14

-Jun-0

221

-Jun-0

228

-Jun-0

212

-Jul-0

226

-Jul-0

229

-Aug

-02 6-Sep

-02 13-S

ep-02 20

-Sep

-02 27-S

ep-02 7-Oct-

0218

-Oct-

0225

-Oct-

02 8-Nov

-02 22-N

ov-02 6-D

ec-02 9-J

an-03 21

-Feb

-03 27-F

eb-03 6-M

ar-03

15-M

ar-03

21-M

ar-03

30-M

ar-03 5-A

pr-03

17-A

pr-03

25-A

pr-03 1-M

ay-03 20

-May

-03 11-Ju

n-03

24-Ju

n-03

10-Ju

l-03

4-Sep

-03

pH

5.0

5.1

5.2

5.3

5.4

5.5

5.6

5.7

5.8

5.9

6.0

6.1

6.2

6.3

6.4

6.5

6.6

.25m

1m 2m

Figu

re 3

.2: T

empo

ral p

atte

rns o

f pH

for t

hree

dep

ths:

.25m

, 1m

, and

2m

23

Sec

chi D

epth

Dat

e

27-Mar-02 5-Apr-02 12-Apr-02 21-Apr-02 26-Apr-02 2-May-0210-May-0215-May-0226-May-02 2-Jun-02 7-Jun-0214-Jun-0228-Jun-02 12-Jul-02 26-Jul-0222-Aug-0229-Aug-02 6-Sep-0213-Sep-0220-Sep-0227-Sep-02 7-Oct-02 18-Oct-02 25-Oct-02 8-Nov-0222-Nov-02 6-Dec-02 9-Jan-0321-Feb-0327-Feb-03 6-Mar-0315-Mar-0321-Mar-0330-Mar-03 5-Apr-03 17-Apr-03 25-Apr-03 1-May-0320-May-0311-Jun-0324-Jun-03 10-Jul-03 4-Sep-03

Depth (m)0.0

0.5

1.0

1.5

2.0

2.5

Figu

re 3

.3: T

empo

ral p

atte

rns i

n Se

cchi

dep

th

24

3.5 Dissolved Inorganic Ions

Table 3.1 presents monthly measurements of nitrate, phosphate,

ammonium, and sulfate for samples from each depth between May 2002 and

March 2003. Entries labeled “ND” represent ion concentrations below the

detection limit (see section 2.7).

Nitrate concentrations were higher in the fall and winter, and were highest

in February (0.12- 0.15 ppm). An average concentration of 0.16 ppm was

recorded in the surface samples from June. Orthophosphate concentrations

remained under the detection limit from June 2002 – September 2002. The

highest concentrations were recorded in fall and spring (0.15- 0.20 ppm). The

highest ammonium concentrations were recorded in September, October, and

November 2002. The highest sulfate concentrations were recorded in winter and

spring.

3.6 Photosynthetic Pigments (chlorophyll a and peridinin)

Table 3.2 presents monthly measurements of chlorophyll a and peridinin

for samples from each depth between June 2002 and February 2003. Entries

labeled “ND” represent samples where the pigment was not detected.

Chlorophyll a concentrations ranged from 0.8- 8.6 ppb, with the highest

concentrations occurring in the summer and fall. Peridinin concentrations were

highest in July, August, and September (1.37- 9.39 ppb). Peridinin was either

detected in low concentrations or not detected in the fall and winter samples.

25

NO

3 (p

pm)

P

O4

(ppm

)

N

H4

(ppm

)

SO4

(ppm

)

.25m

1m2m

.25m

1m2m

.2

5m1m

2m.2

5m1m

2mM

onth

n

May

-02

1 0.

07

ND

0.05

0.

06N

DN

D0.

150.

090.

081.

861.

751.

78Ju

n-02

3

0.16

ND

ND

ND

ND

ND

0.14

0.10

0.07

1.67

1.51

1.97

Jul-0

2 2

ND

ND

0.01

ND

ND

ND

0.12

0.07

0.19

1.05

1.10

1.40

Aug

-02

1N

DN

DN

DN

D0.

190.

150.

920.

17S

ep-0

2 2

ND

0.07

0.04

ND

ND

ND

0.22

0.28

0.42

1.00

1.10

1.16

Oct

-02

10.

020.

030.

09N

DN

D0.

150.

100.

211.

121.

291.

360.

34N

ov-0

2 1

0.15

0.02

ND

0.20

ND

ND

3.94

0.24

0.29

0.86

1.25

1.27

Dec

-02

10.

030.

04N

DN

D0.

090.

191.

531.

56Ja

n-03

10.

030.

020.

040.

05N

DN

D0.

070.

120.

121.

761.

711.

79Fe

b-03

1

0.12

0.15

0.15

0.05

ND

ND

0.15

0.07

ND

2.23

2.28

2.27

Mar

-03

20.

03N

DN

D0.

15N

DN

D0.

150.

130.

161.

962.

022.

13

T

able

3.1

: Mon

thly

mea

sure

men

ts o

f nitr

ate,

pho

spha

te, a

mm

oniu

m, a

nd su

lfate

con

cent

ratio

ns.

Val

ues f

or m

onth

s with

mor

e th

an

one

mea

sure

men

t (n>

1) h

ave

been

ave

rage

d.

26

Chlorophyll a (ppb) Peridinin (ppb) Month n 0.25 1 2 0.25 1 2

Jun-02 5 1.32 1.45 2.36 1.01 1.00 0.93 Jul-02 2 2.04 1.61 8.56 2.66 2.67 9.39 Aug-02 2 1.71 1.91 3.07 1.67 2.63 4.40 Sep-02 4 3.70 1.75 1.19 4.32 1.37 3.01 Oct-02 2 3.26 4.47 2.67 1.03 1.07 0.60 Nov-02 1 1.48 3.11 1.33 ND 0.30 ND Dec-02 1 1.52 2.60 1.96 0.26 0.67 0.23 Jan-03 1 1.39 0.14 1.47 ND ND ND Feb-03 1 0.83 1.86 1.76 ND ND ND

Table 3.2: Monthly measurements of chlorophyll a and peridinin concentration. Values for months with more than one measurement have been averaged.

27

Section 4: Identification of Dinoflagellate Species from Boondoggle Lake

Six species of freshwater armored dinoflagellates were identified from the

water samples taken from Boondoggle Lake. They include Peridinium deflandrei,

Peridinium volzii, Peridinium wisconsinense, Peridinium limbatum, Peridinium

inconspicuum, and Peridiniopsis polonicium. This section highlights the main

morphological features that are unique to each species, and presents data on cell size

and geographical distribution.

4.1 Peridinium deflandrei

P. deflandrei belongs to the subgenus Poroperidinium, meaning it possesses

an apical pore. The general epithecal plate formula for this subgenus according to the

Kofoid system of plate designation is 4/, 3a, 7// (Taylor 1987; Popovsky and Pfiester

1990). However, P. deflandrei has the epithecal plate formula of 4/, 2a, 7// (Popovsky

and Pfiester 1990). Popovsky and Pfiester (1990) classify this species as Peridinium

umbonatum var. deflandrei, grouping it with a number of other species. The

taxonomic schemes of Lefevré (1932) and Starmach (1974) have it listed as a

separate species, Peridinium deflandrei.

Distinguishing features used to positively identify P. deflandrei included

epithecal plate pattern, absence of reticulation on thecal plates, presence of rows of

28

small points on thecal plates, and two antapical spines (Popovsky and Pfiester 1990;

Lefevré 1932, Starmach 1974).

Vegetative cells of P. deflandrei are between 28-35 µm long and 26-32 µm

wide. This species has been reported in Spain and in New York (Lefevré 1932;

CSLAP 2002).

Scanning electron micrographs of P. deflandrei are presented on Plate 4.1.

4.2 Peridinium volzii

P. volzii is the only Peridinium species in this study belonging to the subgenus

Cleistoperidinium. This subgenus name has now been changed to Peridinium and

includes all Peridinium species lacking an apical pore (Popovsky and Pfiester 1990;

Carty 1986). The general epithecal plate formula for this subgenus according to the

Kofoid system of plate designation is 4/, 3a, 7// (Taylor 1987; Popovsky and Pfiester

1990).

Distinguishing features used to positively identify P. volzii included

symmetrical arrangement of the three intercalary plates, a small 1/ plate, sulcal

penetration into the epitheca, and reticulation on the thecal plates (Carty 1986; Susan

Carty pers. Comm.).

P. volzii vegetative cells are between 38-42 µm long and wide. It has been

previously reported in the United States in Oklahoma, North Carolina, Minnesota,

Maryland, and Massachusetts. It has also been reported in Canada, Australia, Europe,

Asia, and Africa (Carty 1986).

Scanning electron micrographs of P. volzii are presented on Plate 4.2.

29

4.3 Peridinium wisconsinense

P. wisconsinense belongs to the subgenus Poroperidinium. Cells of this

species are pointed at each end, giving them a spindle shape. The two bottom plates

on the hypotheca (1//// and 2////) form a horn-like structure, with the 1//// plate extending

to the tip of this structure. The sulcus only penetrates slightly into the epitheca, and

reticulation is present on the thecal plates (Carty 1986; Popovsky and Pfiester 1990).

These features were used to positively identify P. wisconsinense.

Vegetative cells of P. wisconsinense are between 55-64 µm long and 48-56

µm wide (Popovsky and Pfiester 1990). It has previously been reported in the United

States and Canada. Within the United States, there have been reports from Alabama,

Georgia, Louisiana, Massachusetts, Michigan, Minnesota, New Jersey, North

Carolina, South Carolina, Texas, West Virginia, and Wisconsin (Carty 1986).

Scanning electron micrographs of P. wisconsinense are presented on Plate 4.3.

4.4 Peridinium limbatum

P. limbatum belongs to the subgenus Poroperidinium. The most distinctive

features of this species are the pair of horn-like thecal projections on the hypotheca

and the conical, pointed epitheca. The sulcus widens on the hypotheca and reaches

the antapex of the cell. Reticulation is present on the thecal plates (Popovsky and

Pfiester 1990).

P. limbatum is the largest species identified in this study. Vegetative cells are

between 80-93 µM long and 60-82 µm wide (Popovsky and Pfiester 1990). An

30

incomplete list of where it has been documented includes Canada and Oklahoma

(Holt 1981; Yan and Stokes 1978; Yung 1991).

Scanning electron micrographs of P. limbatum are presented on Plate 4.4.

4.5 Peridinium inconspicuum

P. inconspicuum belongs to the subgenus Poroperidinium and is recognized

by its small size as well as shape. The cingulum divides the epitheca into almost

equal halves, and the sulcus penetrates slightly into the epitheca. There is no

reticulation on the thecal plates, but there is ornamentation in the form of longitudinal

rows of dots. Spines may be found on the antapex (Carty 1986). These features,

along with the expertise of Dr. Susan Carty, were used to identify P. inconspicuum

(Susan Carty pers. comm.).

P. inconspicuum is the smallest freshwater dinoflagellates species, with

vegetative cells ranging from 15-30 µm long and 12-25µm wide. It is a widely

reported species. Reports come from most European countries, Cuba, and the United

States. Within the United States, it has been reported in 25 states, including

Mississippi. The range within the continental United States includes far western

(California) to eastern (South Carolina) and southern (Mississippi, Louisiana) to

northern (Wisconsin, North Dakota) (Carty 1986).

Scanning electron micrographs of P. inconspicuum are presented on Plate 4.5.

31

4.6 Peridiniopsis polonicium

The genus Peridiniopsis is distinguished from Peridinium because species

possess 0-1 anterior intercalary plates. Peridiniopsis polonicium was identified by its

oval shape, large 1/ plate, reticulation on thecal plates, and sulcal extension to the

antapex (Popovsky and Pfiester 1990). Identification was also confirmed with Dr.

Susan Carty (Susan Carty pers. comm.). Variations are known to occur in the number

of anterior intercalary plates in this species, and cells observed in this study had two

anterior intercalary plates (Popovsky and Pfiester 1990).

Vegetative cells of Peridiniopsis polonicium are between 33-54 µm long and

30-51 µm wide. Areas where this species has been documented include Europe and

North America (Popovsky and Pfiester 1990).

Scanning electron micrographs of Peridiniopsis polonicium are presented on

Plate 4.5.

4.7 Other Phytoplankton and Zooplankton

Other phytoplankton identified included cryptomonads, diatoms, green algae,

and the chrysophyte Dinobryon. Although it was not quantified, Dinobryon was

found in high densities in the spring. This alga is phagotrophic and feeds on bacteria

(Graham and Wilcox 2000). Scanning electron micrographs of diatoms are presented

on Plate 4.6. Scanning electron micrographs of Dinobryon and the green alga

Micrasterias are presented on Plate 4.7.

The zooplankton identified included rotifers, cladocerans, and copepods. A

scanning electron micrograph of a Keratella rotifer is included on Plate 4.7.

32

Plate Legend Plate 4.1 a. P. deflandrei ventral view………………………………………………....34 b. P. deflandrei epitheca..................................................................................34 Plate 4.2 a. P. volzii ventral view…………………………………………………........35 b. P. volzii epitheca..........................................................................................35 Plate 4.3 a. P. wisconsinense dorsal view………………………………………...……36 b. P. wisconsinense side view………………………………………………..36 Plate 4.4 a. P. limbatum ventral view………………………………………………….37 b. P. limbatum (dorsal) and P. volzii epitheca……………………………….37 Plate 4.5 a. Peridiniopsis polonicium ventral view………………………………..….38 b. P. inconspicuum dorsal view …………………………………………….38 Plate 4.6 Diatoms…………………………………………………………………..…39 Plate 4.7 a. The green alga Micrasterias……………………………………………..40 b. A rotifer, Keratella………………………………………………………40 c. The chrysophyte Dinobryon…………………………………………..…40

33

Plate 4.1

a

b

34

Plate 4.2

a

b

35

Plate 4.3

a

b

36

Plate 4.4

a

b

37

Plate 4.5

a

b

38

Plate 4.6

39

Plate 4.7

a

b

c

40

Section 5: Spatial and Temporal Patterns in Population for Dinoflagellates in Boondoggle Lake

This section presents the results of the counts of total dinoflagellate abundance,

and for five of the six species identified, population proportion of species and estimates

of population sizes for individual species are presented. One species, Peridinium

inconspicuum, could not be quantified because it is smaller than the size of the mesh used

to concentrate the water samples (see methods, section 2.3).

5.1 Total Dinoflagellate Abundance

The temporal changes in combined population density for all dinoflagellates greater

than 20 µm in size is shown for each depth sampled in Figure 5.1. It should be noted that

the X-axis is not to scale.

Maximum population densities occurred between July 26 and September 13, 2002

for all three depths. The maximum population density for 0.25 m occurred on August 22,

2002 and was 2 X 105 cells/L. The maximum density recorded for 1 m occurred two

weeks later on September 6 and was 2.96 X 105 cells/L. For the 2 m samples, the

maximum density occurred on September 13, 2002 and was 1.78 X 105 cells/L.

In addition to the summer maxima, other smaller peaks of density occurred. At

0.25 m, there were two small peaks, one two months before and one a month after the

main summer maximum. These peaks were between 5-7 X 104 cells/L. Two peaks

41

0

50000

100000

150000

200000

250000

300000

.25 MC

ells

/L

0

50000

100000

150000

200000

250000

300000

1M

5-Ap

r-02

12-A

pr-0

221

-Apr

-02

26-A

pr-0

22-

May

-02

10-M

ay-0

215

-May

-02

26-M

ay-0

22-

Jun-

027-

Jun-

0214

-Jun

-02

21-J

un-0

228

-Jun

-02

12-J

ul-0

226

-Jul

-02

22-A

ug-0

229

-Aug

-02

6-Se

p-02

13-S

ep-0

220

-Sep

-02

27-S

ep-0

27-

Oct

-02

18-O

ct-0

225

-Oct

-02

8-No

v-02

22-N

ov-0

26-

Dec-

029-

Jan-

0321

-Feb

-03

27-F

eb-0

315

-Mar

-03

30-M

ar-0

317

-Apr

-03

20-M

ay-0

311

-Jun

-03

0

50000

100000

150000

200000

250000

300000

2M

Date

Figure 5.1: Total dinoflagellate population density

42

between 6-8 X 104 cells/L were seen in the spring at 1 m. Also at 1 m, a peak in June of

1.2 X 105 cells/L and a peak in October of 7 X 104 cells/L were seen.

5.2 Population Proportion

Figures 5.2-5.4 show the patterns in proportion of the total population for each

species at all dates and depths sampled. Clear temporal patterns of population proportion

were observed for all species. However, no clear differences in population proportion

were seen between sample depths.

Individual species showed one of two temporal patterns in percent of the total

population. The first pattern was a higher percentage in the summer and fall, and the

second pattern was a higher percentage in the winter and spring. P. deflandrei, P.

wisconsinense, and Peridiniopsis polonicium all were a higher percentage of the total

population between mid-May and mid-November. P. volzii and P. limbatum were all a

higher percentage between November and April.

The two species that represented the highest proportion at all three depths and for

all dates sampled were P. deflandrei and P. volzii. None of the other three species ever

comprised more than 25 % of the total dinoflagellate abundance. P. deflandrei was 50%

or more of the total dinoflagellates on approximately 63% of the days sampled, especially

in the summer and early fall. P. volzii was 50% or more of the dinoflagellates on

approximately 32% of the days sampled, especially in the late fall, winter, and spring.

Since P. volzii occurs in the winter and spring when total dinoflagellates abundance is

low, its high proportion at these times does not necessarily mean it has a high population

43

P. deflandrei

0

25

50

75

100

P. volzii

0

25

50

75

100

P. limbatum

Perc

ent o

f Tot

al D

inof

lage

llate

s

0

25

50

75

100

P. wisconsinense

0

25

50

75

100

Peridiniopsis polonicium

12-A

pr-02

26-A

pr-02

15-M

ay-02

2-Jun-02

14-Ju

n-02

28-Ju

n-02

12-Ju

l-02

26-Ju

l-02

22-A

ug-02

29-A

ug-02

6-Sep

-02

13-S

ep-02

27-S

ep-02

18-O

ct-02

25-O

ct-02

8-Nov-0

2

22-N

ov-02

6-Dec

-02

9-Jan

-03

21-Feb

-03

15-M

ar-03

30-M

ar-03

17-A

pr-03

1-May

-03

20-M

ay-03

11-Ju

n-03

24-Ju

n-030

25

50

75

100

Date

Figure 5.2: Population proportion at 0.25 m

44

P. deflandrei

0

25

50

75

100

P. volzii

0

25

50

75

100

P. limbatum

Perc

ent o

f Tot

al D

inof

lage

llate

s

0

25

50

75

100

P. wisconsinense

0

25

50

75

100

Peridiniopsis polonicium

12-A

pr-02

26-A

pr-02

15-M

ay-02

2-Jun-02

14-Ju

n-02

28-Ju

n-02

12-Ju

l-02

26-Ju

l-02

22-A

ug-02

29-A

ug-02

6-Sep

-02

13-S

ep-02

27-S

ep-02

18-O

ct-02

25-O

ct-02

8-Nov-0

2

22-N

ov-02

6-Dec

-02

9-Jan

-03

21-Feb

-03

15-M

ar-03

30-M

ar-03

17-A

pr-03

1-May

-03

20-M

ay-03

11-Ju

n-03

24-Ju

n-030

25

50

75

100

Date

Figure 5.3: Population proportion at 1 m

45

P. deflandrei

0

25

50

75

100

P. volzii

0

25

50

75

100

P. limbatum

Per

cent

of T

otal

Din

ofla

gella

tes

0

25

50

75

100

P. wisconsinense

0

25

50

75

100

Peridiniopsis polonicium

12-A

pr-02

26-A

pr-02

15-M

ay-02

2-Jun-02

14-Ju

n-02

28-Ju

n-02

12-Ju

l-02

26-Ju

l-02

22-A

ug-02

29-A

ug-02

6-Sep

-02

13-S

ep-02

27-S

ep-02

18-O

ct-02

25-O

ct-02

8-Nov-0

2

22-N

ov-02

6-Dec

-02

9-Jan

-03

21-Feb

-03

15-M

ar-03

30-M

ar-03

17-A

pr-03

1-May

-03

20-M

ay-03

11-Ju

n-03

24-Ju

n-030

25

50

75

100

Date

Figure 5.4: Population proportion at 2 m (circles represent no data)

46

density. P. deflandrei, however, reaches a maximum proportion of the population in the

summer when total density is high and is responsible for 90% of the summer maximum.

5.3 Individual Population Trends

Calculated estimates of population density for each species over the sampling

period are shown in Figures 5.5-5.7. Y-axes are scaled differently for each species in

order to clearly show trends in population density. The X-axis is not to scale.

Peridinium deflandrei showed a temporal pattern in population density almost

identical to the total abundance of dinoflagellates because it was such a high percentage

of the dinoflagellate assemblage in the summer. Spatial and temporal patterns were seen

in this summer bloom of P. deflandrei that suggested rapid initial growth followed by the

sinking of the bloom. A sharp increase in population at 0.25 m was seen beginning in

July 2002, and an initial maximum density of 1.6 X 105 cells/L occurred at 0.25 m in late

August (see Figure 5.5). By the beginning of September, a maximum density of 2.75 X

105 cells/L was observed at 1 m, accompanied by an 80% decrease in density at 0.25 m

(see Figures 5.5 and 5.6). The maximum at 1 m decreased 98% by mid September, at

which time a maximum of 1.75 X 105 cells/L was seen at 2 m (see Figure 5.7).

Peridinium volzii had maximum densities mostly in the spring. At 0.25 m, it had

a maximum density of 2 X 104 cells/L in April 2002 and another peak of 1.2 X 104

cells/L in August 2002 (see Figure 5.5). At 1 m, a maximum of 5.7 X 104 cells/L

occurred in April 2002. This high density was not seen in samples from April 2003 (see

Figure 5.6). At 2 m, clear peaks were seen in population density in mid to late April for

2002 and 2003 (see Figure 5.7). These peaks were between 2.2 - 2.8 X 104 cells/L.

47

P. deflandrei

0

60000

120000

180000

P. volzii

0

10000

20000

30000

P. limbatum

Cel

ls/ L

0

1000

2000

3000

4000

5000

P. wisconsinense

0

10000

20000

30000

Peridiniopsis polonicium

12-A

pr-02

26-A

pr-02

15-M

ay-02

2-Jun

-02

14-Ju

n-02

28-Ju

n-02

12-Ju

l-02

26-Ju

l-02

22-A

ug-02

29-A

ug-02

6-Sep

-02

13-S

ep-02

27-S

ep-02

18-O

ct-02

25-O

ct-02

8-Nov

-02

22-N

ov-02

6-Dec

-02

9-Jan

-03

21-F

eb-03

15-M

ar-03

30-M

ar-03

17-A

pr-03

20-M

ay-03

11-Ju

n-03

0

10000

20000

30000

Date

Figure 5.5: Individual population trends at 0.25 m

48

P. deflandrei

0

60000

120000

180000

240000

300000

P. volzii

0

20000

40000

60000

P. limbatum

Cel

ls/ L

0

1000

2000

3000

4000

5000

P. wisconsinense

0

10000

20000

30000

Peridiniopsis polonicium

12-A

pr-02

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pr-02

15-M

ay-02

2-Jun

-02

14-Ju

n-02

28-Ju

n-02

12-Ju

l-02

26-Ju

l-02

22-A

ug-02

29-A

ug-02

6-Sep

-02

13-S

ep-02

27-S

ep-02

18-O

ct-02

25-O

ct-02

8-Nov

-02

22-N

ov-02

6-Dec

-02

9-Jan

-03

21-F

eb-03

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ar-03

30-M

ar-03

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pr-03

20-M

ay-03

11-Ju

n-03

0

1000

2000

3000

4000

5000

Date

Figure 5.6: Individual population trends at 1 m

49

P. deflandrei

0

60000

120000

180000

P. volzii

0

10000

20000

30000

P. limbatum

Cel

ls/ L

0

1000

2000

3000

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5000

P. wisconsinense

0

1000

2000

3000

4000

5000

Peridiniopsis polonicium

12-A

pr-02

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ay-02

2-Jun

-02

14-Ju

n-02

28-Ju

n-02

12-Ju

l-02

26-Ju

l-02

22-A

ug-02

29-A

ug-02

6-Sep

-02

13-S

ep-02

27-S

ep-02

18-O

ct-02

25-O

ct-02

8-Nov

-02

22-N

ov-02

6-Dec

-02

9-Jan

-03

21-F

eb-03

15-M

ar-03

30-M

ar-03

17-A

pr-03

20-M

ay-03

11-Ju

n-03

0

1000

2000

3000

4000

5000

Date

Figure 5.7: Individual population trends at 2 m (circles represent no data)

50

Peridinium limbatum stayed at relatively low population densities throughout the

sampling period. At both 0.25 m and 1 m, a peak in population density was seen in late

April with a density of approximately 2 X 103 cells/L (see Figure 5.5 and 5.6). At 1 m,

another peak of 2.5 X 103 cells/L was seen in mid March (see Figure 5.6). P. limbatum

never reached a density higher than 300 cells/L in the 2 m samples.

Peridinium wisconsinense reached maximum population densities in the late

summer. At 0.25 m, maximum density occurred in August and was 1 X104 cells/L (see

Figure 5.5). The maximum population density at 1 m occurred in September and was 2.1

X 104 cells/L (see Figure 5.6). Densities at 2 m were low for P. wisconsinense. The

highest density at 2 m occurred in late July and was 5 X 103 cells/L (see Figure 5.7).

Peridiniopsis polonicium was only found at high density in the summer and in the

surface water. The maximum density at 0.25 m occurred in August and was 1.6 X 104

cells/L (see Figure 5.5). At 1 m, the density was low except in July, when it was 3 X 103

cells/L (see Figure 5.6). At 2 m, a maximum of 3.5 X 103 cells/L occurred in mid

September.

5.4 Correlations Between Temperature and Population Density

Correlations between dinoflagellate population density and temperature are

presented in Figures 5.8 and 5.9. Figure 5.8 shows the correlation of total dinoflagellate

density to temperature for each sampling depth through the entire sampling period.

Figure 5.9 shows the correlation of the most abundant dinoflagellate, P. deflandrei, to

temperature for each sample depth through the entire sampling period.

51

For the total dinoflagellate density, a positive correlation between density and

temperature was seen at all three depths. This correlation was stronger for samples from

1 m (r2= 0.61) than for samples from 0.25 m and 2 m (r2= 0.40 and 0.34, respectively).

P. deflandrei density was also positively correlated with temperature at all three

sample depths. This correlation was slightly stronger for samples from 0.25 m (r2= 0.70)

and 2 m (r2= 0.77) than for samples from 1 m (r2= 0.67).

52

Total Dinoflagellates (.25m)

R2 = 0.40

0.000

1.000

2.000

3.000

4.000

5.000

6.000

0 5 10 15 20 25 30Temperature

Log

Cel

ls/L

Total Dinoflagellates (1m)

R2 = 0.61

0.000

1.000

2.000

3.000

4.000

5.000

6.000

0 5 10 15 20 25 30

Temperature

Log

Cel

ls/L

Total Dinoflagellates (2m)

R2 = 0.34

0.000

1.000

2.000

3.000

4.000

5.000

6.000

0 5 10 15 20 25 30

Temperature

Log

Cel

ls/L

Figure 5.8: Correlation of total dinoflagellate density to temperature

53

P. deflandrei (.25m)

R2 = 0.70

0.0

1.0

2.0

3.0

4.0

5.0

6.0

0.0 10.0 20.0 30.0

Temperature

Log

Cel

ls/L

P. deflandrei (1m)

R2 = 0.67

0.0

1.0

2.0

3.0

4.0

5.0

6.0

0.0 5.0 10.0 15.0 20.0 25.0 30.0

Temperature

Log

Cel

ls/L

P. deflandrei (2m)

R2 = 0.77

0.0

1.0

2.0

3.0

4.0

5.0

6.0

0.0 5.0 10.0 15.0 20.0 25.0 30.0

Temperature

Log

Cel

ls/ L

Figure 5.9: Correlation of P. deflandrei density to temperature

54

Section 6: Discussion Dinoflagellates have potentially large roles in freshwater ecosystems, yet there is

still a paucity of information on the ecology of freshwater dinoflagellates. Many studies

of dinoflagellate ecology have been conducted in areas other than the United States.

Information on dinoflagellate diversity and abundance is especially lacking in the

southeastern United States.

To my knowledge, all species identified in this study, with the exception of P.

inconspicuum, have not been previously reported in Mississippi. There seems to be little

information on the distribution of P. deflandrei. Lefevré (1932) reported this species in

Spain, and a report from Echo Lake in New York was the only occurrence I could find in

the United States (CSLAP 2002). P. volzii is a cosmopolitan species, with reports from

the southeastern, northeastern, and midwestern United States, and it has also been

reported in Europe, Asia, Africa, and Austrailia (Carty 1986; Starmach 1974; Lefevré

1932). Given this information, it is not surprising that it is also found in Mississippi. P.

limbatum has been reported mostly in Canada, but one report comes from Oklahoma

(Holt 1981; Yan and Stokes 1978, Yung 1991). To my knowledge, this is the furthest

south P. limbatum has been identified. P. wisconsinense is a common species in the

eastern United States. It has been documented in at least 11 states, including two that

border Mississippi (Carty 1986). Peridiniopsis polonicium may also be a cosmopolitan

species since it has been documented in Europe, North America, and Japan (Popovsky

and Pfiester 1990; Hashimoto et. al. 1968).

55

By far, the dominant species of dinoflagellate in the summer bloom in

Boondoggle Lake is P. deflandrei. This species comprised an average of 90% of the total

dinoflagellates during the bloom period that began in June and ended in September. The

shift in the population density maximum from 0.25 m in late August to 1 m in early

September and to 2 m in mid September suggests that the bloom formed in the surface

waters and moved downward. Whether this downward movement was due to migration

or sinking is unclear.

P. volzii was the second most abundant species of dinoflagellate in Boondoggle

Lake. However, the maximum density observed for P. volzii was only 20% of the size of

maximum density for P. deflandrei. P. volzii also had a different temporal pattern in

abundance than P. deflandrei; larger populations were observed in months with cooler

temperatures. This pattern is consistent with a previous study that found population

maxima of P. volzii at temperatures between 170 C and 210 C (Olrik 1992). In this study,

P. volzii reached maximum densities in March and April, was mostly absent in the

summer, and was at low densities (< 5000 cells/L) from October through December. One

exception to this pattern was a summer peak of P. volzii at 0.25 m on August 22 (see

Figure 5.5). On this date, water temperature at 0.25 m was 250 C, about 5 degrees cooler

than measurements before and after, which may account for the brief summer peak of P.

volzii.

The other three species exhibited a temporal pattern similar to either P. deflandrei

or P. volzii. P. limbatum was most abundant in the spring and fall. P. wisconsinense and

Peridiniopsis polonicium were most abundant in the summer. These differing patterns

56

may be attributable to a number of dynamic environmental variables or interactions of

those variables.

Many environmental variables contribute to the bottom-up and top-down control

of dinoflagellate populations in freshwater systems. Those that are likely to be important

in determining the temporal and spatial patterns in abundance and species composition of

the dinoflagellates in Boondoggle Lake include, but are not limited to, low nutrient

levels, an acidic pH, summer anoxia, warm temperatures, wind protection, and

interspecific competition.

The low phosphate levels in Boondoggle Lake may be a major contributor to the

relative success of dinoflagellates species. Low concentrations of orthophosphate,

between 1-10 ppb, are often found in freshwater systems where dinoflagellate blooms

occur (Pollingher 1987). The persistence of Peridinium at low phosphate levels is

thought to be due to its ability to take up and store phosphate in an internal pool of

polyphosphates during times of higher phosphate concentrations (Serruya and Berman

1975; Elgavish et. al. 1980). Because the detection limit for orthophosphate in this study

was 10 ppb, it is probable that orthophosphate was not detected simply because it was

below the detection limit.

The pH range of Boondoggle Lake (5.5-6.5) influences species diversity because

it excludes species of dinoflagellates that are not tolerant of acidic environments. Acid

tolerant species are not necessarily acidophilic and may remain at low population sizes

because of reduced growth rates in acidic environments. According to Pollingher (1987),

most freshwater dinoflagellates prefer alkaline environments. Since the pH in

Boondoggle Lake is always acidic, the six species identified in this study must tolerate or

57

prefer acidic environments. The effect of pH on growth rate has been investigated in four

of the six species of this study. Experiments with pure cultures showed P. inconspicuum

did not vary in growth rate across a wide range of pH (5.5- 8.5), but Peridiniopsis

polonicium had reduced growth rates under slightly acidic (6.5) and basic (8.5)

conditions. The same study found that P. volzii and P. limbatum had higher growth rates

under acidic conditions (Holt 1981). A water quality assessment for Echo Lake in New

York found P. deflandrei to be the most abundant species (40% of the phytoplankton),

with lake pH values being between 6.7 and 7.7 (CSLAP 2002). Although there are no

studies of the effect of pH on growth rate of P. deflandrei, it is clear from this study that

it can thrive in at least a slightly acidic environment.

Bottom water anoxia is likely to be one of the key factors controlling vertical

distribution of dinoflagellates in Boondoggle Lake. As mentioned earlier, dinoflagellates

are thought to be intolerant of low oxygen concentrations. If this is true for all species,

then dinoflagellates should be restricted to the epilimnion during the summer when

anoxic conditions are present below the oxygen chemocline. Indeed, the total

dinoflagellate density remained low at 2 m during the anoxic period, with the exception

of a large peak on September 13 (see Figure 5.1). On this date, anoxia was present at

2 m, whereas oxygen at 1.75 m was 4.25 mg/L. This high density value recorded at 2 m

under anoxic conditions is likely due to an error in sampling around the depth of the

oxygen chemocline. This pattern suggests a high density of dinoflagellates at the oxic-

anoxic boundary. If conditions are favorable for growth of dinoflagellates at 2 m before

the onset of anoxia, then development of anoxia in the bottom waters could be forcing an

58

upward vertical migration and subsequent concentration of dinoflagellates in the top 1 m

of the lake.

Temperature likely plays a general role in determining species abundance. Most

dinoflagellates show maximum growth during the summer (Carty 2003). This pattern

was seen for P. deflandrei, P. wisconsinense, and Peridiniopsis polonicium but not for P.

volzii and P. limbatum. P. volzii and P. limbatum may be inhibited by warm summer

temperatures, or other factors may be responsible for their absence in the summer.

Another potential advantage for dinoflagellates in Boondoggle Lake is that the

lake is protected from wind by hills to the west and south. Wind protection favors

dinoflagellates in two ways. First, phytoplankton that do not actively swim are lost more

rapidly from the water column through sedimentation in wind protected lakes, reducing

their level of competition with dinoflagellates (Pollingher 1988). Second, dinoflagellates

are likely to suffer less mortality from turbulence under wind protected conditions

(Pollingher and Zemel 1981).

Interspecific competition between dinoflagellate species may be important in

determining which species dominate the summer bloom. Physiological features may

confer advantages to certain species. For instance, species that are facultative

heterotrophs may be able to obtain carbon and nutrients to which obligate autotrophs

have no access. It is difficult to estimate how much effect interspecific competition may

have since few studies have investigated this topic with freshwater dinoflagellates.

More information on the physiology and ecology of the dinoflagellate species

from Boondoggle Lake is needed to understand which factors are most important in the

development of the large summer population of P. deflandrei and to understand why the

59

other species do not reach such high population densities. Priorities for future research

include more sensitive and comprehensive measurements of nitrogen and phosphorus and

measurements of dissolved organic carbon. Vertical profiles of light intensity are also

needed in order to determine a light extinction coefficient and estimate the depth of the

euphotic zone. A quantification of P. inconspicuum should be made in order to include

its seasonal abundance as part of the total armored dinoflagellates in Boondoggle Lake.

Because horizontal heterogeneity is often observed in freshwater dinoflagellate blooms

(Pollingher 1988), samples from more than one site are needed to describe the horizontal

spatial variation associated with these populations. Lastly, measurements of diel vertical

migration should be made.

Resources permitting, more extensive studies would be able to provide an even

more comprehensive data set on the ecology of Boondoggle Lake dinoflagellates. Using

sediment traps, measurements of the time of encystment and the amount of cysts formed

for each species could be made. Enumeration of cysts from sediment samples and an

attempt to induce excystment in a laboratory could also yield important data. All of these

measurements would be important in determining the role of cyst recruitment in

population dynamics. If these dinoflagellates could be brought into culture, estimates of

peridinin to chlorophyll a ratios within cells could be made, allowing an accurate estimate

of the percent of total algal biomass due to dinoflagellates using HPLC pigment analysis.

60

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