ji^y

EXTRACTION OF ZYGNEMATACEAN ZYGOSPORES
FROM LAKE SEDIMENTS AND THEIR POTENTIAL A5
PALEO-INDICATORS OF LAKE ACIDIFICATION

R. A. C. PROJECT NO. 464G

Prepared for Environment Ontario by:

Environment
Ontario

l^|9/V

ISBN 0-7729-8587-1

EXTRACTION OF ZYGNEMATACEAN ZYGOSPORES
FROM LAKE SEDIMENTS ANT) THEIR POTENTIAL AS
PALEO-INDICATORS OF LAKE ACIDIFICATION

R. A. C. PROJECT NO. 464G

Prepared for Environment Ontario by:

P.A. Zippi, Y-K Yung, P.M. Welboum,

G. Norris, and J.H. McAndrews

University of Toronto, Royal Ontario Museum,

Trent University, and United States Environmental

Protection Agency

AUGUST 1991

o

RECYCLABLE

Cette publication technique n'est disponible qu'en anglais.

Copyright: Queen's printer for Ontario,

1991

This publication may be reproduced for

non-commercial purposes with appropriate

attribution.

pros 1654

log 91-9300-464

ACKNOWLEDGEMENT AND DISCLAIMER

This report was prepared for the Ontario Ministry of the
Environment as part of a Ministry funded project. The views
and ideas expressed in this report are those of the authors
and do not necessarily reflect the views and policies of the
Ministry of the Environment, nor does mention of trade names
or commercial products constitute endorsement or
recommendation for use. The Ministry, however, encourages the
distribution of information and strongly supports technology
transfer and diffusion.

Any person who wishes to republish part or all of this reporr
should apply for permission to do so to the Research and
Technology Branch, Ontario Ministry of the Environment, 135
St. Clair Avenue West, Toronto, Ontario, M4V 1P5, Canada.

Copyright: 1991 - Ontario Ministry of the Environment

TABLE OF CONTENTS

Acknowledgement and disclaimer i

List of Figures and Tables iij

Executive summary 1

Abstract 1

Introduction 2

Methods 6

Field Methods 6

Laboratory Methods 5

A. Sample splitting and measurement 7

B . Addition of marker-species spike 8

C. Removal of carbonates and soluble Fe-oxy-hydroxides 8

D . Removal of silicates 9

E . Removal of fluoride by-products 10

F. Removal of humic acids and fine particulate matter 11

G. Removal of cellulose and hemi-cellulose by acetolysis 11

H. Inspect sample under microscope 12

I. Removal of extraneous sporopollenin by oxidation 13

J. Removal of very fine adhering particulate matter 13

K. Sieving to remove fine dispersed organic debris 13

L . Slide making 14

Processing strategy 16

Microscopy 16

Enumeration 17

Results of analysis 17

Discussion 18

Effects of HCL, HF, NH4OH and acetolysis 18

Concentration of zygnematacean zygospores by oxidation 19

Oxidation trials 20

Conclusions 21

References 23

Figure 1 31

Table 1 32

Table 2 . . . 33

Table 3 34

Appendix 1 - Preparation of solutions and apparatuses 34

Acids and bases 34

Acetolysis solution 35

Glycerine jelly mounting medium 35

Sieve tube construction 35

Vibration sieving 37

Appendix 2 - Morphometric maps 38-47

List of Figures and Tables

Figure 1 - Lake locations map 31

Table 1 - Lake locations and morphometric data 32

Table 2 - Summary of sample processing steps 33

Table 3 - Occurrences of De^barya sp . in Plastic Lake core 34

Zygnematacean zygospores P^9^ ^

EXECUTIVE SUMMARY

Zygnematacean zygospores can be recovered from lake
sediments by a slightly modified palynological technique. In
10 study lakes, zygnematacean zygospores are not practical
paleo-indicators of lake acidification because they are
diluted (approximately 1:10^ to 1:10^) by pollen from the
surrounding forests . Their potential as a paleo-indicator may
be significantly greater in regions with a lower pollen flux.

ABSTRACT

Sediment cores were taken from 10 lakes in the
Haliburton-Muskoka region of Ontario. Zygnematacean
zygospores were recovered and concentrated from the lake
sediments by the progressive removal of inorganic and acid-
base soluble organic components. The final residue contains
the insoluble zygospores and other sporopollenin microfossils
including spores, pollen, dinof lagellate cysts, Pediastrum,
and fungal spores. In most residues, zygnematacean zygospores
are present in concentrations too low for reliable and
efficient quantitative analysis. Pollen from the surrounding
forest dominates the microfossil assemblage. Because
zygnematacean zygospores are less soluble in oxidizing acids
than are most other sporopollenin microfossils, controlled
oxidation of the residue may enhance their concentration by

Zygnematacean zygospores ps-ge 2

removing a. portion of the pollen present in the residue. The
technique may have potential in regions with a low pollen.
flux. In contrast, Peridlnlum dinof lagellate cysts and
Pediastru/n coenobia were found in abundance and show promise
as paleo-indicators of lake water pH .

INTRODUCTION

The purpose of this study is to develop a standard
method for the recovery and concentration of zygnematacean
zygospores from lake sediments and to evaluate their
potential as a paleo-indicator of lake acidification.

Many of the Zygnemataceae are acidophilic and are
abundant in naturally acidic habitats (Transeau, 1951;
Prescott, 1962; Hoshaw, 1968; Yung et al . , 1986).
Zygnemataceae are known to proliferate in some lakes and
streams that have been experimentally acidified (Hendrey,
197 6; Hall et al . , 1980; Turner et al . , 1987; Detenbeck and
Johnson, 1986; Parent et al., 1986). Certain species of the
Zygnemataceae increase in abundance as pH drops below 6.0
(Jack, 1985; Stokes and Howell, 1987) . The increased growth
of certain zygnematacean algae in soft-water lakes during the
early stages of lake acidification has prompted an
investigation of the potential use of zygospores of this
family as paleo-indicators of lake acidification. The main
purpose of the investigation was to augment the transfer

Zygnematacean zygospores page 3

functions developed for diatoms and other siliceous scale-
bearing Chrysophyceae (Battarbee, 1984; Smol, 1986), in order
to provide a more complete overview of past lake dynamics,
especially in lakes where a clear relationship between
diatoms, Chrysophyceae and pH cannot readily be established.

Lakes by their very nature are sediment traps. A large
component of typical lake sediment is organic. This component
contains readily preservable representatives of the biota
that inhabited the lake in the past. Zygospores of the
conjugating filamentous green algae (Zygnemataceae) are
preserved as part of the fossil flora in recent sediments and
in sedimentary rocks as old as 300 million years (Dickman et
al, 1987; Ellis and Van Geel, 1978; Graham, 1971; Head and
Zippi, 1991; Jarzen, 1979; Lindgren, 1980; Rich, et al, 1982;
Van Geel, and Van der Hammen, 1978; Van Geel, 1976, 1979,
1986; Zippi and Norris, 1991; Zippi et al . , 1990).

The zygospores of the Zygnemataceae are thick-walled
reproductive bodies which form in conjugating vegetative
cells or the conjugation tube as a result of isogamous
fertilization. (Transeau, 1951; Randhawa, 1959; Hoshaw, 1968,
and Hoshaw and McCourt, 1988) . Zygospore formation is a
reproductive strategy that seems to enable the Zygnemataceae
to survive the extreme environmental fluctuations of seasons
(Coleman, 1983) . The presence of these zygospores in lake
sediments is indicative of environmental conditions that
were, at least temporarily, conducive for the growth and

Zygnematacean zygospores P^9Q 4

reproduction of the vegetative filaments. Therefore,
occurrences of zygnematacean zygospores in lake sediments
have potential to be used to reconstruct past environmental
conditions. For many species, zygospores have been described
and are diagnostic at the species level (Wei et al . , 1989;
Zippi et al. , 1990) .

A reliable method for the extraction and concentration
of zygospores is necessary before their potential usefulness
as paleoecological indicators can be practically assessed.
Zygnematacean zygospores are composed of a sporopollenin-like
substance (Ashraf and Godward, 1980; Simons et al . , 1982;
Vries et al, 1983) . The term sporopollenin is generally
applied to an organic substance found in the wall of pollen,
spores, and many algal cysts that is insoluble in acetolysis
solution, bases and non-oxidizing acids. Sporopollenin is a
biologically produced group of oxidative polymers of
carotenoids or carotenoid esters (Shaw, 1971; Brooks and
Shaw, 1972) , which is resistant to acetolysis (Atkinson et
al, 1972) . In the laboratory, the sporopollenin of pollen,
spores, and dinof lagellate cysts can be degraded to varying
degrees by oxidizing acids (chromic or nitric acid) . Vries et
al (1983) found that zygospores from species of Spirogyra and
Mougeotia survived one hour of oxidation by chromic acid at
room temperature, whereas pollen of Taxus baccata and Pinus
sylvestris were dissolved.

Zygnematacean zygospores ps-ge 5

Methods exist for the recovery and concentration from
sediments and sedimentary rocks of dinof lagellate cystis,
pollen, spores and several other types of microfossils of
composed of sporopollenin . The established methods of
palynological extraction are suitable for zygnematacean
zygospores because they are similar in size and composition
to other sporopollenin palynomorphs . If present in the
original sample, zygnematacean zygospores will be contained
in the organic residues that were concentrated by the
standard acid-base palynological techniques .

Zygospores may be present in standard palynological
preparations from lake sediments, but in samples from
forested regions they will almost certainly be diluted
several orders of magnitude by the overwhelming abundance of
pollen and spores from the surrounding catchment area (Zippi
et al . , 1990) . Although the presence of several types of
zygospores may be demonstrated with relative ease, it may be
difficult and time consuming to count zygospores in numbers
sufficient for statistical significance in a study restricted
to zygnematacean species .

Controlled oxidation of the acetolysis residue, however,
may remove a sufficient proportion of spores, pollen and
dinof lagellates to effectively concentrate the zygnematacean
zygospores .

Zygnematacean zygospores P^9^ ^

METHODS

Field Methods

Ten lakes in the geologically acid-sensitive region of
southern Ontario were cored (Figure 1, Table 1) . Target lakes
were restricted to small sized, clear water lakes with a pH
in the range of 4 to 8. Core sites were selected where
morphometric maps indicated a centrally located, deep, flat
area of the lake floor (Appendix 2 shows precise core
locations on a morphometric map for each lake) . A modified KB
gravity corer (6.5 cm diameter) and piston corer (5 cm
diameter) were used to recover sediment cores. A clear
plastic core tube allowed immediate visual inspection of the
sediment core to insure that the uppermost sediments had been
recovered. A portable extruding device (Glew, 1988) was used
to subsample the gravity cores at 0.5 cm intervals for the
first 10 cm, then at 1 cm intervals for the remainder of the
core. Only the upper 3 cm of core top material was recovered
from the piston cores .

Laboratory Methods

A slightly modified palynological extraction procedure
was employed for the extraction of zygnematacean zygospores
from lake sediments. The following section describes the

Zygnematacean zygospores P^gs 7

steps used for the extraction and concentration of
sporopollenin microfossils including zygnematacean zygospores
from lake sediments in this study. Table 2 summarizes the
major processing steps and the effect of each step.

Several excellent technical references for the general
palynological processing of sediments and sedimentary rock,
are Gray (1965), Barss and Williams (1976), and Phipps and
Playford (1984) (Caution: Phipps and Playford (1984) list
solutions as percent of concentrated stock solution rather
than molar or weight percent) . Evitt (1984) outlines several
special techniques for preparing and mounting specimens .
These four references give useful advice on dealing with
special problems or difficult samples .

A. Sample splitting and measurement

Al . Split sample or remove a homogeneous fraction of

uncompacted wet sediment and place approximately 6 to 10
ml into a 15 ml centrifuge tube. If sediment is dried add
approximately 5 ml of sediment and fill tube with
distilled water.

A2 . Centrifuge at speeds sufficient to de-water and compact
sediment, (test for differential compaction of sediment
at different centrifugation speeds and times and
standardize)

Zygnematacean zygospores p3.ge

A3. Measure the volume of the compacted sediment (3-4 ml is

the optimum amount of sediment for easy processing in a
15 ml tube) .

B. Addition of marker-species spike

Bl . Decant supernatant water.

B2 . Add aliquot or tablet of known concentration of marker
pollen or spheres (use a species that does not occur in
the study area) .

33. Fill tube with distilled water and mix on a vortex mixe:

Adding water at this stage removes water-soluble
substances .

C. Removal of carbonates and soluble Fe-oxy-hydroxides
CI. Add 4 ml of cold 10% HCl.
C2 . Mix on vortex mixer^

C3 . Once the reaction has slowed, add an additional 6 ml of
HCl.

^ ^ - when mixing on a vortex mixer, fill tube to 4 ml with reagent, use
mixer, then add remaining quantity of reagent. Appropriate care should
be taken not to allow harmful chemicals or valuable residue to splash or
leak out of the tube while mixing.

Zygnematacean zygospores P^ge 9

C4 . Centrifuge and decant supernatant liquid.

If supernatant liquid is bright yellow-green repeat HCl
wash (steps C1-C4) . Follow final HCl wash with at least 3
water washes to dilute the dissolved calcium and
magnesium and the concentration of acid to approach
neutrality.

Caution : samples containing more than 10% calcium carbonate
will effervesce and may overflow a 15 ml tube. Use a larger
container for calcium carbonate-rich samples . Test reactivity
of sample by slowly adding HCl.

At this stage, a homogeneous portion of the sample may
be removed for siliceous microfossil analysis (diatoms) . The
next step will dissolve all siliceous substances .

D. Removal of silicates

Dl. Add 4 ml of cold 50% HF .

Caution: This step should be performed in a fume hood,

D2 . Mix with a plastic or wooden rod.

D3 . Add an additional 6 ml of cold 50% HF .

D4 . Place tubes in a hot water bath at 90-100°C for 30
minutes and stir occasionally.

Zygnematacean zygospores P^ge 10

D5. Centrifuge and decant supernatant liquid. Follow with as
many water washes as needed to dilute the pH to near
neutrality (at least 3 water washes) .

E. Removal of fluoride by-products

Depending on the composition of the mineral component of
the residue, a fine white gel or crystals may precipitate as
a by-product of the reaction with HF . The precipitate may be
CaF2, MgF2, H2SiF6 or H2Si03. These by-products are more
commonly produced from samples containing leached, oxidized
clays or granitic rock fragments. H2SiF6 is soluble in hot
water, H2Si03 is soluble in hot HF, and CaF2, MgF2 are soluble
in hot HCl. Perform the following steps only if necessary.

El. Add 10 ml of appropriate reagent.

E2 . Heat in hot water bath at 90-100°C for 10 minutes.

E3 . Centrifuge and wash with water at least 3 times.

E4 . Repeat steps using various reagents until by-products are
removed .

The composition of the by-products is difficult to
identify. A trial and error approach using the reagents
listed above may be the most expedient method for the removal
of fluoride by-products. Fluorides are the most common by-
product, so start with the addition of HCl, heat, and wash.

Zygnematacean zygospores P^ge 11

If the precipitate persists, add water, heat to 100°C, and
wash. If precipitate is still present add HF and repeat steps
outlined above. The formation of calcium and magnesium
fluorides may often be prevented by thorough HCl washes prior
to the addition of HF .

F. Removal of humic acids and fine particulate matter

Fl. Add 4 ml of 10% NH4OH and mix thoroughly.

F2 . Fill remaining volume with 10% NH4OH and heat at 90°C for
2 minutes .

F3 . Centrifuge and wash with water at least 3 times or until
supernatant liquid clears .

G. Removal of cellulose and hemi-cellulose by acetolysis

Steps Gl and G6 are very important! Adding acetolysis
solution to a tube containing even a small amount of water
results in an explosive reaction. Take extreme care to keep
water away from acetolysis solution. Follow steps carefully.

Gl . Add 10 ml of glacial acetic acid to dehydrate the sample.

G2 . Centrifuge and decant supernatant acid.

G3 . Add 4 ml of acetolysis solution and mix thoroughly.

Zygnematacean zygospores P^9^ 1^

G4 . Add an additional 6 ml of acetolysis solution.

G5 . Place tube in hot water bath at 90-100°C for 10-15 minutes

G5. Fill tube to safe level with glacial acetic acid.

G7 . Centrifuge and decant supernatant liquid (Balance the
tubes with glacial acetic acid) .

G8 . Wash with glacial acetic acid and centrifuge.

G9. Wash with water and centrifuge.

H. Inspect sample under microscope

HI. Decant as much supernatant water as possible without
pouring off any residue.

H2 . Mix residue and remaining water with a micro-pipette.

H3 . Pipette one drop of residue mixture onto a clean
microscope slide.

H4 . Evaluate the success of the preceding processing steps by
examining the residue under a microscope at low
magnification (do not use a coverslip) .

H5 . Carefully return the residue from the microscope slide to
the centrifuge tube.

H6. Repeat any steps (D,E,F,or G) as necessary.

Zygnematacean zygospores ■ P3.ge 13

I. Removal of extraneous sporopollenin by oxidation

Several attempts at implementing this step were
unsuccessful. See the description of oxidation trials in
Discussion section. Further investigation of controlled
oxidation of organic residue to concentrate the zygnematacean
zygospores relative to other sporopollenin microfossils is
necessary .

J. Removal of very fine adhering particulate matter

Include this step only if abundant fine particles (<2|lm)
obscure the sample.

Jl . Add 4 ml of 10% NH4OH and mix thoroughly.

J2 . Fill remaining volume with water and centrifuge.

J3 . Wash with water at least 3 times.

K. Sieving to remove fine dispersed organic debris

Kl . Add 4 ml of 5% (wt/vol) detergent mixture (e.g. Alconox)
and mix thoroughly using vortex mixer.

Zygnematacean zygospores P<3g'e 14

K2 . Pour contents of centrifuge tube into sieve tube fitted
with a 7 (im screen (see Appendix 1 for sieve tube
construction) .

K3 . Spray with a jet of water until tube is 3/4 full then

vibrate sieve tube with a vibrating engraver until it is
nearly empty (Zippi, 1986). See Appendix 1.

K4 . Repeat step 3 until the water coming through the sieve
contains no suspended particles and is not soapy.

L. Slide making

Slide making may take some practice to achieve the
proper density of specimens for efficient inspection and
analysis later. Slide making should be performed on a slide
warming plate at 50°C (See Appendix 1 for details on the
preparation of glycerine jelly) .

LI. Remove as much water as possible from the centrifuge tube
without removing any specimens .

L2 . Thoroughly mix residue with remaining water and

immediately (before any specimens can settle) place a
drop onto the center of a clean glass microscope slide.

L3 . Add 1 drop of liquified glycerine jelly (Liquify

glycerine jelly by placing a glass tube containing a
small portion of jelly into a warm [~50°C] water bath) .

Zygnematacean zygospores P^ge 15

L4 . Mix residue and glycerine jelly with a clean wooden

toothpick or dissecting needle. Allow excess water from
residue to evaporate.

L5 . Place edge of a 22 x 40 mm number 1 coverslip to the
glass slide and lower at an angle onto the residue
mixture .

L6. Allow slide to remain on the warming plate until mixture
has spread to reach all edges of the coverslip.

L7 . Invert the slide, so that the coverslip is down (but

raised up off the slide warmer with toothpicks under the
edges) to allow specimens to settle through the layer of
glycerine jelly closer to the coverslip for 5 minutes.

L8 . Remove from warming plate and allow slides to cool in an
inverted position for at least 1 hour. Slides may be
inspected but should be handled gently (avoid oil
immersion and cleaning) for at least 2 days. After 2-3
days e.xcess glycerine jelly may be cut away from the
edges of the coverslip with a razor blade. Gently clean
with alcohol, then seal edges with a clear plastic or
varnish .

L9. Store in a cool place (not overly hot or humid for

prolonged times) with slides positioned horizontally. If
the slides are stored on edge, over time, the residue
will flow.

Zygnematacean zygospores P^9^ 1^

LIO. Store remainder of residue. Add a few drops of glycerine
and a drop of 5% copper sulphate solution to a vial
containing the residue and tightly cap.

Processing strategy

The overall processing strategy must be: less is better.
The number of steps used in the processing of samples should
be kept to a minimum. Specimens are lost by repeated
centrifugation and decanting or pipetting of supernatant
liquid. The loss is insignificant during the early stages of
processing before the zygospores are concentrated and as long
as enough residue is settling to form a distinct pellet. The
risk of specimen loss increases as the zygospores are
increasingly concentrated. Although the number of processing
steps should be kept to a minimum, all samples should be
processed in a similar manner in order to reduce statistical
bias resulting from different processing techniques.

MICROSCOPY

Strew mounts were examined using light microscopy. Often
only one layer of the zygospore wall is preserved after
paylonological treatment. Although they are tough and durable
as fossils, these cell membranes may be thin, transparent and
of similar optical contrast to most of the common mounting

Zygnematacean zygospores psge 17

media. Many specimens may be difficult or impossible to view
under bright field illumination. Phase contrast or
interference contrast illumination is essential for the
successful identification and enumeration of the fossil
zygospores .

ENUMERATION

Fossil categories were tallied until the count reached a
minimum of 350 planktonic specimens exclusive of Botryococcus
braunii and the Lycopodium marker species, or until 2 entire
strew mounts were completely counted.

RESULTS OF ANALYSIS

Observations of identifiable zygnematacean zygospores
were extremely rare in the organic residues that were
concentrated from the 10 study lakes and the Plastic Lake
core. After thorough reconnaissance and quantitative
enumeration of the residues, identifiable zygnematacean
zygospores were found only in 3 of the 10 study lakes. Only
one zygospore of Zygogonium tunetanum was observed in the
surface sediment sample from Plastic Lake. Two other
specimens of questionable identification were observed in

Zygnematacean zygospores P^9^ ^S

surface sediment samples from Tock Lake and Four Mile Lake.
Identifications were made by comparison to reference slides
of zygospores extracted from field collections of
zygnematacean filaments recovered from the study area and and
by consulting existing taxonomic literature (Transeau, 1951;
Randhawa, 1959; Wei et al . , 1989).

Zygospores from one species of Deharya were found with
greater regularity in samples from the lower portion of the
Plastic Lake core (Head and Zippi, 1991) . However, the
zygospores of Deharya sp . were not found in any surface
sediment samples and do not occur in abundance in samples
above 17 cm in the core (Table 3) .

In contrast, dinof lagellate cysts and Fediastrum
coenobia were found in abundance in the surface sediments of
the 10 study lakes and in the Plastic Lake core samples and
show potential as paleo-indicators of lake water pH (Zippi et
al. , 1990; 1991a; 1991b) .

DISCUSSlOl^

Effects of HCL, HF, NH4OH and acetolysis

Processing with HCL, HF, NH4OH and acetolysis solution
effectively concentrates all sporopollenin microfossils and

Zygnematacean zygospores P^ge 19

other acid-base insoluble particles including zygnematacean
zvaospores. The concentration occurs by the progressive
dissolution of mineral and organic components. The sample is
demineralized with HCl and HF, humic acids are dissolved with
NH4OH, and cellulose is removed by acetolysis . Some of the
sporopollenin microfossils present at this stage are pollen,
spores, dinof lagellate cysts, various algal cysts such as
Pediastrum, fungal spores and hyphae, and Botryococcus . If
zygnematacean zygospores are present in significant numbers
processing can stop without resorting to harsh oxidation.

Concentration of zygnematacean zygospores by oxidation

In Ontario lakes, a problem of concentration arises
because the zygnematacean zygospores are diluted by the
extremely rich pollen flora. The total pollen, spores, and
dinocyst abundance outnumbers the zygnematacean zygospores by
several orders of magnitude. The ratio of zygnematacean
zygospores to pollen and other sporopollenin microfossils is
estimated to be less than 1:100,000. This concentration is
unworkable for practical paleoecological analysis.

A possible solution to this problem is the further
concentration of zygnematacean zygospores from pollen-rich
organic residues by controlled oxidation. In several
experimental trials with zygospores recovered from acid-
acetylation residues of mature zygnematacean filaments,

ZygnemaCacean zygospores P^9^ 20

zygnematacean zygospores proved to be more resistant to
oxidation by chromic acid than spores, pollen and dinocysts.

Oxidation trials

In order to test the relative resistance of
zygnematacean zygospores to oxidation, field samples from the
Haliburton-Muskoka area of Mougeotia laetevirens , Spirogyra
jatobae, S. jaoii, S. corrugata, Zygogonium tunetanuw, plus
pollen, spores and dinof lagellate cysts, were acetolyzed,
treated with hydrofluoric acid (HF) , and subsequently
oxidized with chromic acid for 90 minutes at 50°C. The
residue was periodically examined (at 10 minute intervals)
under the microscope to check the progress of the oxidation.

After 90 minutes at 50°C in chromic acid, the pollen,
spores, and dinof lagellate cysts are mostly dissolved.
Zygospores remain, but they are thinned and lightened to a
point that makes identification difficult. Various factors
such as wall thickness, sculpturing and possibly composition
causes differential susceptibility to dissolution among
species of zygospores . Consequently, not all species are
dissolved at the same rate. Thick, solid-walled species
survive, while thinner and more deeply sculptured wall types
are less resistant (sculpture such as deep pitting increases
surface area for faster reaction) .

Zygnematacean zygospores P^ge 21

If oxidation is to be carried out on a sample, the
marker grains must be insoluble in oxidizing acids . Spores
are among the very first sporopollenin microfossils to be
dissolved by oxidizing acids and are therefore inappropriate
as marker grains.

CONCLUSIONS

The rarity of zygnematacean zygospores in the study
lakes precludes further analysis of their relationship to
lake water pH. The concentration of zygnematacean zygospores
is diluted by the overwhelming supply of pollen from the
surrounding forests. Because the single zygospore species
{Debarya sp . ) that occurs in abundance in some samples of the
the Plastic Lake core was not recovered from any surface
sediments, precise environmental interpretations cannot be
related to its occurrence.

The use of zygnematacean zygospores as a paleo-indicator
for lake acidification in the recent past may prove useful in
acid-sensitive regions where the pollen supply is much lower.
One possible area is The Netherlands, where algae occurs in
abundance in shallow pools and ponds .

The method of concentration outlined in this paper can
be used to concentrate zygnematacean zygospores from lake
sediments .

ZygnemaCacean zygospores P^9^ 22

ACKNOWLEDGEMENTS

Thanks are extended to Takahashi Sawa for supplying
field samples of Zygnemataceae filaments and to Martin Head
for critically reading the manuscript.

Zygnematacean zygospores- P^g^ 23

REFERENCES

Atkinson, Jr., A.W., Cunning, 3.E.S., and John, P.C.L., 1972.
Sporpollenin in the cell wall of Chlorella and other
algae: Ultrastructure, chemistry, and incorporation of
I'^C-acetate, studies in synchronous cultures. Plantae,
107: 1-32.

Ashraf, M. and Godward, M.3.E., 1980. Ultrastructure and
chemistry of the zygospore wall of Spirogyra . Ann. Bot .
(London), 46: 485-487.

Barss, M.S. and Williams, G.L., 1976. Palynology and

nannofossil processing techniques. Geological Survey of
Canada, Paper, 73-26: 1-25.

Battarbee, R.W., 1984. Diatom analysis and the acidification
of lakes. Philos . Trans. R. Soc . Lond. B Biol. Sci . 305:
451-477.

Brooks, J. and Shaw, G., 1968. Chemical structure of the

exine of pollen walls and a new function for carotenoids
in nature. Nature, 219 (5153): 532-533.

Coleman, A.W., 1983. The roles of resting spores and akinetes
in chlorophyte survival. In: G.A. Frixell (editor)
Survival Strategies of the Algae. Cambridge University
Press . 144 p .

Zygnematacean zygospores P^9^ 24

Detenbeck, N.E., and Johnson, M., 1986. Periphyton growth in
experimentally acidified Little Rock Lake, Wisconsin.
Joint Meeting of American Society of Limnology and
Oceanography, Inc. and the Phycological Society of
America, University of Rhode Island, 27 p.

Dickman, M.D., Van Dam, H., Van Geel, B., Klink, A.G., and
Van der Wijk, A., 1987. Acidification of a Dutch moorland
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(3) : 377-408.

Ellis, A.C. and Van Geel, B., 1978. Fossil zygospores of

Debarya glyptosperma (De Bary) Wittr. (Zygnemataceae) in
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Evitt, W.R., 1984. Some techniques for preparing,

manipulating and mounting dinof lagellates . Journal of
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Glew, J.R., 1988. A portable extruding device for close
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Graham, A., 1971. The role of myxomycete spores in palynology
(with a brief note on the morphology of certain algal
zygospores) . Review of Palaeobotany and Palynology, 11:
89-99.

Zygnematacean zygospores ■ P^ge 25

Gray, J., 1965. Extraction techniques. In: B. Kummel and D.
Raup (editors) Handbook of Paleontological techniques . pp.
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Hall, R.J., Likens, G.E., Fiance, S.B., and Hendrey, G.R.,
1980. Experimental acidification on a stream in the
Hubbard Brook Experimental Forest, New Hampshire. Ecology,
61: 976-989.

Head, M. and Zippi, P., 1991. Fossil and modern species of
Dejbarya (Zygnemataceae, Chlorophyta) from Canada and the
United Kingdom. Geological Association of Canada Annual
Meeting, Toronto, Program and abstracts. May 1991

Hendrey, G.R., 1976. Effects of pH on growth of periphyton
algae in artificial stream channels. Sur. Nedhobors
Virkning pa skog og Fisk. Project IR 25/76

Hoshaw, R.W., 1968. Biology of the filamentous conjugating
algae. In: D. Jackson (Editor) Algae, Man, and
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Hoshaw, R.W. and McCourt, R.M., 1988. The Zygnemataceae
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Zygnematacean zygospores P^9^ 26

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Jarzen, D.M., 1979. Zygospores of the Zygnemataceae in the
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Zygnematacean zygospores P^ge 27

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Zygnematacean zygospores P^9S 28

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Zygnematacean zygospores

page 29

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28.

ome

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Abstract .

Zygnewatacean zygospores pag^s 30

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paleo-indicators of recent lake acidification.
Environmental Research, Technology Transfer Conference,
Proceedings, Volume 1: 393-396.

31

CO

c
O

CO
o
w

3

C
O

n
n

I

(0
CO

3

Zygospore extraction methods

page 32

Lake

Leonard

Plastic

Tock

St. Nora

Harp

Little Clear

Rabbit Bay

Kushog

White

4 Mile

pH

5.5
5.8
6.2
6.2
6.3
6.3
6.8
6.3
7.4
7.8

ALK area
(Heq/1) (h)

0.3

14.7

2.2

2.1

61.1

7

3.5

3.1

61 .1

89.4

467

32.1

288

647

71.4

136

17428

1580

432

1942

depth
(m)

13

15.5

14.5

14

13

12

18

15.5

9

20

core
type

location (Twp., lat . /Ion. )

grav. Monck, 45''04 ' /79°27 '

grav. Sherborne, 45°11 ' /78°50 '

pist. McClintock, 45°16 ' /78°53 '

grav. Sherborne, 45''09 ' /7B°50'

pist. Chaffey, 45°23 ' /7 9°07 '

pist. Sinclair, 45°24 ' /79°00 '

pist. Muskoka, 45°15 ' /78°54 '

grav. Stanhope, 45°04 ' /78°47 '

pist. Galway Township, 44°50 ' /78°29 '

pist. Summerville, 44°41 ' /78°31 '

Table 1 - Lake locations and morphometric data for 10 lakes in the
Haliburton-Muskoka region of south-central Ontario. pH and ALK data from
the Ontario Ministry of the Environment, Dorset, Ontario.

Zygospore extraction methods page 33

Step 1

Measure de-watered sediment volume

add known quantity of marker grains
yields density: specimens /ml

Step 2

Carbonate removal

HCL

removes calcareous algae and carbonate rock fragments

Step 3

Humic acid removal
NH4OH

removes humic acid, oxidized cellulose,
amorphous organic particles and some clay-sized minerals

Step 4

Silica and silicate removal
HF

removes silica and silicate microfossils and mineral grains

Step 5

Acetolysis
H2SO4 : (CH3CO) 2O (1:9)

removes cellulose and hemi-cellulose

Step 6

Sieving (7 |im)

removes fine particles

Step 7

Sporopollenin residue

pollen, spores, dinof lagellate cysts, Pediastrum coenobia,and
zygnematacean zygospores

Step 8

Ac id- oxidation
HNO3 or Cr03

limited success concentrating zygnematacean zygospores
from pollen-rich organic residues

Table 2 - Summary flow chart of the steps for concentrating
zygnematacean zygospores from lake sediments.

Zygospore extraction methods P^ge 34

Depth (cm) Debarya sp . Other Zygnemataceae (number of specimens)

Zygogonium tunetanum (1)

0

0

1

0

2

0

3

0

4

0

5

1

6

2

7

1

8

4

9

0

10

0

11

0

12

0

13

1

14

0

15

0

15

0

17

11

18

14

19

20

24

0

29

0

34

0

39-

26

Mougeotia sp . (1)
Mougeotia laetevlrens (1)

Spirogyral sp (i;

Table 3 - Occurrences of Zygnemataceae in the Plastic Lake
core . Zygnematacean zygospores are rare in the surface
sediments of the 10 study lakes . The only commonly occuring
species in the Plastic Lake core was Dejbarya sp . , but it was
not recovered from any of the surface samples .

Zygospore extraction methods P^g^ 3 5

Appendix 1 - Preparation of solutions and apparatuses

Acids and bases

Acid solutions are specified in volume percent. Mixtures
should be prepared with distilled water. Where concentrations
are not specified, use a concentrated stock solution.

Acetolysis solution

Mix 9 parts acetic anhydride [ (CH3CO) 2O] : 1 part sulfuric
acid (H2SO4) . Caution: Keep away from water. Tubes and beakers
should be dry or de-watered by rinsing with glacial acetic
acid before use .

Glycerine jelly mounting medium

Mix 10 g of gelatine powder (e.g. Knox gelatine) into 60
ml of warm (50-80°C) , distilled water until dissolved. Add 60
ml of glycerine and 0.25 g of copper sulfate (fungal growth
inhibitor) and continue to mix gently for approximately 10
minutes. Avoid creating air bubbles while mixing. Filter
mixture while still warm through a fine mesh (20-25 Jim) .
Allow any bubbles to rise while still warm, then cap and
store in a cool place .

Zygospore extraction methods ps.ge 3 6

Repeated reheating of mountant may result in mountant
instability. For slide making, remove only a small portion of
glycerine jelly sufficient for the number of slides about to
be mounted. Keep the remainder refrigerated.

Sieve tube construction

The sieve tube is constructed by mounting fine mesh
Nitex* screening between an outer sleeve and an inner tube
that have been cut from polypropylene centrifuge tubes. The
outer sleeve is cut from a 100 ml round-bottom centrifuge
tube that has an inner diameter equal to the outer diameter
of the inner tube. The inner tube is cut from a 50 ml
centrifuge tube. Several outer sleeves may be cut from a
single 100 ml tube. The sleeves should be cut about 1 cm
(1/2") long. Sleeves that are cut too thin may slip off
during sieving and sleeves that are too long are difficult to
remove once screen is in place. The inner tube should be cut
5-8 cm (2-3") long from a smooth sided (non-graduated) 50 ml
tube. Raised graduated markings will form a gap between the
outer sleeve and the inner tube allowing larger particles to
leak through. Tubes cut too short will allow residue to
easily splash over the side when sieving is enhanced with
vibration, and will limit the volume of residue and water.

Tube cutting: Centrifuge tubes are placed in a vice or
suitable holder (tighten only enough to immobilize) and cut

Zygospore extraction methods P^ge 37

to length with a fine-tooth hack saw blade. Cutting with a
razor yields poor results and is very dangerous . The ragged
edges can be neatly shaved using a razor knife. The ragged
edges of the inner tube further smoothed by passing the
shaved cut end lightly over an open flame. (Do not heat treat
the outer sleeve. The melted plastic rolls inward making the
inner diameter too small.)

Vibration sieving

The use of a vibrating engraver can enhance and speed
the sieving of organic microfossils (Zippi, 1986) . The
necessary hardware is inexpensive and readily available. A
standard vibrating engraver* and a short length of narrow-
diameter rubber tubing are all that is required. Cut a short
piece of tubing so that it fits tightly over, and extends
slightly past, the engraving tip. Holding the top of the
sieve tube between thumb and forefinger and the engraver in
the opposite hand, simply touch the rubber tip to the side of
the sieve tube. While water is in the tube, this vibrating
action disaggregates and suspends the residue, greatly
speeding the sieving process .

Nitex screens - Tetko, 420 Sawmill River Rd., Amelsford, NY 10523, or
B & SH Thompson, 140 Midwest Rd., Unit 11, Scarborough, Ont . , MIP 35:

Vibro-graver- Burgess Vibrocraf ters Inc., Grayslake, Illinois
(available at hardware stores) .

38

Four Mile Lake

Sommen/ille Township
Lat. 44° 41' Long. 78° 45'

after Ontario Ministry of Natural
Resources map, MNR# 9566)

Miles

I

0 Kilometers 1

39

500 m

Harp Lake

Chaffey Township

Lat. 45° 23' Long. 79°07'

(source: Ontario Ministry of the Environment.
Dorset, Ontario)

40

Kushog Lake
Northern Arm
Stanhope Township
Lat. 45° 04" Long. 78° 47

(after Ontario Ministry of Natural
Resources map, [V(NR# 9229)

Kilometers

41

Kilometers

Leonard Lake

Monck Township

Lat. 45° 04' Long. 79°27'

(after Reid and Girard, 1987
Ontario Ministry of the Environment
Data Report DR 87/2)

"^..

^Sr

42

Little Clear Lake

Sinclair Township

Lat. 45°24'Long. 79° 00"

(source: Ontario Ministry of the
Environment, Dorset, Ontario)

PAZigg,

500m

43

Plastic Lake

Haliburton County
Sherbourne Township
Lat. 45°11' Long. 78° 50'

(after Girard at al., 1985

Ontario Ministry of the Environment

Data Report DR 85/1 )

44

0 1

Kilometer

Rabbit Bay
(Lake of Bays)
Muskoka Township
Lat. 45°15' Long. 78°54'

(after Ontario Ministry of Natural
Resources map, MNR# 9239)

45

'\

'*»ZI99^

St. Nora Lake

Haliburton County

Sherbourne and Stanhope Townships

Lat. 45° 09' Long. 78° 50'

(after Ontario Ministry of Natural
Resources map, MNR# 9614)

46

2

3 CO

z a>

CO

.2 E

47

Meters

300m

White Lake

Galway Township

Lat. 44° 50' Long. 78° 29'

(after Ontario Ministry of Natural
Resources map, MNR# 9593)