OBSERVATIONS ON THE ECOLOGY OF PROTOZOA ASSOCIATED

WITH SPHAGNUM

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By

Peter Chacharonis, B. A., M. A

The Ohio State University

1954

Approved by

Advise TABLE OK CONTENTS

INTRODUCTION...... 1

HISTORICAL REVIEW...... 2

NATURE OK THE ENVIRONMENT...... iO

Location and History of the Bog...... 10 Description of the Hog...... 11 Ecological Characteristics of the Bog...... 12 Structure and Growth of Sphagnum...... IB

MATERIALS AND METHODS...... 21

General...... ill Collections...... 21 pH Determinations...... 23 Temperature Determinations...... 24 Methods of Examination...... 25 Staining Methods...... 2b Quantitative Determinations...... iO

OBSERVATIONS...... 32

General...... 32 Distribution and Relative Frequency of Protozoa...... 32 The Position of the Organisms on and in the Sphagnum plant... 4J Seasonal Observations...... 45 Associations...... 48

DISCUSSION...... 50

Significance of Associations at Different Levels...... 50 Succession...... 54 Comparison of the Protozoa of Cranberry Hog with those Found by Other investigators...... 5b

SUMMARY...... 58

REFERENCES...... bl

APPENDUL...... bb

PLATES...... b9

i INTRODUCTION

The unusual protozoan fauna characteristic of sphagnua bogs has

been known for aany years, and nuseroni investigators have studied

it. Very few, however, have studied the actual relationships that

exist between various Protozoa and the sphagnua plant itself, and

none have done so systeaatically. This has been attempted in the

present study of Protozoa associated with sphagnua in an acid bog in

central Ohio. Upon close investigation of the smterial, an entirely

new type of ecological relationship has been observed. The Protozoa

live not only in snail pools on the bog, or in water around the base

of the plant, but also in a variety of locations on and in the sphagnum

plant itself.

The present investigation was suggested by Professor Wencel J.

Kostir of the Department of Zoology and Entoaology, at the Ohio State

University, to whoa 1 wish to express ay sincere appreciation for his

suggestions and guidance, and the tine and interest he has given ae.

I aa also deeply grateful to Dr. J. N. Wolfe for his assistance in the

botanical aspects of this study, and to Dr. A. LsRoy Andrews for his

identification of the sphagnua. Finally, 1 wish to thank the Ohio

Acadeay of Science for financial assistance granted for travel to and

froa Buckeye Lake.

1 HISTORICAL REVIEW

The earlier workers who studied Protosoa froai sphagnum and other

■ossea (aamng them Dnjardin, 1852, Greeff, 18bt>, Bhrenberg, 1874,

Maggi, 1688, Scourfield, 1897), did little no re than emwerate or describe certain species fron noss collections. This was also lsrgeljr true of Leidy, 1879, Penard, 1902, 1909, Cash, Wailes, and Hopkinson,

1905-1921, Wailes and Penard, 1911, whose works were primarily taxonomic and general in scope, but who gave considerable infomation on Protosoa associated with sphagnum.

A review of the literature of the Protosoa fron sphagnim discloses three nain lines of study: 1) those papers, prinarily of a taxonomic nature, which yield some infonsetion concerning ecological relation­ ships ; 2) those papers which attest to classify the Protosoa into ecological associations; and 3) those papers, very few in number, which describe in detail certain species found on or in the sphagnum plant itself.

Leidyvs monograph on Fresh-water Rhixopods of North Aswrica is probably the most outstanding of the taxonomic works which include

Protosoa from sphagnum. Leidy not only described numerous species, but also stated under what conditions he collected them. Moreover* he pointed out that certain of these rhisopods frequently occur in loose associations and in relation to special environments. For example* he

2 stated that "usually the naked forms, and especially the larger ones, the Difflugias and the Arcellas, are found most frequently, abundantly and best developed, in the oose of bodies of water. The Euglyphas,

Nebelas, and their nearer allies, are in like manner most frequent in the moist Sphagnum of bogs; " (1879: p. 12). In the sphagnum material examined by Leidy, 38 species of rhisopods were found, most of which were shell-bearing forms belonging to the order Testacea.

Leidy*s collections were made chiefly in southeastern Pennsylvania and southwestern New Jersey; a few collections were made in Maine,

Florida, Alabama, and Wyoming.

Since Leidy1a day many Protosoa, particularly rhisopods, have been reported from sphagnum by investigators in many parts of the world. In 1900 Levender made a study of sphagnum collected from clefts or hollows between the rocks on the SluLren Islands off the coast of

Finland, near Helsinki, and noted that the sphagnum harbored a characteristic fauna. He coined the term sphagnophilic for those forms which repeatedly appear in sphagnum and which, he said, seem to be "bound to it more or less closely."

Penard (1902) in his Faune Rhisopodique du Bassin du Leman (Lake

Geneva in Switserland) also reported rhisopods which live in sphagnum in that vicinity. In 1909 he made a further study of the rhisopods from various mosses, other than sphagnum, and concluded that their rhisopod fauna was essentially similar throughout, but varied from station to station according to differences in topography and climatic conditions.

3 Penard concluded that the rhisopods of Mosses in general consti­ tute a special fauna and should be divided into two aain categories.

The first of these he called the banal fauna, or coaaonplace foras.

This group included those species which are not restricted to aosses and do not possess structures or stages which facilitate their adapta­ bility to the aoss environaent. In this category he listed 19 species in the order of their frequency or relative abundance. The second group he called the characteristic fauna. This category contained those foras which possess, at soae tine in their life history, certain structures or stages which enable then to survive the varying conditions that nay arise in the aoss environaent. Penard listed 17 species as characteristic fauna of aosses other than sphagnua.

In 1910 Heinis, working in the vicinity of Basel, in Switserland, listed M rhisopods which he found in sphagnua. Although Heinis aade no atteapt to classify these rhisopods ecologically, he pointed out that rhisopods in general are the typical sphagnophilic foras. In addition to this, Heinis also discussed briefly the association that exists between the fauna and the sphagnua plant itself. He found that

rotifers, nesatodes, and Protosoa occurred on the outer cells of the

sphagnua leaf, between the leaves and the stea, and around the decaying

bottaa portion of the plant. Heinis concluded that the sphagnii

suffered no ill effects fron this relationship, but that the aniaals

or organisau benefited only through the space thus aade available to

then for their life activities; he called this Rauaparasitisms.

Kleiber (1911), working in the Black Forest in southwestern

4 Germany, and Steinecke (1913) in east Prussia (now a part of Russia), both studied the Protosoa of sphagnua bogs, and each attempted a classification of these foras according to types of habitat.

The three publications of Harnisch (1925, 1927, and 1929) probably comprise the most complete account of the ecology of the shell-bearing rhisopods of sphagnum bogs. The first of these works presents a monographic treatment of the entire fauna in a large bog territory, the Seefelder at Reiners in Silesia (now a part of Poland).

In a study of the bogs of this region, Harnisch found altogether 10 species of testacean rhisopods, and recorded their relative frequency.

He also studied their frequency in relation to different amounts of moisture.

From his observations and those of other investigators, Harnisch compiled a list of 16 purely sphagnophilic species, belonging to the genera Amphitrema. Arcella. Diffiueia. Euelvpha. Heleopera. Hyalosphe- nia. and We be la. He also designated certain forms as conditionally sphagnophilic and others as sphagnophobic. As conditionally sphagno­ philic he listed 45 species of Testacea, some of which have been marked as purely sphagnophilic by other authors. The sphagnophobic foras, on the other hand, were not identified to species, but included

23 genera of rhisopods, a few of which had been reported occasionally in sphagnum and are referred to as sphagnoxene (foreign to sphagnum, but present accidentally). Most of the sphagnophobic foras were referred to as sphagnecthron (sphagnum "enemies") and are not found in sphagnum material.

5 In 1927, using Material froai the same territory and also collect­ ions sent to bin by investigators frosi other localities, in and near

Germany, Harnisch further classified the rhisopods of sphagnum accord­ ing to frequency of occurrence in various types of bogs and sphagnum stands. From his study of these collections Harnisch discovered that different types of sphagni* bogs or stands are characterised by particular associations of rhisopods. His classification of these associations is as follows:

1. The first or forest moss type of association (tfaldsmostyp) contains rhisopods which are found chiefly in the sphagniai of forests, open fields, borders of pools, and other habitats where acid bog conditions do not yet exist. The species in this type of association are varied and are not necessarily bound to sphagnum. Species of

Difflugia. Centropyxis. Kuglypha, Trinena, Arcella, he be la. Assulina, and Conrthion are the principal forms in this type of habitat. The purely sphagnophilic species, such as Hralosphenia elgggg£, Hral. papilio. Amphitrema flavum. and Aaph. wriahtiaaan. are absent from this type of environment.

2. The second type of association is the Hralosphenia type

(Hyalosphenientyp). Hralosphenia. especially Hral. papilio. is the most abundant form in this type of environment, but many species of the forest moss type are also present. This association is usually found in developing bogs tending toward the typical acid bog formation, and on sphagnum in dying or dead acid bogs.

3. The third type of association is the Amphitrema type

6 (Anphitrementyp), with Anphitrena as the doaunant genua. The fauna of thia type is always rich in species. This type is further sut>- divided according to the species of Anphitrena which are present.

The first subdivision of this category is the flavun type of association (flavuntyp) in which Anphitream flavun doadLnates. This type is characteristic of the raised peat bogs of central Europe.

The second subdivision is the wrichtiamus type of association

(tfrightianisstyp) which contains both Anphitresui flavun and Anphitrena wrightianun. with Anphitrena flaviss being the au>re numerous species in the smjority of cases. Moreover, Anphitrena wrixhtIanun does not appear to occur without Anphitrena flavun. The fauna of this type is found only in old, acid, peat bogs.

In his 1929 book, Harnisch considered the varying moisture re­ lationship as decisive for the occurrence of certain foras in particu­ lar bogs.

Harnisch also suggested that the presence of soochlorellae in nany of these sphagnophilic foras is an important factor in their survival and reproduction in an environment which is low in nutritative materials and oxygen*

In all his work, Harnisch discussed the rest of the Protosoa

(i.e. exclusive of the testacean rhisopods) only briefly, and did not attempt to enumerate all of the species or to classify then in any way* He did, however, note the presence of soochlorella-bearing ciliates in certain localities.

The classification of association types set forth by Harnisch

7 has been used by other investigators since its original appearance in the literature. Hoogenraad (1935) used this classification in his study of the rhisopods fron bogs in the Netherlands, as well as those fron sphagnua ante rial sent to bin fron other localities in Europe and Anerica.

In 193b Olivier also enployed Harnisch*s classification in a study of the Testacea of sone peat bogs in the region of Mt. Dore in

France.

in 1945 Fan than and Porter nade a study of the Protozoa present on sone Canadian aosses, aaong which were twenty sphagnua sanples.

They reported 70 species of rhisopods, 32 species of flagellates, and 30 species of ciliates in the sphagnua examined. Fan than and

Porter aade comparisons of the Protosoa of European and North American sphagnua populations, and also compared the faunas of different species of sphagnua.

Studies of either Protosoa or Algae in relation to the sphagnua plant itself are rare in the literature. There are, however, a few papers in which certain species have been described either fron the

hyaline cells of the sphagnua leaf or fron its surface. The earliest

of these papers is the work of Carter (1875) who described Chlaardo- wrxa labTTinthuloides and stated that it is an endoparasitic growth

inside the hyaline cells of the sphagnua for at least a portion of

its life history. Carter's drawings show young ^h^f*|^onyxa and other

stages within these esg>ty cells. Ceddes (1882) encountered the resting

stage only in the sane location in the sphagnua leaf. Both authors

b obtained their Baterial from Westmeath County, Ireland.

Pascher (1929, 19104, 1910B, and 1940), in his studies of the

Algae in Csechosl evakia, discovered several species which he described as epiphytic and endophytic on the leaves of sphagnum. Although

Pascher*s papers do not deal specifically with Protosoa, they are important in that they illustrate a type of relationship between sphagnum and other organisms that has been neglected by other investi­ gators .

9 NATURE o f t h e environment

Location and History of the Bog

Buckeye Lake is situated in Licking, Fairfield, and Perry Counties in central Ohio. Its long axis runs fron east to west. According to

Detmers (1912) it is approximately seven and one^ight miles long, and varies in width from one— fourth mile in the eastern portion to a nile and one-half at the extreme western end, covering an estimated area of 4,200 acres.

Cranberry Island, or Cranberry Bog, is located in the eastern part of Buckeye Lake and lies close to and parallel with the north shore. It is long and irregular in shape, 1,250 feet long by 750 feet wide in its broadest part, and has an approximate area of 45 acres

(Detmers, 1912).

The whole island is a cranberry-sphagnum bog and is of particular interest because its dominant vegetation is composed of boreal species, which are relicts of early postglacial times. It is similar to bogs of more northern regions, and developed at this relatively low latitude during the Cary substage of the late Wisconsin epoch. When the glacier retreated, the bog was already present in the valley which is now the lake basin. In the 1810*s, Buckeye Lake was formed artificially as a feeder for the Ohio Canal, by forming a dike around the west end and

10 a part of the north aide of what ia now the lake basin. At this time the bog slowly broke loose from the bottom and floated to the top of the newly formed lake. Today it is held in place by loose deposits of peat and other decaying organic material, permeated by water, and extending to the bottom of the lake.

Description of the Bo

The general vegetation of this island is that of a bog meadow surrounded by a border of trees, shrubs, and taller herbs. This marginal sone is in turn surrounded by a marsh tone. The original bog meadow has at places been invaded by thickets of trees and shrubs, and by open pools surrounded by cattails. The island border is also characterised by enclosed pools, coves, and marginal lagoons.

By far the greater portion of this island is a Sphagnum-Vacciniwi

(cranberry) association, most of which forms the large, irregular bog meadow. Other species associated with the cranberry and sphagnum in the smadow are:

CalopQKon pulchellus bog orchid Sriophonw virsinicum cotton sedge Rhynchospora alba white beak rush Sarracenia purpurea pitcher plant Decodon vertlcillatus swamp loosestrife Prose ra rotund if olia sundew Dulichium oruarHnu?*— ^ Dulichium Hcnrantfaes trifoliate Virginia Buckbean Scheuchseria palustris

Present also on the bog meadow are slightly raised areas or hummocks.

The husmocks present different conditions from the bog meadow in general and thus are able to support different species of plants. As a result of their higher elevation they are characterised by better drainage.

11 In addition to better aeration due to increased drainage, there is an

increase of minerals in these hwmeocks. They are built up prinarily

by sphagnun and nay contain cltssps of ferns, shrubs, and other plants

requiring a higher drainage as well as a greater amount of mineral material. The floral composition of the hummocks varies according to their age and site.

Characteristics of the Bog

The survival of this disjunct in a deciduous forest region can

best be explained by the conditions which prevail on the island. The

high acidity, the high water content, the mineral deficiency, the short

growing season, the poor aeration, the high light intensity, and the

low temperatures are all factors which help to prevent invasion by deciduous species, and thus favor the continued existence of this bog.

Another important factor which must not be overlooked is the fact that

Buckeye lake is being continually dredged, hence preventing any filling

of the channel between the mainland and the island. All of these factors acting together give this island an environment which is unique in

central Ohio.

Temperature. Compared with the land area surrounding Buckeye Lake,

Cranberry Island is a relatively cold habitat. It has a microclimate

characterised by relatively low tesqperatures, high light intensity,

high humidity, and a short growing season. The daily variation in

temperature is very great, and the extremes of temperature are of greater

importance to the life of the bog than the means.

12 During the day, especially nid-afternoon, the ataoapheric teapera- ture of the bog aeadow is at its s u i a a , and in soae instances exceeds the ataospheric teaperature of the surrounding lake by one or two degrees centigrade. This is partly due to the large aaount of light and heat reflected and re-radiated from the bog during the day

(Diagraa 1).

Very little light or heat penetrates below the tops of the sphagnua plants because of the extrenely crowded condition of the sphagnua and also because of the high specific heat of water. Thus the teaperatures of the surface sphagnua are higher than teaperatures 5 or b inches below the surface (Table 1.). During the night the heat that was absorbed through the day is rapidly lost by re— radiation fron the sur­ face of the bog aeadow (Diagraa I). These conditions help to produce the characteristically low teaperatures. Rapid transpiration and evaporation of water froa sphagnua is still another factor which aay influence the production of low teaperatures in bogs. Table 1. Teaperature (°c .) gradient at different levels of the bog.

bate Ataoapheric Surface 6 in. down Keaarks

9/25/53 21.5 19.8 18

10/8/53 19.5 16 14 Sphagnua froxen in soae shaded spots. 10/14/53 22 18 15

11/1/53 14.5 8 6.5

2/18/54 12 8 2 4gt 4 inches; frosen below this.)

4/22/54 27 26 10

5/18/54 23 26 20.5

b/22/54 31 32 24 SOLAR RADIATION

Re-radiation

Reflection

Re-radiation Heat lost through (heat lost rapidly transpiration and frost plant surface) f evaporation

•Absorbed by i surface

Subsurface - does not heat up (very little conduction below)

WUht

Diagraa I. A siaplified diagraa of the heat ecooosQr of Cran­ berry Bog. (This diagraa is siailar to one used by J. N. Wolfe for the heat econooQr of the earth)

♦There is relatively little absorption at the surface as a result of the high specific heat of water.

15 Light, Although the open pert of the bog meadow receives maximum sunlight, the illumination of the water inpounded in the sphagnum plant diminishes very rapidly with increased depth. In fact many of the organises living at the base of the sphagnua plant and in the nuck below actually live in greatly reduced light or even darkness.

Aeration and Water Level. The general water level of the bog is constant and is Maintained by the water absorbed fron the surrounding lake. This water level, however, varies at different parts of the island, with a s tiinn amount in pools of the open meadow to a minimum in the larger humsocks and wooded areas of increased drainage, in these latter areas the sphagnum plants are not as moist as those in the meadow, hence there is produced what might be called a semi-terrestrial environment.

The water impounded in the bog is completely imsobilized. This iaom>bilisation reduces aeration to a minimum, causing a striking lack of oxygen (especially at the lower levels) and also an increased amount of carbon dioxide.

Dissolved Oxygen and Carbon Dioxide. For exact oxygen and carbon dioxide determinations specific equipment and procedures are necessary.

Special precautions must be taken to prevent gaseous exchange during the collection process. Since the bog water is impounded in the sphagnum and must be squeesed out, accurate determinations of its oxygen and carbon dioxide content are obviously impossible with present smtbods.

Therefore the determinations described below must be looked upon smrely as approximations.

lb Water obtained fron a depression created by pressing down on the sphagnua was analysed (by the Department of Health of the State of Ohio) and yielded 0.2 parts per million of oxygen and 29.5 parts per million of carbon dioxide. With respect to the oxygon content of bog water, sisdlar results were obtained by Welch (1945) in Michigan, who found that the dissolved oxygen content was usually 0. Welch, however, pointed out that in spite of the very active oxygen-consuming agencies in bog- lake mats, it is inevitable that at least a very thin surface layer of water in contact with the air nust contain some dissolved oxygen.

Furthermore, he stated that a certain amount of oxygen w s t be liberated during the photosynthetic activity of the sphagnum and other plants.

Therefore the oxygen absorbed from these two sources would furnish a supply of oxygen to the uppermost layers of water coating the sphagnum.

These conclusions are believed to apply also to the bog at Buckeye Lake.

Hydrogen Ion Concent rat ion. The hydrogen ion concentration at the various levels of the bog profile is fairly constant, with a narrow range of pH 3.b to pH 4.7 at the surface, and pH 4.9 to pH 5.9 at a depth of about eight inches. The higher pH of the subsurface is pro­ bably due to the penetration of the lake water under the island. The pH of the lake ranges between pH 7.2 and pH 7.8. At the different levels of the sphagnum plant the pH varies slightly fron a low of pH 1.2 at the apex to a high of 4.0 at the bottom. While these values differ from time to time, they always present a gradient which is lower at the top and higher at the bottom.

The acidity of the bog water is undoubtedly due to the formation

17 of tunic acids in the decomposition of the sphagmm plant. The degradap- tion of wood and other plant materials is due to the action of micro­ organisms (Erdtman, 1943). Under aerobic conditions dead plants are quickly decomposed, with carbon dioxide and water being the main pro­ ducts formed. When there is a lack of oxygen this process is slowed

considerably. In the case of sphagnua bogs, the dead sphagnum undei>- neatb decomposes far more slowly than new sphagnum grows above. Thus, with the continued growth of the sphagnum, this decaying layer is pressed down and becomes increasingly anaerobic. This in turn causes a pro­

gressive decrease in the number and activity of microorganisms and an

almost complete termination of decomposition. Hence the formation of

peat.

Structure and Growth of Sphagnum

Because of its structure and mode of growth, sphagnum affords an

excellent habitat in which various species of Protosoa can live. The

sphagnum plant continues to grow at its apex while it dies beneath.

Thus there is a continuous development of new shoots above the accuses*

lation of dead sphagnum.

The sphagnum plant, like other mosses, has a stem-like axis, usually

called the "stem1*, and leaf-like scales, usually called "leaves'*. In

cross section the stem is composed of two principal regions, the cortex

and the central axis. The cortex of the stem is casposed entirely of

hyaline cells uhich may or may not have pores and transverse ridges

similar to those of the hyaline cells of the leaves.

IB Occasionally the ites divides dichotoaously, and the two equal branches grow indefinitely. Besides these sain branches, the sten also possesses snaller branches which are of two types: 1) the short, spreading or divergent branches, and 2) the slender, drooping branches which extend downward and hug the sten. At the apex or susssit of the sten the divergent branches are extresmly short and clustered together, forming with their leaves a terminal tuft called the caoitulun.

The concavo-convex, imbricated leaves of both sten and branches are one cell layer in thickness. They closely invest the axis, form­

ing spaces through which water passes by capillarity. The cells of the leaf are of two types, one a large, clear, hyaline cell, which contains no protoplasm and represents merely the cell walls of a once

living cell; the other a long, slender, living, and chlorophyll*

bearing cell. The green cells are such snaller than the hyaline cells, and lie between the latter. (See Plate 11, figure 2.)

The hyaline cells (cell spaces) are somewhat cylindrical in forn, with tapering ends, and have narrow, ring-like ridges at intervals on

the inner surface of the cell wall. Each cell has one to several

apertures or pores through which water is readily absorbed. The pores

vary in site, shape, number, and position according to species. The

following table illustrates the leaf, cell, and pore sites of the

species used in this investigation.

19 Table 2. Leaf, Cell, and Pore Sizes

Species Size of Leaf Size of Hyal. Size of Aperture Cells

S. pa lust re 2m s . by I.Ib m . L. 12b to 17b 16 to 24 /* W. 24 to 10.4*4.

S. capillaceua 1.4m s . by 0.5m . L. 110 to IbO ^ 4.8 to 14.4/4. W. 16 to 17.6/*-

S . recurvua 1.1m s . by 0.1m s . L. 150 to 192/4. 6.4 to 11.2/t W. 16 m -

Since sphagnua has no systea of roots and no internal conducting system, all the intake of water nust be by direct absorption at the surface of stesu and leaves. The elongate hyaline cells of the sten and branches and the hyaline cells of the leaves offer a systea of reservoirs and capillaries through which water is readily conducted.

Upward movement of water is also aided by the hanging leaves and branches which are closely appressed to the stem. These leaves and branches fora an absorbing and water-conducting mantle. As a result of these relationships the entire surface of the sphagnum plant is almost continuously covered with at least a thin fila of water. This affords an ideal habitat for certain su.croscopic organisms.

20 MATERIALS AND METHODS

General

Fron April, 1951, to August, 1954, a series of collections of sphagnum was amde at Cranberry Island, a floating sphagnum bog at

Buckeye Lake in central Ohio. During this period 24 collections were made. Most of these samples were taken in the spring, stmsaer, and autumn months. Although the island is not easily accessible during the winter, several trips were taken when the weather permitted. The results of these winter collections were exceedingly valuable in determining the general condition of the protozoan population at times of very low temperatures.

Collections

Collections were made from different sites chosen according to the varying conditions which exist on the island. The variables con­ sidered in choosing collecting stations were moisture, light, and temperature. Eight collecting stations were set up on the bog. Four of these were on the bog meadow, of which two consisted of Sphagnum palustre Linn, and two of Sphagnum capiliaceum (Weiss) Schrank. These two are the principal species of Sphagnum making up the bog meadow.

Another collection, consisting of Sphagnum palustre. was made from a

21 M a l l huMM>ck located on the aeadow. This latter location was chosen because of the lower anount of noisture at this point. Two stations were chosen in soanwhat raised areas shaded by shrubs and snail trees, and these consisted entirely of Sphagnua palustre. These latter two stations are characterised by a lower light intensity, by slightly lower teaperatures, and by lesser asmunts of suisture. The final site was located in a shaded area at the edge of a thicket, just outside the bog aeadow. The species at this place was Sphagnua recunrua Beauv.

Table i. The Collecting Stations

Station Light Moisture Teap.r Sp. of Sphagnun

innr «-* 1 high Sphagnun palustre

11 MUM high Sphagnua palustre

III *-*■* ■JHI high Sphagnun capillaceun

IV M M M fr* high Sphagnun capillaceun

V «-» * high Sphagnun palustre

VI ** si. lower Sphagnun palustre

VII ** si. lower Sphagnun palustre

VIII * * * * ■ si. lower Sphagnun recurvun

*** r large anount ** - ntdiun anount * = snail anount

♦The teaperature variations were usually slight (a natter of 1 or 2 degrees C).

In order to keep the sphagnua in its natural condition special precautions were taken in the nethod of collection. The sphagnun was

22 taken up at its base and placed into a pint nason jar containing one or two inches of bog water from the unediately adjacent vicinity•

The bog water was never poured over the sphagnun. Decomposing sphagnun, or nuck, present under the growing sphagnun, was also collected fron the collecting sites. Another collecting nethod employed was that of wrapping iarge quantities of sphagnun in cheese cloth. This was used nerely for extra collections, which were employed for the original

identifications of the organissu.

The sphagnun was put into 4— inch finger bowls (extra into 9-inch

culture dishes) and placed in a north window innediately after it was

brought into the laboratory. About one inch of bog water was put into

the dishes before the sphagnun was added. After the sphagnun was

placed in the containers, the water level was narked on the outer

surface with a wax china-narking pencil. In the case of collections

kept for a period of tine this level was naintained fay adding distilled

water. The naterial was studied within a few days after collection,

but in sone cases, sphagnun cultures prepared in this way have been

kept growing for over a year.

Soaw of the naterial was also preserved in o-3-l preservative

(6 parts distilled water, 3 parts 95X alcohol, and 1 part fonalin)

for future reference.

pH Dcterminations

The pH of the bog water and the water inpounded on an*. n the

sphagnun plant was determined by several anthods. In the field the

23 the La Motte and Hellige colorimetric pH meters were employed. The

results of these readings were later checked by the Beckman glass electrode pH meter.

The pH of the bog water in general (water obtained from a depres­

sion created by pressing down on the sphagnum) was taken with the

La Motte pH smter and recorded in the field. This was done on every trip, at all the collection sites, in addition to this the sphagnum was uprooted and the pH of the water 8 to 12 inches below the surface

of the meadow was taken.

The hydrogen ion concentration of the water held by the sphagmau

plant at different levels was also checked. This was accomplished

by dividing the plant into four equal parts and gently squeexing the

water into separate small containers. The Hellige pH smter was used

for these determinations because of the small asK>unt of water involved.

The levels of the plant were designated as 1) apex, 2) upper, 1) middle,

and 4) bottom.

An attempt was also made to determine the pH of the water within

the hyaline cell spaces of the sphagnum leaf at the different levels.

For these determinations neutral red was used as an indicator, and

several leaves from different levels were observed on a slide under

the microscope. This is not a very exact method, and no differences

were discernible in any case.

Teamerature Determinations

Although a complete record of the year round temperatures was

24 not possible, the atmospheric, surface, and subsurface temperatures of the bog were taken at all the stations on the Majority of the trips.

The surface tenperature was taken by laying the thermoswter horizon­ tally in the surface sphagnum, just under the tips of the plants. For subsurface determinations the bulb of the therswea ter was pushed about b inches down into the bog. These temperatures were recorded in centi­

grade.

Methods of Examination

In order to determine the distribution of the Protozoa on the

sphagnua plant, special methods of examination had to be developed.

The position of the Protosoa on or in the leaves and stem at the

various levels was an essential consideration in the development of a successful method. The individual sphagnum plant was divided into

four levels in the same manner as for pH determinations.

After the plant had been cut into these parts, the water from

each part was gently squeezed onto a slide and examined under the

compound microscope. This procedure gave a general view of the

Protozoa at the different levels. For a more specific determination

of location, however, individual leaves from a given level were put

on a slide with a few drops of distilled water and examined. In this

manner the Protozoa living within the clear cell spaces of the sphagnum

leaf were easily seen. For a more accurate structural study of the

organisms it was necessary to squeeze them free from the leaf. This

was readily accomplished by squeezing them out, as described above.

25 The u t u n l position of i o o b of the organisms, specifically the

Testacea, on the surface of the plant presented a more difficult pro­ blem. This was resolved by placing an individual plant under the stereoscopic microscope and teasing the leaves and stems apart with a dissecting needle. Using this procedure, however, identification was rather difficult and only the larger forms were readily seen.

Staining Methods

For the most part, and as far as possible, living material was studied, especially for the distributional studies. However, for enact identification it was often necessary to use special staining and find­ ing techniques. For general observations the vital stains neutral red (Hartmaa-Loddon Co., 1:75,000 and 1:100,000) and methylene blue

(National Aniline Co., 1:100,000) were often employed to give overall contrast. For exact identification lethal stains and fixatives were sosmtimes necessary. The following techniques proved to be the most satisfactory.

Temporary preparations. 1) A 0.2% solution of methyl green in

L% acetic acid was used as a nuclear stain for colorless forms. The stain was added at one side of the coverglass and then observed as it diffused under, killing the organisms that it reached and staining the nuclei.

2) Lugol's solution (4 grasm iodine, b grams potassium iodide,

100 ml. distilled water) was used extensively to demonstrate flagella, cilia, and the presence of starch. This stain was also helpful in

2b observing nuclei. For best results the stock solution was diluted,

1 part to b parts of distilled water.

3) A 2% aqueous solution of osmium tetroxide (osmic acid) was used to kill and fix the organism for pensanent and temporary prepara­ tions. It was used both in the fans of vapors and in solution. When vapors were used, a drop of mediim was placed on a slide in a petri dish containing a depression slide with several drops of osmic acid solution. Vapors were also used by inverting a slide with a drop of the medium on it over the mouth of a bottle containing the osmic acid solution. Osmic acid killed the organism rapidly and was valuable in preserving the flagella in the case of many of the flagellate form.

A modification of Wager's osmic acid technique (1899) was extreme­ ly useful in demonstrating ciliary patterns and showing the basal granules of both cilia and flagella. This technique involved the impregnation of the organism with osmic acid. The impregnation was accosq>lished gradually by adding a drop of 2% osmic acid to several drops of medium on the slide. A covers!ip ringed with vaseline was then placed over the medium to prevent evaporation and the escape of toxic vapors. The slide was then observed under the sdcroscope at various intervals. Ciliary patterns were well defined after two or three hours. These slides could be kept for at least 46 hours without complete blackening of the organism.

Permanent preparations. The most useful permanent slides were those stained with Heidenhain's iroo haematoxyiin or Delafield's haematoxylin as nuclear stains and eosin as a cytoplasmic counterstain.

27 For such propartions the parlodion trap Method as developed by

Coocannon (1951) proved to be very useful for the handling of Protoxoa preparatory to staining and nounting. The parlodion solution was prepared by dissolving grams of parlodion (highly purefied cellulose nitrate) in 100 ml. of absolute alcohol and 100 ml. of ether. The solution was kept under refrigeration.

The trap is prepared by wrapping a three inch piece of lacquer coated wire (about § 30 gauge) once around a piece of glass tubing, and twisting the free ends around each other to form a handle. To decrease the probability of rupturing the parlodion film, the trap should not exceed 10 am. in diameter.

Two methods were used in preparing the Protosoa for imbedding in parlodion. The first was to fix the organisms in 2% esmic acid before placing them on a slide; the second was to put a drop of medium containing the living organissn on a clean slide and then fixing them with absolute alcohol. This latter method was employed by Pappas

(1952) and Ritter (1953) in cytochemical work on amebas and heliosoans

respectively. The first method had an advantage over the second

because of the rapid killing action of osmic acid, which usually kept

the flagella and cilia intact.

The procedure is as follows. k drop of medium containing the

organisms (living or fixed) is placed on a slide and allowed to stand

until only a thin film remains, k drop of absolute alcohol is then

added to the middle of the film. This forces the excess water to the

periphery and also fixes the organisms if they have not already been

28 fixed. In aoat cases seme of the orgisisss are lost when absolute alcohol is added, but not enough of then to destroy the effectiveness of this net hod.

After nost of the alcohol has evaporated, the trap is placed on the slide with the loop surrounding as many individuals as possible and with the handle extending lengthwise of the slide. One or two drops of parlodion are then added at the center of the loop. Care should be taken not to add too mieh, since an excess of parlodion would nake the film too thick. When the parlodion is nearly dry

(about one minute) the slide is tipped to one side and the excess parlodion is drained down the handle of the trap. Thus a very thin layer remains within the loop.

The slide is then flooded with water while it is still in the horizontal position. After the water has penetrated between the slide and the parlodion, the trap may be removed (without disturbing the film) by gently lifting it off the slide with a pair of forceps.

Shallow reagent and staining dishes are used in subsequent steps.

The procedure that follows is similar to the regular methods alloyed in microtechnical work with a few exceptions. Since parlodion is soluble in absolute alcohol, the final dehydration is effected by using carbol-xyiol (25 ml. melted carbolic acid crystals added to

75 ml. of xylene). The trap is left in this solution until it is completely transparent. After complete dehydration, the trap is cleared in xylene for several sdnutes and placed on a clean slide.

The parlodion membrane is then freed from the loop with a sharp

29 dissecting needle, end Mounted in Canada balsam on the slide, under a No. 0 coverslip.

Rosenbaum* s (1948) negative staining technique with nigrosin was useful in certain cases as a rapid aethod for the deaonstration of ciliary bands, vacuoles, and cytoplasadc inclusions.

Quantitative Peterainations

Since the present study is primarily concerned with the relative numbers of different organisms at various locations on the sphagnua plant, a system was devised in which actual counts were made and then represented by the words rare, few, abundant, and numerous. These terms were given the following values, and in the tables are represen­ ted by one to four asterisks, as shown below:

Maatigophora and Ciliophora

Rare (*) 0 to 25 organisms on a given slide Few (**) 25 to 50 organisms on a given slide Abundant (***) 50 to 100 organisms on a given slide Numerous (****) 100 or over organisms on a given slide

Rhizopodd

Rare (*) 0 to 5 organisms on a given slide Few (**) 5 to 10 organisms on a given slide Abundant (***) 10 to 20 organisms on a given slide Numerous (****) 20 or over organisms on a given slide

In devising a amthod for the quantitative enumeration of the

Protozoa of sphagnum, certain limitations become evident. In the first place not every organism can be squeezed from the sphagnum plant.

Many remain in the hyaline ce'ls, while a few others undoubtedly coin* tiaue to adhere to the outer surface of the leaves. Secondly, if one

iO uses an intact plant under a stereoscopic or regular microscope, a complete count of all organisms, and especially the snaller forms, is optically impossible, for obvious reasons. Therefore, it is important to esg>hasise that the counts are only relative and not exact quanti­ tative determinations. However, even with such limitations, it has been possible to arrive at definite conclusions concerning the distri­

bution of the Protozoa on the sphagnua plant.

11 OBSERVATIONS

General

As already stated, three species of sphagnum were represented at the eight collecting stations, but only one species was present at any one station. Sphagnum palustre was present at five stations,

Sphagnum capillaceusi at two, and sphaIT*11- recurv\m at one. With one exception (the Sphagnum recurvum station), the various stations dis­ closed a fauna that was essentially sisular, but differed somewhat in the frequency of the individual species, in addition there appeared at all the stations a definite distribution of species on the indivi­ dual sphagnum plant, with certain species being characteristic of the different levels.

Distribution and Relative Frequency of the Protosoa

Stations 1 and II will be discussed jointly because of the great similarity between the two. Both stations were in the bog meadow and the sphagnum at both consisted entirely of Sphagnum palustre. The conditions prevailing at these two places included a high degree of suisture and -!-•»— surface illimination. The light intensity below the surface, however, is greatly reduced because of the crowded manner in which sphagnum grows on the bog meadow. The micro-fauna from these two stations was richer than that from any other site visited during this investigation. As Tabie 4 indicates, a large number of forms was found at all levels of the plant, with different species characteristic of different levels. At the apex of the plant (the capitulum), species possessing chloropiasts or symbiotic xoochiorellae dosunated. This was illustrated by the high incidence of Carteria sp., Chlorogonium sp., Heleopera picta.

Kyalosphenia papilio. and the green ciliates. (See Table 4.)

While these forms appeared in greater numbers at the apex, their distribution was not limited to this area, but was extended to the lover levels of the plant. At these lower levels, however, their frequency progressively decreased, and at the lowest levels, where they were few in number, their structure was characterised by shrunken chloropiasts or a decrease in number of xoochiorellae. This was strikingly illustrated accasionally by the complete absence of xoochiorellae in Trichopelma sp. at the extreme bottom.

The colorless forms appeared farther down on the plant and pre­ sented a relationship which might be referred to as the exact opposite of that presented by the colored fores. This was illustrated fay the distribution of Sphenoderia lenta and Opisthotricha sphagni. colorless forms which reached their maximns at the lower levels. An exception to this, however, was the consistent occurrence of Motosolenus sp. at all levels, and the higher incidence of hebela collaria during the autimm months at the apex and upper level. Menoidiim incurvum and ni«»ip m gracilis, on the other hand, consistently remained at the

11 bottom mud m r e never seen mt the higher levels.

Stmt ions III mnd IV possessed m sisdlar fauna mnd will be dis­

cussed together. These stmt ions were mlso from the bog meadow, but

the sphagnua plmnts were mil Sphagnum cmpillmceun. The general condi­

tions mt these two stmtions were the smae ms those described for

stmtions I mnd II.

Inspection of Tmble 5 shows thmt stmtions III mnd IV supported

the same fones ms those found mt stmtions I mnd II, mnd thmt their

distribution wms essentially the sue, despite the difference in the

species of sphagnum. The only difference between the fmunms of these

two species of sphmgnun found in the snadow wms in the totml nun be rs

of the orgmnisas on the pimnt. Sphagnum palustre uniformly showed

sonewhmt gremter numbers then did Sphagnum capiilaceun. This

difference smy be mttributed to the manlier, more delicmte structural

nmture of Sphagnum cap Hi m c cun. A few of the less abundant species,

however, were about equally represented an the two species of sphmgnun.

At Station V on m hummock in the bog meadow only Sphagnum pmiustro wms represented. At this place the sphmgnun grows higher than on the

aemdow proper. Correlated with this elevation is the better drainage

mnd aeration characteristic of these raised areas. As m result of

this higher elevation mnd increased drainage, there is a reduction in

the mswunt of water coating the surface of the plant. However, this

decrease in moisture is not great enough to destroy the aquatic neditai

of the hyaline cell spaces and of the spaces between the imbricated

leaves.

14 Comparing station V (Table b) with the first four stations

(Tables 4 and 5) it becosMS evident that the number of individuals of the larger testaceans at this station was definitely smaller. This was especially true of Hralosphenia papilio. Krai« eleaans. Heleopera picta. and Bunlypha ciliata. On the other hand, the Mastigophora were as abundant as at the other stations. The lesser nunbers of the larger forms may be explained by a reduction in the thickness of the water layer coating the plant. Such reduction in moisture may reduce the amount of space with sufficient water for the support of the

larger organisms.

Stations VI and VII. at which only Sphaggufi palustre was repre­

sented, were located at the outermost part of a thicket. They were characterised by lower moisture and lower light intensity.

The fauna at these stations was again qualitatively sisuiar to

that of the other locations, and differed only in mashers of individuals

of the various species. A cosq»arisoo of Table 7 (stations VI and VII) with the other tables shows a progressive decrease in numbers of

individuals of the Testacea from stations 1 and 11 to stations VI and

VII. Most striking at stations VI and Vll was the nearly coaq»lete

absence of the Testacea from the apex and their reduced numbers through­

out the entire plant. Again in this case the decrease in frequency

of these larger organisms may be attributed to the decrease in sraisture

content.

Station VIII. located in a shaded area at the edge of a thicket,

presented an entirely different set of conditions than those of any

15 of the other collection sites. The station was essentially a snail, shallow pool with completely submerged, but normal, Sphaamas recurvum growing over its entire bottom. Obviously the sphagnua plants at this station were saturated with Moisture. The light intensity was low.

The sphagnua at this location yielded fewer Protosoa than that at any other place visited on the bog. Instead, every plant disclosed large quantities of the blue green alga, Anaboena sp., which was most abundant at the apex, but was also present at ail other levels.

With the exception of Arcelia vulgaris and Euxlrpha ciliata. the Testacea were usually absent. Carter in sp., Chlorogonium sp., and Eufllena mu tab 11 is were the only Mastigophora present on the uppermost portion of the plant, but they were auch less niaerous than at all the other stations. At the bottom level, however, Henoidium incurvum and Pistigma gracilis presented maximum growth, with

M. incurvum being the dominant species.

Jb Table 4. Si— a nr of Organ is— at Stations _I and 11.

Frequency of organisms at;

Apex Upper Middle Bot

Mastigophora Carteria sp. JHHHI ■* M. M M ** * Chlorogonium sp. *** ** tt * Notosolenus sp. ** *■* * Euglena mutabilis * * » Carteria klebsii * ■» Henoidium incurvum ** Distigma gracilis **

Sarcodina Heleopera picta *** * Hyalosphenia papilio ** * Hyalos. elegans »*# *+ » mt. Mt mm Nebela collaris fitw * MM.** Sphenoderia lenta * II m m V* Euglypha ciliata » IHk ** ▲.reella vulgaris * ** * * Buglypha alveolata * * * Nebela dentistoma * *# * ▲ssulina seminulun »* Heleopera petricola #

Ciliophora Green ciliate 1 *** * Green ciliate 11 *-* ** Trichopelma sp. ** * # Opisthotricha sphagni * ** Colpoda sp. » *

Anaboena sp. * * Cylindrocystis sp. * ** Diatoms

Metasoa Rotifera Philodina sp. ** ** ++ ** Rotifer sp. ** ** +* ** Castrotricha Chaetonotus sp. * Water mites » * * * Round worms * fr» »*

17 Table 5. Su— arr of Foraa in Stationa 111 and IV*

Frequency of organiana at;

Apex Upper Middle Bottoa

Maatiaophora Carteria ap. Chlorogoniua ap. Notoaolenua ap. Buglena nutabilia * * Carteria klebaii * * Menoidiua incunrua * Biatigna gracilia *

Sarcodina Heleopera picta Hyaloaphenia papilio Hyaloa. elegana * Nebela collaria * Sphenoderia lenta * # * * Buglypha ciliata * * * * Arcella rulgaria found only occaaionally Euglypha alveolata * * Nebela dentiatoaa t • Aaaulina aeaiaultai Heleopera petricola

Ciliophora Green ciliate £ » Green ciliate II » Trichopelna ap. » Opiathotricha aphagni Colpoda ap.

Algae Anaboena ap. * Cylindrocyatia ap, * Diat<

Hetaaoa Rotifera Philodina ap. Rotifera ap. Gaatretricfaa Chaetonotua ap, Water mitea Round wonaa

IB Table 6. S unwary of Foma in Station V.

Frequency of organiana at:

Apex Upper Middle Bottea

Heat Uo p h ore Carteria ap* » Chlorogonlun ap. * * Notoaolenua ap. * Euglena nutabilia * * Carteria kleball * * Menoidiun incurvun » Diatigpa gracilia *

Sarcodina Heleopera picta * Hyaloaphenia papilio * Hyaloa. elegana * Nebela collaria » Sphenoderla lenta Euglypha c11lata Arcella Tulgaria Euglypha alveolate * Nebela dentiatoaa * Aaauliaa aeninulua Heleopera petricola

Ciliophora Green ciliate ,1 Green ciliate II Trichopelaa ap. Opiathotricha aphagni Colpoda ap.

Algae Aaaboena ap. Cylindrocyatla ap. Diatoaa

Hetaaea Rotifera Philodiaa ap. Rotifer ap. Oastrotricha Chaetonotua ap. Water nltea Round worna

19 Table 7. 5i try of Forna Found in Stationa VI and VII

Frequency of organiiu at:

Apex Upper Middle Botto*

Haatjgophora Carteria ap. * Chlorogoniun ap. * Notoaolenua ap. Euglena nutabilia * * Carteria klebaii * * Menoidiun incunrun * Distigna gracilia *

Sarcodina Heleopera picta Hyaloaphenia papilio Hyaloa. elegana * * Nebela collaria * * Sphenoderia lenta * Euglypha ciliata * Arcella vulgaris Euglypha alveolate # Nebela dentiatoant * Aaaulina seaunwlun Heleopera petricola

Ciliophora Green ciliate 1 * Green ciliate II * Trichopelna ap. * * Opiathotricha aphagni * * Colpoda ap. »

Anaboena ap. Cylindcecyatia ap. Diati

Metaaoa Rotifera Philodina ap. Rotifera ap. Gaatrotricha Chaetonotua ap. * Water nitea # Round worms *

40 Table b. Sunwry of F o m a in Station Vlll

Frequency of organises at:

Apex Upper Middle Bottom

Mastigophora Carteria sp. Chiorogonium sp. Notoaolenua sp. Euglena mutabilis Carteria kiebaii Menoidiun incurvun Diatigma gracilia

Sarcodina Heleopera picta Hyalosphenia papilio Hyaloa. elegans. Nebela collaris Sphenoderia lenta Euglypha ciliata Arcella vulgaris Euglypha alveoiata Nebela dentistona Aasuiina aeninuiun Heleopera petricola

Ciliophora Green ciliate J_ Green ciliate 11 Trichopelna ap. Opisthotricha sphagni Colpoda ap.

Anaboena sp. Cylindrocyatis sp. * ** * Diatoms ****** *

Metaaoa Kotifera Philodina sp. Rotifer sp. Cast rot richa Chaetonotus sp. Water mites Round worms

41 IB addition to the f o n t already diacuaaed, the following table containa apeciea which alao occurred occaaionally at varioua levela on the aphagnw plant.

Table 9. Su— ary of Organiana.

Frequency of organiaaa at:

Apex Upper Middle Bottoa

Haatiaophora Chiloomnaa panuneciua * * Cryptoaonaa orata * * Trichloria ap. *

Sarcodina Acanthocyatia ap. * Actinophrya aol * Anoeba guttula Anoeba ap. * Centropyicia ap. * * Cyphoderia ap. * Difflugia loboatoaa * * Nebela flabellulint * Trinena enchelya *

Ciliophora Vorticella ap. «

41 The Poaitioo of the Organisms on and in the Sphagnum Plant

One of the aost «triking relationship* revealed by this study is the spatial relationship of the sticro-fauaa to the individual sphagnum plant. The various species represented are not only adapted to the general bog environment, but also to the structural units or ecological niches presented by the plant. Many of the smaller forsi actually live and nultiply within the hyaline cells of the leaves and steal. (See Plate 111.) This phenoamnoa occurs consistently and has been observed repeatedly during this investigation. The larger organises (Heleopera. Hvalosphenia. Hebela. rotifers, etc.) live and crawl around on the surfaces of the leaves and steal and within the tiny water pockets formed between the imbricated leaves.

At the apex, upper, and middle levels, Carteria sp., Carteria klebsii. Chloroaoniiss ip.r Notosolenua sp.. Euxlena nutabilia. green ciliates 1 and 11, Trlchopelma sp., and round worms have all been observed within the hyaline ceils of the leaves. Within the hyaline cells of the stem, however, only Carteria sp., Chloroconiii sp.,

Motosolenus sp., and round worms were seen. The number of individuals in a given cell is not necessarily constant. At the apex the number is usually greater and in some instances a single hyaline cell may contain as many as ten individuals of Carteria sp. plus several of

Notoaolenua sp. and Chloroxoniun.

In addition to their presence in the hyaline cells, many of the forms naamd above also occur in the surface film of water coating the

41 sphagnum plant. An exception to this in groan ciliate 1. (See Flat*

IV.) This organism always remains within the hyaline ceils, where it moves around with an unusual amount of agility and exhibits a high degree of flexibility. If the leaf is broken or the organism has been squeesed out of the hyaline cells, it becomes fairly rigid and mores very slowly or not at all. Trichopelma sp., on the other hand, swims quite rapidly and only occasionally lives within the ceil spaces.

This undoubtedly is due to the larger sise of this ciliate.

Sosw of the members of the bottom fauna also live within the hyaline cells in that region, but never in large numbers. While

Menoidiun incurvum. Chiloswnas paramsciiss. Pastime gracilis, and round worms do occur in the hyaline cells occasionally, they usually live at the surface of the base of the dying plant.

Ciliates without symbiotic algae reach their maximum numbers at the lower levels of the sphagmss profile, but are never a dominant group. Moreover, they are not found within the hyaline cell spaces.

The species most frequently encountered at the bottom level are

Qpisthotricha snhasni and Colnoda sp.

The larger forms - Testacea, rotifers, and water mites - live

crawl arotmd on the surfaces of the sphagnim leaf and stem.

Htaloaphenia papilio. Hvalos. slogans. Heleopera picta* Mebela collaris.

Neb, dentistoma. A-cella vulgaris. Sphenoderia lenta. Euglypha ciliata.

Eugly. alveolate. and Assulina seminulum are the Testacea inhabiting these locations. At least some of these species occur at every level.

Similarly, rotifers and water mites also live on these surfaces, but always in smaller numbers. Seasonal Observations

The general micro-populat ion of the bog persists throughout the entire year and exhibits an unusual tolerance to low temperatures.

However, certain forms reach their maximum numbers during different seasons and present what might be tensed a seasonal doaunance. (See

Table 10.)

Aawng the Mastigophora, Carteria sp. and Chlorogonium sp. are always present in great numbers either in the active or the passive stage. During the winter the great majority are passive, most of then within the hyaline cells; but they become active about thirty-six hours after they are brought into the laboratory. Notosolenus sp., on the other hand, has never been seen in a passive condition and appears to be able to survive low temperatures in its normal, active

state.

Of ail the Testacea, Heleopera Dicta is the dominant species

during most of the year, and may be considered the most hardy rhisopod

inhabiting the bog. In late sumaer, autumn, winter, and early spring

this form is always in the passive, encysted condition, and it becosms

active only during late spring and early susmmr. The numbers of this

species, while uniformly high throughout the year, increase slightly

during July and August. Hvalosphenia napilio also increases in

number during the simrnmr months, but never attains the frequency of

Heleopera picta.

Sphenoderia lenta and Euslvpha ciliata remain active throughout

the low temperature of winter, and are the dominant active Testacea

45 during the winter non the. Furthermore, Sphcn. lenta. which nornally reaches its greatest musbers at the lower levels of the plant, and is relatively rare at the apex, exhibits a sonewhat increased frequency at the apex during the winter.

Hralosphenia eleaans and He be la collaris reach their n i s w in late susnmr and autunn, with Hebe la collaris presenting a decided dominance. Arcella vulgaris. on the other hand, never appears as a dominant fom, even though it persists throughout the entire year.

The highest incidence of this latter species occurs during the late

Green ciliate 1 is always present in the hyaline cell spaces, either in the active or in the passive, encysted stage. During the autunn and winter this species lives in a spherical, transparent cyst and nay often be seen noving within cyst wall. (See

Plate 111.) In the spring, as tne active forn, its novenents within the hyaline cells are lively and almost constant.

4b Table 10. Sunaary of Seasonal Occurrence of Certain Species

Spring S u ~ * r Autusn Winter

M M U MU H Carteria sp. i n r w w **** JV H ■

M M 1M M ChlorogonitsB sp. W W m w JHHW *** ***

a j t A Heleopera picta s a dh + + * + n u n **

Hyalosphenia papilio * *-* «-*

Hyalosphenia elegans * **

Nebela collaris • V* *•*

Sphenoderia lenta ** * ** S S R

Euglypha ciliata ** *

Green ciliate 1 * ■ * * *

TrichopeJjaa sp. V

For the purposes of this investigation, Spring was defined as the Months of March, April, and May; Sinner as June, July, August;

A u t w n as September, October, November; and Winter as Decenber,

January, February.

47 Association!

It is clear from the facts brought out above that the species of Protozoa living on and in the sphagnum plant nay be divided into tvo main associations: (1) those living nearer the top of the plant, which are principally (though not entirely) green flagellates, rhixo- pods containing xoochiorellae, and ciliates containing xoochiorellae; and (2) those living nearer the bottom, which are principally color­ less forms belonging to the sane three groups of Protozoa. It is obvious that the relative distribution of these two associations is correlated with the amount of light in these two general regions of the plant.

The green flagellates, Carteria sp. and Chloroaonium sp. are always the dominant forms at the apex of the sphagnum plant and are the most numerous protozoan species on the bog. Next most numerous as a group are the shell-bearing rhixopods with Heleopera picta,

Hyalosphenia papilio. Hvalos. elegans. and Nebela collaris comprising the major part of the rhisopod population at the higher levels. The rhixopods found below (at the middle and bottom levels) are fewer in number and, with the exception of Sphenoderia lenta and Buglypha ciliata. constitute a relatively small percentage of the over-all population. Nevertheless, all of these bottom species are nearly always present.

4b The following lists show the species of Protozoa in these two associations:

Association

Doetinant species Secondary species

Carteria sp. ♦Notosolenus sp. Chlorogonium sp. fiuglena nutabilis Heieopera picta Carteria klebsii Hyalosphenia papilio Green ciliate II *Hyalos. elegaas ♦Trichopelma ep. ♦Nebela coliaris Green ciliate 1

Bottom Association

♦Sphenoderia lenta ♦Arcella vulgaris ♦Euglypha ciliata ♦Kuglypha alveolata Menoidium incurvim Nebela dentistaaa Distigma gracilis Assulina seuinulum ♦Opisthotricha sphagni Heleopera petricoia Colpoda sp.

A few of these species, (those narked with an asterisk) are often

found with the other association, but always in definitely snaller numbers.

With these various observations in nind, the following names are

proposed for the two main associations of Protozoa present on and in

the sphagnum plant: (1) The photophilic association, living nearer

the top of the plant, sost of whose members possess chlorophyll

directly or indirectly; and (2) the non-photophilic association,

living near the bottom, whose members, with rare exception, do not

possess chlorophyll. (See Plate 1.)

49 DISCUSSION

Significance of Associations at Different Levels of the Plant

All the general characteristics of the Cranberry Island bog help to produce the envirotssent in which the over-all nicrofauna lives and

Maintains itself. However, the existence of the two wain associations that have been described strongly indicates that sesw of the factors exert a greater influence than others on the survival, Multiplication, and distribution of specific forsm. Illustrating this point is the consistently high frequency of green forms in the uppermost portion of the plant.

Influence of Light. With the exception of Mebela collaris. all the forms which occur abundantly at the apex of the plant either possess chlorophyll or contain soochlorellae with chlorophyll, and reach opt imam growth only under conditions of high illumination.

There appears to be a direct correlation between the amount of light and their distribution. As the light which penetrates the lower levels of the plant decreases, the relative nus&er of green forms becomes progressively less.

While the chlorophyll-bearing species possess an undoubted advantage at the upper, illuminated levels, they would have no advantage and possibly be at a disadvantage in the lower, uailluminated region. Here the advantage would lie with the colorless forms, and

50 especially so because of the presence of More mseerous bacteria and considerable bacterial decomposition at these lower levels, conditions which would favor saproxoic and snaller phagotrophic forms. This would appear to be true in general, even though most of these bottom dwelling species are capable of living at the top and occasionally do so.

Influence of Oxygon. While the bog under the surface is characterised fay a low oxygen content, it is obvious that this con­ dition does not necessarily apply to the upper levels of the sphagnum plant. Here the water film at the surface of the plant must possess

some dissolved oxygen derived by direct absorption from the air and as the end product of the photosynthetic process. However, it is

safe to assisne that the organisms living at the lower levels must

have an unusual ability to persist under low concentrations of dis­

solved oxygen* Moreover, at the very bottom, the oxygen is almost

completely absent. Hence, the survival of species at this level

strongly indicates an ability to exist under nearly anearobic con­

ditions. This is particularly true of Menoidiiwi incurvum and Pistigma

gracilis, which consistently live at the extreme bottom of the plant

and in the muck below. Pistigma gracilis has even been observed in

the muck as low as twelve inches below the base of the sphagnum plant.

Moisture Content. According to Penard (1909) and Harnisch (1919),

moisture is a factor influencing the occurrence and abundance of

certain Testacea found in sphagntmi. Penard made this statement con­

cerning the distribution of rhisopods in various mosses, while Harnisch

51 correlated moisture with the occurreace of particular specie* in sphagnum. This conclusion is confirmed by the results of the present investigation. The rhisopods were more abundant at stations 1, 11,

111, and IV, having a higher moisture content, and less abundant at stations V, VI, and VII, having a lower moisture content.

Food Relationships. Food supply and food associations are also important factors affecting the maintenance and distribution of species on the sphagnum plant. As stated above, the species possess­ ing chlorophyll have an added advantage in that they are able to pere­ form photosynthesis. In this way, Carteria. Chloroxonium. and Sunlena nutabilis are largely independent of other microorganisms * Holeopora picta. Hvalosphenia papilio. green ciliated 1 and II, and have a similar advantage in that they possess symbiotic soochlorellae which perform photosynthesis. This advantage is specifically illustrated in the survival of Heleopera picta in the encysted condition for great periods of time. In spite of the fact that it does not in­ gest solid food during the long-continued encysted stage, this species appears to be very hardy and is one of the deminent species on the sphagnimi.

The food relationship that exists between holosoic and holophytic foras is readily illustrated by observing the contents of the food vacuoles of many of the Testacea. We be la . tualrpha. Arctlla. and

Hyalosphenia have been seen with food vacuoles containing Carteria and Chlorosonium. Moreover, on several occasions Mebe la collar!* has been observed with a pseudopodiun extended through the aperture

52 Into & hyaline coll of the leaf where groan organism wars present.

No ingestion was seen, but it seems highly probable that it nay occur under these circiasstances.

The bacterial distribution on the sphagnun plant is in accent with what one night expect in an envirouaent of this type. Bacterial counts, aade by the Depart went of Health of the State of Ohio, show an increased ntaeber of bacteria at the botton levels of the sphagnun profile. The exact counts wore: 140,000 per ml. for the upper and niddle (living) portion of the plant and 240,000 per nl. for the lower, deconposing portion.

Correlated with this bacterial distribution is the occurrence of bacterial feeders and saprosoic forms at the bottom of the plant.

Monoidiun incurvua and niatlifri gracilis. both saprosoic forms, and

Sphenoderia lenta. Onisthotricha snhasni. and Colnoda sp., all bacten- ial feeders, occur consistently at the lower levels of the plant.

Cannotition. In the previous discussions, the effects of corn- petit ion anong the various groups of Protosoa are illustrated by their distribution on the sphagnms plant. Light, oxygen, and food are the chief factors influencing the survival or dominance of certain forms in any given space on the plant. Hence, as any one of these factors changes there is a direct reaction of the nicrofauna with respect to specific requirenents of a given group. Thus, the photophilic forms have an advantage at the upper portion of the plant, since their nutrition is directly aided by the presence of light. On the other hand, the soo-photophilic organisms have an advantage at the lower

51 levels because of the more numerous bacteria and increased bacterial decomposition in this region.

The symbiotic relationship resulting fresi the presence of zoochlorellae in sosw of the rhisopods and ciliates is of great interest in that these groups, in their usual form, without symbionts, would not be considered photophilic. The possession of these symbionts not only aids in the nutrition of these fonns, but also contributes additional oxygen as a result of photosynthesis. In this way Heleopera picta. Hralosphenia papilio. green ciliate 1, etc. may be able to compete and survive in an area dosunated by forms which possess a hoiophytic type of nutrition.

Succession

In a general ecological study of the Protozoa of sphagmas the question of succession arises. Does the linear distribution of protosoan species on the sphagnum plant illustrate a succession? In an attempt to answer this question it is necessary to emphasize the fact that the general environment is a dynamic one, and that the mode of growth of sphagmm autoantically creates a continuous change in the physical and chemical factors at the various levels of the plant profile. Hence, as long as the apex of the plant is growing the general conditions along the length of the plant are constantly changing, so that what was the middle level become a new bottom level, and the former upper level becosms the middle level. Therefore, the organisms present at the different levels must either move up with the growth of the plant or remsin behind and eventually die,

54 in this mnner, the xeaenl succession of the Frotoioa on the

sphagnum plant occurs in the following sequence: First, Carteria sp. and Chlorogonium sp. and sometiMs a few individuals of Buglena autabilis appear in the new growth at the top. This fact is particu­

larly evident in sphagnum cultures kept in the laboratory, where, although the general environaental conditions have been changed, the young branches and leaves are literally awarning with these two organ­

ises . As the plant continues to grow and the nuabcr of branches and

leaves increases, Heleopera picta and Hvalosphenia papilio. both of which contain soochlorellae, begin to coae in. Following this, the

continued growth of the uppermost portion eventually shades the part

iMediately below and Testacea without soochlorellae, such as We be la

collaris and Hvalosphenia elegant, tend to replace the two soochlorella—

bearing Testacea that were first on the scene. In the same way, the

other green forma move up on the plant, and their places gradually

taken by colorless forms. Furthermore, in that part of the plant

which continues to deccaq>ote and is pushed farther down into the bog,

the number of living organisms continues to decrease and eventually

only esg>ty tests and corpses remain. Hence, the continuous change

brought about by the growth of the spagnum plant creates a sequence

or succession of organisms along the entire length of the plant.

55 CowHtriioB of the Protoxoa of Crtnbtrrr Bog with Thoic Found in Sphagnun Bogs by Other Investigators

An ovei^-all comparison of protoxoan species found by other investigators shows that certain rhixopods of the order Testacaa are characteristic of sphagnun bogs. These forns have been listed by

Harnisch (1925, 1929) in two groups:

(1) Conditionally sphagnoohilic species. Those species which favor sphagnun, but are also found in other places.

Arcella vulgaris Difflugia arcula A. arenaria Nebela bohemia Assulina seminulun ♦Neb. tubulosa Ass. nuscorun Neb. ninor ♦Nebela collar is Neb. lagenifomis ♦Neb. anericana Difflugia constricts Heieopera petricola Diffl. pyrifomis v. lacustris ♦Hel. rosea Diffl. pyr. v. bryophila Hel. sylvatica Diffl. lucida ♦tjuadrula synnetrica Diff. globulosa Corythion dubiun •Centropyxis aculeata v. discoides *Cor. pulchellun Centr. laevigata Euglypha alveolate Centr. arcelloides Eugl. ciliata ♦Lequereusia spiralis ♦Eugl. strigosa L. nodesta Eugl. laevis ♦Placocystis spinosa ♦Eugl. filifera ♦Plac. jurassica Sphenoderia fissirostris Phasg»hagus nutabills Sph. lenta Trinena enchelys Phryganella hesusphaerica ♦Tri. cosq>lanatun •Cyphoderia asgmlla Tri. linesre Corycia flava

(2) Purely aphacnophilic species. Those forns which are bound to sphagnun and arc found in no other place.

Arcella artocrea Neb. crenulata A. discoides Heieopera picta Hyalosphenia papilio Euglypha cristata Hyaios. eiegaas Eugl. coeq>ressa Nebela carinata Difflugia bacillifera Neb. galeata Anphitrena flavun Neb. nilitaris Anphitrena stenostena Neb. tens11a Anph. wrightianun

•Species considered by sons authors to be purely sphsgnophilic.

5b With the exception of Difflugia lobostoma ail of the species of

shell-bearing rhixopods found on Cranberry Island appear in one or the other of these two lists. Moreover, the dominant species encountered during this investigation are aswng those considered by Harnisch to

be purely sphagnophilic.

A study of the literature of the testacean rhixopods encountered

in sphagnum shows that even though certain forms may be considered

purely sphagnophilic, they do not necessarily appear in eveiy stand

of sphagnum. Some forms, such as Amphitresm flavum and Anph. wrightianum. are often completely absent. This was true of the

Cranberry Island bog during the present study and it mss also true

in the Canadian investigation of Fantham and Porter (1945).

Using the classification set forth by Harnisch, the rhisopod

population of the bog at Buckeye Lake has been found to be more

sisdlar to the HTaiosphenia type than either of the other two association types designated by him. Oar rhisopod population, how­

ever, differs from the Hyalosphenia association of Harnisch in that

Heieopera picta is the dominant rhisopod species.

57 bUMMAKT

1. A study has been Bade of the ecology of Protozoa asso­ ciated with the sphagnun plant in an acid bog on Cranberry Island at

Buckeye Lake in central Ohio. Collections were made from April, 1951 to August, 1954, and 24 collections were made xn all.

2. Eight collecting stations representing three different species of sphagnum were visited on each trip. The species were Sphagnum pa lustre at five stations, Sphagnum capillacttss at two, and Sphagnum recurvum at one.

3. With the exception of that from Sphagnum recurvum. the faunas from the various stations were fairly consistent qualitatively, and differed from station to station chiefly in the frequency of the individual species.

4. As a result of its structure and mode of growth, the sphagnum plant furnishes an aquatic habitat in which many species of Protozoa live. Twenty species of Sarcodina, ten species of Hastigophora, and six species of Ciiiophora were recorded from the water impounded on and in the sphagnum plant.

5. To study the distribution of the Protozoa on the individual sphagnum plant, the latter was cut into four sections, bottom, middle, upper, and apex, and observations were made on the numbers and postion of all the species present on these several parts of the plant.

5b Different species were found to be characteristic at different levels.

b. At all stations except the one with Sphagnun recurvun, practically all the hyaline cell spaces of the sphagnun leaf at the higher levels are inhabited by Carteria sp., Cfalorogoniun sp.,

Notosolenus sp., Buglena nutabiiis. green ciliate* 1 and 11, and round woras. The larger organise*, testacean rhixopods, rotifers, and water uites, live and crawl around on the surfaces of steu and leaves, especially within the tiny water pockets foneed between the imbricated leaves.

7. The species found nay be divided into two nain associations according to their position on the sphagnun plant. Those characteristic of the BK»re highly i 1 Lunina ted upper portion of the plant have been t e m e d photophilic: nost of these either possess chlorophyll or possess xoochlorellae. Those characteristic of the relatively darker lower portion of the plant were terned non-photophilic: all of these are without chlorophyll or xoochlorellae.

b. The general micro-population of the bog persists throughout the entire year and exhibits an unusual tolerance to low temperatures.

Certain forns reach their naxinun growth at particular seasons of the year, and present what night be temed a seasonal dominance.

9. There is evidence that there is an extremeiy low concentra­ tion of dissolved oxygen at the bottom of the sphagnun plant and in

the decanposing muck underneath it. The species here nust therefore

live under nearly anaerobic conditions.

59 10. Moisture is a decisive factor in the distribution of

Testacea at the different stations on the bog.

11. A food relationship was observed between the holophytic and holozoic species. Many of the Testacea were seen with food vacuoles containing Carteria sp. and Chlorogoniusi sp. In addition there appeared to be a correlation between the increase in number of the bacteria at the bottom of the plant and the occurrence of small phagotrophic and saprozoic forms.

12. The changes brought about by the continuous growth of the

sphagnum at the top create a continuous succession of species along the entire length of the plant.

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67 APPEND 11

List of *11 Protozoa encountered in the course of this study

Mastiaophora

Carteria sp. Chlorogonium sp. Notosoienus sp. Euglena sattabilis Schmitz Carteria klebsii (Dangeard) Prance Crypt omonas ovata Ehrenberg Menoidium incurvum (Presenius) Klebs Distigma gracilis Pringsheim ChilaBonas paramecium Ehrenberg Trichloris sp.

Sarcodina Heieopera picta Leidy Hyalosphenia papilio Leidy Hyaios. elegans Leidy Nebela collaris (Ehrenberg) Leidy Sphenoderia lenta Schiumberger Euglypha ciliata (Ehrenberg) Leidy Arcella vulgaris Ehrenberg Euglypha alveolata Dujardin Nebela dentistoma Penard Assulina semimulum Ehrenberg Heieopera petricoia Leidy Centropyxis sp. Cyphoderia sp. Difflugia lobostoma Leidy Nebela flabellulum Leidy Trinema enchelys (Ehrenberg) Leidy Acanthocystis sp. Actinophrys sol Ehrenberg Amoeba guttula Dujardin Amoeba sp.

Ciliophora

Green ciliate 1 Green ciliate II Trichopelma sp. Opisthotricha sphagni Kahi Colpoda sp. Vorticeila sp.

bb P1ATE 1

Sphagnun palustre showing the four levels studied and the

relative frequency of photophiiic and non-photophilic individuals at the different levels. The relative numbers of individual

organisms at the different levels are indicated by the number of circles, and the relative numbers of photophilic and non- photophilic individuals are represented by the relative number of black and white circles* i 2.

69 \ UPPER »MWM» oo

MIDDLE • • oooooo

1 ______

> I30T TOM « oocoo PLATE 11

Fig. 1. Branch— leaf, X 2b.

Fig. 2. Enlarged portion of the branch—leaf showing the large,

clear, tapering, hyaline cells with apertures and

ring—like ridges. The living, much snaller, chloro­

phyll-bearing cells lie between them. X 7 50.

71 PLATE 11

F ig 2

7 J PLATE ill

A camera lucida drawing of a portion of the sphaenin leaf showing the relationship between certain Protozoa and the hyaline cells. X 1200.

1. Chlorogoniun sp., non-motiie form immediately after

division, within a hyaline cell.

2. Cyst of Green Ciliate 1, within a hyaline ceil.

1. Carteria sp., within a hyaline cell.

4. Carteria sp., on the outer surface of a hyaline ceil.

5, Euglena nutabilis. coming out of a hyaline cell

through the aperture.

Chiorogonium ap., within a hyaline ceil.

7. Notosoienus sp., within a hyaline ceil.

71 PLATE II

74 PLATE IV

A camera lucida drawing of a hyaline ceil containing two individuals of Green Ciiiate I. X 120 0 .

1. Individual showing micronucleus and macronucleus, mouth,

contracitile vacuole, and the spiral arrangement of the

ciliary rows.

2. Individual showing xoochloreilae, micronucleus and

macronudeus, mouth, and cilia.

75

AUTOBIOGRAPHY

1, Peter Chacharonis, was born in Zanesville, Ohio, Dec saber

9 , 1 9 2 5 . 1 received qr secondary school education in the public s c h o o l ! of the city of Zanesville, Ohio. My undergraduate training w m a obtained at Marshall College, Huntington, West Virginia, froa w h i c h 1 received the degree Bachelor of Arts in 194b. Froa the

O h i o State University, 1 received the degree Master of Arts in

1 9 5 0 . In 1949 1 received an appointaent as a graduate assistant in t h e Department of Zoology and Entoaology. 1 held this position while c n a y l e t i s g the requireaents for the degree Doctor of Philosophy.

77