Taxonomy, Distribution, and Seasonal Occurrence of the Saprolegniaceae in Aquatic Sites in Northwest Iowa Maren A

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1980 Taxonomy, distribution, and seasonal occurrence of the Saprolegniaceae in aquatic sites in northwest Iowa Maren A. Klich Iowa State University

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KUCH, MAREN A.

TAXONOMY. DISTRIBUTION, AND SEASONAL OCCURRENCE OF THE SAPROLEGNIACEAE IN AQUATIC SITES IN NORTHWEST IOWA

Iowa State University PH.D. 1980

University Microfilms

IntGrnStiOnSi 300 N. Zeeb Road. Ann Arbor, MI 48106 18 Bedford Row, London WCIR 4EJ, England

the Saprolegniaceae in aquatic sites in northwest Iowa

Maren A. Klich

A Dissertation Submitted to the

Graduate Faculty in Partial Fulfillment of the

Requirements for the Degree of

DOCTOR OF PHILOSOPHY

Department: Botany Major: Botany (Mycology)

Approved:

Signature was redacted for privacy. In Charge of Major Work,

Signature was redacted for privacy. For the Major Department

Signature was redacted for privacy.

For the Gradu College

Iowa State University Ames, Iowa 1980

TABLE OF CONTENTS

Page GENERAL INTRODUCTION 1

PART I 3

LITERATURE REVIEW 4

Morphology 4 Collection 4 Culture 7 Ecology 10

MATERIALS AND METHODS 15

Preliminary Laboratory Studies 15 Field Sites and Sampling Techniques 17 Laboratory Culture 23

RESULTS AND DISCUSSION 24

Species Isolation 24 Effects of Cultural Regimes 26 Comparison of Field Sites 30 Seasonal Periodicity and Water Temperatures 36

SUMMARY 63

PART II 65

INTRODUCTION 66

COMPUTERIZED SYNOPTIC KEY TO SPECIES OF ACHLYA AND SAPROLEGNIA 70

LITERATURE CITED 83

ACKNOWLEDGMENTS 92

APPENDIX A. COLLECTION DATES AND WATER TEMPERATURES 93

APPENDIX B. PRECIPITATION AND TEMPERATURE DATA FOR NORTHWEST IOWA, 1976-1978 96

APPENDIX C. SIMILARITY INDICES 100

APPENDIX D. COMPUTER PROGRAM FOR A HYPOTHETICAL SYNOPTIC KEY 103 1

GENERAL INTRODUCTION

The Oomycete family Saprolegniaceae is a group of predominantly aquatic fungi characterized by biflagellate zoospores. Although these fungi are common in freshwater habitats, their role in aquatic ecosys tems is virtually unknown. This may be due largely to isolation and identification difficulties. The purposes of this work were 1) to study the occurrence and distribution of the aquatic species of the Sapro legniaceae in northwest Iowa, 2) to refine isolation and culture methods for these fungi, 3) to devise a workable alternative to existing dichotomous identification keys for species belonging to the common aquatic genera Achlya and Saprolegnia.

Some members of the Saprolegniaceae reportedly display a distinct seasonal occurrence (Coker 1923, Hughes 1962, Dick and Newby 1961,

Roberts 1963, Alabi 1971a,b). The field study was designed to investi gate this ecological aspect of the family by repeatedly sampling several aquatic sites for two years. Differences in the fungal flora between sites were also studied.

To expedite ecological work with the Saprolegniaceae, techniques for isolation and culture should be as simple as possible. The tradi tional isolation substrate has been boiled hemp (Cannabis sativa L.) seeds. These seeds are often not readily accessible, therefore, several more common seeds were compared with hemp seeds as isolation substrates.

The identification process for species of Saprolegniaceae is tedious at best. Every isolate must be observed several times over 2

one to three weeks to study taxonomically important characters as they

develop. Some isolates never produce the structures necessary for identification utilized in standard dichotomous keys, but may possess other characters that could aid in identification. The various sorting systems and random-access keys allow workers to use several characters

to identify an organism, but these keys have not gained acceptance because they are unwieldy. The synoptic key included in the final section of this paper has been computerized for easy use. 3

PART I 4

LITERATURE REVIEW

Morphology

The Saprolegniaceae is characterized by a vegetative thallus com

posed of coenocytic hyphae. The vegetative colony of most aquatic

species is 1-5 cm in diameter on a hemp seed substrate. The vegetative

mycelium, once believed to be haploid (Patterson 1927, Shanor 1937), is

now generally considered to be diploid (Heath and Greenwood 1968, 1970;

Howard and Moore 1970). Sexual reproduction is oogamous, with one to

many oospheres per oogonium. Antheridial hyphae attach to the oogonial

wall and form fertilization tubes through which a single male nucleus

passes to fertilize each oosphere. Two major forms of asexual repro

duction exist within the family. The first is by production of zoospores

characterized by possessing one tinsel type and one whiplash flagellum.

Some members of the family are diplanetic with pip-shaped primary zoo

spores and reniform secondary zoospores. The second type of asexual

reproduction is by formation of chlamydospores or gemmae. These gemmae

may eventually develop into zoosporangia, oogonia with oospores develop

ing parthenogenetically, or germinate directly by a germ tube. A good

account of the morphological characteristics of the family may be found in Dick (1973).

Collection

Aquatic saprolegniaceous fungi are inconspicuous and ephemeral and, therefore, cannot easily be collected from the environment as one would collect vascular plants or mushrooms. Generally, a suitable 5 substrate is placed in the water, or in a water sample in the laboratory, from which one wishes to isolate species of the family. These sub strates, appropriately referred to as "baits," are colonized by fungal propagules in the water. The resultant fungal thalli can then be observed and identified.

To those not familiar with the baiting techniques used for aquatic fungi, a list of isolation substrates would appear to be more appropriate as a recipe for a witch's brew than for any scientific purpose. Among the baits used are dead flies, termites, ants, ant pupae, snake skin, grass blades, mushroom grubs, egg yolk, and various kinds of fruits, twigs and seeds (Pieters 1915, Coker 1923, Harvey 1925, Cejp 1934, Wolf and Wolf 1941, Hamid 1942, Beneke 1948a, Hoffman 1949, Johnson 1956,

Cooke and Bartsch 1960, Scott 1961, Dick 1966, Thakur Ji 1970, Alabi

1971a, Rooney and McKnight 1972, Zebrowska 1976, Padgett 1978a,b).

Various workers have used semi-solid media, but Ho (1975) developed a medium that is reportedly selective for Saprolegnia species. Although

Scott (1961) found boiled snake skin to be the best bait for Aphanomyces, most workers have found boiled hemp seed preferable for most members of the Saprolegniaceae. Zebrowska (1976) compared several isolation sub strates and found that saprolegniaceous fungi appeared more frequently on hemp than on flies, fruits, or twigs. Harvey (1925) noted that the presence of bacteria was "noticeably reduced by use of boiled hemp seeds as substratum." Bacteria compete for some of the same substances in the bait and it seems logical that fungi would establish more readily in the absence of bacteria (Holtje 1943). Johnson (1956) summed up the situation at the time by stating "present day students of the saproleg- 6

niâceous fungi have come to use hemp seed 'bait' almost exclusively for

collection, propagation, and maintenance of their isolates."

Perhaps one of the reasons that hemp seeds and other natural sub

strates have continued to be popular baits is that they are easy to

use. Hemp is no longer a common agricultural crop and its seeds are not readily accessible to all workers. It is interesting, therefore,

that Hoffman (1949) noted that two common crop seeds, alfalfa and clover

seeds, yielded cultures similar to those on hemp.

The two major isolation techniques, situ baiting and removing water samples, each have certain advantages and disadvantages for the research worker. In situ baiting of aquatic fungi has been used success

fully by many workers (Cooke and Bartsch 1959, 1960; Sparrow 1960,

Waterhouse 1942, Padgett 1978a, Rooney and McKnight 1972). Bait is placed in a container such as an aluminum tea ball, nylon mesh bag, or metal screen container, which is then submerged and anchored in place.

After a time, the researcher retrieves the container, carefully washes the baits and observes the colonies established on the bait. From an ecological perspective, iii situ baiting is preferable for nonrepetitive sampling of the environment. Placing bait directly into the environment varies only one factor, availability of a suitable substrate. By remov ing a water sample, however, many factors are changed. Propagules that would be inactive in the natural environment may accidentally be induced to germinate, thus distorting the researcher's data on which fungi are active. ^ situ baiting is not appropriate if the site is to be sampled repeatedly. By placing a suitable substrate into the environment, the environment is changed. Propagule numbers may increase greatly from 7

the colonies on the baits, biasing further sampling data (Dick 1976).

There are also several practical disadvantages to ^ situ baiting. If

the area being investigated is highly populated, there is a great likeli hood that the containers would be disturbed. In situ baiting is incon venient if many sites are being studied, because it is necessary to return to each site to collect the containers. For these reasons, many workers have chosen to collect water and soil samples and bait them in the laboratory (Johnson 1956, Wolf 1944, Beneke 1948a, Coker 1923,

Harvey 1942, Alabi 1971a,b, Dayal and Thakur Ji 1966, Dick and Newby

1961, Roberts 1963).

The propagules infesting the baits are generally assumed to be germinating encysted zoospores (Dick 1968, 1976). Willoughby (1962) substantiates this assumption that zoospores, and not germinating oospores or mycelial filaments, are the invading substances. This could lead to underestimates of active saprolegniaceous fungi that do not produce large numbers of zoospores. It is interesting that the size of the sample does not seem to be important in qualitative studies.

Dick (1966) has shown that a 100-fold dilution of soil and mud samples does not effect estimates of relative abundance.

Culture

Techniques for isolating and culturing of individual species from the initial isolation substrates range widely. The simplest techniques used by Emerson (1958), Wolf (1944), and others is to remove all or a part of the thallus from the initial culture, rinse it several times in distilled water and place it in a petri dish containing fresh bait 8 and distilled water. If more than one species appears to be present, the same process is utilized, but the hyphal inoculum is removed care fully from the mat to insure unihyphal colonies (Dick and Newby 1961).

These "quick and dirty" methods work reasonably well if bacteria-free colonies are not required.

Better methods of isolation of individual species have been developed, but all of these are quite time-consuming. Johnson (1956), Scott et al.

(1963), Seymour (1970), and Raper (1939) collected zoospores, encysted zoospores, or, if the isolate does not readily produce spores, hyphal tips or gemmae and placed them on a semi-solid medium such as corn meal agar, potato dextrose agar, malt agar, yeast extract agar, glucose glutimate agar, water agar, etc. By repeated transfer of the edge of resultant colonies, unifungal, bacteria-free colonies may be obtained.

These may be maintained on semi-solid media or transferred to another bait.

Many taxonomically important morphological characters of the Sapro- legniaceae are influenced by the substrate and environmental conditions in which they are cultured. Johnson et al. (1975) found that "structural modifications in size, density and shape of the oogonial wall could be induced" by growing the fungi on various media under different environ mental conditions. Oospore number and size were not greatly influenced by these changes. Fowles (1976) found that, although a wide range of temperatures and pH had no specific influence on the induction of the reproductive process of Aphanomyces species, light inhibited the forma tion of oogonia. Light also inhibits oospore formation in Saprolegnia 9 diclina Humphrey (Szanlszlo 1965). On the other hand, Ziegler (1948a) found that light was necessary for the germination of oospores. Elliot

(1968) was able to induce "small but significant" changes in the morphol ogy of the gametangia of two species of Saprolegnla by changing the size of the culture vessel and incubation temperature. Age of the culture also influenced the morphology of these fungi. Clausz (1968) also found temperature to be influential on the morphology of sex organs, finding that cultures of Achlya hypogyna Coker and Pemberton incubated at 15 C had "more characteristic" oogonia. Humaydan and Williams (1974) found that streptomycin sulfate suppresses oospore and zoospore production in

Aphanomyces raphani Kendrick. Laboratory studies by Padgett (1978b) and

Harrison and Jones (1975) showed zoospore formation to decrease with an increase in salinity.

A number of studies have been conducted on the optimal temperatures for zoospore formation. Klebs (1899) found the optimal temperatures for zoospore production to be 24-28 C for Saprolegnia mixta deBary. Coker

(1923) reported temperatures ranging from 18 to 30 C as being optimal for various species. Cotner (1930) determined the optimal temperatures for zoospore production for Saprolegnla monoica Pringsheim, Isoachlya paradoxa (Coker) Kauff., Achlya conspicua Coker, and Aphanomyces euteiches

Drechsler to be 26 C, 23-25 C, 23-27 C, and 26-28 C, respectively. The optimal temperature for zoospore production of Aphanomyces raphani is much lower, only 20 C (Humaydan and Williams 1974). Optimal temperatures for zoospore formation obviously differ for the different species. 10

Ecology

Many members of the Saprolegniaceae are saprophytic freshwater forms.

They are transient fungi found on such substrates as plant debris, insect exuviae, fish carcasses, and other debris in the water. Some are parasitic on fish, fish eggs, aquatic animals, algae, and other aquatic fungi. Although early workers (Humphrey 1893) believed the Saproleg niaceae to be restricted to freshwater habitats, some members of the family can survive in estuarine habitats (TeStrake 1959, Padgett 1977,

1978a). Some are soil saprophytes, while a few are root parasites of land vascular plants (Harvey 1930, Coker 1923, Scott 1961).

Seasonality of some species of the Saprolegniaceae was first noted by Coker (1923) and workers have since been trying to determine which environmental factors influence the distribution of these fungi in space and time in natural systems. Season, temperature, latitude, physical location in the aquatic system, pollution, dissolved oxygen, salinity, and altitude have all been studied.

Since Coker's (1923) initial observations, several studies on seasonal periodicity have been carried out (Roberts 1963, Dick and Newby

1961, Hughes 1962, Ziegler 1958, Dick 1963, Alabi 1971a,b, Dayal and

Thakur Ji 1966, Dayal and Tandon 1962, Cooke 1954, Rooney and McKnight

1972). All found that at least some species were more abundant during some times of the year than others. Cooke (1954), Dick and Newby (1961),

Roberts (1963), and others working in temperate zones have found greater species diversity and frequency of occurrence during spring and fall.

However, Rooney and McKnight (1972) in their study of a very cold (maximum temperature 19 C) subalpine lake found the greatest abundance of these 11 fungi in July, August, and September. Temperature appears to have a major role in seasonal periodicity.

Alabi (1971a,b), in Nigeria, and Dayal and Thakur Ji (1966), in

India, working in areas with relatively small seasonal temperature dif ferences still found seasonal differences in the fungal flora between the wet and dry seasons. So, temperature does not seem to be the only factor involved in seasonal periodicity. Perrot (1960) hypothesized that, in addition to temperature, maturation of resting spores is a major factor in seasonal periodicity of aquatic phycomycetes.

Oospore type is the major morphological factor which correlates with seasonal periodicity. Ziegler (1958) noted that in Florida "saprolegniaceous fungi with eccentric oospores tend to dominate collections made during warm weather, while those with centric and subcentric oospores rarely occur in warm weather but are of frequent occurrence in cooler weather." Hughes (1962), also working in the southeastern

USA, found that species with centric and subcentric oospores exhibit seasonal periodicity, but not those with eccentric oospores. Hughes, intrigued by the correlation between oospore type and temperature, rein terpreted data from many studies and found that species with eccentric oospores were most abundant in tropical regions, whereas those with centric and subcentric oospores were relatively more abundant in temper ate zones. Dick (1976) concludes that centric and subcentric species are isolated most commonly in spring and fall in temperate zones and rainy seasons in tropical areas, and eccentric species are isolated relatively more abundantly during the summer and dry seasons. 12

Not all genera of the Saprolegniaceae are common in aquatic habitats, but Achlya and Saprolegnia species exhibit large populations in the water

(Lui and Volz 1976). Of those that occur frequently in lentic systems, most are found in the littoral zones (Dick 1971). Willoughby (1965) reported that the surface muds of this zone contain especially high numbers of fungal propagules. In the Iowa Great Lakes area, the abundance of Saprolegniaceae has also been shown to be greater in the littoral zone than in the limnetic or profundal zones (Arne Skarshaug, Department of

Plant Pathology, University of Georgia, Athens, Georgia, personal com munication, 1978). Zebrowska (1976) compared the mycoflora from lentic and lotie systems and found that, although some species were common to both, others were restricted to one system or the other. The above information indicates that factors other than temperature and season seem to affect the distribution of saprolegniaceous fungi.

Several workers have measured oxygen content of the water in rela tion to the occurrence of Saprolegniaceae. Suzuki (1961) found aquatic phycomycetes to be scarce in low oxygen mud bottoms in Lake Makanuma where temperature varied only three degrees during the year, and the bottoms were anaerobic from May to December. The occurrence of phyco mycetes in this lake exhibited a mid-summer minimum and a mid-winter maximum. Dayal and Tandon (1963), working in a warm lake (minimum temperature 18 C), found that an increase in nutrients and oxygen resulted in an increase in fungal development. Rooney and McKnight (1972) came to the opposite conclusion, the number of species increased with a decrease in oxygen content. The subalpine lake they studied, however, remained quite cool (maximum 19 C) during the summer so dissolved oxygen was 13 probably not at the critical levels reported in the tropical lakes studied by Dayal and Tandon. Alabi (1971a), studying several aquatic sites on the campus of the University of Ibadan in Nigeria, found no correlation between dissolved oxygen and the occurrence of the Sapro- legnlaceae.

The effect of pollution on the distribution of aquatic fungi has been studied by several workers. Research on fungi Involved in sewage has been reviewed by Cooke (1976). Generally Oomycetes are not abundant in streams carrying heavy loads of organic pollution. Harvey (1952) noted that Saprolegnla, Achlya, and Dlctyuchus were consistently absent from heavily polluted water and rare in partially polluted water, whereas Aphanomyces may be associated with polluted water. Cooke (1954) noted that "molds are generally inhibited at or just below the point of outfall in warm seasons, but not noticeably so in cooler seasons."

Salinity appears to limit the distribution of at least some members of the Saprolegniaceae. Studies in estuarine environments by TeStrake

(1959) and Padgett (1977, 1978a,b) show that occurrence of members of the Saprolegniaceae decreases as salinity increases. Laboratory studies by the above authors indicated that these fungi were much more tolerant of salinity in the laboratory than in natural conditions. These findings led TeStrake (1959) to postulate that "nutrition fundamentally and not hydrographie factors limits the estuarine distribution of saprolegnl- aceous fungi."

Altitude has been discussed Incidentally as a factor in the distri bution of water molds. Harvey (1942), in a survey of the western states, found no water molds above an altitude of 5000 ft. He attributed this 14 absence to altitudinal factors. However, Rooney and McKnight (1972) found several water molds, including several members of the Saproleg- niaceae, in a subalpine lake at 10,000 ft. Obviously more information is needed. 15

MATERIALS AND METHODS

Preliminary Laboratory Studies

Substrates

These experiments were designed to determine the substrates most appropriate for rapid production of reproductive structures necessary for identification of isolates of the Saprolegniaceae. The commonly used seed baits can be colonized by fungal propagules only after the seed coat (and/or fruit wall) is broken. This is usually accomplished by boiling the seeds in water. The seeds may also be autoclaved in water, but seeds treated in this manner tend to disintegrate easily.

The most commonly used bait is hemp seed (Cannabis sativa L.). In an effort to find a more suitable "standard" bait, ten other seeds were compared with hemp using three species of fungi as test organisms.

Seeds of velvetleaf (Abutilon theophrasti Medic.), radish (Raphanus sativa L.), alfalfa (Medicago sativa L.), white sweet clover (Melilotus alba Desr.), onion (Allium cepa L.), sugar beet (Beta vulgaris L.), cabbage (Brassica aleracea L.), spinach (Spinacia oleracea L.), lespedeza

(Lespedeza sp.), and yellow foxtail (Setaria lutescens (Weigel) Hubb.) were used as baits. These readily accessible species were chosen to sample a wide variety of plant families.

The fungi used as test organisms were Saprolegnia ferax (Gruithusen)

Thruet, Dictyuchus monosporus Leitgeb., and Achlya dubia Coker. Hyphal tips from well-established thalli were placed in petri plates containing

50 ml glass distilled water and freshly boiled seeds of one species. The petri plates were placed on a laboratory bench and observed every three 16 days. Colony development was compared with hemp as a standard for bacterial infestation, colony diameter, and presence of sporangia, oogonia, antheridia, and gemmae.

From these experiments, fungal colonies established on sweet clover, alfalfa, and sugar beet seeds were most similar to hemp in the morpholog ical characters considered. Sugar beet seeds, however, were readily colonized by bacteria. Sweet clover and alfalfa were the most appropri ate baits to compare with hemp as substrates for isolation and culture of the fungi in the long-term field study.

Temperature

This experiment was designed to determine the temperature most appropriate for the growth and consistent development of taxonomically important morphological characters. The three species of fungi used as test organisms were Saprolegnia ferax, Dictyuchus monosporus, and Achlya dubia. Twelve petri plates containing boiled hemp seeds and 50 ml glass distilled water were inoculated with each of these fungi. Six plates, two of each species, were incubated at each of the following temperatures:

7 C, 13 C, 16 C, 19 C, 22 C, and 34 C. Colony and reproductive struc ture development was observed every three days.

Identification of the fungi was possible in a shorter time period if the fungi were incubated at several temperatures because different structures develop more rapidly than others at a given temperature.

Temperatures between 13 C and 22 C seemed best. Temperatures of 15 C and 22 C (room temperature) were chosen as incubation temperatures for the field portion of the study. 17

Field Sites and Sampling Techniques

All sampling sites were located in the lakes region of northwest

Iowa. This area is characterized by rolling hills and many small natural lakes and marshes associated with the terminal moraine of the Gary sub- stage of the Wisconsin glaciation. All but one site was in Dickinson

County, and this site was in adjacent Clay County. Seven of the nine sites sampled were in lentic environments. These were all subject to heavy blue-green algal blooms during the summer months. The two lotie sites were both on the Little Sioux River. Samples were obtained from approximately the same location every three weeks from April through

November for two years.

Figure 1 contains maps designating the location of the sampling sites. A list of the names, legal locations, and descriptions of the sites follow.

Mud Lake

Clay County, R-35W, T-97N, Sec. 25.

The sampling site was located among the emergent vegetation on the west side of the lake near the outflow dam. Mud Lake is a small lake in the midst of many marshes. It is on the migration route for many kinds of waterfowl, which could be bringing in propagules of aquatic fungi not normally found in this area (Thorton 1971).

Lake Minnewashta

Dickinson County, R-36W, T-99N, Sec. 30.

The sampling site was located at the edge of an open sandy public beach on the west side of the lake. There was no emergent vegetation Figure 1. Maps of collection sites for aquatic fungi in northwest Iowa. A - Dickinson County; B - Clay County; C - Mud Lake; D - Lake Minnewashta; E - East Lake Okoboji; F - Spirit Lake; G - Jeramerson Slough; H - Triboji Lagoon; I - Little Sioux River at Highway 9; J - Silver Lake; K - Little Sioux River at Horseshoe Bend 19

G •

C# 20

at this site.

East Lake Okoboji

Dickinson County, R-36W, T-99N, Sec. 19.

The sampling site was located at the bottom of a very steep, heavily wooded hill on the west side of the lake. The site was heavily shaded by the overhanging trees, mostly Quercus macrocarpa Michx. The rock and sand beach was narrow with a maximum width of 1.25 m. There was no emergent vegetation.

Spirit Lake

Dickinson County, R-36W, T-IOON, Sec. 20.

The sampling site was located along an open, wide sand and pebble beach lined with cabins on the west side of the Lake in North Templar

Park, on property owned by John E. Vanderlinden. There was no emergent vegetation, and, due to the openness of the site, the area was subject to much disturbance by wave action.

Triboji Lagoon

Dickinson County, R-37W, T-99N, Sec. 2.

The sampling site was located at the south edge of a sheltered, tree-covered, sand and mud bottomed lagoon. The lagoon is a man-made extension of West Lake Okoboji, and the sampling site was within 20 m of the lake proper. The site was regularly disturbed by motor boats passing in and out of the lagoon, but not subject to the regular wave action of the lake. The bank at the edge of the lagoon was very steep. 21

Jemmerson Slough

Dickinson County, R-36W, T-99N, Sec. 6.

The site was located in a dredged boat launch area leading from

the shore through the emergent vegetation to the open water. The site

had a mud bottom, and was among a large stand of Typha spp. The slough

is a nesting site for many kinds of waterfowl.

Silver Lake

Dickinson County, R-38W, T-IOON, Sec. 32.

The site was located in a Typha spp. stand on the southwest edge

of the Lake, down a gently sloping hill from Silver Lake Fen State

Preserve. In the drought of 1977, the Lake receded beyond the emergent

vegetation zone and was in open water during most of that year.

Little Sioux River at Horseshoe Bend

Dickinson County, R-37W, T-99N, Sec. 6.

The sampling site was located next to a small suspension bridge in Horseshoe Bend County Park. The bank of the river adjacent to the sampling site was quite steep. The mud-bottomed river was flowing quite rapidly at this point, and there was no emergent vegetation.

Little Sioux River at Highway £

Dickinson County, R-37W, T-99N, Sec. 6.

The sampling site was located just south of the highway bridge over the river. The adjacent land was cattle pasture. The river had a steep bank and was mud-bottomed at this point. When flow was high, the site sampled was not in the main channel of the river; however. 22 in the drier months, the sample was taken from the main channel.

Surface water samples were collected within % m of the shoreline in a clean beaker. All samples were taken between 11 a.m. and 4 p.m. of the sampling day. During the drought of 1977, samples were taken at the shoreline wherever it happened to be as the lakes and rivers receded. If the site had dried entirely, samples were not taken.

The temperature of the water in the beaker was measured and ali- quots of the sample were poured into six 125 ml sterile screw cap bottles containing the baits. These seeds had previously been boiled in glass distilled water until the seeds were soft- The seeds were then placed in the bottles and the bottles with seeds were capped and sterilized in an autoclave for 15 minutes at 15 psi. Two of these bottles contained 5 hemp seeds, two contained 6 alfalfa seeds, and two contained 6 sweet clover seeds. Willoughby (1962) suggested that encysted zoospores may die within 2 hours if a suitable substrate is not available. By placing the sample in contact with the bait immediate ly upon collection of the sample, this loss was hopefully kept to a minimum.

The samples were transported back to the laboratory and incubated.

The time between collection and incubation of the samples was less than two hours during June, July, and August when the laboratory work was conducted at Iowa Lakeside Laboratory, but the time between collection and incubation was up to 9 hours during the rest of the year when the samples were transported to Ames. 23

Laboratory Culture

In the laboratory, the contents of each bottle were poured into a

sterile petri plate and incubated. Half of the six plates from each

collection site, one of each bait type, was placed in a dark 15 C

incubator. The other six plates were placed on a laboratory bench where the temperatures varied from 21-26 C. These plates were exposed

to normal laboratory light.

A sample from each collection site consisted of six isolation

plates containing one of each of the bait types (hemp, alfalfa, or

sweet clover) at each of two temperatures (15 C and room temperature).

Fungal colonies on the seeds were noted after 5 to 7 days and, if more

than one species was present or if the colony was not readily identi

fiable, a hyphal tip from each was transferred to a sterile petri plate

containing 45 ml glass distilled water and fresh sterile bait of the same species as the isolation bait. These unifungal colonies were identified. A photographic record was made of the less common species. 24

RESULTS AND DISCUSSION

Species Isolation

The species identified during this study are listed in Figure 2 as they are recognized by the authors indicated. Several taxonomic changes have been made since the monographs and other works used for identification were written. Saprolegnia parasitica is now synonymous with diclina, and Achlya conspicua and A. flagellata are synonymous with A. debaryana (Johnson 1980).

Achlya and Saprolegnia were the most common genera with nine and eight species, respectively. This was expected as Achlya and Saprolegnia are large genera known to be in aquatic habitats (Lui and Volz 1976).

Three species of Dictyuchus and two species of Aphanomyces were isolated.

Only one species of Protoachlya, P. paradoxa, was identified. eccen trics, terrestris, turfosa, Achlya cambrica, A. inflata, A. oblon gata, A. proliferoides, A. racemosa, D. pseudodictyon, and D. missouriensis were all very rare, occurring in only one to three cultures.

One interesting fungus that occurred several times appeared ini tially to be a mixed culture of Saprolegnia ferax and S^. hypogyna.

However, oogonia characteristic of each of these species formed within

200 um of one another on the same hyphal strand. This finding casts doubt on the validity of hypogyna as a separate species.

Several isolates belonging to the above genera could not be iden tified to species because they did not produce gametangia. These will be designated "ui" throughout the rest of this discussion. Another group of isolates were sterile, producing neither gametangia nor 25

Saprolegnia

S. asterophora deBary australis Elliot diclina Humphrey eccentrica (Coker) Seymour ferax (Gruith.) Thuret. _S. hypogyna (Pringsheim) deBary parasitica Coker S^. terres tris Cookson ex Seymour turfosa (Minden) Gaumann

Achlya

A. americana Humphrey A. bisexualis Coker & Couch A. cambrica (Trow) Johnson A. conspicua Coker A. debaryana Humphrey A. diffusa Harvey ex Johnson A. flagellata Coker A. inflata Coker A. oblongata deBary A. proliferoides Coker A. racemosa Hildebrand

Aphanomyces

A. helicoides Minden A, laevis deBary

Dictyuchus

D. missouriensis Couch D. monosporus Leitgeb D. pseudodictyon Coker & Braxton

Protoachlya

P. paradoxa Coker

Figure 2. Species of Saprolegniaceae isolated from all aquatic sites sampled in northwest Iowa, April through November 1977 and 1978. Saprolegnia (Seymour 1970); Achlya (Johnson 1956); Aphanomyces (Scott 1961); Dictyuchus and Protoachlya (Beneke 1948a) 26 sporangia. These could not even be placed into a genus and will be referred to as "UI" in the following discussion.

Effects of Cultural Regimes

The results of the bait and incubation experiments are summarized in Figure 3. The information reported therein includes all of the species isolated at all collection sites during both sampling years.

The number of isolations of each species on the three baits in each of the light and temperature regimes are reported in columns one through six. The number of isolations of each species on each of the bait types is summarized in columns seven through ten. In the last two columns, the total number of isolates in each of the two temperature and light regimes, on all baits, is presented.

The light and temperature effects on isolation and production of reproductive structures essential for identification are not simple to explain. Although the difference is not large, more unidentifiable

(UI) cultures grew in the 15 C/dark (15D) incubation regime than in the room temperature/light (RTL) regime. However, within a given genus, fewer isolates could not be identified to species in the 15D incubation.

The major reason many members of these genera could not be identified to species was that they never produced gametangia. Szaniszlo (1965) and Fowles (1976) report that oogonial formation is inhibited by light.

This study lends further support to their observations since most of the unidentified species in this study were exposed to light regularly in the laboratory. Room temperature 15 C dark lighted Sweet Sweet Hemp Alfalfa Clover Hemp Alfalfa Clover

Saprolegnia spp. ui^ 47 47 50 27 42 34 S. asterophora 1 1 3 S. australis 2 4 7 6 7 S. diclina 3 1 5 24 22 25 S. eccentrica 1 S. ferax 37 28 30 59 39 56 S. hypogyna 4 2 1 S. terrestris 2 1 S. turfosa 1 1

Achlya spp. ui 8 10 8 3 5 7 A. americana 14 8 6 17 9 7 A. bisexualis 6 1 2 3 3 A. cambrica 1 A. debaryana 7 5 5 8 6 2 A. diffusa 1 2 3 1 1 A. inflata 1 A. oblongata 1 A. proliferoides 1 1 A. racemosa 1 1

Aphanomyces spp. ui 1 2 1 5 4 6 A. helicoides 1 1 3 A. laevis 1 1 6 8 14

Dictyuchus spp. ui 1 2 1 D. missouriensis 1 D. monosporus 11 9 10 6 4 5 D. pseudodictyon 1 2

Protoachlya paradoxa 1 1 1 1 1

UI^ 4 5 8 8 5 10

%i = unidentified species.

^UI = unidentified isolates.

Figure 3. Species of Saprolegniaceae isolated on various baits held at different temperatures and light conditions 28

Total—all temperatures .,. , .. J - , All baits and light conditions Hemp Alfalfa Sweet clover Room temperature 15 C dark lighted

12 23 28

All baits and light conditions Hemp Alfalfa Sweet clover Room temperature 15 c dark lighted

74 89 84 144 103 4 1 2 3 9 6 11 6 20 27 23 30 9 71 1 1 96 67 86 95 154 4 2 1 7 2 1 1

11 15 15 26 15 31 17 13 28 33 9 4 2 9 6 1 1 15 11 7 17 16 5 2 3 5 5 1 1 1 1 1

6 6 7 15 1 1 3 5 7 8 15 28

2 2 5 1 1 1 17 13 15 30 15 1 2 3

1 2 2 3

12 10 18 17 23 29

In general it appears that a dark, cool incubation regime is more conducive to the establishment of colonies and development of taxonomically important morphological characters than a warm lighted regime.

Many more of the colonies identified to species in Saprolegnia and

Aphanomyces were isolated in 15D than RTL. Identifiable species of

Achlya, however, were fairly evenly distributed in the two incubation regimes, and many more Dictyuchus species were identified in the RTL than 15D regime. Dictyuchus monosporus, the most frequently isolated

Dictyuchus species can be identified without the presence of gametangia, and many of the isolates never produced them. Therefore, the light/dark influence is not significant in this case.

Use of only a dark incubation chamber may reduce the chance of isolating the rare species. Of the species isolated only one to three times in the present study, only Saprolegnia turfosa appeared in the

15D incubation. The other nine species all occurred only in the RTL incubation. This could, of course, be due to chance. However, there is some indication that oospores of some species require light to germinate (Ziegler 1948a). If oospores served as the initial inoculum for these rare colonies, perhaps light triggered germination. Further work obviously needs to be done, but perhaps placing the baited water samples in a lighted area for some period of time and then transferring them to dark chambers would result in a greater diversity of readily identifiable colonies than exposing the collections to only one light regime.

Two alternative substrates to the "standard" hemp seed bait, alfalfa seed and sweet clover seed, were used during this study. Baited sweet clover seed is most similar to hemp for isolation of species of Sapro- legnia and Dictyuchus and appears to be a better isolation substrate than alfalfa or hemp for species of Aphanomyces. For isolation and identification of species of Achlya, however, neither alfalfa nor sweet clover were as good as hemp. The rare species, those isolated fewer than four times, were not restricted to any one bait, although these species were found more commonly on baited hemp seeds than the other two baits.

The differences in the number of isolations of the various species on the different baits could be due to several factors. The alfalfa and sweet clover baits seemed to support a slightly higher bacterial flora than hemp. Perhaps the fungi (especially Achlya species) do not compete well with the bacteria. Hemp seed is much larger than alfalfa or sweet clover seed. Perhaps more available substrate affects the establishment and growth of at least some members of the Saprolegniaceae.

Any number of biochemical factors cçuld also be involved in the selec tivity of these baits. Finally, many of the differences in numbers of isolations on the different bait types could be due to chance.

More comparative work needs to be done with these and perhaps other baits, but these results indicate that sweet clover seed could serve as an acceptable alternative to hemp seed for isolation of species of Saprolegnla, Aphanomyces, and Dictyuchus.

Comparison of Field Sites

The species isolated during this study are listed by sampling location and collection trip number for both years in Figure 4. The Figure 4. Species of Saprolegnlaceae Isolated from aquatic sites In northwest Iowa at specific sampling times, April through November, 1977 and 1978 32

Littlu SÎDux River Little Sioux .It Ili^-.liw.nV 9 .jt Horseshoe 1 2 3 4 5 h 7 8 9 10 11 12 3 4 5 6 7

SanroU",;tiia spp. ui ' 1977 XX XX 1978 X . X S^. astcrophora 1977 X , 1978 austral is 1977 XX 1978 X X S. diclina 1977 X X 1978 X ecccntrica 1977 ' 1978 S. Cerax 1977 X X X X XX XX X 1978 X X X X X X XX XX X X X X hvpo^vna 1977 1978 1977 X . 1978 tur t'osa 1977 1978 Ach Iva spp. ui. 1977 1978 X X A. amoricana 1977 X X 1978 X X X X X X X X A. bisoxualis • 1977 X X 1978 • X X A, cainbrica 1977 1978 A. clcbaryana 1977 X X 1978 X X X A. cii 1."Fusa 1977 1978 A. inflata 1977 1978 A. ohloni;ata 1977 1978 A. pro IjL'eroidos 197 7 - 1978 A. racomosa 1977 1978 A M p 1. ! N r, s ]. I Triboji Lai;aon Mud l.;il e JcmtTicrHon Slough

1334567S9:0I1 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 b 9 10

XXX XXX X

X X

X X XX X. X X X X X X X X X X X X X X X X X X XXX X X X' X

X XXX X X X X XXX XX X X X X X

X X X X

X X X X X X X : p L ! N r; si r i: s JemnuTson S.loii^;h Silver I./ike ' I,nke Xinnewnshta

23456769 10 11 1 2 3 4 5 6 7 89 10 LI !2 3 4 3 b 7 80 10 11 1z

XX X X xxxxx x> X X X XXXXXXX)

XX XXX X xxxxxxx

X X X X XX X X x XX xxxxx xxxxx X X XXXX X

X XX

xxxxx XXXX X

XXX XX

X !.nke :!inne\;>'ifj!)ta Spirit I.ake East Lake Ol-oboj t 2 j 4 b 6 7 8 0 10 11 123456789 10 II 1234567891

XX X X X XXXXXXXXX X X XXXXXXXX X XX X X X X X X X X X XX xxxx xxxx

X X X X X

XXX X X X X XX XX X X X X X XXXX X XX X X X - X

XXX X X XX X XXX X X X XXX X X

X Spirit Lake East I.akc Ol oboji

5 6 7 8 9 10 11 123456789 10 11

XXXXXX X X XXXXXXXX X XXX XX XXXX XXXXXX

X X X X X X XXX X X X X X X X X X

X XX A X X XXX X X

X A. cainbrica 1977 1978 A. dcbarvana 1977 1978 A. .diffusa 1977 1978 A. inflata 1977 1978 A. oblongata 1977 1978 A. pro Li tcroidos 1977 1978 A. raccmosa 1977 1978 Aplianomyces spp. ui 1977 1978 A. helicoidcs 1977 1978 A. laovis 1977 1978 Pictyiiclnis spp. ui. 1977 1978 _D. missouriensis 1977 19 78 D. iiionosponis 1977 1978 D. pseudoc! ic t: von 1977 19 78 Protoacli 1 va parndoxa 1977 1978 U' i 1977 19 78

'ui = unidentified spi:cic h = unL'/iDUl: L1 Icil i point; X X

X XX X

X X XX XX X

X X

X X X X X X X X X

X XX X XX XX X XX X XX

X X

X \X X X X X X

X X X X X XX X X X

X XXX X X X X

X X

X K X

X X X X X A X 33

exact collection dates and water temperature of each sample may be

found in Figure A,l. Saprolegnia ferax, diclina, S. australis,

Achlya americana, A. debaryana, Aphanomyces laevis, and Dictyuchus

monosporus were the most frequently isolated species. Of these, only

ferax was isolated from all nine sampling locations. Achlya americana

and diclina were collected from eight locations. australis and

D. monosporus were found at seven sites, Achlya debaryana was isolated

from six locations, and Aphanomyces laevis from five.

Water samples could not be collected if the sites were dry, there

fore, there were some differences in the number of times the sites were

sampled. The sampling location at Jeramerson Slough was dry from the

fifth through the ninth samplings (July through September) of 1977.

The Little Sioux River collection site at Highway 9 was dry during the

sixth, seventh, and eighth samplings (late July, August, and early

September) of 1977, and the collection site on the Little Sioux River

at Horseshoe Bend was dry at the seventh collection (August) of 1977.

One of the difficulties in assessing site variations in open water

systems is determining the source of inoculum (Dick 1976). Willoughby

(1962) and Collins and Willoughby (1962) concluded that since the fre

quency of phycomycete spores in the water decreased with increasing

distance from the shoreline, much of the spore population was related

to leaching by rainfall. The data in Figure 4 reveal some interesting

information regarding inoculum origin. Although most species of Dic

tyuchus and Achlya were present in samples during the drought of 1977,

they were isolated more frequently in 1978 when the rainfall and leaching

were normal. Precipitation and temperature data for northwest Iowa from 34

1976 through 1978 may be found in re B.l. The correlation between rainfall and isolation of these fungi is not as direct as one might expect. The frequency of isolation did not necessarily increase if a collection was made after a period of heavy rainfall (see Figure B.2).

These data do suggest, however, that Achlya and Dictyuchus are more common in the water when there is leaching from the soil.

Although the data for Achlya and Dictyuchus tend to support

Willoughby's hypothesis that the inoculum is being leached into the aquatic system from the soil, this is evidently not the case for all members of the Saprolegniaceae. Several species, especially those in the genera Saprolegnia and Aphanomyces were quite common in the water during the 1977 drought when there was little possibility of leaching from the soil. The source of inoculum was probably at or near the sampling sites in the lentic locations and at or upstream from the sites

Some species were isolated far more frequently from certain habi tats. The most striking example was the common isolation of Saprolegnia diclina in the sand-gravel bottom sites with no emergent vegetation, and the rather infrequent recovery of this species from other locations.

These open-gravel-sand sites, which were subject to great amounts of wave action, included Spirit Lake, East Lake Okoboji, Minnewashta, and

Silver Lake (1977). These locations were also characterized by the infrequent occurrence of species of Achlya and Dictyuchus.

The greatest number of isolates of Achlya were recovered from the mud bottom sites on the Little Sioux River and in Triboji Lagoon. These 35

three sites also had very steep mud banks, so propagules could easily

have washed into the water from the shore. This hypothesis is supported

by the greater frequency of Achlya species at these sites during the

year with normal inflow, 1978.

Aphanomyces laevis was restricted almost entirely to sites in or

near emergent vegetation. These sites were Mud Lake, Silver Lake,

Jemmerson Slough, and Triboji Lagoon.

There appears to be no distinct lotie saprolegniaceous flora. With

the exception of terrestris which was isolated only once, none of

the species were restricted to lotie systems. S^renson's Index of

Similarity and Spatz's Index of Similarity were computed for all possible

pairs of sampling sites (see Figures C.l and C.2). In both analyses,

the two lotie systems showed a higher degree of similarity to other

sites than to each other.

Mud Lake, in spite of heavy use by migrating waterfowl, yielded

no unusual species other than a single collection of Aphanomyces heli-

coides. This fungus was also isolated from the Little Sioux River at

Horseshoe Bend at about the same time of year, and at Jemmerson Slough

during the previous year.

Generally, species which occurred at a given site during the first year of sampling were also found at the same site during the second year, and often at approximately the same time of year. This indicates

that perhaps these fungi are persistent members of the flora in a given

aquatic system, with definite seasonal occurrence. 36

Seasonal Periodicity and Water Temperatures

Because seasonal periodicity is correlated with temperature, the

two will be discussed together. To demonstrate this correlation, graphic representations of the data are presented for both season and water

temperature. There have been several reports of a relationship between seasonal occurrence and the nature of the mature oospore. The data on this subject gathered in this study will be compared to that reported by previous workers. Little relation seems to exist between seasonality and occurrence at the generic level, therefore, occurrence of the common species, Saprolegnia ferax, diclina. Achlya amcricana, A. debaryana,

Dictyuchus monosporus, and Aphanomyces laevis will be discussed in detail.

Species with centric and subcentric oospores appear to have a distinct seasonal periodicity (see Figure 5). They were isolated much more frequently during the spring and fall than during the summer months and were most abundant in samples in the 17-20 C range (see Figure 6).

These data generally support the interpretation of Hughes (1962) using the data from Coker (1923) in North Carolina and Lund (1934) in Denmark.

In the Lund study, however, this oospore type group did not increase greatly in frequency in the fall as it did in this study and in Coker's work. Fox and Wolf (1977), working in Tennessee, also observed the same trend of decreasing frequency of this group during the summer.

Maestres and Nolan (1978) reported that occurrences of species with centric and subcentric oospores peaked in the summer, with the exception of monilifera and S. terrestris which occurred more frequently in

March. The Maestres and Nolan study was conducted on the Broadcove Figure 5. Percentage of total aquatic locations sampled in northwest Iowa from which saprolegniaceous fungi with centric and subcentric oospores were isolated, April through November of 1977 and 1978 38

1977 1978

Apr May June July Aug Sept Oct Nov SAMPLING DATES Figure 6. Frequency of occurrence of species of fungi with centric and subcentric oospores isolated from aquatic locations sampled in northwest Iowa, April through November, 1977 and 1978. Frequency = number of isolates with centric and subcentric oospores isolated drom a specific temper ature range/total number of samples taken in the same temperature range

Figure 7. Frequency of occurrence of species of fungi with eccentric oospores isolated from aquatic locations sampled in northwest Iowa, April through November, 1977 and 1978. Frequency = number of isolates with eccentric oospores isolated in a specific temperature range/total number of samples taken in the same temperature range 40

100

90 80

70 60 o>- z 50 =) 3 40 ce. 30 LL. 20

8-12 13-16 17-20 21-24 25-28 29-32 33-38 TEMPERATURE IN DEGREES C

100 90 g 80 70

> 60 S 50 i» O: 30 20

8-12 13-16 17-20 21-24 25-28 29-32 33-38 TEMPERATURE IN DEGREES C River in Newfoundland, where the highest summer sampling temperature was 17 C. This is about the same temperature range of maximum frequency found in this study. Rooney and McKnight (1972) also reported a greater frequency of centric and subcentric species in July and August in a subalpine lake in Utah. The maximum sampling temperatures in the Rooney and McKnight study were low, 19 C. This information tends to indicate that the occurrence of species with centric and subcentric oospores is temperature dependent.

The seasonal occurrence and temperature relations of species with eccentric oospores is not as distinctive as it is for the centric and subcentric species (see Figure 8). The lack of any distinctive pattern of seasonal occurrence of this group has been noted by several authors including Hughes (1962), Dick and Newby (1961), and Fox and Wolf (1977).

In this study, however, the species with eccentric oospores did tend to occur with slightly greater frequency during the summer months, decreasing somewhat in frequency in the spring and fall. This tendency is supported by the slight peak in the 21-24 C range in Figure 7. The bar in the highest temperature range on this figure is probably somewhat inflated because there were few samples taken in this range. The decrease in occurrence of these species in the 8-12 C range corresponds to the decrease in the early spring and late fall.

Saprolegnia ferax, the most common species isolated in this study, occurs predominantly in the spring and fall (see Figure 9). It is apparently a cool water organism as it was isolated most frequently from water in the 8-12 C range and 17-20 C range (see Figure 10). In

1978, S, ferax was more abundant in the spring than in the fall. This Figure 8. Percentage of total aquatic locations sampled in north west Iowa from which species of saprolegniaceous fungi with eccentric oospores were isolated, April through November, 1977 and 1978 43

100

1977 80

Apr May June July Aug Sept Oct Nov SAMPLING DATES Figure 9. Percentage of total aquatic locations sampled in northwest Iowa from which Saprolegnia ferax was isolated, April through November, 1977 and 1978 45

June July Aug Sept Ocl Nov SAMPLING DATES Figure 10. Frequency of occurrence of Saprolegnia ferax isolated from aquatic locations sampled in northwest Iowa, April through November, 1977 and 1978. Frequency = number of isolates of ferax isolated in a specific temperature/ range/total number of samples taken in the same tempera ture range

Figure 11. Frequency of occurrence of Saprolegnia diclina isolated from aquatic locations sampled in northwest Iowa, April through November, 1977 and 1978. Frequency = number of isolates of diclina isolated in a specific temperature range/total number of samples taken in the same tempera ture range 47

100 • 90 •

g 80

70 ^ 60 • S , S 40 Od ^ 30 ———»

20 •

8-12 13-16 17-20 21-24 25-28 29-32 33-38 TEMPERATURE IN DEGREES C

100

g 80 70

O 60 S 50

S 40 ai ^ 30 •

20 L

8-12 13-16 17-20 21-24 25-28 29-32 33-38 TEMPERATURE IN DEGREES C difference, however, was not evident in 1977. Perhaps the cool, wet

conditions of the spring of 1978 were more conducive to growth and

propagule production than the warm, dry conditions of 1977 (see Figure

B.l for temperature and precipitation data for the years 1976-1978).

Coker (1923) and Hughes (1962), both working in the southeastern U.S.,

classified S^. ferax as a spring occurring fungus. Roberts (1963) in

England called it a summer species because it was absent from her other

collection in January. Fox and Wolf (1977) found that, in Tennessee,

ferax was isolated most frequently from September through December.

In Maestres and Nolan's 1978 study in Newfoundland, S^. ferax had the

greatest relative occurrence in July at a collection temperature of

13 C. In all of these studies, as well as the present study, ferax

characteristically appears in cool waters.

Saprolegnia diclina occurred less frequently in this study than

ferax (see Figure 12). It did not display either the distinct

seasonal periodicity of the latter nor the distinct temperature range

peaks of ferax. although it was isolated more commonly from water

between 13 and 16 C than other temperatures (see Figure 11). In 1977,

it occurred in the spring collections, was not present in June or early

July, then increased through early September. In 1978, it generally increased in frequency throughout the sampling season. In both years, it was most abundant during September. Other workers have reported

S. diclina as a fall species (Fox and Wolf 1977), a winter species

(Roberts 1963), and a summer species (Maestres and Nolan 1978). It appears that temperature and season do not have as strong an influence on the occurrence of S, diclina as they do on ferax and other factors Figure 12. Percentage of total aquatic locations sampled in north west Iowa from which Saprolegnia diclina was isolated, April through November, 1977 and 1978 50

1977 ' 1978

Apr May June July Aug Sept Oct Nov SAMPLING DATES 51

are influencing the occurrence of S^. diclina to a greater extent.

Achlya americana was the most commonly isolated member of this

genus. The relative frequency of isolation was much higher in 1978 than

1977 (see Figure 13). In 1977, it was isolated on only three of the

eleven sampling days, in early May, September, and October. The 1977

data support findings by Fox and Wolf (1977) that A. americana is

absent during the summer. However, the 1978 sampling in this study

showed peaks of occurrence not only in the spring and fall but also a

third peak in mid-summer'. This pattern is more similar to that of a

constant species, which is the way Roberts (1963) described this species.

Differences such as this one between the two sampling years indicate

that workers should exercise caution in drawing any conclusions regard

ing seasonality based on one year's data. The occurrence of this

species appears to be linked to temperature (Figure 14). Temperatures

during July and August of 1978 were relatively low, which could account

for the mid-summer peak in that year (see Figure B.l). Achlya americana

occurred most frequently in samples in the 17-20 C range, and less

commonly at higher and lower temperatures.

A. debarvana. which now includes A. flagellata and A. conspicua,

had a low frequency of occurrence both sampling years (see Figure 16).

It was present during the spring, early summer, and fall of both years,

but absent during August and September. A. debaryana occurred most

frequently in the 21-24 C temperature range (see Figure 15). Hughes

(1962) isolated this species (A. flagellata) all year, but most fre

quently at temperatures between 23 and 25 C. Dick and Newby (1961)

isolated this species frequently from soils in England in June and July Figure 13. Percentage of total aquatic locations sampled in north west Iowa from which Achlya americana was isolated, April through November, 1977 and 1978 53

'1977 1978 M*• « t X • » X X X X \ \ % \ X H X • V X X \ \ • X \ %

Apr May June July Aug Sept Oct Nov SAMPLING DATES Figure 14. Frequency of occurrence of Achlya americana isolated from aquatic locations sampled in northwest Iowa, April through November, 1977 and 1978. Frequency = number of isolates of A. americana isolated in a specific tempera ture range/total number of samples taken in the same temperature range

Figure 15. Frequency of occurrence of Achlya debaryana isolated from aquatic locations sampled in northwest Iowa, April through November, 1977 and 1978. Frequency = number of isolates of A. debaryana isolated in a specific tempera ture range/total number of samples taken in the same temperature range 55

100 90

70 o>- 60 UJz 50 ZD S 40 et: u_ 30

10 mÊmmm^^ammaÊÊmmÊKaàÊÊÊmmÊmmmKmÊ^m^^mmmam^ÊmmmtÊÊmammmàmmmmÊmmÊÊÊmmàÊÊmÊmÊmmÊÊÊÊma 8-12 13-16 17-20 21-24 25-28 29-32 33-38 TEMPERATURE IN DEGREES C

100 90 g 80

O W 50

S 40 a: LL. 30

10 1 8-12 13-16 17-20 21-24 25-28 29-32 33-38 TEMPERATURE IN DEGREES C Figure 16. Percentage of total aquatic locations sampled in north west Iowa from which Achlya debaryana was isolated, April through November, 1977 and 1978 57

""" 1978

Apr May June July Aug Sept Oct Nov SAMPLING DATES 58

as well as the winter months. Roberts (1963) found this species to be

more common in her summer than winter collections. It appears that

temperature rather than season per se influences the occurrence of

A. debaryana.

D. monosporus was most commonly isolated from 13-17 C water (see

Figure 18). During 1977, D. monosporus appeared to be a late spring,

early fall fungus, whereas in 1978 its peak occurrence was during the

mid-summer (see Figure 17). Coker (1923) classified D. monosporus

(D. sterile) as a summer species. Roberts (1963) and Fox and Wolf

(1977) referred to it as a constant species in their studies. Factors

other than temperature and season are influencing the occurrence of

this species.

The results of this study indicate that Aphanomyces laevis is a

summer species, and that the frequency of isolation is greatly influenced

by temperature (Figures 19 and 20). The warmer the water, the greater

the frequency of isolation. Fox and Wolf (1977) and Maestres

and Nolan (1978) isolated this species most frequently during the

summer. Roberts (1963), however, classifies this as a winter species,

and Coker (1923) isolated it most frequently in the fall.

It is interesting that, although A. laevis was isolated only from

warm water samples, it occurred much more frequently in the culture

plates under cool/dark incubation conditions. This may indicate that

the propagules of this species only germinate in cool or dark conditions

or an ecological or physiological displacement of this species in the

natural system. Figure 17. Percentage of total aquatic locations sampled in north west Iowa from which Dictyuchus monosporus was isolated, April through November, 1977 and 1978

Figure 18. Frequency of occurrence of Dictyuchus monosporus isolated from aquatic locations sampled in northwest Iowa, April through November, 1977 and 1978. Frequency = number of isolates of D. monosporus isolated in a specific tempera ture range/total number of samples taken in the same temperature range 60

100

O LU __l û. I 00 CO Z 1977 O 60 1978 < O 3 g 2 H- Z LU O w CL

r June Aùg Sept Oct Nov SAMPLING DATES

70 60 50 o>- 40 ID 30 OLU û£ U. 20 10

8-12 13-16 17-20 21-24 25-28 29-32 33-38 TEMPERATURE IN DEGREES C Figure 19. Percentage of total aquatic locations sampled in north west Iowa from which Aphanomyces laevis was isolated, April through November, 1977 and 1978

Figure 20. Frequency of occurrence of Aphanomyces laevis isolated from aquatic locations samples in northwest Iowa, April through November, 1977 and 1978. Frequency = number of isolates of A. laevis isolated in a specific temperature range/total number of samples taken in the same tempera ture range 62

100

90 «S < 80 CO

Z 70 O H- 60 — 1977 O O 50 ••19 78 I G 40 2 H- 30 g Ë 20 LU ^ 10

Mo y Aug Sept Oct SAMPLING DATES

70 g 60

>- 50 O S 40

O 30 LU S 20 10

8-12 13-16 17-20 21-24 25-28 29-32 33-38 TEMPERATURE IN DEGREES C 63

SUMMARY

The results of the culture experiments indicate that sweet clover

seed could serve as an acceptable alternative to hemp seed bait for

isolation of Saprolegnia, Aphanomyces, and Dictyuchus but not for Achlya.

In addition to an appropriate bait, there appear to be other conditions

necessary for gametangial development. There is some indication that

photoperiod is a governing factor in this process, therefore, placing

freshly baited water samples in a lighted area for several days and

then moving them to dark chambers may be the best means of obtaining

the greatest possible diversity of identifiable colonies.

The data gathered in this study indicate that certain groups of species of saprolegniaceous fungi tend to be associated with certain kinds of littoral aquatic habitats. These associations of fungi were shown to be fairly consistent in a given habitat for two years. The exact physical and chemical factors responsible for these associations remain to be investigated. The nature of the bottom, presence of emergent vegetation, and slope of the adjacent bank, however, all seem to have some influence on the saprolegniaceous fungal flora. Leaching from the soil did not affect populations of Saprolegnia and Aphanomyces in the littoral zone. Achlya and Dictyuchus, however, were isolated more frequently when there was some leaching from the soil.

In this study, members of the Saprolegniaceae possessing centric or subcentric oospores occurred most frequently in the cool (17-20 C) waters of the spring and fall. Species of the Saprolegniaceae with eccentric oospores did not correlate as well with season or temperature. 64

This group tends to be slightly more common in the summer than spring or fall. Saprolegnia ferax and Aphanomyces laevis showed distinct, temperature-related, seasonal periodicity over both years with S^. ferax being most common in the spring and fall and A. laevis most common in the summer. S^. diclina was isolated most frequently during the fall of both years. Achlya americana, A. debaryana, and Dictyuchus monosporus did not show the same seasonal pattern during the two sampling years.

This discrepancy illustrates the necessity of at least two years' data before valid conclusions regarding the seasonal periodicity of these fungi can be made. 65

PART 66

INTRODUCTION

The most difficult aspect of the field study was identification of the isolates. Specimens often did not possess the characters necessary for identification in available dichotomous keys. Isolates which did not fit the key characters exactly lessened my confidence in ability to use the keys. One isolate of Achlya at times produced mature eccen

tric oospores, but at other times the oospores aborted. The isolate could not be placed easily in either A. americana or A. conspicua. One isolate of Saprolegnia had no antheridia on some oogonia as in S. ferax and hypogynous antheridial cells on others as in hypogyna. It was frustrating also to observe a distinctive character, such as coiling antheridial hyphae or an oogonium with distinctive wall decorations, and not to be able to use these in identification.

The generic distinctions in the Saprolegniaceae are not clear-cut.

As early as 1931, Couch noted a Dictyuchus species which produced achlyoid primary sporangia. Many species of Achlya and Saprolegnia occasionally produce sporangia characteristic of other genera (Johnson

1956, Seymour 1970). The same kinds of problems arise at the species level. For example, Milanez (1970) reported an isolate of Achlya flagellata possessing the balloon-like hyphal swellings characteristic of A. diffusa. Within the family there is much variability in the limited number of morphological characters available to the taxonomist

(Dick 1972). For this reason, the worker should use as many characters as possible in identification of a specimen. This cannot be accomplished using a dichotomous key. 67

Several alternatives to dichotomous keys have been developed that

allow the worker to select the available characters for use in identi

fication. These synoptic keys include punch card systems, polyclaves, selective keys, and matching systems. A basic premise of these keys is that the one purpose is to facilitate reliable identification, and

not necessarily to reflect natural relationships or to demonstrate systematically important characters. Synoptic keys are more useful

than dichotomous keys in identifying incomplete specimens, incompletely known taxa, taxa with striking or exclusive characters, and taxa delimited by combinations of characters (Leenhouts 1966).

The oldest of these synoptic keys, a tabular form, was published by Ogden in 1943, to species of Potamogeton. Various workers have since developed punch card identification systems or polyclaves (Locquin

1967, Ola'h 1970, Weber and Nelson 1972). These keys are tedious to use because the worker must constantly shuffle, poke, and shake a packet of cards. Computer-assisted matching systems and other computer- assisted identification systems have been developed. Detailed discussion of many of these may be found in the articles in the text edited by

Pankhurst (1975). Many systems require knowledge of a large number of characters before they will work. For many fungal taxa, not enough characters are known to utilize these systems. Some computerized keys are complex, and taxonomists, naturally, hesitate to use a system if they do not understand how it operates.

In 1966, Leenhouts proposed a new kind of key but did not apply it to any particular group of organisms. Each taxon included in the key is assigned a number. The numbers of the taxa possessing a given character are listed after each character in the key. If a character is not known for a taxon, it is placed in the list in parentheses so that the user is aware of this situation. The user selects the identi fication characters and eliminates the numbers not common to all of the selected characters until only one number remains. This system has been used by Korf (1972) for genera of the Pezizales and by Trappe (1979) for hypogeous Ascomycotina. Korf (1972) noted that "some characters which may vary widely in a genus may still prove of value in keying a specimen to that particular genus when using a Leenhouts key, something that the dichotomous key can only do with great difficulty." It is easy to add new characters and new taxa to a Leenhouts key. A major difficulty with this key is that it is inconvenient to handle over 30 taxa because, beyond that number, it is too cumbersome for the worker to perform the necessary manipulations with the numbers.

The following key is a Leenhouts key, but the computer, rather than the user, manipulates the numbers. Because computers, unlike humans, can easily manipulate many numbers, any number of taxa could be included in the key with no inconvenience to the user.

The most commonly isolated saprolegniaceous genera in aquatic sys tems are Achlya and Saprolegnia. These are relatively large genera and the species are often difficult to identify. For these reasons, the following key to the species belonging to these genera is proposed.

The species included in the key are those which will appear in Johnson's

(1980) forthcoming monograph of the family, with the exception of

blelhamensis and subeccentrica which are included in S^. asterophora and truncata which is excluded because its description has not been 69

published. The characters used in creating the key were compiled from

the descriptions in the sources indicated after each species name in the key.

The brief sample program in Figure D.l demonstrates the nature of

the FORTRAN IV program used in this key. The actual program has been deposited in the Iowa State University Herbarium (I.S.C. 345609). 70

COMPUTERIZED SYNOPTIC KEY TO SPECIES OF ACHLYA AND SAPROLEGNIA

For ease of use, the key is divided into six sections, each dealing with a different morphological character. The most important distin guishing characters are marked by an asterisk. Use as many of these characters as possible.

Write down the three digit number preceding each character possessed by the fungus in question. Then count the number of characters listed and place this number at the front of the list. Repeat this process for each isolate to be identified.

Next, key punch the numbers onto computer cards. The computer has been programmed to read numbers from specific spaces on the computer card, so it is important to key punch the cards exactly as specified.

In the first two spaces on the card, punch the number of characters.

If there are fewer than 10 characters, punch a zero in the first space and the number in the second. Space once and punch one of the three digit numbers. Space again and punch another three digit number. Repeat this process until all of the three digit numbers pertaining to an isolate are punched. If more than 19 characters are used, it is neces sary to use a second card. To do this, begin the second card with a space and then a three digit number and continue as on the first card.

The second card must be placed immediately behind the first in the data deck. Begin a new card for each isolate to be identified. If, for example, an isolate has the following characters: 1) saprolegnoid spore release; 2) sporangia renewed by internal proliferation;

3) hypogynous antheridia; and 4) crenulate projections on the oogonial 71 wall, the computer card should read 04 341 361 613 485.

Replace the "PLACE DATA DECK HERE" card with the punched cards and run the program.

The computer will print out all of the species numbers for species having the designated characters as well as the species numbers of those for which a particular character has not been reported. When the latter is the case, the computer print-out will so indicate. Because the key has not received sufficient use to enable clarification of the possible taxonomic inconsistencies, the program will indicate also those species which disagree in one character from those given by the user. The computer program statement with a number corresponding to the three digit character number in the key will contain all of the species numbers for species possessing that character, if the symbol .EQ. is present in that statement. If the symbol .NE. is present, all species not listed possess the character. VEGETATIVE MYCELIUM Colony diameter at 2 weeks on hemp seed bait (measurements in cm) 101 £0.5 103 >0.5 and ^1.0 105 >1.0 and _<1.5 107 >1.5 and £2.0 109 >2.0 and ^2.5 111 >2.5 and <3.0 113 >3.0 and <3,5 115 >3.5 and _<4.0 117 >4.0 and 4.5 and ^5.0 121 >5.0

Hyphal branching 131 hyphae unbranched or sparingly branched 133 hyphae moderately to extensively branched ^

Hyphal diameter at the base of the colony (measurements in um) 141 < 20 143 > 20 and < 40 145 > 40 and < 60 147 > 60 and < 80 149 > 80 and <100 151 >100 and <120 153 >120 and <140 155 >140 and <160 157 >160

Figure 21. Computerized synoptic key to species of Achlya and Saprolegnia GEMMAE ^ 201 Present *203 spherical gemmae present (la, lb) 205 Absent

SPORANGIA AND SPORES Abundance 301 moderately to very abundant 303 rare or absent Shape 311 clavate (2a), fusiform (2b), naviculate (2c), filiform (2d), or torulose (2e) 313 spherical (2f), pyriform (2g), globular (2h), or barrel-shaped (2i) *321 Sporangia sometimes branched (3) 4 *331 Lateral discharge pores present (4) Spore release *341 saprolegnoid (5a) *343 achlyoid (5b) 345 spore cluster persistent at exit pore 347 spore cluster not persistent at exit pore *349 dictyoid (5c) *351 aplanoid (5d) *353 thraustothecoid (5e) Renewal 361 internal proliferation (6a, 6b) 363 cymose (6c), sympodial (6d), or in basipetalous succession (6e)

Figure 21. (Continued) Sporangia and spores, cont. Diameter of encysted primary zoospores (measurements in ;im) 371 <_ 9 373 > 9 and £12 375 >12 and £13 377 >13 and £14 379 >14 OOGONIA Abundance 401 moderately to very abundant 403 rare or absent Location on hyphae 411 lateral (7a) or terminal ( 7b) 413 intercalary (7c) or catenulate (7d) *415 Balloon-like lateral hyphal swellings present which do not develop into oogonia (8) Stalk characters ratio of stalk length to oogonial diameter 421 <1 423 >1 and <2 425 >2 and <3 427 >3 and <4 429 >4 and <5 431 >5 and <6 433 >6 435 stalks rarely branched 437 stalks commonly branched *439 stalks coiled

Figure 21. (Continued) Oogonia, cont. < Shape of the oogonium 441 spherical (9a), ovate (9b), obovate (9c), pyriform (9d), or globose (9e) 443 fusiform (9f), filiform (9g), doliform (9h), or developing in old sporangia (9i) 445 angular (9j) 447 terminally naviculate (9k) or apiculate (91) Diameter or length of terminar and lateral oogonia including ornamentations (measurements in um) 451 £ 25 453 > 25 and 1 50 455 > 50 and 5 75 457 > 75 and £100 459 >100 and 1125 461 >125 and 1150 463 >150 and 1175 465 >175 and 1200 10 467 >200 Oogonial wall *471 moderately to abundantly pitted (lOa) *473 unpitted or pitted only under antheridia *475 wall thick (usually 2 pm) *477 inner wall irregular *479 wall yellow in color 481 wall decorations absent shape of wall decorations when present: *485 papillate (10b), crenulate (10c), or wavy (lOd) *487 tuberculate (lOe) *489 bullate (lOf) or truncate (lOg) *491 mammiform (lOh) *493 spiny (lOi)

Figure 21. (Continued) OOSPORES *501 Oospores frequently not maturing or breaking down immediately on maturation 503 Oospores usually maturing 11 *505 Mature oospores centric (11a) *507 Mature oospores subcentric (lib, lie) *509 Mature oospores eccentric (lid) or subeccentric (lie) Number of oospores per oogonium *521 1 *523 2-5 *525 6-10 *527 11-15 *529 16-20 *531 21-25 *533 26-30 *535 more than 30 Diameter of oospores (measurements in pm) 541 <15 543 >15 and <20 545 >20 and <25 547 >25 and <30 549 >30 and <35 551 >35 and <40 553 >40

ANTHERIDIA Abundance 601 moderately to very abundant 603 rare or absent

Figure 21. (Continued) Antheridia, cont. 12 Antheridial hyphae source *611 exigynous (12a) 1 *613 hypogynous (12b) — *615 androgynous (12c) *617 monoclinous (12d) *619 diclinous (12e) branching 621 simple or sparingly branched 623 moderately to profusely branched ]3 *631 antheridial hyphae coiling or wrapping around oogonal stalk or vegetative hyphae *633 antheridial hyphae contorted or irregular (13) number of antheridial hyphae per oogonium 635 1 or 2 637 more than 2 Antheridial cells 641 simple, unbranched (14a) 643 branched (14b), compound (14c), or intercalary (14d) shape 651 clavate (15a) or ovate (15b) 653 tubular (15c) 655 lobed (15d) or irregular (15e) attachment to oogonium 661 apical (16a) 663 lateral (16b) 665 attached by projections (16c)

Figure 21. (Continued) Key # Species Descriptive sources

1 Achlya abortiva Coker & Braxton Coker & Matthews (1937), Johnson (1956), Ziegler (1958) 2 A. achlyoides (Coker & Alexander) Johnson^ Coker (1927), Coker & Matthews (1937), Beneke (1948a) 3 A. ambisexualis Raper Raper (1939), Beneke (1948a), Johnson (1956) Chiou et al. (1975) 4 A. americana Humphrey Humphrey (1893), Coker (1923), Beneke (1948a) Johnson (1956), Ziegler (1958), Johnson & Seymour (1974) 5 A. androgyna (Archer) Johnson & Seymour Archer (1867), Humphrey (1893), Coker (1923), Johnson (1956), Ziegler (1958), Howard et al. (1970), Lui & Volz (1976), Johnson (1980) 6 A. apiculata deBary Coker (1923), Johnson (1956), Ziegler (1958), Milanez (1965 & 1970), Beroqui de Martinez (1969a), Howard et al. (1970), Chiou et al. (1975) 7 A. bisexualis Coker & Couch Coker (1927), Coker & Matthews (1937), Beneke (1948a), Johnson (1956) 8 A. bonariensis Beroqui de Martinez Beroqui de Martinez (1969b) 9 A. cambrica (Trow) Johnson Johnson (1956), Howard et al. (1970) 10 A. caroliniana Coker Coker (1910 & 1923), Forbes (1935), Johnson (1956), Ziegler (1958), Elliot (1967), Chiou et al. (1975)

^If the authority is Johnson, and Johnson (1956) is not cited as a descriptive source, the species will be transferred to this genus in his forthcoming monograph, and I have not seen the new description.

Figure 21. (Continued) Key # Species Descriptive sources 11 A. colorata Pringsheim Coker (1923), Beneke (1948a), Johnson (1956), Ziegler (1958) 12 A. crenulata Ziegler Ziegler (1948b & 1958), Johnson (1956) 13 A. debaryana Humphrey Humphrey (1893), Coker (1923), Forbes (1935), Beneke (1948a), Johnson (1956 & 1968), Ziegler (1958), Milanez (1968), Howard et al. (1970), Chiou et al. (1975) 14 A. diffusa Harvey ex Johnson Harvey (1942), Johnson (1956), Howard et al. (1970) 15 A. glomerata Coker Coker (1923), Johnson (1956) 16 A. heterosexualis Whiffen-Barksdale Barksdale (1965) 17 A. irregularis (Coker & Ward) Johnson Ward (1939) 18 A. inflate Coker Coker (1927), Coker & Matthews (1937), Johnson (1956) 19 A. intricata Beneke Beneke (1948b), Johnson (1956), Howard et al. (1970) 20 A. lobata Ziegler & Gilpin Ziegler & Gilpin (1954), Johnson (1956) 21 A. megasperma Humphrey Humphrey (1893), Coker (1923), Johnson (1956), Ziegler (1958) 22 A. oblongata deBary Coker (1923), Johnson (1956), Ziegler (1958) 23 A. oligacantha deBary Coker (1923), Johnson (1956) 24 A. orion Coker & Couch Coker (1923), Beneke (1948a), Johnson (1956), Ziegler (1958) 25 A. ornata (Dick) Johnson Dick (1960)

Figure 21. (Continued) Key ^ Species Descriptive sources

26 A. oviparvula Rogers & Beneke Rogers & Beneke (1962) 27 A. papillosa Humphrey Humphrey (1893, Coker (1923), Johnson (1956 & 1968), Howard et al. (1970) 28 A. polyandra Hildebrand Coker (1923), Johnson (1956) 29 A. prlmoachlya (Coker & Couch) Johnson Coker & Couch (1924), Coker & Matthews (1937), Beneke (1948a) 30 A. proliféra (Nees) deBary Coker (1923), Beneke (1948a), Johnson (1956), Ziegler (1958), Howard et al. (1970) 31 A. proliferoides Coker Coker (1923), Beneke (1948a), Johnson (1956), Ziegler (1958), Chiou et al. (1975) 32 A. pseudoachlyoides (Beneke) Johnson Beneke (1948a) 33 A. racemosa Hildebrand Coker (1923), Beneke (1948a), Reischer (1949), Johnson (1956 & 1973), Ziegler (1958) 34 A. radiosa Maurizio Coker (1923), Johnson (1956), Beroqui de Martinez (1969b), Milanez (1969), Howard et al. (1970), Johnson et al. (1975) 35 A. recurva Cornu Coker (1923), Latham (1935), Johnson (1956), Ziegler (1958), Milanez (1970), Chiou et al. (1975) 36 A. rodrigueziana Wolf Wolf (1941), Beneke (1948a), Johnson (1956), Ziegler (1958) 37 A. spinosa deBary Coker (1923), Johnson (1956 & 1980) 38 A. stellata deBary Coker (1923), Johnson (1956 & 1980, Johnson et al. (1975)

Figure 21. (Continued) Key # Species Descriptive sources 39 A. subterranea Coker & Braxton Coker & Matthews (1937), Johnson (1956) 40 Saprolegnia anisospora deBary Humphrey (1893), Coker (1923), Newby (1948), Seymour (1970), Hallett & Dick (1976) 41 asterophora deBary Humphrey (1893), Coker (1923), Beneke (1948a), Dick (1969), Howard et al. (1970), Seymour (1970), Chiou & Chang (1976) 42 australis Elliot Elliot (1968), Padgett (1976 & 1978b), Nolan & Maestrès (1978) 43 diclina Humphrey Humphrey (1893), Coker (1923), Harvey (1942), Beneke (1948a), Ziegler (1958), Howard et al. (1970), Milanez (1970), Seymour (1970), Thakur Ji (1970), Chiou et al. (1975), Hallett & Dick (1976) 44 accentrica (Coker) Seymour Coker (1923), Seymour (1970) 45 ferax (Gruithusen) Thuret. Coker (1923), Harvey (1942), Beneke (1948a), Howard et al. (1970), Seymour (1970), Chiou et al. (1975), Hallett & Dick (1976) 46 furcata Maurizio Coker (1923), Seymour (1970) 47 glomerata (Tiesenhausen) Lund Seymour (1970) 48 S. hypogyna (Pringsheim) deBary Coker (1923), Seymour (1970), Howard et al. (1970) 49 irregularis Johnson & Seymour Johnson & Seymour (1975) 50 S. itoana (Nagai) Johnson Seymour (1970), Chiou et al. (1975) formerly S^. subterranea (Dissmann) Seymour

Figure 21. (Continued) Key ^ Species Descriptive sources

51 S. litoralis Coker Coker (1923), Beneke (1948a), Ziegler (1958), Howard et al. (1970), Seymour (1970) 52 S^. luxurians (Bhargava & Srivastava) Seymour Seymour (1970) 53 megasperma Coker Coker (1923), Beneke (1948a), Ziegler (1958), Seymour (1970) 54 richteri Richter ex Seymour Seymour (1970) 55 S^. terrestris Cookson ex Seymour Elliot (1968), Howard et al. (1970), Seymour (1970), Chiou et al. (1975) 56 torulosa deBary Humphrey (1893), Coker (1923), Beneke (1948a) Howard et al. (1970), Seymour (1970) 57 £. turfosa (Minden) Gaumann Coker (1923), Beneke (1948a), Johnson (1968), Howard et al. (1970), Seymour (1970) 58 unispora (Coker & Couch) Seymour Coker (1923), Seymour (1970), Volz et al. (1974) 59 uliginosa Johannes Johannes (1950), Howard et al. (1970), Seymour (1970)

Figure 21. (Continued) 83

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ACKNOWLEDGMENTS

I wish to express my sincere gratitude to Dr. Lois H. Tiffany for

her guidance and encouragement throughout this study and, more impor

tantly, for instilling in me a positive attitude toward research and

teaching. I wish to thank Dr. Don Norton for his valuable editorial

assistance and the other members of my committee. Dr. John D. Dodd,

Dr. John Holt, Dr. Robert Chapman, and Dr. Roger Landers, for their

help. Finally, I would like to thank Dr. T. W. Johnson, Jr., for

generously providing so much information on the taxa and for answering my numerous questions. 93

APPENDIX A. COLLECTION DATES AND WATER TEMPERATURES

« Collection trip # 1 2 3 4 1977 4/9 5/6 5/25 6/15 Date 1978 4/8 4/29 5/20 6/10 Site

Mud Lake 1977 17 22 27.5 22.5 1978 9 12 18.5 26

Little Sioux River 1977 17 23 31 22 at Horseshoe Bend 1978 10 11 20 21

Lake Minnewashta 1977 12 21 25 23 1978 10 11 21 22

East Lake Okoboji 1977 10 21 24 23.5 1978 9 10.5 18 21

Spirit Lake 1977 12 22 26 24.5 1978 10 10.5 18.5 21

Jemmerson Slough 1977 23 29 No measure 31 1978 14 14 22 23

Silver Lake 1977 13 22 30 28 1978 11 12 20 24

Little Sioux River 1977 20 25 26 27 at Highway 9 1978 12 12 19 22

Triboji Lagoon 1977 16 22 27 24.5 1978 10 11 21 23

Mean temperature 1977 15.55 23 27.1 25.11 1978 10.55 11.55 19.78 22.55

Temperature range 1977 10-23 21-29 24-31 22-31 1978 9-14 10.5-14 18.5-22 21-26

Figure A.l. Water temperatures on all sampling dates from aquatic sites in northwest Iowa, April through November of 1977 and 1978 95

5 6 7 8 9 10 11 7/7 7/27 8/12 9/3 9/23 10/15 11/5 6/30 7/21 8/11 9/2 9/23 10/14 11/4

38 31 26 21 15 11 11 30 23 26 23.5 17 12 12.5

33 24.5 dry 23 15.5 10 8.5 27 22 22 26 18.5 11 12

32 27 26.5 21 19 11 11 29 26 20 25 21 13.5 13

32 26 27 22 18.5 10.5 11 27 25 20 25 19.5 13.5 11

31 21 26 22.5 17.5 10 12 29 24 18 25 21 13 12

dry dry dry dry dry 9 12 33.5 24.5 17.5 28 19 15 12

30.5 28 31 28 16 11 12 29 20 19 27 18 12 14

25 dry dry dry 15.5 9 9 27 18.5 23 28 20 13 13

34 29.5 29 22 17 9 10 29 20.5 25 26 19.5 12.5 11.5

31.94 26.71 27.58 22.79 16.75 10.06 10.72 28.94 22.61 21.16 25.94 19.28 12.83 12.33

25-38 21-31 26-31 21-28 15-19 9-11 8.5-12 27-33.5 18.5-26 17.5-26 23.5-28 17-21 11-15 11-14 96

APPENDIX B. PRECIPITATION AND TEMPERATURE DATA FOR NORTHWEST IOWA, 1976-1978 97

1976 1977 1978 Average Departure Average Departure Average Departure

Temperatures January 17.5 + 1.7 5.4 -10.0 4.6 -11.2 February 30.0 +10.0 26.5 + 5.6 10.1 -10.8 March 34.2 + 3.2 38.2 + 7.2 31.0 0 April 52.2 + 4.9 54.2 + 6.9 46.2 - 1.1 May 58.0 - 1.0 67.0 + 8.0 59.7 + 0.7 June 69.8 + 1.3 70.6 + 2.1 69.2 + 0.7 July 74.7 + 1.6 74.8 + 1.7 72.7 - 0.4 August 72.9 + 1.4 67.9 - 3.6 71.1 - 0.4 September 63.0 + 1.3 63.1 + 1.4 67.4 + 5.7 October 44.6 - 7.1 48.5 - 3.2 49.7 - 2.0 November 28.7 - 6.0 33.0 - 1.7 31.6 - 3.1 December 16.6 - 5.0 17.9 - 3.7 15.2 - 6.4

Precipitation

January .16 - .43 .22 - .37 .38 - .21 February .67 - .29 .62 - .34 .53 - .43 March 3.13 +1.56 4.13 +2.56 .40 -1.17 April 1.60 - .75 3.18 + .83 3.59 +1.24 May 2.21 -1.66 3.11 - .76 2.61 -1.26 June 2.72 -2.12 3.08 -1.76 3.19 -1.65 July 1.49 -2.10 4.25 + .66 7.94 +4.35 August .82 -2.54 4.03 + .67 3.86 + .50 September 2.19 - .84 4.66 +1.63 2.77 - .26 October .97 - .79 3.36 +1.60 .61 -1.15 November .07 - .88 2.44 +1.49 1.11 + . 16 December .43 - .37 1.00 + .20 .70 - .10

Figure B.l. Average monthly temperature and precipitation for northwest Iowa, 1976 through 1978 (N. 0. A. A. 1976-1978) 1977 Mar Apr May June July Aug Sept Oct Nov

1 ,15 .07 2 .39 .62 .05 .25 3 .27 ,04 ,72 xa 4 T .02 ,32 .05 5 .02 ,06 xa 6 T xa .20 T 7 .04a T 2.72 1.34 .05 8 T .04 .07 .09 .12 9 xa T .62 T 3.00 10 .23 .44 11 1.17 T .62 T T T 12 .36 .18 xa .24 13 T .62 14 T .11 .18 T 15 .27 1.92* .02 2.87 .04a T 16 T T 1,04 .06 17 .07 .06 .24 .52 .09 18 .07 T T T ,06 19 .08 .58 ,08 T 20 .05 .62 .20 T T .09 21 .12 ,32 .23 .02 .15 T T 22 ,13 .02 .04 ,22 T 23 .15 .68* .04 .07 24 .03 .09 .11 .25 25 X= ,15 26 T ,09 T T 27 .04 T .11 .03 xa T 28 .72 ,06 .04 .10 T 29 .11 T T .98 30 .05 .44 .25 2.24 1.25 .04 31 .54

^Sampling day.

Figure B.2. Daily precipitation at the Milford, Iowa, Station from March through November of 1977 and 1978 (T = trace of precipitation; X = sampling day with no precipitation) 99

1978 Mar Apr May June July Aug Sept Oct Nov

1 T .46 T 2 .17. .06 T X® .18 .10 3 T .16 .48 4 T 1.55 xa 5 T .09 .65 .01 6 T 1.00 .75 7 .10 .10 .93 8 .083 .45 .05 T 9 .15 T T .05 10 T .11 Xa T T 11 Xa T 12 T .07 T .13 13 .21 .27 T .70 .05 T 14 T .18 .13 xa 15 .06 1.12 16 .10 .10 17 .33 18 1.22 .07 .23 .09 19 .06 .02 .72 T 20 .06 xa 2.25 .63 .12 .24 21 1.46* .01 22 .16 2.23 T 23 .14 T .02 xa T 24 .09 T

25 00 T .02 26 .45 .08 .45 T 27 .24 .02 28 T .04 .02 .05 .08 29 .02* .14 1.02 T .05 T 30 .09 .21 .043 .30 .06 31 T 100

APPENDIX C. SIMILARITY INDICES 101 River 9 Minnewashta Lake Spirit Lake Jemmerson Slough Silver Lake Lake at Highway at Horseshoe Bend Triboji Lagoon Little Sioux River Little Sioux Mud

East Lake Okoboji 38 44 33 50 24 33 46 61

Spirit Lake 67 59 47 67 50 59 67

Lake Minnewashta 67 59 35 80 50 47

Silver Lake 60 73 64 60 57

Jemmerson Slough 53 67 48 63

Mud Lake 66 80 50

Triboji Lagoon 50 64

Little Sioux River /U at Horseshoe Bend

Figure C.l. S^renson's Index of Similarity for species isolated from all possible pairs of aquatic sites sampled in northwest Iowa from April through November of 1977 and 1978 (Mueller-Dombois and Ellenberg 1974) 102

M 0) a> > > a 42 to •rt . o ss cB Ai f4 « •H (0 (-5 c to c « CO & CA 0) 0) o c Hi Xi CO •H Ai CO Ej 0) U 4J •H fH Ai •H •r) -W •i-l 4J W 3 (U •H to A 1-4 CO n) H S CO •J M

East Lake Okoboji 9.1 9 .4 3.6 14.6 3.4 6.9 22. 23.4

Spirit Lake 14.2 11.8 4.3 19.4 5.6 16.1 31.

Lake Minnewashta 20.2 9.8 3.8 40.5 8.4 8.8

Silver Lake 33.3 33.9 20.9 19.3 22.1

Jemmerson Slough 21.3 35.1 18.7 23.3

Mud Lake 24.2 35.7 14.9

Triboji Lagoon 18.9 21.1

Little Sioux River 27.8 at Horseshoe Bend

Figure C.2. Spats Index of Similarity for species isolated from all possible pairs of aquatic sites sampled in northwest Iowa from April through November of 1977 and 1978 (Mueller- Dombois and Ellenberg 1974) 103

APPENDIX D. COMPUTER PROGRAM FOR A HYPOTHETICAL SYNOPTIC KEY 104

IMPLICIT INTEGER (A-Z) DIMENSION CNTR (20), N0TDEF(20), CHAR(39) 10 DO 12 SAP=1,20 CNTR(SAP)=0 N0TDEF(SAP)=0 12 CONTINUE 14 READ (5,15,END=925) N, (CHAR(I),I=1,N) 15 FORMAT (12, 19(14),/,20(14)) DO 999 1=1,N 100 DO 198 SAP=1,20 IF(CHAR(I).EQ.101) GO TO 101 GO TO 102 101 IF(SAP.EQ.1.0R.SAP.EQ.2,0R.SAP.EQ.3) CNTR(SAP)=CNTR(SAP)+1 IF(SAP.EQ.2) NOTDEF(SAP)=NOTDEF(SAP)+1 GO TO 198 102 IF(CHAR(I).EQ.103) GO TO 103 GO TO 104 103 IF(SAP.EQ.3.0R.SAP.EQ.15.0R.SAP.EQ.5) CNTR(SAP)=CNTR(SAP)+1 GO TO 198 104 IF(CHAR(I),EQ.105) GO TO 105 GO TO 198 105 IF(SAP.EQ.17.0R.SAP.EQ.4.0R.SAP.EQ.3) CNTR(SAP)=CNTR(SAP)+1 IF(SAP.EQ.17) NOTDEF(SAP)=NOTDEF(SAP)+1 198 CONTINUE GO TO 999 999 CONTINUE WRITE (6,902) 902 FORMAT ('0 NEXT ISOLATE') DO 910 SAP=1,20 IF(CNTR(SAP).EQ.N) GO TO 905 GO TO 910 905 WRITE (96,906) SAP, NOTDEF(SAP) 906 FORMAT ('SPECIES NUMBER',13,' FITS ALL CHARACTERS GIVEN WITH', -13,' OF THE CHARACTERS GIVEN NOT DEFINED FOR THIS SPECIES') 910 CONTINUE GO TO 10 925 CONTINUE STOP END

Figure D.l. Portion of a FORTRAN IV computer program for a synoptic key to 20 taxa