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THE BIOLOGY AND ECOLOGY OF CO-EXISTING SPECIES OF POLYCENTROPUS
(TRICHOPTERA: POLYCENTROPODIDAE) IN A MICHIGAN BOG LAKE
Alice J. Loesch Anderson
A Dissertation
Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
June 1979 il
ABSTRACT
An analysis of the life history, morphology, behavior and niche
separation of coexisting species of Polycentropus caddisflies was
performed on data collected over three years, 1976, 1977, and 1978.
Collections were made from a bog lake to simplify the potential inter
actions in the community.
It was found from adult emergence data that three species were
present, P_. interruptus, P_. remotus, and P. flavus, and that differing
emergence time was adequate to separate the niche of only P_. flavus
from the other two species. From analysis of the larval collection
data, it was found that P^ interruptus dominated at stations which were
in open water and free from vegetation, and that P_. remotus larvae
dominated at stations close to shore, or in deep flocculent peat with
vegetation. Further analysis of these differences showed that P_.
remotus actually had two color types, one with spots on the parietal
sclerites of the head, and one without, and that these two color types
were separated by differential utilization of substrate. The light-
colored P_. remotus utilized aquatic plants for net-building almost
exclusively, thus influencing the difference in substrate utilization
between P_. remotus and P_. interruptus considerably.
In multivariate analyses of morphological characters and two
environmental characters, it was found that P^. interruptus and P_. remotus
were most clearly separated morphologically by a difference in setal pattern on the pronotum. P. interruptus, and the two color variants Ill
of P_. remotus were separated most clearly by setal pattern on the
pronotum, color pattern, and collection site. Collection site was
important because transects of lily pads where hand picking was carried
out were included in the analysis, and these were where the light color
variant of P_. remotus were dominant. More data on physical and bio
logical characteristics at different stations, and at different levels
at each station need to be collected to distinguish the exact mechanism of separation between the dark color variant of P_. remotus and P^. inter-
rùptus. From observational data, it is hypothesized that the P_.
interruptus larvae utilized a deeper level within the detritus for net building and feeding as well as pupating than did the dark color variant of 1?. remotus. If 3?. interruptus were able to tolerate lower C>2 conditions found within the detritus, an interesting and unusual niche separating mechanism would be exemplified for lentie species.
Scanning electron microscopy and gut analysis yielded inconclusive results. No differences were found in the net structure and attachment sites between species, but subtle differences in mouthpart structure were found. Gut analysis indicated that P_. interruptus was more exclusively a predator, and more often a predator of Chironomidae.
P_. remotus was found to contain parts of Corrixidae, which were in young stages and plentiful at the time of collection of the larvae. IV
ACKNOWLEDGEMENTS
Many people helped and influenced me during the course of this project. I would like to acknowledge the able assistance of the following people in identifying insects, ecological problems, techniques, and statistical analyses: Dr. Todd Harris, Dr. G. Wiggins, Dr. R.
Mackay, Dr. R. Stein, Dr. J. R. Voshell, Dr. R. Loesch, Dr. R. K.
Tucker, Dr. R. C. Graves, Dr. R. Lowe, Dr. M. H. Hohn, Dr. W. Easterly,
Di. R. E. Crang,, and Dr. G. Heberlein. I also wish to specially thank the following fellow graduate students and friends for encouragement and help: G. Bernon, B. Peck-Lewis, M. Bruno, R. Grimm, B. Walker,
Dr. Win. Baxter, B. Silcox, H. Silcox, and all the Beaver Island crew, particularly P. Cupp. The intangible support of these and many others can only be thanked by my deepest appreciation, and the knowledge that
I have come a long way, which would not have been possible without them. V
TABLE OF CONTENTS
Page
INTRODUCTION...... 1
Life History Importance in Ecological Studies...... 1
Bog Lakes: A Simpler System...... i...... 3
Polycentropus Information in the Literature...... 4
Predation in Polycentropus...... 5
Niche Analysis...... 7
Multivariate Statistical Analysis...... 9
Summary...... 10
MATERIALS AND METHODS...... 11
Field Location...... 11
Field Methods...... 16
Sampling Methods...... 26
Laboratory Methods...... 31
Behavioral Observations...... 35
RESULTS AND DISCUSSION...... 38
Barneys Lake and Greens Lake Comparison...... 39
Adults of P_. interruptus, P_. remotus, and P^. flavus...... 41
Morphology of Polycentropus Adults...... 48
Niche Separation Based on Adult Emergence...... 49
Morphology of Polycentropus Larvae...... 49
Life History Analysis of P_. interruptus and P_. remotus...... 63
Statistical Analysis of Morphological and Ecological Data.... 64
Factor Analysis, Discriminant Analysis of Morphological and Ecological Variables...... 74 VI
Page
Scanning Electron Microscopy: Mouthpart Morphology...... 35
Scanning Electron Microscopy: Net Structure and Attachment Sites...... 114
Feeding Behavior and Territorialism...... 117
Feeding Behavior and Gut Analysis...... 123
Summary of Results...... 130
Discussion of Evolution and Adaptation in Polycentropus.... 133
CONCLUS IONS...... 139
LITERATURE CITED...... 144 Vll
LIST OF TABLES
Table Page
1. Families of Trichoptera found in Barneys Lake and Greens Lake, 1976...... 40
2. Numbers of Polycentropus larvae collected stations in Barneys Lake, 1976...... 42
3. Numbers of Polycentropus adults collected at stations in Greens Lake, 1976...... 44
4. Number of Polycentropus adults collected at stations in Greens Lake, 1977...... 46
5. Larval instars head capsule sizes for interruptus and P_. remotus...... 67
6. Station types and station numbers in GreensL ake, 1977.. 68
7. Numbers of Polycentropus species in four collection types...... 73
8. Principal components analysis factor loadings...... 78
9. Discriminant analysis DF coefficients...... 83
10. Analysis two: Two taxonomic groups...... 84
10a. Density of Polycentropus larvae on artificial substrate 2 samplers (per cm )...... 121
11. Frequency of larvae in lily pad areas...... 122
12. Average spacing distances of Polycentropus larvae.... , . 123
13. Types of food materials in gut of Polycentropus larvae.. 127 viii
LIST OF FIGURES
Figure Page
1. Outline map of Beaver Island with location of the two lakes sampled for Polycentropus: Greens Lake, an acid bog lake, and Barneys Lake, an alkaline bog lake. Inset map shows location of Beaver Island in northern Lake Michigan...... 13
2. Greens Lake showing one depth contour, within which a depth of more than 1 m is reach. An extensive bog mat is also indicated...... 15
3. Barneys Lake showing a less extensive bog mat than Greens Lake. Survey sampling only was done at this lake...... 18
4. Location of stations in Greens Lake, 1976 and 1977. Outlined areas in and around the lake are bog mats.... 20
5. Sampling types and times in 1976 and 1977. Artificial substrates were in place for five weeks. The beginning of the bar represents the set-up time; the end repre sents removal time. Other squares represent weeks during which sampling took place...... 22
6. Detailed habitat map of "Boat Launch Bay" in Greens Lake. Extensive sampling along transects was done in May, 1978...... 25
7a. Artificial substrate samplers were constructed of small and medium cottage cheese containers cut into slats. Two 2x2 cm. squares of conservation webbing were placed inside...... 30
7b. Emergence trap samplers were constructed of plastic and screen cones with removable museum jar tops. These were submerged with a 5-7 cm. bubble of air left in the jar for emerging insects...... 30
8-1. Aerial photograph of Greens Lake "Boat Launch Bay" from the southeast. Note the areas of bog-building and vegetation...... 33
8-2. Aerial photograph of Greens Lake "Boat Launch Bay" from the northwest. Note the bog edge on the east side of the island (top of picture) 33 Figure Page
8- 3. Emergence trap and artificial substrate in place in Barneys Lake. Note the vegetation: Nymphaea, Scripus, and others...... 33
9- 1. P_. remotus in culture vessel in aquarium. Net was built on the side of the vessel near the top...... 38
9-2. P_. remotus in culture vessel after just capturing a chironomid larva (at arrow)...... ’.. 38
9-3. Vessels used for net-building SEM preparations. "Hach kit" chemical containers were lined with coverslips so that larvae would attach nets to be coverslips. These could then be observed for attachment sites...... 38
10. Adult genitalia of three species of Polycentropus found at the Greens Lake in 1976 and 1977. (From Ross, 1944)...... ,...... 52
11. Emergence of P_. remotus, P_. interruptus, and P_. flavus in 1976 and 1977 combined...... 54
12. Generalized emergence pattern of P_. remotus, P_. inter ruptus and P_. flavus. Temporal separation only is adequate to separate P_. flavus from the other two species...... 55
13a. P_. interruptus larval morphology. Head color is dark with dark spots, pronotum is dark, and anal appendages has hairs in a straight light...... 58
13b. P_. remotus larval morphology, spotted type. Head light with dark spots. Main irons setae with dark color at base, anal appendage hairs thick and patchy...... 58
13c. P_. remotus larval morphology, non-spotted type. Head light with no spots except a few on the irons. Hairs on anal appendages thick and patchy...... 58
14-1. Labrum of P_. remotus. Apparent protrusion in the center is due to extreme curvature of the labrum...... 61
14-2. Labrum of P_. interruptus...... 61
14-3. Genitalia and eggs of P^. remotus. Eggs measured .2-. 3 pm corresponding to the head capsule size of the first instar larvae...... 61
14-4. Genitalia of P_. interruptus...... 61 X
Figure '' Page
15. Sizes and numbers of head capsules for P. remotus and P_. interruptus. P. ? is the non-spotted remotus type...... 65
16. Larvae encountered at stations in 1977...... 69
17. Larvae encountered at four general areas in Greens Lake in 1976 and 1977...... 71
18. Plot of factor scores as related to Factor I and Factor II. Two groups can be seen based on relationship to Factor 1...... 79
19. Plot of factor scores as related to Factor I and Factor III. Three groups can be seen in this plot: one in relation to factor I, and another in the right hand Factor I group, based on III...... 81
20-1. Dorsal view of mouthparts of P^. interruptus (lOOx).... 89
20- 2. Ventral view of mouthparts of P. interruptus C200x).... 89
21- 1. Dorsal view of mouthparts of P_. remotus (200x)...... 91
21- 2. Ventral view of mouthparts of P_. remotus (200x)..... 91
22- 1. Ventral view of P_. remotus, non-spotted type (200x).... 93
22- 2. Ventral view of P_. remotus, non-spotted type, detail (500x)...... 93
23- 1 Tip of labium of P_. remotus, non-spotted type, showing labial palps and silk gland opening (_2,000x)...... 95
23- 2. Tip of labium of EL interruptus showing labial palps and silk gland opening (2,000x)...... 95
24- 1. Dorsal view of the tip of the labium of P_. remotus, spotted type, showing labial palps (2,000x)...... 97
24- 2. Ventral view of the tip of the labium of P^. remotus spotted type, showing labial palps (2,000x)...... 97
25- 1. Net of P_. remotus in battery jar, incorporating Utri- cularia (5x).
25-2. Large attachment area of Polycentropus net (500x)..... 99 XI
Figure ' Page
26-1. Pupal case of P_. remotus on the side of a culture vial (approx.lx)...... 101
26- 2. SEM of end piece of pupal case showing perforations and threads (8,000x)...... 101
27- 1. Net of P_. remotus as it appears in the field attached to Dulichium leaves (approx, lx)...... 103
27- 2. Net of P_. interruptus as it appears in the field, attach ed to dead leaves on the bottom (approx, lx)...... 103
28- 1. Nuphar as it appears in Greens Lake...... 105
28-2. Dulichium growing in shallow area in Greens Lake...... 105
28-3. Net of 1?. remotus as typically found under Nuphar leaves...... 105
28-4. Net of P_. remotus attached to dead Dulichium leaves in area like Figure 28-2...... 105
28-5. Air-dried preparation of nets showing flattened threads and mucilaginous background (8,000x)..... 107
28- 6. Critical point dried preparation of nets showing single and double threads, less flattening, and no mucilagin ous background (5,000x)...... 107
29- 1. Attachment site after one hour of building time (4,000x). 109
29-2. Attachment site after four hours building time (2,000).. 109
29-3. Attachment site after six hours building time (1,500)... 109
29-4. Attachment site after six hours building time, detail (4,000x)...... 109
29-5. Attachment site after twelve hours (l,500x)...... Ill
29-6. Attachment site after one day (2,000x)...... Ill
29-7. Detail of attachment site after one day (5,000x)...... Ill
29-8. Small attachment area in one week building time (5,000x) 111
29-9. Threads of nets after one week (2,500x)...... 113 Xll
Figure ' Page
29-10. Non-cleaned net with feathery extensions (l,000x)..... 113
29-11. Detail of feathery extensions (10,000x)...... 113
29- 12. Feathery extensions on cleaned net after six hours (l,500x)...... 113
30- 1. Gut contents of P_. interruptus, Ceratopogonid head (50x)...... 129
30-2. Gut contents of P_. remotus, Corixid tarsus (50x)..... • 129
30- 3. Gut contents of P_. remotus, Corixid compound eyes (lOOx)...... '...... 129
31- 1. Summary of ecological and physiological adaptation of Trichoptera in the superfamily, Hydropsychoideae...... 138 1
INTRODUCTION
In this research the differences between two species of caddisfly
larvae (Polycentropus remotus and EL interruptus) and three species
of adults (EL remotus, P_. interruptus and EL flavus) were investigated.
The morphology of these larvae and adults are described and mor
phological measurements of the larvae analyzed. Ecological aspects
of the distribution of larvae and adults are discussed, as are larval
net-building and feeding behavior.
Life History Importance in Ecological Studies
Most descriptions of generalized Trichoptera life histories are
based on studies of the family Limnephilidae (Resh, 1976a). Recently,
however, departures from this type of life cycle have been published
(Winterbourn, 1971; Solem, 1974; Resh, 1976a; Mackay, 1977; Maias and
Wallace, 1977; Wiggins, 1977). Solem, for example, found that activity
of Trichoptera was not always nocturnal as had been believed, and that
in some species diel rhythms of larvae may differ from those of adults.
Other studies have shown exceptions to the limnephilid pattern of five
stadia with a short emergence time in mid-summer and overwintering .
fifth stadia. Resh (1976a) found 2 cohorts in Ceraclea transversa, with different emergence periods, flight activity, and adult survival
distinctness. Workers whose aim was to illustrate differentiation between closely related species of Trichoptera have indicated that
separation occurs in time of emergence and stadium pattern (Mackay and
Kalf, 1973; Istock, 1973); substrate preference, stream velocity and net mesh size (Maias and Wallace, 1977); substrate, distribution of
food, and stream current velocity (Cummins, 1964). As the goal of
these workers was to show mechanisms for separation of closely related
species, divergence from a generalized life cycle was assumed &_ priori.
The importance of specific and detailed life history description
was stressed by Resh (1976a) as preliminary to any other kind of
ecological work with insects. Campbell and Meadows (1972) stated that
it is important to determine species and exact habitat location before
doing experimental or observational analyses of habitat selection,
distribution, or niche separation. Istock (1973) stated that diffi
culties involved in data-model matching may be "partly overcome through more extensive empirical study of the detailed dynamics, pattern, and natural history of species assemblages." 3
Bog Lakes; A Simpler System
Life history patterns represent a series of selective compromises
to a suite of environmental variables (Wilber, Tinkle and Collins,
1974) . A major problem in analyzing these patterns and comparing them
in terms of specific habitat selection and species differences is the
complexity of the habitat and of the niche (both terms used as defined
by Whittaker, Levin, and Root, 1973). Islands have been utilized as
isolated systems where complexities are reduced (Colwell and Fuentes,
1975) . Similarly, in aquatic studies, bog lakes provide a habitat that
is less complex (McLachlan and McLachlan, 1975; Wetzal, 1975). In a
comparison of acid Lake Gribso and eutrophic Lake Esrom in Norway, Berg and Peterson (1966) found that Lake Gribso contained 3 to 4 times less
species richness and productivity than Lake Esrom. In addition, bog lakes have an allochthonous detritus-based food chain (Griffiths,
1973). This considerably simplifies biological interrelationships as the allochthonous source is a fairly uniform bog mat with comparatively low diversity. Also, decay of detritus is retarded in acid bog water
(Wetzal, 1975) . These factors all result in a reduction of food availability and diversity at the base of the food web, and subsequent reduction in the complexity of the entire food web. For all the pro ceeding reasons a bog lake was chosen for the detailed analysis of species differences in this research. 4
Polycentropus Information in the Literature
Very little work has been done on the fauna of bog lakes including the life history of any species of Polycentropodidae found in bog lakes. Most information of Polycentropus species is found in surveys of lake or stream fauna or in general aquatic insect texts (Philipson and Moorhouse, 1976; Ross, 1954).
Information about the Polycentropodidae is found in many general works (Macan, 1962, 1977; Hilsenhoff, 1975, 1976; Betten, 1934; Wichard,
1974; Anderson, 1976; Berg and Peterson, 1966; Wiggins, 1977; Ross,
1945; Usinger, 1956). Some observations from these are; all species are probably univoltine (Hilsenhoff, 1975); they feed on mayflies
(Berg and Peterson, 1966); they are net-spinning and almost exclu sively carnivorous (Hilsenhoff, 1976); the 40 species are highly specialized predators, build capture nets of several kinds, and occur over a wide range of habitats (Anderson, 1976).
Ross (1944) described the morphology of 6 species of larvae and
15 species of adults. Wiggins (1977) described the general morphology of the genus, and p. sp. was used as the morphological illustration.
The morphology of P. flaVomaculatus was described in detail by Lepneva
(1964). Other information about this "model" Polycentropus species is that it is a lentic species (Philipson and Moorhouse, 1976;
Noyes, 1914; Flannagan and Lawler, 1972), and is predaceous (Bracken and Murray, 1973; Noyes, 1914). Its emergence peak is in September
(Bracken and Murray, 1973). 5
Of the three adult species of lentic caddisflies in my study, two have been partially described. Polycentropus interruptus has been
found to be predaceous (Fahy, 1972; Tachet, 1965) and to emerge from
6 to 24 July (Flannagan and Lawler, 1972). Polycentropus remotus has been described as predaceous and herbivorous (McGaha, 1972); found at depths of 0.9-2.4 m on rock and Nymphaea submerged vegetation (Flannagan and Lawler, 1972); and found to emerge from 24 July to 1 August
(Flannagan and Lawler, 1972).
Other species of lentic Polycentropus which are frequently encoun tered in aquatic surveys are P_. cinereus (Wichard, 1974; Winterbourn,
1971; Hilsenhoff, 1976; Leonard and Leonard, 1949; Corbet and Schmid,
1966), and P_. flavomaculatus (Bracken and Murray, 1973; Noyes, 1914;
Fahy, 1972).
Predation in Polycentropus
Members of the family Polycentropodidae and particularly of the genus Polycentropus are generally regarded as predatory. Specific studies of gut contents for P_. flavomaculatus and 1?. cinereus indicate that these two species are indeed carnivorous. No specific observations of actual feeding behavior were included in these studies, however.
McGaha (1972) noted that E\ remotus consumed leaves of aquatic plants in the laboratory. Mackay (1978 in lit.) noted that lotic species of Polycentropus consumed oligochaetes in the laboratory and "stored" excess worms when overfed. Some other behavioral observations include the catagorization of Polycentropus species at sit-and-wait predators
(Macan, 1977). Macan also noted that "their constant numbers in the 6
absence of fish is due to self-regulating territorialism." He mentioned that the prey for Polycentropus larvae are small Crustacea. In terms of the mechanism for capturing prey, Wetzel (1975) stated: "insects detect light intensity and movements but do not form images, so invertebrate predation is more tactile." Predatory mechanisms for
Polycentropus species therefore are perhaps a function of movement of the prey items themselves, or movement of the net as prey items strike it (Wiggins, 1977). These behavioral aspects are significant in defining the niche of Polycentropus species and have been examined in this research.
The method of predation of Polycentropus species can be called
"sit-and-wait" predation (Anderson, 1976). An interesting generaliza tion from a study of competition among fish is that the foraging procedure for littoral, weed-inhabiting species is a "sit-and-wait" strategy (Werner and Hall, 1977). This could be due to the abundant prey in the richly diverse littoral zone, or to the difficulty of maneuvering in a densely populated area. In addition to Polycentropus, which by its habit of building a capture net in one area must be a sit-and-wait predator, many other aquatic insects which inhabit littoral zones of lakes have adopted this strategy.
A sit-and-wait strategy of predation reduces danger of attack on the predator itself. Polycentropus species occupy a fixed net in lentic situations. These are constructed of loosely spun silk (Philip- son and Moorhouse, 1976) which becomes coated with detritus and provides excellent camouflage for the larva in addition to being a perch from which to attack prey. In this research I examined the structure of 7
the nets and the morphology of net-spinning structures of three species
to see if there are any differences. Scanning electron microscopy
(SEM) was used for this part of the research. Other SEM work on Tri
choptera nets has been done for lotic species only (Wallace, Woodall and Staats, 1976; Maias and Wallace, 1977).
Gut analysis is done to determine diversity of food items.
Devonport and Winterbourn (1976) found no strong relationship between size or species of predator and size of prey in a gut analysis from
¿'collection of lotic predators including Polycentropus species. Very little is known about competition in freshwater communities (Fahy, 1972), and this holds true for Polycentropus. Fahy (1972) found that there was no correlation between feeding intensity and prey density in benthos for Polycentropus: "The prey are devoured in an indiscriminant manner and the behavior of predators can be described as grazing."
Niche Analysis
The niche has been defined as "frequency distribution of utilization or occurrence along dimensions such as prey size, temperature, etc."
(Schoener, 1975). Schoener also stated that qualitative sorting out of biologically important effects of resource dimensions is critical.
Of these resource dimensions, more recently the emphasis has been on biological interactions. Whittaker and Levin (1976) stated that the direction in the evolution of niches is toward refinement of biological interactions. Competition can be the chief mechanism in this category, or as Weins (1977) suggested, a complex interaction of predation and habitat pressures may be a more realistic moving force for niche 8
evolution. In either case, niche definition and differentiation between closely related species requires a multidimensional approach utilizing observations of inter-and intra-species interactions, and of habitat dimensions as well.
In his review article of niche, Schoener (1975) found that the most common number of dimensions separating species is three, which is multidimensional. Other generalizations he found from those studies were that morphological differences are indicative of some resources shch as food size or food hardness, that predators separate more often by being active at different times of the day, and that habitat is
"less often the most important dimension in aquatic animals." In another review article of niche studies, Colwell and Fuentes (1975) reviewed major niche separation mechanisms, but examples from areas other than aquatic ecology were used and generalizations for aquatic systems were not made. My research begins to define the niche of
Polycentropus species in several ways: 1) by "qualitatively sorting out biologically important effects," 2) by utilizing measurements of morphology to define groups, and 3) by utilizing environmental measure ments to describe some resource utilization.
Some specific studies which address the question of important effects in aquatic systems are helpful for later comparison. Allan
(1975) found three separating factors in aquatic niches: (1) feeding differences, (2) size-staggered cohorts, and (3) substratum differences.
Reid (1961) stated that in lakes there is usually a high correlation between nature of substrate and number and distribution of species.
Cummins (1964) found that the distribution of insects in streams 9
depended on (1) substrate particle size, (2) current velocity, and
(3) distribution of food particles. Hilsenhoff (1976), however, stated
that food depended on local habitat for many aquatic organisms, hence
local conditions determined diet. As Polycentropus build nets in one
location, this mechanism of food determination would require that they
be extreme feeding generalists. Diet diversity of larvae from different
locations was examined in this research to see if this is applicable.
Multivariate Statistical Analysis
Considerable work has been published with multivariate analysis
used in taxonomy and niche analysis (Sokal, 1969; Rao, 1952; Sokal and
Sneath, 1963; Bloom, 1976; Green, 1971; Strahler, 1978). Cluster
analysis has been used in taxonomy, but presents a basic problem of
determining just what a cluster is (Rao, 1952) and other problems
(Crovello, 1968). Sokal and Sneath (1963) suggested that discriminant analyses might be a more manageable technique for taxonomists. Blackith and Reyment (1971) classified clustering techniques into four groups, and concluded that multivariate techniques such as principal components pass the "clear-cut clustering" test best. My research used a factor analysis and a discriminant analysis to define important grouping variables and to define important discriminanting variables. 10
Summary
The overall intent of this research, therefore, was to contribute to the understanding of the morphology and ecology of a poorly studied group of insects. It also aims to define ecologically important aspects of niche differentiation in aquatic communities through analysis of differentiation between closely related species of the genus Polycentropus which occur together in a bog lake. As Polycentropus is common in lentic waters when other related genera of net-spinning caddisflies prefer lotic water, this study may be instrumental in determining some evolutionary relationships between genera and between species of net-spinning caddisflies. 11
MATERIALS AND METHODS
Field Location
All field samples were collected from bog lakes on Beaver Island,
Michigan. Beaver Island is located in northern Lake Michigan 51.2 km 2 NW of Charlevoix, Michigan, and has 123.2 km of wooded and old field habitat, seven inland lakes, and two intermittent streams (Fig. 1).
The inland lakes are all glacial in origin: many are kettle, formed from large blocks of ice left behind the retreating glacier. The kettle lake sampled extensively in this investigation. Greens Lake, is a dystrophic bog lake surrounded by beech-maple climax forest
(Fig. 2). Bogs often develop in kettle lakes in northern climates because of the characteristic lack of drainage and the surrounding geologic formations and substrate.
Water chemistry data from Greens Lake in 1976 showed the typical chemical character of acid bog waters, low in calcium, total hardness, alkalinity, pH, and saturation, but high in CO^ and acidity
(Smith, 1976).
Another lake sampled in 1976 for distribution of Polycentropus was Barneys Lake (Fig. 3), which had a contrasting alkaline bog condition. The reasons for the development of different bog types is not well understood (Smith, 1976). Bottom deposits differ dramatically in the two lakes, in Barneys Lake ranging from marl to gravel and sand, and in Greens Lake almost exclusively loose flocculent peat. 12
Figure 1. Outline map of Beaver Island with location of the two lakes
sampled for Polycentropus; Greens Lake, an acid bog lake, and Barneys
Lake, an alkaline bog lake. Inset map shows location of Beaver Island
in northern Lake Michigan. 13 14
Figure 2. Greens Lake showing one depth contour, within which a depth
of more than 1 m is reached. An extensive bog mat is also indicated. 15
V2 kilometer Greens Lake 1
T37N, R10W Another primary variation between the two lakes was found in dominant vegetation. In the acid bog lake, Greens Lake, Sphagnum was dominant in the mat, and in the alkaline bog, Barneys Lake, Scirpus and Juncus were dominant. In the littoral zones of both lakes, away from the bog, vegetative differences were also noted, e.g., Dulichium and Utricularia were prevalent in Greens Lake, and Polygonum, Scirpus and Juncus were prevalent in Barneys Lake. Wiggins (1977) and Ross (1944) stated that Polycentropus species were found in a variety of habitats. Wiggins (1978, in lit.) stated that Polycentropus was the dominant group in deeper waters of lakes. Field Methods Collections of caddisflies were made during the summer of 1976, 1977, and 1978 for qualitative and quantitative studies. During the first season (1976) the following sampling methods and schedules were used in the two lakes: (1) emergence traps, emptied twice weekly (Anderson and Wold, 1972) in July and once weekly in August; (2) Ekman dredge samples (Welch, 1954), taken every ten days to three weeks in June, July and August; and (3) conservation web artificial substrates, removed once in July and once in September after a four week colonizing period. A total of ten stations in a shallow bay and along a bog mat in Greens Lake, and ten stations covering many different habitats in Barneys Lake were utilized in 1976 (Figs. 3 and 4). In 1977 twenty-one stations were chosen in Greens Lake in order to maximize the diversity of habitat types represented in one lake (Fig. 4). Barneys Lake was not sampled. The collections in Greens 17 Figure 3. Barneys Lake showing a less extensive bog mat than Greens Lake. Survey sampling only was done at this lake. 18 19 Figure 4. Location of stations in Greens Lake, 1976 and 1977. Out- lined areas in and around the lake are bog mats. 20 21 Figure 5. Sampling types and times in 1976 and 1977. Artificial sub strates were in place for five weeks. The beginning of the bar represents the set-up time; the end represents removal time. Other squares represent weeks during which sampling took place. CM CM r------¡------r"— ' J. I : 1 ! ¡ Hand ■i P icking 1 1 : , ¡ i I j 1 ! • ! ! ' ! E mergence ■ lililí III 1 T raps ■ : I 1 t 1 ¡ 1 Ekman ■ III 1 Dredges ■ j 1 r 1 ( i i SET^ll* REMOVIO Artific ia I IHHHMi Subst ra tes i ■ i. ! J • 1 - i------1------L i■ Moy 76 June 76 July 76 Aug. 76 Sept. 76 May 77 June 77 July 77 Aug. 77 Sept. 77 Moy 78 23 Lake began in May with the following methods and schedule: (1) emergence traps emptied twice weekly in late May and June, (2) hand-picking in late May, (3) artificial substrates, removed once in June and once in September, and (4) Ekman dredge samples, taken once in June. In addition, light trapping of adult caddisflies was done several nights in late June and July: areas sampled were along the shore and in the bay (Fig. 4). Sampling was done for 4 days in 1978 to locate the exact microhabitats of three species of Polycentropus. Three areas were chosen for concentrated qualitative and quantitative sampling in a large bay in Greens Lake (Fig. 6). Aquatic vascular plants (mostly Nuphar variegatum, Nymphaea ordorata, and Utricularia vulgaris) were 2 removed and examined along a transect line as well as from aim area at the shore end of the line. Plants were removed at convenient intervals along the transect line and at.the end, thus random quanti tative sampling was not the case. For each plant removed, however, each caddisfly larva found was recorded, and its distance from other larvae on the same plant noted. Dip net samples were taken along the transect lines as well, to determine utilization of surface detritus as a habitat for the larvae. Figure 5 is a summary of sampling dates and methods for all sampling done in the three years of this study. A total of 50 Ekman dredge samples, and 20 artificial substrate samples were analyzed in 1976 in addition to emergence trap contents. In 1977, 21 Ekman dredge samples and 42 artificial substrate samples were analyzed. Emergence traps were not used in 1978. 24 Figure 6. Detailed habitat map of "Boat Launch Bay" in Greens Lake Extensive sampling along transects was done in May, 1978. CN Conifers & Deciduous trees Open water Figure 6 26 Sampling Methods Ekman dredge sampling is best suited to lake and pond bottoms which are soft and loose (Merritt and Cummins, 1978). This method was thus most useful at stations which had loose flocculent peat or marl bottoms. However, for those stations which had extensive aquatic vascular plant growth, the Ekman dredge rarely sampled its theoretical 2 31.5 cm . Merritt and Cummins, (1978) recommend the use of Wilding or Stovepipe samplers or removal of natural substrate by SCUBA for habitats like these. In this study quantitative sampling at these stations was accomplished with artificial substrate samplers. SCUBA was not used because retrieval was possible from a boat. Voshell and Simmons (1977) came to three major conclusions about the effectiveness of artificial substrates in lentic collecting of benthic organisms: (1) more taxa are likely to be encountered with artificial substrates than a dredge, (2) "conservation web and/or leaves placed separately in small plastic baskets...are equally as effective as their larger counterparts in regard to individuals and taxa collected," and (3) small leaf and web samplers meet all criteria tested. The criteria were: (1) high density of individuals with as little variability as possible, (2) many different taxa, and (3) reasonable amount of -time for collection and processing. They also suggested that four weeks were optimum for adequate coloni zation. For these reasons, small cottage cheese containers and other plastic containers holding 10 x 20 rectangles of 3M Corporation’s #200 conservation web were used as artificial substrate samplers. Two of these were placed at each station in 1977 and one at each in 1976. 27 Figure 7 a illustrates the slotted design of the containers. This design allowed easy access to the conservation web and also simplified collec tion. Measurements of the containers ranged from 16 cm. top diameter x 11 cm. depth to 12 cm. top diameter x 10 cm. depth. In order to offset any possible bias introduced by container size differences, those of different sizes were randomly selected to be placed at stations in 1976, and in 1977 containers of two different sizes were used at each station. Conservation webbing was selected for use since Dickson and Cairns (1972) and Crossman and Cairns (1974) reported that it was selectively colonized by caddisflies. A problem encountered with the artificial substrate device, however, was that it was colonized almost as heavily on the container as on the conservation webbing. This was probably due to the habits of the predominant caddisfly larva found in the lake, Polycentropus spp. Emergence traps used were built following a modified design of Buscemi (1961). They were made of galvanized screen, polyethylene plastic, and copper wire, with a museum jar for collecting emerging insects (Fig. 7b). Hose clamps held the lid of a museum jar in place at the apex of the cone made of a double layer of wire screen and plastic A halter was made for the jar from plastic coated copper wire, and a loop was fashioned at the top. A clasp was attached to this loop which was in turn attached to a line leading to a float. The museum jar lid secured in the cone had a 3 cm. hold drilled in it to allow insects to emerge in the air pocket of the jar when the trap was in place. When the emergence trap was brought to the surface to be emptied, the process 28 was done slowly so that the air pocket in the submerged jar was not disturbed. The jar was then unscrewed from the cone while the jar was still under water, and a new jar screwed into place on the cone. A new lid was screwed onto the old jar (a lid without a hole) and the jar containing emerged insects brought to the surface. A duplicate set of jars was maintained for this process. Several problems were encountered with the design of this trap. First, some traps were prone to tip over easily. Second, the traps Were not large enough to sample a sufficiently large area in the patchy distribution of the insects, hence very small sample sizes per date were obtained. Third, the plastic and screen provided an excellent substrate for the growth of algae and consequently that surface was covered in less than two weeks. To prevent avoidance by emerging insects, traps should be as transparent as possible (Buscemi, 1961). Although the traps thus did not allow for a quantitative analysis of data, nevertheless the data do give a good indication of the emergence times for the three species encountered. The traps were excellent for collecting emerging Chironomidae and could possibly be suitable for a quantitative study of this group. Hand-picking of aquatic vascular plants was initiated in May, 1977 after observations of many caddisfly larvae using Nuphar and Nymphaea stems for net attachment sites. Three transects were chosen to include both Nuphar and Nymphaea stems and to include bog edges, open shore with grasses, and wooded shore areas. Lily pads in a relatively straight line were then pulled from the bottom and examined for larval nets. The presence of nets and the distance of one net from its 29 Figure 7a. Artificial substrate samplers were constructed of small and medium cottage cheese containers cut into slats. Two 2x2 cm. squares of conservation webbing were placed inside. Figure 7b. Emergence trap samplers were constructed of plastic and screen cones with removable museum jar tops. These were submerged with a 5-7 cm. bubble of air left in the jar for emerging insects. 30 Figure 7a Figure 7b 31 neighbors was recorded. Lily pads were harvested until the lily pad zone ended. All larvae and their nets were preserved and later identified. In 1978 further hand-picking and dip net sampling was carried out to further pinpoint the exact microhabitats of the three species of Polycentropus found. During this sampling other aquatic plants were examined, principally Dulichium sp. Again numbers of larvae and distances between nets were recorded. Four transects were used again in 1978 to cover diverse habitat areas (Fig. 6). In addition to examining harvested plants along the transect, dip net samples were taken along the transect to examine the surface detritus for use as net substrate by Polycentropus. Fig. 8-1 is an aerial photograph from the west of the habitat in this bay. Fig. 8-2 is from the east, and Fig. 8-3 shows the habitat and traps in place in Barneys Lake. Laboratory Methods Identifications of adults and larvae were made using keys (Ross, 1944; Wiggins, 1977). Further species level identification and separa tion were accomplished using a numerical taxonomy technique. Ross's 1944 key includes the three species encountered in this study, but discrepancies were found between specimens and descriptions. Measure ments for numerical taxonomic analysis were made of the following characters: (1) head capsule width across the eyes (Mackay, 1978), (2) irons length (upper and lower sections), (3) anal leg length, (4) number- of setae on pronotal edge, and (4) color pattern on the head. These measurements, with three environmental parameters were entered into two multivariate analyses, factor analysis and discriminant 32 Figure 8-1. Aerial photograph of Greens Lake "Boat Launch Bay" from the southeast. Note the areas of bog-building and vegetation. Figure 8-2. Aerial photograph of Greens Lake "Boat Launch Bay" from the northwest. Note the bog edge on the east side of the island (top of picture). Figure 8-3. Emergence trap and artificial substrate in place in Barneys Lake. Note the vegetation: Nymphaea, Scirpus, and others. 33 34 analysis', to determine differences between species. Environmental parameters used were: vegetation and bottom type at each station, and collection method. Collection method was used because it represented substrate type utilized by the larvae. This will be discussed in a later section. To determine influence of food utilization in the distinction between species, qualitative food content in the gut was analyzed. A method described by Wallace (1975) was used with some modifications: the foregut and midgut contents were excised, the contents mixed with glycerine on a glass slide, and each type of food organism found recorded. Volumes of food were not measured. Other laboratory examinations carried out were electron micro scopic observations of net and mouthpart structure. Maias and Wallace (1976, 1977) have shown species-distinctive net structure for stream caddisflies, and has related net mesh size to habitat utilization and niche separation. There have been no observations of lentic predaceous Polycentropus species. To examine these nets, larvae were allowed to build nets in filtered pond water or distilled water for periods of time from one hour to two weeks. Figure 9-3 shows the culture vessels used. These nets were observed for structural steps and species differences. For the second observation, species differences, larvae were allowed to build nets for two hours (a length of time determined to be optimum for characteristic building in the previous experiment) and then prepared for observation. Mouthparts and other head capsule morphology were also observed for each of the species. Heads were removed and prepared for observation of the silk gland opening, the 35 labrum, and other mouthparts. SEM observations were made with a Hitachi (Model HHS-2R) instrument operating at 20kV. Nets were prepared by dehydration in a graded alcohol series and critical point drying with carbon dioxide, followed by coating with approximately 200 A gold in a sputter coater. Behavioral Observations Beginning in the spring of 1977 behavioral observations were carried out. Six observation jars were set up in May, five more in June, and five more again in July. Animals were put in individual culture vials and observations made of net building, feeding and other behavior. Figures 9-1 and 9-2 show these culture vessels with larvae and nets. Metamorphotypes from larvae in these experiments were used to associate larvae and adults. An attempt was made to rear larvae of P_. remotus from eggs derived by the CO^ method described by Resh (1975). This was only partially successful for P. remotus. Eggs could not be forced from the other species. Experimental behavioral observations were begun in July and again in November 1977. In July, larvae were tested for feeding capacity and time intervals between feeding. Several larvae were fed individual food items continuously for two hours. When one food item had been consumed another would be released in the culture vial in the net area of the larva. This was continued until the larva no longer made any attempt to attack the new food item. Time between food items was recorded for each trial. In November, larvae were tested for spacing behavior and aggressive behavior. For this experiment, 5 larvae were placed in a battery jar 36 with five artifical lily pad stems. These were made of plastic tubing attached to a screen at the top of the battery jar. Observations of behavior were made for two days for each of two sets of tests. 37 Figure 9-1. P_. remotus in culture vessel in aquarium. Net was built on the side of the vessel near the top. Figure 9-2. 1?. remotus in culture vessel after just capturing a chi ronomid larva (at arrow). Figure 9-3. Vessels used for net-building SEM preparations. "Hach kit" chemical containers were lined with coverslips so that larvae would attach nets to the coverslips. These could then be observed for attachment sites. 38 39 RESULTS AND DISCUSSION Barneys Lake and Greens Lake Comparisons In 1976 two lakes were sampled for comparison of Trichopteran families encountered and for the occurrence of Polycentropus species. Table 1 summarizes the major families of Trichoptera found and identified in the two lakes. Rare species were not included. In Barneys Lake the stations close to protected shores most often yielded large numbers of larvae. Station 7 (Fig. 3) was located in deep water (3.5 m) with fine marl on the bottom, and no vegetation. Only Chironomids were collected from this station. Station 10 had a relatively large popu lation; it was nearly 1 meter deep at this location, and aquatic plants were plentiful. The area was protected from wave action by a bog mat and was located on the NE (windward) side of the lake. Other bog stations (stations 1 and 8) were locations of fair populations as were stations 5 and 6. Stations 5 and 6 were in a calm, protected bay, although they were located at the NW end of the lake which was subject to considerable wave action. In this bay thick beds of Chara and other aquatic plants were present. The bottom was sandy and marlaceous, and at station 5 many larvae of the genus Molanna, which builds envelope-like cases of sand grains, were found. Stations 3 and 4 were located where natural vegetation was sparser and where wave action was sometimes quite heavy. Bluegill nests were also common in the sand in these areas. There were 196 Polycentropus larvae collected by artificial sub strate and dredge sampling in the summer of 1976. Table 2 shows the number of larvae collected at each station for these two sampling 40 TABLE 1 Families of Trichoptera found in Barneys Lake and Greens Lake, 1976 Family Genus Species Emergence Barneys Lake Phryganeidae Banksiola sp. Molannidae Molanna sp. Limniphilidae Clostoeca sp. Arctopora sp. Leptoceridae Triaenodes sp. Oecetis sp. Polycentropodidae Polycentropus interruptus July 9-July 21 remotus Greens Lake Leptoceridae Triaenodes sp. June 28-June 30 Oecetis sp. Polycentropodidae Polycentropus interruptus June 5-July 28 remotus May 28-July flavus June 15-Aug. 10 41 methods. ' When a date is not listed for a station, no larvae collected at that station for that collection. Emergence of adults in traps set up at each station was recorded only from July 9 to July 21. Only four adults were trapped in Barneys Lake in 1976 from stations 3, 5 and 6. The difficulties with the emergence trap design as discussed in the Materials and Methods were compounded in Barneys Lake by the exposure of the lake to wind and to recreational use. Greens Lake was located in a more protected.part of the island, and was not disturbed as much by storms or recreational use. In Greens Lake in 1976, in comparison, 62 adults were found in emergence traps; 48 of these were saved and identified. Table 3 is a list of stations, dates, and species of adults collected in Greens Lake in 1976. If no species name is given, the specimen was not identifiable due to mold. All stations except station 1 yielded adults, and two stations in particular had large populations. Station 6 was in a small protected bay, at a depth of about 1 meter. Station 7 was just outside this bay at the same depth. The bottom was not predominately thick flocculent peat as were most stations, but rather more coarse detritus with only a small amount of flocculent peat. Vegetation at both stations consisted of lily pads and Dulichium. Adults of P. interruptus, PL remotus, and P_. flavus In 1977 sampling was restricted to and intensified in Greens Lake. Figure 4 shows the location of stations in Greens Lake in 1977 and 1976. Table 4 is a list of adults collected from emergence traps in 1977. Station 8, 11, and 12 had large populations in 1977. Station 8 was located at the mouth of the large bay where station 5 was located 42 TABLE 2 Numbers of Polycentropus larvae collected Stations in Barneys Lake, 1976. Numbers of Station # Date and collection type Polycentropus larvae collected 1 July 29 dredge 5 Aug. 17 dredge 24 Sept. 1 substrate . 4 2 July 29 dredge 15 Aug. 17 dredge 3 Sept. 1 substrate 1 3 July 29 dredge 5 Aug. 1 substrate 6 Aug. 17 dredge 4 Sept. 1 substrate 3 4 (Samplers July 29 dredge 1 stolen) 5 July 14 dredge 4 July 29 dredge 2 Aug. 1 substrate 7 Aug. 17 dredge 5 Sept. 1 substrate 2 6 Aug. 1 substrate 13 Aug. 17 dredge 18 Sept. 1 substrate 7 7 NONE 8 July 14 dredge 6 July 29 dredge 10 Aug. 17 dredge 10 Sept. 1 substrate 13 9 July 29 dredge 3 Aug. 17 dredge 1 Sept. 1 substrate 7 43 TABLE 2. (Cont.) Numbers of Station # Date and collect type Polycentropus larvae collected 10 July 14 dredge 7 July 29 dredge 5 Aug. 1 substrate 3 Aug. 17 dredge 4 Sept. 1 substrate 3 44 TABLE 3 Numbers of Polycentropus adults collected at stations in Greens Lake, 1976 Station # Date Number Species 1 6/30-8/19 NONE 2 8/7 1 8/19 1 3 7/13 1 8/1 1 4 7/13 1 interruptus 8/8 2 remotus 5 7/3 3 interruptus 6 7/3 5 interruptus 7/7 1 remotus 7/13 1 interruptus 7/13 1 remotus 7/24 1 remotus 8/14 4 remotus 7 6/30 3 interruptus 7/3 8 interruptus 7/10 5 interruptus 7/10 1 flavus 7/7 1 7/13 1 7/17 1 7/24 1 8/1 1 remotus 8/7 6 flavus 8 7/17 1 8/7 3 remotus 9 6/30 1 interruptus 7/3 2 remotus 8/19 1 45 TABLE 3. (Cont.) Station # Date Number Species 10 7/7 1 7/17 1 8/1 1 pupa 8/19 1 remotus 46 TABLE 4 Numbers of Polycentropus adults collected at stations in Greens Lake, 1977 Station # Date Numbers 1 5/31-7/4 NONE 2 5/31 1 remotus 3 5/24 2 6/3 1 remotus 6/27 1 interruptus 4 5/31 1 interruptus 6/27 1 interruptus 5 5/31-7/4 NONE 6 6/27 1 flavus 7 5/29 1 6/27 2 remotus 8 5/29 3 remotus 5/31 1 6/27 5 9 5/29 1 6/27 1 remotus 10 5/31 1 remotus 6/3 2 remotus 11 6/3 1 remotus 6/27 3 remotus 6/27 1 interruptus 6/27 2 remotus 12 6/27 3 flavus 6/27 3 interruptus 13 5/31-7/4 NONE 47 TABLE 4. (Cont.) Station # Date Numbers Species 14 6/2 1 15 6/27 2 flavus 6/27 1 interruptus 16 5/31-7/4 NONE - 17 6/27 1 - remotus 18 5/31-7/4 NONE 19 6/27 1 flavus 20 5/31-7/4 NONE ★ * shore at boat landing 6/2 2 remotus 6/27 1' flavus ** light trapping 6/27 7/4 4 flavus 7/4 30 * remotus 7/4 10 * interruptus 5 * flavus * not saved for collection * * not collected from emergence traps 48 in 1976. Stations 11 and 12 were located at the west end of the lake where station 10 was in 1976. Station 15 was located where station 7 was in the previous year, but in 1977 it yielded fewer adults. Overall the traps were less productive in 1977; this could have been due to their early removal and to the wear and accumulation of dirt on the samplers from the previous year's use, which decreased their transparency 1?. flavus adults were collected at stations 6, 12, 19 and at the boat landing in 1977 in addition to the same location as in 1976 (station 7, now station 15). A large collection of adults was made on June 27 by light trapping in a rowboat on the bay. Adults landed on the boat seats and on the bottom of the boat near the lantern and were picked up by hand. These were later anaesthetized and revived to force egg-laying. Many P_. remotus were forced successfully and many Oecetis adults were forced easily, but the others were not. Only eggs of 1?. remotus hatched, and these did not grow beyond the second stadium. Morphology of Polycentropus Adults Identification of adults was accomplished using the key by Ross (1944). Figure 10 is a reproduction of drawings of genitalia from this key. Figures 14-3 and 14-4 are photographs of the female genitalia of P^. remotus and P^. interruptus. Specimens with genitalia cleared with NAOH were identified easily using these diagrams. A difficulty was encountered only in distinguishing P_. interruptus males from ¡P. flavus males. The different length of "c" was difficult to see, and the different shape of the clasper was not distinct. Female specimens, however, were distinctly different. Identifications were checked by Dr. Todd Harris at Purdue University. 49 Niche Separation Based on Adult Emergence A temporal mechanism is often responsible for the separation of closely related species of aquatic insects living in the same habitat (Allan, 1975). In the case of Polycentropus species living in the littoral zone of a bog lake, Figure 11 illustrates the close range of emergence times, which only would separate adequately P_. flavus from the other two species, IL interruptus and P_. remotus. Figure 12 is a generalized representation of the temporal separation of P_. flavus and the similar emergence pattern of EL remotus and P_. interruptus. Temporal differences in emergence indicate that the larvae are at different instars at the same times of the year, thus probably eating different size food items and utilizing different space resources, which would thereby allow them to avoid competition. In the case of the two species with the same emergence pattern, however, a closer investi gation of the larval ecology was necessary in order to determine the mechanism of separation in the community. Morphology of Polycentropus Larvae The only taxonomic key to species of Polycentropus which includes the adults herein reported is that by Ross (1944). That key, however, is a specific reference to a collection in Indiana. There are no further studies in larval taxonomy of the Polycentropus species; therefore the taxonomy of this group is not clear. In order to approach the problem of niche separation in the two species, P^. remotus and IL interruptus, therefore, it was first necessary to determine a method of taxonomically separating the species during their larval stages. Mackay (1978) pre sented a thorough account of the method she used for this same problem. 50 The following is a modification of her method. First, larvae in the final stage were studied carefully and compared with existing descriptions. Figure 13 is a diagram of the three mor phologically different types of larvae encountered in Greens Lake. I will discuss each one in turn. In the description for P^. interruptus (Figure 13a) Ross (1944) includes the following: Length 15 mm. Head yellowish with distinct dark spots and with most of the dorsum clouded with reddish brown, the major pair of setae of the upper irons with a small,, pale area around base. Pronotum and legs yellowish brown. Remainder of body pale. Other characteristics used by Ross in the key are: 1) upper part of frons shorter than lower portion, and 2) hair on basal segment of anal appendages in regular rows. Figure 13a shows a very clear difference in color of the head of P_. interruptus from the other two larvae. In all stadia it was possible to see this color difference. In the final instar the head was very dark, the sclerites well outline, and the area around the base of the frontal setae very light. However, measure ments of the irons showed that 26% of the specimens had a longer upper section, 20% had a shorter upper section, and 54% had both sections the same. This was thus not a very diagnostic character, and not parti cularly useful to separate the remotus larvae from the interruptus larvae in this community. A characteristic which was constant, in addition to color, and useful for distinguishing final and some earlier larvae was the number of setae on the anterior edge of the pronotum. In P_. interruptus there were two main setae on either side of the center line of the pronotum, and in P. remotus there were always more than 51 Figure 10. Adult genitalia of three species of Polycentropus found at the Greens Lake in 1976 and 1977. (From Ross, 1944) 52 53 Figure 11. Emergence of ~P_. remotus, IP. interruptus, and P_. flavus in 1976 and 1977 combined. 54 P. remotus R interruptus R tluvus 22 i LATE MAT - JUNE JULY AUGUST EARLY J UN C 55 Figure 12. Generalized emergence pattern of P_. remotus, P_. interruptus and P_. flavus. Temporal separation only is adequate to separate P_. flavus from the other two species. kO Lf) t tz> oc co s 3 Z <______time -> 57 Figure 13a. E\ interruptus larval morphology. Head color is dark with dark spots, pronotum is dark, and anal appendage has hairs in a straight light. Figure 13b. P_. remotus larval morphology, spotted type. Head light with dark spots. Main frons setae with dark color at base, anal appendage hairs thick and patchy. Figure 13c. P_. remotus larval morphology, non-spotted type. Head light with no spots except a few on the frons. Hairs on anal appendage thick and patchy. 58 59 two (Fig. 13b) . The last stage larvae of P_. interruptus also had hairs on the anal appendages in a straight line, often with very few setae on the outside of the basal segment (Fig. 13a, bottom). This was not discernable in the early stages, however. The pronotum of IL interruptus was clouded with reddish brown coloration, in contrast to Ross's description, and was also spotted with black in a regular pattern near the posterior portion of the sclerites. This coloration was not found on the other.larvae. The second type of larva that occurred in this community was ]?. remotus. Figure 13b shows the color pattern of this larva; quite different from that of P.. interruptus. Ross (1914) describes this larva in this way: Length 14 mm. Head, pronotum and legs straw color, the head with well-marked spots. Upper portion of irons subequal in length of lower portion. Body pale, without markings. In the measurement of the sections of the irons for this larva, 71% of the specimens had a longer upper irons than lower irons. Usually the difference was between 0.15 and 0.05 mm, which was less difference than between the upper and lower frons of P_. interruptus. The irons sections were equal in 29% of the cases. Spots were well marked in about two-thirds of the cases, but many specimens had no spots on the parietal sclerites of the head (Fig. 13c). The remainder of the body was very light in color for the spotted and non-spotted color types. In the spotted type, leg setae ranged from very patchy and thick, to a few hairs in short rows with some patchiness. The pattern of setae on the anterior edge of the pronotum was usually 6 setae on either side, but different lengths of setae were noted. Both spotted and unspotted 60 Figure 14-1. Labrum of P^. remotus. Apparent protrusion in the center is due to extreme curvature of the labrum. Figure 14-2. Labrum of P. interruptus. Figure 14-3. Genitalia and eggs of P_. remotus. Eggs measured .2-.3 pm corresponding to the head capsule size of the first instar larvae. Figure 14-4. Genitalia of P. interruptus. 62 larvae had at least one very long seta, two medium length and several short ones on each side of the pronotum. In both cases the pronotum was very light in color and not spotted. The irons sections on the unspotted larvae were comparable to the spotted ones: 75% of the specimens had a longer upper irons, and 25% had equal iron sections. Figure 14-1 and 14-2 illustrate a difference between the P_. interruptus labrum and the P_. remotus labrum which will be discussed further in a later section concerning mouthpart morphology as seen by scanning electron microscopy. A second approach to separating species in the larval stage and associating them with the adults was to rear larvae to pharate adults in the laboratory. The metamorphotype method/ (Wiggins, 1977) , described for field collections, was utilized in the laboratory. Pupae did not develop fully in the laboratory, many dying just before adult emergence, or during emergence. These pharate adults had fully formed genitalic structures and the larval sclerites were compressed in a mass in the posterior end of the pupal case. Of 10 of these metamorphotypes observed, 2 were of the non-spotted P_. remotus type. Six were of the spotted I?. remotus type and two were P^. interruptus. There were unfortunately no P_. flavus metamorphotypes reared or collected. Even though the sample of metamorphotypes was limited, the non-spotted larvae appear to be color variations of P_. remotus. Larval sclerites of the other P_. remotus pharate adults had various intensities of the markings seen in figure 13b. More rearing of larvae to adults and more collecting of pupae about to emerge is obviously necessary to obtain positive evidence for this hypothesis, but other results which 63 follows corroborates this hypothesis substantially. Futher morphological analysis of these larvae was carried out using scanning electron microscopy in order to determine net and mouthpart structures; this will be discussed in a later section. Life History Analysis of P_. interruptus and P^. remotus In order to determine number of stadia, a graph was made of larval head capsule sizes were graphed vs. numbers of individuals (Fig. 15) (Benke, 1970). Collections from 1976 and 1977 were combined to cover the entire summer. It appears that there are 5 instars of both P_. remotus were measured at 0.2 mm (Fig. 14-3) as an estimate of the first stadium, and it is presumed that for P_. interruptus a similar first stadium is missing from the data. For hydropsychid larvae Mackay (1978) suggested that head capsule measurements of successive stadia increase in size in a regular geometric progression with a factor of increase of about 1.5. She also suggested that this is a useful tool to separate young stadia of closely related species. Table 5 shows the stadium number, range of sizes for head capsules, and mean head capsule size for P_. remotus and P_. interruptus. The data labeled P. ? on Figure 15 is combined with P_. remotus in Table 5. The specimens used for P. ? measurements were the non-spotted EL remotus. This is further evidence that they are indeed P_. remotus; the size range for the final stadium is nearly the same. The mean for the non-spotted EL remotus is 1.0 because of the lack of larvae on the low end of the range. 64 These data indicate the P. interruptus larvae were slightly larger in all stadia. For the very small stadia, it is possible that ranges and means have somewhat less accuracy due to the small sample size; however, if the factor of increase of 1.5 that Mackay (1978) suggests is operable, measurements are close on all instars except for instar IV for P_. remotus. This was a small sample, and had a wide range with out any clear peaks, which would explain the discrepancy. Perhaps the smaller instar IV larvae were missed, and the 16 collected represented the large end of the range. Statistical Analysis of Morphological and Ecological Data In order to quantitatively and statistically approach the problem of species differences and niche separation, several approaches were taken. First stations were descriptively broken down. The type of vegetation and lake bottoms were characterized for each station, and then the stations were listed in seven categories (Table 6). This was used as one variable in a multivariate analysis to be described later. Another aspect of the habitat of the larvae that was determined from the collections was substrate utilization. It was assumed that larvae from dredge collections were utilizing benthic deposits or benthic/ water interface substrates, such as undecayed leaves or horizontally growing plants. Larvae collected in artificial substrate samplers were assumed to be normally utilizing bottom substrates. Larvae which were collected by hand picking aquatic vascular plants were utilizing vertically growing plants as substrate. Another collection method which was not originally planned was the colonization of emergence trap 65 Figure 15. Sizes and numbers of head capsules for P_. remotus and P. interruptus. P. ? is the non-spotted P. remotus type. m m n ¿ WIDTH OF LARVAI HEAD CAPSULE 9 9 67 TABLE 5 Larval instars head capsule sizes for P_. interruptus and P_. remotus Stadium Number Head capsule Head capsule Number Measured size range (mm) size mean (mm) P_. remotus and P. ? I 10 .18-.21 .2 II 11 .23-.36 .3 HI 28 .39-.54 .48 IV 16 .69-.88 .80 V 163 .9-1.2 .96 P. interruptus I — — — II 16 .26-.46 .34 III 53 .48-.6 .51 IV 85 .67-.9 .82 V 67 .93-1.29 1.1 68 Figure 16. Larvae encountered at stations in 1977. 32 30 28 26 24 22 20 a m 16 CD 3 14 □ 2 12 10 8 6 4 5. 6 7 8 9 1.0 11 12 13 14 1 2 3 4 6 9 STATIONS 70 TABLE 6 Station types and Station numbers in Greens Lake, 1977, .1. Nuphar, flocculent 2, 4, 13, 14 Transect 2, 3 peat bottom 2. Nymphaea, flocculent 19, 20 Transect 1 peat bottom 3. Utricularia, flocculent 21 peat bottom 4. Bog edge or beaver 1, 3 run through flocculent peat bottom (deeper water) 5. Bog edge outside of 9, 10, 16, 18 bay (subject to wave action) 6. Bog-building muck area 5, 6, 7, 11, 12 7. Open water, flocculent 8, 15 peat, some sand on bottom 71 Figure 17. Larvae encountered at four general areas in Greens Lake in 1976 and 1977. 72 34 32 30 28 26 24 22 20 18 16 14 12 10 8 6 4 2 0 1976 1977 1976 19 77 BAY I BOG E DGE.I OPEN BOG emergence hand TOTAL DREDGE SUBSTRATE TRAP PICKING P 158 4 9 19 . - 9 0 1 . 1 P 9 79 4 2 . 19 T 7 - R 0 0 A 6 . B L E P I 43 n 3 10 . 3 T - ? 0 F o u N r u P 3 m 21 6 C . 0 2 7 o 0 1. b l e l e r s c 1977 P 1 t 4 48 i 2 O 2 o R 2 6 1 f 5 n . P T o y P l p y 30 22 . 53 e 0 c s 1 ? e n t r o P p 1 15 . u 0 - - 5 1. s S p 1 P e 9 2 . 22 c 7 — - 0 R i 2 e 8 . s P . 1 1 — - 2 ? 2 4 74 jars by larvae. This was listed as a separate category of substrate utilization in later analyses. To see if station locations or different categories of habitat influenced the occurrence of larvae, numbers of larvae collected by all methods in 1977 were graphed vs. stations. Figure 16 shows that hand-picking at station 2 influences the numbers of individuals found there greatly. Other stations which were only sampled by artificial substrates and dredges showed some interesting results; e.g., station 8, located away from aquatic vegetation in deeper water had very few P_. remotus, but many specimens of P^. interruptus individuals while station 5, in an area of lily pads with a very thick coarse flocculent peat bottom, supported only P^. remotus. Another way to look at this difference is shown in figure 17. If stations are lumped into four categories, Bay I (the "Boat Launch Bay"). Bay II (a smaller one), bog edge, and open water stations, it can be seen that again hand picking influenced the distribution in Bay I. Both species are found in bog edge areas, but in open areas without much vegetation on P_. interruptus is common. This relationship of _P. interruptus to open water stations can be explained further by data broken down by collec tion method. Since it was assumed that collection method could represent differences in substrate utilization, Table 7 represents substrate utilization by each of the species. A dramatic difference can be seen here between J?, remotus and P_. interruptus. For 1?. inter ruptus the relative occurrence is ranked: 1) dredge and artificial substrate and 2) hand-picking. For P_. remotus the ranking would be the opposite: 1) hand-picking and artificial substrate, 2) dredge. 75 In this table P_. ? represents P_. remotus larvae which were non-spotted These were almost never found in dredge samples, but were frequent in hand-picked samples and fairly common in artificial substrate collec tions. This comparison of substrate types utilized is relative; precentages of each species per substrate type is pertinent, not total amounts collected per method. Factor Analysis, Discriminant Analysis of Morphological and Ecological Variables In a discussion of clustering techniques, Blackith and Reyment (1971) concluded that most cluster analyses suffer from many problems, and that the only solution is to use "divisive polythetic" techniques such as principal components analysis. An "agglomerative clustering technique" was attempted with this data, but it was found to form clusters with no relevance to the color differences or any other of the obvious morphological characteristics. For this season, factor analysis was performed instead. The questions which could not be clearly answered by the morphological data alone, or by the distribu tion data alone were; 1) are there two or three distinguishable species in the community? and 2) which morphological and ecological features separate these groups most effectively? In short, what are the groups, and how are the niches of the groups separated? Factor analysis groups individuals by ordination on graphs whose axes are vectors, or linear combinations of variables, termed factors. Factor scores for the individuals are plotted on graphs with each pair of factors used. Factor scores are derived from the standardized 76 score of each individual on each variable times the factor score coefficient matrix. This matrix is derived from the original solution of the factor analysis. The solution of the factor analysis consists of weights or loadings of each variable for each factor derived. The factor vectors, or linear combinations of the variables show relation ships between variables in the population measured. Factors are described in the variables which load heavily on the factor. A heavy loading is one of .30 or more. Table 8 shows the loadings of 9 variables measured in this analysis on three factors. In the first factor, variables related to a coded pattern of taxonomic characters are weighted heavily. In the second factor, variables related to measurements of the head, frons and leg are highly weighted. In the third factor, environmental factors of collection type and collection site are highly weighted. This means that the variation in the group of larvae collected, measured on two environmental and seven morpho logical variables, is characterized most importantly by a taxonomic factor. It is secondly characterized, when the variation of the first factor is removed, by another taxonomic factor. These first two factors (or latent variables) are the axes, and factor scores the points on Fig. 17. The seta pattern, color pattern, and leg seta pattern were coded so that dark color, three or less seta on the pro notum, and straight leg setae were at one end of the continuum, and the opposite types at the other end. Thus, it is on this basis that the factor score plots show two groups based on these characters. There is a distinct division between the two groups in the direction of factor I (Fig. 18), but for factor II a continuum exists. Thus the 77 taxonomic confusion of frons measurements in the Ross (1944) key is substantiated by this factor grouping. Froih this evidence it also appears that there are only two species groups. However, when factor I and factor III are plotted (Fig. 19) three groups emerge. Again the division by seta, color, and leg hair pattern can be seen, but the P_. remotus end of factor I (more than 2 pronotal seta, light head with dark spots or no spots, scattered or patchy leg seta) is divided into two close, somewhat overlapping but distinct groups. At the high end of'the scale for factor III are found larvae recovered from substrates and dredge from open water, bog-building muck, open lake bog edge, and deeper water bog edge in the bay. At the other end are larvae which were found by hand-picking from vegetation in Nuphar, Nymphaea and Utricularia beds in the bay. If anything, this division along environ mental lines probably represents a movement of species at a particular time in the life cycle, although it could represent a developing subspeciation of P_. remotus which is not evident in the morphology of the adult. The individuals falling into the upper right corner of the plot are also ones which were found on aquatic plants or in emergence trap jars, and which had no spots or the color pattern of both light background and light spots. This is the morphological type which does not fit into Ross's key and which has been difficult to place in a group in this study. Discriminant analysis of the nine measured variables corroborates the results obtained by means of factor analysis. In discriminant analysis, groups are formed before the analysis, and the procedure determines which of the variables in combination with each other 78 TABLE 8 Principal Components Analysis Factor Loadings Variable Factor 1 Factor 2 Factor 3 1. Collection type -.107 .241 .821 2. Collection site -.160 -.030 .877 3. Head width -.135 .752 -.004 4. Frons length I -.299 .825 .053 5. Frons length II .090 .800 .090 6. Seta pattern .938 .026 -.164 7. Color pattern .888 -.088 -.127 8. Leg hair pattern .901 -.004 -.063 9. Leg length .214 .519 .131 Variance explained 2.67 2.22 1.51 42% 35% 23% 79 Figure 18. Plot of factor scores as related to Factor I and Factor II. Two groups can be seen based on relationship to Factor I I I R O T C A F Seta,Col or Pattern FACTOR I 8 0 81 Figure 19. Plot of factor scores as related to Factor I and Factor III. Three groups can be seen in this plot; one in relation to factor I, and another in the right hand factor I group, based on factor III. FACTOR 1 I 1 83 TABLE 9 Discriminant Analysis DF Coefficients Analysis One: Three Taxonomic Groups D F I D F II Standardized Standardized Variable Coefficient Coefficient 1. Collection 0.31 0.01 type 2. Collection -0.70 0.24 site 3. Head width 0.14 0.13 4. Frons length I 0.14 -0.25 5. Frons length II 0.01 0.22 6. Seta pattern -0.99 0.65 7. Color pattern -0.42 -0.97 8. Leg hair pattern -0.30 0.19 9. Leg length -0.02 -0.34 Bartlett's Chi Square Test for P < .0001 P < .0001 Significance Percent of 94.68 5.32 Variation 84 TABLE 10 Analysis Two: Two taxonomic Groups D F I Standardized Variable Coefficient 1. Collection type 0.22 2. Collection site 0.14 3. Head width 0.07 4. Frons length I 0.18 5. Frons length II -0.14 6. Seta pattern -0.80 7. Color pattern -0.22 8. Leg hair pattern -0.37 9. Leg length 0.004 Bartlett's Chi Square Test for P < -0001 Significance Percent of Variation 100 85 separates the groups best. The resulting discriminant functions (DF's) are somewhat comparable to the factors of factor analysis in that they consist of weighted linear relationships of the variables. They can also be named according to the variables which are weighted most in the analysis. For the discriminant analysis with individuals sorted into three groups, P_. interruptus, P_. remotus, and P. ? (the non-spotted) two DF's are the result. The first is a combination of the first two factors from factor analysis. Seta pattern is the most influential variable separating the groups, with collection site second (Table 9). Along with seta pattern, color pattern and leg hair pattern are weighted sufficiently to be included in the discriminant function, and, along with collection site, collection type is sufficiently weighted. The second DF, which accounts for only 5% of the variance, consists of some aspect of color and seta pattern along with leg length. It was noted in the morphological analysis of these larvae that the general shape of the body of the light colored larvae was typically longer and less compressed than the P_. interruptus type, with the dark-spotted P_. remotus larvae intermediate. For this reason, the second DF reflects the three variables including leg length as a discriminating combination Table 10 shows the DF. values which result when both P^. remotus types are combined, and two groups are used in the analysis. In this case, the most discriminating variable is seta pattern, with leg seta pattern contributing enough to be included. This is a very clear-cut taxonomic character difference between the two groups, and is not surprising. Other confusing characters, like frons measurements and head capsule 86 measurements, which frequently overlap are not important discriminators. In discriminant analysis, DF's are found only to one less than the number of groups, and thus only one DF is possible for the analysis with two groups. It does, however, corroborate the grouping in the plot of factor I (seta pattern, color pattern, leg hair pattern) vs. factor II. That is, there are two groups with these two factors plotted, and three groups when the first factor is plotted against the factor for collection site and collection type (Fig. 19). Scanning Electron Microscopy: Mouthpart Morphology Figure 20-1 is the dorsal surface of the anterior portion of the head capsule of 3?. interruptus. The lower figure (20-2) is the ventral surface. Figure 21-1 is the dorsal surface of P_. remotus, and 21-2 is the ventral surface. Several comparisons can be made between these two views obtained by means of the scanning electron microscope (SEM) and in comparison with the light micrographs presented earlier (Figs. 14-3, 14-4). From data from 5 preparations of each species it is clear that the labrum in P_. remotus is curved inward more toward the other mouth parts than it is in the case of P_. interruptus (Figs. 20-1 and 21-1. This can be seen in the light micrographs of the labrums (Figs. 14-3 and 14-4) as an apparent projection of the center portion of the labrum in P_. remotus. SEM micrographs of P_. remotus non-spotted larvae show this same type of curvature. Further morphological similarities (labral setal pattern, and prementum shape) indicate that these larvae represent the same species. A second comparison between P^. interruptus and P. remotus can be seen in the lower set of micrographs (20-2 and 87 21-2). In these views it is evident that the shape of the submentum is different in the two species, and that the general shape of the prementum is different. In P_. remotus the submentum and prementum are tapered anteriorly, whereas in P. interruptus the submentum is squared off, and the tip of the prementum is more blunt. Figure 22-1 and 22-2 are ventral views of the tip of the labium of the non-spotted P_. remotus type. In micrograph 22-1 the toothed mandible can be seen. Wiggins (1977) suggested that the two basic types of mandibles (toothed and flat) are probably correlated with larval feeding type. In this study, both species have the toothed mandible which evidently functions in the carnivorous eating habit of the larva. The tapered aspect of the submentum of the non-spotted larva is clear in the top micrograph, and the tapering of the tip of the labium is evident in both micrographs. In figure 23 the labial tips of a P^. remotus non-spotted larva (top) and a P^. interruptus larvae are compared. A very clear difference between the shapes of the labial palpi can be seen in the two pictures. Labial palpi are positioned very close to the silk gland opening and probably are used to guide and perhaps even control the size of the silk being used to make a net. In P. remotus four processes are apparent, one with two smaller processes attached. One of the four, the most dorsal, is shorter and squared. Adjacent to that is a slightly notched process, and a third which is smaller in diameter and also slightly notched. The most ventral process has an olblique edge and two smaller thin processes. These structural differ ences do not appear to cause variation in the structure of the nets built in the laboratory by these two species. In 25 preparations of 88 nets for SEM no species specific differences in net structure were seen. For both species attachment sites like the one seen in Figure 25-2 were found. Nets like the one seen in Figure 25-1 were built in the laboratory by P_. remotus larvae. P.. interruptus larvae had a less elaborate network of threads in laboratory-built nets, but used the same design as P_. remotus. Figure 26-2 shows the end piece of a pupal case having the perforated design held in place by threads of silk. Figure 27-1 and 27-2 illustrate nets of P_. remotus and P^. interruptus respec tively as they are found in the field. Nets of P_. remotus larvae (Fig. 27-1) were found on dead Dulichium leaves in abundant numbers (every plant out of 10 sampled had at least one larva and up to six were found on some plants). Figure 28-2 shows the habitat of Dulichium in water ca. 0.3 m deep. Figure 28-4 illustrates the actual net attached to a dead Dulichium plant. Other common attachment sites for P_. remotus nets were lily pad leaves and stems. Figure 28-1 shows a group of Nuphar leaves and 28-3 illustrates the common use of the notch between the stem and the leaf base for a net. Much floating-type debris was found to be incorporated in these nets. Some of these fragments were exuvia of other emerging insects, particularly mosquitoes. Polycentropus interruptus nets in the field were jelly-like masses of threads incorporated with fine black detritus from the bottom (Figure 27-2). In the intensive sampling of 1978, P_. interruputus larvae were found in nets of this type. When dredge samples were taken, the habitat was so disturbed that nets were never recovered, even though many P_. interruptus larvae were collected. When larvae were cultured in the laboratory, nets of both species were not readily distinguishable. 89 Figure 20-1. Dorsal view of mouthparts of interruptus (lOOx). Figure 20-2. Ventral view of mouthparts of EL interruptus (200x). % 91 Figure 21-1. Dorsal view of mouthparts of P_. remotus (200x). Figure 21-2. Ventral view of mouthparts of remotus (200x) % 93 Figure 22-1. Ventral view of ]?. remotus, non-spotted type (200x). Figure 22-2. Ventral view of P. remotus, non-spotted type, detail (500x). s 95 Figure 23-1. Tip of labium of P_. remotus, non-spotted type, showing labial palps and silk gland opening (2,000x). Figure 23-2. Tip of labium of P_. interruptus showing labial palps and silk gland opening (2,000x). % 97 Figure 24-1. Dorsal view of the tip of the labium of P_. remotus, spotted type, showing labial palps (2,000x). Figure 24-2. Ventral view of the tip of the labium of P_. remotus spotted type, showing labial palps (2,000x). <8 99 Figure 25-1. Net of P_. remotus in battery jar, incorporating Utri cularia (5x). Figure 25-2. Large attachment area of Polycentropus net (500x). /oo 101 Figure 26-1. Pupal case of EL remotus on the side of a culture vial (approx, lx). Figure 26-2. SEM of end piece of pupal case showing perforations and threads (8,000x). 103 Figure 27-1. Net of P_. remotus as it appears in the field attached to Dulichium leaves (approx, lx). Figure 27-2. Net of P_. interruptus as it appears in the field, attached to dead leaves on the bottom (approx, lx). 104 105 Figure 28-1. Nuphar as it appears in Greens lake. Figure 28-2. Dulichium growing in shallow area in Greens lake. Figure 28-3. Net of P. remotus as typically found under Nuphar leaves. Figure 28-4. Net of P_. remotus attached to dead Dulichium leaves in area like Figure 28-2. 107 Figure 28-5. Air-dried preparation of nets showing flattened threads and mucilaginous background (8,000x). Figure 28-6. Critical point dried preparation of nets showing single and double threads, less flattening, and no mucilaginous background (5,000x). i 109 Figure 29-1. Attachment site after one hour of building time (4,000x). Figure 29-2. Attachment site after four hours building time (2,000x). Figure 29-3. Attachment site after six hours building time (l,500x). Figure 29-4. Attachment site after six hours building time, detail (4,000x). //Q Ill Figure 29-5. Attachment site after twelve hours (l,500x). Figure 29-6. Attachment site after one day (2,000x). Figure 29-7. Detail of attachment site after one day (5,000x). Figure 29-8. Small attachment area in one week building time (5,000x). HA. 113 Figure 29-9. Threads of nets after one week (2,500x). Figure 29-10. Non-cleaned net with feathery extensions (l,000x). Figure 29-11. Detail of feathery extensions (10,000x). Figure 29-12. Feathery extensions on cleaned net after six hours (l,500x). 115 However, when larvae were transported in large plastic tubs and battery jars including detritus and Utricularia from the lake, larvae of P. remotus were recovered from the sides of the container and from the Utricularia. Polycentropus interruptus larvae were usually found in nets attached to the bottom of the container, the nets with an appearance like Figure 27-2. Scanning Electron Microscopy: Net Structure and Attachment Sites Scanning electron microscopy was utilized in order to examine the fine structure of the nets of Polycentropus species. Results of an experiment to test preparation methods can be seen in Figs. 28-5 and 28-6. Figure 28-5 is a net prepared by air-drying with no other preser vation. A flattened appearance and mucilagenous background appearance is distracting compared with the preparation in 28-6. In this prepara tion the net was dehydrated with a graded alcohol series and critical point dried. Thread size and attachment site configuration were much easier to distinguish when nets were prepared in this way. In the only previous study of Trichopteran net structure (Wallace, 1976) the nets were air-dried only. Next, net building was compared for a progression of time periods. Nets were examined after building times of 1-12 hours, 1-5 days, one week, and two weeks. Figures 29-1 through 29-8 show threads and attach ment sites for the following building times: one hour, four hours, six hours, detail of six hours, twelve hours, one day, detail of one day, and one week. Size of attachment sites increased as the time period increased; attachment sites of 100 pm were only found in nets more than one day old. 116 Size of attachment sites ranged from 2 pm to as large as 100 pm in width. Apparently the first step in building nets for Polycentropus larvae is to form a triangulation of small attachment sites; in the culture vessels used for this experiment this involved an area of ca. 8x3x3 cm. From this first structure of outlying threads with small attachment areas, the larvae worked closer and closer to an eventual tube-shaped retreat. After four hours, a tube-shaped structure could usually be distinguished in culture vessels. Largely because the water was filtered, the net strands and tubes were nearly invisible. The only way of determining the position of the net in many cases was to assume that the tube-shaped portion of the net had been built when and where the larva rested (often ventral side up) on the bottom of the vessel. Threads could then be detected on other parts of the vessel lining when the coverslips were removed. Figures 29-1, 29-2, 29-3, and and 29-4 show an increasing size of attach ment sites taken from the main tube-shaped portion of the net. Figures 29-5 and 29-6 respectively, show attachment sites in the main tube area after 12 hours, and after one day. Figure 29-7 shows the one-day attach ment area in detail, illustrating how some large attachment areas break apart. The small attachment area formed after one week of building time and shown in Fig. 29-8 is still intact, which may indicate that larvae continuously attach new threads to maintain the net. Figure 29-9 illustrates the increase in bacterial load noticed on all net preparations as time progressed. Figures 29-10, 29-11, and 29-12 are of feathery structures encountered in two net preparations. Figures 29-10 and 29-11 are micrographs from a net that was not built in clean, filtered water on coverslips. This elaborate network normally was not observed in 117 filtered water cultures. One instance of a similar structure being found on a cultured net is seen in Figure 29-12. No function has been determined for these structures, and further examination of this type of net structure should be made in order to determine their source and function. The difference between the nets of the two species appears not to be in size or pattern of silk threads used. For both species net silk strands were from .25-.1 pm and the pattern random (Loesch, 1978; Loesch, 1978; Loesch and Crang, 1978), nor was it in attachment site configuration or in building pattern, but rather in substrate incorpora tion. The difference in nets is therefore a habitat difference rather than a difference in silk size or instinctive building pattern. IP. interruptus nets, when built in the detritus of the bottom of the lake or battery jar are more compact and appear to have less area. P_. remotus nets are more variable in shape and size depending on the type of aquatic plant used for substrate. In some cases net strands attached to lily pads could be seen streaming 30 cm. or more from a stem. The significance of the small diameter of the threads has been discussed by Maias and Wallace (1976). They calculated that with . support strands of 0.3 pm and mesh strands of a smaller size, only 1/38 3 . mm of silk is required to spin the silk required to build the posterior two-thirds of a net 60 mm long. The small diameter of the strands of silk in these nets thus allows the larva to conserve silk and energy expenditures. The same certainly appears to be equally true of Polycentropus larvae. Even though no pattern of building of the tube 118 like portion of net could be determined, the size of the individual threads composing the net were comparable to the ones in the Maias and Wallace study. The function of the peripheral threads will be discussed in the following section. Feeding Behavior and Territorialism Macan, (1977) stated that Polycentropus larvae exhibited self regulating territorialism in the absence of predators. Pianka (1978) stated that the most widespread type of territory is the feeding territory. In Polycentropus larvae territorialism would obviously not be for mating, so the question of which limiting resource might be respons ible can be narrowed. Also, Pianka stated that dispersed spacing systems which result from territorial defense are indicative of competition for some resource in short supply. To determine if the spacing system is dispersed, and thus if the organisms are territorial, Pianka referred to a statistical technique where a ratio of variance to.mean in the numbers of individuals per quadrat is found. If this ratio is unity, spacing is random. If the ratio is less than unity the spacing is dispersed; when it is greater than unity the spacing system is clumped. Pianka (1978) then suggested that dispersed spatial distributions are indicative of competition and K selection, but that random and clumped distributions do not indicate much at all about the factors influencing the distribution. The question of territorialism in Polycentropus was investigated by finding the density of larvae on artificial substrate samplers, by finding the frequency of larvae on lily pads, and by measuring spacing distances on these two. In addition, laboratory 119 experiments with artificial lily pads were carried out. In this population the density of larvae per station as determined by artificial substrate samplers is seen in Table 10a. The variance of these density values is 2.41 and the mean is .048. The ratio of 2.41/.048 is 50.2. This is far in excess of unity and indicates a clumped distribution of larvae. However, this does not necessarily mean that territorial spacing does not occur within the clumps. If the quadrats are smaller or of a different type, a situation closer to dispered spacing can be seen. For example, if lily pads in a transect are examined for larvae, the frequency of these larvae (Table 1) has a variance of 2.8 and a mean of 0.48 individuals per stem. The ratio is 28/0.48 or 5.8, less clumped than when quadrats at widely spaced stations are used. When two or more Polycentropus larvae are found in the same vicinity, as on a single aquatic plant or on a piece of artificial substrate, spacing distances are somewhat predictable. Table 12 shows spacing distances in several circumstances: on lily pad stems, on other aquatic plants, on artificial substrates, and on laboratory equipment. In the last three categories, distances averaged close to 5 cm. On lily pad stems, however, distances averaged 8.3 cm. It appears that the greater spacing distance on the lily pad stems is due to the paucity of attachment angles on the cylindrical lily pad stem as com pared with the numerous notches on Dulichium and in artificial substrate. It may be, therefore, that spacing for a feeding territory may be the case in optimal net-building areas, such as in the beds of dead Dulichium, or in Utricularia masses. Overall, there does not appear 120 to be a large enough population for this to be an operable mechanism of competitive avoidance either intra-or interspecifically. In the laboratory aggressive encounters were seen between five larvae placed together in a three liter batter jar. Wiggins (1978) stated that larvae tend to be cannibalistic in captivity, thus the artificiality of captivity could be an explanation for this behavior. The battery jar had been set up with five lengths of plastic tubing extending from a piece of screening fitted in the top of the jar. This was to simulate the microhabitat conditions in the lily pad areas of the lake. In the first 24-hour period aggressive encounters were observed between larvae #3 and #4, #1 and #5, #3 and #5, and #3 and #5 a second time. At the end of the first 24- hour period #5 was dead, and numbers 1 through 4 had established nets at an average distance of 9.1 cm. apart. Larvae did not establish nets on the plastic tubing, but rather on the sides and bottom of the battery jar, and one on the underside of the screen where it dipped into the water and was partially submerged. Six days later the original four were still alive and feeding from the nets they had built. Larva #5 was dead and exemplified the occurrence of cannibalism that Wiggins (1978) suggested. A second trail of this experiment resulted in two of the five larvae being killed. Thus, in a laboratory situation, aggressive behavior which might indicate territorial defense occurred. Whether this was simply due to captivity, or whether it occurs in the field is not known. Thus, because of the low density and clumped distribution in the field, it does not seem warranted to say that Polycentropus larvae are terrestrial. TAB L E 10 Q. Densi ty Of Polycentropus Larvae On Artificial Substrate Samplers (per cm2) 1976 1977 STATION STATION 1. 1. 018 11. .006 2. 10 2. 12. .018 3 09 3. .03 13. .006 4. 09 4. .006 14 - 5 — 5 .042 15 .03 6 23 6. .012 16. .024 7. .04 7. .078 17. .018 8. 025 8. .078 18. .018 9 02 9. .024 19 08 10 .09 10. .072 20 .03 1 2 1 TABLE 11, Frequency Of Larvae Lily Pad Areas 1978 1977 S ample T ype Frequency Ot Stems S amp 1e Frequency Inhabit ed 1 MIXED .5 5 r 1.4-1,62/sfem 2 M IXED .5 5 2 1.00-4.6/ ■■ 3 MIXED .4 6 3 1.00 - 1.09/ - . 4 M 1 XED .80 5 NY'1 .16 • All 6 NY . 05 Mixed 7 NU 2 . 75 1* Nymphaea 8 N U . 60 2. Nuphar 9 N U . 8 7 10 N U . 1 6 11 NU . 40 12 DULICHIUM TA BLE12.Average Spacing Distances OI Polycent ropus Larvae Field La bo r a t o r y ARTIFICIAL AQUATIC PLANTS SUBSTRATES L i 1 f Pads 01 her Plant s sample d i s lance sample d i slance sample distance sample d i s tance no. no- no- no- 1. 10 cm 1. 6 cm 1. 5cm 1. 5cm 2. 8 2. 5 2. 4.8 3. • 7 3. 4.5 A V G 83 5.3 4.9 5 S> v > s HX 1 P 2 32 L E 8 44 1 0 E 124 Feeding Behavior and Gut Analysis Larvae brought to the laboratory in .1977 were maintained in aquaria with natural vegetation and some detritus. Some larvae were separated and reared in culture vials within the aquaria in such a manner that metamorphotypes or adults could be used for associations. It was necessary to feed these larvae by hand, and cladocerans were the main food item used. Obervations were made of feeding behavior of these larvae. The most typical response of the larva was a quick movement of the entire body when the food item moved one of the peripheral threads of the net. If the food item did not struggle or continue to move the net threads, the larva also remained stationary. Once the net was moved sufficiently, however, the larvae moved rapidly to the source of disturbance and caught the prey. They would then dislodge the prey from the net and return to the main tube-like portion of the net to consume all of the prey. If the net was disturbed artificially with a probe the larva would also react by moving quickly to the area of disturbance. No differences were noted between the two species of larvae in their feeding behavior. In an experiment to test satiation, one larva was fed 17 Clado cerans in a four hour period. Within this time various lengths of time between feedings were observed. The most rapid time for captur ing, consuming and capturing a second prey was one minute. In a series of six prey items the time lapse betwen feeding was: 1 minute, 2 minutes, 1 minute, 2 minutes, 3 minutes, and 3 minutes. Each new food item was introduced as soon as the larva had captured the previous one. 125 In 1978 larvae were fed Oligochaetes. These were readily avail able from ditches and rivers in the Bowling Green area. Larvae could also easily capture these, since they did not swim and were not able to extract themselves from a net once caught. Another food item that was used in 1978 was brine shrimp (Artemia). Twenty-four spotted EL remotus, 10 jP. remotus of the non-spotted type, and 5 P_. interruptus larvae were analysed in 1978 for gut contents Table 13 summarizes the types of food materials in the guts of,these larvae. For IL remotus larvae, 70% had animal food in the gut. For P_. remotus of the non-spotted type, 70% also had animal food in the gut. For P_. interruptus 80% had animal food in the gut. Table 13 also lists the types of animal food which were possible to identify. A commonly occurring food item was corixid parts. At this time in the spring eggs of corixids had just hatched and they were abundant in the sheltered bay. There were also many places with dense populations of Ostracods. These were not identifiable in the gut contents, but probably were present listed here as arthropod parts. There were many cases of segmented antennae, legs, etc. which were not classifiable. Figure 30-1 is the head of a ceratopogonid found in a P_. interruptus gut. Figure 30-2 and 30-3 are parts of a corixid. The spatulate tarsus is clearly that of a corixid, and the compound eyes would locically be those of the most plentiful adult or hemimetabolous young prey. From the above data, it appears that previous workers were correct in stating that net-building Trichoptera are not selective feeders. From the gut contents analysis it does appear that species are 126 are predaceous. From the laboratory feeding experiments, it further seems that Polycentropus larvae will eat whatever is available. 127 TABLE 13 Types of food materials in guts of Polycentropus larvae. No. of cases of No. of cases of No. of cases of plant detritus animal parts unidentified animal parts and plant parts P. remotus 3-100? 3-cladoceran-like 6^-arthropod parts, claws crustaceans 1-95% 3-cladocera 7-insect leg sections, other parts 1-50% 2-corixidae claws 3-compound eyes, tarsi, probable of corixidae -vascular plant 2-mayfly larvae leaf parts -desmid 1-oligochaete 1-midge larva 24 observed, 17 with animal food P. ? 1-100% 1-Clodocera 2-insect antennae 1-90% 1-Corixidae claw 2-arthropod bristles claws 2-80% 3-mayfly larvae 3-insect body parts, legs 1-50% l-midge larvae 10 observed, 7 with animal food 128 TABLE 13? (Cont.) No. of cases of No. of cases of No. of cases of plant detritus animal parts unidentified animal parts and plant parts P_. interruptus 1-100% 1-Cladoceran-like 1-large insect parts legs 1- Ceratopogonidae head 2- midge parts 5 observed, 4 with animal food 129 Figure 30-1. Gut contents of P. interruptus, Ceratopogonid head (50x) Figure 30-2. Gut contents of P. remotus, Corixid tarsus (50x). Figure 30-3. Gut contents of P. remotus, Corixid compound eyes (100x) /3ö 131 Summary of Results Six general statements can be made about the biology and ecology of Polycentropus species from these results. (1) They are found in even a very limited habitat of an acid bog lake and make up a major portion of the Trichopteran fauna there, in comparison to being a smaller portion of the Trichopteran fauna in a similar, but more diverse lake type, an alkaline bog lake withsome sandy shores. (2) Adults of three species were found emerging in May, June, July, and August: Polycentropus flavus, EL remotus, and IL interruptus. A common mechanism for niche separation of aquatic insects, temporal life history differences, only adequately separated P_. flavus from the other two species. (3) Larval life history of the two larval species found, IL remotus and IL interruptus, showed that both had five larval instars, and that EL remotus was slightly smaller than P_. interruptus in all instars. Morphological observations and measurements showed that a good character for separation of the two species in young instars was the number of setae on the pronotum, and color difference of the head. P_. remotus was found to have two color types, one with dark muscle scar spots and dark patches on the head, the other with no muscle scar spots. Wiggins (1978) stated that the Polycentropus speciments he had indicated that speciments could have muscle scars spots lighter than the ground or darker as some 132 diagnoses stipulated. This was true for EL remotus in this collection. (4) Analysis of the larvae found at different collection sites indicated that EL interruptus was dominant in stations away from vegetation, though common at stations with vegetation and a fine, loose detritus bottom; and that P_. remotus was dominant in stations with extremely thick bog/flocculent peat bottoms, but with floating vegetation, and at stations very close to shore with submerged vegetation. Multivariate analysis of morphological characters with ecological measures indicated several things: a) the best distinguishing character for two species was pronotal setal pattern and leg hair pattern, b) if three groups were formed, distinguishing characters were, in order: setal pattern, collection site, color pattern, substrate, and finally leg hair pattern; c) factor analysis showed two groups in one comparison, and three groups in another comparison: if factor scores were plotted on a graph with factor I (seta, color and leg hair paterns) and factor II (head measurements) two groups form; if the axes were Factor I and factor III (collection site and type), three groups emerged. (5) Scanning electron microscopic observation of nets and labial structures indicated very little difference between larvae of the two species. Nets had the same range of thread sizes and no pattern in either species. Attachment sites seen for nets of both species were the same, and increase in 133 size as the net-building process progresses were observed. Silk gland openings in both species measured ca. 1.2pm and each had specific "tools" on the ends of the labial palps. (6) An analysis to determine territorial behavior found that Polycentropus larvae in the field are clumped in distribution, not dispersed as would be required for territorial organisms. Aggressive cannibalistic behavior observed in the laboratory was probably due to the laboratory conditions rather than to territorial defense. Even though overall distribution in the field was clumped, in a field microhabitat situation, such as on aquatic plants, spacing ranged from 5 cm. to 9 cm. Thus in areas where optimal net sites are limited perhaps territorialism did occur. Feeding was found to occur when threads of a net were disturbed, that food was 70% - 80% animal material, and that when larvae were satiated, further movement of the net threads elicited no response. 134 Discussion of Evolution and Adaptation in Polycentropus Lentic Polycentropus are adapted to life in the most extreme of aquatic conditions. There are species adapted to temporary pools which have evolved a diapausing strategy to avoid dessication (Wiggins, 1978). There are also species of Polycentropus which live in detritus in the benthos with chironomids (Wiggins, 1978). The two species of Polycen tropus in this study were found in an acid bog lake; one species occupying a niche including aquatic vegetation as substrate, and aquatic arthropods as food,.and the other occupying a niche which included fine detritus in the benthos as substrate and aquatic arthropods as food. Both larvae build retreat nets with a large netwook radiating from the retreat. A potential prey item disturbs a thread in the catch-network and the larvae is alerted by the movement. These two species are probably taking advantage of diurnal movement of aquatic arthropods by occupying different levels of the water column. They may also be taking advantage of different prey items indigenous to each microhabi tat, e.g. chironomids vs. corixids. This second hypothesis was partially shown by stomach analysis in this study, but requires further analysis to be fully substantiated. The position of the Polycentropodidae in the phylogeny of Trichop tera families relfects this ability to diversify in a community. Figure 31 is a summary of ecological and physiological adaptations in the superfamily Hydropsychoideae. Most of the information is gathered from Wiggins (1978) and Ross (19682_. The family Polycentropodidae is pivotal in the successful diversification of this superfamily. With the Polycentropodidae twice as many habitats are exploited, and twice 135 as much food type is exploited. There are lotic as well as lentic polycentropodids, and predators as well as detritus-feeders. Adapta tion for respiration has also taken a path of maximum exploitation. Larvae have developed the ability to undulate the abdomen, which creates a current of fresh water: this frees them from dependence upon a current. A fringe of adominal hairs provides spacing between the larva and the net to facilitate passage of water. In larvae of species found in the benthos (P_. interruptus in this study) the fringe is thicker, and the abdomen shorter and flatter, allowing for effective undulations, and preventing the abdominal surface from being clogged with the fine detritus of its surroundings. A puzzling adaptation is that of diverse "tools" on the labial palps. In some polycentropodids, the labium is extended in length and the labial palps absent, as is the case in the Psychomyiidae. These polycentropodids builds compact tubes as do the psychomyiids. In Polycentropus larvae, however, the labium is pointed and the labial palps have variously shaped processes on their ends. The bottom- dweller (P_. interruptus) has stockier processes than the vegetation- dweller (1?. remotus) . The reason for this difference is not clear. The color differences in P_. remotus and P. interruptus also show interesting adaptations to the diverse habitats occupied by these species. Polycentropus interruptus has a dark colored head, camouflag ing it on the bottom of the lake; P_. remotus has two color variations, and occupies two types of substrate. With very light colored heads, the larvae are best camouflaged for life on vegetation near the surface. With the intermediate spotted coloration, larvae are camouflaged for 136 life near the bottom. The data in this research indicate that the two color types correspond to these camouflage ideas. Genetic evolutionary divergence or environmentally stimulated color dimorphism may be responsible for these color types. A test of these alternate hypotheses might be to rear larvae of each color type in two conditions; one simulating the dark bottom the other the light vegetation zone. Figure 31 also highlights the adaptations in the superfamily Hydro psychoidae which lead to the wide diversification in the polycentropodids. It is interesting to note that one genus of the family Psychomiidae has been found to occupy lake margins when all the others in the family are restricted to lotic habitats. This ability was incorporated in the polycentropodids and expanded to include all lake conditions. An impor tant aspect of this adaptive change in Polycentropodidae was the behavioral ability to undulate the abdomen to increase respiration efficiency. When the Hydropsychoidae are considered, it is seen that another adaptive route was taken. This family, which developed from the same ancestor as the Polycentropodidae, turned to a specialization of abdominal gills to cope with respiration problems in an increased body size. This adaptation allowed the larvae to increase in size, but still limited them to lotic water for sufficient oxygenated flow. It seems, then, that the polycentropodids introduced the evolu tionary line which was followed by case-making Trichoptera: abdominal undulation which creates a flow of fresh, oxygenated water enabled larval Trichoptera to exploit all aquatic habitats, to build protective cases, and to become mobile. 137 The two species of larvae in this study also are examples of exploitation by this family of range of habitats in a harsh bog environment. 138 Figure 31. Summary of ecological and physiological adaptation of Trichoptera in the superfamily, Hydropsychoideae. PHYLOGENY OF HABITAT NET/FEEDING RESPIRATION FAMILIES LOTIC LENTIC / Philopotamidae fast tube nets with cutaneous, no running very fine mesh fringe of hairs water eat fine organic particles fast Tinodes detritus covered cutaneous, no running lake tubes fringe of hairs water margins eat detritus various branched tubes cutaneous, fringe currents------^-filter food of hairs + trumpet tubes undulations filter food all lake extensive tubes on cutaneous, fringe conditions aquatic vegetation of hairs + undulations predaceous tubes in detritus cutaneous, fringe of hairs + predaceous undulations various some lake various, but with abdominal gills currents margins specialized mesh catch net 1 3 eat organic particles 9 140 CONCLUSIONS The literature suggests that there is a temporal difference in the emergence of IL remotus and EL interruptus (Flannagan and Lawler, 1972). Polycentropus remotus emerged later than EL interruptus with no overlapping time. Thus study, however, shows that both P_. remotus and ]?. interruptus emerge at the same times throughout the summer, and that a third species, EL flavus emerges at a later time. The larvae of P_. remotus and EL interruptus are ecologically separated by utilization of different substrates. Multivariate statistical analysis shows that with the specimens used and the variables measured, only two morphological groups were discernable. These groups were: dark colored heads, two setae on the pronotum, leg setae straight, and light colored heads, 3+ setae on the pronotum, leg setae patchy. This analysis also shows that two environmental variables (substrate and habitat type) further separate the specimens in the light colored group into two subgroups: one hand-picked from emergent and floating vegetation, and the other from vegetation and bottom deposits in open water and bog edges. 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