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University Microfilms International 300 North Zeeb Road Ann Arbor, Michigan 48106 USA St. John's Road, Tyler's Green High Wycombe, Bucks, England HP10 0HR 7 9 0 2 2 3 3

STQFFER» RICHARD LAWRENCE A COMPARATIVE BEHAVIORAL STUDY OF THE DIPTERA FAMILY CHIRONOMIDAE.

• THE OHIO STATE UNIVERSITY• PH»D*« 1978

University M icrofilm s international 300 n zeeb r o a d, a n n a r b o r, mi 4bio6

© Copyright by Richard Lawrence Stoffer 1978 A COMPARATIVE BEHAVIORAL STUDY OF

THE DIPTERA FAMILY CHIRONOMIDAE

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

BY

Richard Lawrence Stoffer, B.S., M.S.

The Ohio State University

1978

Reading Committees Approved By

Dr. Barry D . Valentine

Dr. N. Wilson Britt

Dr. David H. Stanshery

'.M \. v iisor

Department of Zoology ACKNOWLEDGMENTS

I am indebted to a number of people who have influ­ enced this study. To my advisor Dr. Barry D. Valentine I owe much for his guidance and confidence in advisees to encourage original research.

I thank Dr. Donald E. Johnston for his assistance with the NTSYS computer program; Dr. James E. Sublette of

Eastern New Mexico University for his time spent checking the identification of the Tanytarsini; and Dr. Ole A.

Saether, formerly of the Freshwater Institute in Canada, for his verification of several Orthocladiinae. To

Dr. Saether, Dr. Andrew L. Hamilton, also of the Freshwater

Institute, and Dr. D. R. Oliver of the Biosystematics

Research Institute in Canada, I am indebted for the use of their unpublished keys to Chironomidae. I also thank

Water Resources for the use of one of their Ekman dredges.

I thank Ohio State University and the Zoology

Department for their financial assistance, mainly in the form of a University Fellowship and Teaching and Research

Associateships.

I wish to thank the reading committee for their time and comments concerning the preparation of this work. To my fellow graduate students, especially those who shared room 373 BZ , I am indebted for their friendship, intellectual discussion, and many cherished remembrances.

Most of all I wish to acknowledge my wife Cathy for her assistance in the collection of specimens and data, her editorial and typing time on this manuscript, and her patience during the course of this study. VITA

December 13, 19^8.... Born - Cleveland, Ohio'

1970...... B .S ., Ashland College, Ashland, Ohio

1972-1976...... University Fellowship, The Ohio State University, Columbus, Ohio

1975...... M.S., The Ohio State University, Columbus, Ohio

1978...... Research Assistant, Zoology Department, The Ohio State University, Columbus, Ohio TABLE OF CONTENTS

Page ACKNOWLEDGEMENTS ii

VITA iv

LIST OF TABLES vii

LIST OF FIGURES ix

LIST OF ABBREVIATIONS xi

INTRODUCTION 1

Survey of Behavioral Systematic Literature 2

General Information About Chironomidae 12

Larval Lifestyles 13

Larval Feeding Behavior 19

Larval Irrigation 24

Larval Grooming 25

Behavior Associated with the Pupae 25

Fore Leg Positioning in Adults 27

Adult Grooming Behavior 27

STATEMENT OF PURPOSE 30

METHODS AND MATERIALS 31

Collection and Sorting of Specimens 31

Rearing of Specimens 35

Observation of Specimens 39

v Page Mounting Procedures 44

Analysis of Data 48

TAXONOMIC TREATMENT 50

RESULTS 68

Larval Habitation 68

Larval Feeding Behavior 83

Larval Irrigation 85

Larval Grooming 88

Behavior Associated with the Pupae 89

Fore Leg Positioning in Adults 91

Adult Grooming Behavior 106

ANALYSIS OF DATA AND CONCLUSIONS 125

Selection of Characters 125

Character Analysis 136

Comparison of Taxa 144

Discussion of Behavioral Relationships of Taxa 160

Significance of this Study 167

REFERENCES 169

APPENDIX A: ADDITIONAL TABLES 193

APPENDIX B: COLLECTION LOCALITIES AND NOTES 221

vi LIST OF TABLES

Table Page 1 . Taxa Examined 51

2. Adult Grooming Frequencies 107

3. Number of Observed Grooming Combinations 122

4. Behavior of the Larvae and Pupae 126

5- Fore Leg Positioning in Adults 129

6. Product-moment Correlation Coefficients for

Characters 194

7. Principal Component Analysis of Characters 196

8. Product-moment Correlation Coefficients for

Chironominae 198

9. Principal Component Analysis of Chironominae

Characters 200

10. Product-moment Correlation Coefficients for

OTU's 202.

11. Distance Matrix of OTU's 205

12. Principal Component Analysis of OTU's 208

13- Five Dimensional MDS of OTU's 210

14. Distance Matrix of OTU's Based on Characters

from Immatures 211

15* Distance Matrix of OTU's Based on Adult Fore

Leg Positioning Characters 213 vii Table Page 16. Distance Matrix of OTU's Based on

Characters of Adult Self-grooming 217

17- Three Dimensional MDS of OTU's Based on

Characters of Adult Self-grooming 220 LIST OF FIGURES

Figure 1. Frame for Collecting Tent

2. Section from a Test Tube Rearing Apparatus

3- A. Longitudinal Cross-section of a Typical

Tanypodinae Burrow

B. Tube and Feeding Net of Rheotanytarsus,

redrawn from Walshe (1950)

U. Tubes of Polypedilum digitifer

5. Leaf-miner Nets

6. Substrate Leg Position of Psectrotanypus

dyari

7- Fore Leg Positioning in Chironomus

attenuatus

8. Positioning of FFA in Chironomus attenuatus

9. Fore Leg Positioning in Dicrotendipes

nervosus

10. Fore Leg Positioning in Stenochironomus

hilaris

11. Plot of the first three PCA factors using

all OTU's and characters

12. Plot of the first three MDS dimensions

using all OTU's and characters Figure Page 13. Phenogram showing distances in the behavior

of immatures 15^

14. Phenogram showing distances in adult fore

leg positioning behavior 155

15- Plot of the first two MDS dimensions using

all OTU's andgrooming characters 157

x LIST OF ABBREVIATIONS

Ab - grooming movement in which the abdomen is rubbed by

one or both hind legs.

ABLMAL - OTU abbreviation for Ablabesmyia mallochi ,j

ACRSEN - OTU abbreviation for Acricotopus senex.

An - grooming movement in which one or both antennae are

rubbed by one or both fore legs.

BL - a line bisecting the body longitudinally as seenjfrom

above .

CHIATT - OTU abbreviation for Chironomus attenuatus.

CHIPLU - OTU abbreviation for Chironomus plumosus.

CHIRIP - OTU abbreviation for Chironomus riparius.

CHIRON - OTU abbreviation for Chironomus summary.

CLADOT - OTU abbreviation for Cladotanytarsus summary.

CLIPIN - OTU abbreviation for Clinotanypus pinguis.

COELOT - OTU abbreviation for Coelotanypus summary.

CONCHA - OTU abbreviation for Conchapelopia summary.

CORYNO - OTU abbreviation for Corynoneura summary.

CRIBIC - OTU abbreviation for Cricotopus bicinctus.

CRICOT - OTU abbreviation for Cricotopus summary.

CRISP3 - OTU abbreviation for Cricotopus sp. 3*

CRISP4 - OTU abbreviation for Cricotopus sp. 4.

CRISP5 - OTU abbreviation for Cricotopus sp. 5* xi CRITRI - OTU abbreviation for Cricotopus trifasciatus.

CRYGAL - OTU abbreviation for Cryptocladopelma galeator.

CRYPTO - OTU abbreviation for Cryptochironomus summary.

DEMBRA - OTU abbreviation for Demei.jerea brachial is .

DICFUM - OTU abbreviation for Dicrotendipes fumidus.

DICMOD - OTU abbreviation for Dicrotendipes modestus.

DICNER - OTU abbreviation for Dicrotendipes nervosus.

DICROT - OTU abbreviation for Dicrotendipes summary. dvW - grooming movement in which the ventral wing surfaces

are rubbed by the contralateral hind leg in conjunc­

tion with dWH. dWH - grooming movement in which the dorsal wing surfaces

are rubbed by the ipsilateral hind legs. dWM - grooming movement in which the dorsal wing surfaces

are rubbed by the ipsilateral mid legs.

EINCHE - OTU abbreviation for Einfeldia chelonia.

ENDNIG - OTU abbreviation for Endochironomus nigricans.

FF - grooming movement reported by Heinz (19^9) in which

the fore legs rub each other.

FFA - in adult fore leg positioning the angle produced by

the fore femora from a vertical line through the coxa

as viewed from the front.

FH - grooming movement in which a fore leg may be rubbed

by the hind legs.

FMs - grooming movement in which a fore leg is rubbed by

its ipsilateral mid leg as the mid leg steps down on

it. xii GLYLOB - OTU abbreviation for Glyptotendipes lobiferus.

GLYPAR - OTU abbreviation for Glyptotendipes paripes.

GLYPTO - OTU abbreviation for Glyptotendipes summary.

HARINC - OTU abbreviation for Harnischia incidata.

HH - grooming movement in which the hind legs mutually rub

. each other.

HMs - grooming movement in which a hind leg is rubbed by

its ipsilateral mid leg as the mid leg steps down on

it.

LENCRU - OTU abbreviation for Lenziella cruscula.

MDS - Multidimensional scaling.

MH - grooming movement in which the hind legs are rubbed

by their ipsilateral mid legs.

MHH - grooming movement in which a mid leg is rubbed by

both hind legs.

MICNIG - OTU abbreviation for Microchironomus

nigrovattatus.

MTOPED - OTU abbreviation for Microtendipes pedellus.

MICROP - OTU abbreviation for Micropsectra summary.

NANDIS - OTU abbreviation for Nanocladius distinctus.

ORTHOC - OTU abbreviation for Orthocladius summary.

OTU - Operational taxonomic unit.

P - grooming movement in which one or both palps are rubbed

by one or both fore legs.

PARACH - OTU abbreviation for Parachironomus summary.

PARATA - OTU abbreviation for Paratanytarsus summary.

PCA - Principal component analysis. xiii PHAFLA - OTU abbreviation for Phaenopsectra flavipes.

POLYPE - OTU abbreviation for Polypedilum summary.

PROBEL - OTU abbreviation for Procladius bellus.

PROCLA - OTU abbreviation for Procladius summary.

PROSUB - OTU abbreviation for Procladius sublettei.

PSEDYA - OTU abbreviation for Psectrocladius dyari.

SFA - in adult fore leg positioning the angle produced by

the fore femora anterior or posterior to a perpendic­

ular through the coxa to a line along the dorsal sur­

face of the mesonotum as viewed from the side .

SMISP1 - OTU abbreviation for Smittia sp. 1.

SMISP2 - OTU abbreviation for Smittia sp . 2.

SMITTI - OTU abbreviation for Smittia summary.

STEHIL - OTU abbreviation for Stenochironomus hilaris.

STEPOE - OTU abbreviation for Stenochironomus

poecilopterus.

STICTO - OTU abbreviation for Stictochironomus summary.

T - grooming movement in which the dorsum of the thorax

and head are rubbed by the fore legs.

TANSTE - OTU abbreviation for Tanypus stellatus.

TANYTA - OTU abbreviation for Tanytarsus summary.

THIENE - OTU abbreviation for Thienemanniella summary.

TiTEA - in adult fore leg positioning the tibia-tarsal

extreme angle produced by the angle of a line through

the proximal tip of the tibia and apex of the tarsus

to the anterior section of a line bisecting the body

longitudinally as seen from above (BL) . xiv UPGMA - cluster analysis using unweighed pair-group

method using arithmetic averages. vW - used to represent the presence of any grooming of the

ventral wing surfaces. vWH - grooming movement in which the ventral wing surfaces

are rubbed by the ipsilateral hind legs. vWp - grooming movement in which the ventral wing surface

is positioned over the hind leg.

WH - grooming movement in which the wing is rubbed by the

hind leg(s), but details are not specified by Heinz

( 1 9 W • XENXEN - OTU abbreviation for Xenochironomus xenolabis.

xv INTRODUCTION

The works of Konrad Lorenz, Karl von Frisch, and

Nikolaas Tinbergen have advanced the use of behavior as

a systematic tool. While they pointed to the works of

Heinroth (1911) and Whitman (1919) as being the first to

actually use behavior in drawing taxonomic conclusions, it was their combined efforts that put ethology alongside morphology in actual systematic usage.

Lorenz (1957a) related how Charles Otis Whitman wrote,

in I898, "Instincts and organs are to be studied from the

common viewpoint of phyletic descent." Tinbergen (1951) pointed out that innate motor patterns behave much like morphological characters and can be treated in much the

same manner. Behavioral patterns are a part of complex

systems also involving morphological and chemical compo­ nents . Each component is the result of a lengthy evolu­ tionary history and is adaptive, adaptable, and closely attuned to the niche by constant interplay between the genome, phenome, and environment (Evans, 1966a). As with morphological characters, successful use of the comparative method depends on the selection of suitable characters to be compared. The characters must not be indicative of environmental variation, but of genetic diversity. Hinde 1 2 and Tinbergen (1958) emphasize that the characters chosen

for a study must have variability suitable for the problem.

Characters which are too conservative or too divergent within a group are of little value in assessing relation­

ships within the group, although conservative characters

may give an indication of relationships with other groups.

The best classifications are based on a large number of

characters. Relationships become more reliable as individ­

ual and environmental variations are minimized with in­

creasing numbers of characters (Mayr, 1958; Evans, 1953)-

Survey of Behavioral Systematic Literature

Behavioral characters have been used successfully in a

number of systematic studies. The following survey omits

primarily descriptive studies, not because they are unim­

portant, but because they are too numerous for the scope of

this survey, and the emphasis is intended to be on studies which develop some indication of relationships within or

between groups of taxa.

Birds have probably had the most behavioral systematic work of any group. Heinroth (1911) was the first to look

at the family Anatidae and analyze it taxonomically based

on innate behavior patterns. Additional analyses of ducks followed. Lorenz (19^1) utilized various display compo­

nents in his revision of the family. Delacour and Mayr

(19^5) emphasized the value of pair formation, displays, nesting, and feeding habits in forming a classification for

their revision of the family. In the most recent revision, 3 Veselovsky (1975) not only pointed out the importance of

ethology in determining phylogenetic relationships, but also determined at what taxonomic levels various types of behavior were meaningful. Whitman (1919) found that one single character, drinking behavior, can separate the pigeon family. This type of drinking behavior is found in only one other family, that of sandgrouse, which along with the pigeon family comprise an order. Stonor (1936) used male displays and morphological characters in discussing the relationships and phylogeny of six species of birds-of- paradise. Davis (19^-2) proposed a phylogeny for a group of social nesting cuckoos based primarily on their nesting be­ havior. Mayr and Bond (19^3) contributed to the generic classification of swallows using both morphological and behavioral characters, such as nesting habits. They point­ ed out that behavior is often more conservative than mor­ phology. Wing (19^6) compared seven genera of grouse with respect to various components of courtship and constructed a phylogenetic tree based on them. Daanje (1951) made a comparative study on intention movements derived from loco- motory movements that have been ritualized in bird display, threat, and begging movements; and showed that this ritual- ization parallels phylogenetic lines. In Lack's (1956) re­ view of the nesting habits of swifts, he found that certain nesting characters are characteristic for each genus.

Andrew (1956a) quantified ritualized intention movements in a number of passerine birds and found that sub-families can 4 be defined by certain behavioral characters, especially tail-flicking. Andrew (1956b), narrowing his study to finches, studied a number of behavioral components of threat postures, calls, courtship displays, mating, and nest site displays. From these he formed four behavioral phenotypes and speculated on the geographical migration of their ancestors. Hinde (1956) studied the presence and frequencies of occurrence of behavioral patterns in hybrid weaverbirds. He found that patterns which occur in both parents are unchanged in hybrids, frequencies occur at in­ termediate levels in hybrids of parents with unequal fre­ quencies, and patterns found in only one parent also tend to be intermediate in hybrids. Tinbergen (1959)« studying the various behavioral patterns in gulls, found the pat­ terns rather similar throughout the family, except for many species-specific differences. He decided that this strengthened the conclusion that gulls are a monophyletic group. Kunkel (I967) showed that social displays are valu­ able taxonomic characters in studying the relationships of two genera of wax bills. Bergmann (1972) analyzed the alarm calls of four Mediterranean warblers of the genus

Sylvia. He found that the calls are highly species-specif­ ic, although the various elements of the calls are identi­ cal. Kahl (1972a, 1972b) found that many components of. displays are very useful in determining the relationships within various groups of storks. 5 Several works have used behavior in mammalian studies.

Eisenberg (I963) traced sandbathing in five genera of heteromyid rodents and speculated on the phylogeny at the subfamily level. Fabri (1972) looked at the reliability of using behavior for taxonomic and phylogenetic conclusions within the Bovinae. Somers (197-3) found that geographic variations, or dialects, in vocalizations are present in pikas of the southern Rocky Mountains and speculated that acoustical characteristics of vocalizations could be a use­ ful taxonomic tool in Ochotona. Two works have studied the use of agonistic and play behavior in canids. Bekoff

(1975) used discriminant function analyses to show that quantified agonistic and play behavior in infant canids can be used in deriving taxonomic relationships. Fox et. a l .

(1976) used pure and hybrid individuals to speculate on the taxonomic and phylogenetic relationships between Canis familiaris and Canis lupus.

Components of call structure have been used in the

Anura for systematic studies. Littlejohn (1959) found that components of the male mating calls of two genera of

Australian burrowing frogs can be used in distinguishing between the genera and similar sympatric species.

Fouquette (i960) analyzed the structure of breeding calls of twelve species of frogs from five genera to study the relationships between the groups. Barrio (I963) used both morphological characters and characters of "aggresive call" behavior to support the separation of three genera of frogs from the family in which they had previously been placed.

Littlejohn and Oldham (1968) found that there were four distinct allopatric populations, with no clinal variabili­ ty, of the Rana pipiens complex in central United States based on the structure of the mating call. Heyer (1971) found that the mating calls of two families of frogs from

Thailand each appear to have been derived from a separate prototype, which has diverged in frequency or pulse. He pointed out, however, that divergence of song characters may not always correlate with genetic relationships.

Martin (1972) used male mating calls in the genus Bufo, along with morphological evidence from larynges, to produce an evolutionary scheme of the development of vocalization and stated that the results may be of taxonomic use.

Brown and Littlejohn (1972) used male release call struc­ ture to develop a classification of the genus Bufo. They believed the male release call to be superior to the mating call as a phylogenetic tool because it is not known to function as an isolating mechanism or sex attractant; and, hence, it should not be exposed to the same divergent adaptive selection pressures that influence mating call structure. Any selection should be for relative uniformity in release call structure among sympatric species. They argued that this character should, therefore, be conserva­ tive in its evolution. They, in fact, found that sympatric species have more uniform call structure and hybrids show intermediate calls. They determined that pulse rates are 7 the only useful attribute in determining evolutionary rela­ tionships, and a phylogeny based on these agrees closely with the evolutionary history proposed by Blair in 1963*

The insect order Hymenoptera is another group in which behavioral studies have yielded much systematic informa­ tion. One of the most prolific authors using behavioral data to study this group is H. E. Evans. Evans' studies

(1955> 1957. 1963. 1966a, 1966b) on a number of taxa within the Hymenoptera have successfully used a variety of behav­ ioral components in discussions on the phylogeny and evolu­ tion of the various taxa. These include nest construction, prey type, provisioning, and oviposition habits. He found that behavioral data parallels the results previously based on morphological characters. One of the first studies on

Hymenoptera to use behavioral characters was that of Ducke

(1913), who used nesting behavior in his discussion of the classification and phylogeny of social vespids . Plath

(193*0 also used nesting behavior in his proposed classifi­ cation of bumblebees. Bugbee (1936) and Rau (19^2) dis­ cussed their belief in the value of using nesting behavior in working out the taxonomy and phylogeny of polistine wasps. One of the most successful uses of behavioral char­ acters in separating two morphologically similar species was the use by Adriaanse (l9**-7) of various components of nest construction, prey type, provisioning habits, and nesting season to distinguish two species of Ammophila.

Beaumont (1952) discussed the close correlation between classifications of aculeate Hymenoptera based on morpholog ical and those based on behavioral characters. Jander and

Jander (1970) split the genus Apis into two groups accord­ ing to the type of orientation to gravity, which coincides with the transfer of angles into the dance language of honey bees. Farish (1972) used grooming behavior of 115 species of Hymenoptera to develop a pattern of grooming which parallels an existing phylogeny; and emphasized the value of using grooming data in phylogenetic interpreta­ tions. Other recent studies which used behavioral charac­ ters in the Hymenoptera include those of Peckham et. a l .

(1973) > who used nesting behavior to propose the reevalua­ tion of the classification of the genus Oxybelus, and

Goodpasture (1975)> who proposed a phylogeny for four spe­ cies in the genus Monodontomerus based on a combination of chromosome number and courtship behavior.

Three groups within the Orthoptera have seen systema­ tic studies using behavioral characters. Mantids were studied by Crane (1952) using innate defensive behavior of fifteen species from Trinidad. She concluded that the de­ fensive behavioral patterns agree well with taxonomic con­ clusions drawn previously using comparative morphology.

Edmunds (1972) also used defensive behavioral patterns in mantids as evidence for taxonomic and phylogenetic rela­ tionships in twenty-five species from Ghana. Ethology has proved useful in studies on crickets. Fulton (1952) showed, using habitat preferences, seasonal history, and type of song, that four distinct populations of field crickets, formerly considered a single species, were pres­ ent in North Carolina. Alexander (1957) and Alexander and

Thomas (1959) analyzed calling and courtship songs in the genus Nemobius to confirm classifications based on morphol­ ogical characters and discussed the behavioral distance be­ tween species. Mays (1971) found that mating behavior is distinct between three genera of nemobiine crickets and speculated on the evolution of behavior within them.

Gangwere (1958) studied grooming behavior in Orthoptera and described two basic types. He found that short-horned grasshoppers groom relatively infrequently when compared to non-acridoid Orthoptera and that the two types of grooming correlate with two types of mouthparts and follow the evo­ lutionary patterns believed to have occurred within the

Orthoptera.

Termite nests have been found valuable in studying be­ havioral relationships of termites. Emerson (I938, 1956) was one of the first to point out that termite nests are morphological expressions of behavioral patterns and that individual variation is cancelled, since a termite nest is the work of a colony. He proposed phylogenies for termites based on nest construction. In further studies with the termite genus Apicotermes, Schmidt (I955a» 1955h» 1958) stated that nest characters show phylogenetic relationships more clearly than do morphological characters. 10 Within the Diptera, studies have been confined to the genus Drosophila. Spieth (19^7) studied courtship behavior in one species group and found that the behavioral patterns and evolution parallel the morphological evolution within the group. Spieth (1951) then studied a second species group, then (1952) a third group covering over one hundred species and subspecies within the genus, and (1966) pub­ lished a study on the origins and relationships of Hawaiian

Drosophila based on mating behavior. In his 1952 work, also based on mating behavior, he found that qualitative differences are few, if any, between closely related spe­ cies. More distantly related species possess more qualita­ tive differences. Quantitative differences are often visi­ ble and measurable between closely related species and are sometimes detectable even in intraspecific forms. He spec­ ulated on the ancestral mating behavior and the probable relationships between certain subgenera and species groups.

In general, his findings confirmed the validity of the ex­ isting classification. Weidmann (1951) also used mating behavior and found that the patterns are similar to the classification of the nine species based on morphology.

Brown (1965) found that behavioral divergence correlates well with morphological divergence in his comparison of male courtship behavior within a species group. Ewing and

Bennet-Clark (I968) found that courtship songs are more re­ liable characters in the identification of species than the wing displays that produce them in their study of two 11 species groups. In a most recent study on Drosophila,

Lipps (1973) analyzed the grooming in twelve species. She found that grooming patterns are species-specific and that the results closely paralleled the most recently developed phylogeny.

Other behavioral systematic studies have been done on a wide variety of organisms. After Petrunkevitch (1926) pointed out the value of using instinctive behavioral char­ acters as taxonomic characters in spiders, Crane (19^8,

19^9a, 19^9i>) utilized display behavior as a taxonomic character in her study of salticid spiders from Venezuela.

She also emphasized components of agonistic and courtship displays in her revisions of fiddler crabs (Crane, 19^-1,

1975)* Barber (1951) classified fireflies of the genus

Photuris according to their lighting displays. Berg (197^) examined ten species from two genera of strombid gastropods according to various types of behavior. He found that, al­ though there is a great variation in shell shapes, behavior within the Strombacea is remarkably similar and species within a genus behave similarly, while there are some dif­ ferences between the genera. Purdue and Carpenter (1972a,

1972b) quantified components of body motions in displaying male iguanid lizards to use in determining similarities and possible relationships within the groups studied. Ewing

(1975) analyzed aggressive behavior in eighteen species of

Old World Cyprinodont fish and found minor frequency varia­ tions between closely related species, while more distantly 12 related species show greater fundamental differences. Two final studies are similar in comparing grooming behavior of

a wide variety of insects. Heinz (19^9) found that, in

general, similarity of grooming movements in different

groups increases with closer relationship. Jander (1966),

in a more in depth study of grooming in insects, speculated

on the phylogenetic similarities and evolutionary trends within insects. She also found that grooming patterns fol­

low or parallel morphological evidence.

From this brief survey, it can be seen that a great variety of innate behavioral patterns have been used in

meaningful systematic studies. A survey of behavioral

patterns that have been successfully utilized in the past

is valuable for indicating what characters may yield mean­

ingful results in a group which has not seen such previous

study, such as the Chironomidae.

General Information About Chironomidae

The Diptera family Chironomidae is one of the most

ecologically diverse insect groups in the world. The fami­

ly has a world-wide distribution with members found on eve­

ry continent including Antarctica (Brundin, 1966, 1970).

The larvae inhabit primarily freshwater habitats, although

marine (Hashimoto, 1976) and terrestrial (Strenzke, 1950) habitats are also utilized. Freshwater habitats may be of

such extremes as glacial pools (Dumbleton, 1973)1 hot

springs (Sheppe, 1975)1 the murky liquid of the traps of

pitcher plants (Swales, 1969» 1972). The Chironomidae have 13 shown promise both as indicators for biogeographic studies

(Brundin, 1966) and environmental conditions (Brundin,

19^9; Mason, 1975)* The species of Chironomidae are holo- metabolus with four life stages, egg, larva, pupa, and adult. Unless otherwise stated, this discussion pertains to freshwater forms. Literature on the behavior of the

Chironomidae is scattered through a great many sources, and, except for Thienemann (195*0 » there has been little effort to condense this information.

Eggs are laid by the females in bodies of water or at­ tached to various objects in or along sources of water.

The eggs are contained in a gelatinous matrix, which ex­ pands in water to several times its original size. Upon hatching, the larvae usually remain in the matrix for about a day, presumably feeding on matrix material. Upon leaving the egg mass, the larvae are positively phototactic and planktonic for one to several days before settling in a suitable habitat. This larval behavior has been studied by a number of authors (Sadler, 1935; Lieux and Mulrennan,

1956; Hilsenhoff, 1966; Spence, 1971).

Larval Lifestyles

Chironomid lifestyles become quite varied once the larvae have settled into their microhabitats. Larvae of the Tanypodinae, Corynoneurini (Orthocladiinae), and

Cryptochironomus (Chironominae) are usually considered to remain free-living. Although Johannsen (1937) stated that

Corynoneura larvae sometimes are found protected by a loose Ik tube of debris, other authors support their remaining free- living (Brundin, 1956; Darby, 1962; Forsyth, 1971). Many references to the Tanypodinae state that they do not build tubes and move about freely on the substrate (Johannsen,

1937; Hindle, 19^3; Thut, 1969; Sturgess and Goulding,

1969). Robaek (1969) stated that Tanypus prefer a soft mud bottom. Malloch (1915) found that Tanypodini and

Macropelopiini invariably prefer to remain under whatever debris is present, only leaving shelter for a short time.

Lindegaard-Petersen (1972) regarded species of the two tanypodine tribes Tanypodini and Macropelopiini as sub­ strate burrowers. Mitchell (I906) stated that Tanypus dyari may construct tubes in algae by fastening bits to­ gether with silk, or construct burrows that are open at both ends in soft mud, either by wriggling about on the surface until they form one, or burrowing in head first.

The literature is contradictory regarding these lifestyles.

Most of the rest of the Orthocladiinae and the

Chironominae build tubes by cementing various materials to­ gether with salivary secretions, hereafter referred to as silk. Many references describe these tubes, and most tubes are similar in general construction. Edgar and Meadows

(1969) described the tube building behavior of Chironomus riparius. The larva begins by cementing small bundles of suitable material (algae in the case described) together with silk, aided by movements of the anterior prolegs.

When several bundles have been formed, the larva tunnels 15 through them forming a preliminary tube. The larva occa­ sionally emerges to manipulate various pieces of algae and collect and add more bundles to the ends. Tubes take long­ er to build using mud, probably because it requires more effort to cement the mud into a structure. Cavanaugh and

Tilden (1930) described the tube building behavior of

Paratanytarsus dissimilis. They indicated that the larva was first able to form a small ring or collar around its body just behind the head. It then cemented additional material to the leading ends until a complete tube was formed. Other descriptions are basically similar, depend­ ing on the amounts and type of material present. Miall and

Hammond (1900) stated that Chironomus will eventually build an entire silken tube if no debris is available. Larvae extend and expand their tubes with age and growth, and apparently continue construction in the direction of the best food supply (Cavanaugh and Tilden, 1930).

The most unique tube is constructed by larvae of the genus Kheotanytarsus. Mundy (I909) described the construc­ tion of a typical tube. The larva begins by gathering par­ ticles together until a short strap is formed across the body and adds to this until a simple straight tube is con­ structed. The larva always remains inside the strap as it is added to. Having completed the straight tube, the larva continues adding material at almost a right angle to the substrate and the tube. The larva tears particles from the end of the straight tube to add to the vertical tube 16 projecting into the water column until the entire horizon­ tal tube is destroyed. Eventually, only a narrow strip or stalk is left attaching the vertical tube to the substrate.

The larva then constructs up to seven slender arms project­ ing at right angles from the anterior tube opening. The arms are connected by silken webs to collect particles from the passing water currents upon which the larva feeds.

Mundy described a congeneric larva which does not destroy its horizontal tube to build a stalk. It does, however, build the arms as extensions of reinforced ribs which run the length of the tube, Figure 3B. Mundy stated that with­ out a current of water flowing over the tube the larva will not construct this elaborate tube. Similar descriptions of

Rheotanytarsus tubes have also been published by Walshe

(1950b) and Scott (1967). Thienemann (195^) believed this type of tube construction to be typical of Rheotanytarsus.

Some species of Orthocladiini have been reported to construct gelatinous tubes (Miall and Hammond, 1900;

Thienemann, 195^; Darby, 1962); however, the details and circumstances of this construction are not known. Species have also been reported which carry movable tubes with them much in the same manner as some Trichoptera (Lauterborn,

1905; Thienemann, 195^)* Certain species of Orthocladiinae, genus Cricotopus, are leaf-miners between the two epidermal layers of aquatic plants and apparently do not line these substitute tubes with silk (Berg, 1950; Brock, i960) . 17 Very little is known about the actual lifestyle of terres­

trial larvae.

Under certain stress conditions larval Chironomidae

have been known to construct special cocoons in which they

may curl up and wait for better conditions. These cocoons

are. semicircular, completely enclosed, and translucent.

Cocoons have been reported to serve as an overwintering

case in various Endochironomus (Buscemi, 1957; Saether,

1962), Eukiefferiella claripennis (Madder et. al ., 1977).

Einfeldia synchrona (Danks, 1971). and Dicrotendipes

(=Limnochironomus) (Oliver, 1971); as a drought-resistant

case in two species of Phaenopsectra (Grodhaus, 1976) and

Parakiefferiella (Hudson, 1971); and to aestivate in hot weather in Hydrobaenus pilipes (Hudson, 1971)* The true

extent of this phenomenon in the Chironomidae is probably

not known.

Under normal conditions chironomid larvae seldom com­

pletely leave their tubes. However, numerous accounts of

post-first instar larvae swimming in the water column are

available (Miall and Hammond, 1900; Pause, 1919; Sadler,

1935; Hilsenhoff, 1966). Spence (1971) best explains this

habit as caused by a variety of factors including lack of

suitable substrate, over-crowding, and food or oxygen

shortage.

As already mentioned, environmental factors may influ­

ence aspects of tube construction, such as the need for a flow of water over the tube for characteristic construction 18 of Rheotanytarsus tubes. A few species that are usually tube builders have occasionally been reported living in gastropods with no visible tube or lining construction

(Hoffmann, 1931; van Benthem Jutting, 1938). Jonasson

(1972) described how Chironomus anthracinus builds conical tubes, which extend above the surface.of the substrate as a chimney, during the summer stagnation period in Lake Esrom,

Denmark. These tubes are moderately lined with silk. In oxygen rich water the tubes are more U-shaped with a thick, solid, and tough web of silk. McLachlan and Cantrell

(1976) found that sediment depth influences the shape of tubes in Chironomus plumosus. In shallow sediment they are more likely to build horizontal tubes, open at both ends.

In deeper sediments, U-shaped tubes open at both ends, or

J-shaped tubes with only one opening at the surface, are usually constructed. In a subsequent paper, McLachlan

(1977) found that the shape of the tube is also dependent on the slope of the substrate. In level substrate in the middle of a lake, U-shaped tubes are usually constructed, while J-shaped blind tubes are more common on the slopes of lakes. Hilsenhoff (1966) found that fourth instar larvae of Chironomus plumosus build deeper and more distinctly

U-shaped tubes than earlier instars. A good example of the versatility of various larvae is that of Glyptotendipes lobiferus. Leathers (1922) mentioned that this species may build its tubes on floating logs, at the bottom of ponds, or attached to stems and other perpendicular surfaces. It 19 may also be found in the stems of aquatic plants or mining their leaves.

Larval Feeding Behavior

Feeding behavior in larval Chironomidae is almost as diverse as the types of habitation. Larvae of the

Tanypodinae, Corynoneurini, and Cryptochironomus. all of which are considered free-living, actively search for their food (Chernovskii, 19^9). The remaining Chironomidae have a variety of types of feeding behavior.

One of the most common feeding habits is that of col­ lecting food in the vicinity of their habitation. Larvae which mine in plant tissues merely feed on the plant tissue as they extend their channels (Berg, 1950; Brock, i960).

Walshe (1950a) described the collecting behavior of

Chironomus plumosus. The larva extends its body from its tube entrance, always retaining a firm hold on the inside of the tube with its posterior prolegs to permit rapid re­ entry if needed. As it extends along the substrate surface it scrapes food particles from the substrate with the mouth parts and spreads silk over the surface at the same time.

Fine particles become attached to the silk; and when the larva withdraws into its tube, it drags any adhering parti­ cles with it to consume in the seclusion of its tube. As

Cavanaugh and Tilden (1930) mentioned, chironomid larvae will sometimes put their heads through the walls of their tubes to feed on material either attached to the outer tube walls or on the substrate surrounding the tube. Larvae 20 will often feed on portions of debris cemented into their

tubes, especially if food is scarce and they are abandoning

their old tube to find a new habitation site (Mackey, I976).

The most interesting feeding behavior found in the

larvae is their construction of nets of silk for filter-

feeding. Three types of nets are constructed by larval

Chironomidae. The first is the net, already mentioned,

which Rheotanytarsus spin between the arms which extend

from the anterior ends of their tubes. Mundy (I909) de­

scribed the nets as forming a kind of shallow cup in which

floating particles are entrapped. To spin the net, the

larva begins at the apex of one of the arms and swings

across to the next arm, drawing a strand of webbing which

it attaches to the second arm. It draws webbing back to

the first arm again, attaching it slightly proximal to the

original attachment by retreating slightly into its tube.

This zigzag movement is repeated until the webbing reaches

the base of the two arms. Nets are built between the re­

maining arms in the same manner until the net is completed.

Occasionally, the larva tears down the net between two arms,

using its mouthparts and anterior prolegs to work it into a

compact mass. It may consume this mass along with any col­

lected particles or use it for additional building or repair

of the tube. The larva will then reconstruct the net be­

tween the two arms. This type of net requires a flow of water over it to provide particulate material, but no work

by the larva to draw the particles into the net. 21 Walshe (19^7a) described a net constructed by

Chironomus plumosus inside its tube. With its posterior

prolegs firmly anchored, the larva performs a number of ro­

tary movements with the anterior region of its body; the head describing complete circles and alternating direction with each pass. A sheet of silk is. thus spun across the tube. The larva then retreats slightly from the net and

begins dorso-ventral undulations of the body, or irrigation,

to draw a current of water into the entrance of the tube.

The water then passes through the net, depositing particles

on it, then past the larva, and out the exit opening of the tube. After irrigating for usually only a few minutes, the

larva moves forward, grasps the net with its anterior pro­

legs and mouthparts, and twists the net into a compact mass

along with any entangled particles. As with Rheotanytarsus

larvae, the larva then has the option of either consuming

the mass, using it as building material, or expelling it or

its remains from the tube with irrigation. The larva usu­ ally rebuilds the net and repeats the entire process.

The third type of net constucted by Chironomidae is very similar to that described for Chironomus plumosus, ex­

cept that the net is spun posterior to the larva. The first published account of this type of net was by Leathers (1922) for Glyptotendipes (Phytotendipes) lobiferus. According to

Walshe (1951) the larva extends its body forward and spins a crossbar of silk across the tube to act as a suspension for the apex of the net. The larva then draws itself 22 backwards a short distance with its posterior prolegs and fixes the webbing to the wall of the tube before slowly re­ extending its body, alternately expanding and retracting its anterior prolegs as the silk is applied to the net.

With each pass the larva twists its body alternately to the right or left to lay the threads along different planes to complete the net. Unlike Chironomus plumosus, the larva turns around once the net is complete, so that the net is posterior to the larva. The larva then irrigates for up to ten minutes before again turning around, twisting up, and consuming the net with its contents. The irrigation time before consuming nets is indirectly proportional to the speed of irrigation in all species and does not correlate with material in the net. This type of net has been report­ ed in a variety of leaf-mining species within the genera

Endochironomus, Glyptotendipes, and Polypedilum

(Pentapedilum) (Leathers, 1922; Burtt, 19^0; Berg, 1950;

Lieux and Mulrennan, 1956). Glyptotendipes nets are either a short, asymmetrical cone or, as a variation of the cone, an oblique sheet across the tube. Glyptotendipes nets are not completely conical because their posterior prolegs are always fixed when they alternately twist to the right or left after each pass. The body cannot twist a full 180° with the prolegs fixed, which would be necessary to con­ struct a symmetrical net. Endochironomus and Polypedilum

(Pentapedilum) nets are symmetrical cones, with the length in Endochironomus about a third the length of the body, 23 while P. (Pentapedilum) nets are always a perfect cone about half the length of its body. Sometimes Endochironomus move the hind prolegs slightly posteriorly after drawing out webbing on a pass resulting in a net consisting of a series of half-cones (Walshe, 1951).

Feeding behavior is also variable in chironomid larvae.

The larvae that construct nets filter-feed, and they may also be collectors. Berg (1950) described both the mining and net-feeding behavior of several species. Mention of both filter-feeding and non-mining collecting of food in species of Glyptotendipes is found in the literature

(Leathers, 1922; Walshe, 1951; Lieux and Mulrennan, 1956).

Leathers (1922) also reported Rheotanytarsus collecting as well as filter-feeding. McLachlan (1977) found that the type of feeding used in Chironomus plumosus is, to some extent, dependent on the site and construction of the tube.

U-shaped tubes, constructed more often in the middle of a lake, are utilized more for filter-feeding; while J-tubes, constructed more often on the lake slopes with only one opening, are used more for collecting food. Walshe (19^7a) also mentioned that the nets constructed by Chironomus plumosus are only constructed when the water is well oxygen­ ated. This was confirmed by Jonasson (1972), who also noted that larval feeding habits are related to environ­ mental conditions and the type of tube inhabited. Again larval behavior appears to be flexible and dependent on a variety of parameters. 24 It might be mentioned that the larvae are equally flexible in the type of food which they consume. Even in the Tanypodinae, which are usually considered predacious,

accounts of rearing on total vegetarian diets are not un­

common (Mitchell, 1906; Biever, 19?1; Mackey, 1976).

Oliver (1971) believed that perhaps only a few Tanypodinae

are obligate carnivores. Loden (1974) found that many

species of usually non-carnivorous larvae will, on occasion, feed on oligochaetes.

Larval Irrigation

A larval behavior that has already been mentioned re­

garding net construction is that of irrigation. The term

"irrigation" was first used for chironomids by Walshe

(1950a) for the rhythmic dorso-ventral undulations seen passing backwards along the body that drive water posteri­

orly past the body. Its respiratory function was quickly

established as it renews the water around larvae in tubes

and maintains a fresh oxygen supply. The time a larva

spends irrigating has been shown to be inversely propor­

tional to the oxygen pressure of the water, and under very

low oxygen levels larvae may irrigate continuously. Under normal oxygen levels irrigation is only periodic (Lindroth,

1942). The flow of water can be reversed so that water flows anteriorly over the larva (Leathers, 1922), although this evidently is not common. Tube-living larvae line their tubes with silk throughout their larval life. This

continuous addition is probably essential for maintaining 25 a clear passage for the water current, especially in soft mud (Walshe, 1951)* Another function of irrigation is to purge tubes of fecal pellets when these are deposited in a tube (Berg, 1950; Walshe, 1951)* A violent, short burst of irrigation serves to expel fecal material from the tube.

Although usually mentioned with larvae that inhabit tubes,

Mitchell (1906) mentioned that the larvae of Psectrotanypus d.yari keep "up a constant waving motion" while in a mud burrow.

Larval Grooming

A behavior which is seldom mentioned in the literature is that of larval cleaning behavior. Burtt (19^0) mentioned a Glyptotendipes larva cleaning its body with its mouth- parts as it turned around within its tube. Walshe (1951) observed this behavior in many tube-living larvae, includ­ ing the three leaf-mining net-builders . This behavior is usually done when larvae bend double as they turn around in their tubes . The ventral body surface and areas around the posterior prolegs usually get the most attention. She speculated that this kept the body surface free of epizo- ites; because when they do occur, they are usually found on the back of the head or dorsal to the anus, the only places the larvae cannot clean or rub against the tube walls.

Behavior Associated with the Pupae

Pupal behavior is restricted owing to the shortness of this life stage. Pupae of Tanypodinae are free-living and 26 strong swimmers (Oliver, 1971). Darby (1962) found that the pupae of Cryptochironomus fulvus lie free on the sub­ strate. Pupae of larvae which inhabit tubes are usually found in the larval tubes until ready to emerge

(Chernovskii, 19^9). In some cases the usual larval tube is somewhat modified by the prepupa before actual pupation takes place. Lauterborn (I905) reported that Tanytarsini pupation tubes are modified with both ends constricted and partially closed by a membranous operculum that has a small circular opening in the center. Darby (1962) reported the pupation tubes for Cladotanytarsus viridiventris to be some­ what shorter, with its openings more constricted, than the larval tube. Scott (1967) reported that the prepupa of

Rheotanytarsus usually remove the arms from their tube and seal the opening with an operculum like that described by

Lauterborn. The pupal respiratory organs then project through the central hole in the operculum. Mackey (I976) described the pupation tube of Cricotopus bicinctus as being more robust than that of the larva, firmly attached to the substrate, and closed at both ends. One free-living larva whose pupa is found in a protective shelter is Corynoneura.

Johannsen (1937) stated that the pupae are found in ellip­ soidal gelatinous masses, while Forsyth (1971) described how the prepupa builds a silken sheet over itself in which to pupate. Pupae themselves are not entirely inactive.

Miall and Hammond (1900) reported that, like the larvae, pupae will irrigate as a respiratory function. In a similar 27 account, Scott (1967) reported that Rheotanytarsus will ro­ tate within the pupal tube, apparently also a respiratory function.

Fore Leg Positioning in Adults

An aspect of individual behavior in Chironomidae that has sometimes been mentioned, but never studied, is the curious adult habit of holding the fore legs raised above the substrate (Miall and Hammond, 1900; Johannsen, 1905;

Heinz, 19^-9). It is interesting that this habit gave the

Chironomidae their name. Higginson (1868) stated that the name is derived from the Greek Chironomeo, to gesticulate, since they carry the fore legs pointed forwards both in flying and walking. Although he added that this position­ ing "may have reference to its mode of catching its prey, it being said to feed on the Aphis," no other reference has reported chironomid adults to prey on other organisms.

Positioning has also been reported in Culicidae, which po­ sition with their hind legs (Johannsen, 1905), and Diopsidae

(Seibt, 1972), although no comparative studies on this be­ havior are available.

Adult Grooming Behavior

Another behavior in adults which has seen use in com­ parative studies is self-grooming. The only comparative study to use Chironomidae was that of Heinz (19^9)* He re­ corded four rubbing movements: F F , MHH, HH, and W H . He also catagorized chironomids as being low frequency groomers. 28 Other studies on grooming have contributed much to our understanding of the use of self-grooming as a systematic tool. Wilson (1962) emphasized that it is not the basic movements themselves that vary greatly, but their pattern of presence or absence. A majority of the studies utiliz­ ing grooming behavior have dealt with this type of qualita­ tive difference (Szymanski, I9I8 ; Heinz, 19^9; Jander,

1966; Farish, 1972; Valentine, 1973)- Self-grooming ap­ pears to be somewhat independent of external stimulation.

Several amputation studies have shown that attempts are made to groom the missing appendage as often as before am­ putation or as often as remaining appendages are groomed

(Heinz, 19^9; Laudien, 1970). Thus, external stimulation does not appear necessary for grooming to take place.

Other studies have shown that there is internal neural in­ hibition of grooming and that destruction of these inhibit­ ing ganglia result in spontaneous grooming (Huber, 1955;

Eisner, 1961; Eaton and Farley, 1969). This also shows that grooming can be independent of external stimulation.

However, external stimulation has been shown to increase the frequency of grooming. Both Heinz (19^9) and Eaton and

Farley (1969) found that grooming frequencies can be in­ creased with external stimulation of the sensory organs.

Acker (1966) found that grooming increases when Raphidiidae become initially excited by the presence of a member of the opposite sex. Connolly (I968) theorized that grooming serves as a signaling device facilitating the spacing of 29 animals after finding that grooming increased with the presence of other individuals in Drosophila. Bastock (1955)

observed grooming in Drosophila courtship phases and consid­

ered it a displacement activity. Baerends (1959) best ex­ plained this balance between internal and external control

of grooming in theorizing that grooming activity is composed

of two parts. The first is a centrally coordinated fixed pattern which is relatively independent of external stimu­ lation. The second is a steering component which is contin­ ually influenced by external stimuli and adjusts the first

component to the environment. Thus, the frequencies are

somewhat influenced by the environment. Jander (1966) found that relatively active species tend to have higher grooming frequencies than less active species, thus, indi­

cating that frequencies of grooming may still be of some systematic value. Lorenz (1957b) stated that with a central innate component the threshold value for releasing an innate movement decreases during a period of non-use. This period of non-use literally becomes a "motive" and causes unrest within the organism as a whole until the movement is releas­ ed. If this is true, frequency data for grooming could be of some systematic value. Lipps (1973) used frequencies of the amount of time spent performing various grooming acts as taxonomic characters in twelve species of Drosophila. Her results paralleled the most recently developed phylogeny at that time. Thus, the pattern of presence or absence and the frequency of grooming can be utilized in systematic studies. STATEMENT OF PURPOSE

The purposes of this study are to examine the behavior of the Diptera family Chironomidae; to describe the behav­ ioral patterns found within the family; to determine what behavioral characters may be of value in systematic studies and to form a classification, using those characters found to be of systematic value, that may be compared with exist­ ing arrangements of the family.

As emphasized by both Evans (1953) and Mayr (1958), good classifications are based on a large number of charac­ ters. Only then may taxa agreeing in a majority of charac­ ters be considered closely related. Characters used in this study will be taken from immatures, including types of habitation, general feeding behavior, and use of irriga­ tion; as well as from adults, including the positioning of the fore legs and patterns of self-grooming. Examination of behavior in life stages with differing ecologies should maximize emerging patterns based on genetic similarity and minimize patterns based on similar environmental influences

Care has also been taken to avoid characters which may show displacement due to divergent selection pressures, since these might distort relationships.

30 METHODS AND MATERIALS

Collection and Sorting of Specimens

For observation of larval and pupal behavior, larval chironomids were collected from a variety of habitats (see

Appendix B for collection site information) to insure the greatest variety of species for this study. Larvae inhab­ iting shallow substrates were collected by shoveling some of the substrate, to a depth of up to 10 cm, into a five gallon polyethylene bucket. The substrate was then gently agitated with water several times to separate the larvae from the substrate. Water, larvae, and various debris were then poured through a kitchen sieve with approximately a

0.3 m toe section from a nylon stocking attached over the bottom. Larger larvae and debris collected in the sieve, while smaller, more delicate larvae, such as Corynoneura and Tanytarsini, passed through and collected in the stock­ ing toe. A stocking toe was found to be superior to metal sieves because its small mesh collected the smallest of chironomid larvae, while preventing injury with its softer, flexible texture. Contents from the strainer and stocking were then placed into white enamel trays containing a small amount of water from which the larvae could be removed by pipette and separated into groups. Very small larvae and 31 32 predacious larvae were usually kept in separate containers.

Larvae adhering to rocks were gently brushed loose in the water-filled collecting bucket. Larvae collected in algae or other plant growth were placed, along with the plant material, in water-filled 32 ounce jars, shaken vigorously to dislodge the larvae, and the water with larvae was then poured through the sieve and stocking as above. Larvae from deeper water were collected with the use of an Ekman dredge and then sorted as above.

Adults were collected during 197^ and 1975 by attract­ ing them to a white sheet with a Travel Lite TL15 12 volt fluorescent light by McLean Electronics to which a black- light was added. Adults were collected individually from the sheet and placed in individual ten dram, snap-cap vials. Each vial had a piece of white tissue or lens paper, approximately 1 x 7 cm, adhered to the inside with water. The moisture in the tissue aided in the maintenance of a high humidity, since Hilsenhoff (I966) showed that adults kept in 100$ humidity lived an average of 1.5 days longer than those at 60$ humidity. A few crystals of sugar were placed at the upper end of the tissue, since Goff

(1972) showed with Chironomus riparius that chironomids will feed on sugar. Most adults were observed to pause longer while lapping moisture around the site of sugar dep­ osition, and a few were even observed feeding directly on undissolved sugar crystals. The tissue served both as a medium for moisture and a light background during 33 observation. To prevent condensation from forming on the

inside of vials during observations and allow excess mois­ ture to exit, a small square of nylon stocking was attached over the open end of the vial with a rubber band. This still allowed the snap-cap to be placed on the vial to pre­ vent excessive moisture loss.-

In 1976 a collecting tent, Figure 1, was used to at­ tract adults. This four foot square tent consisted of four, four foot tall, one inch diameter dowels at the cor­ ners with a one inch spike on the bottom that could be pushed into the ground. A 7 x 16 foot piece of white sheet was attached around the corner stakes, forming the walls of the tent with eighteen inches extra at the top and bottom acting, respectively, as a partial roofing and ground cover to catch insects that dropped to the ground from the walls .

A five foot, eight inch long, one inch diameter, dowel formed a diagonal at the top of the tent to provide sup­ port. The diagonal was fitted with spikes at both ends, bent at right angles to fit into holes in the tops of two opposing corner stakes. Two straightened coat hanger wires looped around the center of the diagonal dowel and extended at right angles to hook into the tops of the other two op­ posing corner stakes. If windy, two opposing corner stakes could be staked to provide additional stability. A two- mantle Coleman lantern was then hung on the middle of the diagonal dowel to attract insects to the tent. With this collecting tent chironomids could be attracted from a 360° 34

Figure 1. Frame for Collecting Tent a. Corner stake b. Spike to anchor corner stake in the ground c. Diagonal dowel d. Wire diagonal supports

A 7 x 16 foot piece of white sheet (not shown) is attached to the corner stakes. A light source (not shown) hangs inside the tent from the diagonal dowel. 35 field and collected from all four sides of the tent. Indi­ vidual chironomids were collected in the same way from the collecting tent as described above.

During collections, observations were made on the be­ havior of immatures and adults in the field so that labora­ tory observations could be compared with field observations to get an indication of whether laboratory conditions were influencing observed behavior.

Larvae were later placed in petri dishes and sorted by means of a stereo-dissecting microscope into separate

60 x 15 mm glass or polystyrene petri dishes, each contain­ ing those individuals appearing morphologically alike. In most cases genera, and often species within genera, could be separated in this gross morphological examination. From each group one larva was placed into a labeled 60 x 15 mm petri dish for behavioral observations. Another group, the size depending on the number of individuals in the group, the number of groups in the collection, space available, and how much data had already been collected on the group, were placed into rearing containers to be reared to the adult. Remaining larvae were preserved in 70fo ethanol.

Rearing of Specimens

Larvae were reared using one of two methods. The first method, Figure 2, was to rear individual larvae in 1 x 6 inch test tubes slanted 30° from the vertical, similar to that used by Leathers (1922). The tubes were placed in wooden racks that would hold thirty such tubes. To each 36

b.

Figure 2. Section from a Test Tube Rearing Apparatus a. 1 x 6 inch test tube b. Section of wooden rack c. Styrofoam divider d. Pasteur pipet e. Cotton plug f . Adaptor tubing g. Tygon tubing to air pump 3? tube was added a small amount of organic debris, algae, or silt, depending on the type of larva. Each tube was fitted with an oval styrofoam divider braced to one side near the bottom to allow the larva complete use of the bottom, while partitioning the water column and surface so that pupae moving to the surface to emerge would not be subjected to turbulence from aeration. Aeration was accomplished by in­ serting a disposable Pasteur pipet into the chamber parti­ tioned by the styrofoam divider. The divider left spaces between the larval and aeration chambers enabling a gentle flow of oxygenated water to pass into the larval chamber.

The pipet passed through a cotton plug, that allowed excess humidity and air to escape, while providing the adult a place to rest upon emergence. The pipet was connected to an air pump through an adaptor piece of 3/80D, l/^ID Tygon tubing to l/^OD, 1/8ID Tygon tubing and a battery of brass air valves. The valves could be adjusted to allow the pro­ per flow of air through each pipet into the aeration chamber. This rearing method showed good emergence results for most groups, but problems were found in the rearing of burrowing larvae and the ability to locate exuviae upon the emergence of the adult. Nonpredaceous larvae were fed small amounts of dried fish food, while predaceous larvae were fed oligochaetes from a mixed culture that was main­ tained by adding oligochaetes from larval chironomid col­ lections and dried fish food. Algae and organic debris 38 from larval collections were also saved to be used as rear­

ing material.

The second rearing method used 4 3/4 x 3 1/4 x 2 3/4

inch and 7 i/2 x 3 3/4 x 3 1/4 inch plastic refrigerator

crispers divided into four compartments each by styrofoam

dividers. The larger crispers were filled to a depth of

1/4 to 1/2 inch with silt and mud and occasionally stocked with oligochaetes from the mixed culture for burrowing, predaceous, chironomid larvae. This rearing method worked

better than the test tube method for burrowing larvae, al­

though larval exuviae were still sometimes difficult to lo­

cate because of the amount of substrate present. The

smaller crispers were supplied with small amounts of organ­

ic debris or algae and used for larger larvae, larvae that

did well without oxygenation, and larvae that lived in and

fed on algae. Small amounts of dried fish food were added

to feed larvae in crispers, also. Amounts of food added varied from very little in compartments where the larvae were feeding on algae, to larger amounts for both larvae

and oligochaetes in the larger crispers for burrowing lar­ vae. Crispers could be surveyed under a stereo-dissecting

microscope when it was necessary to locate larval exuviae,

somewhat increasing the percentage of exuviae found. Once

an adult emerged, it was placed in a ten dram vial for be­

havioral observations as were adults collected in the field.

The larval and pupal exuviae were removed from the rearing

chamber and preserved in 70fo ethanol in a one dram vial, 39 which was labeled to associate it with the adult vial.

Upon the death of the adult, or termination of observations

on it, it was preserved along with the exuviae.

Rearing enabled the larval and pupal exuviae to be

associated with the adult for identification purposes,

since many species are described only from the adult male.

It also enabled females, which were not listed in keys or

described, to be identified by backtracking to their exu­ viae which could be compared with the exuviae of identified males. Adults from species field collected only as larvae

could also be obtained through rearing the larvae, thus

permitting behavioral observations on the adult. This was

especially valuable with adults that were very short lived

and for obtaining adults in the lab when seasonal tempera­ tures reduced adult emergence in the field.

Observation of Specimens

Larvae being observed were reared in 60 x 15 mm petri dishes supplied with a small amount of algae, silt and mud,

sand, or organic debris depending on the larval preference.

If the larval preference was not known, various substrates were tried until its preference was determined. Many lar­ vae were found to be adaptable to more than one substrate, especially algae, sand, and organic debris. Sand was used when it was desired to observe the actions of a larva with­ in a tube, since the outline or silhouette of the larva could usually be seen through the sand grains incorporated 40 in the tube. Sand grains could also be most easily removed from the tube without causing severe structural damage.

Larvae and pupae were checked once a day, during which observations were made and recorded as to their general wellbeing, type of habitation (if any), feeding behavior, changes since the previous record, and any other pertinent information. Any larvae or pupae found dead or almost dead and exuviae were preserved in 70% ethanol. Observations were made with a stereo-dissecting microscope. Tubes were periodically measured, using larval length and width as the units of measure. Besides size, notes on the shape and construction of tubes, as well as burrows, were recorded.

Information such as type of food on which larvae were seen feeding and how they fed was also recorded. Powdered char­ coal was put into suspension and added to the water near the anterior tube opening when larvae constructed food nets, so that the irrigation of the larvae would cause the charcoal to collect in the net, making its shape readily visible.

Adults were observed over fifteen minute blocks of time spread out during the day. A total of two hours of observation per individual was sought. One of a kind spec­ imens or specimens from a species or genus for which little behavior was observed were often observed for longer total times. Because of the short lifespan of the adults (an average of only several days), it was often impossible to get a total of two hours of observation time, especially 41 from adults of unknown age collected in the field. Obser­ vations at night were made with red plastic over the micro­ scope lights, since bright white light caused adults to go into frenzied activity and flight, which often led to phys­ ical damage to the specimens. Observations were recorded using a cassette tape recorder and later transcribed for permanent storage in a notebook along with observations on the immatures. The procedure was basically that described by Szebenyi (1969), in which verbal symbols were recorded to represent individual grooming movements. The use of the tape recorder was necessitated by the rapidity with which grooming movements could procede from one to another; at times, verbal recording could not keep up with some groom­ ing sequences, limiting the precision of direct visual ob­ servations. In such cases only high speed filming could show the complete sequence. Because adult chironomids seem to prefer resting positions other than dorsal surface up­ right and most have poor traction on glass surfaces, it was sometimes necessary to observe them while they hung upside- down under the tissue in the vials. To view specimens in this position, a mirror was proped at an angle enabling the underside of the tissue to be viewed from above.

In the adults, the number of each type of grooming movement in the order in which they occurred was recorded.

The grooming terminology used here is basically that of

Valentine (1973)* Since each grooming act often includes a number of strokes of the same type, as in leg rubs other 42 than the two step-rubs done by the mesothoracic legs on

either the ipsilateral pro- or metathoracic legs, a single

movement was defined as occurring until there was a pause

or termination in a particular act, regardless of the num­

ber of continuous strokes that took place during its pro­

gress. No attempt was made to keep the specimens active

(Farish, 1972), or to discourage activity (Hay, 1972;

Heinz, 1949), since it was desired that grooming patterns

be as natural as possible under laboratory conditions.

Several times, however, either powdered charcoal, flour, or

mineral oil was placed on chironomids that were showing no

grooming pattern. The only results from these attempts were that specimens seemed to die very quickly when fouled

by fine particular contaminants, without showing any in­

crease in grooming activity, or became so mired from even

small amounts of liquid contamination that observation

proved useless.

Leg positioning was not recognized as significant

until late in the second year of study after which the na­

ture of positioning activity was measured in the adults

during each fifteen minute observation block. Observations

included whether positioning of any type occurred, whether

the fore legs were held aerial or on the substrate while at

rest, what type of motion was used during activity, the

angles of the tarsal segments with respect to the rest of

the body, the angles of the fore femora with respect to the

rest of the body, and what parameters might produce changes in these positions. A single measurement was taken of the

fore tarsal segments with respect to the body. This was

the tibia-tarsal extreme angle (TiTEA), Figure 7A and B,

which is the angle produced by a line through the proximal

tip of the tibia and the apex of the tarsal segment to the

anterior section of a line bisecting the body longitudinal­

ly (BL) as seen from above. Two measurements were taken of

the fore femora. These were: 1.) the angle anterior or

posterior that it produced with a perpendicular (vertical)

through the coxa to a line along the dorsal surface of the

mesonotum as viewed from the side (SFA), Figure 7C and D;

and 2 .) the angle that it produced from a vertical line

through the coxa as viewed from the front (FFA), Figure 8 , which can be considered the angle at which the femur di­ verges from the body.

Because of the ephemeral adult expectancy, at night or

after finishing observations for the day, adults that were

being observed were often kept in a refrigerator at 3° C or

in a cooler with just enough ice to lower the temperature

below ambient summer temperatures. This was done since

Hilsenhoff (1966) found that adults lived up to eleven days

at 16° C, but a maximum of only three days at 32° C.

Sadler (1935) stated that females of Chironomus tentans

lived an average of 3-5 days and males an average of five

days. Similar figures are given by Leathers (1922) and

Nasr (1971). Refrigeration seemed to work well in prolong­

ing the adult expectancy, especially during the heat of 44 summer. Chironomids of the genus Tanypus, however, were usually uncoordinated or dead after refrigeration, and were usually placed in a cooler between observations. Chirono­ mids of the Harnischia complex usually required about two minutes to recover after refrigeration since they were often found on their backs with their legs in a tangle when first removed from the refrigerator.

Mounting Procedures

For identification purposes specimens were mounted on

1 x 3 inch microscope slides using Hoyer's Mounting Medium.

Because Hoyer's contains a clearing agent and is water sol­ uble, specimens could be placed directly from 70$ ethanol

into Hoyer's. Its primary fault is that it is not in it­ self a permanent medium, in that it shrinks with drying and must be ringed to make a permanent mount.

When both larval and pupal exuviae were present with the adult, the exuviae were mounted together under half

(11 x 22 mm) of a No. 1 thickness, square cover glass; and the adult was mounted alone under a separate whole cover glass to the right of the exuvial cover glass. In all other cases mounting was done using a single 22 x 22 mm,

No. 1 thickness cover glass. Cover glasses were centered on slides in all cases to leave an equal space for labeling at both ends of the slides. The exuviae were taken directly from 70$ ethanol and placed into a drop of Hoyer's. Larval exuviae were oriented in ventral view, with the anterior end towards the top of the slide, and to the left of the /+5 pupal exuviae . Pupal exuviae were oriented in dorsal view with the anterior end towards the top of the slide. Once oriented, the cover glass was gently added. Usually drift­ ing was not a problem, since the exuviae contacted the cover glass and were compressed, while being held in place by the contact with the-cover glass. Adults were first dissected by removing the wings, head, and abdomen from the thorax. In large specimens the thorax was longitudinally bisected along a dorsal-ventral line. The parts were then transferred to a drop of Hoyer's to the right of the exu- vial mounts and oriented with the head anterior in view to­ ward the top of the slide; the wings one above the other under the head; the halves of the thorax flanking the wings

(or to the left of the wings if not bisected) with lateral view and anterior toward the top of the slide with dorsal facing the wings; and the abdomen dorsal in view with the anterior end toward the top of the slide. If the genitalia would not stay in a clear dorsal view, they were removed from the abdomen along with the penultimate abdominal seg­ ment and oriented to the right of the abdomen with the an­ terior end toward the top of the slide. The abdomens of some females were oriented with a lateral view for conven­ ience. While this arrangement does not yield a dorsal view of the pronotum, which is often used in keys, it caused no problems. Before applying the cover glass over the adult, the slide was placed on a slide warming tray for several hours until the Hoyer's began to solidify. This usually 46 minimized drifting when the cover glass was applied. Addi­ tional Hoyer's was added and the cover glass carefully ap­ plied. A penny was placed on the cover glass as a weight to aid in the distribution of the Hoyer's until no further settling was noticed (usually 10-15 minutes), after which additional Hoyer's was added as necessary to fill the space under the cover glass.

Larvae were mounted by removing them from 70$ ethanol and placing them directly into a drop of Hoyer's centered on a slide. With a pair of fine probes, each head capsule was removed and oriented to the right of the rest of the body and in ventral view with the anterior end toward the top of the slide . The rest of the body was either oriented in ventral view or, if the body was dorso-ventrally curved, as usual, in lateral view with dorsal to the left.

Pupae were mounted by removing them from 70$ ethanol and placing them directly into a drop of Hoyer's centered on a slide. Each abdomen was removed and oriented in dor­ sal view, with the anterior end toward the top of the slide.

The rest of the body was in lateral view with anterior to­ ward the top of the slide and dorsal to the right, except in large pupae where the rest of the body was longitudinal­ ly bisected along a dorso-ventral line with ventral aspects facing toward the center. Larval exuviae were mounted as described above, to the left of the pupae.

For labeling, the slides were inverted so that when the specimens were viewed under a compound microscope the 47 images would appear as the specimens were oriented above.

Collection labels were placed to the left of the specimens.

Besides listing locality, date, and collector, they also give the specimen number corresponding to the behavior sheet for that specimen. Preparation and determination la­ bels were placed to the right of the specimens for listing materials such as clearing agents, staining agents, and mounting medium used in the slide's preparation, as well as the specimen's determination. Each slide was also given a number following the author's initials in the lower left corner of the preparation and determination label to cor­ respond to any measurements or other data taken from the mounted specimen and recorded under that number in a log for such data.

After a drying period of approximately six months, or less during periods of low humidity, the slides were ringed.

Using a N o . 1 paint brush, two coats, about a week apart, of piccolyte mounting medium, followed by two coats, about one day apart, of polyurethane varnish were applied around the cover glasses. This in effect seals the Hoyer's medium in plastic. Neither the piccolyte nor the polyurethane var­ nish worked well alone, since the piccolyte seemed to crack with age and the polyurethane varnish did not harden prop­ erly when in direct contact with the Hoyer's medium.

Slides ringed in this manner show no sign of deterioration after five years. Analysis of Data

For analysis of behavioral data, characters to be used in forming a classification were coded for computer analy­ sis. Presence-absence characters include all immature characters of habitation, feeding types, and irrigation use, and adult characters of various grooming movements and substrate vs. aerial rest position of the fore legs. I.iul- tistate characters include grooming frequency data and the remainder of the positioning data. Fifty-two total behav­ ioral characters were initially used in the analysis.

Forty-four OTU's (Operational Taxonomic Units) were used in the analysis using all characters. Only those taxa with at least five hours of observation time or a total of fifty observed grooming movements in the adult were used in the computer analysis using all characters, since the adult characters make up forty-three of the total characters and the grooming characters make up thirty-three of those.

Where more than one species within a genus was present as an OTU and behavior differed between them, a summary OTU for the genus was also included as a check of the analyses.

Some OTU's had to be deleted, while others could be added, when individual types of behavior were analyzed.

Correlation matrices were generated and used to pro­ duce phenograms and principal component analyses (PCA) of both characters and OTU's. Distance matrices were gener­ ated and used to produce phenograms and various dimensional representations of the OTU's by multidimensional scaling (MDS), using all characters or different sets of characters individually. The various programs used in the analysis are contained in the Numerical Taxonomy System of

Multivariate Statistical Programs (NTSYS) of Rohlf,

Kishpaugh, and Kirk (197^).

Figures of adult chironomids were drawn from 35 mm' slides of living adults and mounted specimens. Figures were transferred to two-ply Bristol Board and photograph­ ically reduced to produce the final figures. TAXONOMIC TREATMENT

Table 1 of taxa examined during the course of this study follows the general classification of Hamilton e t . a l . (1969) except that the subfamily Orthocladiinae is di­ vided into tribes as in the classification of Sublette and

Sublette (1965); the subfamily Tanypodinae follows the classification set forth by Roback (1971); and the

Harnischia complex, within the tribe Chironomini, has undergone futher change at the generic level as detailed by Saether (1977) •

The specimens observed in this study were identified using a variety of sources, since no single reference is available for the identification of the family as a whole.

The immatures, especially the larvae, are difficult to identify both because of their variability and the paucity of taxonomic work on these stages. The females are also difficult to identify because of the almost exclusive use of male genitalia and secondary sexual characters in keys.

Because of the above, reared males with associated larval and pupal exuviae were important in identifying immatures and females with associated larval and pupal exuviae.

Voucher collections of duplicate specimens will be de­ posited in the National Museuin of Natural History and the ‘ 50 ' Table 1. Taxa Examined

CO CD CD CD CO r —1 > CD f r i P h i— 1 £ 3 ccJ CD i- q G n S

Chironominae

Chironomini

Chironomus attenuatus Walker, 1848 13 9 19 12

Chironomus crassicaudatus Malloch, 1915 1 - 4 -

Chironomus plumosus (Linnaeus, 17 58) 4 4 7 2

Chironomus riparius Meigen, 1804 1 1 1 6

Chironomus staegeri Lundbeck, 1898 1 1 2 -

Chironomus spp. - -- 3

Cryptochironomus fulvus (Johannsen, 1905) 6 2 4 1

Cryptochironomus ponderosus Sublette, 1964 3 2 1 2

Cryptochironomus sp. 1 2 2 - 2

Cryptochironomus spp. 1 1 1 3

Cryptocladopelma galeator (Townes, 1945) 4 3 2 3

Cryptotendipes casuarius (Townes, 1945) - - 1 - Table 1. Taxa Examined (cont.)

Cryptotendipes emorsus (Townes, 1945)

Demei.jerea brachialis (Coquillett, 1901)

Dicrotendipes fumidus (Johannsen, 1905)

Dicrotendipes modestus (Say, 1823)

Dicrotendipes nervosus (Staeger, 1839)

Dicrotendipes spp.

Einfeldia chelonia (Townes, 1945)

Endochironomus nigricans (Johannsen, 1905)

Glyptotendipes (Ph.ytotendipes) lobiferus (Say, 1823)

Glyptotendipes (Phytotendipes) paripes (Edwards, 1929)

Harnischia incidata Townes, 1945

Microchironomus nigrovittatus (Malloch, 1915)

Microtendipes pedellus (DeGeer, 1776)

Parachironomus monochromus (Wulp, 1874) Table 1. Taxa Examined (cont.)

Parachironomus potamogeti (Townes, 19^5)

Parachironomus tenuicaudatus (Malloch, 1915)

Parachironomus spp.

Paracladopelma undine (Townes, 19^5)

Paralauterborniella nigrohalterale (Malloch, 1915)

Phaenopsectra flavipes (Meigen, 1818)

Polypedilum (Pentapedilum) tritum (Walker, 1856)

Polypedilum (P.) convictum (Walker, 1856)

Polypedilum (P.) illinoense (Malloch, 1915)

Polypedilum (Tripodura) digitifer Townes, 19^5

Polypedilum spp.

Pseudochironomus pseudoviridis (Malloch, 1915)

Stenochironomus hilaris (Walker, 1848)

Stenochironomus poecilopterus (Mitchell, 1908) Table 1. Taxa Examined (cont.)

Stictochironomus near varius (Townes, 19^5)

Xenochironomus xenolabis Kieffer, 1916

Tanytarsini

Cladotanytarsus viridiventris (Malloch, 1915)

Cladotanytarsus sp. 1

Cladotanytarsus sp. 2

Lenziella cruscula Saether, 1971

Micropsectra nigripila (Johannsen, 1905)

Micropsectra sp. 1

Paratanytarsus dissimilis (Johannsen, 1905)

Paratanytarsus sp. 1

Rheotanytarsus exiguus (Johannsen, 1905)

Stempellina sp.

Tanytarsus (Calopsectra) dendyi Sublette, 196^- Table 1. Taxa Examined (cont.) Females

Tanytarsus (Calopsectra) sp . 1

Tanytarsus (Calopsectra) sp. 2

Tanytarsus (Calopsectra) sp . 3

Tanytarsus (T.) sp. 4

Orthocladiinae

Corynoneurini

Corynoneura lobata Edwards, 192^

Corynoneura sp. 1

Corynoneura spp.

Thienemanniella spp.

Metriocnemini

Limnophyes hudsoni Saether, 1975

Smittia sp. 1

Smittia sp. 2 Table 1. Taxa Examined ( c o n t.)

CTj CD ft r—1 cti 3

ft ft Females

Smittia sp. 3 1

Orthocladiini

Acricotopus senex (Johannsen, 1937) 2 2 1 2

Cricotopus bicinctus (Meigen, 1818) 7 6 3 2 Cricotopus exilis Johannsen, 1905 2

Cricotopus slossonae Malloch, 1915 1

Cricotopus trifasciatus (Panzer, 1813) 5 5 7

Cricotopus sp. 1 3 1

Cricotopus sp. 2 1

Cricotopus sp. 3 1 1 2 2

Cricotopus sp . ^ 2

Cricotopus sp. 5 1

Eukiefferiella sp. 1 1 1

Nanocladius distinctus (Malloch, 1915) 3 1 3 -V_n ON Table 1. Taxa Examined (cont.)

Orthocladius sp. 1

Orthocladius spp.

Rheocricotopus sp.

Tanypodinae

Coelotanypodini

Clinotanypus (C.) pinguis (Loew, 1861)

Coelotanypus concinnus (Coquillett, 1895)

Coelotanypus scapularis (Loew, 1866)

Macropelopiini

Natarsia baitimoreus (Macquart, 1855)

Procladius (P.) sublettei Roback, 1971

Procladius (Psilotanypus) bellus (Loew, 1866)

Psectrotanypus (P.) dyari (Coquillett, 1902) Table 1. Taxa Examined (cont.) Larvae

Pentaneurini

Ablabesm.yia (A.) mallochi (Walley, 1925) 5

Ablabesmyia (Karelia) sp .

Ablabesmyia sp. 2

Arctopelopia (Meropelopia) sp.

Conchapelopia (C.) telema Roback, 1971 1

Conchapelopia (Helopelopia) cornuticaudata (Walley, 1925) 1

Conchapelopia (Mesopelopia) aleta Roback, 1971 1

Conchapelopia spp. 3

Labrundinia pilosella (Loew, 1866) 1

Larsia decolorata (Malloch, 1915)

Larsia sp. 1 1

Nilotanypus fimbriatus (Walker, 1828)

Thienemannimyia (T.) senata (Walley, 1925) Table 1. Taxa Examined (cont.)

to 0 0 rt 0 to i—l > Oj 0 prt ft ft £ d 0 nJ 0 ft ft S ft

Tanypodini

Tanypus (Apelopia) neopunctipennis Sublette , 1964 1 1 1 1

Tanypus (T.) coneavus Roback, 1971 - 1 -

Tanypus (T.) punctipennis Meigen, 1818 1 1 2 -

Tanypus (T.) stellatus Coquillett, 1902 5 3 7 5

Totals 199 151 227 132

vO 60 Ohio State University Museum of Entomology. Unique spec­ imens and undescribed material will remain in the author's collection until they can be studied and described further.

New species and undescribed stages will be described else­ where .

Most species fit their published descriptions and keys within expected ranges of variation. However, some species differ from their descriptions in one or more variables.

Rearing in at least one case resulted in variation from characteristics seen in wild caught individuals and their descriptions. The following information will describe ob­ served variation and clarify those characters believed to separate possible new species from similar described spe­ cies .

Chironominae

Chironomini: Most of the Chironomini were identified

using Johannsen and Townes (1952), which reflects

the work of Townes (19^-5) on this tribe.

Chironomus attenuatus: Specimens of this species

reared from larvae tended to be darker and have a

higher fore leg ratio (ratio of the length of the

first tarsomere to the length of the tibia) than

wild caught adults.

Cryptochironomus sp. 1: This species is similar to

C. fulvus Johannsen except that the pupal cephalic

tubercles are simple, forked structures. In the 61 adults, the fore tibiae and tarsi are lighter

brown and the dististyle of the male is not as ex­

panded apically as in C. fulvus.

Cryptotendipes emorsus; While the one male keys out

to C. emorsus in Saether (1977)» the tubercles on

either side of the base of the anal point, which

are not described or figured in the literature,

have one to several bristles each; and the pupal

characters are closer to Cryptotendipes sp. of

Beck and Beck (1969) than to Johannsen's (1937)

description of Chironomus (Limnochironomus) sp.

which Townes (19^5) suggests may be C. emorsus.

Stictochironomus near varius: The specimens in­

cluded here are close to S. varius Townes in

having a fuscous halter knob and middle tibiae

lacking a central dark annulus. However, the num­

ber of dorso-lateral bristles varies, the tarsal

beard is sparse to absent instead of long, spec­

imens are smaller, leg ratios are higher (espe­

cially in females), and the antenna ratios (ratio

of the length of the last segment of the flagellum

to the combined length of the rest of the flagel­

lar segments) are lower. These characteristics

also show similarities with S. annulicrus (Townes)

and S . lutosus (Townes) .

Tanytarsini: The Tanytarsini are one of the least

known groups within the family, as can be seen by 62 comparing the number of described species in this

study with the total number studied in the above list.

No adequate source is available for the identifica­

tion of individuals from this tribe in North America.

Species from this study were confirmed and deter­

mined by Dr. James E. Sublette of Eastern New Mexico

University, who has been working on the monumental

task of revising this tribe.

Micropsectra sp . 1: This species was represented by

a single female which differs greatly from those

of M. nigripila.

Stempellina sp.: This genus, represented by a sin­

gle male, is very poorly represented in described

North American species.

Orthocladiinae: The Orthocladiinae as a whole are poorly

worked in North America, and no single source is ade­

quate for identification. Manuscript keys to the imma-

tures were kindly provided by A . L. Hamilton and

0. A. Saether.

Corynoneurini: The Corynoneurini are not well known in

North America, due in part to their extremely small

size. Schlee (1968) has improved the taxonomy of

this group.

Corynoneura lobata: Although this species has not

been recorded from North America, the specimens

included here fit very well with C. lobata in

Schlee (1968). The femora are pale instead of 63 light brown as stated by Edwards (19240, and they

are of slightly smaller size, although this could

be a variable induced by being reared.

Corynoneura sp. 1: This species is represented by a

single male which does not agree with any of the

species included in Schlee (1968). The antenna

ratio (O.73) will separate it from C. taris

Roback (0.38) and C. diara Roback (0.55) from

North America.

Thienemanniella spp.: The lone male included in this

group does not fit any species in Schlee (I9 6 8 ).

It will key to T. xena (Roback) in Sublette (1970)

due to its 13-segmented.antenna. However, this

specimen is larger, has a more clubbed antenna, a

more distinct lobe on the basistyle, and a much

stouter dististyle than described for T . xena in

Roback (1957)* The lone female and the immatures

are unassociated.

Metriocnemini:

Smittia spp.: This genus is not well known, due in

part to the small size of the adults and the ter­

restrial habitat of many, if not most, of the im­

matures. None of the species within this study

seemed to adequately match described species.

Orthocladiini:

Acricotopus senex: Individuals belonging to this

species varied extremely in size, some being almost 100fo larger than others. All specimens

matched Johannsen's (1937) description well, how­

ever .

Cricotopus sp. 1: This species will key to C.

bicinctus in Johannsen and Townes (1952) because

abdominal tergites one and four and the termi-

nalia are yellowish. However, a lower fore leg

ratio, a lower antenna ratio, shorter wing length,

and numerous immature and genitalic differences

with C. bicinctus will clearly establish this as

an undescribed species.

Cricotopus sp. 2: This species is similar to C.

politus Coquillett in general structure and color­

ation of the genitalia, but differs in having

darker body coloration and uniform brown legs,

although Sublette (I967) mentioned a specimen of

C. politus from Georgia with darker coloration.

The basistylar lobe of the genitalia also differs

from that of C. politus.

Cricotopus sp. 3- This species will key to C. edurus

Sublette and Sublette in Sublette and Sublette

(I971)i but differs in having the mid and hind

femora almost entirely dark and more infuscate

yellow banding on the abdominal tergites.

Cricotopus sp. This species is similar to

Cricotopus sp. 2 above except for minor differ­

ences in the genitalia, a higher antenna ratio, 65 lower fore leg ratio, and larger size. The abdo­

men darkens posteriorly as in C. politus.

Cricotopus sp. 5: This is a very distinctive spe­

cies owing to its smaller size; lower antenna

ratio and fore leg ratio; yellow banding on the

abdomen; stout triangular basistylar lobe; short,

stout dististyle; and pale genitalia.

Eukiefferiella sp. 1: This species is distinguish­

able from other Eukiefferiella in having glabrous

eyes, fourth abdominal tergite and terminalia

pale, no anal point, and the pupal respiratory

organ not napiform.

Orthocladius (0.) sp. 1: This species is similar to

Orthocladius (Pogonocladius) consobrinus

(Holmgren) in size, coloration, and presence of a

tarsal beard. The antenna ratio is similar to the

lower ratio (2.0) found in West German material

of 0. consobrinus by Pinder and Cranston (1976).

However, the basal lobe of the genitalia has both

a dorsal and ventral lobe, as in the subgenus

Orthocladius, and triangular basal median lobes.

The dististyle is similar to Orthocladius (0.)

glabripennis Goetghebuer, although other features

of the genitalia differ.

Orthocladius spp.: The remaining specimens of this

genus appear similar to nigritus Malloch, dorenus

(Roback), and obumbratus Johannsen; however, I was not able to adequately determine them using

Soponis (1977) due to the variation present

within the characters. While Soponis (1977)

points out the difficulty of determining these

species using quantitative measurements, many of

the qualitative characters also appeared inade­

quate .

Rheocricotopus sp .: This female specimen was con­

firmed by Dr. 0. A. Saether, but specific identi­

fication is difficult without associated males or

immatures.

Tanypodinae: This subfamily has been monographed by

Roback (1971), which was used exclusively for the iden­

tification of specimens.

Pentaneurini:

Arctopelopia (Meropelopia) sp .: The single specimen

of this genus appears closest to A. (M.)

flavifrons (Johannsen) except for its pale color­

ation, nine teeth on the spur of the fore tibia,

and ten teeth on the spur of the middle tibia.

Larsia sp. 1: The immatures and female are similar

to L. decolorata (Malloch) except for their much

smaller size and lower leg ratios. They are sim­

ilar in size to L. berneri Beck and Beck but are

entirely pale except for the slightly darker yel­

low vittae. Nilotanypus fimbriatus: The antenna ratio (0.75) is

slightly above the range given by Roback (0.58-

0 .7 1 ), and the fore leg ratio (0 .7 1 ) is slightly

lower (0.75-0.80). Otherwise, this specimen

matches the description for this species.

Thienemannimyia (T.) senata; The specimens included

here match the description given by Roback in

every character except for having small mesonotal

tubercles. Roback uses the absence of mesonotal

"spurs" in his key to genera as a characteristic

of Thienemannimyia. However, given the other

characteristics of the specimens in question,

there can be no doubt as to their being T. senata. RESULTS

The following material is a description of behavioral patterns found in the taxa observed during this study.

Analysis and discussion of these patterns will be confined to a later section.

Larval Habitation

Larval behavior associated with habitation will be arranged in three categories; free-living, burrow-forming, and tube-building. Free-living larvae construct or form no temporary or permanent habitations, although they may seek natural shelter such as in algae or hollow stems. Burrow- forming larvae form temporary cavities in loose substrate,

such as mud or silt, without lining these cavities with * - silk. Tube-building larvae normally construct somewhat

cylindrical, tunnel-like tubes, which usually assimilate various debris, using salivary secretion to cement the de­ bris together and line the inside walls of the tube. Al­ though no obligate leaf-miners were observed in this study, their tunnels would be considered substitute tubes, even though they do not line the tunnels with salivary secretion according to the literature (Berg, 1950; Brock, i960).

68 69 Free-living larvae include all observed species within

the tribes Pentaneurini (Tanypodinae) and Corynoneurini

(Orthocladiinae). The Pentaneurini are usually found

crawling along the substrate surface. Occasionally, they

were found to rest in a natural shelter such as a hollow

stem or vacant tube. They were also occasionally observed

tunneling into loose mud or silt as they prowled in search

of food. Only once was a prepupa of Conchapelopia

cornuticaudata observed making a chamber by irrigating in a

mat of filamentous algae as it was about to pupate. How­

ever, since no chamber was formed prior to the start of

pupation, this was not considered a burrow. The

Pentaneurini possess long, well-developed prolegs with

which they move about quite easily. On level substrate

they amble forward, propelled primarily by the posterior

prolegs, which are drawn anterio-ventrally onto the sub­

strate under the body and propel the body forward by

thrusting posteriorly while grasping the substrate. When

disturbed, they retreat rapidly backwards to shelter using

rapid anterio-ventral thrusts of the posterior prolegs.

They may often be seen holding onto vegetation with only

the posterior prolegs, while the rest of the body waves

searchingly about.

The Corynoneurini were observed continually crawling

over the substrate or vegetation. These active larvae ap­

pear nervous due to the almost continuous bobbing and weav­

ing of not only the head, but also the antennae, which may 70 approach twice the length of the head in some Corynoneura.

These motions make the tiny larvae appear even more active as they graze on whatever they might find. These observa­ tions support those of Darby (1962) and Forsyth (1971)-

No evidence was found to substantiate the report of

Johannsen (1937) that the larvae are sometimes found pro­ tected by a loose tube of debris.

Burrow-forming larvae include all observed species of the three remaining tanypodine tribes; Coelotanypodini,

Macropelopiini, and Tanypodini. Published accounts of the lifestyles of these larvae may differ, since it appears that they form only temporary burrows and seem free-living when observed prowling for food. These larvae often tun­ nel through loose mud or silt. When not actively tunnel­ ing, they are usually found in shallow burrows, Figure 3A.

The burrows usually consist of a chamber slightly longer than the larva with openings to the surface at both ends, although because of the loose nature of soft mud or silt, the openings are not always visible and are often partially blocked. The chamber, in which the larva is usually found, is often no more than 5 111111 below the surface, even for large larvae. The chambers in Coelotanypodini appear to be deeper, if the sediment depth allows, although attempts to measure this depth failed due to the usual destruction of the chamber from probing the loose sediment and the hasty evacuation of the chamber by the larva. The larva begins a burrow by tunneling head-first into the sediment to the 71

B

Figure 3- A. Longitudinal Cross-section of a Typical Tanypodinae Burrow

B. Tube and Feeding Net of Rheotanytarsus. redrawn from Walshe (19501 depth of the final chamber. Some irrigation-like motion

is used to press the sediment into place and widen the

chamber. This irrigation will often create a second open­

ing at the anterior end of the chamber if the sediment is

loose. If the sediment is harder-packed, the larva will

tunnel to the surface to create the second opening. • The

larva will continue to irrigate, and bits of loose sediment will be carried out the exit opening by the water current

created by the irrigation. The larva will continue to ir­

rigate periodically as in tube-building larvae. A larva

that is already below the substrate surface may begin a

burrow by just tunneling to the surface to make the open­

ings for water circulation. This is probably done often,

since these larvae tunnel through the substrate in search

of food. It is possible that in some cases a chamber is

formed without any openings to the surface, although this

has not been observed. One specimen of Psectrotanypus

dyari formed its chamber close enough to the surface that

it could be observed turning around within the chamber. It

turned its head ventrally under the body and curled poste­

riorly while the posterior body section moved the opposite

direction, almost like doing a forward somersault. Turning

around may allow the larva to check on any disturbance that

occurs at the opposite end of the chamber. Larvae were

often seen extending their heads from a burrow opening as

if looking around. If a probe is used to disturb the sub­

strate in the vicinity of an opening, a larva will often 73 extend from the burrow and strike at the probe. When ready to search for food, the larva simply tunnels out of its chamber without having to expose itself at the surface.

Because of the temporary nature of these burrows, the sub­ strate surface is often left with a number of openings, most belonging to abandoned burrows. One problem with ob­ servations on these burrows is that the larvae often aban­ don them when disturbed. Being only a temporary habita­ tion, the larvae do not seem to defend their burrows, al­ though they will feed on food in the vicinity of burrow openings. When crawling over the surface of the substrate, the motion is like that of the Pentaneurini, as is the rapid backwards movement when disturbed. References to the lifestyle of this group of larvae appear to all be at least partly correct, because much time is spent prowling in search of food. The report by Mitchell (1906) of

Psectrotanypus dyari constructing tubes by fastening bits of algae together with silk was not substantiated, however.

Tube-building larvae include the subfamilies

Chironominae and Orthocladiinae except for the tribe

Corynoneurini. Many of the larvae of the genus Smittia

(Orthocladiinae) are terrestrial (Johannsen, 1937;

Laurence, 195^) and are not dealt with under larval behav­ ior because the larval behavior is not well known and no larvae were observed in this study. In general, the con­ struction of tubes was found to be highly variable . Typi­ cal tubes are straight structures 1 .5 - 2X the larval 74 length or U-shaped tubes in sediment several times the lar­ val length. Actual tubes may be of any shape between these two typical shapes or range up to a complex network.

Tube length may vary from just slightly longer than the larva to many times longer. The amount and type of mate­ rial incorporated into tubes in most cases depends on the material available.

The general behavior in constructing horizontal tubes is basically that described by Cavanaugh and Tilden (1930) and Edgar and Meadows (1969)- The larva applies silk to various materials, pulling debris from the surrounding area. Once enough material has been pulled together, the larva begins to tunnel into it until at least a small loop or strap will hold around its body. If after several tun­ neling attempts the larva is unsuccessful, it will either add more material or seek out a new site. Once a loop is obtained, it is used as the foundation for new material, which is applied to its leading edges. The larva keeps its body within the loop at all times to act as the template.

The larva extends from its elongating tube, always keeping at least the hind prolegs and penultimate segment within or through the structure; it then applies silk to outlying material, and hauls it back to be cemented into place. In spreading silk, the anterior prolegs extend anteriorly along the ventral surface of the head capsule and mouth- parts, then extend away from the head capsule ventrally, making contact with the surface to which the secretion is 75 to be applied, as if pressing it into place, before being retracted to go through the cycle again. This rotary mo­ tion is done very rapidly with several passes being com­ pleted in a second. Once the tube is near completion, the larva spends more time lining the inside walls of the tube with silk. This lining closes up any small holes between the building material. As Walshe (1951) observed, this process of adding silk to the inner walls of the tubes con­ tinues throughout larval life. After applying silk to the tube lining, the larva often irrigates for a short period.

Besides serving a respiratory function, this irrigation may help shape the tube and its salivary lining as done by burrow-forming larvae. Irrigation at this time may also give the larva some indication of how well the tube is con­ structed based on the flow of water from irrigation. With large holes the larva will often extend its body through the opening until it finds some suitable material, which it drags back and cements into the hole. Larvae are often seen chewing open sections of their tubes, and then mending the site. The larva may extend from its tube to apply silk between the outside walls of the tube and the substrate .

This is more often seen in larvae which construct their tubes on vegetation, such as Endochironomus nigricans, and makes the tube appear like a tent with numerous irregular support ropes to the substrate.

Although many types of material are used by chirono- mid larvae to construct their tubes, Edgar and Meadows 76 (1969) reported they prefer to use material other than sand

and gravel. Chironomid larvae such as Acricotopus and

Cricotopus will often tunnel into mats of algae to build

tubes. Any filaments that get in the way are quickly cut with the mandibles and cemented into a more suitable posi­

tion. These tubes are lined with silk as described above.

Larvae which build U-shaped tubes in the substrate, such as

Chironomus plumosus, begin much of their initial construc­

tion by simply tunneling into the substrate. When little

suitable material is available, many larvae will construct

tubes entirely of silk, as reported by Miall and Hammond

(1900) . Larvae which often build tubes in or on vegetation will construct silken tubes more readily than substrate

dwellers.

Tubes constructed by the Tanytarsini tend to be more

compact and rigid than tubes of the Chironominae. Instead

of loose debris, these larvae prefer finer particulate types of material. Consequently, their tubes are often

less densely matted with debris and appear more cylindrical

The one Rheotanytarsus larva that was observed constructed a simple, horizontal tube without any of the arms or net

construction described in the literature, Figure JB. This was probably due to the lack of sufficient current as ex­ plained by Mundy (I909) • Shortly before pupation, however, the anterior section of the tube was extended up into the water column at a 45° angle. 77 Larvae of the genus Cryptochironomus have been consid­

ered free-living and not to construct even temporary tubes.

They have been reported to inhabit a medium lacking in

stability and slither through sand or silt like a snake as

they search for prey (Chernovskii, 19^9) • Chernovskii's

account of the- larvae slithering like a snake is indeed

accurate. The larvae are often found tunneling through mud,

silt, and other loose substrate using very characteristic

bobbing movements of the head and rapid extensions and re­

tractions of the anterior prolegs to pull themselves

through the substrate. The posterior prolegs are reduced

and do not usually function in this locomotion. The ac­

counts of these larvae not building even temporary tubes were found to be inaccurate, however. While a few larvae

observed in this study did not construct any tubes, the ma­

jority, and members of each species, were found to con­

struct temporary tubes. These tubes appear to be only a

temporary resting shelter, similar to the burrows formed

by some Tanypodinae. Indeed, the entire lifestyle of

Cryptochironomus seems to parallel that of the burrow- forming Tanypodinae. Cryptochironomus tubes are very

loose, flexible structures thickly matted with the silt and

mud of the substrate. The silk is so loosely woven that

the tubes often fall apart when picked up with a probe.

During the course of one day's activity, a single larva may

construct and later abandon several temporary tubes . No

openings to the substrate surface were ever seen, although the larvae were often observed irrigating within their tubes. Observing larvae within these tubes was very diffi­ cult, since there are no openings to the surface to indi­ cate where such a tube might be present, and with the slightest disturbance the larvae usually abandon the tube by tunneling out into the substrate, as do burrow-forming

Tanypodinae. These tubes are more often built in loose mud or silt substrates than in substrates of coarser debris.

It was not determined to what extent various substrate types influence the construction of these tubes or what the effect of food sources is, since a larva that has to spend all its time searching for food might not construct a tem­ porary tube. On one occasion a larva was consistently found in a hollow stem when checked.

Another larva observed in this study, which tends to have a somewhat similar habit to that of Cryptochironomus, is Stictochironomus. Stictochironomus will build regular tubes both under and at the surface of the substrate.

Their tubes under the substrate are like those of

Cryptochironomus in that there are no openings to the sur­ face . But the tubes are more permanent, and the larvae do not abandon them quite so readily. Thus, this species of

Stictochironomus appears quite adaptable to various habi­ tats .

Tube shape and size are highly variable for most chi- ronomid larvae. Variations in tube shape may include sim­ ple curves, multiple curves, branches, loops, chimneys, and 79 complex networks. Since tubes are continually added to,

their lengths are variable depending on how rapidly sec­

tions are added while others are allowed to deteriorate.

Most species of tube-building chironomids will construct

curves in tubes, especially if the tube needs to curve

around an object in the substrate or along plant material.

Branches and chimneys have been seen most frequently in

species of Dicrotendipes and Polypedilum. A chimney is a

branch which extends almost vertically from the substrate

and main tube into the water column. Polypedilum digitifer

is a master when it comes to complex tubes, Figure 4. The

tube illustrated in Figure 4B was constructed over several

days time in loose silt and mud. Not all parts of the com­

plex were in use at the time of its excavation, as indi­

cated by the dashed lines of abandoned sections that were

in various stages of decay. This single complex illus­

trates all the possible variations found in chironomid

tubes.

One larva of Nanocladius distinctus was found with a

gelatinous extension attached to a normal tube. The regu­

lar tube was about the length of the larva with a moderate

amount of debris incorporated into it. The gelatinous ex­

tension was also about as long as the larval body and ele­ vated at a 30° angle from the substrate and silken tube.

The walls of the extension ranged from about the thickness

of the larva at the point of attachment to the regular tube

to about half a larval width at its apex. It was Figure k. Tubes of Polypedilum digitifer

A. Branched tube B. Tube network a. Pupation chamber b. Pupal exit opening c. Chimney Dashed lines represent portions of tube abandoned at the time of excavation and in various stages of decay. 81

Figure 4. translucent and very flexible. The larva could bend its

body at a right angle and the gelatinous tube would bend

with it. This larva built several similar tubes and one of

only the gelatinous material, but the actual construction

of the gelatinous portion was not observed. Since this

larva also constructed a regular silken tube and two other

larvae which were observed only built regular tubes, the

significance of the gelatinous tube is not known.

Larvae were often seen defending their tubes against various intrusions. Types of defense include turning to

face a disturbance, exaggerated irrigation, and striking

out with the mandibles. When organisms such as ostracods

and cladocerans accidently get into the posterior tube

openings, the larvae can usually expel them with a rapid burst of irrigation. Intrusions in front of the larva are

often met with striking mandibles. Disturbances outside

the tube are responded to aggressively by the larva first

turning to face the disturbance, if not already doing so.

Additional agonistic behavior then includes either exagger­

ated irrigation, striking with the mandibles, or both if the first failed. Different groups of larvae respond dif­ ferently to disturbances, and individual variation is pre­

sent. Tanytarsini were more often observed to just move away from any disturbances. Orthocladiinae will either

move away or turn to face a disturbance but do nothing more. Substrate dwelling larvae such as Chironomus will

usually turn to face a disturbance but do nothing more except to back away if the disturbance persists. The most aggressive larvae were found to be those which are often found on vegetation. These larvae include the chironomine genera Dicrotendipes, Endochironomus, Glyptotendipes, and

Parachironomus. These larvae will turn to face a disturb­ ance and use irrigation, mandible strikes, or both at the point of disturbance. Larvae which build temporary tubes, like Cryptochironomus. and those which form temporary bur­ rows, like Tanypodinae, show no defense of their habita­ tions, but abandon them readily when disturbed.

Larval Feeding Behavior

The larvae of Cryptochironomus (Chironominae),

Corynoneurini (Orthocladiinae), and Tanypodinae were found to search for their food. Cryptochironomus and Tanypodinae are facultative carnivores and actively prowl through or on the substrate in search of prey. Pentaneurini can often be found crawling along the substrate in search of prey. They may hold onto vegetation with their hind prolegs as if waiting for prey. As described by Leathers (1922), the larvae of the Tanypodinae suck their prey into their mouth opening with a rapid dorso-posterior flip of the muscular lingua. Small prey are sucked in and swallowed whole.

Larger prey are pierced and held by the mandibles . Then the lingua scoops the body contents into the alimentary canal as the mandibles work to keep the prey properly in­ serted. The Corynoneurini might be considered grazers, as 84 they continually scour the substrate and vegetation for bits of algae, diatoms, and debris.

Little can be added to what has already been published regarding collecting behavior in chironomids. All tube- building larvae were found to utilize this method of feed­ ing to some extent. It might be added that larvae will usually move to a new site once food material is exhausted within reach of their old tube, although they will attempt to extend their old tube to new food sources as reported by Cavanaugh and Tilden (1930), providing ample tube- building material is present. Even net-building larvae often use this method to feed. Numerous larvae of

Glyptotendipes lobiferus were observed to feed exclusively by this method and did not construct nets. The only tube- building larvae that very seldom use this method are those of Cryptochironomus. which do almost all their feeding while prowling through the substrate. Only a couple of ob­ servations indicated that these larvae will utilize the collecting method to feed.

The only type of filter-feeding net observed during this study was the type spun by leaf-miners behind the lar­ va. The lone Rheotanytarsus larva observed fed exclusively by the collecting method, possibly because of the lack of current in its rearing container. None of the numerous

Chironomus larvae observed constructed the type of net de­ scribed by Walshe (1947a). Walshe (1951) stated that she observed numerous Chironomus but only found this type of net in C. plumosus. With C. plumosus having a world-wide distribution, it would be possible that geographic varia­ tion exists in this behavior. Leaf-mining nets were ob­ served in both species of Glyptotendipes (Phytotendipes),

G. lobiferus and G. paripes, and the one species of

Endochironomus, E. nigricans, used during this study. No larvae of Polypedilum (Pentapedilum) tritum were collected.

The larvae of both species of Glyptotendipes build nets consisting of oblique sheets, Figure 5A, while

Endochironomus nigricans nets are conical, Figure 5B, as stated by Walshe (1951)* These larvae did not always con­ struct nets but often fed by collecting. Numerous

Glyptotendipes lobiferus larvae did not build nets during the periods of observation. The extent of individual vari­ ation and effect of environmental conditions need to be studied to fully understand net-building behavior in

Chironomidae .

The larvae of chironomids were found to be flexible in their actual choice of food. Larvae were usually very op­ portunistic and fed on whatever was available, although most had distinct preferences as to animal versus vegetable and detritus diets. Many general statements in the liter­ ature support these observations.

Larval Irrigation

Irrigation is a rythmic, dorso-ventral undulation that passes backwards along the body of larval or pupal 86

B

Figure 5* Leaf-miner Nets A. Glyptotendipes

B . Endochironomus nigricans 87 chironomids. To accomplish this, the larvae hold the in­ side walls of their tube with both sets of prolegs to pre­ vent any forward body movement, and a series of up to two sine wave-like undulations move back along their body like the waves that move along a rope fixed at one end and being vibrated at the other. Tanypodinae usually hold on with only their posterior prolegs, however. As reported by both

Lindroth (19^-2) and Walshe (1950a), these movements are periodic, occurring in pulses with pauses between, serving to change the water within the tube and bring in a fresh oxygen supply. A typical pulse begins at a higher fre­ quency and then drops quickly to a frequency that is usu­ ally maintained for the duration of the pulse, although variations in this frequency may occur. The usual range for larval irrigation is between 50 and 200 cycles/minute depending on the environmental conditions. Although the usual flow of water from irrigation is from anterior to posterior, the larva may reverse this flow as reported by

Leathers (1922). This flow reversal is usually a short, rapid pulse used to expel something from the tube which is anterior to the larva. It was sometimes observed when leaf-mining larvae twisted up their nets only to find them full of particles of powdered charcoal, which were used to make the shape of the net visible. As if in a rage, the larvae would expel the mass of net and charcoal from their tube, like a cannon ball fired from a cannon, with the use of a sudden jet of reverse-flow irrigation. 88 While irrigation is usually associated with living in a confined space, such as a tube or burrow, several

Pentaneurini prepupae were observed irrigating slightly as they were about to pupate. This could serve as a respira­ tory function in these larvae, since the respiratory phys­ iology is in the state of changing to that of the pupa at this time. However, undulatory actions similar to irriga­ tion are also used by other chironomid larvae to aid in splitting and removing the exuvial cuticle, so these

Pentaneurini may have been moving for this purpose.

Other functions of irrigation that have already been described are defense of a tube, filter-feeding with nets, expelling fecal material from tubes, and shaping the tube or burrow. The frequency for the defense irrigation usu­ ally surpasses 300 cycles/minute; and its exaggerated, al­ though still rythmical, pulse shakes the entire tube. In

Chironominae, irrigation is used to expel fecal material that is deposited in their tubes. This can be done with either a rapid pulse or regular irrigation. Orthocladiinae generally extend posteriorly and deposit their fecal mate­ rial outside their tubes.

Larval Grooming

Burtt (19^-0) and Walshe (1951) reported tube-building larvae cleaning their bodies with their mouthparts when they turn around in their tubes. This behavior appears to be most common in tube building larvae, but is not confined to them. One Thienemanniella larva was observed to turn 89 back and chew at one spot along its body like a dog might bite at a spot giving it irritation. Details of what goes on during this cleaning process are difficult to see, espe­ cially with larvae inside tubes. One Glyptotendipes lobiferus was observed to pause during its turn and groom along its- body long enough so that it was possible to see that, at least in this one case, the mandibles were not in­ volved. Another type of larval grooming was seen only once in a pentaneurine larva believed to be Labrundinia, although the specimen was lost to roundworm parasitism. In this grooming the anterior prolegs were extended along the ventral surface of the head capsule and along the partially retracted antennae, brushing away any material that might collect around the head capsule where the antennae retract into the head. This was the only time any larval grooming was observed other than the cleaning of the body by the mouthparts as larvae turn around.

Behavior Associated with the Pupae

In general, pupal behavior parallels that of larval behavior. Pupation usually occurs in the larval tube of tube-builders, although there is often some modification of the tube by the prepupa. Various authors have mentioned modification of larval tubes in a variety of taxa (i.e.

Lauterborn, 1905; Darby, 1962; Scott, 1967; Mackey, 1976).

These tube modifications were found to be highly variable even between individuals of the same species. Some indi­ viduals will modify their larval tube during the prepupal 90 stage, while other individuals of the same species will

show no noticeable tube modification. This variation prob­

ably led to the discussion by Darby (1962) of the different

kinds of pupation tubes in Tanytarsini mentioned in the

literature. One of the most common modifications is the widening of the tube to accomodate the enlarged pupal tho­

rax. This was occasionally seen in a variety of

Chironominae including, Dicrotendipes, Glyptotendipes,

Paralauterborniella, Polypedilum, and genera of

Tanytarsini. It was most frequently observed in the

Tanytarsini. Openings to tubes are occasionally sealed.

This again was most often observed in Tanytarsini. It is

possible that tubes are sometimes sealed internally, since

this was found once in Microtendipes. Seals of this type would be more difficult to detect than the opercula often

found in Tanytarsini. Some prepupae add more material to

their tubes, making them appear more robust (Mackey, 1976).

Acricotopus, Cricotopus, and Cryptochironomus pupation

tubes are often more densely covered with debris than their

larval tubes.

The prepupae of Corynoneurini construct a loose silken

case of salivary secretion in which to pupate. This case

consists of a loose rectangular sheet covering a more

structured, oval chamber, which surrounds the pupa. This

silken chamber is enclosed posteriorly and loosely closed

anteriorly, but allows the pupa to exit when ready. This

confirms the description of the pupal case by Forsyth 91 (1971), and may be what Johhannsen (1937) referred to in

stating that the larvae are sometimes found protected by a

loose tube of debris.

Pupae of Tanypodinae are free-living. Pentaneurini will pupate on most substrates; the pupae being found

loosely attached to the larval skin unless disturbed, at which time they swim away in a jerky motion much like that

of mosquito pupae. Burrow-forming Tanypodinae prepupae will come just to the substrate surface to pupate. Before

emergence, they may be found sitting on the substrate in

the characteristic C-shape of Tanypodinae and Culicidae, in which the pupal caudal fin is curled ventrally until it al­

most touches the pupal head. On closer inspection the pupa

may be found still loosely attached to the larval skin, which is still hidden in the burrow, unless the pupa has been disturbed.

Pupae residing in tubes or the cases of Corynoneurini

may be observed irrigating as in the larval stage. Only

the pupal abdomen undulates to force water through the

tubes. Pupal irrigation may be one reason why most pupal

tubes in this study were found open at the ends. The pupal

irrigation frequency is usually lower than that of their

larval counterparts. Frequencies less than 50 cycles/ minute were common in the pupae observed in this study.

Fore Leg Positioning in Adults

Adult fore leg positioning refers to the normal eleva­

tion of the forelegs in other than a walking function. 92 This may vary from a slight tapping on the substrate with the legs normally resting on the substrate, to the contin­

ual maintenance of the fore legs in an elevated position.

There is a progression within Chironomidae from taxa that use no positioning to those in which it is well devel­

oped. -Tanypodinae and Corynoneurini observed in this study display no use of this behavior, Figure 6. Smittia

(Metriocnemini) show a rare tapping of the substrate consist­

ing of a very slight vertical motion and may leave the fore

legs elevated for short periods of time. Most Orthocladiini

show an occasional up and down motion, sometimes tapping on

the substrate; while Cricotopus (Orthocladiini) and

Chironominae use frequent to continual elevation of the fore

legs coupled with frequent gesticulations.

One aspect of fore leg positioning that can be exam­

ined is whether the legs rest in the aerial position or on the substrate. The fore legs of Orthocladiinae rest on the

substrate, although they may remain aerial for a period of time after activity such as active positioning, grooming,

or drinking. Cricotopus maintain their fore legs in the aerial position for long periodsof time and only gradually drop them to the substrate. Sometimes when they make con­ tact with the substrate, they are re-elevated. The usual rest position for Chironominae is with the legs held above the substrate and the tarsal segments about parrallel to the

substrate, Figures 70 and 90. Chironominae have been ob­

served using their fore legs to grasp the substrate if that 93

Figure o. Substrate Leg Position of Psectrotanypus dyari

Male at 15X 94 substrate is slippery and they are having trouble maintain­ ing their hold with the other legs. Stictochironomus adults may rest their fore legs on the substrate equally as often as aerially, although they are considered to rest aerial for the purpose of this study.

There are two basic types of motion used in fore leg positioning depending on whether the motion begins with the legs on the substrate or in the aerial position. When starting from the substrate, the usual motion is a series of up and down strokes, sometimes tapping on the substrate, although a rotary motion may result if there is no contact with the substrate. The basic aerial motion is a rotary movement. The legs begin moving dorso-anteriorly and change to a dorso-posterior motion. As the legs reach the posterior extent of their rotation, they begin to move more ventrally and then anteriorly to continue the cycle. The slower the motion, the more distinct the rotary configura­ tion. However, with quick motions, such as when disturbed, this rotary motion appears more vertical or horizontal.

These more rapid components of rotary motion have several variations. In many of the larger species, the fore legs frequently sweep posteriorly and are tapped to the sub­ strate up to several times before swinging anteriorly again. Many Chironominae will vibrate their fore legs up and down very rapidly preceding or following walking or other activity. While walking, the Chironominae will usu­ ally tap their fore legs to the substrate as a blind person 95 might a cane, but they do not seem to function in the actual walking motion. Most of the motion in these legs is due to movement around the coxal articulations. Both fore legs will sometimes start motion together, but they seldom remain in synchrony. Often the legs are observed using different amounts and types of motion, and it is not uncom­ mon for one leg to be motionless while the other is in con­ tinuous motion. Motion is greatest during periods of ac­ tivity or when disturbed. If one passes a hand along one side of a chironomid with both fore legs still, creating a shadow that passes over it, the leg on the side facing the movement will usually start into motion. Motion and activ­

ity are greatest during crepuscular periods. Members of the Harnischia complex, Tanytarsini, Dicrotendipes,

Xenochironomus, Polypedilum, and Cricotopus tend to be more active throughout the day. These taxa are easily disturbed and proved difficult to observe and take measurements of fore leg positions.

Measurements of the tibia-tarsal extreme angle (TiTEA) were taken while the specimens were still, as were the

other measurements discussed below. These measurements usually produced a range for each specimen. Upon examina­ tion of these ranges and noting the activity of the speci­ men when measurements were taken, it became clear that the extremes in TiTEA measurements corresponded with the activ­

ity extremes of the specimen. One extreme in the range was most often seen while the specimen was at rest, usually most noticeable during mid-afternoon; while the other ex­ treme usually corresponded to the time of most activity, usually crepuscular. Between these two extremes were a variety of measurements that could be found at different times during the day. Measurements during activity were often difficult, especially in the more-active taxa listed above, since they seldom remained still long enough to get an accurate fix. Their measurements were also more uni­ form, since they usually appeared active, making it diffi­ cult to correlate the extremes with rest or activity. How­ ever, two basic patterns emerged from these measurements.

The first, and most common, found the TiTEA close to paral­ lel to the body line (BL) in front of the body at rest and swinging out to between 30° to 60° from the BL when active,

Figures 7A and B, 9A and B. In other words, in this group, as activity increases, the legs swing out from in front of the body to a position further to the side. This group in­ cludes Cricotopus and all the Chironominae except

Phaenopsectra, Stenochironomus, Tanytarsus, and possibly

Stempellina. In these genera, making up the second group, the TiTEA is greater at rest and decreases to an angle closer to the BL, again usually in the 30° to 60° range.

The most extreme member of this second group is

Stenochironomus, Figure 10A and B. In Stenochironomus the tibiae and tarsal segments slant posteriorly along the body at rest, giving a TiTEA of about 140°. When active, how­ ever, they swing forward into the usual position. It 97

Figure 7. Fore Leg Positioning in Chironomus attenuatus

A. Rest TiTEA B. Active TiTEA C. Rest SFA

D. Active SFA Male at 10X 98

F ig u r e 7 99

A

O

Figure 8. Positioning of FFA in Chironomus attenuatus

A. Rest FFA B. Active FFA Male at 60X 100

Figure 9 Fore Leg Positioning in Dicrotendipes nervosus

A. Rest TiTEA B. Active TiTEA C. Rest SFA

D. Active SFA Male at 20X 101

F i g u r e 9> 102

Figure 10. Fore Leg Positioning in Stenochironomus hilaris

A. Rest TiTEA B. Active TiTEA C. Rest SFA

D. Active SFA Female at 10X 103

F ig u r e 10 104 should be noted that the active position is maintained while it is the rest position that has seen some divergence.

In most cases the tarsal segments are held parallel to the

substrate, but at times the generally more active taxa will

swing the fore legs up so that, for an instant, they will point straight overhead.

Measurements on the fore femora from the side (SFA) were also diverse with the extremes corresponding to either

the rest or the activity position. Again generally active

taxa caused problems because measurements were continually

changing throughout an observation period. In the first

pattern the fore femora begin posterior to the vertical at

rest and move forward to up to 30° anterior to the vertical when active, Figure 7C and D. The taxa showing this pat­

tern included Chironomus, Einf eldia, Demei.jerea,

Endochironomus, Glyptotendipes, Microtendipes,

Phaenopsectra, Polypedilum, Stenochironomus, and

Stictochironomus. In the second pattern the fore femora

begin at least 45° anterior to the vertical at rest and

move posterior when more active, Figure 9C and D. The taxa

showing this pattern included Cricotopus. the Harnischia

complex plus Dicrotendipes and Xenochironomus, and

Tanytarsini. Again it should be noted that the same gen­

eral active range is maintained in all taxa, while the rest

positions designating the two groups fall on either side of

the active position. 105 Measurements on the fore femora from the front (FFA) were also found to range between extremes corresponding to

either the rest or activity position. In the first pattern

the fore femora begin closely appressed to the thorax at

rest and move out away when more active, Figure 8. In the

second pattern they begin at a greater angle away from the

thorax at rest and decrease toward the same active range found in the first FFA pattern. The taxa included in these

two groups are identical to those in the same group meas­

ured by the SFA. Thus, these two measurements produce the

same similarity pattern between the taxa observed in this

study.

The significance of the activity ranges being similar

in all observed positioning chironomids, while rest ranges

diverge to either extreme, probably indicates that the se­

lection pressures which led to the use of positioning act

strongest on the active position, keeping it conservative, while the rest positions have shown divergence. Thus, the function of positioning is probably to be found in the ac­ tive positioning. Positioning may serve to attract the at­ tention of possible predators to the legs rather than the body of the insect, sacrificing the fore legs to save the rest of the insect. Support for this theory may come from the fact that the fore tarsal segments are very easily lost

in Chironominae. Adult Grooming Behavior 1Ub

Grooming in Chironomidae consists entirely of leg rub­ bing, with the antennae, palps, wings, abdomen and legs being rubbed in various grooming movements. All rubbing proceeds from proximal to distal on the groomed structure.

No cleaning behavior (use of the mouthparts in grooming) was observed, although one specimen of Cricotopus trifasciatus was observed to touch the tips of both fore tibiae to the mouthparts once. At times, specimens would attempt to do a certain act, but unless actual contact was made, this was not recorded as a grooming act. Fifteen basic grooming movements were observed in the Chironomidae,

Table 2. In general, frequencies of grooming are low, often less than ten movements per hour, except in the

Orthocladiinae, where one specimen of Cricotopus sp. 5 was observed to groom at a rate of 20^ movements per hour. Be­ cause grooming occurs in bouts, individual variation within species was high depending on whether a bout occurred during an observation time. It was not uncommon to have specimens with two hours of observation and no observed grooming in the Tanypodinae and Chironominae. It was probably for this reason that Heinz (19^9) recorded only four different groom­ ing movements for the Chironomidae and considered them in­ frequent groomers.

One of the most frequently used grooming movements is the grooming of the antennae with the fore legs (An). In this movement the head is tilted to one side so that one Table 2. Adult Crooming Frequencies

T ime An r T FMs ■II Mil MHII IIMs Ill) Ab dWM dWII vWH dvW vWp Tot . Chironominae

Ch ironomini

ChironomuB attenuatus 59 <30 1 .80 0 .2h . 0.81 _ _ 1 .03 1.33 0.87 0.22 0.44 . . . 6.72

Chironomus crassicaudatus hi 00 0.25 ------0.25 ---- - 0.5"

Chironomus plumosus 13*^5 3-85 0.36 - 0.51 -- - 0.51 0.07 0.29 - 0.22 -- - 5.82

Chironomus riparius 12i 00 0.17 0.08 - 0.33 - -- 0.08 0.25 0.08 ----- 1 .00

Chironomus staegeri 3 ,1*5 0.53 ------0.53

Chironomus spp. hi 00 0.25 ------0.25

Chironomus summary 97t00 1.71 0.21 - 0.61 --- 0.71 0.86 0.60 0.13 0.30 --- 5.12

Cryptochironomus fulvus 5 2.96 0.52 - 0.87 - - - 0.87 1.22 0.87 - 0.17 --- 7. '17

Cryptochironomus ponderosus 3 ,00 2-33 1-33 -- --- 0.33 1.00 6.00 ----- 11 .00

Cryptochironomus sp. 1 2il5 3 -U 1-33 -- - - - O.bb 1-33 0.44 ----- 6 .67 Cryptochironomus spp. 61 <15 1 .oh 0.15 ------0.15 0.44 - --- - 1 .78

Cryptochironomus summary 17i'<5 2.08 0.62 - 0.28 - -- 0.39 0.79 1.52 - 0.06 --- 5.80 CO XPl 0 Cryptocladopelma galea tor 8 1 30 1-53 - 0 .U7 --- 0.12 1.76 0.35 - 0.47 --- 5.2" Cryptotendlpes casuarlus 1 lOO 3.00 2.00 ------5.00

Cryptotendipes emorsus 0|25 ------0.00

Demel.jerea brachial is 2i 15 18.22 0.89 ------10.11 so CO Dlcrotendipes fumidus 9<30 p~\ 0.71* - 0.7't --- 2.42 0.63 0.8 4 - 0.63 - - - 9.68

Dicrotendip.es modes tus 81 30 2.12 ------b .58 2.82 8.00 - 0.35 --- 17.88

Dlcrotendipes nervosus 10th5 3 .hh 1.17 - O .56 --- 1.30 5.02 0.65 - 0.19 - - - 12.28 Dlcrotendipes spp. 6ih5 o.7h -- 0.15 --- 0.30 1.93 0.44 - 0.15 - - - 3.70

Dlcrotendipes summary 35i30 2.68 0 .5 b - O .39 --- 2 .19 2.73 2.42 - 0.33 - -- 11 .29 Elnfeldla chelonia 5.60 0.80 ------6 .hO

1.15 107

Endochironomus nigricans 2I1I5 0.61 0.09 ------0.05 ------0.75 Table 2. Adult Grooming Frequencies (cont.)

Time An P T FMs Fll Mil Mllll MMs 111! Ab dWM dWtl vWH dvW vWp Tot.

Glyptotendipes lobiferus 9 >45 1 .64 0.21 - - - 0.10 0.31 0.62 - 0.21 - 3.07

Glyptotendipes parlpes 9 >00 4.11 0.11 - O .67 0.11 0.22 - 0.33 - 5.56

Glyptotendipes summary 18.45 2.83 0.16 - O .32 - 0.15 0.21 0.43 - 0.27 - 4.27

Harniscliia incldata 2i45 1.45 1 .82 - - - 3.27 ------6.55 Microchironomus nigrovittatus 3> 30 1 .14 0.86 - 1 .43 - 0.29 0.29 ------4 .00

Microtendipes pedellus Oi45 - - - 1.33 6.67 8.00

Paracbironomus monochromus 4 i45 3-37 1 .89 - 1 .47 3.79 0.21 0.63 0.63 12.00

Paruchironomus potamogetl Oi45 1 .33 - - - 1 .33 Paracbironomus tenuicaudatus 4i 15 1 .41 0.47 - - - 0.47 5.18 - - - 7.52

Paracbironomus spp. 1145 1.14 0.57 - - - O .57 2.29

rarachironomus summary 111 30 2.17 1.04 - 0.61 - 0.17 3.57 0.09 0.26 0.26 8.61

Paracladopelma undine llOO ---- 0.00

Paralauterbornlella nigrohalterale 11 20 2.25 - - - - 0.75 4.50 1.50 - 15.75 24.75 Pbaenopsectra flavipes 4 150 0.21 -- - - 0.41 - 0.62

Polypedilum tritum 0 i30 -- - - 0.00 Polvpedilum illinoense 4,45 0.21 - - 0.21 0.42

Polypedilum digitifer 14145 1.36 0.20 - 0 . 0 7 0.41 0.34 0.20 0.07 2.64 Polypedilum summary 20i 30 1 .02 0.15 - 0.10 0.29 0.24 0.15 0.05 2.00

Pseudochlronomus pseudoviridis 11 30 ---- 4.00 4.00

Stenochironomus hilaris 11 30 2.67 0.67 - - - 1.33 4.6?

Stenochironomus poecilopterus 1145 - - - - 0.00

Stictocbironomus near varius 10i25 0.58 0.10 - 0.29 0.29 - 0.10 - 0.48 1 .82

Xenochironomus xenolabis 3 >45 17.06 3.73 - 2 .40 6.13 1.07 1.60 0.27 2.13 35.47 nytarsini 108 Cladotanytarsus virldlventris It 45 6.86 - - - 6.66

Cladotanvtarsus sp. 1 4i00 1.00 0.25 -- - 0.25 1 .50 Table 2. Adult Grooming Frequencies (cont.)

Time An P T FHs Fll Mil MIDI IIMs Illl Ab dWM dWH vWll dvW vWp Tot.

Cladotanytarsus sp. 2 1.15 3.20 1.60 ------0.80 ------5 .6 O Cladotanytarsus summary 7.00 2.86 0.29 - - - -- 0.10 0.14 ------3-57

Iienziella cruscula 3.15 4.31 ------0.62 ------4.92

Micronsectra nleripila 6.30 0.92 1.69 -- - -- 1.23 - 0.31 - 0.31 - - - 4.46 Micropsectra sp. 1 1.45 ------0.00

Micropsectra summary 8.15 0.72 1.33 ----- 0.97 - 0.24 - 0.24 -- - 3.52 Paratanytarsus summary 3.30 ------0.00 Idieotanvtarsus exleuuo 1130 ------0.00 Tanvtarsus dendyi 1.45 0.57 ------0.57

Tanvtarsus sp. 1 0.30 - - ~ ------0.00

Tanvtarsus sp. 2 8.15 0.85 ------0.24 0.12 0.12 - -- - - 1.33

Tanvtarsus sp. 3 1.45 ------0.00

Tanvtarsus sp. 4 5.15 0.57 - - 0.38 -- - 0.19 ------1.14

Tanytarsus summary 17.30 0.62 -- 0.11 - - - 0.17 0.06 0.06 ---- - 1 .02

Stempellina I.30 1.33 ------1.33 Orthocladijnae

Corynoneurini

Corynoneura lobata 0.50 - -- - 10.80 - 6.00 - 22.80 -- 2.40 -- - 42.00

Corynoneura sp. 1 0.30 ------2.00 - 34.00 ------36.00

Corynoneura s p p . 1 *15 0.80 0.80 - - 4.00 - 6 .4o - 8.80 3.20 - - 0.80 -- 24 .80

Corynoneura summary 2.35 0.39 0.39 - - 5.42 - 5.42 - 18.19 1.55 - 0.77 0.39 - - 32-52

Thienemanniella spp. 3.00 2.67 2.33 - -- - 5.00 5.67 6.67 0.33 12.67 11 .67 - 5-67 - 53.00 Metriocnemini

Limnophyes hudsoni 1.20 - -- -- 0.75 I .50 - 2.25 -- 1.50 0.75 - - 6.75

Smittia sp. 1 6.45 1.70 0.74 --- - 2.81 - 14.37 0.44 - 10.07 - 1.33 - 31.56 60 T 60 Smittia sp. 2 4.15 0.94 0.24 - - - - 3.53 - 18.12 1.18 - 10.59 2.12 - 0.47 37-16 Table 2. Adult Grooming Frequencies (cont.)

T iine An PT FMs FI! Mil MHII HMs Mil Ab dWM dWH vMII dvW vWp Tot.

Smittia sp. 3 Ii45 - - - - 1 .14 - 4.57 0.57 0.57 - 6.86

Smittia summary 12i45 1.25 0.47 - - 2.82 - 14.27 0.71 8.94 0.71 0.71 0.71 30.04 Orthocladlini

Acricotopu3 senex 6 i00 8.67 - 1.50 - 1.17 - - 6.00 2.50 - 8.50 1.83 - - 30.17

Cricotopus bicinctus 8i 30 1.53 1 .89 - - - 2.35 - 24.00 3-29 - 21 .8% 6.25 + - 63-29

Cricotopus exills 1130 1.33 2.67 - -- 2.67 - 21 -33 -- 1.33 - -- 54 .00 Cricotopus slosBonae 1130 0.67 0.67 - - - 3-33 - 22.00 -- 12.00 12 .00 -- 50.67

Cricotopus trifasciatus a . 5 5 3.70 1.79 - - 0.11 2.58 - 8.75 0.34 - 6.39 2.13 0.56 - 26.58 Cricotopus sp. 1 liOO 1.00 1 .00 - -- 3.00 - 3.00 3.00 - 2.00 2.00 1.00 - 16.00 Cricotopus sp. 2 2.15 0.44 - - -- 1.33 - 4.89 1.33 - 4.44 0.44 -- 12.89 Cricotopus sp. 3 5.00 5.80 4 .00 - -- 1.40 - 12.60 2.20 - 8.4o 3.20 -- 37.60

Cricotopus sp. 4 3.00 18.33 1.33 - -- 4.67 - 45.67 10. oS - 21 .oS 27.6? 0.67 - 131 .oS

Cricotopus sp. 5 o.'»5 1.33 5.33 - __ 16.00 _ 42.67 20.08 - 72.00 26.67 13.33 - 200.00

Cricotopus summary 32.25 4.19 2.04 - - 0.03 2.81 - 18.29 2.87 - 14.50 7 .1 % 0.5& - 52.66

Nanocladius distinctus 2.25 5.79 2.48 - 2.07 - - 8.69 - 11.17 2.07 - 6.62 3-31 0.83 - 43.03 Orthocladius sp. 1 3.20 7.20 ------O.90 ------8.10 £ o Orthocladius spp. o 7 .06 0.59 - - 0.06 1.76 - 4.82 0.76 - 1 .00 0.71 - - 16.76

Ortliocladius summary 20.20 7.08 0.49 - - 0.05 1 .48 - 4.18 0.64 - 0.84 0.59 - - 15.34

Rheocricotopus 1.15 4.00 0.80 - 1 _ 2.40 _ 16.00 _ _ - -_ - 23.20 Tanypodinae Coelotanypodini

Clinotanvpus pinguis 3.35 0.28 - - - 0.28 0.28 4.47 2.23 5.02 - - 1.95 14.23

Coelotanypus concinnus 8.15 1 .'*5 0.12 - 0.12 0.12 0.73 - 1.45 1 .21 - 3.03 0.61 - 2.55 11.39

Coelotanypus scapularis 7.00 0.14 -- - 0.14 0.86 - 0.43 - - 0.29 1.86 110 Coelotanypus summary 15.15 0.85 0.07 - 0.07 0.07 0.46 1.18 0.66 1.84 0.33 - 1.51 7 .02 Die 2. Adult Grooming Frequencies (c o n t .)

Time An FMs FH Mil MHII HMs III! Ab dWM dWIl vWH dvW vWp T o t .

Macropelopiini

Procladius sublettei 29t 15 0.88 - 0.07 0.14 - 0.48 0.54 - 0.64 0.24 - - 3-35

Procladius bellus 13«35 0.52 0.59 - 0.96 0.44 0.22 0.07 3-09

Procladius summary 42i 50 0.77 - 0.05 0.20 - O.63 0.37 - 0.58 0.23 0.02 3.26

Psectrocladius dvarl 18115 0.16 0.05 0.11 O.38 - 0.27 1.37 - 0.11 0.33 2.85

Pentaneurlni

Ablabesmyia mallochi I 81OO 0.28 0.28

Ablabesmyla (Karelia) It 00 5.00 1 .00 6.00

Ablabesmyia summary 19 >oo 0.52 0.05 0.58

Arctopelopia 0115 - 0.00

Conchapelopia telema 4 115 - 0.00

Conchapelopia cornuticaudata O130 - 0.00

Labrundinla pilosella 2>15 - 0.00

Larsia decolorata l l 30 - 0.00

Nilotanypus fimbriatus 2130 - 0.80 0.80

Thienemannimyia senata 4i00 - 0.00

Tanypodini

Tanypus neopunctipennis liOO - 0.00

Tanypus concavus 0130 - 0.00

Tanypus punctipennis Oi45 - 0.00

Tanypus stellatus 17*45 0.62 0.06 - 0.06 0.11 0.79

I-1 112 antenna becomes lower than the other. The antennae are also spread further apart so that they appear one above the other in orientation. The apical tips of one or both fore tibiae are then rubbed along the lower antenna, usually making contact with the antenna only on the strokes going away from the body. These movements are usually bilateral, occasionally unilateral, or rarely the fore legs may alter­ nate. The head and antennae sometimes are not positioned as above, and then each fore leg strikes its ipsilateral antenna. There is sometimes contact between the fore legs in this movement, but to what extent and whether it is only incidental is difficult to determine. This movement may be the FF observed by Heinz (19^-9)» since no contact between fore legs was ever observed besides this possible inciden­ tal contact while rubbing another structure. The palps are usually extended during the antenna rub, and contact has been observed between the fore legs and palps during this movement. However, when the majority of the motion and contact was along the antenna, this was considered an anten­ na rub. This movement was observed throughout the tribes of the Chironomidae and was one of only three movements ob­ served in the Pentaneurini.

The palps are also rubbed by the fore legs (P) . The head may be tilted, but not as much as in the antenna rub.

Both palps are extended anterior to the head, and the apical tips of the fore tibiae stroke either the more ven­ tral palp if the head is tilted or their ipsilateral palp 113 if the head is not tilted in a motion very similar to that of the antenna rub. This movement may also be unilateral.

Frequently the legs strike some of the fine hairs of the large, plumose male antennae in this movement. This move­ ment is also widespread in the Chironomidae, although it was not observed in the Pentaneurini or Tanypodini.

The dorsum of the thorax and head are rubbed by the fore legs (T) in Acricotopus senex. This movement is bas­ ically similar to the antenna or palp rub described above except that the fore leg begins more posteriorly and strikes either the dorsum of the head or the dorsum of the thorax and head before proceeding on to rub the antenna or palp in the final contact. It is usually a unilateral movement.

The fore legs have been observed being groomed in sev­ eral ways. A fore leg may be rubbed by its ipsilateral mid leg as the mid leg steps down on the fore tarsus when the specimen begins to walk (FMs). The fore leg is placed on the substrate as the specimen begins to move forward, and the mid leg steps down on the fore leg, rubbing the fore leg with the apex of the tibia. The fore leg may be drawn up from the substrate as the mid leg rubs along it. This movement was suspected for some time, but it took numerous observations before it could be confirmed as an actual grooming movement and not a clumsy walking start, which it usually resembles. Even when it was recognized as a groom­ ing movement, it was still difficult to detect at times. 114 This movement is a single stroke and is usually unilateral; however, bilateral rubs have been observed. It can some­ times be observed following An. This movement may be sim­

ilar to a movement in the Satyridae (Lepidoptera)f discus­ sed by Jander (1966), in which the mid legs brush against the antennae while walking, since the fore legs are reduced in this family. This movement has been observed in most

Chironominae and Nanocladius distinctus. Because of its low frequency of use and the difficulty detecting it, it is probably found in more of the Chironominae than the data indicate. One specimen of Glyptotendipes paripes was ob­ served stepping down on its right fore leg with both mid legs. The left fore tarsus was missing. The right fore leg was held on the substrate by the right mid leg with the body elevated anteriorly to allow the left mid leg to step across to the right fore leg. This movement was awkward and seen only once in this one individual.

One fore leg may be extended posteriorly, ventral to the body between the mid legs, and rubbed by the hind legs

(FH). In this movement the body is elevated by extending the remaining fore leg and mid legs, which together form a tripod stance allowing the groomed fore leg to extend ventro-posteriorly. The tips of the hind tibiae are used to rub the tibial apex and the entire tarsus of a fore leg.

This movement has been observed in Corynoneura, once in

Coelotanypus concinnus, and once in Psectrotanypus dyari while it hung from a probe with one fore and one mid leg 115 and groomed the other fore leg. This latter observation may not be in the regular repertoire for Psectrotanypus.

A mid leg may be groomed by an ipsilateral hind leg

(MH), but this movement is usually mutual. The body is supported by the other four legs, and the ipsilateral fore leg usually moves posterior to provide more balance and tilt the body somewhat toward the other side, giving the grooming legs more room. This movement is usually aerial, although one of the grooming legs may contact the substrate occasionally. This movement has been observed in

Coelotanypodini, Macropelopiini, and Orthocladiini .

A mid leg may also be groomed by both hind legs (MHH).

In this movement the mid leg receives most of the grooming.

The body is elevated posteriorly, and a tripod stance is maintained by the other three legs. The movement is mainly aerial, with occasional contact with the substrate. This movement is commonly observed in Coelotanypodini,

Macropelopiini, and Orthocladiinae.

The two mid legs were observed to groom each other in a specimen of Xenochironomus xenolabis when it rested in a position with the fore legs on a vertical surface and the hind legs on a horizontal surface. The mid legs were sus­ pended above the substrate and probably groomed in what may be a displacement activity. This is not considered a nor­ mal grooming movement.

A hind leg may be groomed by its ipsilateral mid leg in the mutual grooming movement, MH, described above. 116 A hind leg may also be groomed by its ipsilateral mid leg as the hind leg steps forward and the mid leg steps down along it (HMs) . This is usually a single stroke as the specimen begins to walk and may be bilateral. As with the similar rubbing of the fore leg, this movement was not recognized until numerous observations had been made and was still difficult to detect once recognized. This move­ ment also appears like a clumsy walking start. The wings are often positioned during this movement by simply elevat­ ing them out of the way of the grooming legs or vibrating them in the extended flight position, without flight. This movement has been observed in many Chironominae and

Thienemanniella.

The most common grooming movement involving the hind legs is the mutual grooming of both hind legs (HH). The body is supported by the fore and mid legs, and the move­ ment is usually aerial. In Chironominae this movement is quite awkward and abrupt. The fore legs tend to continue aerial positioning or be tapped to the substrate during this movement. The result is that the body is supported mainly by the mid legs. The hind legs extend and elevate the abdomen and then quickly try to make contact before the abdomen returns to its regular position. Often, the hind legs only strike the substrate in their attempt. This movement is less awkward for Chironominae resting upside- down . As with the movement above, the wings may be 117 positioned in Chironominae as well as other taxa. This movement has been observed in all tribes except the

Pentaneurini.

The abdomen may be groomed by either or both hind legs

(Ab) . In the usual position the body is supported by the fore and mid legs while- the hind legs rub the abdomen using primarily the tibial apices. In Chironominae this support is more on the mid legs and sometimes the abdomen itself may rest on the substrate to provide support. The fore legs may be tapped on the substrate for additional support.

The wings are often positioned out of the way during this movement, especially in Chironominae. This movement was observed in all tribes except Tanypodini.

The dorsal wing surfaces are occasionally groomed by their ipsilateral mid legs (dWM). The wing is usually po­ sitioned by extending it a short way out from the body.

The mid leg is then raised above the wing and sweeps across it with its anterior tibial surface against the wing sur­ face, usually pulling the wing down alongside the body.

This type of grooming has been observed in only Chironomus attenuatus, Parachironomus monochromus, and Xenochironomus xenolabis.

The dorsal wing surfaces may be groomed by the ipsi­ lateral hind legs (dWH). This may be unilateral, bilateral, or the hind legs may alternate on their ipsilateral wings.

The usual body stance is as described above for grooming the abdomen. The hind legs are elevated above the wings 118 and then sweep distally across them, sometimes pulling them

down alongside the abdomen. Contact with the wing is made

by the tip of the tibia and the anterior tibial surface.

Often the abdomen is hit as the legs proceed further after

passing completely over the wing. This movement was ob­

served in all tribes except Pentaneurini.

The ventral wing surfaces are usually groomed by the

ipsilateral hind legs (vWH) . This may be either unilateral

or bilateral. The hind leg is elevated, bringing the pos­

terior tibial surface and the tip of the tibia in contact with the ventral wing surface. The leg continues to ele­ vate and extend posteriorly so that its force pushes the wing upward relative to the body, and it rubs across the ventral surface of the wing until contact is lost. The wings may be vibrated against the leg during this contact.

The body is supported by the first two pairs of legs. This

movement was observed in Tanypodinae, except for

Pentaneurini, and in Orthocladiinae.

The ventral wing surfaces may be groomed by the con­ tralateral hind leg in conjunction with dWH (dvVJ). In this movement the ipsilateral hind leg pushes the wing and

costal margin down so that the ventral wing surface faces the contralateral hind leg, which swings across under the abdomen to rub the ventral surface of the wing with its apex. Usually only the tip of the ventral wing surface can be reached by the contralateral leg. Once each in

Cricotopus sp. 1 and Thienemanniella the ipsilateral leg 119 pressed the wing down to the substrate so that its ventral surface was against the substrate, causing the contralater­ al leg to rub the dorsal wing surface along with the ipsi­ lateral leg. This movement was observed in Procladius and various Orthocladiinae.

The ventral wing surface is positioned over the hind legs (vWp) in an unusual grooming movement in which the wing is extended as the body tilts toward the same side.

This tilting causes the ipsilateral legs to extend, with respect to the body, and lay close to the substrate. The wing rubs across the hind leg and sometimes the mid leg or substrate as the body tilts. The body then straightens, drawing the wing back over the legs . The wings do most of the movement that results in rubbing. This movement has been observed in Coelotanypodini and Smittia .

In a totally different type of rubbing, adults rub against the substrate. In this, the body is dipped to the substrate and rubbed there briefly. The usual area rubbed is the coxal area of the legs, although the palps and geni­ talia may also be rubbed against the substrate. All major taxa have been observed to do this.

An aspect of grooming observed numerous times in

Cricotopus is asynchronous rubbing by the hind legs. This usually beings as bilateral grooming of the dorsal wing surface, ventral wing surface, abdomen, or other hind leg.

After grooming for a short period, each leg begins grooming a separate structure. The result is that both legs are 120 grooming simultaneously, but each is proceeding on a dif­ ferent pattern. When Cricotopus get into a grooming bout,

it is difficult enough to record the series of movements when both legs are together. The only way to record a

total sequence with asynchronous rubbing would be with high

speed photography. When this was observed during a bout,

the usual procedure was to record that asynchronous groom­

ing was taking place, the type of rubbing that was observed

and the elapsed time. This rubbing sometimes lasted for

thirty seconds. Each leg may be rubbing the dorsal surface

of the ipsilateral wing, the ventral surface of the ipsi­ lateral wing, the ventral surface of the contralateral wing, or the abdomen. This phenomenon is usually observed toward the middle of a grooming bout, as if a threshold is reached after grooming steadily for a certain time, and the

legs proceed as if no longer under total control. Since

Cricotopus showed the highest frequency of grooming, this type of threshold might not manifest itself in other chiron

omids as often. However, a few chironomine specimens, mainly Chironomus attenuatus and Xenochironomus xenolabis, that were observed during exceptionally long bouts, showed unique movements during periods of peak grooming intensity.

This type of phenomenon might explain some of the rare movements that were seen only in specimens with higher frequencies of grooming.

A variety of grooming combinations was also observed

in chironomids. Grooming combinations are two or more 121 basic grooming movements done simultaneously. Like the asynchronous grooming described above, these movements were usually observed during longer bouts, when the specimens appeared to be at a peak in grooming activity. The com­ plexity of these combinations is great, since each movement may be unilateral or bilateral, while in relation to each other the two may be ipsilateral or contralateral. Table 3 lists the nine observed combinations by taxa. Only those taxa which used combinations are listed in this table.

From this description of various behaviors within the

Chironomidae, it can be seen that the behavioral patterns present within the family are diverse. Certain behavior, such as immature habitation and feeding and adult position­ ing of the fore legs, generally appears to follow major taxonomic divisions, while in adult grooming the pattern is very complex. Part of this complexity may be due to move­ ments being present in an organism's repertoire but not being observed because of the low grooming frequencies. Table 3* Number of Observed Grooming Combinations

S X X 3 X X 3 3 s s X 3 > 3 X < T3 X 3 3 3 X) x f > XX X) + + + + + + + + + + to m m CO S S X s 2 S s X & 3 3 3 XX X XX <

Chironomini

Chironomus attenuatus 5 3 - 3 - 1 - 9 - -

Chironomus plumosus

Cryptochironomus fulvus

Cryptochironomus sp. 1

Cryptocladopelma galeator

Dicrotendipes fumidus 12- 1------

Dicrotendipes modestus

Dicrotendipes nervosus 42------

Dicrotendipes spp. 1 ------Table 3- Number of Observed Grooming Combinations(cont.)

S X X 3 W rO 3 3 s s X 3 > 3 < 3 3 3 • T> TJ > TJ XX + + + + + + + + + + CO CO CO CO s m X S S SS X 3 3 3 XX X X < < T3

Parachironomus monochromus ______1

Parachironomus tenuicaudatus

Polypedilum digitifer

Xenochironomus xenolabis 2 3 1 3 ------

Tanytarsini

Micropsectra nigripila 1 ------

Orthocladiinae

Corynoneurini

Thienemanniella spp. 2 - - 14 15

Metriocnemini

Smittia sp. 2 123 Table 3. Number of Observed Grooming Combinations(cont.)

SX X 3 : X Xi s s s X 3 > x < X X 35 3 3 X X X X + + + + + + + + + tn CO m to s s S S m § X x X s 3

X X X X X < < XX vWHdWH +

Orthocladiini

Cricotopus bicinctus 2+ - - 2+

Cricotopus sp. 2 1

Cricotopus sp. 3 1

Cricotopus sp. 4 1+2 - 4+

Cricotopus sp. 5 1 + - - 1 +

Nanocladius distinctus 2

Tanypodinae

Coelotanypodini

Clinotanypus pinguis

+ indicates additional combinations during asynchronous grooming.

rv> ANALYSIS OF DATA AND CONCLUSIONS

Selection of Characters

The first step in the analysis of behavioral data is the selection of characters to be used in forming the even­ tual classification. Initially, fifty-two characters were selected for analysis.

Nine characters were selected from the immature behav­ ior patterns and coded only for presence or absence, Table

The first three of these characters were based on the type of larval habitation, including no habitation, forma­ tion of burrows, and construction of tubes. Three more characters were based on feeding behavior, including col­ lecting, construction of leaf-miner nets, and free-living feeding. Only the leaf-miner net building was used because it was the only type of net observed in this study. The use of irrigation was used as the seventh character. Con­ struction of a pupal case when no larval habitation was present and the presence of pupal irrigation were the re­ maining characters selected. Characters were coded "1", if present, and "0 ", if absent.

The remaining forty-three characters came from adult behavior. Ten characters were from positioning of the fore legs, Table 5* Presence of any type of positioning was

125 126

Table 4. Behavior of the Larvae and Pupae

Columns represent: 1. No larval habitation

2. Burrow-former 3. Tube-builder

4. Collector 5* Leaf-mining net-builder

6 . Free-living feeder 7 . Use of larval

irrigation 8 . Construction of prepupal case

9. Use of pupal irrigation Table 4. Behavior of the Larvae and Pupae 127

1 2 3 4 5 6 7 8 9

Chironominae

Chironomini

Chironomus 0 0 1 1 0 0 1 0 1

Cryptochironomus 0 0 1 1 0 1 1 0 1

Cryptocladopelma 0 0 1 1 0 0 1 0 1

Dicrotendipes 0 0 1 1 0 0 1 0 1

Einfeldia 0 0 1

Endochironomus 0 0 1 1 1 0 1 0 1

Glyptotendipes 0 0 1 1 1 0 1 0 1

Microtendipes 0 0 1 1 0 0 1 0 1

Parachironomus 0 0 1 1 0 0 1 0 1

Paralaut erborniella 0 0 1 1 0 0 1 0 1

Polypedilum 0 0 1 1 0 0 1 0 1

Pseudochironomus 0 0 1 1 0 0 1 0 1

S t i ct o ch ir onomus 0 0 1 1 0 0 1 0 1

Tanytarsini

Cladotanytarsus 0 0 1 1 0 0 1 0 1

Lenziella 0 0 1 1 0 0 1 0 1

Micropsectra 0 0 1 1 0 0 1 0 1

Paratanytarsus 0 0 1 1 0 0 1 0 1

Rheotanytarsus 0 0 1 1 0 0 1 0 1

Tanytarsus 0 0 1 1 0 0 1 0 1

Orthocladiinae

Corynoneurini

Corynoneura 1 0 0 0 0 1 0 1 1 Table . Behavior of the Larvae and Pupae(cont.) 128

1 2 3 k 5 6 7 8 9

Thienemanniella 1 0 0 0 0 1 0 1 1

Orthocladiini

Acricotopus 0 0 1 1 0 0 1 0 1

Cricotopus 0 0 1 1 0 0 1 0 1

Eukiefferiella 0 0 1

Nanocladius 0 0 1 1 0 0 1 0 1

Orthocladius 0 0 1 1 0 0 1 0 1

Tanypodinae

Coelotanypodini

Clinotanypus 0 1 0 0 0 1 1 0 0

Coelotanypus 0 1 0 0 0 1 1 0 0

Macropelopiini

Natarsia 1? - - - - 1 - - -

Procladius 0 1 0 0 0 1 1 0 0

Psectrocladius 0 1 0 0 0 1 1 0 0

Pentaneurini

Ablabesmyia 1 0 0 0 0 1 0 0 0

Conchapelopia 1 0 0 0 0 1 0 0 0

Labrundinia 1 0 0 0 0 1 0 0 0

Larsia 1 0 0 0 0 1 0 0 0

Tanypodini

Tanypus 0 1 0 0 0 1 1 0 0 Table 5- Fore Leg Positioning in Adults

Columns represent: 1. Degree of positioning

2. Substrate or Aerial rest position

3. TiTEA rest position b . SFA rest position

5* FFA rest position 6. TiTEA active position

7. SFA active position 8. FFA active position

9. Direction of TiTEA motion 10. Direction of

SFA and FFA motion Table 5» Fore Leg Positioning in Adults

7 8 9 10

Chironominae

Chironomini

Chironomus attenuatus 2 0 0 0 1 1 1 1

Chironomus crassicaudatus 2 0 0 0 0 - - -

Chironomus plumosus 2 0 0 0 0 1 1 1

Chironomus riparius 2 0 0 0 1 1 1 1

Chironomus staegeri 2 - 0 0 - 1 0 1

Cryptochironomus fulvus 2 0 3 2 1 2 1 0

Cryptochironomus ponderosus 2 0 2 2 -- - -

Cryptochironomus sp. 1 2 0 3 2 1 2 1 0

Cryptocladopelma galeator 2 0 3 2 1 2 1 0

Cryptotendipes 2

Demei.jerea brachialis 2 0 0 0 0 1 0 1

Dicrotendipes fumidus 2 0 3 2 1 2 1 0

Dicrotendipes modestus 2 0 2 2 1 1 1 0 130 Dicrotendipes nervosus 2 0 2 2 1 1 1 0 Table 5» Fore Leg Positioning in Adults (cont

1 2 3 4 5 6 7 8 9 10

Einfeldia chelonia 2 1 0 0 0 1 1 1 1 1

Endochironomus nigricans 2 1 0 0 0 0 1 1 1 1

Glyptotendipes lobiferus 2 1 0 0 0 0 1 1 1 1

Glyptotendipes paripes 2 1 0 0 0 1 1 1 1

Harnischia incidata 2 1 0 3 2 1 2 1 1 0

Microchironomus nigrovittus 2 1 0 3 2 1 2 1 1 0

Microtendipes pedellus 2 1 0 0 0 1 1 1 1

Parachironomus monochromus 2 1 0 2 2 1 1 1 1 0

Parachironomus tenuicaudatus 2 1 0 3 2 1 1 1 1 0

Paralauterborniella nigrohalterale 2 1 - 0 0 - - -

Phaenopsectra flavipes 2 1 2 1 1 1 1 1 0 1

Polypedilum illinoense 2 1 - 0 0 1 1 1 - 1

Polypedilum digitifer 2 1 0 0 0 1 1 1 1 1

Stenochironomus hilaris 2 1 3 1 0 0 2 0 0 1

Stenochironomus poecilopterus 2 1 3 1 0 1 2 0 0 1 Table 5- Fore Leg Positioning in Adults (cont

1 2 3 4 5 6 7 8 9 10

Stictochironomus near varius 2 1 0 0 0 1 1 0 1 1

Xenochironomus xenolabis 2 1 0 2 1 1 1 0 1 0

Tanytarsini

Cladotanytarsus sp. 1 2 1 0 2 1 1 1 0 1 0

Lenziella cruscula 2 1 0 2 1 1 1 1 1 0

Micropsectra nigripila 2 1 0 2 1 1 1 0 1 0

Paratanytarsus dissimilis 2 1 0 2 1 1 1 0 1 0

Stempellina 2 1 3

Tanytarsus 2 1 2 1 1 1 1 1 0 0

Orthocladiinae

Corynoneurini

Corynoneura 0 0

Thienemanniella 0 0

Metriocnemini

Smittia 1 0 132 Table 5- Fore Leg Positioning in Adults(cont.)

1 2 3 4 5

Orthocladiini

Acricotopus senex 0 0

Cricotopus bicinctus 2 0 0 3 2

Cricotopus trifasciatus 2 0 - 3 2

Cricotopus sp. 2 2 0 0 3 2

Nanocladius distinctus 2 0

Orthocladius 2 0

Rheorthocladius 2 0

Tanypodinae

Coelotanypodini

Clinotanypus pinguis 0 0

Coelotanypus 0 0

Macropelopiini

Procladius 0 0

Psectrocladius dyari 0 0 133 Table 5* Fore Leg Positioning in Adults (cont)

123^-56789 10

Pentaneurini

Ablabesmyia 0 0

Arctopelopia 0 0

Conchapelopia 0 0

Labrundinia 0 0

Larsia 0 0

Nilotanypus 0 0

Thienemannimyia 0 0

Tanypodini

Tanypus 0 0 135 coded according to the three types observed: "0" for no positioning, "1" for the presence of very rare positioning, and "2" for the presence of very common and frequent use of positioning. The rest position was coded "0" if substrate and "1" if aerial. The TiTEA was coded "0" if < 30° from the BL, "1" if >30° but <60° from the BL, "2" if 2:60° but <90° from the BL, and "3" if >90° from the BL. The

SFA was coded "0" if posterior to the vertical, "1" if

< 30° anterior to the vertical, "2" if > 30° but < 60° an­ terior to the vertical, and "3" if ^60° anterior to the vertical. The FFA was coded "0" if <30° from the verti­ cal, "1" if > 30° but < 60° from the vertical, and "2" if

> 60° from the vertical. The TiTEA, SFA, and FFA each had one character for the rest position and another for the active position. Two additional characters indicated the direction of movement of the fore femora as they went from the rest position to the active position. The direction of

TiTEA motion was coded "0" if motion is toward the body and

"1" if motion is away from the body. The direction of SFA and FFA motion is one character, since both follow the same pattern, and was coded as in TiTEA motion above.

Thirty-three characters come from adult grooming be­ havior. The fifteen basic grooming movements, Table 2, plus asynchronous grooming, the presence or absence of any grooming of the mid legs, and the presence or absence of any ventral wing grooming were coded "1" if present and "0" if absent from a repertoire. Grooming frequencies for all 136 basic movements except T, since it was observed in only one

taxon, and total grooming frequencies were coded "1" for

frequencies > 0 but < 1 movement/hour, "2" when > 1 but

< 1 0 movements/hour, and "3" when >10 movements/hour. If

a particular grooming act had not been observed, a "no code" was used. This prevented the possibility of both the presence-absence and the frequency characters giving double weight in a case where a particular movement may be part of

a taxon's repertoire but was not observed, since the ab­

sence of an observation of a particular grooming movement does not guarantee the absence of that movement from that taxon's repertoire. This is especially important in move­ ments which show a very low frequency of use but may be

important systematically.

Character Analysis

Thirty characters were initially analyzed using product-moment correlation coefficients to determine rela­ tionships between characters. These included all immature characters, presence of fore-leg positioning, aerial vs. substrate rest position, all presence-absence grooming characters, and total grooming frequency. Individual grooming movement frequencies and positioning measurements could not be used because their correlations with "no code" entries resulted in division by zero in calculating certain product-moment correlation coefficients. Product-moment correlation coefficients range from -1.0 to 1.0, with simi­ larity increasing as the coefficient approaches 1.0. This analysis emphasized the one-to-one relationship between the construction of tubes and the collecting feeding behavior.

It was determined, therefore, that collecting, as a feeding behavior, is a function of living in a tube; and was delet­ ed as a character to be used in forming a classification, since it only repeated the tube-building pattern. Likewise, larval irrigation was found to have a -1.0 correlation with lacking habitation. This emphasized the primary function of irrigation as providing an exchange of water in tubes or burrows. It also showed that irrigation correlates with living in a confined space, which is already accounted for by type of habitation. Pupal irrigation showed a similar correlation with living in a confined space. Consequently, both types of irrigation were also deleted from further analysis.

The remaining twenty-seven characters were again ana­ lyzed using product-moment correlation coefficients. Of the 351 correlations produced, Table 6, only 19, or 5*^$ showed correlations beyond the ±0.707 range indicating that at least 50$ of the variation in one variable can be ex­ plained by another. This showed that the characters used in this study measure a number of different behavioral pat­ terns and that their redundancy is low.

The one pattern which did appear from the character correlation is a particularly interesting one. This pat­ tern is the relationship between positioning, specifically the use of the aerial rest position, and chironomid 138 grooming. High positive correlations are found where taxa which use the aerial rest position use FMs and HMs, the rubbing movements where the mid leg steps down on the ipsi- lateral fore or hind legs as the individual begins to walk.

High negative correlations are found where taxa which use the aerial rest position do not use rubbing of the mid legs by the hind legs, MHH or M; and also do not groom the ven­ tral surface of the wings with the hind legs, vWH or v W .

Additional high correlations between the grooming movements mentioned in this pattern also exist.

The significance of this pattern can be speculated on by examining the taxa involved and the description of their grooming. The Chironominae utilize the aerial rest posi­ tion and the mid legs in step grooming of the ipsilateral fore and hind legs. No other chironomids use the aerial rest position, and the only step grooming outside the

Chironominae was observed in Nanocladius distinctus, FMs, and Thienemanniella, HMs. The Chironominae do not groom the mid legs with the hind legs as in most other chirono­ mids . The Chironominae also show no grooming of the ven­ tral wing surface, which is common in the other taxa. In reexamining the other grooming movements found in the

Chironominae, it may be recalled that grooming done with the hind legs was often awkward. In Tanypodinae and

Orthocladiinae, the body is supported equally by the fore and mid legs when grooming is occurring with the hind legs.

In the Chironominae, however, the fore legs primary 139 function appears to be positioning, and they are seldom left on the substrate for any period of time. As the hind legs groom, the fore legs often tap the substrate, but do not maintain contact long enough to provide the necessary support. Consequently, grooming by the hind legs in

Chironominae is more awkward than in other groups, appar­ ently due to the positioning of the fore legs.

While direct observations indicate that grooming by the hind legs in Chironominae may be less efficient than the rest of the family, the correlations of the aerial rest position with the grooming movements of the rest of the family indicate that more drastic changes may have occurred in the coevolution of positioning and grooming in the

Chironominae. The fact that no grooming of the mid legs or ventral wing surfaces was found in this subfamily may indi­ cate that these movements were dropped from the repertoire at some time. While the grooming of the mid legs is con­ ceivably a more difficult movement for the hind legs, the explanation for the absence of ventral wing grooming is less understandable. The Chironominae rub the dorsal wing surfaces with the ipsilateral hind legs, which would appear to be a more difficult movement than the ventral wing rub, since the hind legs must be raised above the wings before being brought down in contact with the dorsal wing surface and extended to rub along the wing. In a ventral wing rub, the hind legs must only be raised into contact with the ven­ tral surface of the wing and extended to rub its surface. 140 Thus, the absence of the ventral wing rub is not as easily explained by the difficulty created by the aerial rest pos­ ition of the fore legs considering the presence of the dor­ sal wing rub in the repertoire of Chironominae.

The absence of mid leg and ventral wing surface rub­ bing by the hind legs in the grooming repertoire of

Chironominae is accompanied by the presence of the two step grooming rubs, FMs and HMs. These two rubs appear to fill part of the grooming gap in Chironominae left by the ab­ sence of the rubs usually performed by the hind legs.

Grooming by the mid legs is not a common movement in midges although its occurrence is probably more frequent than indi cated by the data because of the difficulty in observing it as a specimen walks. It should also be mentioned that the rubbing of the dorsal wing surface by the mid legs, dWM, was observed only in the Chironominae and Thienemanniella.

Thus, the development of the aerial rest position in

Chironominae appears to coincide with the loss of some movements of the hind legs, the awkward nature of the re­ maining rubs by the hind legs, and the increased use of the mid legs in grooming.

In a further analysis of the characters used in this study, principal component analysis (PCA).was used to de­ termine the characters that contribute the most to the major behavioral patterns. Five factors were generated for this analysis, Table 7- The five factors accounted for a total of 72.7°f° of the variation, again indicating that the 141 characters used show a number of behavioral patterns. The individual factors respectively accounted for 3 2 .327S,

1 7 »56$, 8 .67^1 8 .26$, and 5 -89$ of the total variation.

The characters which contributed over 50?o of their variation to the first factor, in order of their importance, included use of the aerial rest position when positioning . the fore legs; use of ventral wing surface grooming, vW; rubbing of the mid legs by the hind legs, M; rubbing a mid leg with both hind legs, MHH; step grooming of the hind leg by a mid leg, HMs; rubbing the ventral wing surface with the ipsilateral hind leg,vWH; step grooming of a fore leg by an ipsilateral mid leg, FMs; the construction of tubes; and the use of fore leg positioning. The construction of tubes is the only character listed above which does not ap­ pear to be involved in the major behavioral pattern involv­ ing the use of the aerial rest position in positioning of the fore legs, although its pattern of variation follows that involving the aerial rest position.

Two characters contributed over 50?° of their variation to the second factor. These were abdominal grooming by the hind legs, Ab, and total grooming frequency. In examining the original data, taxa which do not show Ab also have the lowest grooming frequencies found in the family.

The major character of importance in the third factor proved to be construction of a prepupal case as found in the Corynoneurini. No character contributed more than 50?° of its variation to the fourth factor. The use of T, found Ik2 in Acricotopus senex, was a major contributor to factor f ive .

These factors will be discussed further in the forma­ tion of a classification using PCA, since it will be these major patterns that will be heavily utilized in the con­ struction of this classification.

Positioning measurements could not be analyzed using

character analysis of all OTU's, since the correlations with "no code" entries in OTU's that did not show aerial positioning resulted in division by zero when calculating the correlation coefficients. However, by running only the

Chironominae, the positioning measurements would not cor­ relate with "no code" entries, and a character analysis

could be produced. The initial analysis showed that the direction of TiTEA motion followed the same pattern as the

TiTEA rest position. This indicated that the direction of

TiTEA motion is dependent on the TiTEA rest position.

Since both characters gave the identical pattern, the dir­ ection of TiTEA motion was deleted from further analysis.

Strong correlations remained between the SFA rest position and both the FFA rest position and direction of motion of

SFA and FFA, and between the FFA rest position and direc­ tion of motion of SFA and FFA. However, these characters were maintained since some variation is present between them.

The final character correlation using just the

Chironominae, for the purpose of analyzing the 143 relationships between the positioning characters and vari­ ous other characters, compared seventeen characters which varied within the family, Table 8. Invariate characters within the subfamily were removed for this analysis. Only

3 of the 136 correlations, or 2.2$, showed correlations beyond the ± O .707 range. Thus, the redundancy was lower when positioning measurements were considered along with the other characters.

Analysis of these characters using PCA was also done,

Table 9* Again five factors were generated, accounting for a total of 74.39$ of the variation. The variation ac­ counted for by the factors is very similar to the analysis discussed above.

The characters which contributed over 50$ of their variation to the first factor included the FFA rest posi­ tion, SFA rest position, and the direction of motion of the

SFA and FFA. This indicates the importance of these char­ acters as a major behavioral pattern within the

Chironominae.

Two major contributors to the second factor were the

TiTEA rest position and presence of the palp rub by the fore legs, P. The presence of these two characters in this factor is probably coincidental, since both characters are almost invariant, and it is probably coincidence that one of two OTU's where P was not observed happened to be the only variant OTU with respect to the TiTEA. 144 Factor three had only FMs as its major contributor, while the remaining two factors showed no character con­ tributed over 50% of its variation.

These character analyses indicate, first, that behav­ ioral patterns may be of importance in forming a classifi­ cation, and second, which characters will be used most by the PCA to produce a classification.

Comparison of Taxa

In the first step in forming a classification, product-moment correlation coefficients were generated,

Table 10. These indicate the relative similarity between

OTU's using all characters. A distance matrix was also generated, Table 11. These indicate the distance between

OTU's using all characters. In comparing these two types of matrices with the original data matrix, it was found that less distortion was present in the distance matrix.

For this reason, distance matrices were used for most com­ parisons between OTU's.

In the only analysis using the product-moment correla­ tion matrix, a five factor PCA was generated, Table 12.

The five factors accounted for 6 7 .04$ of the total varia­ tion. The individual factors accounted for 33-32%» 14.60%,

7.51%, 6.78fo, and 4.82% of the variation. This indicated the robustness of the original data matrix. Ideally, a higher percent of the total variation would be desired.

However, the factors obtained here may be compared with the factors obtained in the character analysis discussed above 145 to determine which characters are being used most to sepa­ rate various OTU's. Figure 11 shows a three dimensional plot of the first three factor loadings for each OTU. The third factor is given by a line representing the height from the base . From this plot it can be seen that the first factor has separated the subfamily Chironominae from the Tanypodinae and Orthocladiinae. The characters that were most important in distinguishing the Chironominae from the other two subfamilies can be determined from the most

important characters in the first factor of the character analysis. These characters are the use of the aerial rest position when positioning the fore legs, the absence of vW and M grooming movements, the presence of FMs and HMs, the construction of tubes, and the use of positioning. These

characters together effectively separate the Chironominae from the other subfamilies.

When the second factor is considered in the plot, it can be seen that the subfamilies have been further divided.

The Chironominae begin to show two or three groups. The first group contains the genera Chironomus, Glyptotendipes,

Polypedilum, and Stictochironomus, with Endochironomus being somewhat separated from the main group. This group will be referred to as the Chironomus complex. The rest of the Chironominae appear as one or two groups. The

Tanytars ini appear as a group of three somewhat on the periphery of the main group, which includes the Harnischia

complex. The Tanytarsini are separated from the other 146 K

a5 6y o u

ul<

q S

-0.5 o 0.5

Figure 11. Plot of the first three PCA factors using all OTU's and characters.

Coelotanypodini O—□ Corynoneurini Macropelopiini ■—■ Metriocnemini Pentaneurini Orthocladiini o— o Tanypodini ► Tanytarsini i i Chironomini h— h Chironominae by elements of both the first and second fac­ tors. The remaining group includes the genera

Cr.yptochironomus , Cryptocladopelma, and Parachironomus, which belong to the Harnischia complex, plus the genera

Dicrotendipes and Xenochironomus. This group will be re­ ferred to, hereafter, as the Harnischia complex and will

also include Dicrotendipes and Xenochironomus unless other­ wise stated. From the character analysis, it can be shown

that the characters which distinguish these groups are frequency of grooming, FFA rest position, SFA rest posi­ tion, and the direction of motion of SFA and FFA. The mem­ bers of the Harnischia complex usually have higher grooming frequencies, FFA rest positions are away from the body, SFA rest positions are anterior to the SFA active positions,

and the direction of motion of SFA and FFA is toward the

body. The Chironomus complex usually has lower grooming frequencies, FFA rest positions are closely appressed to the thorax, SFA rest positions are posterior to the SFA active position, and the direction of motion of SFA and FFA

is away from the body. The Tanytarsini tend to follow the

Chironomus complex in having a lower grooming frequency; but their positioning is closer to the Harnischia complex, although their rest positions are not as far from the body; and Tanytarsus shows a TiTEA rest position and movement

opposite most Chironominae. Endochironomus nigricans is separated from the rest of the Chironomus complex by the first factor due to its very reduced grooming repertoire, 148 Table 3« It is interesting that two groups within the

Chironomini appear less similar behaviorally than the other

tribe, Tanytarsini, is to each group.

The Tanypodinae and Orthocladiinae are separated by

factor two. The major character separating these two sub­

families is grooming frequency, although a number of other

characters contribute lesser amounts of variation to this

separation. The Pentaneurini appear as a distinct group

within the Tanypodinae due to their reduced grooming reper­

toire. The other tribes within the Tanypodinae appear as a

group, although the Tanypodini lie at the edge toward the

Pentaneurini and also have a somewhat reduced grooming

repertoire. The Orthocladiinae are spread over a wide area,

indicative of the behavioral diversity present within this

group. The taxa within the separate tribes tend to be

within the same general area, but are not in distinct clus­

ters. The species of Corynoneura appear closest to the

Tanypodinae when considering these two factors. They have

behavioral similarities with Tanypodinae in that they have

no larval habitation and feed while moving about. They

also have no positioning of the fore legs and FH in common with Tanypodinae. These characters contribute some of

their variation to the first two factors. Smittia make up

a group of three OTU's at the high end of factor one and in

the middle of the range of factor two. The Orthocladiini

are distributed over the widest area, with 149 Acricotopus senex and Orthocladius being widely separated from the remaining OTU's.

The only character having a majority of its variation used in the third factor is the construction of a prepupal case. This is found only in Corynoneurini and serves, along with the other variation used in the third factor, to further differentiate the two corynoneurine OTU's from both

Tanypodinae and the remaining Orthocladiinae. The third factor also widens the separation of Pentaneurini and

Tanypodini from the rest of the Tanypodinae and the

Harnischia complex and Tanytarsini from the Chironomus com­ plex within the Chironominae.

Thus, while only 67-04% of the variation is considered after five factors and only 55*43% in the three plotted factors, PCA does separate the OTU's fairly well to the tribe level and, in conjunction with the PCA character analysis, gives a good indication of the most important characters that were used to obtain the clustering in at least the first and second factors. After the second fac­ tor, major separation of OTU's is minimal and the ability to identify important characters within the factors de­ creases .

Another technique that may be used to yield a classi­ fication is multidimensional scaling, MDS. Multidimension­ al scaling is an attempt to represent N objects, in this case the OTU's, geometrically as N points in a set dimen­ sional space, so that the interpoint distances in the set 150 dimensional space correspond to the observed distances between the objects. In this analysis, the distance matrix,

Table 11, is used to produce a multidimensional config­ uration in which the interpoint distances in the config­ uration should correspond to the distances in the distance matrix. A measure of the distortion created when this con­ figuration is generated is given by the stress factor.

Ideally, a configuration should be in as few dimensions as possible to maintain a low stress. Multidimensional scal­

ing uses all the variation present between OTU's, unlike PCA.

A five dimensional MDS was generated from the distance matrix in Table 11. The coordinates are given in Table 13-

The stress was 1^.9°/° at five dimensions. Figure 12 gives a plot of the first three dimensions from Table 13. As in

PCA, the Chironomini are represented by two distinct groups.

In this MDS plot, however, the other tribe, Tanytarsini, lies between the Chironomus and Harnischia complexes. As in PCA, Endochironomus nigricans lies outside the main cluster of the Chironomus complex. Joining E. nigricans outside the main cluster is Stictochironomus. The three taxa of Tanytarsini comprise a scattered group intermediate between the Chironomus and Harnischia complexes in both the second and third dimensions. Xenochironomus xenolabis plots well outside the remaining Harnischia complex cluster in the second dimension, although the first and third dimen­ sions are within the range of the Harnischia complex clus­ ter. Three of the Dicrotendipes OTU's plot higher in the 151

t

ip

Figure 12 Plot of the first three MDS dimensions using all OTU's and characters.

Coelotanypodini □— O Corynoneurini Macropelopiini ■— ■ Metriocnemini Pentaneurini <—> Orthocladiini o— o Tanypodini Tanytarsini i 1 Chironomini 152 third dimension than the remaining Harnischia complex and

Dicrotendipes fumidus.

The Orthocladiinae are distributed over a wide area in the plot, as they were in the PCA. No distinct cluster is present within this subfamily, indicating the diverse be­ havioral patterns within it. Smittia (Metriocnemini) and

Corynoneurini are represented by scattered groups on the periphery between the Orthocladiini and Coelotanypodini

(Tanypodinae) .

MDS separates the Tanypodinae into four distinct clus­ ters that correspond to the four tribes within this sub­ family. The four tribes are differentiated by each of the five dimensions. Within the Macropelopiini, MDS also sep­ arates Psectrotanypus dyari from the Procladius OTU's begin­ ning in the third dimension.

This MDS plot clearly shows that the behavioral pat­ terns in this study tend to follow the present classifica­ tion. The similarity between this plot and that of the PCA is obvious; however, MDS appears to distinguish between existing taxa with more precision than PCA. This may be due to the amount of variation accounted for by PCA being considerably less than that in MDS . In comparing the five factors generated by PCA with the five dimensions in MDS, a correlation coefficient of 0.839 was obtained. Thus, even though PCA accounted for only 6 7 .04$ of the total var­ iation in five factors, the correlation between these two methods indicates the similarity in their final product. Additional analyses using distance matrices were used separately on immature, positioning, and grooming behavior in order to determine what patterns these three behaviors produce.

A phenogram, Figure 13, based on the distance matrix,

Table 14, was produced to show the immature behavioral pat­ tern. Coelotanypodini, Macropelopiini, and Tanypodini can be characterized as burrow-forming, free-living feeders.

Pentaneurini and Corynoneurini build no larval habitation and are free-living feeders, while Corynoneurini construct pupal cases as prepupae. The remaining immatures construct larval tubes, including Cryptochironomus which contains free-living feeders and Endochironomus and Glyptotendipes which construct leaf-miner nets while feeding. Tanypodinae can be distinguished from the other two subfamilies using the behavior of the immatures, although the Corynoneurini are divergent from the remaining Orthocladiinae and are grouped with the Pentaneurini because both build no larval habitation. The Orthocladiini cannot be separated from the

Chironominae with these behavioral characters.

A second phenogram, Figure 14, based on the distance matrix, Table 15, shows the pattern of fore leg position­ ing behavior in adults. The Tanypodinae, Corynoneurini and

Acricotopus senex are characterized by the absence of ob­ served positioning. Smittia use a slight positioning on rare occasions. All other chironomids use frequent posi­ tioning. The remaining Orthocladiinae use the substrate Coelotanypodini

------Macropelopiini

Tanypodini

—— ------Pentaneurini

----- — ------Corynoneurini

Orthocladiini

Chironomus

Cryptocladopelma

Dicrotendipes

Microtendipes

------— ------Parachironomus

Paralauterborniella

Polypedilum

r ---- Pseudo chironomus

Stictochironomus

Tanytarsini

------Cryptochironomus

Endochironomus

Glyptotendipes

0.75 0-50 0.25 0

Figure 13- Phenogram showing distances in the behavior of immatures. Correlation with the distance matrix in Table 1^- is O.99I. Based on UPGMA cluster analysis. 155

Tanypodinae ------Corynoneurini ------ACRSEN ------Smittia ------Orthocladiini CRYPTO CRYGAL P ----DICFUM XENXEN ____MICNIG ----HARINC ------PARACH DICMOD ------DICNER — ---- DICROT ------LENCRU CLADGT ------MICROP ---- PARATA ______TANYTA ______PHAFLA ------Stenochironomus CHIATT CHIRIP CHIRON ---- GLYPTO POLYPE r ------EINCHE CHIPLU ENDNIG ------GLYLOB MICPED ______GLYPAR I ---- STICTO I------DEMBRA

1.5 1.0 0.5 0

Figure 14. Phenogram showing distances in adult fore leg positioning behavior. Correlation with the distance matrix in Table 15 is 0.897* Based on UPGMA cluster analysis. rest position, although Cricotopus may hold their fore legs aerial for long periods of time and have a similar motion pattern to that of the Harnischia complex. The

Chironominae use the aerial rest position of the fore legs.

This aerial rest position correlates with the higher fore leg ratio found- in Chironominae. The fore first tarsomere is elongated in these. The Harnischia complex and

Tanytarsini, except for Tanytarsus, form another group based on their SFA and FFA rest position being away from the body with movement toward the body when active. Again, three Dicrotendipes OTU's separate from the remaining

Harnischia complex and Dicrotendipes fumidus, and show similarities to the Tanytarsini in that both the rest and active SFA's are closer to the vertical. The Chironomus complex forms another distinct group based on SFA and FFA rest position being up against the body with movement away from the body when active. Tanytarsus and Phaenopsectra flavipes form a side group within the Chironominae based on their TiTEA rest position, and Stenochironomus forms another side group based on extreme TiTEA rest position.

Thus, positioning of the fore legs can be useful not only at the subfamily level, but also in determining relation­ ships below the tribe level.

Figure 15 represents a plot of the first two dimen­ sions of a three dimensional MDS analysis of grooming be­ havior in adult Chironomidae from data in Table 17* The first dimension separates the Chironominae from the 157

& CONCHA

& ABLMAL ^TANSTE + E N D N 1G

T tanyta c l a d o t T O n'tRopT + C H IR 1 P

apROBEL STICTO + ■ PROSu.fi 6 LYLO0+ J 2 ^ ° La CCORYNOps!LDtAB«PROCU? GLYPAR + -4- + CRYPTO Polype 4 - , 4-c h ia t t CRY6 A L + t CR1R0N QCOtLOT D\CFUM

CTHlENE »SM

+ DtCRoT (S M IT T 1 QORTHOC D1CM004- +WCNE(,

a o c r .? rC.r , s p 3 •o OCRlBlt I OCRISPiT ONANDIS QCR'COT X E N X E H f

OACRSEN CRISPH O

-1 .0 •0.5 0.5

Figure 15* Plot of the first two MDS dimensions using all OTU's and grooming characters.

Coelotanypodini □ Corynoneurini c Macropelopiini ■ Metriocnemini • Pentaneurini A Orthocladiini o Tanypodini ▲ Tanytarsini T Chironomini +

* 158 Orthocladiinae and Tanypodinae. The second dimension scat­ ters the Chironominae without clustering the three groups discussed above, although each group tends to occupy a dis­ tinct area within the entire cluster. The first dimension appears to utilize the presence of grooming movements, while the second appears to correspond to grooming frequen­ cies. This is similar to the variation accounted for in the first two factors of PCA, Figure 11. Xenochironomus xenolabis occupies an isolated point at the lower right be­ cause of its high frequency of grooming for Chironominae.

Another group occupying an isolated cluster because of high er grooming frequencies is formed by three of the four

Dicrotendipes OTU's. Dicrotendipes fumidus lies with

Cryptochironomus and Cryptocladopelma galeator adjacent to the Chironomus complex. Townes (19^5) considered

D. fumidus belonging to a different group of Dicrotendipes than D. modestus and D. nervosus. This may explain the separation of D. fumidus from the cluster formed by the re­ maining Dicrotendipes OTU's, and indicates that this type of analysis may yield evidence as to subgeneric relation­ ships. Endochironomus nigricans is represented by an iso­ lated point above the rest of the Chironominae because of its reduced grooming repertoire. Grooming frequency differ ences appear to be the reason Xenochironomus xenolabis and

Endochironomus nigricans appear as isolated points in

Figure 12. 159 The second dimension also separates the Orthocladiinae and Tanypodinae, although this separation is not distinct since the Corynoneurini, Metriocnemini, and Coelotanypodini overlap between the two subfamilies, as the Corynoneurini do with behavior in the immatures and adult positioning.

Clinotanypua pinguis lies well within the Orthocladiinae due to having higher grooming frequencies than other

Tanypodinae. The Macropelopiini form a distinct cluster based on grooming behavior; and the third dimension will separate the three Procladius OTU's from Psectrocladius dyari, indicating the value of grooming behavior to separ­ ate genera. Pentaneurini and Tanypodini, like

Endochironomus nigricans, occupy isolated points due to their reduced grooming. It is interesting that reduced grooming was observed in a variety of taxa. The

Pentaneurini and Tanypodini represent entire tribes that show reduced grooming, while the other two tribes within the Tanypodinae show average to moderate grooming. The

Pentaneurini and Tanypodini tend to have longer legs in re­ lation to body size than most other chironomids, and this may account for some of the grooming reduction. Within the

Chironominae and Orthocladiinae individual species can be found with reduced grooming, such as Endochironomus nigricans. The significance of this reduction in grooming is not known. Discussion of Behavioral Relationships of Taxa 160

The Chironominae are clearly behaviorally distinct

from the Orthocladiinae and Tanypodinae. The Chironominae

are distinguished from the Orthocladiinae by the habit of

keeping the fore legs aerial at rest, the absence of groom­

ing of the mid legs by the hind legs, the absence of ven­

tral wing grooming, the more extensive use of the mid leg

in the two step grooming movements (FMs and HMs), and gen­

erally lower grooming frequencies. The Chironominae are

distinguished from the Tanypodinae by the construction of

larval tubes with their associated feeding by collecting,

the use of fore leg positioning, and the same presence or

absence of grooming movements that distinguish them from

the Orthocladiinae. Cryptochironomus, however, appear to

have converged toward the tanypodine type of feeding in

that they are free-living while searching for food, possi­

bly because of their predacious feeding, which is similar

to most of the Tanypodinae.

Within the Chironominae three behavioral groups are

indicated. The tribe Tanytarsini appears intermediate be­

tween the two groups comprising the Chironomini when all

behavioral characters are considered. Positioning behavior

is closest to the Hamischia complex in that the SFA and

FFA are held away from the body at rest, and motion is to­ ward the body as they become more active; although, again

like the Harnischia complex, the Tanytarsini are more gen­

erally active. Tanytarsus positioning is most similar to 161 Phaenopsectra flavipes because of their TiTEA rest position

being almost perpendicular to the B L . This, along with

their reduced SFA and FFA movement, results in Tanytarsus

and Phaenopsectra being placed closest to the Chironomus

complex in Figure 14. The one Stempellina observed showed a TiTEA rest position of 135° from the BL, which would be more extreme than in Tanytarsus and similar to that observ­

ed in Stenochironomus. This may show additional similarity between some of the Tanytarsini and certain members of the

Chironomus complex. Until additional material can be ob­ served, the full extent of this relationship is unclear.

Thus, positioning behavior within the Tanytarsini appears variable, and different taxa show similarity to each of the two complexes within the Chironomini, indicating that the

Tanytarsini may occupy an intermediate position between the two complexes with respect to positioning behavior. In grooming behavior the Tanytarsini show a reduced grooming repertoire and grooming frequency. Only Endochironomus nigricans shows more reduced grooming within the

Chironominae. Thus, the Tanytarsini occupy a position closer to the Chironomus complex and at one end of the

Chironominae with regards to grooming behavior.

The members of the Chironomus complex appear distinct from the other groups within the Chironominae. They differ from the Harnischia complex primarily in that the SFA and

FFA rest position is posterior to the vertical and against the body, with the direction of motion being away from the 162 body as activity increases. They also tend to show lower grooming frequencies, although there is some overlap with the Harnischia complex. This group includes the genera

Chironomus , Demei.jerea, Einf eldia, Endochironomus ,

Glyptotendipes, Microtendipes, Polypedilum, and

Stictochironomus. Paralauterborniella appears to belong here, based on incomplete positioning measurements, al­ though the grooming from the one individual that did groom had a higher frequency than is usually found in the complex.

The genera Phaenopsectra and Stenochironomus may also be­ long here, although their positioning behavior appears in­ termediate between some of the Tanytarsini.

The Harnischia complex may be characterized by having the SFA and FFA rest position away from the body anteriorly with the direction of motion being toward the body as ac­ tivity increases. This group includes the usual Harnischia complex genera:Cryptochironomus, Cryptocladopelma,

Harnischia, Microchironomus, and Parachironomus.

Cryptotendipes and Paracladopelma are also Harnischia com­ plex genera (Saether, 1971), although their positioning be­ havior was not determined adequately in this study to show that they follow the regular pattern. For the purposes of this study, Dicrotendipes and Xenochironomus are also in­ cluded in the Harnischia complex. Similarities between these two genera and the usual usage of the Harnischia com­ plex are indicated in the literature. Beck and Beck (I969) mentioned that this complex has many characters in common with Dicrotendipes immatures, but do not elaborate.

Saether (1977) gave a key to female Chironomidae, in which

Xenochironomus falls in the usual Harnischia complex, al­

though Dicrotendipes falls in the Chironomus complex.

Freeman (1956) considered Xenochironomus morphologically

intermediate between Dicrotendipes and the .Harnischia com­

plex. The possibility exists that the Harnischia complex

should be considered a separate tribe from the remaining

Chironomini.

The Chironominae are usually considered derived from

the Orthocladiinae (Brundin, 1956; Schlee, 1968a). Brundin

(1956) placed their origins within the Orthocladiini. This

could explain the well-developed use of positioning in

Orthocladiini and Chironominae. Brundin (1966) later

placed the origin of the Chironominae much earlier than

this, as the apomorph sister group of a group comprising

Diamesinae + Telmatogetoninae + Orthocladiinae. This view

of the origin of the Chironominae being before that of the

most primitive Orthocladiinae was also advocated by

Lindeberg (1962). While the distance between the

Orthocladiinae and Chironominae is distinct in all behav­

ioral plots, the behavioral similarity is greatest between

the Chironominae and Orthocladiini, owing to their similar­

ity in immature behavior and use of positioning. As dis­

cussed earlier in this study, the primary differences be­

tween these two groups may be due to the establishment of

the aerial rest position, which causes modification in 164 grooming behavior, resulting in the distinct behavioral gap between the Chironominae and Orthocladiini.

The Orthocladiinae may be behaviorally distinguished from the Tanypodinae primarily in having higher grooming frequencies. Many other characters may be found in both subfamilies, especially between the Tanypodinae and

Corynoneurini or Metriocnemini. Because of this sharing of many characters and the behavioral diversity within the sub­ family, each tribe will be discussed separately. It might be mentioned that Hamilton et. a l . (1969) did not divide the Orthocladiinae into tribes because of the confusion created by the morphological diversity that exists within the subfamily.

Although the Corynoneurini have high grooming frequen­ cies, they possess a number of characters which are also found in other taxa. Their behavioral similarity with the

Tanypodinae can be seen in their larvae building no habita­ tion, as in Pentaneurini; being free-living feeders; the absence of positioning in the adults; and the use of FH in

Corynoneura. Thienemanniella shows the grooming movements

HMs and dWM in common with the Chironominae. A comparison of grooming movements between Corynoneura and

Thienemanniella reveals distinct differences. This diver­ sity can also be seen in morphology and has led to this group being placed differently in various classifications.

Edwards (1929) placed Corynoneura, with Thienemanniella as a subgenus, in the Orthocladiinae. Goetghebuer (1932) considered them a subfamily, as did Freemen (1956).

Brundin (1956) considered the Corynoneura group the most derived of Orthocladiinae and suggested that they be a sis­ ter group of Pseudosmittia (Metriocnemini). Lindeberg

(1962) stated that the Corynoneura group being considered a subfamily was not entirely unwarranted, although they could be included as a tribe in Tanypodinae based on his study of the abdominal spiracles in Chironomidae. Schlee

(1968b) considered the Corynoneura group belonging to the

Metriocnemini and rejected its separation as a subfamily.

The behavioral evidence from this study indicates that the

Corynoneurini not only show characters intermediate between

Orthocladiinae and Tanypodinae, but also show behavior com­ mon to the Chironominae. It is, therefore, possible that the Corynoneurini are derived from primitive, unknown an­ cestors before or near the divergence of the ancestors of the three subfamilies studied here. If so, their separa­ tion as a subfamily is partly justified.

The Metriocnemini are represented in this study only by Limnoohyes hudsoni, for which insufficient behavioral data was obtained, and Smittia. Smittia, like the

Corynoneurini discussed above, are usually behaviorally intermediate between the Tanypodinae and Orthocladiini.

While their grooming frequencies and most movements appear like the Orthocladiini, they possess only a rare use of slight fore leg positioning and have the use of vWp in com­ mon with the Coelotanypodini. 166 The Orthocladiini are a behaviorally diverse group, as

shown by their scattered distribution in Figure 12. As dis

cussed under Chironominae, the Orthocladiini show similar

behavioral patterns to the Chironominae in behavior of im-

matures and well-developed fore leg positioning. The dis­

similarities appear to correlate with the development of

the aerial rest position in the Chironominae. However, the

Orthocladiini, or their ancestors, cannot be ruled out as

the origin of the Chironominae. The lone Eukiefferiella

and Rheocricotopus observed in this study conform to the

general Orthocladiini behavioral patterns where data is

present.

The Tanypodinae, although showing some behavioral

similarity to certain of the Corynoneurini and Smittia, can

be distinguished behaviorally by a variety of characters

including free-living feeding behavior, absence of fore leg

positioning, and a lower grooming frequency. The

Coelotanypodini show the most similarity to the

Orthocladiinae with their higher grooming frequencies than

other Tanypodini, and the presence of FH, as in Corynoneura

and vWp, as in Smittia. The most reduced grooming reper­

toire found in Chironomidae is in the Pentaneurini. Most

specimens from this tribe showed no grooming during the

usual observation period, and the only rubbing observed was

An, Ab, and occasional rubbing of the substrate. Schlee

(1968a) considered the Tanypodinae to be the sister group

of the Orthocladiinae plus Chironominae. While some 167 behavioral similarity is evident between the Tanypodinae

and Orthocladiinae, little similarity exists between the

Tanypodinae and Chironominae. This indicates that the

origin of the Tanypodinae was either before that of the

Chironominae, as indicated by Schlee, or from different

Orthocladiinae stock, since the Orthocladiinae show very

diverse behavioral patterns.

Significance of this Study

This study clearly shows that behavioral characters from not only adult Chironomidae, but also from the imma-

tures, have a place in determining the systematics of this family. As was expected, not all characters are of equal

importance, and characters are not always important at the

same taxonomic level.

Evidence from this study illustrates how the evolution

of one type of behavior may result in changes to another.

For example, the development of the aerial rest position of the fore legs has resulted in grooming with the hind legs being awkward, the possible loss of other grooming by the hind legs, and the increased usage of grooming by the mid legs as adults begin to walk.

Behavioral characters indicate that two distinct be­ havioral groups exist within the tribe Chironomini. The

Tanytarsini, the other tribe in the Chironominae, actually show an intermediate position between these two groups based on behavioral characters of grooming and fore leg positioning. The separation of these two groups into two 1 6 8 tribes is a possibility that needs further consideration.

Otherwise, behavioral evidence closely parallels existing classifications. Thus, in Chironomidae, behavioral char­ acters show promise in forming or checking classifications.

Behavioral evidence may be used to speculate on the evolutionary origins of taxa. Only when a variety of evi­ dence from different studies jointly point to a certain origin, can we be more confident of these origins. Behav­ ioral patterns may shed light on such groups as the

Corynoneura group discussed above.

Thus, the use of behavior in systematic studies can yield a variety of information about taxa and can and should be used with other data to more completely analyze these taxa. REFERENCES

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(Latreille) with a classification of self-grooming

movements in ants. Bulletin of the Museum of

Comparative Zoology 127 (7): 401-422. 192 Wing, L. 1946. Drumming flight in the blue grouse and

courtship characters of the Tetraonidae. Condor

48 (4): 154-159. APPENDIX A: ADDITIONAL TABLES

193 Table 6. Product-moment Correlation Coefficients for Characters

Rows and Columns represent: 1. No larval habitation

2. Burrow-former 3* Tube-builder 4. Leaf-mining net-builder

5. Free-living feeder 6. Construction of prepupal case

7. Degree of positioning 8. Substrate or Aerial rest position

9. An 10. P 11. T 12. FMs 13. FH 14. MH 15. KHH

16. MH or MHH present 17. HMs 18. HH 19. Ab 20. dWM

21. dWH 22. vWH 23. dvW 24. vWp 25. vW

26. Asynchronous grooming 27. Total grooming frequency Table 6. Product-moment Correlation Coefficients for Characters

1 2 3 4 5 6 7 10 11 1? 13 14

1 1 .000 2 -0.163 1 .000 3 -0.537 -o .7kk 1 .000 4 -0.118 -0.163 0.219 1 .000 5 0.505 0.699 -0.940 -0.233 1 .000 6 0.687 -0.112 -0.369 -0.081 0.347 1 .000 7 -0.505 -0.699 0.940 0.233 -0.878 -0.347 1 .000 8 -0.362 -0.501 0.673 0.325 -0.603 -0.248 0.663 1 .000 9 -0.479 0.078 0.258 0.056 -0.242 0.039 0.239 0.146 1 .000 10 -0 .279 -0.12k 0.295 0.163 -0 .261 0.112 0 .401 0.167 0.351 1 .000 11 -0.056 -0.078 0.105 -0.056 -0.112 -0.039 -0.239 -0 .146 0.023 -0.351 1 .000 12 -0.293 -0.405 0.544 0.055 -0.465 -0.201 0.550 0-737 0.121 0.218 -0.121 1 .000 13 0.218 0.364 -0.459 -0 .100 0.431 0-368 -0.424 -0.258 0.041 0.118 -0.041 -0.215 1.000 14 -0.191 0.534 -0.327 -0.191 0.287 -0.131 -0.413 -0.485 0.077 -0.088 0.301 -0.402 0.310 1 .000 15 0.094 0.481 -0.475 -0.262 0.420 0.309 -O.38I -0.833 0.133 0.254 -0.133 -0.598 0.310 0.468 16 0 .07k 0.450 -0.434 -0.277 0.378 0.292 -0.450 -0.872 0.139 0.147 0.167 -0.631 0.296 0.555 17 -0.171 -0.475 0.522 -0 .000-0.453 -0.000 0.530 0.864 0.139 0.147 -0.119 0.682 -0.247 -0.463 18 -0.536 0.139 0 .243 0.100 -0.221 0.069 0 .220 0 .078 0.564 0.375 o.o4i 0.215 0.073 0.137 19 -0.322 -0.167 0.360 -0.087 -0.327 0.102 0.323 0.115 0.384 0.370 0.061 0.315 0.107 0 .201 20 0.162 -0.163 0.030 -0.118 -0.049 0.303 0.086 0.231 0.055 O.I56 -0.055 0.304 -0.097 -0.182 21 -0.322 0.206 0.042 -0.087 -0.016 0.102 0.024 -0.151 0.384 0.370 0.061 0.043 0.107 0.201 22 -0.08k ■ 0.481 -0.355 -0.262 0.303 0.064 -0.363 -0.795 0.127 0.109 0.183 -0.565 0.325 0.495 23 0.087 O.I67 -0.201 -0.149 0.172 0.221 -0.114 -0.518 O.O83 0.236 -0.083 -0.319 -0.147 0.128 24 -0.081 0.496 -0.369 -0 .081 0.347 -0.056 -0.317 -0.302 0.048 -0.079 -0.048 -0.251 0.228 0.232 25 0.055 0.557 -0.513 -0.293 0.453 0.276 -0.520 -0.913 0.146 0.042 0.160 -0.665 0.283 0.531 26 -0.081 -0.112 0.150 -0.081 -0 .I60 -0.056 0.219 -0.302 0.048 0.138 -0.048 -0.251 -0.086 0.036 27 -0.130 -0.180 0 .242 -0.267 -0.257 0.288 0.169 -0.297 0.321 0.233 0.153 -0.131 -0.003 0.171

15 16 17 18 19 20 21 22 23 24 25 26 27

15 1.000 16 0.955 1 .000 17 -0 .70k -0.742 1 .000 18 0.236 0.247 0.066 1 .000 19 0.213 0.230 0.230 0.418 1 .000 20 -0.168 -0.183 0.392 0.097 0.142 1 .000 21 0.346 0.363 -O.036 0.418 0.614 0.142 1 .000 22 0.768 O.8I9 -O.76O 0.225 0.061 -0.298 0.331 1 .000 23 0.622 0.594 -O.386 0.147 0.057 -0.023 0.215 0.431 1 .000 24 0.363 0.346 -0.289 0 .086 0.126 -0.113 0.126 0.210 0.017 1 .000 0.912 -0.781 25 0.955 0 .258 0.115 -0.199 O.38O 0.871 0.568 0.331 1 .000 195 26 0.363 0.346 -0.289 0 .086 0.126 -0.113 0.126 0.380 0.394 -0.100 0.331 1 .000 27 0.541 0.586 -0.130 0.427 0 .628 0.033 0.524 0.351 O.39I 0.203 0.488 0.32? 1 .000 196

Table 7. Principal Component Analysis of Characters

Rows represent: 1. No larval habitation 2. Burrow- former 3* Tube-builder 4. Leaf-mining net-builder

5. Free-living feeder 6. Construction of prepupal case 7- Degree of positioning 8. Substrate or

Aerial rest position 9* An 10. P 11. T

12. FMs 13* FH 14. MH 15- MHH 16. MH or

MHH present 17. HMs 18. HH 19* Ab 20. dWM

21. dWH 22. vWH 23- dvW 24. vWp 25. vW

26. Asynchronous grooming 27. Total grooming frequency Column 6 represents total variation accounted for by all five factors for each character. 197 Table 7. Principal Component Analysis of Characters

Factor 1 Factor 2 Factor 3 Factor 4 Factor 5 6

1 0 .246 -0.623 0.637 -0.273 0.099 0.9378 2 0.674 -0.162 -0.318 0.529 -0.210 0.9049 3 -0.742 0.560 -0.159 -0.267 0.113 0.9736 4 -0.303 -0.013 -0.180 -0.004 -0.379 0.2674 5 0.681 -0 .544 0.167 0.298 -0.120 0.8917 6 0.297 -0 .068 0.853 0 .014 0.086 0.8283 7 -0.731 0.545 -0.065 -0.264 -0.179 0.9377 8 -0.949 0.058 -0.002 0.240 -0.058 0.9651 9 -0 .020 0.619 -0.021 0.337 -0.066 0.5015 10 -0.079 0.589 0.276 0.109 -0.510 0.7018 11 0.106 0.038 -0.259 -0.081 0.805 0.73^5 12 -0.745 0.195 0.125 0.282 0 . 028 0.6899 13 0.424 -0.102 0.197 0.451 -0.038 0.4339 14 0.583 0.135 -0.366 0 .240 0.275 0.6256 15 0.880 0.338 0.127 -0.092 -0.192 0.9500 16 0.905 0.350 0.047 -0.118 0.051 0.9608 17 -0.820 0.072 0.28 7 0 .261 0 .101 0.8391 18 0.055 0.692 -0.014 0 .346 -0.067 0.6071 19 -0.034 0.736 0 .216 0.248 0 .262 0.7197 20 -0.230 0.047 0.582 0.206 0.136 0.4550 21 0.246 0.635 0 .147 0.311 0.094 0.5909 22 0.811 0.296 -0.198 -0.125 -0.016 0.8000 23 0.501 0.309 0.199 -0.390 -0.243 0.5977 24 0.418 0.014 -0.160 0 .394 -0.044 0 .3580 25 0.937 0.275 -0 .014 -0.082 0.030 0.9619 26 0.229 0.383 -0.050 -0.585 -0.160 0 .5704 27 O .303 0.733 0.262 -0.176 0.308 0.8239

Percent Accumulative Eigenvalue of Trace Percent

Factor 1 8 .72710 32.32 32.32 Factor 2 4.73998 17.56 49 .88 Factor 3 2.33955 8.67 58 .5^ Factor 4 2.23078 8.26 66.81 Factor 5 1 .59071 5-89 72.70 Table 8. Product-moment Correlation Coefficients for Chironominae Characters

Rows and Columns represent: 1. Leaf-miner net-builder 2. Free-living feeder

3. TiTEA rest position U-. SFA rest position 5« FFA rest position

6. TiTEA active position 7- SFA active position 8. FFA active position

9. Direction of SFA and FFA motion 10. P 11. FMs 12. KMs 13* HH

1^. Ab 15- dWM 16. dWH 17* Total grooming frequency Table 8. Product-moment Correlation Coefficients for Chironominae Characters

1 2 3 4 5

1 1 .000 2 -0.115 1 .000 3 -0.115 -0.053 1 .000 4 -o .465 0.343 -0.042 1 .000 5 -0.467 0.290 0.023 0.957 1 .000 6 -0.490 0.096 0.091 0.398 0.401 1 .000 7 -0 .210 0.546 -0 .108 0.674 0.577 0 .198 1 .000 8 -0.062 0.115 0 .108 0.091 0.213 -0.198 0.235 1 .000 9 0.500 -0.229 -0.213 -0.930 -0.937 -0.428 -0.462 -0.023 1.000 10 0.167 0.076 -0.689 -0.067 -0.210 -0.132 0.157 -0.157 0.309 11 -0.289 0.132 0.125 0.017 0.064 0.411 0.271 0.298 0.085 12 -0.667 0.076 0.073 0.317 0.319 0.331 0.157 0.256 -0.340 13 0.115 0.053 0.050 -0.134 -0.023 -0.091 0.108 0 .461 0.213 14 -0.250 0.076 0.073 0.061 0.143 0.331 0.157 0.256 -0.015 15 -0.210 -0.096 -0.108 0.100 0.050 0.198 0.074 0.235 0.023 16 -0.062 0.115 -0.461 0.187 0.213 0 .149 0.235 0.074 -0.023 17 -o.4oi -0.053 -0.068 0.425 0.586 0 .417 0.111 0.149 -0.496

10 11 12 13 14 15 16 1-7 10 1.000 11 0.200 1 .000 12 -0.105 0.200 1 .000 13 -0.073 0.400 -0.073 1 .000 14 -0.105 O.58O 0.447 -0.073 1 .000 15 0.157 0.271 0.157 0.108 0.157 1.000 16 0.256 0.298 0.256 -0.108 0.669 0.235 1.000 -0.248 17 0.171 0.447 0.068 0 .447 0.111 0.408 1.000 199 200

Table 9- Principal Component Analysis of Chironominae Characters

Rows represent: 1. Leaf-miner net-builder 2. Free- living feeder 3* TiTEA rest position 4. SFA rest position 5. FFA rest position 6. TiTEA active position 7« SFA active position 8. FFA active position 9* Direction of SFA and FFA motion 10. P

11. FMs 12. HMs 13- HH 14. Ab 15- dWM

16. dWH 17- Total grooming frequency

Column 6 represents total variation accounted for by all five factors for each character. 201 T a b le 9 . Principal Component Analysis of Chironominae C h a r a c te r s

Factor 1 Factor 2 Factor 3 Factor 4 Factor

1 0.689 -0.110 0.086 -0.252 0.092 0.5665 2 -0.334 -0.136 0.194 -0.513 -0.438 0.6223 3 -0.089 0.915 -0.251 -0.062 -0.186 0.9468 4 -0.843 -0.024 0.466 -0.149 0 .045 0.9526 5 -0.877 0.052 0.347 -0.143 0.193 0 ..9496 6 -0.611 0.037 -0.106 0.322 -0.375 0.6297 7 -0.596 -0.233 0 .202 -0 .548 -0 .240 0.8081 8 -0.248 0 .028 -0.454 -0.556 0.458 0.7873 9 0.815 -0.273 -0.468 -0.011 -0.086 0.9642 10 0.206 -0.798 0.104 -0.089 -0.244 0.7577 11 -0.350 -0.175 -0.706 -0.214 -0.382 0.8425 12 -0.614 0 .001 -0.270 0.268 0.095 0.5303 13 0.033 0.020 -0.434 -0.630 0.282 0.6669 14 -0.469 -0.190 -0.639 0.234 -0.143 0.7392 15 -0.208 -0.261 -0.352 0 .008 0.127 0.2511 16 -0.372 -0 .662 -0.258 0.206 0.045 0.6879 17 -0 .674 -0.068 -0.140 0.298 0 .418 0 .7418

Percent Accumulative Eigenvalue of Trace Percent

Factor 1 4.93227 27-40 27.40 Factor 2 3.05494 16.97 44.37 Factor 3 2.37393 13.19 57-56 Factor 4 1.79795 9-99 67-55 Factor 5 1.23201 6.84 74.39 ^snxw^-nQcr-mccocoooon n h3oC'iwM020ooonn>“ -3n>t3i3‘Tjno OTU’ for Profluct-momrnt Coefficients Correlation 10. Tablo >Mrn>-3o>r!rr2HMHH3)3Xs: = s:2:o322:o>3o»ro:«5o:oo:.*>oco5o:c;oot"' 2C2>!Zl-1tr,a3'-PlKHMMirnMMMH3CD«C:302ncBWNl KXDXnK>'0“r25323Tn(T3a3n3*-a OOPl^tin^XDHQCilOOHOHr H22H*t3‘T30HQ,T;'m^HCrK^2:>rrnCQH >-uHzowiox®aH»a2cHo z ,iic puj H02>m>^>r,wH2

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202 Table 10. Froduct-moment Correlation Coefficients for OTU's (cont.)

CRISP4 CRTSP5 CRICOT NANDIS ORTHOC SMISP1 SMISP2 SMITTI CORYNO THIENE CH1ATT CHI FLU CHIRIP

CRISP4 1 .000 CRISP5 0.788 1 .000 CRICOT 0.772 0.648 1 .000 NANDIS 0.557 0.423 0.546 1 .000 ORTHOC 0.009 0.003 0.418 0.294 1.000 SM1SP1 0 0 6 9 0.436 0.375 0.451 0.266 1 .000 SMISP2 0.186 0.294 0.188 0.241 0.201 0.395 1 .000 SMITTI 0.190 0.104 0.252 0.392 0.358 0.560 0.717 1 .000 CORYNO 0.020 0.117 -0.106 -0.038 0.103 0.219 0.377 0.281 1 .000 TII1ENE 0.120 0.162 -0.052 -0.035 -0.174 0.555 0.062 O.I96 0.492 1 .000 c m ATT -0.535 -0 .604 -0.533 -0.327 -0.251 -0 .440 -0.528 -0.469 -O.398 -0.072 1 .000 CHIPLU -0.519 -0.612 -0.459 -0.281 -0.230 -0.459 -0.572 -0.481 -0.433 -0.345 0.643 1.000 CHIRIP -0.513 -0.458 -0.453 -0.246 -0.323 -0.452 -0.343 -0.424 -0.302 -0.270 0.588 0.557 1 .000 CHIRON -0.535 -0.604 -0.533 -0.327 -0.251 -0.440 -0.528 -0.469 -O.398 -0.072 1 .000 0.643 0.588 CRYPTO -0.33(i -0.410 -0.275 -0.325 -0.388 -0.542 -0 .489 -0.567 -O.276 -0.313 0.274 0.303 0.248 CRYUAL-0.430 -O.568 -0.178 -0.208 -0.219 -0.358 -0.500 -0.408 -O.389 -0.364 0.351 0.368 0.305 DICFUM -0.519 -0.612 -0.223 -0.281 -0.230 -0.459 -0.572 -0.481 -0.433 -0.244 0.323 0.344 0.286 D1CM0D -0.038 -0.098 -0.084 -0.140 -0.157 -O.269 -0 .249 -0.307 -O.219 -0.159 0.091 0.082 0.017 D1CNER -0.333 -0.395 -0.176 -0.020 -0.206 -0.354 -0.500 -0.427 -0.387 -0.150 0.288 0.321 0.264 DICROT -0.258 -0.320 -0.233 -0.089 -0.195 -0.346 -0.343 -0.421 -0.289 -0.292 O .307 0.338 0.279 ENDNIG -0.233 -0.192 -0.276 -0.323 -0.255 -0.357 -0.208 -O.256 -0.191 -0.262 0.194 0.400 0.440 GLYLOB -0.385 -0.486 -0.398 -0.441 -0.160 -0.351 -0.481 -0.370 -0.339 -0.277 0.428 0.735 0.352 GLYPAR -0.365 -0.462 -0.40? -0.210 -0.147 -0.327 -0.456 -0.348 -0.329 -0.377 0.445 0.432 0.372 GLYFTO -0.519 -0.612 -0.527 -0.323 -0.247 -0.459 -0.572 -0.481 -0.392 -0.346 0.615 0.636 0.582 PARACH -0.428 -0.482 -0.252 -0.165 -0.319 -0.446 -0.548 -0.498 -0.412 -0.027 0.643 0.293 0.242 POLYPE -0.519 -0.612 -0.537 -0.281 -0.230 -0.459 -0.572 -0.481 -0.433 -0.345 0.795 0.801 0.730 ST1CT0 -0.657 -0.586 -0.485 -0.289 -0.281 -0.491 -0.401 -0.489 -O.32 I -0.340 0.614 0.583 0.657 XEHXEN -0.092 -0.238 -0.190 0.077 -0.441 -0.288 -0.451 -0.464 -0.451 0.193 0.365 0.125 -0.001 CLADOT -0.186 -0.285 -0.180 -0.342 -0.230 -0.350 -0.374 -0.310 -0.304 -0.180 0.183 0.096 0.181 MICROP -0.222 -O.I67 -O.O89 -0.162 -0.229 -0 .342 -0.257 -0.314 -0.208 -0.101 0.123 0.055 0.111 TANYTA -0 .Mu -0.398 -0.226 -0.221 -O.368 -0.490 -O.36 O -0.427 -0.297 -0.238 0.152 0.111 0.370 203 : 1 •/ . 1-1 'j<:! c.'-ii’. t.'jl I : i\ i'.ri Go' 11 joiori'.:: 1 01 I . 1

CHIRON CRYPTO CRYGAL DICFUM DICMOD DICNFR DICROT ENDNIG GLYLOB GLYPAR GLYPTO PARACH POLYPE CHIRON 1 .000 CRYPTO 0.274 1 .000 CRYGAL 0.351 0.786 1.000 DICFUM 0.323 0.724 0.851 1 .000 DICMOD 0.091 0.320 0.343 0.457 1 .000 D1CNKR 0 .288 0.3H2 0.571 0.671 0.649 1 .000 UICROT O .307 0.547 0.601 0.?04 0.730 0.810 1 .000 ENDN1G 0.194 -0.044 -0.052 -0.028 -0.181 -0.138 -0.124 1 .000 GLYLOB 0.428 0.116 0.164 0.153 0.093 0 .124 0.135 0.677 1 .000 GLYPAR 0.445 0.122 0.174 0.210 -0.028 0.179 0.194 0.448 0.607 1 .000 GLYPTO 0.615 0.271 0.347 O.3 I9 0.090 0.284 0.303 0.521 0.790 0.817 1 .000 PARACH 0.643 0.414 0.586 0.474 0.313 0.625 0.452 -0.038 0.118 0.124 0.259 1 .000 POLYPE 0.795 0.396 0.483 0.449 0.159 0.417 0.440 0.225 0.541 0.565 0.786 O .378 1 .000 STICTO 0.614 0.267 0.326 O .306 0.033 0.283 0.299 0.171 0.372 O .663 0.608 0.261 0.760 XENXEN 0.365 0.451 0.422 0.488 0.468 0.506 0.471 -0.133 0.082 0.095 0.131 0.607 0.131 CLAUOT 0.183 0.291 0.308 O .293 0.179 0.232 0.273 0.354 0.118 0.231 0.183 0.260 0.220 MICROP 0.123 0.181 0.248 0.210 0.179 O.36 I 0.186 -0.087 0.077 0.177 0.123 O .366 0.154 TANYTA 0.152 0.152 0.182 0.172 0.196 0.176 0.155 O.O83 0.022 0.029 0.151 0.173 0.188

STICTO XENXEN CLADOT MTCROP TANYTA

DTICTO 1 .000 XENXEN -0.008 1 .000 CLADOT 0.326 0.241 1 .000 MICROP 0.394 0.175 0.259 1 .000 TANYTA 0.161 0.043 0.159 0.031 1 .000 +702 Table 11 . Distance Matrix of OTU’s

CLIPIN COELOT PROSUB PROBEL PROCLA ABLMAL :o n c h a TANSTE rSEDYA ACRSEN CRIBIC CRITRI CRISF3 CLIf’Ill 0.000 COELOT 0.338 0.000 PROSUB 0 . A 2 0 0.333 0.000 FROBEL 0.530 0.454 0.297 0.000 PROCLA 0.454 0.373 0.167 0.239 0 .000 ABLMAL 0.670 0.670 0.616 0.587 0.643 0 .000 CONCHA 0.707 0.707 0.655 0.627 0.681 0 .189 0.000 TANSTE 0.596 0.530 0-395 0.354 0.433 0.455 0.500 0.000 PGEDYA 0.485 0.329 0.236 0.343 0.289 0.643 0.681 0.433 0.000 ACRSEN 0.603 0.664 0.664 O .707 0.686 0.766 0.779 O .707 0.664 0.000 CRIBIC 0.759 0.775 0.845 0.857 0.862 0.871 0.886 0.919 0.845 0.577 0.000 CRITRI 0.664 0.687 0.687 0.717 0.658 0.891 0.906 0.829 0.726 0.542 0.392 0.000 CRISP3 0.718 0.737 0.775 0.786 0.793 0.851 0.866 0.848 0.775 0.522 0.229 0.329 0 .000 CRISPS 0-935 1 .000 1.074 0 .98I 1 .036 1 .000 0.946 1.123 1.038 0.791 0.392 0.609 0 .480 CRISP5 0.935 1 .000 1.074 1.056 1.106 0.922 0 .946 1.063 1.038 0.764 0.392 0.720 0 .480 CRICOT 0.748 0.764 0.833 0 .845 0.805 0.910 0.926 0.952 0.833 0.594 0.213 0.312 0 .324 NANDIS 0.759 0.775 0.811 0.775 0.782 0.891 0.906 0.884 0.811 0.577 0.338 0.333 0 .239 0.664 0.645 ORTHOC 0.601 0.686 0 .624 0.871 0.886 0.771 0.645 0.569 0.535 0-333 0 .414 SM1SP1 0.612 0.663 0-775 0.775 0.760 0.775 0.795 0.929 0.825 0.659 0.490 0.519 0 .490 SMISF2 0.600 0.609 O.76O 0.775 0.784 0.775 0.827 0.885 0.760 0.612 0.439 0.588 0 .4 39 SMITTI 0.600 0.577 0.679 0.650 0.638 0.837 0.858 0.834 0.734 0.645 0.555 0.471 0 .480 CORYNO 0.601 0.642 0.651 0.632 0.655 0.670 0.707 0.729 0.601 0.674 0.737 0.737 0 .655 THIENE 0.651 0.707 0.748 0.748 0.737 0.719 0.732 0.842 0.786 0.707 0.707 O.676 0 .707 CUiATT 0.810 0.798 O.7I8 0.750 0.739 0.830 0.845 O .762 0.759 0.707 0.900 0.854 0 .866 CHIPLU 0.791 0.778 0.696 0 .729 0.718 0.809 0.824 0.741 0.739 0.685 0.900 0.838 0 .850 CHIRIP 0.783 0.771 O .707 0.741 0.729 0.766 0.802 0.753 0.750 0.718 0.870 0.866 0 .878 CHIRON 0.810 0.798 0.718 0.750 0.739 0.830 0.845 O .762 0.759 0.707 0.900 0.854 0 .866 CRYPTO 0.771 0.759 0.674 O .685 0.696 0 .809 0.824 O .696 O .674 0 .707 0.655 0.615 0 .624 CRYGAL 0.771 0.759 O .718 0.750 0.739 0.809 0.824 0.762 0.759 0.661 0.577 0.520 0 .527 DICRUM 0.791 0.778 0.696 0.729 0.718 0.809 0.824 0.741 0.739 0.685 0.636 0.545 0 .601 DICMOD 0.729 0.810 0.771 0.783 0.791 0.830 0.84 5 0.753 O./fi 0.559 0.563 0.553 0 .478 D1CNER 0.750 0.798 0.759 0.791 0.778 0.871 0.886 0.823 0.798 0.637 0.577 0.520 0 .500 UICROT 0.771 0.798 0.759 0.771 0.778 0.871 0.886 0.823 0.759 0.612 0.577 O .569 0 .500 ENDNIG 0.856 0.803 0.741 0.718 0.762 0.695 0.732 0.683 0.762 0.775 0.922 0.926 0 .924 GLYLOB 0.791 0.778 0.696 O .729 O.7I8 0.809 0.824 0.741 0.739 0.68 5 0.900 0.838 0 .860 GLYPAR 0.791 0.778 0.696 0.729 0.718 0.809 0.824 0.741 0.739 0.685 0.900 0.8^8 0 .85 0 GLYPTO 0.810 0.798 0.718 0.750 0.739 0.830 0.845 0.762 0.759 O .707 0.900 0.854 0 .866 PARACH 0.791 0.798 O .759 0.791 0.778 0.830 0.845 0.783 0.798 0.685 0.598 0.545 0 .527 POLYPE 0.791 0.778 O .696 0.729 O.7I8 0.809 0.824 0.741 0.739 0.685 0.886 0.838 0 .850 STICTO 0.750 0.739 0.651 O .685 0.674 0.809 0.845 0.6o6 0.696 0.729 O .926 0.870 0 .882 XENXEN 0.860 O .913 O .913 0.933 0.935 1 .000 0.946 0.977 0.913 0.752 0.550 O .508 0 .544 CLADOT 0.816 O .803 0.741 0.718 0.762 0.743 0.756 0.730 0.762 0.683 0.652 0.632 0 .642 MICROP 0.762 0.771 0.707 0.741 0.729 0.74 3 0.770 0.730 0.750 O .696 0.644 0.601 0 .561 TANYTA 0.762 0.803 0.741 0.775 O .762 0.74 3 0.779 0.730 0.696 0.806 0 .822 0.783 0.717 O Table 11. Distance Matrix of OTU's (cont.)

CRIGP4 CR1GF5 CRICOT NANDIG ORTHOC SM1SF1 SMISP? SMITTI CORYNO THIENE C.H1ATT CHIPLU CHIHir CRISF4 0.000 CRISP5 0.471 0.000 CRICOT 0.385 0.544 0.000 NANI) JS 0.471 0.609 0.333 0.000 ORTHOC 0.8 32 0.8 32 0.527 O.R78 0.000 3MISP1 0.620 0.620 0.519 0.519 0.600 0.000 SMISP2 0.650 0.588 0.519 0.519 0.650 0 .447 0.000 SMITTI 0.720 0.816 0.544 0.471 0.480 0.392 0.^30 0.000 CORYNO 0.899 0.855 0.775 0.697 0.655 0.632 0-555 0.519 0.000 THIENE 0.760 0.760 0.717 0.717 0.767 0.439 0.632 0.588 0.618 0.000 CI1IATT 0.979 0.957 0.926 0 .664 0.522 0.816 0.890 0.791 0.853 0.862 0.000 CHIPLU 0.957 0.935 O .926 0.642 0.492 0.791 0.866 0.764 0.835 0.822 0.213 0.000 CHIRIP 0.978 0.909 0.897 O.67H 0.559 0.752 0.780 0.808 0.848 0.798 0.264 0.264 0.000 CHIRON 0.979 0.957 0 .926 0.664 0.522 0.816 O.89O O.79I 0.853 0.862 0.000 0.213 0.264 CRYPTO 0.890 0.866 O .690 0.664 0.577 0.816 0.842 0.791 0.816 0.840 0.674 0.674 O .890 0.699 CRYOAL 0.866 0.617 0.569 0 .461 O .707 0-791 O .677 0.778 0.804 0.640 0.640 DICFUM 0.665 0.957 0.935 0.673 0.642 0.492 0.791 0.866 0.764 0.835 0.804 0.640 0.640 DICMOD O.78O 0.605 0.665 0.752 O .530 0.468 0.722 0.752 O .692 0.750 0.778 0.636 O .636 D1CNER 0.842 0.816 0.514 0.663 0.617 0.461 0.707 0.791 O .677 0.778 0.748 0.584 0.584 0.610 DICROT 0.764 0.514 0.791 0.617 0.461 0.707 0.736 0.677 0.739 0.786 0.584 0.584 0.610 ENDNIG 0-953 0.879 0.949 0.762 0.648 0.826 0.826 O .879 0.898 0.898 0.474 0,418 GLYLOB 0.387 0-935 0.913 0.926 0.674 0.492 0.764 0.842 O .736 0.835 0.822 0.305 0.216 0.345 GLYPAR O .926 0.642 0.764 0.935 0.913 0.492 0.842 O .736 0.835 0.853 0.305 0.305 0.345 GLYPTO O .957 0.935 0.926 0.664 0.522 0.791 0.866 0.764 0.853 0.840 0.213 0 . 2 P 0.264 PARACH 0.890 0.866 0.636 O .569 0.522 0.764 0.842 O .736 0.816 0.828 0.615 0.657 0.682 POLYPE 0-957 0.935 0.913 0.642 0 .H92 0.791 0.866 0.764 0.835 0.822 0.151 0.151 0.216 3T1CT0 1 .021 0.957 0.951 0.686 0.550 0.816 0.842 O.79I G.G35 0.857 0.302 0.302 0.264 XENXEN 0.677 0.707 0.603 O .566 0.677 0.707 0.791 0.736 0.866 0.760 0.816 . 0.862 0.907 CLADOT 0.826 0.798 0.689 0.672 0.539 0.739 0.798 0.798 0.861 0.791 0.541 0.541 0.518 MICROP 0-933 0.860 0.681 0.586 O .530 0.752 0.780 0.722 O.77I 0.816 0.556 0.556 0.541 TANYTA 1 .000 0.929 O .837 0.685 0.596 0.798 0.826 O .853 0.880 0.810 0.556 0.556 0.488 206 Table 11. Distance Matrix of OTU's (cont.)

CHIRON CRYPTO CRYGAL DICFUM DICMOD DICNER D1CR0T ENDNIG GLYLOB GLYFAR GLYPTO PARACH FOLYPE CHIRON 0 .000 CRYPTO 0.6?4 0.000 CRYCAL 0.640 0.302 0.000 DICFUM 0.640 0.302 0.213 0.000 DICMOD 0.636 0.463 0 .408 0 .408 0.000 IIIONER 0.584 0.452 0.337 0.337 0.267 0 .000 DICHOT 0 .584 0.399 0.337 0.337 0.218 0.213 0.000 ENDIIIG 0.474 0.806 0.791 0.775 0.751 0.758 0.742 0.000 CL 71.OB 0.305 0.715 0.682 0.682 0.636 0.629 0.629 0.354 0.000 CI.YPAR O .305 0.715 0.682 0.665 0.661 0 .610 0.610 0.418 0.309 0.000 GLYPTO 0.213 0.674 0.640 0.640 0.636 0.584 0.584 0.418 0.216 0.216 0.000 PARACH 0.615 0.399 0.261 0.337 0.408 0.302 0.369 0 .806 0.699 0.699 0.657 0.000 POLYPE 0.151 0.657 0.622 0.622 0.617 0.564 0.564 0.447 0.264 0.264 0.151 0.640 0.000 STICTO 0.302 0.674 0.674 0.674 0.673 0.622 0.622 0.474 0.374 0.305 0.302 0.691 0.261 XENXEN 0.816 0.478 0.478 0.478 0.461 0 .447 0.447 1 .016 0.874 0.874 0.845 0.441 0.845 CLADOT 0.541 0.469 0.442 0.442 0.447 0.469 0 .442 0.570 0.541 0.548 0.541 0.469 0.518 MICROP 0.556 O .512 0.436 0.463 0.442 0 .408 0.463 o.64l 0.556 0.563 0.556 0.408 0.535 TANYTA 0.556 0.636 0.598 0.598 0.584 0.556 0.577 0.641 0.605 0.605 0.556 0.598 0.535

STICTO XENXEN CLADOT MICROP TANYTA

STICTO 0.000 XENXEN 0.910 0 .000 CLADOT 0.541 0.637 0.000 MICROP 0.512 0.674 0.354 0.000 TANYTA 0.556 0.835 0.524 0.524 0.000 207 Table 12. Principal Component Analysis of OTU'

Column 6 represents total variation accounted for by all five factors for each character. 209

Table 12. Principal Component Analysis of OTU's

Factor 1 Factor 2 Factor 3 Factor 4 Factor 5 6

CLIPIN 0 .621 -0.354 0 .040 -O.306 -0.078 0 .6126 COELOT 0 .600 -0.550 -0.065 -0.359 0.020 0.7965 PROSUB 0.642 -0.531 -0.186 -0.391 0 .083 0 .8384 PROBEL 0 .674 -0.425 -0.030 -0.035 0.043 0.6392 PROCLA 0 .678 -0.479 -0.200 -0.302 0.032 0.8218 ABLRlAL 0.207 -0.462 0.659 0.315 0 .088 0.7975 CONCHA 0 .208 -0.339 0.568 0.307 0 .001 0.5752 TANSTE 0 .442 -0.542 0.238 -0.056 0.238 0.6061 PSEDYA 0.563 -0.532 -0.039 -0.363 0 .182 0.7664 ACRSEN 0 .264 0.056 0 .014 -0.056 0.207 0.1191 CRIBIC o .430 0.726 -0.054 0 .138 0.252 0.7975 CRITRI 0.504 0.506 -0.291 -0.279 0.073 0.6784 CRISP3 0 .462 0.728 -0 .216 -0 .026 0 .018 0.7913 CRISP4 0.550 0.579 0 .066 0 .458 0 .169 0 .3800 CRISP5 0.594 0.525 0.122 0.514 0.142 0.9270 CRICOT 0.527 0.617 -0 .146 0.056 0 .294 0 .7694 NANDIS 0.371 0 .627 -0.313 0.105 -0.139 0 .6585 ORTHOC 0.407 0.181 -0.532 -0 .297 0.037 0.6264 S MIS PI 0.598 0.300 -0.219 0.152 -0.498 0.7676 SMISP2 0.665 0.055 -0.104 0.026 -0.185 0.4904 SMITTI 0.658 0.026 -0.225 -0.013 -0.339 0.5987 CORYNO 0.492 -0.246 0.233 0.027 -0 .287 0 .4400 THIENE 0.302 -0.031 0.420 0.190 -0.716 0.8178 CHIATT -0.771 -0.197 -0.155 0.058 -0.350 0.7332 CHIPLU -0.771 -0.158 -0.358 0 .134 -0.044 0.7669 CHIRIP -0 .674 -0.242 -0.171 0 .174 0.010 0 .5724 CHIRON -0.771 -0.197 -0.155 0 .053 -0.350 0.7832 CRYPTO-0.596 0.084 0.292 -0.371 0 .242 0 .6442 CRYGAL -0 .699 0.286 0.073 -0 .411 0 .082 0.75U DICFUM -0.704 0.245 0 .128 -0 .473 0.100 0.8054 DICMOD -0.408 0.434 0.312 -0.240 0.077 0.5156 DICNER -0.655 0.450 0.107 -0.331 -0 .088 0.7605 DICROT -0 .661 0 .404 0 .110 -0.315 -0.015 0.7122 ENDNIG -0 .324 -0.367 -0.167 0.547 0.349 0.6898 GLYLOB -0.601 -0.242 -0.365 0.333 0.113 0.6766 GLYPAR-0.598 -0.207 -0.381 0.267 0 .094 0.6254 GLYPTO -0.765 -0.263 -0.344 0 .216 0 .049 0.8215 PARACH -0 .061 0.217 0.219 -0.223 -0.224 0.6321 POLYPE -0.848 -0.154 -0.335 0.036 -0.113 0.8694 STICTO -0.722 -0.251 -0.292 0 .021 -0.033 0.6713 XENXEN -0.533 C .429 0 .427 -0.031 -0.240 0.7088 CLADOT -0.450 0 .024 0.222 0.131 0.285 0.3512 MICROP -0.350 0.130 0.228 -0 .027 0 .074 0.1976 TANYTA -0.40L -C.063 0.266 0.007 0.232 0.2915

Percent Accumulative Eigenvalue of Trace Percent

Factor' 1 14. 66117 33.32 33-32 Factor 2 6 .42355 14 .60 47 .92 Factor 5 3 ■30612 7.51 55-43 Factor 4 2.98526 6.78 62.22 Factor 5 2.11982 4 .82 67 .04 210 Table 13* Five Dimensional MDS of OTU's. Stress is 0.149.

Dim. 1 Dim. 2 Dim. 3 D i m . 4 Dim. 5

CLIPIN -0.888 0.271 -0.263 -0.189 -0.384 COELOT -0 .854 0.474 -0.388 -0.251 -0.112 PROSUB -0.551 0.707 -0.459 -0.060 -0.080 PROBEL -0 .617 0.752 -0.404 0.127 -0.054 PROCLA -0 .604 0.702 -0.483 -0.127 -0 .110 ABLMAL -o .670 0.594 0.201 0.877 0.111 CONCHA -0.659 0.556 0.227 0.971 0.147 TANSTE -0.507 0.915 -0.339 0.482 -0.071 PSEDYA -0.606 0.706 -0.576 -0 .101 0.033 ACRSEN -0.228 -0.221 -0.044 0.043 -0.795 CRIBIC -0.236 -0.974 0.029 0.052 -0 .094 CRITRI -0 .234 -0.704 -0.370 -0 .142 -0.155 CRISP3 -0 .208 -0.841 -0.124 -0.003 -0 .038 CRISP4 -0 .3^0 -1.486 0 .460 0 .001 -0.035 CRISP5 -0.194 -1.239 0.779 0-353 -0.081 CRICOT -0.294 -1.039 -0.093 -0.036 -0 .140 NANDIS 0.005 - O .698 0 .121 -0 .218 -0.134 ORTHOC 0.177 -0.128 -0.136 -0.371 -0.125 SMISP1 -0 .606 -0.530 0 .460 -0.313 0.156 SMISP2 -0.807 -0.552 0.327 -0.205 -0.252 SMITTI -0.624 -0.361 0.029 -0.618 0 .094 CORYNO -0.887 0.044 0 .000 -0.217 0.691 THIENE -0.786 -0.196 0 .684 0 .054 0.515 CHIATT 0.670 0.545 0.277 -0.274 -0.046 CHIPLU 0.599 0 .548 0.306 -0.258 -0.071 CHIRIP 0.566 0.574 0 .427 -0.113 -0 .012 CHIRON 0.664 0.539 0.273 -0.271 -0.047 CRYPTO 0.376 -0.136 -0.677 0.320 0.217 CRYGAL 0 .428 -0.260 -0.527 0 .144 0.116 DICFUM 0.516 -0.123 -0.598 0.175 0.182 DICMOD 0.479 -0.421 -0 .182 0.081 0.202 DICNER 0.549 -0.310 -0.263 0.001 0.122 DICROT 0.528 -0.320 -0.248 -0.019 0.134 ENDNIG 0.339 0.730 0.773 0 .080 -0.155 GLYLOB 0.517 0.531 0.450 -0.274 -O.I36 GLYPAR 0.519 0.540 0.379 -0.384 -0.070 GLYPTO O.636 0.533 0.333 -0.272 -0.046 PARACH 0.561 -0.347 -0.451 0.223 0 .094 POLYPE 0.616 0.531 0 .280 -0.228 -0.048 STICTO 0.545 0.706 0.225 -0.266 -0.013 XENXEN 0.399 -0.945 -0.475 O.O83 0.421 CLADOT 0.593 0.020 0.034 0.377 0.155 MICROP 0.511 0.027 -0.039 0.242 0.365 TANYTA 0.609 0 .286 0.065 0.522 -0.450 al 1. itne arx f T’ Bsd n hrces rm Immatures from on Characters Based OTU’s of Matrix Distance 14.Table >Mt-HO>tr'tr'tr'2HHMM33i2:2:asr:o3>x^33330C/)>oD3!D^^c:r^3nwT3^ncopicDDCoonoon-isnoznnnn>>DHn>,T3^hDoa ^ajcnK^^^fZ^zsTnTj^aTjxr^sonoiHtjaMcwosotstiin20>*-«r*siHSKKaoonoM;M;i-HMh-^HOn2O^C0OHxC3t-OZ‘T3CTROnM^VjHO2>n>eH>t-OH2“i00HhC0H>0MOn0C>H0MtH-;Z20HQ^XHtilKHZ>^^dCM o oooooooooooooooooooooooooooooooooooooo r* '0-v3-0''J>JN CDCD03 05'J^'>Jn3'0'-'\>0'0*v3'0'«:'vJ-n3'>0'0'v]'n]'s]'nJ 0 0 ^ ^ O O O OO "73 OOOOOOH*^H-f-oOOOO^JOOOOOOOOOOOOOOO-vJ'0OOOOO -O M-s] >3 NJ 0'0\0^0'*0*0>J>J^^J*v]M^'>0>3"\}'v3-'J^N>J*v]'0 00*^^00000 Z

0000000000000000000000000000000000000 ori ,N3*s3-sJ*sJ*v3~OCDCOCDCO"N3'0'vJ*s3"N3V-f'~s3'>0'<3'>J*Nj*N:-0'0'0",0“^^0'^100'-AW\0000 C OOOOOOOOOh-oOOOO-'OOOOOOOOOOOOOOOO'O-'JOOOO Q Off'0'0N'0'v3*^N-s3''J'^N'Nj'0-vJ>JSl'0,v]M*vJ>0'v3 00*v3^0000 “d

0000000000000000 OOOOCOOOOOOOOOOOOOOO -O -v] 'O *vj -O “vj CX3COCDC23-v3-v:-N:'sJ'NlV_n''J-s3-s}-sl-s:'Nj-s3-Nj-N3-sl-v3'vJ-vJ O OU\U\0 O O OOOOOOOOH-f—oOOOO-OOOOOOOOOOOOOOOO-O-'OOOO “sj "sj -s] -sj -sj “nJ 0 s C '0 N 0 v -s3 * 0 ' n }‘n J ~ ^ ‘^J 'v 3*v 3 's}‘n 3 '0 * vJ- s3"s3*nJ*n 3*v 3 ‘n 3 “nJ O O “nJ O O O

00000000000000000000000000000000000 O -Nj-sJ-sJ-sJ-vJ*vJC0C0C0CD-sJ-v3*vJ's]-OVJ,‘,,0*s}-s3'N3-s:*Nj's3'vl'Nj>J-,sJ-N3-s3OOV-ri'-nOO cd '^-vj~vO'-J-sJ~sOCrsCNONON~^]'N3‘NJ~^'0'sO'sJ-sJ~sJ*^*sJ'N3'Nj*^0-vO'Ni'^J~^~<10O O O O O O ^ * — |- * h-' 00000- s l 000000000000000 0- “0'vJO0' s j 00 O tr

OOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOO 33

■^J'SjA jA o-sJ-sj CD CO CO CO-sJ -Nj'N3 - 0 *sJV-f\*v3 -vJ''.J-sJ-v3 -N j-s3 -vJ'N3 ''J-0 -vJ-«JOOU>U\ 0 O 000000OH-»— h-00000-0000000000000000' n)-nJ0 tr -vJ-nJ-vJ-O-O-sJ O O "nJ *nJ O >

000000000000000000000000000000000 CD> ^0 -v3 - v : ^0 ^ 0 -vJ C0 0 3 COCO-'0 -'0 -'0 -'0 ''-JV»n'-0 ~'J-vO'-0 XT p *-0 'n J 'v J '\3 -nJ*n3 -s}U>V-a O O 3 OOOOOOOl—■ o>-»00000*'J0000000000000->J'000 > "J-vJ-'J-nJ-sJ-'J on o On ON“nJ "0-«J-v:J~'J''0-'0-n3'>0''0 CCCD~'0-0*''J~'J'-0-0*'0~'0-nJ O O o

o 00000000000000000000000000000000 o -nJ~0-s3-nJ-v3-nJ CDO0COC0*'O-n]'O'vJ'>vJU\-nJ-sJ-s3-vJ .p. O OOOOOO^^^^OOOOO- nJOOOOOOOOOOOOO- nJ-OO — -*s3'^-sJ-J*vJ-s3C7\OnOnO\-*J*sJ'nJ'nJ'O‘nJ*nJ*nJ*nJ'sJCDCD'nJ'0*,nJ'nJ*sJ*,nJ^J*sJ'nJO >

0000000000000000000000000000000 C0C0 00 03*nJ~>0'n3'nJ"vJV/>"nJ"v3-v3*n1"nJ*nJ-v3‘n3-*'J*v3'vJ"sJ*nJ O O OOOOOOO^-oooOOOO-JOOOOOOOOOOOOOOO -vj-vj-vj-vj-sj-sO OnO«OnC7n"sJ*sJ's]‘nJ~n1~n3"^3'sn]*n3"sJ'n3'nJ*s]*n]'nJ'nJ”,nJ',n3~vO O O

000000000000000000000000000000 -O-nJ-vJ-'J-'O-nJ COCDCDCO'*‘J-nJ-v3*nO'n3VjX“nJ-n3‘n3-s3*,n1*nJ-sJ-nJ'0*n3*vJ-s3-sJ O C00000OOOO00000-N300000000000000 r* ^j-vJ-*vJ-sJ-n3-n30n0nOn0n-nJ-s3*s3-vJ-n3-nJ*nJ-n3-0-*0-nJ-n0-sJ*vJ*nJ-nJ-nJ*sJ*nJO >

> 00000000000000000000000000000 o o o o o o o W b-p-o o o o o o o-c* o ooo ©o o o o o o o 22 OOOOOOOOOOOOOOOOOOOOOK-»QOOOOOO w OOOOOOCOCOCDCOOOOOOCOOOOOONCNOOOOOOO 3

o OOOOOOOOOOOOOOOOOOOOOOOOOOOO ^0 bobbbopVpPoooobPobboaJCDbooooo * © OOOOOOOOOOOOOOOOOOOOOOOOOOOO o OOOOOOfflCDCOCOOOOOOCOOOOOO'ONOOOOOO o

o OOOOOOOOOOOOOOOOOOOOOOOOOOO » bobbboWVpooooo000000000000000000000*-*00000 toooocDcoooooo © ^ OOOOOOOOCDCOOOOOOOOCDOOOOaNONOOOOO o

n OOOOOOOOOOOOOOOOOOOOOOOOOO 2 bbbbbbb'bb-bbbobb-P'obbbboobbbo w OOOOOOOOOOOOOOOOOOOOOOOOOO TJ OOOOOO'XfflCDWOOOOOUOOOOO'a'OOOO '-j

TT2 Table 14. Distance Matrix of OTll's Baaed on Characters from Immatures (cont o "3 tr t o 70 zz ►< a > cr* o 3 o

a > r. 2 c 1-000 — -a — . . . — -a —

»—*—00 ^ - O O O O I I —3 n GO

> m k zkm > ) - ^ 2 Z O t CO C'C'O * O O O

V—h-^OO v n CO •— ICHPIOOMH h c r.c-^2ZOHC

o o o o CO o

* p - -

...... --- 300000000000 :t*o: O 0-0 O O O0-0O n O Os Os n O 0-0000000 0-0000000 - 0 - Ui Ui J UU U (M>C'C'C''0 ^ J UU^ J U(M>C'C'C''0 0 j - V^J -C-C'Pr.CroOOOC o o n n o c a ~ n r. o c n o H n o n H ocn o r. n ~ cao n n o o n "3 lo^coa^^c-' c-siKKKConnr O O O c~* 333 »-<; »—«; V—^ UU U k i K M ;i—i HV—^ i - H ■ »—«; »-<; 333 c~* n 1 ^ T O ‘4>OHQ>10C>',50 i O n -- C O C\V O n n O n C0CDCDC0C00DvOv-O'*O VO CO CO CD CD CD-0 CO CD CO CD O CD CO CD CO CD-0 CD CD CO CO VO 000000000000000000000C0CDCDC0C00DvOv-O'*O 00000000000000000000 OOOOOOOOOOOOOOOOOOOOOOOOO-C'Pr^T^roOOOO-^'OOOOOOOOOOOOOOOOOOOOOOOOOOOOCDCOCDCDOOOOOCDOOO bboboo^^^^oooco^oooocDaooooooooooooooooooooooOOOOOOOOOOOOOOOOOOOOOOObbbooo-e'*r-=*-J=*ooooo-e-oooococDoOOOOOOOOOOOOOOOOOOOOOOOoooooocomcacoooooocoooooNCNO o OOOOOOOOOOOOOOOOOOOOOOOO 00000000000000000000ooooooppppooooo O OOOOOOCOCDODCOOOOOOCOOOOO CN CT CN OOOOOOCOCDCOCOOOOOOCOOOOOONO'OO 00-0 0000000000000000000000COC3COCOCOCO'«OsOvOvOOOCOCDCD Z O >O Z OOOOOOOOOOOOOO'bbboooi’^^tooooo-oooococDooo00000000000000000000 OOOOOOOOCDOOOOOCCSOCOOO'OOOO OOOOOOOOOOOOOCCOOO h s n M Tj Tj sM n h >Mtr"-3o>rtr,tr,2HHHi-.3:~3:3ss2:o3D>Ti br b- b V -Cr VOv UN VJN v»A .p-Vr ■£-V O ■£-V v»A .p-Vr VJN UN VOv V-Cr b b- br OOOOOOOOOOOOOOO OOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOCOODCOOOOO booooopt^toooOOOOOOOOOOOOOOOOOOOCOCOOOCDOOO ^ O O OOOOOOOOOOOOOOOOOOOOOOOCOCOCOCDOOOOOCDO -O-O-OCOODCOCDOOO CO-O OOOOOOOOOOOOOOOO00 00 00 000000 03 00 0 0 0OOOOOOOOOOOOOOO 0 0 0 OOOOOOCOCDCOOOOOOOO ooooooP'PPPooooOOOOOOOOOOOOO 000000000000000000OOOOOOOOOOOOOOOOObbooboPPPPooooo boooooV-P*-5*4=‘ooooo-£'oo OOOOOOCDC0C0C0OOOOOCDOO o o ra s 70 o o r1 <-< 3 o -TJ ca £ a o o IT OT H o H a o o 0 O n V w w -S* -S* a cu cu a V a t V W bo o o ■cr O o O ■cr ■ £ - 0 0 0 0 -£• o -£• O O O O O o oo o CO CD O O O 0 0 0CD CD O O O O O O O CD oo -pro 0 0 0 0 0 0 0 0 0 O CO CO CD CD O CD CD CO 0CO 0 O 0 0 0 0 0 0 0 0o 0 0 0 -S’ O 0000000 CO o o o o o CO *33 v > r r3JKKK r1-3 O 2 H H33 33 OO X O CO CD CD CO O 0 0 0 0 0 0 n O B;> H Q n f’Ol/YrE 0.000 0.000 o.4o8 o.4o8 o.4o8 0.408 0.000 0.000 STICTO 0.000 0.000 0.408 0.408 0.408 0.408 0.000 0.000 0.000 OI.AUOT 0.000 0.000 0.408 0.408 0.408 0.408 0.000 0.000 0.000 0.000 MI CROP 0.000 0.000 0.408 0.408 0.408 0.408 0.000 0.000 0.000 0.000 0.000 TANYTA 0.000 0.000 0.408 0.408 0.406 0.408 0.000 0.000 0.000 0.000 0.000 0.000 212 213 0 * 0 0 0 SZ/.'o S/./.-o (./.?• i i tt?-1 /.?•• i / / - I- /??• o OIR-0 S/./.-o (//.i-1f/.-■• i non non ‘ o 0 00(1" 0 00(1- ooo" "OOO (1 I" 0/.O/.-o•’! Zo/.’OZIP" ? 0 - 0 t/.-r / t- ir•tIBS" iizr 1 • 1 " tt?-1 t /.9 1 ’/re i L/,1- 1 (/.'I-1 i.o/: r./.o-1 r U S/./.-o I /.()/.' ll-0 ,l|l|- / ciro /.dr //

i do-1 1 /.o/.-o o 0 0 on ro o i i i 1 . t ||ZI ■ ctr t ft?-1 ooo- o ooo • o I* Si|/.-o 9IB*0 (19" 1 Sii/.-o 910*0 01)0- 1 OOI)-0 0 * 0 1 9 omro o l . f ifZC • rr?-1 1 551-1 0(10* 0 0(10* ooo -oooo'o -0 0 1 01 0 " 0 0 0 rr 1ntiIBS" 1 i t n 1 T U /./. S-0 II 9" 1 /.O/.-O •f/.r. 1.1.9" it?*i ttt-o poro ooro /.O/.-O /.OZ‘0 •ill)-1 >- B2S-1 161 u>e-1 f-1 i6e-1 ri.i.l (in ?-1 - 1 1 ooo-o 0 * 0 0 0 ttc-o C IBS-1 CCC 1 1 IBS'- ftf 0 - C 1 • . l t 1 Hi -0 0 * 0 0 0 'O O O O ooo" o ooo-o ooo-o ttr-1 9 fit • l t 0IS IBS - IBS' 1 l ttl -0 SSI-1 in/.- ZoZ'o (100* 0 (100* / S 0IBS -1 Z/.S-0 /./.s-o ZoZ o Sn/.-o Z/S-o 1.1 - f If l I iii ii iii n/.t • i l|U|- 1 i//.f i i/ro r riIBS' 1 or i C .- 1 • i - I o m:i 1 1 1 B-lIBS' 1 IBS- l 1

OOO’O ooo-o -0 0 1 ( 0 •ini' i ZoZ’O /.O/.-O ZO/.-O I I I ' M . ' l V •iiif-1• i i i n r f i ■ lii i r i n •Mr •Mr i III nir ifi tfiri*)-1If-1 i nir i B ' •IBS’ 1 IBS' • B " lIBS' I IBS" l IBS - IBSIBS' I -1 1 in5" I IBS’ 1 IBS- l IBS' 1 IBS' 1 IBS' 1 IBS' 1 ins ins IPSIBS' -1 l IBS' I IBS' 1 IBS- 1 IBS' IBS' 1 IBS 1 - 1 B IBS"IBS I - l IBS' 1 ms ms 105 -1 IBS’ 1 IBS’ 1 IBS' 1 IBS' I I8S- I 185* 1 B ' 1IS IBS’ 1 IPS’ 1 IBS' 1 IBS’ l IBS' l -1 -1

o- ooo’o 000-0 ooo-o . /- /.O/.-O /.O/.-O 0 - 0 0 0 O OOO’ ./ - /.o/.’o /.o/.-o . .O/O/.-O /.O /.-O /.O •dir i iinr I r i • M r i M •ini-1 ifl|M|- 1 iinr nr i i Bur i v/n v/n r-.i IBS' 1 B’ IIBS' l IBS’ I IBS' 1 IBS- I IBS’ IBS' tos I • I IBS' I IBS1 I IBS' I IBS -1 P 1IBS- I IPS -1 IBS - 1 P’ IIBS' 1 IPS’ I ms IBS' 1 P * 1IBS- 1 IPS* 1 B IBS’IBS I - 1 P- IBS' 1 IPS-1 B 1IBS' I IBS -1 -1 1

0 - 0 0 0 ooo'o O 'O O O O 0 - 0 0 0 •in r i r-i.i.BMV. mi mi i-1 'ini-1 nir i IBS' IBS- IBS’ 1 IBS" I ms ms IBS" 1 IBS- IBS’I IBS' 1 IBS- 1 IBS - I IBS - IBS' IBS- I IBS' 1 IBS - 1 -1 -1 1 1 1 1 1

I'll I'll ooo-o O OOO’ 000-0 000-0 00(1-0 'O O O O /.Ol'O Zo/.-o n i r i r i n • l •(Hr 1 i*1 • ' I ( - • 1 Hi-1 1'i •l •ill1 ll• (■ 1 I /.o/.-o I r n •l I ff-1 IBS- 1 IBS’ I IBS' 1 IBS’ I IBS’ IBS-1 I IBS ’ I IBS" 1 IBS’ 1 1 IBS* IBS' OS1 •1 IBS" I IBS' 1 IBS 1 ’IBS I -IBS' 1 1 IBS’ 1 IBS' IBS' 1 IBS"1 IBS" 1 IBS' IBS'1 1 I IBS" 1 IBS' 1 IBS-IBS I -IBS' 1 IBS' IBS"I I IBS'1 IBS' 1 1 IBS’ 1 IBS' IBS’1 1 IBS' I IBS' 1 B’ 1IBS' 1 IBS’ 1 IBS' 1 IBS' 1 IBS' 1 IBS'IBS" 1 1 IBS' I IBS’ I 1,11

11 1 o n , | v | n .ln .l .l .ln n | v , | n o O’O OOO’ 'O O O O 'O O O O ooo -01)0-0 o 'O O O O /.o/,- 0 »f iif-1 000-0 i •f r n /.?/,'0 in!.- IPS' 1 IBS-1 IBS -1IBS-1 ••iff-1•f Hi-1 i r i 'i i M r •• - ms IBS' ! IBS - 1 IBS' 1 IBS - I IBS' 1 IBS' 1 IBS' IBS'1 1 ibs IBS' 1 iif- -

0 1 i 1 ooo-o OOO'O 000-0 000-0 0 000- O OOO’ 000-0 'O O O O /.o/.-o ooo’o 000-0 I f f111f 1 I f I -1 f • II H l f f - - 1it 1 /.O/.-O /.O/.-O IBS" 1 IBS-1 IBS -1 111 If-1 i i iir • If Ilf-1 If Ilf-1 IBS -1 IBS' 1 IBS' 1 IBS’ 1 IBS' 1 IBS-1 IBS' 1 ios IBS - 1 If II IBS - 1 IBS' IBS1 -1IBS’ IBS’1 I IBS’ IBS’l 1 IBS' I IBS’ 1 IBS-IBS' 1 1 IBS' 1 IBS' 1 IDS • IBS -1 viikim IBS' 1 IBS' IBS- IBS-1 I 1 IBS' 1 IBS' IBS’1 IBS'1 IBS'1 1 1 r |.Or| i Vr. V r 11 |i, l ) | , | f , r r|| |i.iO or. f n | n | (■ - I 1 . i ini imiio-i.i -iinoiM .i.orioo OOO’O 000*0 /.O/.-O ooo'o 'O O O O ooo'o 000-0 000" 0 /»/.- /.O/.'O if •fI PIif" Iff-if-1 1 1 IBS’ 1 IBS - I IBS -IBS- 1 1 IBS- 1 IBS - 1 •ri r i i i I ifiir* 1 if 1 if- 1 IBS’ I IBS' I IOS' 1 IBS' 1 IBS-1 IBS' 1 IBS' 1 IBS' 1 - s io IBS-1 IBS' I IBS- 1 IBS-1 IBS- 1 IBS-I IBS" I IBS' IBS'1 1 IBS’ IBS'1 1 •(• 0 1 l 000" ooo- 000- ini' /.O/.- /.o/.-0 0 000 000 0 000 00(1 0 000 0 000 000 it Hi* •(Hi- 000 000 r •M (i i n IBS- s- in IBS- ni 6 ni it i if- IBS- t IBS- IBS- I it i if " IBS’I IBS' IBS' IBS’1 IPS’ IBS- 1 IBS- IBS' 1 IBS- I n i v* IBS- IBS- IBS' IBS’ IBS' IBS’I IBS' I IBS’I IBS' IBS' 1 IBS' IBS' I ­1 L K IBS" 1 IBS" IBS-1 IBS’ IBS -I IBS' 1 0 0 0 0 0 0 (I 0 0 1 1 I 1 IBS- 1 I 1 I I I 1 1 1 1 1 I I I 1 I I 1 1 1 OOP- 000-0 /,?/.- lol- 000 /.(I/.-0 ()()(> 000 000 000 r n n n in- n in IBS- IBS- IBS- 000 ii 0 0 000 •fin IBS’1 IBS' 1 IBS’ IBS' IBS' IBS' IOS’ IBS’1 n in IBS' IPS- 1 IBS' 1 IBS- 1 IBS •1 ni n ni n i if- IBS- t IBS- 1 IBS- IBS' 1 IBS" IBS' IBS ‘ IBS' IBS' I IBS' ins-

in »’1|» 0 0 0 (} f) 0 0 ■:ll|fi • M'' . 'l'M M | • i| .■■:illl'|Of 0 t 0 0 0 1 1 1 I I I I 1 t I I 1 I t I I 1 1 I 1 1 1 I I o o o - o oil). oil). >ion OHM Mi l, o - o o o OOO'O non-o o w i r .’ . /.-o /.o ooo-o VIOOHI000-0 000-0 VIIOIKIOooo-o ono-o H:l'.;ilOV 0 " 0 0 0 0 - 0 0 1 ( l .T..I.I /.-o *i::/.o /.-o /.o ooo" o o- m.irio ooo-o OMiioiw 1 - 5 0 1 m u i i i i i i ’ iS K Mo-i -I in IHKHCTI 1 ' S IB 11)5-1 'I S B I *f VMIIVIIIHr I I •OO I 1 I , I " I . I I I I I ld O oH B -1 5 0 1 1 HV.IVIO IIOVOVl -1 5 0 1 -1 5 0 1 | - V.t.AirV 5 o I, i VMini:itt l MIIOUl I -IBS 1 - il 5 iv 0 1 iir o I ' 5 i'i 0 1 in (i.o 1 - 5 0 1 -1 5 0 1 miiio i IIIIIino lour i - i i i i i if i ■ i ill)' 5-• 1 1 1 | - r I : l IIK) ll:i l i . l l i -1 5 0 1 111? Boil l ' S IB IB W IV 11)5* I 105" -1 5 in 0 o o 1 o M m n m / .i OltlMIH -1 5 8 1 i - S n i I ' S IB i i i i | - 1 f-i i . i t i y | - 1 I 1 ll.l-l 11)* 105- .l m -1 l.v 5 O 0 1 •1 105 5 o i VI.-1VII.1 B 'llll.U I IBS" -1 5 0 1 O.MJH:i i in .u n I w - 5 0 1 I " 5 o l ' S B I i f-1 i|tt:i : t |.t i if 1 - -1 I I I x n i x i r i Imillo i -I m 'I!) U O H i).l:'I.I.B v 00 .i.onvio - - | ' | l | ( ) ) | . | : i , i , r : i i v . i . .| ,Dr|:ino iiiiiioh VJ.IIOIII m ivwiiiv i i . i . i v h v :: : h i i h i : h d h i . :; ; :> : i j i Table 15. Distance Matrix of OTU's Based on Adult Fore Leg Positioning Characters (cont.)

CRISP4 CRISP5 CRICOT NANI) IS ORTHOC > IMG PI SMISr2 SMITTI CORYNO TH1ENE CHIATT CHIPLU CHI RIF CRISP4 0.000 CRISP5 0.000 0.000 CRICOT 0.000 0.000 0.000 NANDIS 0.000 0.000 0.000 0.000 ORTHOC 0.000 0.000 0.000 0.000 0.000 SMISP1 0.707 0.707 0.707 0.707 0.707 0.000 SHISI’2 0.707 0.707 0.707 0.707 0.707 0.000 0.000 SMITTI O .707 0.707 0.707 0.707 0.707 0.000 0.000 0.000 CORYNO 1.414 1.414 1.414 1.414 1.414 0.707 0.707 0.707 0.000 THIENE 1.414 1.414 1.414 l .414 1.414 0.707 0.707 0.707 0.000 0.000 CHIATT 0.707 0.707 1.333 0.707 O .707 1 .000 1 .000 1 .000 1.581 1.581 0.000 CHIPLU 0.707 0.707 1 .37'+ 0.707 1 .000 1 .000 0.707 1 .000 1.581 1.581 0.333 0.000 CHIRIP 0.707 0.707 1-333 0.707 O .707 1 .000 1 .000 1 .000 1.581 CHIRON 1.581 0.000 0.333 0.000 0.707 0.707 1.333 0.707 0.707 1 .000 1 .000 1 .000 1.581 1.581 0 .000 0.333 0.000 CRYPTO 0.707 0.707 0.333 0.707 1 .000 0.707 1 .000 1 .000 1 .581 1.581 1.291 1 -333 1 .291 CRYGAL 0.707 0.707 0.707 0.333 0.707 1 .000 1 .000 1 .000 1.581 1.581 1 .291 1-333 1.291 DICFUM 0.707 0.707 0.333 0.707 0.707 1 .000 1 .000 1 .000 1.581 1.581 1.291 DICMOD 0.707 1-333 1 .291 0.707 0.577 0.707 0.707 1 .000 1 .000 1 .000 1.581 1.581 1 .000 1 1 .000 DICNER 0.707 .05!) 0.707 0.577 0.707 0.707 1 .000 1 .000 1 .000 1.581 1.581 1 .000 1 .051* 1 .000 DICROT 0.707 0.707 0.577 0.707 0.707 1 .000 1 .000 1 .000 1.581 1.581 1 .000 1.0511 1 .000 ENDNIG 0.707 0.707 l .374 0.707 0.707 1 .000 1 .000 1 .000 1.581 1.581 0.000 GLYLOB 0.707 0.333 0-333 0.707 1.37'* 0.707 O .707 1 .000 1 .000 1 .000 1.581 1.581 0.000 GLYPAR 0.707 0.333 O .333 0.707 l .374 0.707 0.707 1 .000 1 .000 1 .000 1.581 1.581 0.333 GLYPTO 0.707 0.471 0.333 0.707 1-333 0.707 0.707 1 .000 1 .000 1 .000 1.581 l .581 0.000 PARACII 0.707 0-333 0.000 0.707 0.471 0.707 O .707 1 .000 1 .000 1 .000 1.581 l .581 1.247 POLYPE 0.707 1.291 1.247 0.707 1-333 0.707 O .707 1 .000 1 .000 1 .000 1.581 1.581 0.000 STICTO 0.707 0-333 0.000 0.707 1.374 0.707 0.707 1 .000 1 .000 1 .000 1.581 1.581 0.4-1 XENXEN 0.707 0.333 0.333 0.707 0-333 0.707 0.707 1 .000 1 .000 1 .000 1.581 l .581 CLADOT 0.707 0.707 1.291 1-333 1.291 0.745 0.707 O .707 1 .000 1 .000 1 .000 1.581 1.581 0.882 MICROF 0.707 0.707 0.745 0.943 0.882 0.707 0.707 1 .000 1 .000 1 .000 1.581 1.581 0.882 0.943 0.882 TANYTA 0.707 0.707 1.106 0.707 O .707 1 .000 1 .000 1 .000 1.581 1.581 0.882 0.943 0.882 DEMBRA 0.707 0.707 1.414 0.707 0.707 1 .000 1 .000 1 .000 1.581 1.581 0.471 EINCHE 0.707 0.707 0-333 0.471 1-333 0.707 O .707 1 .000 1 .000 1 .000 1.581 1 .581 0.000 MICNIG 0.707 0.707 O .333 0.333 0.000 0.707 0.707 1 .000 1 .000 1 .000 1.581 1.581 1 .291 IIARINC 0.707 0.707 O .333 1-333 1 .291 0.707 0.707 1 .000 1 .000 1 .000 1.581 1.581 1.291 1 .291 MICPED 0.707 0.707 1-333 1.374 0.707 0.707 1 .000 1 .000 1 .000 1.581 1.581 0.000 PHAFLA 0.707 0-333 0-333 0.707 1.155 0.707 O .707 1 .000 1.000 1 .000 1.581 1.581 0.816 0.816 STEHIL 0.707 0 .882 0.707 1.528 0.707 O .707 1 .000 1 .000 1 .000 1.581 1.581 1 .202 STEPOE 0.707 1.155 1 .202 0.707 1.491 0.707 O .707 1 .000 1 .000 1 .000 1.581 PARATA 1.5 81 1.155 1 .202 1.155 0.707 0.707 0.745 0.707 O .707 1 .000 1 .000 1 .000 1.581 1.581 0.882 LENCPII 0.707 0.943 0.882 0.707 0.667 0.707 0.707 1 .000 1 .000 1 .000 1 .581 1 -581 0.816 0.882 0.816

1—*ro -p- Table 15* Distance Matrix of OTU's Dasod on Adult Foro Lnr, Positioning Characters (cont.)

CHIRON CRYPTO CRYGAL DICFUM DieMOD DICKER DICROT ENDNIG GLYLOB GLYPAR GLYPTO PARACH POLYPE CHIRON 0.000 CRYPTO 1 .291 0.000 CRYGAL 1 .291 0.000 0 .000 DICFUM 1 .291 0.000 0.000 0.000 DICMOD 1 .000 0.1*71 0.'*71 0 .'*71 0.000 DICKER 1 .000 0.1*71 0.1*71 0 .'*71 0.000 0.000 DICROT 1 .000 0,'*71 0.'*71 0.1*71 0.000 0 .000 0.000 ENDNIG 0-333 1 -333 1.333 1 -333 1 .051* 1 .051* 1 .05'* 0.000 GLYLOB 0.333 1 -333 1.333 1 -333 1 .051* 1 .051* 1 .051* 0.000 0 .000 GLYPAR 0-333 1 -333 1 -333 1 -333 1 .051* 1 .051* 1 .051* 0.1*71 0.1*71 0.000 GLYPTO 0.000 1 .291 1.291 1 .291 1 .000 1 .000 1 .000 0-333 0-333 0-333 0.000 rARACH 1.21*7 0-333 0.333 0.333 0.333 0.333 0-333 1 .291 1 .291 1 .291 1 .21+7 0.000 POLYPE 0.000 1 .291 1.291 1 .291 1 .000 1 .000 1 .000 0.333 0.333 0-333 0.000 1 .21*7 0.000 STICTO 0.333 1 -333 1-333 1.333 1 .051* 1 .051* 1 .051* O.1+71 0.1*71 0.000 0.333 1 .291 0.333 XENXEN 1.291 0.000 0.000 0.000 0.1*71 0.1*71 0 .1*71 1.333 1-333 1-333 1 .291 0.333 1 .291 C LA DOT 0.882 0.667 0.667 0.667 0 .1*71 0 .1*71 0.1*71 0 .91*3 0 .91*3 0.816 0.882 0.577 0.882 MICROP 0.882 0.667 0.667 0.667 0 .1*71 0 .1*71 0 .1*71 0 .91*3 0 .91*3 0.816 0.882 0.577 0.882 TANYTA 0.882 1 .051* 1 .051* 1.0511 0.816 0.816 0.816 0 .91*3 0 .91*3 0 .91*3 0.882 1 .000 0.882 DENiBRA 0.471 1 .371* l .3711 1 .371* 1.106 1 .106 1.106 0.333 0-333 O .333 0.1*71 1.333 0.471 EINCHE 0.000 1.291 1.291 1.291 1 .000 1 .000 1 .000 0.333 0.333 0-333 0.000 1.21*7 0.000 MICNIG 1 .291 0.000 0.000 0.000 0.1*71 0 .1+71 0.1+71 1-333 1-333 1-333 1.291 0.333 1 .291 HARINC 1 .291 0.000 0.000 0.000 0.1*71 0 .1*71 0 .1*71 1.333 1-333 1-333 1.291 0.333 1 .291 MICPEP 0-333 1-333 1-333 1.333 1 .051* 1 .051* 1 .051* 0.000 0.000 0 .1*71 0.333 1.291 0.333 PHAFLA 0.816 1.106 1.106 1.106 0.882 0.882 0.882 0.882 0.882 0.882 0.816 1 .051* 0.816 STEHIL 1 .202 1 .**91 1 .1*91 1 .1*91 1.1*11* 1.1*11* 1 .1+11* 1.155 1.155 1.155 1 .202 1 .528 1 .202 STEPOE 1.155 1.453 1.1*53 l .1153 1.3711 1 .371* 1-371* 1 .202 1 .202 1.106 1 .155 1 .491 1.155 PARATA 0.882 0.667 0.667 0.667 0.1*71 0 .1+71 0.1*71 0.91*3 0.91*3 0.816 0.882 0.577 0.882 LENCRU 0.816 0.577 0.577 0.577 0.333 0.333 0.333 0.882 0.882 0.882 0.816 0.1*71 0.816

M- Table 15. Distance Matrix of OTU's Based on Adult Fore Leg Positioning Characters (cont.)

ST I CTO XENXEN CLADOT M1CR0P TANYTA DEMBRA K1NCHE MICNIG IIARINC MICPED PHAFLA STEHIL CTErOE

STICTO 0.000 XENXEN 1.333 0.000 CLADOT 0.816 0.667 0.000 MICRO!’ 0.816 0.667 0.000 0.000 TANYTA 0 .963 1.054 0.816 0.816 0.000 1JEMURA 0.333 1.37'* 0.882 0.882 1.000 0.000 EINCKE 0.333 1.291 0.882 0.882 0.882 0.471 0.000 MICNIG 1-333 0.000 0.667 0.667 1.054 1.374 1.291 0 .000 I1ARINC 1.333 0.000 0.667 0.667 1.054 1 .374 1.291 0.000 0.000 MIC I'Ell 0.471 1 . 333 O.9A 3 O .943 0.94 3 0.333 0.333 1.333 1.333 0.000 MIAEl.A 0.882 1 .106 0.802 0.882 O .333 0.943 0.816 1.106 1.106 0.882 0.000 GTEHIL 1.155 1.491 1.247 1.247 0.816 1.106 1.202 1.491 1.491 1.155 0.745 0.000 STEPOE 1.106 1.453 1.202 1.202 0.745 1.155 1.155 1.453 1.453 1.202 0.667 0.333 0.000 PARATA 0.816 0.667 0.000 0.000 0.816 0.882 0.882 0.667 0.667 0.943 0.882 1.247 1.202 LENCRU 0.882 0.577 0.333 0.333 0.745 0.943 0.816 0.577 0.577 0.882 0.816 1.291 1.247

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217 Table 16. Distance Matrix of OTU'g Based on Characters of Adult Self-groominp (cont.)

CRISP4 CRJSP5 CRICOT NANDIS ORTHOC S MIS PI SMISP2 SMITTI CORYNO THIENE CHIATT . CHIPLU CHIRir CRISPS 0 .000 CRISP5 0 .[[71 0.000 CRICOT 0.385 0.544 0.000 [IAMBIC 0.471 0.609 0.385 0.000 ORTHOC 0.832 0.832 0.609 0-555 0.000 SM13P1 0.588 0.588 0 .480 0 .480 0.566 0.000 SM1SP2 0.620 0.555 0.480 0 .480 0.620 0.447 0 .000 SMITTI 0.69k 0.793 0.509 0.430 0.439 0.392 0.430 0.000 CORYNO 0.809 0.760 0.650 0.519 0.439 0.600 0.5i9 0.480 0.000 TUIENE 0.650 0.650 0.555 0-555 0.632 0.392 0.600 0.555 0.721 0.000 CHIATT 1 .000 0.979 0.935 0 .800 0.645 0.816 0.890 0.791 0.791 0.809 0.000 CHIP1.U 0.979 0.957 0.913 0.775 0 .612 0.791 0.866 0 .764 0.764 0.748 0.196 0.000 CIIIR1P 1 .000 0.933 0.885 0.816 O .692 0.752 0.780 0 .808 0.780 0.707 0.346 0.283 0.000 CHIRON 1 .000 0.979 0.935 0.800 0.645 0.816 0.890 0.791 0.791 0 .809 0.000 O.I96 0.346 CRYPTO 0.913 0.890 0.890 0.748 0.645 0.816 0.842 0.791 0.736 0.775 0.277 0.196 0.346 CRYGAL 0.913 0.890 0.842 0.693 0.577 0.707 0.791 0.677 0.677 0.721 0.277 0.196 0.346 DICFUM 0.979 0.957 0.913 0.775 0.612 0.791 0.866 0.764 0.764 0.721 0.277 0.196 0.346 DICMOD 0.808 0.780 0.780 0.659 0.590 0.722 0.752 0.692 0.626 0.677 0.540 0.500 0.590 DICNER 0.866 0.842 0.791 0.632 0.577 0.707 0.791 0.677 0.677 0.632 0.439 0.392 0.490 DICROT 0.816 0.791 0.791 0.632 0.577 0.707 0.736 0.677 0.612 0.693 0.439 0.392 0.490 ENDNIG 0.977 0.905 O .929 O .905 0.769 0.826 0.826 0.879 0.826 0.826 0.564 0.522 0.426 GLYLOB 0.957 0.935 0.890 0.791 0.577 0.764 0.842 0.736 0.736 0.721 0.283 0.200 0.354 GLYPAR 0.957 0.935 0.890 0.748 0.577 0.764 0.842 0.736 0.736 0.764 0.283 0.200 0.354 GLYPTO 0.979 0.957 0.913 0.775 0.612 0.791 0.866 0.764 0.764 0.748 O .196 0 .000 0.283 PARACH 0.913 0.890 0.842 0.693 0.645 0.764 0.842 0.736 0.736 O.76O 0.272 0.340 0.447 POLYPE 0 . 979 0.957 0.913 0.775 0.612 0.791 0.866 0.764 0.764 0.748 O.I96 0.000 0.283 STICTO 1.041 0.979 0.935 0.800 0.645 0.816 0.842 0.791 0.736 0.775 0.277 0.196 0.200 XENXEN 0.707 0.736 0.736 0.600 O .707 0.707 0.791 0.736 0.791 0.679 0.544 0.588 0.691 CLADOT 0.853 0.826 0.853 0.826 0.674 0.739 0.798 0.798 0.798 0.692 0.417 0.361 0.361 MICROP 0.956 0.885 0.834 0.722 0.659 0.752 0.780 0.722 0.659 O .736 0.45b 0.408 0.417 TANYTA 1 .022 0.953 0.905 O .834 0.739 0.798 0.826 0.853 0.82b 0.722 0.408 0.354 0.204 218 Table 16. Distance Matrix of OTU's Based on Characters of Self-grooming (cont.)

CHIRON CRYPTO CRYGAL DICFUM DICIWOD D ]CMFR DICROT ENDNIG GLYLOB GLYPAR GLYPTO FARACH FOLYFE CHIRON 0.000 CRYPTO 0.277 0.000 CRYGAL 0.277 0.277 0.000 DICFUM 0.277 0.277 0.277 0.000 DICMOD 0.590 0.956 0.956 0.956 0.000 D1CNER 0.939 0.939 0.390 0.390 0.359 0.000 DICROT 0.939 0.390 0.390 0 . 390 0 .289 0.277 0.000 ENDNIG 0.569 0.522 0.569 0.522 0.690 0.707 0.679 0.000 GLYLOB 0.283 0.283 0.283 0.283 0.956 0.997 0.997 0.977 0.000 GLYPAR 0.283 0.283 0.283 0.200 0.511 o.9oo 0.900 0.977 0.289 0.000 GLYPTO 0.196 O.I96 O.I96 0.196 0.500 0.392 0.392 0.522 0.200 0.200 0.000 FARACH O .272 0.392 0.277 0.392 0.500 0.3M0 0.9 39 0.690 0.900 0.900 0 .390 0.000 POLYPE 0.196 O.I96 0.196 O.I96 0.500 0.392 0.392 0.522 0.200 0.200 0.000 0.390 0.000 STICTO 0.277 0.277 0.277 0.277 0.590 0.939 0.939 0.9?? 0.283 0.283 0.196 0.392 0.196 XENXEN 0.599 0.555 0.555 0-555 0.956 0.9 39 0.919 0.826 0.600 0,600 0.588 0.971 0.588 CLADOT 0 .917 0.361 0.91? 0.917 0.522 0.552 0.511 0.369 0.295 0.926 0.361 0.511 O.36 I MICROP 0.956 0.956 0 .908 0.956 0.511 0.956 0.590 0.535 0.359 0 .966 0.908 0.908 0.908 TANYTA 0 .908 0.908 0 .908 0.908 0.552 0.500 0.590 0.988 0.917 0.917 0.359 0 .956 0.359

STICTOXENXEN CIA DOT M1CROF TANYTA

STICTO 0.000 XENXEN 0.679 0 .000 CLADOT 0 .917 0 .626 0 .000 MICROP 0.359 0.677 0.977 0.000 TANYTA 0.289 0.707 0.926 0.926 0.000

DO VO 220 Table 17. Three Dimensional MDS of OTU's Based on Characters of Adult Self-grooming. Stress is 0.181.

Dim. 1 D i m . 2 Dim. 3

CLIPIN -0.795 -0.495 -0.337 COELOT -0.935 -0.001 -0.460 PROSUB -0.500 0.165 - O .696 PROBEL -0.568 0.426 -0.603 PROCLA -0.599 0.129 -0.706 ABLMAL 0.285 1 .184 0 .028 CONCHA 0.44-7 1.235 0.175 TANSTE -0.113 0.977 -0.697 PSEDYA -0.641 0.119 -0.822 ACRSEN -0.392 -0.868 0.099 CRIBIC -0.670 -0.538 0.999 CRITRI -0.658 -0.506 0 .240 CRISP3 -0.549 -0.473 0.660 CRISP4 -0.555 -0 .880 1 .507 CRISP5 -0.272 -0.633 1 .638 CRICOT -0.928 -0.691 0.917 NANDIS -0.575 -0.661 0.643 ORTHOC -0.565 -0.274 -0.206 S MIS PI -0.628 -0.117 0 .909 SMISP2 -1.006 -0.129 0.801 SMITTI -0.942 -0.277 0.395 CORYNO -1.001 0.133 0.227 THIENE -0.231 -0.072 1.042 CHIATT 0.716 0.071 -0.487 CHIPLU 0.613 0.136 -0.426 CHIRIP 0.589 0.360 -0.410 CHIRON 0.713 0.067 -0.480 CRYPTO 0.642 0.094 -0.362 CRYGAL 0.550 0.033 -0.277 DICFUM 0.603 0.068 -0.423 DICMOD 0.480 - 0.298 0.121 DICNER 0.565 -0.314 -0.093 DICROT 0.546 -0.265 -0.067 ENDNIG 0.378 0.945 -0.370 GLYLOB 0.505 0.179 -0.386 GLYPAR 0.499 0.122 - 0.448 GLYPTO 0.616 0.139 -0.425 PARACH 0.752 -0.116 -0.213 POLYPE 0.615 0.138 -0 .424 STICTO 0.564 0.235 -0.503 XENXEN 0.735 -0.679 0.398 CLADOT O .613 0.455 -0.037 MICROP O.38O o.4io -0.149 TANYTA 0.715 0 .469 - 0.320 APPENDIX B: COLLECTION LOCALITIES AND NOTES

Clark Hollow tributary of Pike Run in Tar Hollow State

Forest; Ross Co., Ohio; on south side of Forest Road

#3, 3-^ km west of its junction with S.R. 327, and 15

km south of Adelphi; stream 1 .5 m wide, 0.1 m deep; wa­

ter clear with moderate flow; substrate rock and gravel

with leaves and organic debris along its banks; no

aquatic vegetation; fauna included Ephemeroptera nymphs

and Trichoptera larvae; Corynoneura larvae found among

the leaves; March 3» 197^-

Ohio Canal; Licking Co., Ohio; 3 km SW of Hebron and the

intersection of U.S. 40 and S.R. 79; canal about 25 m

wide with unknown depth, collected at depths of 1 m or

less; water with moderate turbidity and current unappar-

ent; substrate rock, gravel, and silt; Typha along west

bank; fauna included Oligochaeta, Hirudinea, Copepoda,

Cladocera, Amphipoda, Ephemeroptera nymphs, and

Coleoptera adults; Chironomidae included six species

from six genera; March 20, 197^ (larvae), water temper­

ature 5° C .

O.S.U. Farm Pond; Franklin Co., Ohio; 5-7 km NW of the

intersection of Broad and High Streets in Columbus;

pond 60 m long, 25 m wide, with a maximum depth of 2 m; 221 water with a distinct brownish coloration; substrate

sand, gravel, and organic debris; flora included Typha,

Salix, and dense mats consisting of Lenina, Spirogyra,

Ulothrix, Oedogonium, and other filamentous green

algae; fauna included Oligochaeta, Hirudinea,

Gastropoda, Ostracoda, Cladocera, Amphipoda, Odonata

nymphs, Dytiscidae, Corixidae, Naucoridae, and

Notonectidae; Chironomidae included fifteen species

from fourteen genera; May 12, 1975 (larvae), water tem­

perature 19*5° C; March 2, 1976 (adults), air tempera­

ture 19° C; March 19 $ 1976 (larvae), water temperature

7° C; August 31i 1976 (larvae), water temperature 20° C.

Scioto River at Hoover Park (now Griggs Reservoir Park);

Franklin Co., Ohio; 8.75 km WNW of the intersection of

Broad and High Streets in Columbus; river about 60 m

wide at this point and of unknown depth; water moder­

ately turbid with a slow current; substrate of sand,

gravel, rock, and boulders; beds of Justicia in shallow

areas with filamentous algal growth on most rocks;

fauna included Oligochaeta, Tricladida, Gastropoda,

Pelecypoda, Copepoda, Ostracoda, Amphipoda,

Ephemeroptera nymphs, Zygoptera nymphs, Elmidae, and

Ceratopogonidae larvae; Chironomidae included twenty-one

species from seventeen genera; October 6, 1975 (larvae),

water temperature 19-5° C; October 27 f 1975 (adults),

air temperature 12° C; March 2, 1976 (adults), air tem­

perature 20° C; May 19 f 1976 (larvae), water temperature 14 C; September 20, 1976 (larvae), water

temperature 20° C .

Miller-Antrim Quarry lake; Franklin Co., Ohio; 1.75 km SW

of Worthington; lake 700 m long, 275 m wide, and of

unknown depth; very little turbidity; substrate sand,

mud, organic silt, gravel, and rock; beds of

Potamogeton, Chara, and filamentous green algae growing

from the substrate in shallow water; fauna included

Oligochaeta, Tricladida, Copepoda, Ostracoda, Cladocera,

Ephemeroptera nymphs, Odonata nymphs, Ceratopogonidae

larvae, and Chaoboridae larvae; Chironomidae included

thirty species from twenty-three genera; June 11, 1974

(larvae), water temperature 23*5° C; July 11, 1974 (lar­

vae), water temperature 28° C; July 16, 1974 (adults);

June 3, 1975 (larvae), water temperature 21.5° C; June

30, 1975 (larvae); May 10, 1976 (larvae), water temper­

ature 16° C; June 6 , 1976 (adults), air temperature 16°

C; June 20, 1976 (adults); August 26, 1976 (adults),

air temperature 21° C; September 7 , 1976 (larvae),

water temperature 23° C; October 4, 1976 (adults).

Olentangy River; Franklin Co., Ohio; 1.75 km SW of

Worthington; riffle area varies from less than 10 m to

30 m wide with a maximum depth of 0.5 m; water with low

turbidity and rapid flow; substrate rock, gravel, and

sand; shallow areas with large beds of rooted Justicia;

fauna included Oligochaeta, Hirudinea, Tricladida,

Gastropoda, Pelecypoda, Copepoda, Amphipoda, Isopoda, 22k Ephemeroptera nymphs, Plecoptera nymphs, Trichoptera

larvae, Zygoptera nymphs, Elmidae, Psephenidae larvae,

Ceratopogonidae larvae, and Simuliidae larvae;

Chironomidae included seventeen species from fourteen

genera; July 15, 1975 (larvae), water temperature 22.5°

C; April 29, 1976 (larvae), water temperature 10° C;

September 20, 1976 (larvae), water temperature 19*5° C.

Alum Creek at Schrock Road; Franklin Co., Ohio; 1.5 km SW

of Westerville; creek divides south of bridge, spur k m

wide and 0.25 m deep in the riffle area, while the main

creek is 20 m wide and 2 m maximum depth; water moder­

ately turbid with rapid flow in the riffle and slower

in the main creek; Westerville sewage treatment outflow

is 0.5 km upstream; beds of Justicia along the riffle;

fauna included Oligochaeta, Hirudinea, Tricladida,

Gastropoda, Pelecypoda, Copepoda, Decapoda,

Ephemeroptera nymphs, Elmidae, Tipulidae larvae, and

Simuliidae larvae; Chironomidae included thirty-one spe­

cies from twenty-three genera; March 19, 1976 (larvae),

water temperature 5-5° C; April 5, 1976 (larvae), water

temperature 7° C; April 15, 1976 (larvae), water temper­

ature 12° C; April 20, 1976 (adults), air temperature

18° C; May 27, 1976 (adults), air temperature 16° C.

Bay Point Marina on Lake Erie; Ottawa Co., Ohio; sampled

at 3 m depth; water very turbid from algal blooms; sub­

strate organic silt and ooze; Oligochaeta and

Chironomidae the only fauna; Chironomidae included 225 eleven species from eight genera; August 19. 1974 (lar­

vae), water temperature 28° C; August 5, 1975 (larvae),

water temperature 27° C; August 15, 1975 (larvae); June

29, 1976 (larvae), water temperature 25° C; July 3,

1976 (larvae), water temperature 21° C; August 4, 1976

(larvae), water temperature 23*5° G.

Bay Point Trailer Court on Lake Erie; Ottawa Co., Ohio;

ideal location for collecting adult Chironomidae around

lights; Chironomidae included twenty-three species from

fifteen genera; August 22, 1974; August 3, 1975; August

5, 1975; August 6, 1975; August 11, 1975; June 27, 1976;

July 2, 1976; July 3, 1976; August 1, 1976; August 3,

1976.

Bay Point lagoon on Lake Erie; Ottawa Co., Ohio; lagoon

480 m long, 120 m wide, with a maximum depth of 2 m;

water with a high turbidity; substrate sand, organic

silt, and ooze; flora consisted of Nelumbo, Nuphar,

Sagittaria, Typha, and Lemna; fauna included

Oligochaeta, Copepoda, Zygoptera nymphs, and

Chaoboridae larvae; Chironomidae included seven species

from five genera; August 11, 1975 (larvae), water tem­

perature 29° C; July 1, 1976 (larvae), water tempera­

ture 22° C .

Bay Point swamp forest on Lake Erie; Ottawa Co., Ohio;

water area 280 m long, 180 m wide, with a depth usually

no more than 0.5 m; water moderately turbid; substrate

sand and organic silt; flora included Cabomba and Lemna; 226 Chironomidae included two species of Tanypus; August 13»

1975 (larvae); August 4, 1976 (larvae).

Sandusky Bay at Bay Point; Ottawa Co., Ohio; collected

along west shore of Bay Point at a depth of 1 m; water

moderately turbid from wave action; substrate sand with

some organic silt; no aquatic vegetation; Oligochaeta

and Chironomidae the only fauna; Chironomidae included

eight species from six genera; August 13» 1975 (larvae),

water temperature 28° C; August 4-, 1976 (larvae), water

temperature 23*5° C.

Clear Fork of the Mohican River, under covered bridge in

Mohican State Forest; Ashland Co., Ohio; 7*25 km WSW of

Loudonville; 15 m wide and 0.5 m deep; water usually

clear with a moderate flow; substrate rock, sand, and

gravel with silt and algae usually covering the tops of

the rocks; beds of Anacharis (=Elodea) common; fauna

included Hirudinea, Tricladida, Gastropoda, Amphipoda,

Ephemeroptera nymphs, Zygoptera nymphs, Elmidae larvae

and adults, Gyrinidae, and Corixidae; Chironomidae in­

cluded nine species from nine genera; September 9> 197^

(larvae); November 6, 1975 (larvae), water temperature

14° C.

Ohio Agricultural Research and Development Center cattle

spring; Wayne Co., Ohio; 3 km SE of Wooster; cement en­

closed spring for cattle 1.5 m in diameter and 0.5 m

deep with clear, cool water; substrate an organic ooze

that is periodically cleaned out; fauna included Oligochaeta, Ephemeroptera nymphs, and Dytiscidae;

Chironomidae included nine species from eight generaj

September 21, 1975 (larvae); July 25, 1976 (larvae),

water temperature 20.5° C; August 21, 1976 (adults),

air temperature 1 ^.5° C.

Open Pond Campsite in Conecuh National Forest; Covington

Co., Alabama; collected adult chironomids at lights

while camping overnight; Chironomidae included five spe­

cies from four genera; December 7, 1975*