<7
BEHAVIOURAL ECOLOGY OF THE LONGNOSE DACE,
RHINICHTHYS CATARACTAE (PISCES, CYPRINIDAE):
SIGNIFICANCE OF DACE SOCIAL ORGANIZATION
by
VICTOR GEORGE BARTNIK
B.Sc. (Hons.), University of Manitoba, 1968 M.Sc, University of Manitoba, 1970
A THESIS SUBMITTED IN PARTIAL FULFILMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
,in the Department
of
ZOOLOGY
We accept this thesis as conforming to the
required standard
THE UNIVERSITY OF BRITISH COLUMBIA
December, 1973 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study.
I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.
Department of ZOOLOGY
The University of British Columbia Vancouver 8, Canada
Date ii
ABSTRACT
The social behaviour of a stream population of longnose dace,
Rhinichthys cataractae dulcis, is described. Both males and females
occupy the same riffle areas and defend territories (approx. 10 cm in
diameter) during the breeding phase. In addition, male dace are
evidently attracted to and interact agonistically with other males
resulting in the formation of clusters of male territories. During the night, females leave their territories to court and spawn with terri•
torial males. After spawning, males show strong nest site attachment
and are inhibited from eating eggs. Parental males remain directly over
the nest site and frequently probe the substrate with their snouts.
They defend the area against all fish with the exception of receptive
females with which they may spawn.
Comparison with a stream population of the sub-species, R. cataractae
cataractae, reveals differences in phenotypic appearance, diurnal rhythm of breeding activity, and female territoriality. Unlike R.c.. dulcis,
R.c^. cataractae are reproductively active during daylight hours and males display bright nuptial coloration. Breeding coloration in males of
R.£. dulcis is faint or absent. Unlike R.c. dulcis, males and females of R.c. cataractae are segregated into different habitats and females remain non-territorial.
Experimental analyses of the physical and biotic factors involved in the causation of territorial behaviour were conducted in a laboratory stream tank. The major physical factor involved in the selection of territories by male dace is the presence of coarse gravel suitable for iii
spawning. Female dace select and defend only enclosed areas which
provide both overhead cover and shelter from current. Females prefer
shelters which contain food and thus the presence of food may be a
proximate factor involved in the selection of territories by female
dace. Laboratory tests indicate that female territorial behaviour is
elicited by interaction with breeding males. Outside the breeding phase,
when territoriality is relaxed, male and female dace differ little in
the areas they choose to occupy.
The experimental evidence suggests that the functions of male
territory are: i) provision of space in which males can court and spawn with females with minimal interference from other males, and ii) protection
of eggs from intraspecific predation. For females, territoriality
apparently functions to reduce conflicts with males (i.e., reduce court•
ship harassment and attacks from males). Changes in the behaviour of males occur after spawning and coincide with periods of greatest egg vulnerability. These post spawning behavioural activities of males protect
freshly deposited eggs while making them less accessible to predators.
The evidence also suggests that a major function of territory in both sexes is the provision of shelter from current. Dace not defending a
shelter may be forced to make frequent movements in the strong current of
the riffle habitat. Dace swimming against strong currents for even short periods (i.e., 5 min) become fatigued and lose their ability for co• ordinated locomotion. Such stressed individuals may be vulnerable to predation. The data also suggest that dace territorial behaviour may act as a dispersing mechanism, thereby limiting the density of breeding fish in a localized area. iv
Territorial males of clustered groupings remain behaviourally synchronized. They display strong site attachment and interfere little with the reproductive activities of neighbouring males. Consequently, these social groupings function to further reduce interference from conspecifics. Assemblages of more widely spaced territorial males,
created in the laboratory, experienced greater intraspecific interference and egg predation than did males of clustered territorial groupings. Thus it seems most probable that intraspecific interference and egg predation have provided a major selection pressure favouring both male territori• ality and territory clustering in longnose dace. :.v
TABLE OF CONTENTS
Page
LIST OF TABLES xi
LIST OF FIGURES . .... xiii
ACKNOWLEDGEMENTS xvli
INTRODUCTION 1
MATERIALS AND METHODS . 4
A. Study Area 4
B. Holding Conditions ^
C. Description of Stream Tank 6
D. Experimental Channels 6
E. Experimental Conditions 8
F. Experimental Fish 9
SECTION I: THE SOCIAL BEHAVIOUR OF LONGNOSE DACE • H
A. Description of the Behaviour of R.c_. dulcis H
a. Reproductive Behaviour H
b. Agonistic Behaviour Patterns Recorded During 1*
Observations
c. Courtship Behaviour Patterns Recorded During
Observations ; ^
d. Egg Eating Inhibition ^
i. Outline of Experiment ^
ii. Procedure 17
iii. Experimental/Fish 18
iv. Results and Discussion 18
e. Comparison with R. £. cataractae 20 Page
B. Summary and Discussion . 27
SECTION II: FACTORS INVOLVED IN THE CAUSATION OF TERRITORIAL
BEHAVIOUR AND THE SELECTION OF A TERRITORY 31
A. Breeding Phase 32
a. Experimental Analysis of the Factors Involved
in the Selection of Territories by Male and
Female Dace 32
i. Outline of Experiment ;. 32
ii; Procedure. 35
iii. Experimental Fish...... 37
iv. Results and Discussion 37
b. Social Factors Affecting Territorial Behaviour
in Female R. c_._ dulcis 45
i. Outline of Experiment 45
ii. Procedure...... 45
iii. Experimental Fish . 46
iv. Results and Discussion 46
c. Social Factors Affecting Territorial Behaviour
in Female R.c:. cataractae ...... 49
i. Outline of Experiment.... 49
ii. Procedure 49
iii. Experimental Fish 51
iv. Results and Discussion 51
d. Social Factors Affecting Territorial Behaviour
in Male R.c. dulcis 56 via.
Page
e. Clustering of Male Territories... 58
i. Outline of Experiment 58
ii. Procedure . ... ,. 60
iii. Experimental Fish 62
iv. Results and Discussion .. ... 62
f. Behaviour of Clustered Territorial Male Dace... 66
i. Outline of Experiment 66
ii. Procedure 67
iii. Experimental Fish • ^
iv. Results and Discussion ... 69
g. The Role of Food in the Distribution of Dace
Territories 73
i. Outline of Experiment... 73
ii. Procedure 73
iii. Experimental Fish 75
iv. Results and Discussion , 75
B. Non-Breeding Phase .... 80
a. Site Preferences of Male and Female Dace in
Late Summer and Winter...... 80
i. ' Outline of Experiment 80
ii. Procedure 82
iii. Experimental Fish 82
iv. Results and Discussion .... 82 viii
Page
b. Seasonal Variation in the Spacing Patterns
of Dace 88
i. Outline of Experiment...... 88
ii. Procedure 88
89
iii. Experimental Fish
iv. Results and Discussion 89
c. Role of Food in the Distribution of Dace in
LateSSummer 93
i. Outline of Experiment 93
ii. Procedure .... 93
iii. Experimental Fish 93
iv. Results and Discussion • 94
C. Spacing Patterns of Dace Fry/ 94
i. Outline of Experiment 94
ii. Procedure 97
iii. Results and Discussion...... 97
D. Discussion of the Causation of Territorial Behaviour 99
SECTION III: THE FUNCTIONAL SIGNIFICANCE OF THE SOCIAL
ORGANIZATION OF DACE.. 104
A. Introduction 104
B. Inferences from Observations and Experiments in
Section II as to the Function of Various Aspects
of Territorial Behaviour 105 ix
Page
C. Experimental Tests of Hypotheses Regarding Function.... 106
a. Significance of Territory Clusters 106
i. Outline of Experiment.. .. 106
ii. Procedure.....:. ••• 107
iii. Experimental Fish •••• 109
iv. Results and Discussion 110
b. Significance of Post Spawning Changes in Male
Behaviour 118
c. Significance of Male Territory as Determined by
Vulnerability of Dace Eggs to Intraspecific
Predation .. . 122
i. Outline of Experiment ...... 122
ii. Procedure...... 122
iii. Experimental Fish 124
iv. Results and Discussion 125
d. Other Predators ... 129
i. Trout...... 129
ii. Sculpins • • 130
iii. Egg Predation... 131
e. Significance of Female Territory as Determined
by Changes in Blood Lactate in Dace after
Exercise 134
i. Outline of Experiment 135
ii. Procedure . 136
iii. Experimental Fish 138
iv. Results and Discussion 139 X
Page
f. Territorial Behaviour as a Dispersing
Mechanism 141
i. Outline of Experiment 141
ii. Procedure..... •• 142
iii. Experimental Fish 144
iv. Results and Discussion... 144
D. Discussion of the Significance of Social Organization
in Dace • • • • 148
LITERATURE CITED • 154 XX
LIST OF TABLES
Table Page
I. . Number of eggs eaten by different types of dace
presented with recently fertilized eggs 19
II. Agonistic behaviour patterns commonly performed by
R.-c. cataractae and R._c. dulcis during the breeding phase.. 24
III. Environmental features incorporated into experimental
structures.... 34
IV. Mean number of agonistic acts won per 30 min by male
and female dace against the same and opposite sex during
day and night • 39
V. Total number of agonistic acts won by males against other
males at coarse substrate structures during night 56
VI. Actual mean distance between four territorial male dace
as compared with maximum and minimum mean distances
attainable 63
VII. Actual mean distance between four territorial male dace
as compared with maximum and minimum mean distances
attainable before and after screen divider was removed 63
VIII. Mean number of agonistic acts won per food and no food
area by male and female dace against the same and opposite
sex during day and night 78
IX. Mean number of agonistic acts won per area per 30 min
by male and female dace during late summer 83
X. Agonistic behaviour patterns commonly performed by
breeding and non-breeding dace '. 84 xii
Table Page
XI. Mean number of agonistic acts won and feeding acts
performed by dace per food, no food, and open area
during late summer.... 95
XII. Number of substrate contacts, agonistic acts won,
and eggs eaten by different types of dace at nests
spawned in closely and widely spaced territorial
groupings 112
XIII. Number of eggs eaten by dace in Experiments A and B.... 117
XIV. Number of substrate contacts made, and eggs eaten
by transient and parental dace at 0, 4, 24, and 72
hr old nests • 127
XV. Frequency of occurrence of eggs in Cbttus asper. Cj.
aleuticus, and Salmo gairdheri stomachs when only
Rhinichthys eggs (1971) or Mylocheilus eggs (1972)
were abundant in riffles from where sculpins and
trout were collected 133
XVI. Frequency of occurrence of eggs in Rhinichthys
cataractae stomachs when Rhinichthys eggs were
abundant in riffles from where dace were collected
(1971) 133
XVII. Blood lactate values for (A) Non-exercised and (B)
Exercised dace 140 xiii
LIST OF FIGURES
Figure Page
1. South Alouette River and locations of stations . ... 5
2A. Top view of the stream tank 7
2B. Observational channel of the stream tank 7
3. Diel periodicity of agonistic and courtship activity
of R._c. dulcis 12
4. Time intervals over which dace spawnings were observed
or occurred but were not observed • 13
5. Rhinichthys cataractae cataractae and R.c dulcis males
from Mink River, Manitoba, and Alouette River, British
Columbia . .. 22
6. Distribution of adult dace in rock riffles during pre-
breeding and breeding periods •.. 26
7. Diagramatic sketches of six combinations of structures
and bottom substrates placed in the experimental channel. 33
8. Mean number of agonistic acts won per area per 30 min
by breeding male and female dace during day and night.... 38
9. Total number of agonistic acts won and lost by
individual male and female dace at different areas in
Replicates I, II, and III ..... 42
10. Mean number of courtship acts performed per area per
30 min by male and female dace during day and night 44
11. Percent occurrence of different group sizes in Channel
1 and Channel 2 . . 47 xiv
Figure Page
12. Total number of agonistic acts won by R.c^. dulcis
females in Channel 1 and Channel 2 ...... 47
13. Number of agonistic acts won by male and female
R.J;. cataractae against the same and opposite sex
during 3 days of 'forced' interaction and a fourth
day not forced , 52
14. Frequency of occurrence of agonistic behaviour
patterns performed by R.cataractae females during
3 days of 'forced' interaction with males 54
15. Pattern of agonistic and courtship activity performed
by dace during the 72 hr before spawning at preferred
structures 57
16. Typical sequence of agonistic and courtship activity
performed at a preferred structure 59
17A. Arrangement of structures in channel (top view)... 61
17B. Pattern of agonistic activity performed by territorial
males during eight successive night observation
periods 61
18. Frequency of substrate probing and time spent on nest
by parental males with and without interaction with
conspecific males • 7u
19. Latency to attack by parental males for each of five
successive interactions with intruding conspecific
males ^2 XV.
Figure Page
20. Mean number of agonistic, courtship, and feeding
acts performed per food and no food area by male
and female dace during day and night 76
21. Percent occurrence and total number of agonistic
acts won at food, no food, and open areas by male
dace during day and night...... 81
22. - Percent occurrence of male and female dace at
different areas during late summer...... 85
23. Percent occurrence of male and female dace at
different areas during winter 87
24. Percent occurrence of different group sizes of dace
during spring, late summer, and winter seasons 90
25A. Relationship between total number of agonistic acts
performed and mean group sizes of dace during spring,
late summer, and winter seasons..... 91
25B. Relationship between total number of agonistic acts
performed, number of agonistic acts won, and different
group sizes of dace during spring, late summer, and
winter seasons...... 91
26. Mean percent occurrence of male and female dace per
food, no food, and open area during late summer 96
27. Arrangement of BC structures in channel to induce
(A) closely spaced or clustered male territories and
(B) more widely spaced male territories 108 xvi.
Figure Page
28. Mean number of substrate contacts, agonistic acts
won against transient males, and eggs eaten by
different types of male dace at nests spawned in
clustered and widely spaced territorial groupings...... m
29A. Total number of agonistic acts won by male and female
dace in experiments A and B before and after spawning... 115
29B. Levels of agonistic activity performed at the nest
site in experiments A and B before and after spawning... 115
30. Agonistic activity performed by a territorial male
before and after spawning .... 120
31. Number of eggs eaten and substrate contacts made by
transient males at 0, 4, 24, and 72 hr old nests...... 126
32. Diagram of swim tunnel used to exercise dace...... 137
33A. Diagram of experimental channel illustrating riffle
and pool zones 143
33B. Group sizes and distribution of dace at increasing
densities during breeding and non-breeding phases...... 143
34. Relationship, between percent occurrence of dace in
pool and mean number of agonistic acts won per 30
min at increasing densities during breeding and non-
breeding phases ...... 146 r xvii.
ACKNOWLEDGEMENTS
I thank Dr. N.R. Liley, my supervisor, for his encouragement
during the study and his many helpful criticisms of the manuscript.
I am most grateful to Dr. J. M. Cullen for his stimulating discussions with me. Drs. J.R. Krebs, T.G. Northcote,and J.D. McPhail also
provided valuable suggestions and guidance.
I am indebted to all the following people who helped me pull
a seine: N. Stacey, B. Wishlow, J. MacLean, J. McClurg,and E. Kliewer.
Blood lactate analyses were done with the help of Mr. R. Stanley.
Finally I wish to thank my wife, Gwen, whose assistance both in the
field and lab made this study possible.
Financial support for both the research and the author was
provided through a Fisheries Research Board Block Grant. 1
INTRODUCTION
Fishes show all types of dispersion from basically solitary to highly organized schooling, and further, the dispersion frequently
changes from one type to another during development. Seasonal cycles modify social behaviour markedly. The seasonal temperature change
and the sexual cycle cause perhaps the most prominent differences in
the attitudes of individuals.
Although social organizations have been studied intensively in
a variety of fish species, attention has usually been given to des•
criptive aspects alone. Such studies give rise to interesting questions
concerning the biological significance of these social responses in
nature. Answers are frequently suggested but rarely accompanied by
objective evidence. A notable exception is the recent study of the
significance of territoriality in three-spined sticklebacks conducted
by van den Assem (1967).
The longnose dace, Rhinichthys cataractae (Valenciennes), has the
widest distribution of any North American cyprinid. It occurs from
coast to coast in Canada, as far south as the Rocky Mountains in Mexico,
and as far north as the Mackenzie River near the Arctic Circle (Carl
et al. , 1959). Longnose dace are found in swiftly flowing streams and
occasionally in lakes. Ecological studies conducted by Becker (MS,
1962) , Gee and Northcote (1963) , and Bartnik (1970) have revealed that
the adults of this cyprinid occur mainly in spaces between stones in the
rapids areas of streams where they feed primarily on bottom insects. 2
As a consequence of a strong preference for fast water habitats, longnose exhibit highly contagious distributions within streams. The terete shape and negative buoyancy of older dace are adaptations to life in swift flowing waters. Newly emerged longnose are neutrally buoyant and aggregations occupy shallow still waters along shores.
Later in their first year, these fish move into areas of higher water velocities (Gee, 1968).
Carl e_t al. , (1959) state that the adult longnose dace is apparently a solitary rather than a schooling fish. However, with exception of recent studies of breeding habits (Bartnik, 1970, 1972), there is virtually nothing in the literature on the behaviour of this ubiquitous species. A substantial population of Khinichthys cataractae dulcis near the University of British Columbia at Vancouver made this species suitable for experimental investigation of several aspects of social behaviour.
The aims of this study were: 1) to provide an accurate description of dace social behaviour, 2) to determine the causal factors involved in the particular social organization observed, and 3) to present ob• jective evidence as to the functional significance of dace social organi• zation.
Dace were studied during both breeding and non-breeding phases of their life cycle. The physical and biotic factors involved in the causation of territorial behaviour and the selection of a territory by dace were first determined through experimentation. These experiments on causation gave rise to hypotheses about the function of dace 3 territorial behaviour which were then tested. The results are related to work on other species and the social organization of dace as an adaptive and adaptable system is discussed. 4
MATERIALS AND METHODS
A. Study Area
The South Alouette River originates at the west end of Alouette
Lake and flows westward, to Pitt River, a tributary of the Fraser.
Figure 1 shows stations along the portion of the river from which fish were collected. Upper reaches of the river are characterized by large pools and stretches strewn with large boulders. Lower sections lie in meadow land where the bottom is composed of fine gravel, sand, and mud.
Only the high gradient middle portion, characterized by a series of
fast flowing rapids areas with rock and gravel bottom, supports a
sizeable population of longnose dace, Rhinichthys cataractae dulcis.
Highest mean monthly flows in the Alouette generally occur in
January and the lowest discharges in July and August. Detailed informa•
tion on patterns of zonation of fishes, temperature, discharge patterns,
and gradient characteristics of the South Alouette River have been pub•
lished by Withler (1966), Hartman (1968), and Hartman and Gill (1968).
B. Holding Conditions
Collections were made with a two-man seine (3 meshes/cm). Dace
were taken from riffles by kicking the substrate thus driving fishes
into a seine held downstream. Dace collected were returned to the
laboratory where they were held in 20, 40, and 60 1 glass aquaria, large
plastic containers (40 X 59 X 49 cm), and 400 1 wood and glass aquaria.
All holding tanks contained one or more 19 cm long air-diffusor stone.
These were located on the bottom at one end to produce a circular current 5 6.
on a vertical plane as described by Gee and Bartnik (1969) . The bottom of each tank was covered with a number of rocks (5-15 cm diameter).
Dace were held under temperature and photoperiod regimes comparable to those in nature. Laboratory temperature, controlled by a thermostat, had a lower limit of 14C. Additional cooling for holding tanks was provided by cooling coils or baths. Photoperiod was regulated by a time
clock connected to six ceiling-mounted fluorescent light fixtures each holding two 1.22 m tubes. Dace were fed Tetramin flakes, frozen brine shrimp, and tubifex worms.
C. Description of Stream Tank
Since shallow turbulent waters make field observations nearly
impossible, a flow tank was used for observation and experimentation.
The stream tank was a closed system with circulation on a horizontal
plane. The fiberglas tank consisted of two straight troughs (1.83 X .46
X .38 m) joined together by semi-circular elbows (0.91 m radius) at either
end (Fig. 2A) . The straight troughs contained glass windows (25 X 168 cm)
on both sides through which observations could be made. Flow was created
by four submersible water pumps each with a capacity of 2536 1 per hr.
Water temperature was controlled by a 7.3 m length of 2 cm diameter glass
cooling coil located on the floor of the tank.
D. Experimental Channels
Most experiments were carried out in the straight glass walled
sections of the stream tank (Fig. 2B). With the aid of stainless steel 7.
Fig. 2A. Top view of the stream tank showing symmetrical
channels where experiments were conducted.
1 = submersible pumps; 2 = screen dividers.
Fig. 2B. Simple line drawing of an observational channel
of the stream tank.. 1 = screens; 2 = false bottom;
3 = concrete blocks; 4 = glass windows. Submersible
pumps and cooling coil have been omitted. 7a. 8
screens (1.6 meshes/cm, 0.12 cm diam) and a plexiglas 'false bottom'
(1.27 X 0.45 m) supported on concrete blocks, dace were confined to an 2 area of approximately 0.6 m . Water depth was adjusted to 15.2 cm and submersible pumps created an average channel velocity of 0.6 m/sec.
Except where otherwise stated, food was always available throughout the experimental channels.
E. Experimental Conditions
All breeding phase experiments were conducted under a 16 hr light -
8 hr dark photoperiod and with 14-19C temperatures. A 12 hr light- 12 hr
dark photoperiod and 13-15C temperatures were maintained for late summer
experiments. During winter replicates, an 8 hr light- 16 hr dark photo•
period and 6-10C temperatures prevailed. Densities of dace encountered
in nature were used as guidelines for experimental densities.
Daily photoperiod was regulated by a time clock. Daylight illumina•
tion was provided by six ceiling-mounted fluorescent light fixtures each
holding two 1.22 m tubes. During the night period, the tank was illumina•
ted by four 60-w red incandescent light bulbs held in gooseneck lamps.
Observations were either immediately written down or tape recorded and
later transferred to data sheets. Day observations always were made both
before and after night periods.
Special experimental environments and methods will be described in
specific experimental sections. 9
F. Experimental Fish
Dace in breeding condition were collected from the Alouette River during the spring breeding period. This seasonal constraint along with • the relatively long periods of time required for experiment replication did not allow observation of large numbers of similar animals. Instead, relatively few individuals were studied intensively. Non-breeding dace
for late summer experiments were collected during the first week of
September, while dace for winter replicates were taken in January. Adult longnose dace from the Mink River, Manitoba, were collected by Dr. J.H.
Gee, University of Manitoba, and flown to the University of British
Columbia, Vancouver.
Dace usually were discarded at the completion of each experiment.
However, after some experiments (i.e. , breeding phase experiments) dace were returned to holding tanks and used in later experiments in which the
experimental history of the test fish was irrelevant. Both field and
laboratory fish retained for stomach analyses were killed immediately by
immersion in 10% formalin. They were dissected later.
Small fin clips on different regions of the caudal fin were used so
that individuals could be identified during observations?;. Dace marked by this method showed no observable differences in behaviour from non-
clipped fish. All fish used for breeding experiments were tested for
ripeness to ensure that the condition of fish was relatively constant between replicates. Female dace used for spawning experiments were checked
for ovulation by stroking the abdomen backwards. Breeding dace were
sexed by abdomen size, presence of tuberculation, and easily extruded 10
sex products. Sexing of non-breeding dace, however, relied on the sexually dimorphic trait of pectoral fin length. Male pectoral length is greater than that of females, but occasional exceptions exist. For
this reason, dissections for sex verification were performed once ex• periments were terminated. However, all such autopsies confirmed
external sexing. 11
SECTION 1
THE SOCIAL BEHAVIOUR OF LONGNOSE DACE
A. Description of the Behaviour of R. c_. dulcis
a. Reproductive Behaviour
Breeding longnose males defend territories (approx. 10 cm in diameter) into which they entice females to spawn. Spawning occurs in riffles where the substrate is coarse and provides natural depressions between pieces of substrate for egg deposition. Under simulated riffle conditions, R. c_. dulcis males and females display most agonistic and courtship activity during the night (Fig. 3), and spawning occurs under darkness (Fig. 4).
Initially, males court females entering their territories, by
'nudging' and 'following'. Territorial males also frequently perform
'substrate probing' during which the snout is pushed between pieces of
substrate. Although males perform substrate probing in the absence of
females, it is intensified once a female enters the territory. Because male dace perform this behaviour pattern in the absence of females, it
has been suggested that in addition to being a vital link in the courtship
sequence (i.e., nest site demonstration) this behaviour pattern also may
be important in nest site preparation (Bartnik, MS, 1970).
Once the female has entered the territory, males further court females by
'trembling' during which males vibrate their bodies at high frequencies.
Males, however, may react aggressively to females which fail to respond to courtship within the territory. Ripe male dace without territories dqy wwawwrnnishi JMHIWM dav v.tbimM n.aht-mmmm 357
Fig. 3. Diel periodicity of agonistic and courtship activity of R. c_. dulcis as recorded in the stream tank. A total of 8 dace (4 female, 4 male) were tested. (LD 16:8) 13
Fig. 4. Time intervals over which dace spawnings were observed
(solid lines) or occurred but were not observed (broken
lines). The finding of eggs in the tank verified un•
observed spawnings. Numbers in parentheses indicate the
number of spawnings performed over that interval of
time. (LD 16:8). Data from Bartnik (1970, MS, 1972).
A total of ten R. c_. cataractae (five female, five male)
and 36 R. c. dul cxs (18 female, 18 male) were tested.
14
persistently follow, nudge, and 'quiver' against females in an attempt to
spawn with them. Females, however, never respond to such courtship from non-territorial males.
Once females are receptive, they enter the male's territory and probe the substrate in the same manner as males. The male often quivers
parallel to the female as she does so. Both fish then assume a position
on the bottom over the depression they have probed and go through a spawn•
ing act in which both quiver for 1-2 sec. Eggs and milt are released and
the fertilized eggs fall between rocks, adhering to the surface of under•
lying stones. This concludes the spawning sequence and the female leaves
the nest site.
Once spawning is complete, the parental male displays a high degree
of site attachment. He remains directly over the nest site, probes the
substrate frequently, and defends the area against all fish with the ex•
ception of receptive females with which he may spawn. The term "parental"
males used herein refers to territorial males with eggs. Males spawn with
several females or the same female a number of times.
b. Agonistic Behaviour Patterns Recorded During Observations
Butt: The fish thrusts with the snout or side of the head.
Observed in both sexes.
Bite: One fish quickly closes its mouth over some part of
the body of a second fish. Observed in both sexes.
Dart: One fish swims rapidly toward a second fish. Observed
in both sexes but performed more frequently by males. 15
Chase: One fish follows a second fish closely, biting
whenever the fleeing fish slows or stops. Ob•
served in both sexes.
Fight: Two fish parallel to one another, with their
heads together, go through a series of quick,
sweeping, lateral head butts, the majority of
which are glancing blows causing the fish to
slide, over or under the. opponent's head. When
this crossing over takes place combatants have
merely reversed positions and they immediately
butt each other again. Observed in both sexes.
c. Courtship Behaviour Patterns Recorded During Observations
Follow: A male swims behind a female without making
contact.
Nudge: In lateral nudging, a male while parallel to a
female, pushes his side against that of the
female in a swaying motion. In nose-nudging,
a male pushes his snout against the abdomen of
a female.
Nibble: A male gently opens and closes its mouth on
the dorsal fin, caudal fin, or dorsal surface
of the head of a female. 16
Substrate Probing: The fish pushes its snout, in a
quivering motion, between pieces of substrate ;
the long axis of the body making an angle of
about 45-90° with the bottom. Although the
orientation of the body is often similar during
feeding acts, the distinctive quivering motion
is lacking. Observed in both sexes but per•
formed more frequently by males.
Quiver: While parallel to a female, the male goes
through a number of forceful muscular contractions
which cause lateral undulations to pass down the
length of the body. Females perform quivering
only during actual spawning.
Tremble: The tremble is different from the quiver and .
is performed only by males. The male goes
through a series of high frequency vibrations in
which the entire body trembles. These trembles
last 1/2-2 sec and are repeated every 1/2-1 sec
in the presence of a female.
d. Egg Eating Inhibition
i. Outline of Experiment
The term "egg eating inhibition" has been used in the behavioural literafeuree with respect to a parental state during which fishes are inhibited from eating eggs. Since parental longnose males have never 17
been observed to eat the eggs they guarded either in the laboratory or field, it seems likely that males are inhibited from eating their own eggs. Induction of a state of egg eating inhibition by the presence of eggs and recent spawning experience has been observed in the stickleback
(Van Iersel, 1953) and the blue gourami (Kramer and Liley, 1971). To determine if such a phenomenon exists in dace, territorial males with both eggs and recent spawning experience and territorial and non-terri• torial males without either eggs or recent spawning experience, were exposed to recently fertilized eggs. Numbers of eggs eaten by experimental dace were determined by direct observation and stomach analyses.
ii. Procedure
Tests were carried out in pairs within two parallel channels (60 X
23 cm) separated by a black plexiglas divider. The bottom of each channel 2 was covered with fine gravel (0.5-1.0 cm diam) except for a 10 cm patch of coarse gravel (3-5 cm diam) suitable for spawning. A small enclosure 3
(approx. 1000 cm ) made from flat rocks and consisting of three vertical walls and a horizontal roof was positioned over this coarse gravel. Dace were attracted to such enclosures and took up residence in them.
One channel held a parental male with either 3,12, 24, or 72 hr having elapsed since he spawned. The other channel held a non-parental male. Parental males were provided by allowing males to spawn with a female in the coarse gravel under the enclosure. Females were removed immediately after spawning. The eggs spawned by the female were left in the gravel where they were guarded by the parental male. Territorial and 18
non-territorial types of males were distinguished by the brief introduction
of a second (intruder) male. Territorial males defended the enclosure while non-territorial ones allowed the intruder to enter it and share it.
Each male was presented with 100 eggs from a batch produced through artifi•
cial fertilization. Presented eggs were all viable and batches varied in
age from 12 to 24 hr. Eggs from the same batch were used for each pair of
test dace. Eggs were presented by means of a large-bore eyedropper, after water velocity was reduced to prevent drift. Eggs were dropped directly
into the enclosures. Although some eggs fell between crevices in the sub•
strate and became inacessible, a large number remained available to the fish
in each test. After a 3 hr period of irregularly spaced observations, dace were removed and killed. Their stomachs were examined later.
iii. Experimental Fish
Eight males, dace measuring 87 to 101 mm in fork length were tested
for egg eating inhibition.
iv. Results and Discussion
Some eggs were eaten by dace in each of the four tests (Table I).
Parental males tested 3, 12, and 24 hr after spawning did not eat any eggs
although each was observed to probe the substrate where eggs were present.
Only the parental male with spawning experience 72 hr before egg presenta•
tion, ate eggs. This parental male no longer defended his nest site and was
classified as a non-territorial parental male. All other territorial and
non-territorial dace ate some of the eggs presented (Table I). These
results agree with later experiments (Tables XII, XIII and XIV)in which 19
Table I. Number of eggs eaten by different types of dace presented with recently fertilized eggs
Number of Eggs Eaten Parental Male Non-Parental Male (Hours after spawning) ^ Egg Batch 3hr 12hr 24hr 72hr territorial non-territorial
1- 0 10
2- 0 2
3- 0 11
4- 6 7
non-territorial parental male with spawning experience 72 hr before egg presentation 20
parental males did not eat eggs while territorial and non-territorial
ones did.
The results indicate that parental male dace display egg eating
inhibition for up to a day after spawning. Existence of the egg eating
inhibition phenomenon would prevent egg predation between closely grouped parental male dace where the spread of eggs from neighbouring nest sites might overlap. In three=spined sticklebacks, egg eating inhibition is not place specific, since nest raiding males do not eat stolen eggs but carry
them back to their own nest (Wootton, 1971a). Whether egg eating inhibi•
tion in parental male longnose dace is site specific is not known. However,
this would appear to be somewhat irrelevant since nest site tenacity is strongest at this time.
e. Comparison with R. c_. cataractae
The longnose dace, Rhinichthys cataractae, forms a number of geographic races whose sub-specific status is in doubt (McPhail and Lindsey, 1970).
Two such sub-species are R. cataractae cataractae (Valenciennes), which occurs east of the Continental Divide, and R. cataractae dulcis (Girard), which occurs on both sides of the Continental Divide (Jordan and Evermann,
1896).
For purposes of convenience, Mink River, Manitoba, and Alouette River,
British Columbia, populations of longnose dace are referred to in the present study as sub-species cataractae and dulcis respectively. However, it should be understood that when I refer to behavioural differences between R. c_. cataractae and R. _c. dulcis, the data and conclusions apply only to my 21
population samples and are not necessarily sub-species specific. Observa•
tions of more populations of each sub-species over their geographic ranges will be necessary before we can know whether results given here apply to
different populations.
The reproductive biology of the eastern sub-species, R. c_. cataractae,
has been described (Bartnik, 1970). But little was known of the reproductive
habits of the western sub-species, R. c_. dulcis, until the present study.
Under simulated riffle conditions, observations of breeding R. c_. dulcis
from Alouette River revealed that although patterns of breeding behaviour
of the two sub-species are almost identical, there are striking differences
in phenotypic appearance, diurnal rhythm of breeding activity, and female
territoriality.
Unlike R. £. dulcis, R. c_. cataractae are reproductively active during
daylight hours (Fig. 4), and males display bright crimson patches on the ventral surface (Fig. 5) which function as a visual cue for sex discrimination
(Bartnik, MS., 1970). Breeding coloration in dulcis males is faint or
absent (Fig. 5), and it seems unlikely that the difference in coloration
forms the basis for sexual discrimination in either sex under night condi•
tions .
The difference in time of breeding of the two sub-species (Fig. 4)
continues to be puzzling. One selection pressure suggested which may have
resulted in nocturnal breeding in R. c_. dulcis is that due to predation by
sculpins. Cottus sp., (Bartnik, 1972). But field evidence does not
support the suggestion, since of 38 sculpin stomachs examined none contained 22
Fig. 5. Rhinichthys cataractae cataractae and R. c_. dulcis males
from Mink River, Manitoba, and Alouette River, British
Columbia, respectively. Arranged in order from top to
bottom are lateral view of R. c_. cataractae male, ventral
view of same male, lateral view of R. c_. dulcis male, and
ventral view of same male.
23
dace. However, studies of the role of predation in the evolution of fish species often lack both direct observation of predation and recoveries of the evolving prey from predator stomachs (McPhail, 1969). Eight juvenile steelhead trout, Salmo gairdneri, dissected also produced no dace, although one trout stomach did contain six fish identified as salmonid fry. Infor• mation on dace egg predation and observations on behavioural interactions between juvenile steelhead trout, sculpins, and dace are presented in a later section.
Another difference between the two sub-species is the distance at which males first begin to court females. During R. c_. dulcis courtship, the distance between male and female is almost always less than 5 cm.R. c_. cataractae males, however, generally begin to court females at a distance of up to 12 cm. These slight qualitative differences in breeding behaviour may be attributed to the presence or absence of daylight. Agonistic be• haviour patterns performed by R. c_. cataractae and R. £. dulcis during defence of territory are essentially identical.
By far the most interesting difference to be exposed by the comparison of Alouette and Mink river populations of longnose dace deals with female territoriality. During the breeding period, only male R. c_. cataractae vigorously defend territories, while females are more retiring (Bartnik,
1970). In sharp contrast, both male and female R_. c_. dulcis actively defend territories before and after breeding. Agonistic behaviour patterns observed only in males of cataractae are performed by both sexes of dulcis
(Table II). 24
Table II. Agonistic behaviour patterns commonly performed (X) by R. c_. cataractae and R. c_. dulcis during the breeding phase.
Behaviour Pattern R. c_. cataractae R. £. dulcis mallej-.;-. f emal-e fhal€jc female
Butt X - XX
Bite X XX
Dart X - XX
Chase X - XX
Fight X XX
During the breeding phase, territoriality persists even in dulcis
females that are shown by dissection to be completely spent. Intensive
territorial behaviour by male dace, however, is restricted to ripe indivi•
duals. A comparison of the two populations of longnose daee described
here, in which both males and females are territorial in one and only males in the other, may be useful in providing information about the causa•
tion and possibly the function of breeding territory.
It is suggested that this difference in female territoriality between
the two sub-species (R. £. dulcis from Alouette River, British Columbia
and R. c_. cataractae from Mink River, Manitoba) is related to differences in adult distribution during the breeding phase. A recent study by Gibbons and Gee (1972) of seasonal distribution and abundance changes in R. c_.
cataractae in Mink River, Manitoba, has documented the segregation of adult 25 male and female dace into different environments during pre-breeding and breeding periods. Female dace were more abundant in large rock riffles
(substrate size :>75% large rocks (>15 cm diam)) while males were predominant
in small rock riffles (substrate size :>75% small rocks (5-15 cm diam))
(Fig. 6A) . Since spawning in the Mink River occurs primarily in the small rock riffle environment (Bartnik, 1970), females occupy these areas only while breeding. Outside the breeding phase, both sexes occupy the same environments.
Differences in bottom substrates of the riffle environments (i.e., those areas where velocity exceeds 45 cm/sec) of the Alouette and Mink rivers exist. Gibbons and Gee's (1972) classification scheme of the riffle environments in the Mink River is not entirely applicable to the Alouette
River. Unlike the Mink River, rock riffle areas of Alouette River are not easily divisable into small and large rock types. Using their criteria, based on substrate composition, the majority of rock riffles in Alouette
River fall into a new category. These areas are generally heterogeneous with respect to small and large rock substrate types and homogeneous zones of either are lacking. Such environments might be referred to as "mixed" rock riffles (substrate size :<>50% large rocks (>15 cm diam) :<>50% small rocks (5-15 cm diam).
A series of collections of dace from mixed rock riffles in Alouette
River, during pre-breeding and breeding periods (March 28 - April 19, 1971), 2 revealed no significant difference (X = 2.60, p>0.10) in abundance of adult male and female dace (Fig. 6B). Because of the physical environment, males and females occur in close proximity. Since there is no marked 26
A R.C.CATARACTAE B R.C. DULCIS 1.0 B males • females 0.8
0.6
~ 0.4 o
o J3 E = 0.2
0.0 h KKa—I
sma II large mixed rock rock rock riffles riffles riffles
Fig. 6. Distribution of adult dace in rock riffles during pre-breeding and breeding periods. (A) Densities of male, and female R. c_. cataractae in small and large.rock riffles in Mink River (May 23-June 15). Data from Gibb ons and Gee (1972). (B) Densities of male.and female.R. c_. dulcis in mixed rock riffles in Alouette River (March 28-April 19). 27
habitat difference between sexes during spawning, a potential for frequent encounters between breeding males and unreceptive females exists. On this basis, the hypothesis is proposed that the close proximity of males and females elicits territorial behaviour in females. Experimental tests of this hypothesis follow in.later sections.
B. Summary and Discussion
Both males and females of R.c dulcis occupy the "mixed" rock riffles in Alouette River and defend territories during the breeding phase. During the night, dulcis females leave their territories to court and spawn with territorial males. After spawning, parental males (i.e., those with eggs) show strong nest site attachment and are inhibited from eating eggs.
Males and females of R. The females remain non-territorial and share the male-occupied small rock riffles only when taking part in the spawning activities, which occur during the day. The different social structures of the two populations of longnose dace described here are therefore to some extent dependent on the environ• mental conditions under which each is found. Nowadays it is generally accepted that social structure is not a rigid instinctive feature of species, but that it is quite malleable and influenced by local environmental conditions. Several studies of fish species (Lindroth, 1955; Kawanabe, 1957, 1958; Kalleberg, 1958; Hartman, 1965) have shown that the habit of holding territories in salmonids is regu• lated and modified by the physical factors in the stream. Recent studies of 28 intra-specific variations in social organization in ungulates (Estes, 1966) and primates (Crook, 1970) correlate with contrasts in the ecology of the demes concerned. Crook (1968) has stated that ecological and social condi• tions are important in determining whether a population does or does not exhibit territorial behaviour. Gartlan and Brian (1968) found vervet monkeys to be territorial in one habitat but to move in groups in another. Certain rodents may be highly territorial in some environments, at certain popula• tion concentrations, but fail to show such behaviour under different condi• tions (Anderson, 1961). Data on herds of gazelles, Gazella gazella, at the eastern and western ends of their range, where landscape differs markedly, suggest how profoundly social structure may be influenced by substrate (Klopfer, 1972). It appears conclusive then that the physical and social environment of a population may impose direct constraints on social structure, and that behavioural characteristics of species often show great phenotypic plasticity. Breeding territoriality in both sexes (i.e. , a heterosexual territorial system) as described here'for R. c^. dulcis is a phenomenon rarely described in fishes. A noteworthy exception, however, is documentation by Morris (1958) of a heterosexual system of territories in the ten-spined stickleback, Pygosteus pungitius. Because the female stickleback neither nests nor per• forms parental behaviour of any kind, Morris hardly expected that she would hold a territory. Yet this is what occurs; all the aggressive actions and postures occurring in both sexes, in exactly the same way. Although entirely speculative, Morris offered a possible explanation for the existence of i a heterosexual territorial system in Pygosteus. He suggested that since nest- 29 building Pygosteus males keep hidden amongst dense vegetation because of their small spines which provide an inefficient anti-predator device (Hoogland e_t al. , 1956) , the later arriving Pygosteus females would find it difficult to locate these males. But, if Pygosteus females moved into the breeding grounds with males and had no aggressive tendencies, they would be driven off by the males when the latter were in their pre-sexual condition. Morris therefore concluded that the only solution would be for males and females to move into the breeding grounds together, and to both defend territories. Although Morris did not pursue his hypothesis further, he believed that it was quite possible that under certain ecological conditions of, say, sparse vegetation or high population density, the aggressiveness of the females of a Pygosteus population may not develop. Twelve years after Morris did his Pygosteus work, McKenzie and Keenleyside (1970) published a paper on a population of the North American form of Pygosteus(called Pungitius pungitius) which provides support for Morris' hypothesis. Unlike the European form, the North American population . • M d nests in relatively open water away from dense plant growth. Mckenzie and Keenleyside never observed females to defend territories in this breeding habitat. The likeli• hood that female longnose dace territoriality serves a similar function as that hypothesized by Morris for the sticklebacks (i.e., allows males and females to coexist within the same area) is dealt with in later sections. Many important ideas on the adaptiveness of social structures have been made by induction from extensive field studies of the higher vertebrates, particularly birds and mammals. Crook's (1964, 1965), Lack's (1968) and 30 Goss-Custard's (1970) studies on avian social systems and Crook and Gartlan's (1966) studies of primate social systems provide examples. In these studies, possible factors suggested as selective agents for observed social structures include food exploitation, anti-predator defence, and the gross nature of the habitat. Some of these factors, as they pertain to dace social structure, are considered in a later section of this thesis. 31 SECTION II FACTORS INVOLVED IN THE CAUSATION OF TERRITORIAL BEHAVIOUR AND THE SELECTION OF A TERRITORY In this section I ask the questions: 'What causes or elicits territorial behaviour?' and 'What areas do dace select to defend?' In keeping with the ecological approach of the present study, only external (i.e., environmental)factors are dealt with in the investigation of the causation of dace territorial behaviour. Internal factors (i.e., hormonal) are not discussed. Presumably habitat selection (i.e. , territory selection) has evolved as a means for obtaining the most favourable environment for survival and procreation. Experimental evidence elucidating the relevant proximate factors involved in habitat selection by animals is relatively rare. Note• worthy exceptions are Klopfer (1963, 1965) for birds, Wecker (1963, 1964) for mice, and Hughs (1966) for insects. Some fish species have also been investigated in this regard (Kalleberg, 1958; Norris, 1963; Hartman, 1965; Hagen, 1967; Sale, 1969). Both Lindroth (1955) and Hartman (1965) have stated that the behaviour of choosing and defending territories in fish appears to be a reactive type of behaviour which is governed?: by a complex of environmental stimuli. In separate experimental studies, Kalleberg (1958) and Hartman (1963) found that they could elicit territorial behaviour in young brown trout simply by presenting certain stimuli. Hartman induced trout to take up and defend positions by giving them visual reference points, while Kalleberg showed that this territorial behaviour was initiated by running water. 32 Included in the following section are a number of experimental procedures that were designed to determine what physical and biotic factors might be involved in the causation of dace territorial behaviour and in the selection of territories by male and female dace. A. Breeding Phase a. Experimental Analysis of the Factors Involved in the Selection of Territories by Male and Female Dace The following experimentawas designed to answer several questions concerning dace territoriality. Breeding dace were held under test condi• tions which offered them a choice between various combinations of environ• mental features. The behavioural data gathered were thus capable of revealing the characteristics of sites most frequently chosen by male and female dace to establish territories. i. Outline of Experiment The experimental channel was provided with a number of structures incorporating environmental features common to rock riffles. Collections of thin flat rocks (2.5 cm high) from Alouette River, carefully selected for uniformity of size, were used to make these structures. Sizes of experi• mental structures were decided upon through field observations of spaces utilized by dace. Individual structures placed in the channel were associated with either fine (0.5-1.0 cm diam) or coarse (3-5 cm diam) bottom substrate. Figure 7 shows diagramatic sketches of the different combinations of structures and bottom substrates used. Throughout the thesis each type of experimental structure is referred to by a two letter abbreviation which 33 Fig. 7. Diagramatic sketches of six combinations of structures and bottom substrates placed in the experimental channel. Accompanying abbreviations are used for reference purposes. First letters in abbreviations indicate whether both overhead cover and shelter from current (B), only overhead cover (C), or only shelter from current (S) was provided. Second letters indicate whether the underlying substrate was coarse (C) or fine (F). Arrow denotes direction of current. 34 indicates the features that particular structure incorporates. The first letter of each abbreviation indicates whether both overhead cover and shelter from current (B), only overhead cover (C), or only shelter from current (S) was provided. The second letter indicates whether the under• lying substrate was coarse (C) or fine (F). Each experimental structure incorporated two or more environmental features. Vertical and horizontal rocks of BC and BF provided enclosed areas of both overhead cover and low water velocity. Horizontal rocks of CC and CF provided overhead cover while vertical rocks of SC and SF provided areas of low water velocity (i.e., shelter from current). Table III lists environmental features incorporated into each experimental structure. Table III. Environmental features incorporated into (x) experimental structures. Environmen tal Struc ture Feature BC BF CC CF SC SF Shelter from current X X - - X X Overhead cover X X X X - - Coarse bottom substrate X - X - X - Fine bottom substrate - X - X - X Since vertical and horizontal rocks were uniform in size (Fig. 7), the area of enclosed space in BC and BF, the area of overhead cover in BC, BF, CC, and CF, and the depth of sheltered zone in BC, BF, SC, and SF were 35 constant. Enclosed areas of BC and BF produced a considerable shading effect, but the amount of shading provided by the suspended overhead rocks of CC and CF was considerably less. Vertical rocks of structures SC and SF produced almost no shading. In this and in subsequent experiments the assumption was made that levels of agonistic activity at various areas would be indicative of prefer• ences for those areas. Preliminary tests revealed that structures providing both overhead cover and shelter from current (BC and BF)were occupied quickly by newly introduced dace. Thus dace crowded together in the two enclosures provided. Under these crowded conditions, dace attempting to remove other dace from an enclosure were unable to mount a successful attack. Consequently, dace ceased to perform agonistic behaviour and the development of a competitive situation was retarded. This effect was remedied by providing an additional BF structure which reduced the crowding effect and thereby allowed individuals to establish territories more easily. Once some dace held territories, the level of agonistic activity remained high and the competitive situation was achieved. ii. Procedure The following procedure was repeated for each of three replicates. The experimental structures with their underlying substrate types were uni• formly spaced on the channel bottom. All areas between structures were covered with a layer of fine gravel (0.5-1.0 cm diam). In each replicate, six fish (three female, three male) were placed in the channel. Following a 12 hr adjustment period, 30 min observations were made at 2 hr intervals over a 12-1/2 hr period each day. Four observations were made under night 36 conditions (i.e., red light) and three during daylight. Night observations were more frequent since reproductive activity occurs at this time. Number of agonistic acts won and courtship acts performed were recorded. An agonistic act won is defined as one in which one fish displays one or more agonistic behaviour patterns to a second, resulting in the second fish or "loser" leaving the area where the agonistic behaviour was performed. The "winner", however, remains at the site. Although a measure of the total number of agonistic interactions occurring at various areas would also have served to indicate which sites were most highly contested (i.e., preferred) by dace, the more specific measurement of "agonistic acts won" was used in this and in subsequent experiments. It was felt that measuring agonistic acts won would emphasize the fact that territories were being defended. Noble (1939) defined territory as "any defended area", while Tinbergen (1957) defined territoriality as "a combination of intraspecific hostility and site attachment". Therefore, dace winning agonistic bouts in the present study were said to be territorial. Generally winners were fish already established at the site (i.e., resident). Agonistic acts between unestablished dace, which often ended in a draw with both fish either leaving or remaining at the site, were not recorded. A courtship act refers to any courtship behaviour pattern performed. Courtship activities were recorded to provide an indication of sexual motivation. Observations in each replicate were terminated once the first spawning acts occurred. Only data collected during the 72 hr prior to spawning was used. After each replicate all gravel and rocks were removed from the channel, cleaned, and replaced. Structures were rearranged for each replicate. 37 iii. Experimental Fish Eighteen dace (nine female, nine male) measuring 86 to 101 mm in fork length were used. All fish were held in bare tanks for 24 hr before testing. iv. Results and Discussion The data from all replicates were combined and statistical analyses made with the Wilcoxon matched-pairs signed-ranks test (Siegel, 1956). Since each dace was marked differently, data for individual fish were available. Therefore, in the matched pairs test each dace was compared against itself. The results show that females defended only structures which incorpor• ated both overhead cover and sheltered area components (Fig. 8). Although such enclosures were over either fine (BF) or coarse (BC) bottom substrate, one type was not defended more than (p>.05) the other (Fig. 8). Territorial dace at enclosures (BC and BF) defended the area inside the enclosure but not the area around it. Females showed no significant difference (p>.05) between the total number of agonistic acts won during day and night periods (Table IV). A significant majority of these agonistic acts by females (p<.005) were against the opposite sex (Table IV). Males moved about the channel considerably more than females and thus intruded frequently on the more sedentary females who responded agonistically. Males, however, won significantly more agonistic acts (p<.025) at night (Table IV). At this time, three different types of structures were defended strongly by males. Each defended structure incorporated the components of overhead cover (BC and CC) and/or a sheltered area (SC). The only common component in all three was coarse bottom substrate (Fig. 8). 38 Fig. 8. Mean number of agonistic acts won per area per 30 min by breeding male and female dace during day and night. Only frequencies larger than 0.05 are shown. Replicates I-III are combined. A total of 18 dace (nine female, nine male) were tested. 39 Table IV. Mean number of agonistic acts won per 30 min by male and female dace against the same and opposite sex during day and night. Numbers in parentheses indicate total number of wins Replicates I-III are combined. A total of 18 dace (nine male, nine female) were tested Mean No. Agonistic Acts Won/30 min Day Night Combined by female against female 0.52 1.58 1.12 (14) (57) (71) by female against male 5.66 5.61 5.63 (153) (202) (355) by female against both female -> andemale 6.18 7.19 6.76 (167) (259) (426) by male against matbe 8.18 23.00 16.65 (221) (828) (1049) by male against female 0.92 4.13 2.76 (25) (149) (174) by male against both male and female 9.11 27.13 19.41 (246) (977) (1223) 40 A significantly greater number of these agonistic acts by territorial males (p=.005) were won against other males than females (Table IV). Females generally remained at BC or BF areas, while males moved about the channel competing for the remaining coarse substrate areas. During daylight periods, males also defended three different types of structures (BC, BF, and CC). However, at this time the only common component in each was overhead cover (Fig. 8). Texture of bottom substrate was either fine or coarse. Males rarely defended BF structures at night, but defended them often during the day (Fig. 8). Although CF structures provided the overhead cover component, they were never defended (Fig. 8). Observations showed that since this site was not sheltered from current, position maintenance over the even surface of fine gravel was difficult. At the CC structure, however, fish remained stationary on the bottom with the aid of outstretched pectoral and pelvic fins placed in crevices between pieces of coarse gravel. Dace of opposite sexes were involved in a total of 529 agonistic acts (Table IV) of which females won 67.1% and males 32.9%. Although these results suggest female dominance, a closer examination of the recordings revealed that winners (either male or female) in almost every interaction were also the resident fish. A resident fish is defined as one which has most recently won an agonistic act at a particular site and/or has spent the majority of its time occupying that area. The behavioural phenomena associated with prior residency have been described with respect to fish territoriality by Baerends and Baerends-van Rooh (1950). That a period of prior residency gives an advantage to individual fish in defending their 41 territory has been reported for several species of fish (Braddock, 1949; Chapman, 1962; Jenkins, 1969; Phillips ,1971; Myrberg, 1972). The greater win-loss ratio in favour of female dace may be attributable, therefore, to their more sedentary behaviour. The question which naturally follows is whether males would display a greater preference for BC and BF areas if they were not already occupied by territorial females. The answer is not evident from these results but later experiments (see page 77 ) demonstra• ted that, although vacant enclosures were available, male dace occupied and defended open areas of coarse substrate void of any overhead structure. Enclosures (BC) were, however, usually preferred. Since dace interacted continually during each test period, win-loss records are a good indication of 1) movements of individual male and female dace, and 2) areas at which resident fish were most frequently challenged. A comprehensive record of agonistic activity of individual dace in the ex• perimental channel is shown in Figure 9. The graphs illustrate that dace experiencing little success in agonistic interactions continually interacted with fish at different areas (Fig. 9). No dace remained faithful to a par• ticular site and went unchallenged. These graphs (Fig. 9) also reveal the exchangeability of territories. During each experiment, • dace (particularly males) frequently defended two or more different areas. Although the competitive nature of the test situa• tion undoubtedly enhanced this exchangeability of territories, occasional field observations of deserted territories also has suggested that this phenomenon exists. Symons (1971) found juvenile Atlantic salmon to voluntarily won lost REPLICATE J REPLICATE NK4HI [J REPLICATE IJJ 30 - TJ7TT I NIUHl of o 102 30j 30 10- n II tn 10 30- of 10 J JL 89 301 o7| 30 2 Ql84 Ml 10- < pre 10 10 V 30- 1 30 30 c o 10 • 0) 10| < 30 101 ?1l6 a D_ n n 1 0 301 ! 301 ?- . r k_ 10 J«2 10 10 u m 30 ?2 Z n • 1 30 30 10 ft 10 10 30 ?3 Ll 10 101 10 CC: Cf SC 5f & CC Cf SC Si : ' tf, BF2 Cf BC BF, :BF2 CC CF SC SF vrse 1 BC BF, BFJ CC BC BF, BF2 CC CF SC i>K J "2 Fig. 9. Total number of agonistic acts won and lost by individual male and female dace.at different areas in Replicates I, II, and III. -p- 43 shift location of their territories from time to time in an artificial stream, resulting in part of the population constantly being in transit. Territory locations of Asiatic Barbus fish shift rather often and the same location may be occupied by different males in succession within a few days (Kortmulder, 1972). In the field, breeding male dace have been observed at the same location for up to 3 successive days. A brief mark recapture experiment conducted with 25 breeding adult dace showed 15.4% of the marked fish to 2 be within the same m 4 days later. During the breeding phase, several areas of riffle habitat in the Alouette River were seined repeatedly until no dace were taken on five successive attempts. Reseining of these same areas as little as 4 days later often yielded densities of dace equal to or exceeding those initially.taken. Thus movements of dace within and probably between riffle areas are common. Courtship acts were displayed significantly more at night by both males (p<.005) and females (p<.005; Fig. 10). Males courted females almost exclusively over coarse bottom substrate. Instances of courtship by males over fine substrate (BF) were performed only by non-territorial males. Females reciprocated to courting males only at structures with coarse bottom substrate (Fig. 10). In each of the three replicates, spawning occurred in association with a different structure. The only common component in all three spawning or nest sites was once againscoarse bottom substrate. Unlike the detailed graphs of Figure 9, which show records of indivi• dual dace, Figure 10 and the remaining graphs are presented only in the more condensed form such as Figure 8. 44 NIGHT BF CC CF SC SF • females • males DAY Fig. 10.- Mean number of courtship acts performed per area per 30 min by male and female dace during day and night. Only frequencies larger than 0.05 are shown. Repli• cates I-III are combined. A total of 18 dace (nine male, nine female) were tested. 45 b. Social Factors Affecting Territorial Behaviour in Female R. c_. dulcis i. Outline of Experiment Since male and female R. c_. dulcis occupy the same riffles during the pre-breeding and breeding periods, it seems likely that frequent inter• actions between the sexes occur. The objective of the following experiment was to determine whether such interaction between males and females acts as a causative agent for the expression of R. c_. dulcis female territoriality. Manipulations were carried out to determine whether first the addition,and then the removal of a ripe male brought about any significant changes in the level of agonistic activity and spacing pattern of experimental females. ii. Procedure Several adult female dace, collected in September, were held under laboratory conditions which corresponded to the changing seasons outside. Under these conditions, laboratory held females became gravid at a time corresponding to the normal field breeding phase. These females were held in all-female groups and had had no experience of male courtship since the previous spring. Both experimental channels of the stream tank were used simultaneously. Each channel contained five uniformly spaced enclosures (BC) with fine gravel covering the remaining bottom area. Five gravid experimental females were placed in each of the two channels. After a 12 hr adjustment period, eight 30 min observations (four during the day and four at night) were made 46 at 2 hr intervals to record agonistic activity. In addition, the position of each female was plotted at the beginning, middle, and end of each 30 min period to provide information on the spacing pattern. After the eighth observation period, a recently field-collected ripe male was added to one channel and a gravid experimental female from the lab held all-female group was added to the other channel. A further eight recordings were made. At the completion of the second day's recordings, the ripe male and gravid female added the previous day were removed from their respective channels and were placed in the opposite channel. The addition and removal of the female provided the control. Recordings made on the second and third days included instances of female dace abandoning enclosures after being courted by the male. The data shown in Figure 11 were arrived at by combining the frequen• cies of occurrence of different group sizes for the 24 position checks and then calculating the percent occurrence of each group size (i.e. % occurrence of group size 1 = number of groups of size 1 observed 7 total number of groups observed x 100%). When performing statistical tests on combined position check data in this and in subsequent experiments, the assumption was made that the position check data were independent from one another. iii. Experimental fish Twelve dace (one male, eleven female) measuring 87 to 107 mm in fork length were used. iv. Results and Discussion The results indicate that the introduction of a ripe male dace was 47 Fig. 11. Percent occurrence of different group sizes in Channel 1 on day 1 (five female), day 2 (five female, 6ne7imale),.and day 3 (six female), and in Channel 2 on day 1 (five female), day 2 (six female), and day 3 (five female, one male). Fig. 12. Total number of agonistic acts won by R. £. dulcis females in Channel 1 on day 1 (five female), day 2 (five female, one male), and day 3 (six female), and in Channel 2 on day 1 (five female), day 2 (six female), and day 3 (five female, one male). Numbers in circles indicate number of occasions female dace abandoned enclosures (BC) after being courted by the male. CHANNEL 1 CHANNEL 2 DAY 1 60- 5?? 5?? 20- DAY 2 100- 5??.lo* 6?? 60- 20 • DAY 3 100- 6?? 5??,lo" 60- 20 • 2 3 3+1 2 3^ Group Size 50 h ..CHANNEL 1 c 40 o 5 _ 6?? £ 30 ® ,° 5??.lo* c 20 o CD < /CHANNEL 2 10 o' 6?? 2 DAY 48 instrumental in bringing about a rapid change in the spacing pattern of test females (Fig. 11). With the addition of the male to channel 1 on day 2, females were displaced from previously occupied enclosures by the courting male on 14 oecasions (Fig. 12). The previously non-aggressive females displayed agonistic behaviour patterns to other females and to the added male. As a result of this territorial behaviour, the spacing pattern of the females changed significantly (p<. 001) from a clumped dis• tribution to a near uniform one (Fig. 11). Comparisons of spacing pat• terns are made with Contingency Chi-square tests (Siegel, 1956). The addition of the female to channel 2 on day 2, however, did not have the same effect. Although the occurrence of the larger groupings of three or more females decreased, the spacing pattern of females in channel 2 remained clumped (p<. 001) compared with that of channel 1. Unlike the females in channel 1, channel 2 females performed no agonistic activity. However, when the male was transferred to channel 2 on day 3, the spacing pattern changed significantly (p<.001) as it did when this male was added to channel 1 on day 2 (Fig. 11). Channel 2 females were displaced by the courting male and the females in turn began to defend enclosures (Fig. 12). Other all-female groups held in the experimental channel for up to 6 days failed to show any significant changes in agonistic activity or spacing pattern. Therefore the observed changes in female R. c_. dulcis behaviour (Fig. 11 and Fig. 12) were definitely related to the interaction with the male. When the male was removed from channel 1 and replaced with the female, the amount of agonistic activity (i.e., territorial behaviour) performed 49 decreased only slightly (Fig. 11). However, the spacing pattern took on a more clumped form (one, two, or three fish per enclosure) resembling that of day 1 (p>. 05). It is interesting to note that in channel 1 on day 3 the added female, which had not yet experienced cohabitation with the male, was involved in six agonistic acts with other females. The first five were lost but the sixth won. This observation suggests that frequent displacement by already territorial females may elicit territorial behaviour in other females. c. Social Factors Affecting Territorial Behaviour in Female R_. c_. cataractae i. Outline of Experiment The foregoing data support the suggestion that interaction with males is a causative agent for the expression of R. c_. dulcis female territoriality. Therefore, since R. c_. cataractae females are segregated from their males, it seems possible that this difference in social factors (i.e. , no inter• action with males) accounts for the observed difference in female territori• ality. If this is true, then R.' c_. cataractae females would be expected to display territorial behaviour if they were forced to interact with their males. The following experiment created an atypical grouping of breeding male and female R. c_. cataractae to determine whether the predicted response occurs. ii. Procedure Adult R. c_. cataractae were collected from Mink River, Manitoba, during October and transported by air to the Vancouver laboratory. Dace 50 were held under three different conditions of temperature and photoperiod comparable to late summer, winter, and spring (breeding) seasons (see page 8). Both sexes were kept together in holding tanks except for the spring period when sexes were separated. Dace responded physiologically to in• creasing day length and temperatures by coming into breeding condition. Both ripe males and gravid females (three males, three females) were placed in an experimental channel in which segregation of sexes was impossible. Six BC structures were uniformly spaced on the channel bottom and all areas between structures covered with fine gravel. Following a 12 hr adjustment period, 30 min observations were made at 2 hr intervals over a 14-1/2 hr period each day. Four observations were made under both day and night conditions. Since dace frequently moved about in the experimental channel, inter• actions between males and females were inevitable. For this reason, experi• mental fish were described as being under "forced interaction". During 3 days of such 'forced' interaction, types and frequency of agonistic behaviour patterns performed by females were recorded. In addition, agonistic acts won by each sex and courtship activity performed by males which displaced females from previously occupied enclosures were scored. Position checks of each fish were made at the beginning, middle, and end of each 30 min recording period to determine occupancy rate of enclosures (BC) and "open" (i.e., free of structures) areas. In this and in subsequent experiments, this occupancy rate is expressed as percent occurrence (i.e., % occurrence of dace at enclosures (BC) = number of dace occupying enclosures during the 51 day's position checks -r total number of observations of dace (number:of dace x number of position checks) x 100%). After the final observation on day 3, several thin flat rocks (2.5 cm high) were piled in each of two corners of the channel. These two areas provided small spaces where dace could wedge themselves and thus isolate themselves from other fish. Since dace could avoid interactions with other fish, this condition was referred to as "not forced interaction". Recordings were continued for this fourth and final day. iii. Experimental Fish Six dace (three male, three female) measuring 67 to 87 mm in fork length were used. iv. Results and Discussion Although a shortage of R. c_. cataractae females did not allow repli• cation of this experiment, the results are quite suggestive. Early in the experiment it became obvious that a female would share an enclosure with a male, but would abandon the site or try to defend it should the male persist in courting her. The most common courtship behaviour patterns performed by males were nudging and quivering, both of which moved the female slightly each time. During each of the first 2 days of forced interaction, female dace showed little success in agonistic interactions with males, winning only 25%. However, by the third day this percentage reached 66% (Fig. 13). During the 3 days of interaction with males, females defended enclosures 2 (BC) against other females as readily as they did against males (X = 0.28, 52 Fig. 13. Number of agonistic acts won by male and female R. £. cataractae against the same sex ( Q ) and opposite sex ( • ) during 3 days of 'forced' interaction and a fourth day not forced. Numbers in circles indicate number of occasions female dace abandoned enclosures (BC) after being courted by a male. Small inset graphs indicate percent occurrence ( E3 ) of dace at enclosures (BC), open areas (0), and isolation areas (I). Six dace (three male, three female) were tested. 52a MALES FEMALES DAY 1 .60 © 60] 30 20 O30 1 n BC BC 80 DAY 2 60 f 60 ® 60 30 30 40 1BC BC 20 c o 5 n < DAY 3 » 80 c © J> 60 [ 60 60 30 30] 40 jg. 1 i BC BC 201 ~i— DAY 4 40\ Tl 60Y © 60 • O 30- 20\ 30r I R BC 53 p >.50; Fig. 13). On the first day, 37.5% of all position checks made found female dace sharing enclosures with other dace (either male or female). On days 2 and 3, however, only 4.2% of all such checks found this same situation. Males occurred predominantly at enclosures (BC) which contained the appropriate spawning gravel, and agonistic interactions between males were frequent (Fig. 13). Corresponding with the increase in female aggressiveness on day 3 2 was a significant increase (X =14.0, p<.001) in the occurrence of females at enclosures compared with that of day 2 (Fig. 13). Throughout the period of forced interaction, females abandoned enclosures when continually harassed by courting males (Fig. 13). On day 2, both displacements of females and frequency of occurrence of females at open areas peaked at 19 occasions and 76.4% respectively. However, when cataractae females began to defend enclosures on day 3, the number of displacements of females by courting males decreased to 11. When defending enclosures cataractae females display agonistic behaviour patterns usually common only to cataractae males (Fig. 14). Therefore both sexes are genetically programmed for the full complement of agonistic be• haviour patterns, but females express them only when interacting with breed• ing males. Franck (1969) suggests that behaviour patterns, instead of being genetically fully reduced, may be incorporated by means of different mechanisms into a reservoir of behaviour patterns which are latent under certain condi• tions. Present findings lend themselves well to such an explanation. On day 4, the addition of isolation areas did little to alter the behaviour of male dace, but brought about a marked change in female behaviour. Females almost immediately sought out small spaces in the rock piles where 54 Fig. 14. Frequency of occurrence of agonistic behaviour patterns performed .by R. c_. cataractae females during 3 days of 'forced' interaction with males. I 55 they wedged themselves. This action avoided interactions with males and displacements of females by males were almost entirely eliminated (Fig. 13). Although female dace behaved differently on days 3 and 4, a similar reduction in conflicts with males was observed on both days. During day 3 of forced interaction, females were able to reduce displacement and remain relatively sedentary through territorial behaviour. Similarly, when able to spatially segregate themselves from males on day 4, females avoided almost all agonistic activity and courtship harassmenfct from males. Unanswered by the two foregoing experiments on social factors affect• ing territorial behaviour in female dace is the question of the genotypic or phenotypic nature of the difference between the two sub-species. Such a problem is beyond the scope of this study but the data here would suggest that females of the two populations do differ genetically. From earlier observations we know that under laboratory conditions simulating a natural riffle, R. c:. dulcis females do not choose to isolate themselves from males, but immediately establish territories. However, the present experiment as well as earlier work (Bartnik, 1970) demonstrates that R. c_. cataractae females isolate themselves from males when given the opportunity. But when unable to do so, R. c^. cataractae females will establish territories. There• fore, females of the Mink River population of R. c_. cataractae exhibit con• siderable behavioural plasticity. The adaptation to environment in this case is phenotypic. Hopefully, further comparative studies on different populations of Rhinichthys cataractae will help to support or refute the conclusions reached here. 56 d. Social Factors Affecting Territorial Behaviour in Male R. c_. dulcis The results of the earlier experiments on factors involved in territory selection (Fig. 8) showed that during the night males defended all three types of structures with coarse bottom substrate. However, within individual replicates only two of the three structures were defended. In each replicate, one structure was defended significantly more than the other (p<.001; Table V). Closer examination of the pattern of activity suggested why one particular structure was preferred over others. Figure 15 illustrates fluctuating levels of agonistic and courtship activity recorded during the 72 hr before spawning at each of the preferred struc• tures in replicates I, II, and III. The graphs illustrate a strong Table V. Total number of agonistic acts won by males against other males at coarse substrate structures during night Replicate Number of Agonistic Acts Won „ BC CC SC Total X (Chi-square) I 21 0 231 252 p<.001 II 16 389 0 405 p<.001 III 111 0 60 171 p<.001 66rrelaEI6n betTween levels of male and female courtship and male agonistic activity. Without fail, increases in courtship activity at a site were accompanied by sharp increases in the number of agonistic acts won by the resident male. Replicate I (SO Replicate II • 119 (CC) *_• agonistic by d* 0- -o courtship byo^ 1— (Courtship by°. Replicate III (BC) 7% •JfmgMllHZZZ day day •JPighL Fig. 15. Pattern of agonistic and courtship activity performed by dace during the 72 hr before spawning at preferred structures. Abbreviations in parentheses indicate type of structure in each replicate. (LD 16:8). 58 A detailed temporal examination of a typical sequence of agonistic and courtship activity at one preferred (i.e., highly defended) structure (Fig. 16) illustrates the role of courtship by the female in inducing the resident male to perform courtship behaviour. The subsequent increases in agonistic activity were due to the repeated removal of intruding males which were attracted to the site. Although such a sequence of events was the general case, occasionally the female visited a male already engaged in defence of a site. The attraction of males to sites where females perform courtship behaviour may affect the spacing of territorial males. This possibility is examined in the following experiment. e. Clustering of Male Territories Many groups of closely spaced nests and territorial males have been located in the Mink River (Bartnik, MS, 1970) and Alouette River (unpubl. data). Spatial distribution (i.e., inter-nest distances) of such clusters of nests were smaller than seemed to be required by the available spawning resources. The attraction of males to sites where females perform court• ship behaviour, as described under the preceding heading, at first seemed to be a plausible explanation for the occurrence of such clusters of territories. However, since male dace come into breeding condition before females (Bartnik, 1970), males are usually already holding territories when females become sexually receptive. Further experimental investigation of the formation and maintenance of male territory clusters follows. i. Outline of Experiment To further examine the suggestion that clustering of male territories may be a result of female attention to particular areas, two sets of tests 59 Mr- 12 •_e Agonistic by A /KM' y\/K\/\ v\ J I I L I i I I I I L I I I I I I I I I I I L K) 15 20 TIME (minutes) Fig. 16. Typical sequence of agonistic and courtship activity performed at a preferred structure. 60 were made. Firstly, male dace were introduced into a pre-courtship test situation (i.e., without females) to determine the distribution of terri• tories. In a second experiment, territorial males were purposely spaced widely apart, and then a sexually responsive female was introduced to determine what influence she would have on the territory spacing pattern. ii. Procedure In the first experiment, eight structures were placed on the channel bottom such as to create two zones. Four BC structures were uniformly spaced on the bottom of both upstream and downstream ends of the channel (Fig. 17A). Minimum distances between structures in the experimental channel were comparable with distances between territorial males and nests located in the field. All bottom areas between structures were covered with fine gravel. In each of three replicates, four males were placed in the channel 12 hr before the commencement of a night period. During each of the two following night periods, four equally spaced 30 min observations recorded location and frequency of agonistic acts won. After the final night observation, the position of each territorial male was noted. In the second experiment, the same arrangement of structures was used (Fig. 17A), but a dividing screen was added to separate upstream and downstream areas. In each of three replicates, two males were placed in both upstream and downstream portions of the channel. Casual observations were made over two successive night periods to verify the development of territorial behaviour and site attachment. At the end of the second night, positions of territorial males were noted. Immediately before the 61 Fig. 17A. Arrangement of structures in channel (top view). Numbers refer to BC structures. Arrow denotes direction of current. Fig. 17B. Pattern of agonistic activity performed by territorial males during eight successive night observation periods. Four males were tested in each replicate. LTJ LH LH 0 A m 0 Replicate 1 Replicate II Replicate III - - i ,ll 1 • • • . l1 l I i 1 l • • • . i 1 . 1 1 | 1 . . 1 1 1 ill 1 - • n 1 . 1 • it. 1 . I 1 1 1 1 1 • 1 • • . • 12 3 4 5 6 7 8 12 3 4 5 6 7 8 12 3 4 5 6 7 8 STRUCTURES 62 commencement of the third night period, the screen divider was removed and a recently ovulated female was added. Ovulated females almost always spawned within several hours after introduction. Observations were con• tinued for the third night. Positions of each male were again noted after the final observation was made. iii. Experimental Fish Twelve male dace, measuring 89 to 102 mm in fork length, were used in the first experiment. The twelve male dace used in the second experiment measured 84 to 97 mm in fork length. iv. Results and Discussion In the first experiment, the males held in the experimental channel (Fig. 17A) over two successive nights displayed an unexpected clustering of territories. Actual mean distance between territories in two replicates was equivalent to the minimum mean distance attainable. For the third replicate, it was considerably less than the maximum distance (Table VI). Although female courtship or attention, thought to be instrumental in the attraction of males to localized areas, had been eliminated, the clustering effect still occurred. The importance of male interaction in the formation of territory clusters became most evident during the first experiment. Formation of clusters consisted of a trespassing or intruding male interacting with a newly established territorial male. The aggressive behaviour of the territorial male not only repelled the trespassing male from the territory, but also elicited territorial behaviour in the trespassing fish. Consequently, the trespassing male began to defend a nearby site against other males as 63 Table VI. Actual mean distance between four territorial male dace as compared with maximum and minimum mean distances attainable. Distances are measured from centers of each BC structure. Replicate Mean Distance (cm) Actual Maximum Minimum I 23 107.3 23 II 23 107.3 23 III 70.5 107.3 23 Table VII. Actual mean distance between four territorial male dace as compared with maximum and minimum mean distances ' attainable before and after screen divider was removed and a female was introduced WITH SCREEN (NO FEMALE) NO SCREEN (WITH FEMALE) Mean Distance (cm) Mean Distance (cm) Replicate Actual Maximum Minimum Actual Maximum Minimum I 95.5 107.3 80.6 95.5 107.3 23 II 100.6 107.3 80.6 100.6 107.3 23 III 87.3 107.3 80.6 94.0 10.7.3 23 64 well as the Initial territorial male. Two males engaged in agonistic activity attracted other males which also interacted with them. Therefore, it appeared that the effect of this behaviour was not to increase the dis• tance between males, but to attract other males td the vicinity of terri• torial males. During the first five observations, territorial males of replicates I, II, and III won an average of 10.2 agonistic acts each (Fig. 17B). However, this level dropped to 5.6 agonistic acts won per territorial male for the last three observations. The overall amount of agonistic activity did not change dramatically from the first to the second night, but this activity was distributed between more territorial males during the second night (Fig. 17B). The observed reduction in fighting by individual males was related to the restriction of male activity to the territory. After acquisition of a territory, dace began to confine their movements to a limited area and antagonism between neighbours was reduced. Males of one replicate, left in the channel a third night, continued to interact at this lower level. With the addition of the screen partition in the second experiment, a more widely spaced distribution of male territories was created than that which occurred in the unscreened channel (compare actual mean distances in Table VI and Table VII). After removal of the screen and addition of an ovulated female, the distribution of dace remained essentially the same (Table VII). Only in replicate III did a shift occur. It involved one male and amounted to an increase in the actual mean distance to 94 cm from 87.3 cm. 65 In each replicate, the ovulated female swam throughout the channel encountering all males before restricting the majority of her courtship activity to one particular male. After each visual and/or physical con• tact made with the female, territorial males immediately commenced sub• strate probing in their territories. A male that had strayed outside its territory quickly returned to it when a female entered the vicinity. Unexpectedly, other territorial males were not attracted to the site where the female performed courtship behaviour. Instead, territorial males re• mained faithful to their territories and almost no rearrangement of terri• tory spacing occurred. As a result, a courting male received little in• terference from neighbouring territorial males. In earlier experiments (see page 58 ), which suggested female attention to particular sites attracted other males, both sexes were always placed into the tank simul• taneously. Unlike in the present experiment, male territories were not yet established and/or stable in those earlier experiments. Under those un• stable conditions, males followed females and aggregated around sites at which females performed courtship behaviour. These males often occupied and defended suitable areas near by. Since males generally precede females in breeding readiness, the situation described in the present experiment would appear to be more comparable with field conditions. The present findings show that ripe male dace are attracted to and interact with other males resulting in the formation of territory clusters. As each interacting male claims a territory, the agonistic interactions become limited to the defence of the territory and levels of agonistic activity shown by individual males decrease. In the presence of a receptive 66 female, territorial males of such clusters remain faithful to their chosen territories and intrude little upon neighbouring fish involved in court• ship and spawning. Once these males spawn they show even stronger nest site tenacity, thus enhancing the stability of the assemblage. Many bird species gather on arenas or leks where males compete for females, e.g., the ruff, grouse, and blackcock. Male longear sunfish con• gregate along river edges and nests are grouped closely together in colonies (Keenleyside, 1970). Each nest is guarded by a strongly territorial male. On the other hand, van den Assem (1967) and Jenni (1972) report that ex• perimental pairs of male sticklebacks space themselves out widely in large tanks (100 x 600 cm) by selecting nest sites at an average distance of 85% and 83 to 96% respectively oftthe maximum possible distance from rival nests. f. Behaviour of Clustered Territorial Male Dace i. Outline of Experiment It seems reasonable to infer from the foregoing data that the beha• viour shown by territorial male dace assembled close together may have selective value. The following tests were conducted to determine whether any behavioural differences between clustered males and more isolated ones exist. An obvious difference between the two types is in the amount of interaction each might experience with other males. The foregoing experi• ment suggests that clustered territorial males which interact frequently with other dace might remain faithful to their chosen sites longer (i.e., be less likely to abandon a territory or nest site) than would isolated males. 67 In the following experiment the behaviour of 1) parental males experiencing frequent interactions with other males, and 2) parental males which do not experience such interaction is compared. ii. Procedure Territorial males with eggs were provided by introducing a single male into a small sectioned off area of the experimental channel (60 x 23 cm) from which no other fish were visible. A BC structure was located at one end and a BF structure at the opposite end. The remaining bottom area was covered with fine gravel. The BC structure provided the necessary spawning substrate while the BF structure acted as an alternate site, providing the same environmental features except for coarse gravel. At the commencement of the night period, 12 hr after the male's introduction, an ovulated female was added to the channel. Spawning invariably took place at the BC area with several spawning acts occurring. Once the female's abdomen was noticeably reduced in size, she was removed without disturbing the male. Two treatments were used for such males with eggs. One set of three replicates left the male unmolested for 83 to 90 hr, while making 12 to 14 irregularly spaced 15 min recordings of substrate probing frequency and time spent on the nest. Although a consistent spacing of recordings between replicates is preferable for comparative purposes, the fact that spawnings occurred at different times of the night made this difficult to achieve. In the second set of replicates, the territorial male was allowed to interact with intruding males at irregularly spaced intervals for three or 68 more days. Immediately after a 15 min recording period, the top (horizon• tal rock) of the BF structure was removed. This was done so that when a ripe male (intruder) was added to the channel it would seek out the only available cover (i.e., the BC structure-nest site), attempt to enter that enclosure, and encounter the parental male. During any one interaction period, a total of five interactions were allowed between the parental male and the intruder male. For each interaction, the time between intrusion and agonistic response of the parental male (i.e., latency to attack) was measured. Since male intruders sometimes became reluctant to enter the nest site after being attacked by the defending parental male, it was necessary to replace such intruders with another male in order to accumulate five interactions. Therefore, during each replicate, several different intruderr males were used. After the fifth interaction, the intruder male was removed and the.top of the BF structure was replaced. Although a total of 27 interaction bouts were staged, only in 24 bouts did five successive interactions take place between the territorial male and the intruder; In the other three bouts, which all took place late in the test period, the five interactions did not occur either because the territorial male failed to respond aggressively to the intruder or because he merely abandoned the enclosure after the intruder entered it. Immediately after each interaction period, a final 15 min observation was made to once again record substrate probes and time spent on the nest by the parental male. It was felt that the above described procedure would best mimic the situation in which a territorial resident would interact with adjacently 69 territorial males or intruders over intermittent periods. Unlike mirror images, confined live conspecifics, or models which can neither interact with nor flee from test fish, the present test conditions provided a realistic analog of a territorial intrusion. iii. Experimental Fish A total of 16 males were used. Parental males measured 90 to 101 mm in fork length while intruder males measured 87 to 99 mm in fork length. iv. Results and Discussion Although a certain amount of variability occurred between replicates within each treatment, different trends were still evident in each of the two treatments. Parental males interacting with conspecifics displayed a high degree of site attachment as indicated by the prolonged period of site faithfulness (i.e., time spent on nest) (Fig. 18). Twenty-four hr after spawning all interacting parental males still spent 100% or nearly 100% of their time over the nest site, but males with no interaction were already absent from their nest sites for short intervals. These latter males were usually located at the alternate enclosure (BE). Frequency of substrate probing in both groups was at a peak immediately following spawning, but declined within several hours (Fig. 18). Interacting parental males continued to probe the nest substrate throughout the test period. Substrate probing activity in parental males without interaction, however, usually became nil within 24 hr of spawning (Fig. 18). Parental males always performed more substrate probing during the period following an interaction compared to the period preceding it. After 70 Fig. 18. Frequency.of substrate probing (solid lines) and time spent on nest, (broken lines) by parental males with and without interaction with conspecific males. Points " connected by dotted lines are the 15 min periods before and after interaction with intruding males. 70a INTERACTION 20 0 20 0 40 20 0 J 1 1 1 I I I I I L 20 0 40 20 0 40 20 0 J I I I I I I I I L_ 10 20 30 40 50 60 70 80 90 HOURS AFTER SPAWNING 71 interacting with intruder males, parental males also spent more time over the nest (Fig. 18). For the first interaction in each series, parental males were slow in responding aggressively to intruders. How• ever, the latency period become progressively shorter after the first interaction (Fig. 19). In both interaction and no interaction groups, parental male dace occasionally left the nest site for short periods of 1-3 sec shortly after spawning. During these movements the male moved approximately 5-10 cm from the territory, only to quickly return to the nest site again. Such brief movements in nature may expose males to nearby females and/or bring the parental male into visual contact . with.other territorial males. Possible effects of such movements, are increased sexual and aggressive arousal of both the parental male and his neighbours. Although the sample size is very small, the results indicate that parental male dace interacting frequently with other dace remain at and defend their nest sites longer than parental males not experiencing such interaction. The data also show that parental males take longer to respond aggressively to intruding males when they have not seen or interacted with other fish for several hours. Therefore, it seems reasonable to conclude that interacting parental males (i.e. , closely spaced males) would provide greater protection for their nest sites than more isolated males. Parental males interacting with other males also perform considerably more substrate probing than those males without interaction. The signifi• cance of this difference in substrate probing activity is discussed after a later set of experiments. 72 95 35 30 -* 25! o 20 £ 15i o 10r Oh 1 2 3 4 5 Interaction Fig. 19. Latency to attack by parental males for each of five successive interactions with intruding conspecific males. Points represent means while vertical lines represent range. Sample size for each of the five successive inter• actions is 24. Replicates I-III are combined. 73 g. The Role of Food in the Distribution of Dace Territories i. Outline of Experiment In order to determine whether the presence of food plays a signifi• cant role in the selection and positioning of dace territories, an experimental environment was created in which food was available at some places but not at others. A satisfactory method was devised which allowed tubifex worms to be confined to particular bottom areas of the experimental channel. Tubifex worms located in such a manner simulated the natural prey of longnose dace, which are aquatic insect larvae and nymphs found on, under, and between stones. The following experiment created a competitive situation in which only two food areas were available to four dace. As before, levels of agonistic activity were assumed to be a valid indicator of site preference. ii. Procedure The following procedure was repeated for each of six replicates. Five petri dishes (11 cm diameter, 1.5 cm deep) were uniformly spaced on the channel bottom and filled with a layer of coarse gravel (3-5 cm diam). Surrounding bottom areas were covered with a layer of similarly sized gravel. A BC structure with both vertical and horizontal walls (see Fig. 7) was placed over each petri dish. Water level in the tank was adjusted to 15.2 cm and then tubifex worms (3 ml volume) were introduced into two of the five plates. Worms were added by means of a large-bore eyedropper. Thev were squirted into crevices between pieces of substrate and sank to the bottom of the plate entangling into small clumps. After 15 min water pumps were engaged. Preliminary tests showed that although worms moved about on 74 the bottom of the petri plate, the vertical edges restricted dispersal to the area of the plate itself. Both the layer of rocks in the plate and the vertical and horizontal walls of the overhead structure created an area of low water velocity. Consequently, worms in the plate were not subjected to displacement as a result of currents. Fish fed upon tubifex by inserting their long snouts into crevices between rocks in the petri plate and making rapid thrusts at worms. This activity occasionally displaced small quantities of worms which moved downstream with the current. Such drift passed through the downstream screen and out of the experimental channel. Once reaching the deeper and slower flowing elbow portions of the stream tank such worms sank to the bottom and were not recirculated. In each replicate, four fish (two male, two female) were placed in the channel. Following a 12 hr adjustment period, 30 min observations were made at 2 hr intervals over a 14-1/2 hr period. Four observations were made at night and four during the day. Replicates were of short duration since continued observation might have been misleading in the face of a diminishing food supply. Preliminary work showed that replenishing food sources without disengaging pumps and creating considerable disturbance was impossible. In most replicates 30-40% of the worms remained in the food plates at the completion of the test. Number of agonistic acts won and courtship acts performed (as defined earlier) were recorded to enable site preferences and sexual motivation to be gauged. Number of feeding acts, defined as the number of successful feeding movements, were also recorded. Feeding movements involved an orientation of the body at about 45° to the horizontal with the long snout inserted into narrow crevices between pieces of substrate; rapid thrusts in the direction of the bottom; and a biting movement as the snout neared the bottom. Feeding movements were recorded only when worms were ingested. After each replicate, pumps were disengaged and petri plates, gravel, and rocks were removed. Positions of the petri plates containing worms were varied for each replicate. iii. Experimental Fish Twenty-four dace (twelve male, twelve female) measuring 85 to 107 mm in fork length were used. All fish were held for 1-3 days before testing in tanks with tubifex worms located on the bottom. iv. Results and Discussion The data from all replicates were combined and analyzed with the Wilcoxon matched-pairs signed-ranks test. Each dace was compared against itself in performing the matched pairs tests. Since there were more empty areas than food areas, statistical comparisons were made on a per area basis. The results show that although male dace fed from the petri plates containing tubifex worms, they performed a significantly greater number of defences at empty plates (p<.025) than at those with worms (Fig. 20). In agreement with prior results (Fig. 8), a significantly greater proportion of male agonistic activity (p<.025) occurred at night (Fig. 20). During night hours, males courted females (Fig. 20) and frequently allowed females 76 Fig. 20. Mean number of agonistic, courtship, and feeding acts performed per food and no food area by male and female dace during day and night. Replicates I-VI are combined. Twenty-four dace (twelve male, twelve female) were tested. (Agonistic and courtship acts occurred at areas free of structures also, see text) 76a Ma les Females 55 B agonistic (won) D courtship E3 feeding FOOD NO FOOD FOO1D NO FOOD 1 77 to enter their territories, even when the holding was over a food area. Females, however, showed a significantly greater number of defences at petri plates containing food (p = .01) than at empty ones (Fig. 20). Most of these, defences (p<.005) were during the day (Fig. 20) and were performed almost equally (p>.05) against males and females (Table VIII). A female was observed to feed outside the area she defended only once, whereas a male was observed to leave its territory to feed elsewhere on five separate occasions. One replicate had to be repeated since three spawning acts occurred during the test. However, it is interesting to note that the spawnings took place over an empty petri plate. In addition to defending the areas over petri plates, male dace also defended areas free of enclosures. Although adequate structures of the BC type were available (5 structures: 4 fish), males nevertheless defended these "open" areas 62 times. These defences represented 26.7% of the total number of agonistic acts won by males. Of these agonistic acts won over open areas, 79% were at night and all night defences were against other males. Females never defended such open areas. Since open areas consisted of coarse gravel, this information supports earlier findings (Fig. 8) which showed that male territoriality was restricted to areas with coarse bottom substrate. As in earlier experiments (see Table IV), females displayed better success during agonistic interactions with males, winning 76.7% compared with 23.3% for males (Table VIII). Once again, however, this difference is attributable to the fact that females were more faithful to their occupied areas, and thus were usually residents.being intruded upon by males. 78 Table VIII. Mean number of agonistic acts won per food and no food area by male and female dace against the same and opposite sex during day and night. Numbers in parentheses indicate total number of wins. Replicates I-VI are combined. Twenty-four dace (twelve male, twelve female) were tested. Mean No. Agonistic Acts Food No Food Combined won/area Day Night Day Night Day Night by female against female 29.0 2.5 5.0 8.3 14.6 6.0 (73) (30) by female against male 26.0 7.5 15.0 6.7 19.4 7.0 (97) (35) by female.against both female and male 55.0 10.0 20.0 15.0 34.0 13.0 (170) (65) by male against male 6.5 10.0 12.7 19.7 10.2 15.8 (51) (79) by male against female 5.0 3.0 3.3 4.7 4.0 4.0 (20) (20) by male against both male and female 11.5 13.0 16.0 24.3 14.2 19.8 (71) (99) 79 The results indicate that the presence of food is not necessary for the expression of territoriality by either male or female dace. However, females did select and defend shelters containing food and thus the presence of food may be a proximate factor involved in the selection of territories by females. For males, evidence instead points to the coarse bottom substrate as the factor determining where they select territories.. The brief experi• ments reported on here leave unresolved the question of whether food provi• sion may be an ultimate factor in male and/or female territory. What hope• fully has been demonstrated is the likelihood that, at least for female dace, the distribution of food appears to influence the selection of terri• tory sites. Although food is abundant and relatively uniformly dispersed throughout the riffle habitat, it seems plausible that specific areas may be especially attractive to dace if drift patterns continually replenish food supplies there. The results of the foregoing experiment harbour an uncertainty. The competitive nature of the tests (two food areas: four fish) coupled with the 'dominance' of females over males may create a somewhat misleading situation with respect to male preferences for food and no food areas. To remedy this problem it was resolved to examine males and females separately. However, attempts to test females alone (three food areas: three no food areas: three females) failed to provide any additional information on the role of food in female territory. Female territorial behaviour was sporadic and the measures of agonistic acts won, used to indicate selection of territories,' were not attainable. These results were unexpected and actually prompted the earlier reported experiments on social factors affecting 80 territorial behaviour in female R. c_. dulcis (page 45 ) . Nine male dace (89 to 105 mm in fork length) tested alone in three replicates (three food areas: three no food areas: three males) behaved similarly to when tested with females (compare Fig. 20 and Fig. 21). Position checks made at 15 min intervals during the 30 min observations showed that males occurred more frequently at no food areas than at food areas (Fig. 21A). During both.day and night, these males defended no food areas significantly more (p<.01) than, food areas (Fig. 21B) . Although vacant shelters were available, dace often defended open areas at night (Fig. 21B) . This agrees with previous results and once again stresses the importance of the coarse spawning gravel component in the selection of territories"byiamale B. Non-Breeding Phase a. Site Preferences of Male and Female Dace in Late Summer and Winter i. Outline of Experiment Outside the breeding phase, longnose dace territoriality is consider• ably relaxed. This alone suggests that longnose territories are related primarily to reproductive activities. The requirements of dace certainly differ from breeding to non-breeding phases, and it is to be expected that these differences will be reflected in the social structure. For compara• tive purposes, experiments on site preferences conducted during the spring with breeding dace were repeated during the late summer (September) and winter (January) of the non-breeding phase. 81 50 c 4) 30 o 40 • day C o B night 5 30 20 h c o 10 O) < FOOD NO FOOD OPEN Fig. 21. Percent occurrence (A) and total number of agonistic acts won (B) at food, no food, and open areas by male dace during day and night. Replicates I-III are combined. A total of nine males were tested. 82 ii. Procedure The experiment conducted to determine what factors are involved in the selection of a territory by dace was repeated with late summer and winter collected fish. Four recordings were made under both day and night conditions. However, since agonistic acts by non-breeding dace were infrequent, position checks were also made at 15 min intervals during the 30 min recording periods. If dace.were in motion during a position check their position was recorded once they remained stationary for 10 sec or more. In addition to the six types of structures described earlier (see Fig. 7), the position referred to as "open" and abbreviated "0" was used to designate dace not positioned at any structure. Only two instead of three winter replicates were performed, since it is exceedingly difficult to collect large numbers of dace at this time. iii. Experimental Fish Eighteen dace (nine male, nine female), measuring 82 to 105 mm in fork length, were used for late summer replicates and twelve (six male, six female), measuring 80 to 117 mm in fork.length, were used for winter replicates. iv. Results and Discussion Unlike breeding individuals, dace collected in late summer displayed little agonistic behaviour (Table IX). In addition to quantitative differ• ences, qualitative differences also existed between the agonistic behaviour performed by dace during breeding and non-breeding phases. When agonistic activity occurred during the non-breeding phase, it was in the form of the Table IX.. Mean number of agonistic acts won per area per 30 min by male and female dace during late summer. Replicates I-III are combined. Eighteen dace (nine male, nine female) were tested. Mean Number of Agonistic Acts Won/area/30 min BC BF CC CF SC SF 0 Time of Day Female Male Female Male Female Male Female Male Female Male Female Male Female Male Day 0.16 0 0.07 0 0.13 0.05 0 0 0 0 0 0 0 0 Night 0.02 0 0 0 0 0 0 0 0 0 0 0 0 0 Day and Night 0.18 0 0.07 0 0.13 0.05 0 0 0 0 0 0 0 0 84 weaker behaviour patterns of 'butting' and 'biting'. The agonistic behaviour patterns of 'darting', 'chasing', and 'fighting', however, were not common outside the breeding phase (Table X). Table X. Agonistic behaviour patterns commonly performed (X) by breeding and non-breeding dace. Behaviour Pattern Breeding Non-Breeding Butt X X Bite X X Dart X - Chase X - Fight X — Figure 22 shows a striking similarity in male and female site preferences during late summer. Females were not restricted to BC and BF areas, and males no longer showed rigid preferences for coarse bottom substrate at night (compare Fig. 22 with Fig. 8). Both sexes occupied BF structures most frequently (33 - 54.6%) during both day and night and more than 80% of all position checks found two or more dace (of the same or both sexes) sharing such structures. BC and BF structures together were the preferred sites accounting for 76.4% occurrence of females and 64.2% occurrence of males during the day and 51.1% occurrence of females and 38.8% occurrence of males at night. Marked behavioural differences 0 85 DAY 45 D females B males 30 o » < 15 « a c « BC BF CF SC IF If 15 30 NIGHT Fig. 22. Percent occurrence of male and female dace at different areas during late summer. Only frequencies larger than 1% are shown. Replicates I-III are combined. Eighteen dace (nine male, nine female) were tested. 86 between male and female dace are therefore restricted to the breeding phase. Vertebrate populations commonly show seasonal changes in social organization involving alterations in the relations between the sexes. The changing social grouping of fishes during reproductive activities is complex and varies widely with the species involved (Breder, 1959). Fishelson (1970) has shown that the sergeant major fish change from feeding schools of mature fish to breeding colonies of males that court females which remain in schools. Out of the breeding season both sexes of three- spined stickleback foarm aggregations or schools, but when the males develop their breeding colors they become aggressive and disperse. Females con• tinue to school until egg laying begins. Dace collected during winter displayed no observable agonistic behaviour. Dace were lethargic and position changes which were common in spring and late summer were infrequent under winter conditions. Unlike site preferences of dace during.spring and late summer periods, all winter dace restricted themselves to enclosures (BC and BF) (Fig. 23). Occasional movements outside the enclosures accounted for the occurrence at open areas. Although food was available throughout the channel, dace fed infrequently. Reduction in metabolic rate and activity are general phenomena of poikilo- therms which account for the reduced food intake. Densities at enclosures were high and dace were often pressed firmly against other fish. Under these conditions, dace were able to remain stationary without any movement of the fins or body. During winter, field dace are generally located under large rocks several layers deep in the substrate. Such behaviour may prevent downstream DAY G females B males BC BF CC CF SC" SF NIGHT Fig. 23. Percent occurrence of male and female dace at different areas during winter. Replicates I and II are combined. Twelve dace (six male, six female) were tested. 88 displacement and ice-scouring during the winter period when highest discharges occur in the river. The chances of predation by the winter run of adult steelhead trout and salmon would also be reduced by such hiding behaviour. Edmundson et al., (1968) found that winter locations of young chinook salmon and steelhead trout were primarily under or between rubble particles. Hartman (1965) reported that juvenile steelhead trout hide during winter while juvenile coho salmon form dense groups. He suggested that these responses protected the fish against predation, downstream displacement, and ice-scouring. b. Seasonal Variation in the Spacing Patterns of Dace i. Outline of Experiment The preceding section has shown that site preferences of male and female dace change significantly from season to season. It also suggests that the grouping tendencies or spacing patterns of dace change markedly as well. The objective of the following experiment was to assess the seasonal changes in spacing patterns of adult dace, and to determine what role the relations between individuals (i.e., interactions) played in the observed spacing patterns. Spacing patterns of spring, late summer, and winter collected dace were compared by placing a constant number of dace into a standardized experimental riffle. Agonistic interactions between dace were recorded and the grouping conditions under which they occurred were noted. ii. Procedure Two 3-day replicates were performed with dace collected during the 89 spring (breeding phase), late summer, and winter. The standardized riffle in all tests consisted of six enclosures (BC) uniformly spaced on the experimental channel bottom. All remaining bottom areas were covered with coarse gravel. In each replicate, six dace (three male, three female) collected during a particular season were placed in the channel. After a 12 hr adjustment period, four 30 min recordings were made during both day and night. Position checks of individual dace were made at 15 min intervals. Number of agonistic acts performed by solitary dace and dace in groups were scored. i iii. Experimental Fish Spring (May) collected dace (six male, six female) ranged from 85 to 106 mm in fork length, late summer (September) collected dace (six male, six female), from 87 to 103 mm in fork length, and winter (January) col• lected dace (six male, six female), from 77 to 107 mm in fork length. iv. Results and Discussion The data on spacing patterns of dace were compared with Contingency Chi-square tests. Frequency of occurrence of different group sizes were calculated as shown earlier (see page 46). Spacing patterns of dace (i.e., occurrence of group sizes.oone, two, and three or more) in the standardized experimental riffle changed signifi• cantly from spring to late summer (p<. 001) and from late summer to winter (p<.001; Fig. 24). Breeding dace were characterized by a predominance of solitary individuals (both male and female), a small percentage of pairs, and progressively fewer groups of three and four. Mean group size for this phase was only slightly more than one (Fig. 25A). Outside the breeding 90 1 2 3 4 4+ GROUP SIZE Fig. 24. Percent of occurrence of different group sizes of dace during spring, late summer, and winter seasons. Replicates I and II are combined. Twelve dace (six male, six female) were tested for each season. 91 Fig. 25A. Relationship between total number of agonistic acts per• formed and mean group sizes of dace during spring, late summer, and winter seasons. Replicates I and II are combined. Twelve dace (six male, six female) were tested, for each season. Fig. 25B. Relationship between total number of agonistic acts per• formed, number of agonistic acts won (in parentheses), and different group sizes of dace during spring, late summer, and winter seasons. Replicates I and II are combined. Twelve dace (six male, six female) were tested for each season. 91a A. 200 • © Spring O Late Summer < •Winter y IOO zo oh -A MEAN GROUP SIZE J I 1 I l_ 1 2 3 4 4+ GROUP SIZE phase, numbers of single dace occupying enclosures decreased while groups of two, three, four, and four plus became more common (Fig. 24). Mean group size during the late summer exceeded two (Fig. 25A) . During winter, clumps or larger group sizes were most prominent (Fig. 24) as mean group size approached three. The data on interactions between dace suggest that agonistic beha• viour is the mechanism promoting the observed spacing pattern of breeding dace. Solitary dace account for the majority -of agonistic acts performed during the spring breeding season, while those dace in pairs and in groups of three or four show progressively less agonistic behaviour (Fig. 25B). Frequency of success during agonistic interactions (i.e., agonistic acts won) follows a similar pattern for each group size (Fig. 25B). Solitary dace are almost always successful in defending their territories against intruders, but agonistic behaviour performed by dace in larger groupings often passes with no change in group size occurring. During late summer and winter seasons, agonistic behaviour occurs infrequently (Fig. 25). Therefore, it is.suggested that this difference in the amount of agonistic behaviour performed by breeding and non-breeding dace accounts for the establishment and maintenance of different group sizes. In the experimental channel, winter dace often crowded into one of the available shelters (i.e., more than four dace at one enclosure). In late summer, shelter sites similarly formed loci for the formation of aggregations. Unlike winter groupings, however, such late summer groups were continually changing in size and location. 93 Seasonal changes in spacing patterns of dace correspond with fluctuations in water flow. Such fluctuations in flow ultimately deter• mine the amount of riffle habitat available to dace. In the spring, when dace space out by claiming individual territories, flows are high and riffle areas are plentiful. During late summer, water flows diminish and dace are concentrated into riffle habitats of reduced area. At this time of year dace are able to remain close to one another without consider• able antagonism. c. Role of Food in the Distribution of Dace in Late Summer i. Outline of Experiment Food localization experiments identical with those conducted with breeding dace were repeated with dace collected during the late summer. Tests were made to determine what role the presence of food might play in the distribution of dace within riffles during the non-breeding phase. ii. Procedure Procedures were essentially identical to those already described, except for appropriate temperature and photoperiod changes. Once again, however, position checks were made (three per 30 min) and calculated per• cent occurrences were assumed to be indicative of site preference. As in previous experiments the "open" position was used for recording pur• poses. Feeding acts as defined earlier were also recorded. iii. Experimental Fish Twenty-four dace (twelve male, twelve female) measuring 80 to 101 mm in fork length were used. 94 iv. Results and Discussion - Agonistic acts, although infrequent, were won at both food and no food areas (Table XI). Both males and females fed from petri plates containing tubifex worms (Table XI), but occurrence at food areas was not significant for either males (p>.05; Wilcoxon matched pairs test) or females (p>.05; Fig. 26). Once again each fish was compared against itself in performing the matched pairs tests. Two or more dace (same or both sexes) were found sharing shelters over.food plates on 30% of the position checks, while 51% of the checks recorded this situation at no food areas. During the earlier breeding phase food experiments, male dace but never females were observed, to defend areas free of any structures. The present experiment found both sexes to occur frequently at these open areas, particularly at night (Fig. 26). During observations, dace of both sexes often swam throughout the experimental channel, testing the substrate for the presence of food. The results suggest that it is unlikely that food determines the distribution of dace during the non-breeding phase. Within the riffle environment food is generally uniformly abundant and accessible by forays from posi• tions of cover and shelter. C. Spacing Patterns of Dace Fry i. Outline of Experiment To observe movements and spacing patterns of dace fry, eggs spawned in the stream tank were left to hatch. Fry were allowed to grow to 20 to 30 mm in length. 95 Table XI. Mean number of agonistic acts won and feeding acts performed by dace per food, no food, and open area during late summer. Replicates I-VI are combined. Twenty-four dace (12 male, 12 female) were tested. Mean number of acts/area Food No Food Open Combined Behavioural Act female male female male female male female male Agonistic - won (day) 4.5 0.5 2.7 2.7 0 0 3.4 1.8 Agonistic - won (night) 0 0 0 0 0 0 0 0 Agonistic - won (day and night) 4.5 0.5 2.7 2.7 0 0 3.4 1.8 Feeding (day) 6.0 1.5 0 0 0 0 2.4 0.6 Feeding (night) 0 0.5 0 0 0 0 0 0.2 Feeding (day and night) 6.0 2.0 0 0 0 0 2.4 0.8 96 50 DAY • fe males 30 @ males at < 10h UJ a. UJ FOOD U NO FOOD OPEN Z UJ Of U 10 O 30 50 NIGHT Fig. 26. Mean percent occurrence of male and female dace per food, no food, and open area during late summer. Replicates I-VI are combined. Twenty-four dace (twelve male, twelve female) were tested. 97 ii. Procedure After eggs were spawned in the experimental riffle areas of the stream tank, upstream and downstream screens were removed and sloping floors werers added to each end of the false bottoms. In this manner, an alter- ating riffle-pool environment was created in which the elbows of the tank provided the deeper and slower flowing pool zones. Several rocks and a layer of gravel were placed on the bottom of the pool zones. Once eggs hatched, fry were observed at irregularly spaced intervals for several weeks. iii. Results and Discussion Fry were first noticed in the pool areas 16 days after eggs were spawned. At this time, fry were approximately 10 to 20 mm in length and formed large loose aggregations near the water surface. Not all indivi• duals in such aggregations were uniformly spaced nor oriented in the same direction. The majority of fry within any particular grouping, however, were spaced one to three fish lengths apart (i.e., 10 to 30 mm) and were oriented in approximately the same direction. Aggregations of dace fry observed in the field were similarly spaced. Such aggregations of a hundred or more fry were not uncommon. As fry in the stream tank grew, they appeared in smaller groups (less than 12) near the bottom in the pool zone. Occasionally individual fry were observed foraging alone. Once dace reached 20-30 mm in length, they began moving over the fast flowing riffle areas and maintained posi• tion over the bottom. Gibbons and Gee (1972) reported that young of the year longnose dace move into fast water in July and August when they are 98 between 25 and 30 mm in length. Field collections made in the present study produced similar findings. Dace fry in the experimental riffle areas displayed a preference for areas with overhead cover, adjusting their positions in response to experimental relocation of this environmental feature. Since adult dace show this same association with overhead cover, especially during the day, it seems possible that overhead objects serve as a visual cue upon which dace fix. The somewhat dorsally located eyes of longnose dace would be well adapted in this case. The agonistic behaviour patterns of darting and biting were observed among several closely spaced fry in the riffle area. Although speculative, it seemed that agonistic activity in these small groups served to disperse fry throughout the riffle, particularly upstream. Fry fed almost exclusive• ly on the algae encrusted rocks. Field observations of juvenile dace, 25 to 45 mm in length, made by Gibbons (MS, 1971) found these fish usually not more than 1 cm off the bottom and in groups of three to ten. Spaces occupied by yearling dace are small. Laboratory observations indicate that these younger fish do not enter into competition with older dace until they attain a larger size and require larger spaces. Habitat requirements of Atlantic salmon are also known to change as the fish grow (McCrimmon, 1954). Hynes (1970) suggests that a similar pattern of habitat adjustment occurs in aquatic insect nymphs in rapid water environments where shelter is critical and growing nymphs require increasingly large crevices. 99 D. Discussion of the Causation of Territorial Behaviour The data on territory selection by longnose dace, Rhinichthys £. dulcis, demonstrate a sharp contrast in the behaviour of the sexes. Males are most territorial at night, defending coarse substrate areas against other males. Male courtship is also nocturnal and occurs over these same areas. During the day, however, reproductive activity is relaxed, and males no longer show a rigid preference for coarse substrate areas. Therefore it is concluded that the major factor involved in the selection of territories by male dace is coarse spawning substrate. Females, unlike males, are not more territorial at night. They do not exhibit any bottom substrate preferences, but defend only enclosed areas. Once receptive, females leave preferred areas and court and spawn with territorial males at preferred areas for courtship and spawning. These latter areas are preferred for both defence and courtship by males. Females select and defend shelters containing food and thus the presence of food may be a proximate factor involved in the selection of territories by female dace. Although females feed on the territory this does not necessarily show that the presence of food is a significant and advantageous consequence of female territory, unless proximate and ultimate factors correspond. Similarly, collection of food outside the territory by males is not proof that male territory has no significance in relation to food. In the riffle environment it may be that once a suitable site is secured,food sources within the immediate vicinity can be harvested. For example, dace might base the selection of a territory on the type of sub• strate and/or velocity in an area (proximate factors), but the abundance 100 of food found in the area might provide the selective advantage for making this choice and thus be the ultimate factor involved. Habitat selection tests by Sale (1969) with juvenile surgeon fish and by Baker (1971) with four-spined sticklebacks have led them to agree with Hilden's (1965) conclusions that site selection is not likely to be a response directly to ultimate factors, but to a series of proximate fac• tors. Thus by responding to a series of proximate factors the ultimate factors are acquired. Circumstances in which an adequate food source is available within the confines of the territory may be preferable for female dace. Under these circumstances,frequent feeding movements outside the territory possibly involving encounters and conflicts with males would not be re• quired. The amount of feeding that may be done within the territory is certainly not inconsiderable and may be important during the brief period prior to ovulation when food intake is markedly increased (Bartnik, pers. observation). Symons (1968) hypothesized that if the function of territorial be• haviour of some species of fish is food related, the size of the territories should increase when food is scarce and decrease when food is abundant. Changes in territory size would be brought about by appropriate increases and decreases in aggression. Such increased aggression upon deprivation, of food has been observed in Atlantic salmon (Symons, 1968), in coho salmon (Mason and Chapman, 1965), and in medaka (Magnuson, 1962). Dace held in the stream tank and deprived of food for 4-1/2 days failed to show any significant increase in aggressive behaviour. 101 Earlier experiments (see pages 45-55 ) indicated that interaction with male dace is a causative agent for the expression of territorial behaviour in female dace. It is therefore argued that the difference in social structure (i.e. , female territoriality) between the two populations of dace described here is attributable to the biotic or social factors of either segregation or coexistence of the sexes. Where the sexes coexist, the phenomenon of female territoriality apparently serves to reduce con• flicts between males and females. Male dace in breeding condition are attracted to and interact with other males. This behaviour results in the formation of territory clusters. The fact that some territorial song birds seem to "clump together" at low population densities has been suggested as evidence that displaying to neighbours has a'reward' value (pers. comm., J.R. Krebs). Perhaps a simi• lar reason exists for the observed clustering of male dace territories. The importance of social interaction for the expression of territorial behaviour and the formation.of territorial groupings has been reported for various fish species. Van den Assem (1967) found that under certain condi• tions, settling and nest-building of three-spined sticklebacks is activated by interaction with conspecifics. Abel (1961) and Myrberg et al., (1967) have emphasized that group nesting by pomacentrid fish seems to result from social responses. Keenleyside (1970) has described male sunfish as being strongly drawn towards each other when establishing breeding territories. In the behavioural literature, the term habituation is often used to describe the reduction of aggressive interaction between territorial neighbours. Several recent papers have demonstrated that the aggressive 102 responses of territorial fish will habituate when the fish are exposed repeatedly to conspecifics confined to glass tubes (Peeke, 1969; Peeke and Peeke, 1970), free-swimming adjacently territorial neighbours (van den Assem and van der Molen, 1969; Peeke et al., 1971; Gallagher et al., 1972), or crude models of conspecifics (Peeke et al., 1969; Peeke, 1969). In the present study, however, the aggressive behaviour of interacting clustered territorial male dace did not habituate. Instead, such males continued to attack intruders from neighbouring territories. Although these findings appeared.to be at odds with the literature cited above, they are more tenable in the light of the most recent investigations (Peeke and Veno, 1973) of habituation. In their laboratory tests, Peeke and Veno clearly demonstrated that territorial fish can discriminate individual morphological cues as well as geographic position cues. This led them to conclude that widespread generali• zation of habituation does not occur. These findings actually confirmed the belief held by the authors of the earlier habituation studies that both the variability of the releasing stimuli and the occurrence of inter• mittent consummatory acts (i.e., reinforcement through victory) in nature would protect fish against habituation. Outside the breeding phase, the cues used by dace in site selection change markedly and male and female dace differ little in the areas they choose to occupy. Agonistic behaviour is infrequent among non-breeding dace and as a result the distribution of dace within riffles becomes con• tagious or clumped, particularly during winter. 103 During the non-breeding phase (i.e., late summer) shelters serve as loci for the formation of aggregations of dace. However, at this time neither sex appears to respond to the presence of food in selecting particular sites. Many bird species form territories in the breeding season but occur in flocks at other times of the year, and these changes correlate with a change in food dispersion from an evenly dispersed supply to one that is patchy in distribution (Crook and Goss-Custard, 1972). Changes in spacing patterns of longnose dace from breeding to non-breeding seasons, however, appear unrelated to food dispersion in riffles which probably remains relatively constant through spring and summer months. 104 SECTION III THE FUNCTIONAL SIGNIFICANCE OF THE SOCIAL ORGANIZATION OF DACE A. Introduction Hinde (1956, 1969) has pointed out that two rather different meanings of "function" are sometimes confused. The term is often used to mean any advantageous consequence. Alternatively, other authors (Tinbergen, 1957, 1965; Brown and Orians, 1970) restrict the meaning to biologically signi• ficant consequences—that is, those through which selection in favour of the behaviour can act. Tinbergen states that to conclude that one has demonstrated or determined what the function of a certain behaviour is, one must experimentally demonstrate selection pressures that prevent the species from deviating from its present state. In his paper "Behaviour and Natural Selection", Tinbergen (1965) discusses a number of studies that have taken the necessary experimental approach in attempting to determine the function of various behavioural traits (i.e. , colonial nesting, voca• lizations, parental fanning, etc.). The comparative method has proved particularly fertile to the study of the adaptive significance of social organizations. This approach makes use of closely related species which may differ strikingly in their beha• viour. In extensive field studies, Crook (1965) has shown that by corre• lating ecological variables with specific population dispersion types the adaptive significance of social organizations can be inferred. Since Howard (1920) wrote his classic book on bird territoriality, biologists have sought an answer to the question: "What is the function of 105 territorial behaviour?" Many naturalists have ascribed functions to territoriality mainly on inferential bases, but controlled experimentation (i.e., direct evidence) is lacking. Few studies of territorial behaviour have actually answered the question: "Would deviations from the observed norm be penalized, and if so, how?". One of the basic problems confronting behaviourists studying function is the fact that, unlike structural characters, behavioural ones are more difficult to isolate and experimentally manipulate. However, Tinbergen (1965) argues that perfectly good methods are available for the study of environmental pressures imposing demands on an animal's behaviour. Ex• perimental tests of hypotheses on various aspects of dace territorial be• haviour follow in later sections. B. Inferences from Observations and Experiments in Section II as to the Function of Various Aspects of Territorial Behaviour The observed relationship (see Section II) between courtship, defence, suitable spawning substrate, and time of day leads me to infer that an important function of male dace territory is the provision of a site in which to court and spawn with females. Since longnose dace do not actively bury their eggs by moving the substrate (Bartnik, 1970), only coarse sub• strate areas which provide natural depressions are suitable for egg deposi• tion. Eggs deposited on fine gravel would be exposed to siltation and mechanical damage from a shifting substrate. In addition, necessary re• fuges for newly hatched alevins would be lacking. A probable second function of male territory is the protection of newly deposited eggs from 106 predation. Because territorial male dace continue to defend the territory after spawning, this would seem to be a reasonable inference to make. The observations and experiments in Section II, however, lead me to make a very different inference as to the function of female territory. The experiments on territory selection and social factors affecting female territorial behaviour suggest that female territory functions, at least in part, in maintaining a sheltered area and thereby reducing conflicts with males. Such conflicts (i.e., courtship harassment and agonistic inter• action) might cause females to be frequently displaced from shelters. Females continually swimming about in the strong current of the riffle habitat may experience stressful conditions, and perhaps become vulnerable to predation. Other inferences that can be drawn from the preceding section deal with the clustering of male territories. It seems possible that clustered territorial males experience greater reproductive success than more isolated males. Finally, it seems likely that dace territorial behaviour may also limit the density of breeding fish in a given area, and therefore act as a dispersing mechanism. Experimental tests of some of the inferences drawn on the functions of territorial behaviour are presented in the following pages. C. Experimental Tests of Hypotheses Regarding Function a. Significance of Territory Clusters i. Outline of Experiment Predation of eggs by longnose males has been observed under natural conditions (Bartnik, MS, 1970). In these instances, males with eggs were 107 never seen to eat their own eggs, but when their nest sites were disturbed by shifting the substrate other males entered the nest site and devoured exposed eggs. Egg losses due to intraspecific predation were also evident in laboratory tests when the parental male was removed immediately after spawning, but not when the parental male was present under otherwise identical conditions (Bartnik, pers. observation). Although the above information might lead one to suspect that the risk of egg predation would be great when males are grouped closely toge• ther, the results of preceding experiments suggest that quite the opposite may be true. Since males of territory clusters appear to remain faithful to their chosen sites (see page 65 ) and interacting parental males show prolonged site attachment, at the nest site (Fig. 18), it seems possible that intraspecific interference and egg predation would be reduced.in territory clusters. The following experiment compares closely spaced groupings of terri• tories with more widely spaced territories to determine whether the terri• tory clusters function to reduce intraspecific interference and egg predation. ii. Procedure Two different spacings of territorial males were induced by placing BC structures, in which the underlying coarse gravel was 8 cm in depth, in either a cluster (mean distance between each - 23 cm; Fig. 27A) or spread out in the experimental channel (mean distance between each = 93-6 cm; Fig. 27B). Remaining bottom areas were covered with fine gravel and in each channel one BF structure was placed downstream of the clustered 108 s < A. til H • lm 1 1 1 [BC] B IE1 — El Fig. 27. Arrangement of BC structures in channel (top view) to induce (A) closely spaced or clustered male territories and (B) more widely spaced male territories. Arrows denote direction of current. 109 structures (Fig. 27A) or midway between, the spread structures (Fig. 27B). In each of three replicates for each type of spacing, four ripe males were introduced into the channel and allowed to interact for 48 hr. Observations showed that during this period a stable territorial grouping was formed. At the beginning of the third night period, an ovulated female and three ripe 'transient' males from holding tanks were released into the channel. Once spawning occurred.at one of the BC structures, 30 min recordings were made at 0-30 min, 1-1/2-2 hr, and 2-1/2-3 hr. Number of substrate contacts, defined as contact of the snout region with the nest site substrate, were scored for both transient and territorial males. A substrate contact was considered to be an indication of egg predation potential. Number of agonistic acts won against transients by both the parental male and other territorial males were also recorded. After the last recording, all dace were removed, killed, and preserved in formalin for stomach analyses. Pumps were disengaged and a count was made of all viable eggs remaining in the nest. iii. Experimental Fish A total of 48 dace were used. The six parental males measured 85 to 93 mm in fork length, while the remaining territorial male dace ranged from 81 to 107 mm in fork length. The 18 transient males meausred 79 to 100 mm in fork length and the six females 85 to 109 mm in fork length. Sizes of transient males used in closely spaced and widely spaced groupings did not differ significantly (p».05; Kolmogorov-Smirnov two sample test; Siegel, 1956). Prior to each test, transient males were kept in holding tanks where food was constantly available. 110 iv. Results and Discussion In clustered groupings of territorial males, entries into the nest site by transient males during which egg predation may have occurred (i.e., substrate contacts) were few, and almost no egg predation occurred (Fig. 28). In these groupings, territorial males remained faithful to their chosen sites and frequently defended them against transient males (Fig. 28). In the more widely spaced groupings, considerable egg predation occurred (Fig. 28). Transient males contacted the nest site substrate numerous times and ate 6.3 eggs per transient male (Fig. 28). In one replicate with widely spaced territories, one territorial male abandoned his territory to join transient males in following the female. This male entered the nest site during the spawning act and ate several eggs (see Replicate II; Table XII). Although this particular male initially defended his territory against transient males, his behaviour changed to resemble that of a transient male. However, all other territorial males tested under these more widely spaced conditions displayed strong site faithful• ness . They did not enter the nest site, but remained on their territories and defended them against trespassing transient males. Parental males of the clustered groupings were required to defend 2 their nests less frequently (X ='A'A.5, p<.001 ) than parental males of the widely spaced groupings (Fig. 28). Furthermore, a significantly smaller percentage of these defences against transients by parental males of clus- 2 tered groupings (X = 20.5, p<. 001) were made in the presence of the female (Table XII). Intrusions by male dace on courting pairs usually disrupted Ill CLUSTERED WIDELY SPACED 18 I transiento'd' 16 D territorial0^5* S parental (f(f 14 12 1 _2 o 2 io| « a 8 rl « E Z 61 substrate agonistic eggs substrate agonistic eggs contacts acts won eaten contacts acts won eaten against against transient Jl/1 transientJcP Fig. 28. Mean number of substrate contacts, agonistic acts won against transient males, and eggs eaten by different types of male dace at nests spawned in clustered and widely spaced terri• torial groupings. Replicates I-III are combined. For both experimental groupings, N = 9 for transient and territorial males, and N = 3 for parental males. Table XII. Number of substrate contacts and eggs eaten by different types of dace at nests spawned in (A) closely spaced, and (B) widely spaced territorial groupings. Number of agonistic acts won against transient males and number of viable eggs remaining in the nest at the completion of tests are also shown for each replicate. # Agonistic Acts Won Against Spacing Pattern # Substrate Contacts trans males # Eggs Eaten by # Eggs Left of Territories by by trans terr par par in Nest trans males terr males terr males par males tmalesmalemalesrr immales female Closely Spaced (Clustered) Replicate I 2 0 21(1) 14(2) 2 0 0 0 254 Replicate II 0 1 36(4) 9(0) 0 0 0 0 289 Replicate III 7 0 23(4) 18(4) 0 0 0 0 427 Mean No. per male 1/ 0.1/ 8.8(.l;)/ 13.6(2)/ 0.2/ trans male terr male terr male par male trans male B. Widely Spaced Replicate I 17 0 18(6) 16(5) 26 0 0 0 273 Replicate II 12 19* 10(2) 33(19) 12 19* 0 0 380 Replicate III 21 0 11(1) 29(11) 19 0 0 0 208 Mean No. per male 5.5/ 2.1/ 4.3(1)/ 26(11.6)/ 6.3/ 2.1/ trans male terr male terr male par male trans male terr male ) = in presence of female * = all by one male M ho 113 the courtship sequence with the female leaving the territory. This would suggest that interference with spawning activities is reduced in the closely spaced distribution. Observations indicated that transient males could not enter those nest sites surrounded closely by other territories as readily as they could the more isolated ones. It was quite evident from the observations made that when several male dace intruded simultaneously, the parental male's success at defence was rather poor. Clustered groupings, however, had the effect of splitting up transient males and eliminating the "ganging-up" effect. The behaviour of the territorial males in both types of groupings was, with the one noted exception, similar. This suggested that the actual distance between territories was not necessarily the most crucial factor in determin• ing the success of spawning males. What appeared to be just as important was the type of males occupying a local area in the riffle. Proximity of males possibly promotes synchrony through visual and/or physical contacts. Such synchronized behaviour apparently reduces the likelihood of territorial males abandoning territories to interfere with spawning and/or prey on.eggs of neighbouring males. In addition, it appears that transient males are less likely to intrude into clusters of territories. When receptive females move within a cluster of territorial males, each male remains faithful to its site. Each territorial male defends its terri• tory against transient males and intrudes infrequently on neighbouring fish. An assemblage of closely spaced active males may be of further value in attracting receptive females which may spawn with several males in the cluster. As each, male spawns, the increased nest site tenacity and egg 114 eating inhibition of parental dace would help to further reduce the likelihood of males in the cluster interfering with each others spawning activities and/or eating each others eggs. Male three-spined sticklebacks well established on a territory and heavily involved in parental activities also show little intrusion or egg raiding behaviour (Wootton, 1971b). Although van den Assem (1967) recognized that females might be more attracted to a group of males than a solitary one, he also recognized certain disadvantages for a stickleback breeding in a territory cluster. Unlike longnose dace, whose territories are fixed in size by the topography of the rock riffle bottom, stickleback territories become reduced in size when crowded together. As a result, stickleback males in such groupings act as competitors, interacting aggressively and interfering with one another. Van den Assem found the stealing of eggs, interruption of fanning bouts, and insufficient development of fanning to be common in these rival situations. The foregoing conclusions on dace territory clusters are strongly supported by a two-part experiment done with similarly closely spaced males .eh, The availability of spawning gravel was used to control the beha• viour of the males. In both experiments A and B, five structures were placed in the channel (mean distance between each = 30 cm) and the remaining r bottom area wasacoveredhwlth-if ±ne' -gravel'v. eln ;experiment"A?- onevstructure was of the BC type and the other four of the BF type. In experiment B, all five structures were of the BC type (Fig. 29A). Thus only one suitable area for a male territory existed in experiment A, but there were five in experiment B. Four ripe males and one ripe female were tested in each 115 Fig. 29A. Total number of agonistic acts won by male and female. dace in experiments A and B before and after spawning. Abbreviations refer to structure-substrate combinations. Fig. 29B. Levels of agonistic activity performed at the nest site in experiments A and B before and after spawning. (LD 16:8) 116 experiment. After a 12 hr adjustment period, four 30 min observations were made during both day and night to record agonistic activity. Once spawning began, two additional ripe males referred to as "transients" were added. Two 30 min recordings we-re,mSde,ionedimmediately:'and one .1 hr later. In experiment A, only one male territory existed throughout the test, while several existed in experiment B (Fig. 29A). Experiment A's one suitable spawning site was defended over 500 times during the 6 hr of observation. The number of defences (i.e., agonistic acts won) at experi• ment B's spawning site, however, was considerably lower (Fig. 29A). Al• though the level of agonistic activity was higher in experiment A (12 to 119/30 min) than in experiment B (3 to 23/30 min), similar trends occurred in both before and after spawning (Fig. 29B). The parental male in experi• ment A was intruded on repeatedly (29 to 72/30 min) as his level of agonistic activity indicates (Fig. 29B) . Eggs were eaten, in this experiment but not in experiment B (Table XIII), in which the parental male received consider• ably fewer intrusions from other males (Fig. 29B). Therefore, differences in success at nest defence in the two situa• tions would appear to be attributable to the difference in the number of intrusions from other males. Unlike the test males without territories in experiment A, behaviourally synchronized ones of experiment B spent the majority of time defending their territories and intruded less upon neigh• bouring males. Experimental males in. experiment A, however, continually entered the BG area before the spawning act and attempted to court and spawn with the female. Consequently, the test males without territories 117 Table XIII. Number of eggs eaten by dace in Experiments A and B Number of Eggs Eaten Type of Dace Experiment A Experiment B parental male 0 0 parental female 0 0 experimental male 10 0 experimental male 2 19 0 experimental male 3 9 0 transient male 1 0 0 transient male 2 0 0 118 in experiment A were in reality transient males. Although the density of males and the spacing of shelters was identical in both experiments, a difference in the success of the spawning males occurred. This difference was related to the behaviour of the males surrounding the spawning site. These results reemphasized the fact that the simultaneous intrusion of several males was the most disruptive. Under these conditions, the parental male was not able to prevent entry, sub• strate contacts, and subsequent egg predation. b. Significance of Post Spawning Changes in Male Behaviour Although evidence already collected argues that an important function of male territory is to provide a site for courtship and egg deposition, a possible second function is investigated. This investigation was prompted by the fact that once territorial males have spawned they continue to defend the nest site. It seems likely that territoriality by males with eggs, although still reserving the nest site for possible future spawnings, would also serve to protect already deposited eggs from intraspecific pre• dation. Laboratory observations of numerous males have revealed that both quantitative and qualitative changes occur in the behaviour of territorial males immediately after spawning. Levels of agonistic activity at the nest site generally peak at this time, and the nature of the aggressive behaviour changes. Observations indicate that these changes in male behaviour last from one to several hours. After that time, parental male behaviour be• comes indistinguishable from normal pre-spawning territorial behaviour. 119 Changes in the behaviour of territorial fish during the reproductive phase are well documented. We know from the work of several authors, especially Sevenster (1961) and Wootton (1970), that the behaviour of male three-spined sticklebacks changes significantly at the time they begin nest-building and again when they finish nest-building. Further changes in the behaviour of male sticklebacks are observed after fertiliza• tion of eggs (Sevenster-Bol, 1962; Black, 1971; Wootton, 1972), with peaks of the U-shaped temporal pattern in aggressive behaviour coinciding with periods immediately after egg fertilization and fry hatching. A detailed analysis of the agonistic behaviour of a typical male dace before and after spawning is shown in Figure 30. An increase in the frequency of occurrence of the darting behaviour pattern is observed after spawning. Before spawning, territorial males usually react to an intruding male only once it enters the territory. Thus the resident and intruding fish are in close proximity, and the behaviour patterns performed in defence are usually butting and biting (Fig. 30). However, once an attack is initiated by a territorial male it is continued until the intruder is driven outside the territory. After spawning, parental males respond to dace approaching or even skirting along the territory boundary. Parental males dart out towards such intruders. If the intruder does not flee in response to the owner's dart, but continues to move towards the territory, then the defending male will bite or butt the intruder as it attempts to cross the border. Once such an attack is initiated, the parental male often gives chase until the intruder retreats away from the territory. With this apparent increase 120 HOURS Fig. 30. Agonistic activity performed by a territorial male before and after spawning. Histograms indicate frequency of . occurrence (%) of different agonistic behaviour patterns for each 15 min recording. Three other males were present in addition to the spawning pair. 121 in aggressive motivation, trespassing fish are attacked more readily and levels of agonistic activity at the nest site are higher relative to later in the post spawning period. Stickleback studies (van den Assem, 1967; Black, 1971; Wootton, 1971b, 1972) have shown that territory size actually increases as a function of aggressive levels. Sizes of dace territories were not measured in the present study, since the shelters defended by dace were fixed in size. Unlike stickleback territories, sizes of dace territories observed in nature are fixed by the topography of the bottom. Any increases in dace territory size which may occur in the rock riffle habitat would be slight and difficult if not impossible to quantify. Topography of the habitat is known to influence the size or borders of the territories of a number of fish species (Greenberg, 1947; Fabricius, 1951; Kalleberg, 1958). The actual level of agonistic activity performed by a parental male dace is, of course, dependent on the number of trespassing or intruding dace interacting with the parental male. However, qualitative changes in aggressive behaviour, as described here, are common to all territorial males after spawning. Furthermore, males observed to spawn several times over a number of days display this behavioural change after each successive spawning. Therefore, each subsequent clutch of eggs would appear to be afforded the same degree of protection as the first. A second change in male behaviour also occurs after spawning. The rate of substrate probing by parental male dace remains high, but then declines quickly within several hours. Data for this behaviour change were presented earlier (see Fig. 18). Although substrate probing is 122 considered to be an important component of courtship behaviour, it also appears to be important in nest site preparation and/or maintenance. A more detailed analysis of the significance of this post spawning change in male substrate probing behaviour follows. c. Significance of Male Territory as Determined by Vulnerability of Dace Eggs to Intraspecific Predation i. Outline of Experiment Since post spawning changes in male dace behaviour are temporary and most pronounced during the first few hours after spawning, the likelihood that this period coincides with periods of greatest egg vulnerability was examined. The following series of replicates tested the vulnerability of dace eggs to intraspecific predation at varying times after spawning. ii. Procedure For each replicate, a parental male was provided by introducing a single male into a small channel (60 x 23 cm). Except for one BC struc• ture, in which the underlying coarse gravel was 8 cm in depth, the bottom area was covered with fine gravel. At the commencement of a night period, 12 hr after the male's introduction, an ovulated female was added to the channel. Spawning invariably took place over the coarse substrate of the BC structure. For each egg vulnerability test, three ripe male dace held in a transparent plastic box (12.5 x 7.5 x 7.5 cm) with a plastic screen top (3 meshes/cm) were placed slightly downstream from the spawning site. To commence an egg vulnerability test, the screen was removed and these transient males-were' otales released. Vulnerability tests were of 3 hr duration 123 with three 30 min recordings made at 0-30 min, 1-1/2-2 hr, and 2-1/2-3 hr. Number of substrate contacts were scored for all dace. Substrate probing, of course, was included in this category. Agonistic acts per• formed by the parental male were also recorded. After the last recording all dace were removed, killed, and preserved for stomach analyses. Pumps were disengaged and a count was made of all viable eggs remaining in the nest. Vulnerability tests were made at four different times after spawning (0,4,24, and 72 hr) and all took place during the night period. Times of 0, 4, 24, and 72 hr after spawning were chosen because of their position relative to the period of post spawning changes in male dace behaviour. The most marked change occurs immediately after spawning, while 4 hr falls somewhere after this period. Twenty-four hr. is well outside this period as is 72 hr. A shortage of both time and fish prompted the decision not to replicate the 72 hr test. For the three replicates at 0 hr after spawning, the plastic box con• taining transient dace was placed in the channel shortly after the first courtship behaviour patterns were performed by the female. Once spawning began, transients were released. Thus the transient fish were present during the spawning acts. In the remaining sets of replicates, the female was removed as soon as her abdomen was noticeably reduced in size. She was stripped of any remaining ripe eggs and was placed in a 40 1 aquarium. This tank always held some ovulated females. The box containing transient dace was placed in the channel 2 hr before the transient males were to be released. Just before either 4, 24, or 72 hr had elapsed since spawning 124 occurred, the parental (i.e., recently spawned) female was taken from the holding aquarium, checked for complete stripping, and returned to the channel. This was followed by the release of the transient males approximately 5 min later. Parental females were stripped of ripe eggs to ensure that no further spawning took place after their return to the channel. Such females were held in aquaria with ovulated females since it was observed earlier that females and even males kept in such water were attractive to territorial males (i.e., there was an increase in the courtship level). These find• ings, along with observations of ripe males synchronizing movements with ovulated females while on the opposite side of an opaque partition, suggest that an olfactory stimulus may induce sexual responsiveness in male dace. In the goldfish, the brief introduction of a recently ovulated female goldfish into water containing males increases the courtship level of males which persists even in the absence of the female (pers. comm., N. Stacey). Male channel catfish demonstrate a strong attraction to the source of a sexual pheiBmptfereleased bv ripe females of that species (Timms and Kleerekoper, 1972). The importance of pheromones in the social behaviour of yellow bullheads also has been demonstrated (Todd e_t al. , 1967). iii. Experimental Fish A total of forty males and ten females were used. Parental males (10) measured 87 to 96 mm in fork length, while parental females (10) measured 81 to 101 mm in fork length. Transient males (30) ranged from 75 to 101 mm in fork length. Before each test, transient males were held in holding 125 tanks with food constantly available. iv. Results and Discussion The procedure used proved to be a satisfactory method for attracting transient dace to the nest site where egg predation could occur. When the spent female was returned to the channel in the 4, 24, and 72 hr tests, she attempted to enter the only available enclosure (i.e., the nest site). The parental male immediately began courting her by frequent substrate probing, trembling, and nudging. The.released transient males were also attracted to the female and courted her by following, nudging, and quiver• ing behaviour patterns. These transient males frequently entered the nest site where the parental male was courting intensely. The parental male frequently interrupted his courtship activities to defend the nest site against the intruding males. In the presence of the female and the sub• strate probing parental male, transient males also probed in the substrate. Transient males performed considerably more.substrate contacts during ac• tual spawning (i.e., 0 hr) than they did for the other test times (Fig. 31). This is attributable, at least in part, to the increased stimulus of spawning behaviour, during which both the parental male and female dis• played considerable substrate probing (Table XIV). During 4, 24, and 72 hr tests, snout contacts with the nest site substrate (used as an indicator of egg predation potential) though less frequent,were still performed by transient males. The results indicate that eggs were most vulnerable to predation immediately after being spawned, less so after 4 hr, and apparently not at all in 24 and 72 hr old nests (Fig. 31). Since a comparatively large number of eggs were present in all tests (Table XIV) and the likelihood of 126 Hours After Spawning g. 31. Number of eggs eaten and substrate contacts made by transient males at 0, 4, 24, and 72 hr old nests. Vertical lines above and below means (#,A ) represent ranges. Sample size is three except for 72 hr where N = 1.. Table XIV. Number of substrate contacts made and eggs eaten by transient and parental dace at 0, 4, 24, and 72 hr old nests. Amount of agonistic activity performed by parental males during test periods and number of viable eggs remaining in nests at com• pletion of vulnerability tests are also shown for each replicate. Hours After # Agonistic Acts # Substrate Contacts by # Eggs Eaten by # Eggs Left eggs spawned by par maler trans males par male£ par females trans males par male par in Nest female I 32 34 20 17 36 0 0 239 II 46 60 32 19 18 0 0 362 III 28 18 39 23 14 0 0 177 Mean 37.3/replicate 22.7/replicate I 24 15 32 2 14 0 0 395 II 18 20 29 0 17 0 0 270 III 30 17 19 5 4 0 0 388 Mean 17.3/replicate 11.7/replicate I 31 11 24 0 0 0 0 410 II 19 18 33 3 0 0 0 187 III 14 7 20 0 0 0 0 201 Mean 12/replicate 14 I 12 11 273 128 locating eggs existed, as indicated by substrate contacts, differences in accessibility appears to have accounted for varying amounts of predation observed. Previous observations have shown that nests attended by parental males for longer periods differ in the total amount of substrate probing the gravel containing the eggs has undergone. Differences in appearance of nests of varying ages are apparent when viewed from above with a face mask. In the field, older nests generally are more.difficult to locate by means of mask and snorkel viewing than are freshly spawned ones. In new nests, several eggs adhering to the surface layer of rocks are usually visible. However, in older nests, eggs are located on the under surfaces of stones and down among the interstices of gravel. In these older nests, surface pieces of gravel generally must first be moved before any eggs are. visible. Under stream tank conditions, it has been observed that movements of attending parental males (i.e., substrate probing and fin scraping over the nest substrate) frequently break adhesive points of newly attached eggs, causing them to fall deeper into crevices. Since the period of heightened male activity (see Post Spawning Changes in Male Behaviour, page 118 ) is most marked within the first few hours after spawning, it apparently serves to bury freshly deposited eggs deeper in the substrate. Although egg vulnerability tests suggest that eggs are still vulnerable 4 hr after spawning, they are considerably less so than immediately afterwards. The possibility of changes in egg odor being correlated with vulnerability has not been examined. Although eggs in older nests (24 and 72 hr) were shown not to be vulnerable in these tests, such eggs are eaten if the nest is 129 physically disrupted and the eggs are exposed (observed under both field and laboratory conditions). The results lead one to conclude that dace eggs are most vulnerable to intraspecific predation from 0 to 4 hr after spawning. Vulnerability then decreases to a very low level within 24 hr. The period of heightened parental male aggressiveness, frequent substrate probing, and increased site faithfulness coincides closely with this period of vulnerability. Furthermore, as mentioned earlier, post spawning changes in male behaviour are renewed with each successive spawning bout. Thus the data suggest that parental male territorial behaviour, along with its related activities, renders the eggs in the nest less vulnerable to intraspecific predation. d. Other Predators To determine whether dace are preyed on by the other riffle inhabiting species of fish in the Alouette River, a collection of sculpins, Cottus asper and C_. aleuticus, (90 -to 155 mm in fork length) , juvenile steelhead trout, Salmo gairdneri, (83 to 110 mm in fork length), and dace (41 to 94 mm in fork length) were held together under simulated riffle conditions. Irregularly spaced observations were made during both day and night periods over a 2 wk period. i. Trout Juvenile steelhead trout generally maintained position off the bottom feeding on drifting objects. They were never observed to pick stationary items off the substratum. The only interaction observed between trout and dace consisted of a parental male dace biting the caudal fin of trout which 130 drifted downstream into the male's territory. After each bite'the; trout moved upstream. ii. Sculpins In the simulated riffle, adult sculpins captured and consumed juvenile dace and on occasion pursued and attacked adult dace. The larger adult dace (80 to 94 mm in fork length), although often injured by such attacks, were never consumed. More seriously injured fish, however, fell prey to cray• fish in the tank. For the most part, sculpins showed no apparent attraction towards territorial adult dace or towards a defended nest site. Both laboratory observations and analyses of stomachs of field collected scul• pins have shown aquatic insect larvae to be their predominant prey. Upon entering a dace nest site, sculpins were never observed to orient to or feed upon the buried eggs.' Parental male dace occasionally butted or bit a sculpin which entered the nest site, but such agonistic behaviour was mild, not persistent, and generally unsuccessful in removing sculpins. Although territorial dace were often observed to share an enclo• sure with a sculpin, conspecific males were always attacked when attempting to enter the same site. On six separate occasions, a sculpin and a male dace attempted to enter the territory of a male dace simultaneously. Each time the parental male attacked the dace, but not the sculpin. Since we know that longnose eggs are eaten by conspecifics, it would appear that the main danger to a dace territory and to any eggs within the territory comes from other dace rather than other species. Wootton (1970) comes to a similar conclusion about sticklebacks. He states that under such circumstances one would expect to find that conspecific males are attacked more readily than other fish. 131 iii. Egg Predation Initially it was thought that sculpins and/or trout might prey on dace eggs, but neither field data nor laboratory observations have sub• stantiated this. In the laboratory, sculpins never attempted to burrow in substrate containing dace eggs nor did they appear to be attracted to such sites. Trout held with dace did not feed off the bottom but concen• trated on drift. Field data on species which I suspected might be egg predators was collected by examining the stomachs of field collected fish for the pre• sence of eggs. In the spring of 1971, when freshly spawned eggs were abundant in riffles, sculpins, trout, and dace were seined. The collected fish were killed instantly by immersing them into formalin and they were returned to the laboratory for stomach analyses. At the same time, a sam• ple of several hundred eggs was taken from the entire collection area and was returned to the laboratory for incubation. After hatching, fry were reared until a positive identification could be made (20 to 30 mm). The results showed that the entire sample was comprised of Rhinichthys eggs. This same procedure (i.e., collection of fishes and eggs) was repeated in the early spring of 1972, after a spawning run of Peamouth chub, Mylocheilus caurinum, had occupied a section of river immediately upstream of a large riffle. Unlike dace, the chub is a broadcast spawner releasing large masses of eggs into shallow areas near shore where they adhere to the gravel and rock bottom. However, the current carried much of the chub spawn into the riffle area. Here it attached to rocks, like mats of algae. Unlike the previous year's sample of eggs, all eggs taken from the collection riffle in 1972 and reared in the laboratory were identified as peamouth chub eggs. 132 Differences between the two springs in the number of sculpin and trout stomachs containing eggs would appear to be attributable to the distribution of eggs in the riffle substrate. When Rhinichthys eggs were abundant (1971) , none of the sculpins and trout examined were found to contain eggs (Table XV). However, when Mylocheilus eggs were abundant (1972), 71.4% of sculpins and 80% of trout examined contained eggs (Table XV). Individual sculpins contained up to 65 eggs. The four trout with eggs produced one, two, three, and nine eggs which suggests they may have been taken as drift. The results therefore suggest that dace spawning habits appear to be effective in rendering eggs inaccessible to sculpin and trout predation. Peamouth eggs, however, which for the most part were exposed, were accessible to bottom feeding sculpins and to trout as drift. Hunter (1959), Patten (1962), and Phillips and Claire (1966) have all reported sculpin predation on salmonid fry. Clary (1972) exposed slimy sculpins to brown trout eggs either scattered over the bottom or buried under artificial redds. He found sculpins never preyed on non-hatching trout eggs, but preyed consistently on hatching eggs and sac fry. Since his analyses of stomachs of field collected sculpins verified his experi• mental findings, Clary suggested that the probable mechanism permitting sculpins to locate redds is olfaction, triggered by some compounds released by hatching eggs or emerging fry. Territorial behaviour of parental dace ceases well before hatching occurs and emerging dace fry are not afforded any protection from predation through parental territoriality. No field evidence of dace fry predation by sculpins has been found. This aspect of predation was not pursued 133 Table XV. Frequency of occurrence of eggs in Cottus asper, C_. aleuticus, and Salmo gairdneri stomachs when only Rhinichthys eggs (1971) or Mylocheilus eggs (1972) were abundant in riffles from where sculpins and trout were collected. Numbers in parentheses are percentages of the number of fish examined. Table XVI. Frequency of occurrence of eggs in Rhinichthys cataractae stomachs when Rhinichthys eggs were abundant in riffles from where dace were collected (1971). The number in parentheses is the percentage of the number of dace ex• amined . Table XV Rhinichthys Eggs Abundant (1971) Mylochellus Eggs Abundant (1972) # stomachs # stomachs fork length cocontaining fork length containing Species # examined range (mm) eggs # examined range (mm) eggs Cottus asper male 8 93-130 0 14 78-132 12 female 1 159 0 2 82-107 1 Cottus aleuticus male 4 98-112 0 5 90-111 2 female 2 110-118 0 0 0 Total Cottus sp. 15 0(0) 21 15(71.4) Salmo gairdneri 3 80-119 0(0) 5 84-130 4(80) Table XVI # stomachs fork length containing Dace # examined range (mm) eggs Adult male 27 75-95 1 Adult female 24 77-110 0 Juveniles 8 52-71 0 Total 59 1(1.7) 134 further as it is somewhat superfluous to the overall study. Stomach content data of field collected dace indicate that intra• specif ic egg predation in nature is slight. Of 59 dace stomachs examined only one contained eggs (Table XVI). However, both laboratory and field observations reported herein indicate that intraspecific egg predation does occur. e. Significance of Female Territory as Determined by Changes in Blood Lactate in Dace After Exercise If dace experience some form of stress during prolonged periods of swimming in or holding position against strong currents, provision of a shelter from current would be an advantageous consequence of territoriality. Acting on this conjecture, an initial set of experiments was conducted to determine whether dace without access to areas of reduced flow experienced greater weight loss (assumed to be a valid indicator of energy expenditure) than 1) dace with access to such areas, and 2) control dace which were in still water. No significant differences in weight loss were revealed in tests made over 24 hr, 48 hr, 3 days, and 7 days. However, behavioural differences between dace with access and those without access to areas of reduced flow were apparent. No access dace usually ceased swimming after the first or second day, and remained pressed against the sides of the tank. It was suspected that these behavioural changes under no access conditions may have confounded the measures of energy expenditure. Physiological findings by Erickson (1967) and Stevens (1968, 1972) have suggested that weight loss is a poor indicator of exercise or stress in freshwater fishes. Apparently both handling and exercise can cause small but consistent increases in body weight as a result of an increase 135 in osmotic movement of water into the animal. In the following experiment, the significance of maintaining a sheltered site in the riffle environment was tested by examining the effects of muscular activity on dace blood physiology. i. Outline of Experiment Several authors (Parker and Black, 1959; Parker et al., 1959; Beamish, 1966) have observed a correlation between mortality and blood lactate. They have suggested that many fish die from fatigue, the lethal agent being lactic acid, its action directed against the blood ph. Movements in lotic environments require active swimming against the current. Fishes accumulate lactic acid in their tissues very rapidly so they tire easily; they thus need shelter fairly often, even though they can swim well in short bursts. Miller (1958) observed that newly intro• duced trout encountered competition from resident trout and were forced to remain in the current. Here they suffered from accumulation of blood lactate due to the muscular effort in maintaining their position. He suggested that they died of simple exhaustion resulting from continuously being chased from occupied territories. Symons (1971) suggested that salmon parr in transit between territories are probably more susceptible than are territorial parr to mortality through a build-up in lactic acid or through predation. Jenkins (1969) found that stable (i.e., territorial) brown and rainbow trout were able to avoid excessive swimming. In an earlier experiment, it was demonstrated that female dace not defending a territory against other fish are often forced to make frequent movements within the riffle. Since experimental female dace defended only 136 areas of reduced water velocity, the possibility that such behaviour functions to prevent stressful conditions was tested. ii. Procedure The test chamber or swim tunnel resembled closely in design that used by Kutty and Saunders (1973) to measure swimming of young salmon. It consisted of a cylindrical plexiglas tube (7.5 cm diam) with a removable stainless steel screen (1.6 meshes/cm, 0.12 cm diam) over the upstream opening. Electrified (2-3 volts) stainless steel bars (0.12 cm diam, spread 0.6 cm apart) covered the downstream opening (Fig. 32). The swim tunnel was submerged in the stream tank containing water at 14 + 2C. Each test fish was introduced into the tube and the screen placed over the upstream opening. After a short adjustment period (2-3 min), the test fish was exercised for 5 min. Water was passed through the tube by two water pumps, each with a capacity of 2530 1/hr. Preliminary work determined 5 min to be the maximum time dace would actively resist shock by swimming against the current. Actual flow passing through the tube varied between 51 cm/ sec and 87 cm/sec, depending on whether one or two pumps were engaged. Although longnose dace have a remarkable ability to remain stationary on the bottom in flowing water, both the curvature of the tube and the occasional use of increased flow (i.e., the second pump engaged) prevented them from so doing. When dace stopped swimming vigorously, they were swept downstream by the current. Upon contacting the electrified bars, dace received a shock. When experimental fish were noticeably fatigued (i.e., disoriented), they avoided swimming and gained resting time by remaining pressed against the electrified bars. During instances of prolonged 55 cm Fig. 32. Diagram of swim tunnel used to exercise dace. 1 - plexiglas tube; 2 = inlets from pumps; 3 = screen; 4 = electrified bars. Wires from bars to electrical source have been omitted. 138 contact with the electrified grid (>5 sec), the electric current was turned off and dace were allowed to move off the bars. To compare blood lactate levels of exercised and non-exercised dace, a control group was included to ascertain a resting state. These dace were placed in the swim tunnel as outlined above, but with the velocity of water passing through decreased to 2 cm/sec. These dace were not forced to swim and generally remained on the bottom. Treatments were otherwise identical. After the test period, dace were removed from the swim tunnel, anesthetized with MS-222, and blood samples taken. Fish were sectioned at the caudal peduncle and blood was collected in heparinized capillary tubes. Since dace contain only a small volume of blood, it was necessary to pool samples from two test fish for blood lactate analyses. Two parts of 8% perchloric acid were added to one part blood, the whole blood was centrifuged (2000 rpm, 10 min), and the serum was held in refrigerated bottles. Analysis of lactate was made spectrophotometrically. The procedure used was that published by the Boehringer Mannheim Co. (BMC TLAA 2972). One alteration was made. In order to obtain replicates of samples, serum samples were diluted in a 1:1 ratio with distilled water before analysis. iii. Experimental Fish Twenty-four dace (both sexes represented) measuring 77 to 95 mm in fork length were used. Since demands for breeding dace were great, only dace in post spawning condition were sacrificed. All dace had been starved for 24 hr before testing. 139 iv. Results and Discussion Differences between lactate values of control (non-exercised) and experimental (excercised) fish (Table XVII) were compared with the 1-tailed Mann-Whitney U test (Siegel, 1956) and found to be significantly different (p = .008). Since all dace were handled identically with exception of the excercise period, observed differences could not have been due to handling but must reflect a real difference in excercise stress. Several dace re• turned to holding tanks suffered no lasting ill-effects. Therefore, lactate build-ups reached by exercised dace were obviously less than lethal levels. In salmonid fishes, resting lactate levels vary from approximately 5 to 24 mg% (Parker et al., 1959; Black, 1955, 1956, 1957 abc). After 15 min of vigorous exercise, Black (1955) found blood lactate in Kamloops trout yearlings varied from 99.5 to 100.2 mg%. Lactate levels of 125 mg% for chinook salmon (Parker and Black, 1959) and 100 mg% for haddock (Beamish, 1966) have been suggested as being lethal. The small amount of blood in dace necessitated a rather crude extrac• tion method. Therefore, what is listed in Table XVII as blood lactate is in reality some combination of arterial and venous blood plus a small amount of body fluids. These sources of error are one possible reason for the seemingly high control level of lactate (Table XVII). A second explanation may be that permanently high lactate levels are common to dace. Hochachka (1961) showed that rainbow trout conditioned to fast water showed such high levels of blood lactate. In any case, neither exercised nor control means should be regarded as absolute terms. What, however, can be accepted possibly from the results is that lactate appears to be higher in exercised dace, whether in terms of blood or muscle lactate. 140 Table XVII. Blood lactate values for (A) Non-exercised and (B) Exercised dace. Blood lactate is expressed in milligrams lactate per 100 ml whole blood (mg %). Fork Time Time Total Time Blood Wt. Length Swimming Resting in Tunnel Lactate Dace (gm) (mm) (sec) (sec) (sec) (mg%) A. NON-EXERCISED (CONTROL) 1 2.6 79 _ 300 48.3 2 6.5 90 - - 300 _ 3 4.1 78 300 48.4 4 7.0 95 - - 300 _ 5 6.2 86 300 36.8 6 4.7 86 - - 300 7 5.3 85 _ _ 300 35.3 8 4.5 80 - - 300 9 4.7 85 _ 300 47.3 10 5.7 87 - - 300 _ _ 11 6.6 88 300 41.3 12 5.3 85 - - 300 x= 5.3 x- 85.3 x= 42.9 (gm) (mm) (mg%) B. EXERCISED 1 5.3 81 275 25 300 46.3 2 6.1 85 259 41 300 3 5.8 84 199 101 300 70.3 4 4.1 75 238 62 300 5 5.8 87 250 50 300 71.3 6 4.1 77 187 113 300 7 4.1 79 151 149 300 73.8 8 4.9 81 242 58 300 9 5.0 89 147 153 300 60.7 10 6.2 90 253 47 300 11 5.2 80 138 162 300 12 60.7 8.5 95 182 118 300 x= 5.5(gm) x= 83.6 (mm) . x= 210(sec) x= 89.9(sec) x= 63.8(mg%) U - 3, p - .008 Blood lactate levels of exercised dace are significantly greater than those of non-exercised dace. (1-tailed Mann Whitney U test). Kp-1, p » .05 Body weights of exercised and non-exercised groups of dace did not differ significantly. (Kolmogorov-Smirnov two sample test). 141 Observations of individuals being exercised in the swim tunnel indicated that dace displayed an inability to actively swim into the current for the duration of the 5 min test period. Exercised dace showed visible signs of fatigue as they lost their ability for co-ordinated locomotion. They often turned ventral side up and were swept sideways against the downstream bars. Upon contacting the bars, dace usually re• mained in a semi-circular posture, making rapid respiratory movements. A similar state of fatigue in nature may render dace vulnerable to predation. Such dace may be swept downstream into open areas or pools. Dace unable to make rapid co-ordinated movements may be susceptible to sculpin and even crayfish predation. An example of the latter, involving an injured adult dace, has been observed in the stream tank. Healthy dace able to react quickly to stalking crayfish were never captured. The results show that dace cannot hold against strong currents (51- 87 cm/sec) for any great length of time. Dace forced to do so experience a rise in blood lactate, and must sooner or later pay off this oxygen debt. The fact that dace 'give up' and fall back may or may not be associated with the blood lactate levels recorded. Dace which become fatigued, disoriented, and drop downstream may be vulnerable to predation. Thus a possible function of territory in both male and female dace is that it provides a refuge from the current. f. Territorial Behaviour as a Dispersing Mechanism i. Outline of Experiment Although breeding dace aggregate in clusters (Fig. 17), they continue to defend individual territories. Thus it seems possible that the number 142 of dace breeding within localized areas might be limited if suitable space is in short supply. The following experiment was conducted to determine whether terri• torial behaviour acts as a mechanism for dispersing surplus fish away from densely populated areas. Breeding (territorial) and non-breeding (non- territorial) dace were tested in an experimental channel in which the density of dace was increased in a sequential fashion. The replicate with non-breeding dace was included for comparative purposes. ii. Procedure The experimental set up consisted of a "riffle" zone and a deeper slower flowing "pool" zone (Fig. 33A). A brown rubber mat was used to create the sloping floor between the riffle and pool. The riffle area held six uniformly spaced enclosures (BC) with coarse gravel covering the remaining bottom area. The pool area was devoid of any overhead cover or shelter components, containing only a layer of fine gravel. Both field and laboratory observations have shown that dace generally avoid this type of habitat. Both replicates were 4 days in duration. Six dace (three male, three female) were introduced into the channel initially and an additional six (three male, three female) were added on each of the second, third,and fourth days. Recordings were begun 12 hr after each group was introduced. During the four day and four night 30 min observations, records were kept of 1) positions of individual dace at 15 min intervals, and 2) number and location of agonistic acts won by solitary and grouped dace. Dace were scored as occupying the pool zone if they came in contact with the pool sub• strate or swam about in the water column of the pool zone for a minimum of 10 sec. 143 Fig. 33 A. Diagram of experimental channel illustrating riffle and pool zones. Fig. 33 B. Group sizes and distribution of dace at increasing densities during breeding and non-breeding phases. Above: Percent occurrence of dace in enclosures (BC), open areas (0), and pool (P). Below: Percent occurrence of different group sizes. Percent occurrence is calcula• ted as shown on pages 46 and 50. B. (Day i) 6cjace (Day 2) 12 dace (Day 3) 18 dace (Day 4) 24 dace 90 60 30 « 0 11 ii t, BC O P BC O P UJ BC O P BC O P oc 3 + (Breeding O 75 JNon-Breeding 50 25r ii n n il n JI n lllll. 1234 4+ 1234 4+ 123ULa4 l4+ 1234 4+ GROUP SIZE 144 iii. Experimental Fish The 24 breeding dace (twelve male, twelve female) measured 78 to 106 mm in fork length. The 24 non-breeding dace (twelve male., twelve female) were collected in September and measured 76 to 105 mm in fork length. iv. Results and Discussion The occurrence of different group sizes of breeding and non-breeding dace on day 1 was consistent with previous findings (compare Fig. 33B with Fig. 24). Both breeding and non-breeding dace were predominant at enclosures (BC) , occurring infrequently in the pool zone (Fig. 33B). With the doubling of density, however, breeding dace responded differently from dace in the non-breeding replicate. Solitary breeding dace remained the predominant group size with occasional pairs and even fewer groups of three and four. The occurrence of fish at enclosures (BC) dropped while the occurrence of fish in the pool rose markedly from 4.8 to 23.3% (Fig. 33B). In the non-breeding replicate, the occurrence of solitary dace fell steadily, and larger groupings (i.e., three, four,and four plus) predomina• ted as densities were increased. As a result, the occurrence of dace at enclosures remained high throughout the 4 day period and no marked increase in the occurrence of dace in the pool was observed. In the breeding replicate, only on days 3 and 4 when densities were three and four times the initial density did a noticeable change in the group sizes of dace take place. On these final two days, the occurrence of single dace was reduced and larger group sizes (two, three, four, and four plus) became more frequent. Enclosures were still occupied, but 145 increasing numbers of dace were found in open areas between enclosures on days 3 and 4. The occurrence of dace in the pool zone did not increase, but fell slightly on these last two days. When levels of agonistic activity in the riffle zone are related to the percent occurrence of dace in the pool, spacing patterns and distribu• tion of experimental fish are more easily explained. Breeding individuals displayed a marked increase in levels of agonistic activity with the doub• ling of fish density (Fig. 34). This increase corresponded with the increase in occurrence of dace in the pool zone. With the subsequent density increase, agonistic activity rose further. However, there was no increase in the percent occurrence of dace in the pool. The final density change brought about a marked fall in agonistic activity and a decrease in the percent occurrence of dace in the pool. Keenleyside and Yamamoto (1962) found aggressiveness of wild salmon to increase and peak at intermediate densities but then to decrease at still higher densities. Fenderson and Carpenter (1971) reported a similar response for hatchery Atlantic salmon. Agonistic or territorial behaviour of breeding dace appeared to be most effective in dispersing surplus individuals into the pool zone when the density was doubled. However, tripling density reduced the effective• ness of territorial defence and a clumped distribution resulted. It has long been known that social structure of a group of animals can be altered by changing the density of the group. Kalleberg (1958) observed that very high population densities of hatchery trout resulted in the.disruption of territorial behaviour and the formation of schools. Similar changes occur in natural populations of the Japanese salmonid, Plecoglossus, (Kawanabe, 1958). Field data suggest that the densities of dace produced on day 1 most 146 Relationship between percent occurrence of dace in pool and mean number of agonistic acts won per 30 min at increasing densities during breeding (•) and non-breeding (A) phases. 147 closely approximated those in nature. Those of day 2 are encountered only occasionally. Densities on the third and fourth days, however, were some• what unrealistic. Outside the breeding season, dace displayed little agonistic behaviour and the sequential addition of dace to the channel produced no noticeable dispersal effect. However, since the spacing requirements of dace change markedly when they are breeding, the dispersal of dace throughout the opti• mum habitat available may be an advantageous consequence of breeding territoriality. Chapman (1962), Mason and Chapman (1965), and Hartman (1965) have stated that for coho salmon aggressive behaviour appears to be a key- factor in causing downstream "drift". A recent study by Sale (1972) has provided evidence which suggests that the agonistic behaviour occurring among members of the pomacentrid fish, Dascyllus aruanus, is responsible for the efficient dispersion of this species over the available coral. Observations made immediately after additional groups of dace were added provided some further information. In the breeding replicate, newly introduced individuals encountered aggression from resident dace. New dace moved about from enclosure to enclosure until finding a large group of non- aggressive dace which they joined. Keenleyside and Yamamoto (1962) found that less dominant Atlantic salmon schooled in mid-water and acted as a nucleus to attract more of the less successful competitors. Thus, the clumping effect observed here with dace is somewhat analagous to the shift toward mid-water schooling shown by crowded salmon (Keenleyside and Yamamoto, 1962) and coral reef fish (Sale, 1972). On seven separate occasions during day 2 of the breeding replicate, 148 agonistic interactions resulted in the loser swimming downstream to the pool zone. Such dispersal of dace into the pool zone of the experimental channel was interpreted as dispersal away from already saturated areas. In nature, such dispersal could, of course, be either upstream, downstream, or lateral within the riffle or to another riffle area. In the non-breed• ing replicate, newly introduced dace moved from one enclosure to another, encountering little or no., aggression from resident fish. D. Discussion of the Significance of Social Organization in Dace It has long been stressed that the social organization of a species is fashioned by a complex web of selection pressures and not by just one or a few (Crook, 1965). Thus the social life of the longnose dace as we observe it, is a complex response to numerous variables in its environment. Each different behavioural response of this species contributes in some way to its overall survival. Nice (1941) , Hinde (1956) , Tinbergen (1957), Carpenter (1958), Wynne-Edwards (1962), and Ardrey (1966) among others, have discussed at length the importance of territoriality. However, the only detailed review of this subject in fishes has been by van den Assem (1967). He is also one of the few authors who has carried out an experimental investigation of the function of fish territoriality. His laboratory experiments with the three-spined stickleback led him to conclude that the significance of the male stickleback territory has to be found in a reduction of interference by conspecifics during reproductive behaviour. Since territoriality in longnose dace is restricted to the breeding phase, it would seem fair to assume that it serves a primarily reproductive 149 function. For males, the major functions of territory appear to be, 1) provision of space in which males can court and spawn with females with minimal interference from other males 2) protection of eggs from intra• specif ic predation, and 3) refuge from current. For females, the data suggest that the functions of territory are, 1) refuge from current, and 2) refuge from male attacks. Evidence shows that dace without access to a refuge from the current experience stressful conditions which may render them vulnerable to preda• tion. In addition, female dace defending a territory are able to avoid conflicts with breeding males (i.e., courtship harassment and attacks from males). Male territoriality along with its related behavioural activities serves to protect freshly deposited eggs while making them less accessible to predators. Conclusions reached here as to the functions of dace territoriality would appear to be open to the same criticism that Tinbergen (1968) voiced in his review of van den Assem's stickleback study. Tinbergen's main ob• jection was that van den Assem merely demonstrated a set of adaptive cor• relations within the species rather than a functional relation between territorial behaviour and extra-specific pressures. However, I take issue with this view. Surely failure to demonstrate extra-specific selection pressures for the evolution of territorial behaviour does not necessarily indicate failure to demonstrate a function of territory. Intraspecific nest intrusion behaviour, during which conspecifics enter a nest or nest site where eggs are either in the process of being deposited or are already present, has been described for sticklebacks 150 (Morris, 1952; van den Assem, 1967; Wootton, 1971a), darters (Reeves, 1907), Atlantic salmon parr (Jones and King, 1952), cyprinodonts (Itzkowitz, 1970), and sunfishes (Keenleyside, 1972). Such behaviour in longnose dace is disruptive as some intruders (i.e., transient or non-territorial males) interfere with spawning activities and eat recently spawned eggs. In longear sunfish, although nest intrusion is performed by males without nesting territories, it is also common in those males with both a territory and recently spawned eggs (Keenleyside, 1972). Therefore, it would seem that the breeding system of territory clustering in sunfish and in longnose dace are associated with very different functions. Keenleyside suggests that longear sunfish nest close together so that males can ferti• lize eggs in neighbouring nests, thereby increasing the numbers of their own progeny. By being closely spaced, sunfish males can intrude into neighbouring nests without hazard to their own eggs. The entire intrusion act lasts no more than 2-3 seconds. Unlike sunfish, closely spaced territorial male longnose dace remain faithful to their territories. Interference and egg predation between neighbours is reduced. Since the degree of interruption during courtship and spawning may be an important factor in determining where a female chooses to spawn, the reduction in intraspecific interference may be of equal (if not greater) importance to egg predationt. in the evolution of dace territory clustering. Clustered territorial male dace interact frequently with other males, especially during the formation of the cluster. Such interaction with other males arouses territorial males sexually (i.e., nest site tenacity and 151 substrate probing are interpreted as sexual behaviour). Territorial stickleback males intruded on by other male sticklebacks are also sexually aroused (Wilz, 1972). Wilz suggests that male sticklebacks bear some resemblance to females and may slightly activate the male stickleback's sexual-control mechanism directly. A similar explanation for sexual arousal in territorial male dace seems possible. Sustained sexual arousal (i.e., site attachment and substrate probing) in clustered male dace would appear advantageous in 1) attracting receptive female dace, and 2) burying vulnerable eggs within a shorter period of time. The continued presence of an aggressive male on the nest site, of course, is always a good deferent to trespassing conspecifics likely to prey on eggs. In some of van den Assem's (1967) experiments, he observed a beha• vioural difference between social and solitary stickleback males; the former performed more sexual behaviour. Aubin (1972) found that male ruffed grouse clustered in groups drummed more frequently than did more randomly or uniformly distributed males. Since hens are attracted to drumming logs for mating, the more actively drumming clustered males may attract more hens. Darling (1952) has long held that a group of territories has more chance of attracting females than has an isolated territory. Experiments conducted in the present study have indicated that territorial behaviour may limit the number of dace breeding in localized areas, and therefore serve as a dispersing mechanism. However, attempts to determine whether territorial behaviour of dace limits breeding stocks and ultimately regulates population size were not undertaken in the present study. Watson and Moss (1970) have stated that to demonstrate that terri• torial behaviour limits a breeding population, it is necessary to show that 152 a substantial part of the population does not breed. Non-breeders should be capable of breeding if the more dominant (i.e., territorial) animals are removed. In birds, reproductive territories are quite stable over the entire breeding phase and more recent evidence supports the hypothesis of regula• tion of numbers by territorial behaviour (Watson, 1967; Krebs, 1971). Klomp (1972) provides a comprehensive review of this controversial subject pertaining to bird populations. Unlike birds, the territorial phase in many fish species is brief and successive waves of spawners utilize the same substrate. Longnose dace fit into such a group and determination of whether territorial behaviour prevents part of the population from breeding would be most difficult. Most of the available literature on fish populations and territorial behaviour deals with the regulation of population densities rather than population size. However, even this evidence of whether or not territorial behaviour functions to regulate fish densities is at odds. Macan (1963) believes that fish populations are prevented from becoming too dense by territoriality. Le Gren (1965), in fact, states that territorial behaviour acts as a density-determining mechanism in salmonids. Trout introduced into an unfamiliar section of stream inhabitated by other trout are known to be displaced downstream by the territorial residents (Miller, 1958; Jenkins, 1969). Van den Assem's (1967) experimental results with three-spined sticklebacks also supports the hypothesis that territorial behaviour is a potential agent for limiting the numbers of breeding individuals. On the other hand, other fish behaviourists describe the aggressive behaviour 153 mechanism of the medaka (Magnuson, 1962) and the ayu (Kawanabe, 1958) as being too flexible to limit density at higher population densities. Longnose dace have adopted a breeding social organization which allows them to breed within densely populated riffles without deleterious effects to reproductive success. Male dace coming into breeding condition at the same time cluster together. Males of such groupings remain behaviourally synchronized and exclude non-territorial males. Such non-territorial or transient males apparently pose the greatest single threat to reproductive success through interference and egg predation. Clustered territorial males show strong site attachment and interfere little with the reproductive ac• tivities of neighbouring fish. This clustering phenomenon makes excellent functional sense within the usually densely occupied riffles. 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