NATURAL HISTORY AND SOCIOGENETIC ORGANIZATION OF THE BEE
HALICTUS FARINOSUS (HYMENOPTERA: HALICTIDAE) IN NORTHERN UTAH
JENNIFER ALBERT
A THESIS SUBMITTED TO
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MASTER OF SCIENCE
GRADUATE PROGRAM IN BIOLOGY
YORK UNIVERSITY
TORONTO, ONTARIO
AUGUST 2012
©JENNIFER ALBERT, 2012
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Abstract
Nesting biology, phenology and sociobiology were studied in an aggregation of the
primitively eusocial ground-nesting bee Halictus farinosus in Northern Utah. Nest
architecture was typical of the genus but key phenological events were delayed up to
two weeks when compared to earlier studies of the same population. Bees were
genotyped at six variable microsatellite loci to reveal kin structure within each nest.
Polyandry was uncommon in H. farinosus queens whose population wide effective
mating frequency was 1.07. The queen produced the vast majority of the brood (98%)
while she was present and workers took over reproduction upon being orphaned. There
were significant differences in sex ratios between female-biased queenright and malebiased queenless nests (t = -3.72, p = 0.003). Together these results generally agree with
the predictions of inclusive fitness theory and support the view that haplodiploidy is
important in the evolution of eusociality.
ii
Acknowledgements
This work was funded by an NSERC discovery grant awarded to Dr. Laurence Packer. I
would like to sincerely thank a number of people for their contribution to the project. I
am grateful to Terry Griswold for sharing his expertise and helping me initiate my field
study. He and his wife Rhonda also graciously welcomed me into their home during my
field season. I would like to thank David Trew for his help with nest excavations and for
answering my many questions over the last few years. I am grateful to Dr. Scott Tarof,
Dr. Amro Zayed, Dr. Robert Paxton and Pat Kramer for their lab related advice and
assistance and to Dr. Cory Sheffield for his assistance with the analysis. A special thanks
to Stefanie Cargnelii for all of her hard work assisting me in the lab. My thanks also go to
Jessica Albert, whose skillful editing has vastly improved the work. Finally, I am ever
grateful to my supervisor Dr. Laurence Packer for his help with my field study and for his
comments on the many versions of my thesis. His advice, encouragement and guidance
over the years have been invaluable to me.
To Mom, Dad and Tom who supported me emotionally and financially during this
process, I dedicate this thesis to you.
iii
Table of Contents
Abstract
ii
Acknowledgements
iii
Table of Contents
Iv
List of Tables
v
List of Figures
vi
Chapter One: Introduction
1
Chapter Two: Nesting biology and phenology of Halictus farinosus (Hymenoptera:
Halictidae) in Northern Utah
12
Abstract
13
Introduction
13
Methods
16
Results
20
Discussion
26
Tables and Figures
31
Chapter Three: Sociogenetic organization in the primitively eusocial bee Halictus
farinosus (Hymenoptera: Halictidae)
50
Abstract
51
Introduction
51
Methods
55
Results
60
Discussion
66
Conclusion
73
Tables and Figures
75
References
82
iv
List of Tables
Table 2-1: List of all excavated nests
31
Table 2-2: Comparison of phenological events between years of study
33
Table 2-3: Comparison of H.farinosus nest sizes between 2002 and 2012
34
Table 2-4: Ovary development and matedness in queens and workers
35
Table 2-5: Comparison of social and life history traits between social Halictus
36
Table 3-1: Microsatellite primers and variability in H.farinosus
75
Table 3-2: Estimates of relatedness in worker and reproductive brood
76
Table 3-3: Summary of kin structure in reproductive brood nests
77
Table 3-4: Sociobiological data in all excavated nests
78
v
List of Figures
Figure 2-1: Percentage of brood in different developmental stages
37
Figure 2-2: Halictus farinosus nests (external)
38
Figure 2-3: Internal structure of a typical H.farinosus nest (Or4b)
39
Figure 2-4: Proportion of nests where the queen was present at excavation
40
Figure 2-5: Head widths of H. farinosus workers and queens
41
Figure 2-6: Wing lengths of H. farinosus workers and queens
42
Figure 2-7: Head width vs. wing length in adult females
43
Figure 2-8: Difference in mandible and wing wear between each queen and her workers
44
Figure 2-9: Mandible wear in queens and workers
45
Figure 2-10: Wing wear in queens and workers
46
Figure 2-11: Average daily maximum and minimum temperatures in the summer months for the
years of study in Logan, Utah
47
Figure 2-12: Total rainfall per summer month for the years of study in Logan Utah
48
Figure 2-13: Cumulative degree days between March and August in years of study with key
phenological markers
49
Figure 3-1: Genetic relatedness among females in all genotyped nests
80
Figure 3-2: Size difference between replacement queen and workers
81
vi
Chapter One: Introduction
1
In nature there are numerous examples of cooperation among individuals,
including altruistic behaviour. An action is considered altruistic when it comes at the
expense of the actor's personal fitness in terms of breeding potential but enhances the
fitness of another individual. Eusociality is essentially an extreme example of altruism. A
eusocial society is defined by three characteristics: a reproductive division of labour
between members, overlapping generations, and intergenerational help with rearing
the brood (Wilson, 1971). In eusocial societies the helpers often form a sterile worker
caste that exist to help the queen produce the brood by provisioning resources and
helping to construct and defend the nest. Within the context of Darwin's theory of
evolution by natural selection the origin of a sterile caste that does not contribute
directly to the next generation becomes all the more difficult to explain: how do some
individuals forgo reproduction if it is through reproducing that the genes for such
behaviour would be passed on? Darwin himself recognized this problem, admitting that
it initially seems "insuperable and actually fatal to the whole theory" (Darwin, 1859).
There are two essential difficulties with the evolution of sterility in workers. First is the
paradox of the inheritance of sterility when the trait is necessarily carried by individuals
who do not produce progeny. Secondly, it seems that sterility would never be selectively
advantageous (Crozier, 2008). Darwin attempts to explain inheritance of the trait,
saying that "selection may be applied to the family as well as the individual" (Darwin,
1859) indicating that selection can occur on multiple levels, not just at the level of the
individual but at the colony level as well. Modern group selection emphasizes the
2
competition between groups as a more significant driving force in selection than the
competition between members of the group (Wilson and Holldobler, 2005; Nowak at al.,
2010). Group selection theory stands in opposition to kin selection theory, which is
discussed further in this review, although it has been suggested that the two do not
differ empirically in their expectations or mathematical framework (Queller, 1992;
Gardiner et al., 2007; West et al., 2007a).
In 1964 William D. Hamilton published two papers that contained the first
mathematical proof that altruism can be explained by evolutionary theory without
invoking selection operating at a level "above" the individual (Hamilton, 1964 a, b).
Hamilton's rule states that a particular behaviour is selectively favoured when it leads to
a net increase in the inclusive fitness of the actor. Mathematically it is represented as
follows: br — c > 0. Where c is the lifetime cost to the productivity of the actor
behaving altruistically, b is the lifetime benefit of the behaviour to the recipient and r is
the coefficient of relatedness between the two individuals. This statistic describes in
genetic terms how closely related the actor is to the beneficiary relative to the genetic
similarity in the general population (Frank, 1998; West et al., 2007b). The expression br
is the indirect fitness benefit on the actor calculated as the product of the benefit to the
recipient and its relatedness to the actor. The cost must be less than the indirect fitness
in order for the altruistic behaviour to be selectively advantageous (Hamilton, 1964a).
Hamilton's rule of inclusive fitness emphasizes the importance of close relatedness
3
between individuals in the evolution of altruistic behaviour. Essentially an individual's
genes can be passed onto the next generation in an indirect way, through a family
member who carries genes that are identical by descent. Behaviour that promotes
productivity of those family members can be favoured, even when the cost of
performing the act is quite high. The selective force that acts to increase inclusive fitness
through direct and indirect fitness effects is known as kin selection (Maynard Smith,
1964).
Ensuring maintenance of an altruistic trait would require that organisms are able
to direct their altruistic actions towards other altruists either through kin recognition or
limited dispersal (Hamilton 1964 a, b). The ability of an altruist to recognize kin or other
carriers of an altruistic trait should be selected for since it allows them to direct altruistic
behaviour towards those individuals who are likely to share the trait (Hamilton, 1964b).
Kin discrimination has been demonstrated in a variety of cooperative animal groups
including humans (Jacob et al., 2002), ground squirrels (Mateo, 2002), mice (Manning et
al., 1992), long tailed tits (Russell and Hatchwell, 2001; Sharp et al., 2005), carpenter
ants (Ozaki, 2005), and even in a species of unicellular slime mould (Mehdiabadi et al.,
2006). However it is thought that discrimination of this nature is most often based on
environmental cues such as prior association rather than genetic ones (West et al.,
2007b). This is partly because genetic cues are predicted to be evolutionarily unstable,
often inspiring cheating (acquisition of the discriminatory cue but not the altruistic
behaviours) and becoming fixed as a result (Keller, 1997; Downs and Ratnieks, 1999;
4
Ratnieks et al., 2006; West et al., 2007b). If dispersal in the species is limited and the
social group is mainly composed of close relatives who share genes by descent, altruistic
actions may be performed indiscriminately towards the members of the group who
share the same propensity to perform the altruistic action (Hamilton; 1964ab, Hamilton,
1972). While high population viscosity (i.e. low levels of gene flow) may cause increased
competition between relatives (Taylor, 1992; Wilson et al., 1992; West et al., 2002)
there are a number of environmental and behavioural factors that promote selection of
kin cooperation when dispersal is limited (Queller, 1994; West et al. 2007b). These
forces can work together in some instances. For example, in some bird species kin
recognition is stronger when dispersal is more common (Cornwallis et al., 2010).
Eusociality is expressed in highly diverse ways in different taxonomic groups that
converge on its defining characteristics. Because of the specificity of these
characteristics it is presumed that there is a general evolutionary pathway leading from
solitary or primitive social behaviour toward obligate eusociality. However, the specific
steps in this pathway and the biological and environmental factors that promote its
evolution are still under debate (Wilson, 2005; Nowak et al., 2010; Abbot et al., 2010).
The conventional model for the evolution of eusociality relies on inclusive fitness theory:
close relatedness between nest mates means that the benefit of helping need not be as
great in comparison to its cost. The close relatedness between siblings which results
from monogamy facilitates the evolution of sib-social care likely by manipulation of the
expression of maternal care genes in insect workers (Linksvayer and Wade, 2005). It is
generally accepted that relatedness between siblings must be high in order to support
the evolution and early maintenance of eusociality (Boomsma, 2009). In fact, it is
thought that all current eusocial species come from monogamous ancestors and it is
only after passing through this "monogamy window" and through fixation of obligate
castes in highly eusocial organisms that reduced relatedness due to polygamy becomes
possible (Hughes et al., 2008).
Eusociality is thought to have evolved at least nine times in the Hymenoptera: six
times in bees (Cameron and Mardulyn, 2001; Coelho, 2002; Danforth 2002; Brady et al.,
2006; Schwarz et al., 2007; Cardinal and Danforth, 2011), twice in vespid wasps (Schmitz
and Moritz 1998; Hines et al., 2007) and once in ants (Moreau et al., 2006). It has been
suggested that the propensity for this trait to evolve in Hymenoptera might be due to
their haplodiploid genetic system (Hamilton 1964 b). This hypothesis is supported by
studies conducted on gall thrips (order Thysanoptera), which too are haplodiploid and
also have an evolutionary origin of eusocial behaviour (Crespi, 1992; McLeish et al.,
2006; McLeish and Chapman, 2007). In haplodiploid organisms females are diploid and
have two copies of each gene while healthy males develop from unfertilized eggs and,
having only one copy, are thus haploid. The relatedness values between family members
are different in haplodiploid organisms when compared with diploid ones. Because,
barring mutation, each sperm a haploid male produces is genetically identical to all
others, females on average share V* of their genetic material with their full-sibling sisters
as opposed to
in diploid organisms. This means that worker females are more closely
6
related to their sisters than they are to their own daughters. On the other hand,
daughters will share only % of their genetic material with their brothers which develop
from unfertilized eggs laid by their mother. The average relatedness between siblings in
haplodiploid organisms is therefore Vi, the same as it is in diploid organisms. This
dynamic has led theory to predict conflict between queens and workers over sex
allocation in the nest (Trivers and Hare, 1976). As workers are three times more closely
related to sisters than to brothers, those who remain to help in a nest headed by a
monogamous queen would be expected to attempt to skew the queen's brood sex ratio
in favour of females and/or to lay haploid male-destined eggs themselves (Trivers and
Hare, 1976; Pamilo, 1991). There is evidence of both sex ratio biasing and male
production by workers in eusocial Hymenoptera (Trivers and Hare, 1976; Bourke, 1988;
Packer and Owen, 1994; Schwarz et al., 1994; Bourke and Franks, 1995; Sundstrom et
al., 1996; for reviews see Queller and Strassmann, 1998; Chapuisat and Keller, 1999;
Ratnieks et al., 2006; Crozier 2008).
Whether male haploidy (haplodiploidy) has a role to play in the evolution of
eusociality has become a somewhat controversial topic (Queller and Strassmann, 1998;
Wilson and Holldobler, 2005; Wilson, 2008; Nowak et al., 2010). Critics have pointed to
low relatedness values in many eusocial colonies facilitated by multiple mating
(Muralidharan et al., 1986; Gadagkar, 1990), however, eusociality may have become
established in these groups before the evolution of polygamy (Hughes et al., 2008;
Boomsma, 2009). The haplodiploid hypothesis is also challenged by the fact that many
7
diploid species, including termites, some beetles, shrimp, and naked mole rats, have
been shown to exhibit eusociality (Wilson, 2008; Nowak, 2010), however, diploid
organisms too are predicted to evolve eusociality under inclusive fitness theory. Under
Hamilton's rule it is possible for eusociality to evolve even when relatedness is low so
long as the benefits are great enough to outweigh the cost of forgoing reproduction
(Queller and Strassmann 1998; Boomsma, 2009). Still, more haplodiploid groups exhibit
eusociality that would be expected by chance (Crozier, 2008) and models suggest that it
is easier for eusociality to evolve with male haploidy compared to diploidy (Pamilo,
1991; Linksvayer and Wade, 2005). At the very least haplodiploidy is likely to be one of
the promoting factors in the evolution of eusociality and the predictions of this
hypothesis warrant further investigation.
Empirical evidence does not line up with simplistic predictions based on
relatedness and male haploidy (Richards et al., 1995; Hammond and Keller, 2004).
Confounding behavioural and ecological factors related to colony efficiency, conflict
resolution and reproductive control in the Hymenoptera must be considered when
modelling the predicted evolutionary outcome (Chapuisat and Keller, 1999; Ratnieks et
al., 2006). Numerous mathematical models which are based on inclusive fitness and
take into account ecological and behavioural interactions have been developed to
explain intergenerational cooperation and the tendency for the queen to produce most
of the offspring (high reproductive skew). Reeve and Keller (2001) gave an overview of
these models, dividing them into several different types and showing how they are
8
described by Hamilton's rule. First, transactional models where either: the dominant
individual has control and concedes reproduction to subordinates as staying or peace
incentives; or, where the subordinate has control over reproduction and chooses to
restrain themselves and allow the dominant to breed (Johnstone and Cant, 1999).
Second, tug of war models assume no complete control over reproduction by either the
dominant or the subordinate. This predicts an amount of reproductive skew
intermediate to concession and restraint models (Reeve et al., 1998). According to
Reeve and Keller (2001) empirical data suggest that the transactional models are most
accurate in their predictions and are therefore the most useful, however, evidence can
be presented to support various models and the assumptions of each model should be
tested along with its predictions (Johnstone, 2000; Gardiner and Foster, 2008).
If we are to study the origins and maintenance of eusocial behaviour it is
important that we look at weak forms of eusociality, such as those displayed in the bee
family Halictidae, instead of advanced forms where distinct anatomical differences exist
between castes from early development (Packer, 1992; Bourke and Franks, 1995;
Schwarz et al., 2007). In the Halictinae, eusociality has evolved at least three times and
these events have taken place relatively recently compared to the origin of this trait in
highly eusocial insects such as the corbiculate bees which have been social since the
Cretaceous (Michener and Grimaldi, 1988; Danforth, 2002; Brady et al. 2006; Cardinal
and Danforth, 2011). Members of the Halictinae display considerable variability in their
9
social behaviour (Sakagami and Munakata, 1972; Eickwort et al., 1996; Soucy, 2002;
Richards et al, 2003; Boesi, 2009; Soro et al., 2010; and see Schwarz et al., 2007). In
addition, social behaviour in many halictines shows a high degree of flexibility; for
example, there have been multiple transitions from eusocial to solitary or parasitic
behaviour in some clades (Danforth, 2002; Danforth et al., 2003, Schwarz et al., 2007;
Gibbs et al., 2012). Furthermore, some prospective workers may have the ability to
reproduce instead of working (Yanega, 1988). The existence of socially polymorphic
species in the Halictinae (Eickwort, 1996; Richards et al, 2003) indicates that eusocial
behaviour in some members of the group may not be obligatory. Helping in these
groups persists presumably because remaining in the natal nest to assist in rearing the
brood is in some way beneficial to the helpers themselves. Thus, the halictines are an
ideal clade in which to study the origins of eusociality.
The primitively eusocial bee Halictusfarinosus (Hymenoptera: Halictidae) is
common in western North America. While it is known to be a eusocial species, social
parameters of H. farinosus have never been examined in detail, for example it is not yet
known whether workers lay eggs that contribute to the reproductive brood. This species
was selected for study because it is common, yet rarely studied, it is large, its nest sites
are conspicuous and a large aggregation was available to study. More importantly, it is
closely related to Halictus species that are well-known for their interesting social
behaviour and which include solitary as well as socially polymorphic species (Yanega,
10
1988; Richards et al., 1995; Eickwort et al., 1996; Packer, 1997; Richards, 2001). In this
study I have compared natural history, nesting biology, dissection, measurement and
emergence data in H. farinosus with previous studies on this bee in the same area (Nye,
1980; Sellars, 2004). In addition, highly variable microsatellite loci were used to
investigate relatedness among nestmates, allowing us to determine parentage in the
reproductive brood and compare predictions of models of social evolution to empirical
data in a social halictid species.
11
Chapter Two: Nesting biology and phenology of Halictus farinosus Smith
(Hymenoptera: Halictidae) in Northern Utah.
12
Abstract
Nesting biology and phenology in an aggregation of the primitively eusocial
ground-nesting bee Halictus farinosus was studied at Green Canyon, Utah from May to
August, 2010. Nest architecture was typical of the genus. Nests were small with an
average of 3.5 workers and 13.5 reproductives per colony. Most workers were mated
(77.5%) and had ovary development (71.4%) and the queen-worker size differential was
moderate (8.8% for head width and 5.6% for wing length), indicating that sociality in this
species is weaker than in some other social Halictus species. The study's findings were
compared with those of two previous studies on the same population (from 1977/1978
and 2002). Varying weather patterns in the years of study led to changes in phenological
milestones: in the comparatively cold and wet spring season of 2010, nests were
delayed by up to two weeks. While nest productivity was comparable between years, in
2010 the size difference between queens and workers was significantly larger than in
2002 indicating that weather conditions may have an effect on social parameters in this
species.
Introduction
It is of the upmost importance that we have a detailed understanding of the biology
and natural history of our native pollinators (Sheffield et al., 2012). Repeat field studies
13
in the same population can allow us to make comparisons between years to determine
how weather patterns might affect phenology, productivity and other biological
attributes which will help us to better predict how a group might respond to changing
ecological conditions (Richards and Packer, 1995; Bartomeus et al., 2011). investigating
the natural history and life cycle of a bee species inevitably means studying their nesting
behaviour. Most bees burrow into the ground and construct brood cells off the side of
one or many main tunnels (Michener, 2007, pp. 23-29). Most Halictinae (the sweat
bees) are ground nesters and there is considerable diversity in the size and structure of
these nests (see Sakagami and Michener, 1962 for an early review). These bees are also
mass provisioning: enough pollen and nectar are collected before the egg is laid on the
pollen mass within the cell and the brood cell entrance is then sealed, usually until the
individual is fully developed (Sakagami and Michener, 1962). The size of an adult bee
depends on the quality (Roulston and Cane, 2002) and size (Plateaux-Quenu, 1983;
Richards and Packer, 1994) of the pollen mass provided for it. The size of the pollen
mass will depend on multiple environmental and biological factors including the size and
activity levels (Gathmann and Tscharntke, 2002; Pereboom and Biesmeijer, 2003) of
active foragers in the nest and the amount of available resources which, in turn, are
highly dependent on weather and other environmental factors (Packer, 1990; Minckley
et al., 1994; Richards, 2004).
14
Halictus farinosus Smith is a non-metallic halictine species that is readily identified
by its large size (12 -14 mm) and strong apical bands of yellowish pubescence on the
metasomal terga. It is found in the North American West ranging from British Colombia
to California and has been reported as far east as Nebraska (Ascher and Pickering, 2011).
Halictus farinosus is polylectic: Nye (1980) reports foraging by H.farinosus on 43 plant
species from 14 different families. It is thought to be an important pollinator of carrot
(Nye, 1980), onion (Parker, 1982), and sunflower (Parker, 1981). In Utah, H. farinosus
nests in dry, sandy soil in areas of sparse vegetation and their simple, usually
unbranched, nests extend up to 65 cm into the ground (Nye, 1980; Seliars, 2004).
Halictus farinosus has a two-phase life cycle and exhibits primitive eusociality similar to
other primitively eusocial halictines (Michener, 1974, pp. 269-300; Packer, 1986; Packer,
1992; Richards, 2001; Soucy, 2002; Schwarz et al., 2007; Richards et al., 2010). Queens
found nests independently in April to May in north Utah (Nye, 1980; Seliars, 2004), and
produce a worker brood that help to produce a second, reproductive, brood which
begins to emerge in mid August. The behaviour of this bee is similar in California: they
nest in sandy soil in open areas of sparse vegetation and have a two-phase nesting cycle
but with the warmer climate the nests tend to extend deeper (up to 80 cm) and more
workers are produced (Eickwort, 1985). The nesting season is also longer in California,
lasting from mid February to October (Eickwort, 1985).
15
In this study we present phenological, sociobiological and nest architecture data
from field studies on H.farinosus in North Logan, Utah. As this population has been
studied previously (Nye, 1980; Sellars, 2004) we compare phenological and nest
productivity data among years of study. We draw similar comparisons between Halictus
farinosus in Utah and other closely related Halictus species.
Methods
Study sites and nest excavations
All nests were excavated in July and August 2010 at Green Canyon, North Logan,
Utah (41.769° N, 111.773° W, 1589 m). This site was selected because it was the largest
known H.farinosus nesting aggregation (Nye, 1980; Sellars, 2004). The site was a 0.25
km2 field near the mouth of the canyon, just before the trail-head. Six 2 x 2 m study
sites, marked B to G, were divided into quadrants of 1square meter each, nests
beginning with N were outside of these areas. Each site was monitored during daylight
hours for at least two hours every second day from June 13th until July 30th excepting
days with exceptionally bad weather. Nests referred to as first brood nests are those
excavated on or before July 17th, the date when the first workers emerged. Second
brood nests were any nests excavated after this date and late summer nests were
excavated after August 7th. Nests were excavated throughout the season but because
16
we wanted to maximize sociobiologically relevant data the majority of second brood
nests were excavated during a short period in early to mid August, just before the
reproductive brood started to emerge (see Table 2-1for excavation dates).
Nest entrances were blocked before sunrise on the day of excavation to prevent
escape of any inhabitants. Talcum powder was blown down the entrance and a plant
stalk or wire was inserted into the tunnel to help follow it during excavation. A 30 cm
deep hole was dug in a clear area next to each nest and careful excavation continued
from the hole toward the nest until the tunnel was uncovered. All juvenile and adult
bees were removed from the nests and stored directly in 95% ethanol; no adults present
in the nests during excavations were lost. All components of the nests were recorded
during excavation including unfinished pollen balls, mouldy cell contents and empty
cells. Nest depth measurements were taken for nests by running a measuring tape from
the ground level and following along the main tunnel. Activity in most excavated nests
was monitored from June 13th onwards. Foundresses in these nests, with the exception
of those nests from areas B and N were marked in the spring with unique combinations
of three colours of Testors™model paint on their thorax. Observations of nests from
areas B and N began as the first brood emerged because high mortality at other sites led
to a low number of viable nests for excavation. Queens in B and N nests were
determined using variable microsatellite loci following methods outlined in Chapter 3.
17
Dissections and measurements
Ovary dissections and measurements were performed on all adult females using
a Leica WILD M3B light microscope at 16 x magnification with an ocular micrometer
following the general methods of Ordway (1965). Wing and mandible wear were each
given a score ranging from 0 to 5 where 0 was no wear and 5 was extremely worn. Wear
scores, head width and wing length measurements followed the methods of Richards
(2010). Ovary development was assessed based on the size of developing oocytes: 0
when none were present and 1for each fully developed oocyte. Intermediate conditions
were scored to the nearest quarter based on size relative to a complete egg. These data
were summed to give an overall development score (Packer, 1986). Development and
wear scores were averaged for each group analysed and for the entire population.
Matedness was determined by inspection of the spermatheca when it was found intact
(it was not found in 9 of the 86 bees dissected). Sexing of juvenile brood was
accomplished by genotyping at six microsatellite loci (see chapter 3) and confirmed in
pupae by morphological inspection. Gut contents including pollen, nectar and in some
cases ingested oocytes were also recorded during dissections.
Weather
18
Weather data were gathered from the Utah State University climate centre
website (2011) from a weather station 5.5 km away from the study site for 2010 and all
previous years of study in the population (1976,1977,2002).
Calculations and statistical analyses
Size differences between castes were calculated following (Packer 1992) as
D=m-sw1
SQ
* '
where S^and Sw are the sizes (head width or wing length in mm) of the queen and
worker respectively. First brood productivity was measured as the average number of
individuals in first brood nests after the foundress stops foraging, indicating that all
brood had been produced. Productivity was calculated as brood per working female in
each nest. Because worker mortality was high, minimum productivity was also
calculated as the number of brood divided by the average number of spring (worker)
brood individuals found in nests multiplied by the proportion of spring brood that was
female. The sex ratio presented is the number of males divided by all brood.
Degree days (base 10) were calculated for each day by the basic equation:
DD = ————————— - 10
(2)
19
Where Tmin and Tmax are the minimum and maximum temperatures reached that day,
and cumulative degree days are calculated (Zalom et al., 1983). Correlation between
wing length and head width were calculated using the standard coefficient of
correlation calculation (r). All tests of significance were 2 tailed unpaired t-tests using
Minitab* Statistical Software.
Results
Phenology
All nests studied were singly founded and foraging had commenced in some
cases by the initiation of the study on June 13th. Only one H.farinosus nest founding
event was witnessed after this point but it was outside of the areas under observation.
Brood production by the foundress alone occurred until early July with the last foraging
trip observed on July 12th. In early July, foundresses were frequently seen guarding nest
entrances and rarely left the nest. Nest excavations began on June 28th, the first fully
developed adult worker found in a cell was excavated on July 15th and the first adult
worker brood individual to leave a nest was observed on July 17th. No new nests were
founded after their emergence in the area of observation. Worker foraging slowed
noticeably after the first week of August. The first fully developed second brood male
was found on August 11th, the first adult female in the second brood was excavated on
20
August 12th and second brood individuals continued to emerge throughout August and
into September. Figure 2-1 shows nest contents by developmental stage over 6 day
periods throughout the season.
Nest surveys conducted in mid-June and repeated in early August indicated a
nest mortality of 59.5%, although nests of very low activity may have been missed in
either survey.
Nest architecture
Halictus farinosus nests in Green Canyon were easily identified by their large
tumuli (Figure 2-2). Bees entered the nest through a tunnel that ran horizontally
through the tumulus to the main tunnel which typically extended approximately
vertically. Tunnels changed course occasionally to avoid obstacles (i.e. roots or rocks).
Two nests had a single brood-containing branch off the side of the main tunnel and one
had two brood-containing branches. During wet weather conditions in the spring, the
nest entrances were plugged with soil, but otherwise remained open daily and overnight
throughout the foraging season.
21
Nest depth for first brood nests ranged from 11to 41cm (x = 22.10 cm, SD =
7.44, N = 21), however the shallowest and deepest brood cells at this time were 7 cm
and 20 cm respectively (x = 14.47 cm, SD = 2.64, N = 21). Second brood nests excavated
ranged from 12 cm to 44.5 cm in depth (x = 30.3 cm, SD = 6.10, N = 37), were
significantly larger (t = 4.54, p < 0.001) than first brood nests and had cells reaching to or
very near to the end of the burrow.
All cells were constructed directly off the side of the main tunnel (Figure 2-3) and
measured approximately 1.4 cm (x = 1.40, SD = 0.29, N = 7) long and 0.7 cm (x = 0.66, SD
= 0.14, N = 7) wide with a slightly constricted neck.
The average number of cells in first brood nests was 4.41(SD = 2.04, N = 29).
There were significantly more cells in second brood nests with an average of 20.06 in
late summer nests (SD = 10.08, N = 32, t = 8.2, p < 0.0001) including empty cells that
were previously occupied by first or second brood individuals and cells with mouldy
contents. The percentage of cells containing mouldy contents (mostly pollen) over all
nests was 6.69%.
Spring and summer brood
22
Sixty-six nests were excavated in Green Canyon throughout the season, 29
contained the juvenile first brood and 37 contained mostly the second, reproductive
brood. The first brood nests contained between 1and 6 individuals with an average of
3.52 (SD = 1.96, N = 29). There were fewer brood than brood cells because in some
cases cells were unfinished or contained mouldy pollen instead of live brood. At the
time of excavation an average of only 1.74 workers were present within second brood
nests. Active summer brood nests produced between 1and 46 individuals with an
average of 13.49 (SD = 9.93, N = 37). In both broods, individuals at later developmental
stages tended to be closer to the nest entrance and cells containing only fresh pollen
were always among the deepest excavated indicating that shallower cells were
completed first.
A breakdown of all nest contents in the first and second brood nests is presented
in Table 2-1. A total of 22 of 29 first brood and 12 of 32 second brood nests contained
the founding individual at the time of excavation (Figure 2-4). It could not be
determined if the foundress was present in the remaining five nests because genotype
data were not available. Mean productivity of the queen alone (first brood nests) was
3.52. Mean productivity of worker brood females was calculated using an estimate of
the number of first brood females over the lifetime of the nest. As there was an average
of 3.52 individual bees produced in first brood nests and the proportion of the first
23
brood that was female was 0.851, this suggests there were 2.99 first brood females per
nest on average. As second brood productivity was 13.48 individuals per nest, this gives
4.51 reproductive brood per worker female. Mean maximum second brood productivity
using the actual number of workers found in the nest (not including the queen) was
6.99. The sex ratio in the first brood was 0.149 (SD = 0.31, N = 16) weighting nests
equally and 0.159 with individuals weighted equally. The sex ratio in the second brood
was 0.464 (SD = 0.352, N = 27) averaged over nests and 0.445 in the population as a
whole; neither of these estimates were significantly different from a 1:1sex ratio.
Caste dimorphism
Dissections of workers revealed that 71.4% (N = 43) had developed ovaries and
77.5% (N = 40) were mated. All queens dissected were mated (N = 31) and all but one
that was found dead in the nest with desiccated ovaries had some level of ovary
development (N = 31). The level of ovary development in queens (x = 1.185, SD = 0.668,
N = 31) was significantly greater than in workers (x = 0.648, SD = 0.673, N = 49, t = 3.49,
p = 0.0008). Workers in nests where the queen was present had lower but not
significantly less egg development (x = 0.519, SD = 0.457, N = 26) than workers in
orphaned colonies (x = 0.793, SD = 0.841, N = 23, t = 1.44, p = 0.157).
24
Queens averaged significantly larger than workers in both head width (x = 3.52 mm, SD
= 0.15, N = 33 in queens, x = 3.30 mm, SD = 0.15, N = 55 in workers, t = 6.61, p < 0.0001)
and wing length (x = 3.14 mm, SD = 0.15, N = 27 in queens, x = 2.94 mm, SD = 0.13, N =
45 in workers, t = 5.80, p < 0.0001) (Figures 2-5 and 2-6). This gives a population-wide
caste size difference of 6.12% for head width and 6.11% for wing length. Queens were
also generally larger in both measures than their own workers with a mean percent
difference of 8.79% for head width and 5.63% for wing length. There was a strong
positive correlation between head width and wing length over the whole population
(Figure 2-7, r = 0.695). Queens were not generally more worn than workers in either
mandible (x = 3.75, SD = 1.21, N = 28 for queens and x = 3.57, SD = 1.53, N = 45 for
workers, t = 0.51, p = 0.61) or wing wear (x = 3.13, SD = 1.36, N = 30 for queens and x =
2.81, SD = 1.64, N = 43 for workers, t = 0.88, p = 0.38). Queens were generally more
worn than their own workers (Figure 2-8). Both wing and mandible wear increased
throughout the season in queens, and especially in workers (Figures 2-9 and 2-10).
Temperature and rainfall
Minimum and maximum temperatures were low in the spring of 2010 compared
to the 65 year average but were comparable to the average throughout the summer
(Figure 2-11). Spring rainfall was high in 2010 compared to average, but was close to the
average throughout the rest of the season (Figure 2-12).
25
Discussion
Comparisons of nesting biology of H. farinosus among years
Halictus farinosus has been studied at Green Canyon in the 1970s by Nye (1980)
and in 2001 and 2002 by Sellars (2004). It has also been studied, though for a shorter
duration, in Davis, California by Eickwort (1985). In this section we compare the results
obtained among years and between localities.
Phenological events in H. farinosus 2002 and 2010 coincide well with the number
of degree days accumulated in these years, with the first adult brood emerging at
around 500 days (by equation (2), Figure 2-13). Thus, spring temperature likely had an
impact on phenological events relating to the life history of this bee species. However, in
1976 and 1977 minimum temperatures were much higher and a higher number of
degree days had accumulated before workers emerged (800 and 600 days respectively,
Figure 2-13).
The amount of rainfall may have been responsible for the differences in
emergence dates between years and the poor correlation with degree day
26
accumulation. Brood development and emergence dates (Table 2-2) were delayed
several weeks in 2010 compared to 2002 (Sellars, 2004) but matched those reported by
Nye (1980) for 1977. Timing in 1976 was intermediate (Table 2-2). The amount of spring
rainfall followed the same pattern: high in May of 1977 and 2010 low in 2002 and
intermediate in 1976. The drier weather in May of 1976 and 2002 likely allowed queens
to initiate foraging earlier, to forage more often than in 1977 and 2010 and to produce
brood sooner.
Nests were shallower in both brood producing periods in 2010 (Brood 1: x = 22.1, SD
= 7.4, N = 21, t = 2.30, p = 0.026, Brood 2: x = 30.3, SD = 6.1, N = 37, t = 6.60, p < 0.0001)
compared to 2002 (Brood 1: x = 26.7, SD = 6.8, N = 30, Brood 2: x = 42.7, SD = 10.6, N =
86). However, the number of brood produced per nest in both worker and reproductive
phases was comparable between years. The number of first brood individuals produced,
x = 3.52, (SD = 1.96, N = 29) in 2010 and x = 3.20 (SD = 2.64, N = 30) in 2002, was not
significantly different between years (t = 0.52, p = 0.60), although excavations
commenced much earlier in 2002. Also, the number of second brood individuals
produced per nest was not significantly different between years (2010: x = 13.49, SD =
9.93, N = 37, 2002: x = 13.06, SD = 13.36, N = 81, t = 0.17, p = 0.86) (Table 2-3).
Productivity estimations (brood per working female) were not significantly different
27
between years with an average productivity of 6.99 (SD = 4.21, N = 35) in 2010 and 6.09
(SD = 7.11, N = 70) in 2002 (t = 0.692, p = 0.491).
The percent difference in head width between queens and workers was significantly
larger in 2010 (t = 2.01, p = 0.050) at 8.79%, compared to only 5.79% in 2002. Weather
was much harsher in the spring of 2010 with more rain and colder temperatures
compared to 2002 (Figures 2-11,2-12). Poor spring weather conditions may have led to
smaller workers being produced in 2010 compared to 2002. It is generally thought that
smaller individuals are less efficient at foraging compared to larger individuals because
they cannot forage as far or carry as much pollen (Gathmann and Tscharntke, 2002;
Richards, 2004). Floral resources in Green Canyon are generally very sparse and foraging
trips in the H.farinosus population there are typically very long, often well over an hour
(Nye, 1980; Sellars, 2004; personal observations). Thus, foraging in this population is
likely energetically taxing. Despite this, reproductive nest productivity between the two
years was not significantly different.
The proportion of workers that were mated also differed greatly between years and
a greater percentage of workers in 2010 had developed ovaries but the average level of
egg development in workers was not significantly different between years (Table 2-4).
This result is unexpected as the workers would be predicted to be less fertile when the
28
queen-worker size differential is larger and queens presumably have more control
(Richards et al., 1995; Richards and Packer, 1996). However, excavations in 2002 were
performed at regular intervals throughout the season as opposed to 2010 where many
were excavated in a short period of time late in the season. The difference in sampling
dates between years could account for the greater number of mated and ovarially
developed workers in 2010.
Comparisons to other species
Phylogenetic data, both morphological (Pesenko, 1984) and molecular (Danforth et
al., 1999) have been used to understand the systematics of Halictus. Halictus farinosus
forms a sibling species pair with H. parallelus which, although stated by Knerer (1980) to
be solitary, is probably a social species (Packard, 1868; Taylor and Packer, unpublished
observations). These two are then closely related to the socially polymorphic H.
rubicundus (Soucy, 2002; Soro et al., 2010) and the solitary, occasionally communal, H.
quadricinctus (Knerer, 1980; Sitdikov, 1987). It is more distantly related to the other
Halictus species that are known to be eusocial such as H. ligatus and H. sexcinctus
(Packer, 1986; Richards, 2001), though the latter exhibits some additional social
complexities (Richards et al., 2003).
29
Halictus farinosus produces a small number of workers and has low to moderate
worker-queen size dimorphism compared to other social Halictus species. It also has a
comparatively large number of worker females that are mated and/or have ovarian
development, but this varies widely between years. Small size dimorphism, the small
number of workers and high degree of mating and ovarian development in workers
indicates that the level of sociality in H. farinosus is relatively weak compared to that in
other social Halictus species (Packer, 1992). The nesting biology and behaviour of social
populations of Halictus rubicundus are similar to those of H.farinosus in brood size, nest
architecture and the level of caste size dimorphism (Table 2-5, Soucy, 2002). However,
unlike H. farinosus, H. rubicundus is socially polymorphic (Eickwort et al., 1996; Soucy
and Danforth, 2002; Soro et al., 2010). While solitary and social populations of H.
rubicundus may form phylogenetically separate lineages (Soucy and Danforth, 2002)
some halictines exhibit social polymorphism even within the same population (Packer,
1990; Richards et al., 2003; Hirata and Higashi, 2008). While there are no known solitary
H. farinosus populations, seasonal climatic differences might affect its sociality through
the queen-worker size differential (Richards and Packer, 1996). The biology of H.
farinosus should be studied in locations throughout its geographic distribution, including
populations at high altitude and latitude where the species is more likely to be solitary,
to fully understand the effect of climate conditions on life history and sociality in this
species and determine whether solitary behaviour is within the species' repertoire.
30
Tables and figures
Table 2-1: List of all excavated nests, the date of excavation and a breakdown of their brood
contents by developmental stage. Adults listed were found in the tunnel; callow adults found
cells are included with pupae.
Nest
Number
C301
C302
C307
C308
Excavation
Date
C311
N0702-1
N0702-2
N0702-3
N0702-4
N0702-5
N0704-1
28-Jun-10
28-Jun-10
28-Jun-10
28-Jun-10
28-Jun-10
02-Jul-10
02-Jul-10
02-Jul-10
02-Jul-10
02-Jul-10
04-Jul-10
N0704-2
N0704-3
N0706-1
N0708-1
N0708-2
N0709-1
N0709-4
N0709-5
N0712-1
N0712-2
N0712-3
N0712-4
04-Jul-10
04-Jul-10
06-Jul-10
08-Jul-10
08-Jul-10
09-Jul-10
09-Jul-10
09-Jul-10
12-Jul-10
12-Jul-10
12-Jul-10
12-Jul-10
N0715-1
N0715-2
15-Jul-10
15-Jul-10
N0715-3
N0717-1
15-Jul-10
17-Jul-10
17-Jul-10
17-Jul-10
N0717-2
N0717-3
Adults
Pupae
Larvae
0
0
1
1
1
1
1
2
0
0
1
2
3
0
0
0
0
0
0
0
0
2
3
2
5
1
1
2
3
1
1
3
4
2
1
2
2
2
2
2
2
1
4
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
0
0
1
3
0
0
3
0
4
2
2
1
4
2
5
1
1
3
5
0
4
0
1
0
Eggs
0
0
1
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Pollen
balls
0
1
2
1
0
3
0
0
0
1
2
1
0
0
2
1
0
0
1
2
0
0
0
0
0
0
0
0
0
N0725-1
N0728-1
N0728-2
N0728-3
OrlOb
Or4b
B105
G402
G408
F203
F211
F205
F101
N0811-1
E411
E407
E401
N0811-3
B101
B108
B211
B102
B106
B107
B301
B302
D303
D401
C208
C209
B311
B304
B311-2
B303
B306
B313
F310
25-Jul-lO
28-Jul-lO
28-Jul-lO
28-Jul-lO
29-Jul-10
31-Jul-lO
08-Aug-10
09-Aug-10
09-Aug-10
10-Aug-10
10-Aug-10
10-Aug-10
10-Aug-10
10-Aug-10
ll-Aug-10
ll-Aug-10
ll-Aug-10
ll-Aug-10
12-Aug-lO
12-Aug-lO
12-Aug-lO
12-Aug-lO
12-Aug-lO
13-Aug-10
13-Aug-lO
13-Aug-10
14-Aug-10
14-Aug-10
15-Aug-10
15-Aug-10
16-Aug-10
16-Aug-10
18-Aug-10
18-Aug-10
18-Aug-10
18-Aug-10
18-Aug-10
3
3
0
2
5
1
1
0
0
0
0
3
2
2
3
2
2
2
2
4
1
8
1
2
3
1
1
1
3
2
1
2
3
8
5
2
2
2
5
3
1
1
1
1
1
1
1
5
2
1
2
0
4
2
12
6
1
1
2
11
18
11
8
4
11
6
1
9
9
11
5
6
17
8
2
15
2
3
4
10
11
8
4
10
0
12
9
11
11
7
35
14
7
7
19
22
2
8
8
1
12
3
2
6
7
6
11
11
0
0
0
0
0
0
1
0
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
2
2
4
0
0
4
0
0
0
1
1
1
0
0
0
1
2
0
2
0
0
0
0
0
1
0
0
0
0
0
1
0
0
Table 2-2: Summary of phenological data for all H. farinosus studies inGreen Canyon. N/A
indicates that the information in the column was not given in the study. In Sellars (2004) female
and male reproductive emergence dates were not differentiated so the same date was given for
each.
First adult
worker
First
reproductive
brood cells
First
First
reproductive reproductive
adult female
adult male
N/A
Late June
N/A
Late July
Early August
1977
N/A
July 8
N/A
Early August
Mid August
2002
June 28
July 2
July 3
July 30
July 30
2010
July 12
July 15
July 17
August 11
August 12
Year
1976
Queen
foraging
slows
Source
Nye
(1980)
Nye
(1980)
Sellars
(2004)
Present
study
33
Table 2-3: Comparison of H.farinosus nest size, number of cells and mean offspring number
between 2002 and 2010.
First brood
Depth (cm)
X
SD
6.78
26.71
2010 22.10
7.44
Compari t = 2.30, p = 0.026
son of
means
2002
N
30
21
Cells per
nest
Mean offspring
1-12
1-9
2.64
3.20
1.95
3.52
t = 0.52, p = 0.60
Cells per
nest
Mean offspring
3-56
3-45
13.06
13.36
9.93
13.49
t = 0.17, p = 0.86
X
SD
N
30
29
Second brood
Depth (cm)
X
SD
10.64
2002 42.67
6.14
2010 30.32
Compari t = 6.60, p < 0.0001
son of
means
N
86
38
X
SD
N
81
37
Table 2-4: Summary of egg development and matedness in queens and workers in 2002 and
2010. Numbers in brackets are percentages, averages are presented with standard deviations.
2002
2010
Queens
N
% with 1 egg 50% of
Workers
Queens
Workers
Workers:
Queen
present
Workers:
Queen not
present
46
91
31
49
26
23
full size
(80.4)
(41.8)
(90.3)
(44.9)
(42.3)
(47.8)
% with 1egg 75% of
full size
(50.0)
(28.6)
(41.9)
(26.5)
(26.9)
(26.1)
% with 1egg of full
size
(130)
(11.0)
(25.8)
(10.2)
(7.7)
(13.0)
% with developed
ovaries
(97.8)
(42.10)
(96.8)
(71.4)
(73.1)
(69.6)
% mated
(91.3)
(46.10)
(100)
(77.5)
(75.0)
(80.0)
1.429 +/0.765
0.913 +/1.070
1.185 +/0.668
0.648+/0.673
0.625 +/0.458
0.793 +/0.842
Average amount of
ovary development
35
Table 2-5: Comparison of aspects of sociality and natural history in social species of the
subgenus Halictus. A species with more advanced sociality would be expected to have higher
brood sizes, a lower brood 1sex ratio, fewer workers that were mated or with developed
ovaries and a larger queen-worker size dimorphism (Packer and Knerer, 1985).
Second
brood
size
13.013.3
4.4
10.5
10.3%
n/a
5.8->
9
10-16
5.5%14.6%
In
H. farinosus
4.9 - 5.8
23.0%
»
H. sexcinctus
0°
H. rubicundus
H. ligatus
% workers
Sex
with
ratio
develope
brood 1 d ovaries
5.042.115.0%
77.4%
First
brood
size
3.53.6
%
workers
mated
46.177.5%
Queenworker size
dimorphism
n/a
9.6%
57.16%
42%
12.7%
Source
Present study;
Sellars (2004)
Soucy, 2002;
(social
populations)
Packer, 1986;
Richards and
Packer (1995);
Richards et al..
1995
73.7%
56%
6.9 -11.8%
Richards, 2001
6.4 - 8.6%
36
100%
Pollen
• Eggs
8 40%
c
§ 30%
a
Larvae
Pupae
20%
Jun 28- July 4-9 July 10- July 16- July 22- July 28- Aug 3- Aug 9July 3
15
21
27
Aug 2
8
14
Date (2010)
Figure 2-1: Percentage of brood in different developmental stages throughout the season,
taken every six days. The rate of nest excavation throughout the season was not consistent.
Number of nests excavated during the periods: June 28-July 3, N= 10; July 4-9, N=8; July 10-15,
N=7; July 16-21, N=3; July 22-27, N=l; July 28-Aug 2, N=5; Aug 3-8, N=l; Aug 9-14, N=23; Aug 1520, N=9.
37
Figure 2-2: Four H.farinosus nests. Tumuli are apparent, markers designate nest number.
1 cm
Spr4b.17 dead pupa
)0r4b.01 - empty
0r4b.02 emptyZy»-2'
Or4b.19 empty
0r4b.03 mouldy pollen
0r4b.04 mouldy pollen
0r4b.05 pupa
0r4b.06 larva
Or4b.24 mouldy pollen
0r4b,07 larva
Or4b.21 - mouldy pollen
Or4b.18 pollen
Or4b 23 larva
0r4b.20 larva
15 larva
~iOr4b.16 larva
Or4b.l4 larva
0r4b.08 mouldy pollen
Or4b.22 pollen
_Or4b.l3 mouldy pollen
Or4b.12
larva
Or4b.11 larva
0r4b.09 - adult
0r4b.10 adult
Figure 2-3: A typical second brood nest (Or4b) showing all cells with brood contents.
100%
• Queen not present
• Queen present
Figure 2-4: Proportion of nests with queens in the nest at the time of excavation, organized in 6
day intervals. Very few excavations took place between July 16th and August 8th so data are
lumped together to show overall trend over this period. Nests in which the queen could not be
determined conclusively were omitted. Number of nests: June 28-July 3, N= 10; July 4-9, N=9;
July 10-15, N=8; July 16-Aug 8, N=7; July 9-14, N=20; August 15-20, N=6.
40
• Workers
• Queens
Figure 2-5: Histogram of head widths in millimetres for adult H. farinosus females (workers and
queens).
41
• Workers
• Queens
wing length (mm)
Figure 2-6: Histogram of wing lengths in millimetres for H. farinosus adult females (workers and
queens).
42
y = 0.626x + 0.894
Queens
Workers
Reproductives
- Linear (all)
3.4
Head width (mm)
Figure 2-7: Head width vs. wing length in workers, queens and adult reproductives (brood 2
adults) in the H.farinosus population in Green Canyon showing a strong correlation between the
two variables (r = 0.665).
43
3.5
3
2.5
2
I Mandible wear
1.5
c
Wing wear
1
2
£
2 0.5
0
-0.5
o
-Q JO
o
_
~rq—"
r- r>»
o o
-1
Nest
Figure 2-8: Differences between mandible and wing wear scores in each nest between the
queen and the average wear in her workers for each nest. Queen mandibles were always more
worn than their workers and wing wear in queens was nearly always higher except in two nests.
Only nests where the queen and at least one worker were present are presented. Nests are
listed in order of excavation date showing decreasing differences between the amount of wear
in workers and queens as the season progressed.
44
A W
A
W
01
ft.
•••
§ft.
••
• wear in workers
(9
01
i
V
JB
•o
c
•••
ra
E
••
w
w
A.
w
A
Ml
A
w
• wear in queens
/
m /
••
// ^
0
•—
21-Jun-10
.—AA AA i—
ll-Jul-10
31-Jul-10
AA
Jfc
A
m
4
m
A
- Linear (wear in
workers)
- Linear (wear in
queens)
.
20-Aug-10
Date
Figure 2-9: Wear on the mandibles of queens (red) and workers (blue) throughout the season
showing an increase in the amount of wear over time in both groups.
45
• wear in
workers
• wear in queens
- Linear (wear in
workers)
- Linear (wear in
queens)
^—i' A A
21-Jun-10
06-Jul-10
21-Jul-10
r05-Aug-10
20-Aug-10
04-Sep-10
Date
Figure 2-10: Wear on the wings of queens (red) and workers (blue) throughout the season
showing an increase in the amount of wear over time in both groups.
46
Average daily maximum temperature
• 1976
w 20
• 1977
5b 15
• Average
• 2002
• 2010
July
August
Average daily minimum temperature
25
20
11976
15
w
3
11977
10
i Average
12002
12010
April
May
June
July
August
Month
Figure 2-11: Average daily minimum and maximum temperatures for the four years of study on
H.farinosus in Green Canyon and the seasonal average over the previous 65 years.
47
• 1977
Average
2002
August
Figure 2-12: Total rainfall per month for the four years of study on H.farinosus in Green Canyon
and the monthly average over the previous 65 years.
48
lioe
• Quwwtongnaiaw
om
Mb
9J?
1000 +
• FMopoductovbrooa
•saaas• FjrVKMM*
rapTMUOMt
9?
J
2°
!§
Dale
Date
Figure 2-13: Cumulative degree days between March and August in 1977,1978, 2002 and 2010.
Phenological markers (queen foraging slows, first adult worker, first reproductive brood cell,
first adult male reproductive, first adult female reproductive) have been included as coloured
bars to note the dates of the events. Male and female reproductives were not differentiated
between in the 2002 study, and some events were not recorded in 1977 and 1978.
49
Chapter 3: Sociogenetic organization in the primitively eusociai bee
Halictus farinosus Smith (Hymenoptera: Halictidae)
50
Abstract
Halictus farinosus is a primitively eusocial species of sweat bee that is common in
Western North America. Adult and juvenile bees from thirty-seven nests within an H.
farinosus nesting aggregation in Northern Utah were collected and genotyped at six
highly variable microsateliite loci to reconstruct kin structure and estimate relatedness.
Polyandry was uncommon in H. farinosus queens whose population wide effective
mating frequency was 1.07, with a second mate confirmed in four of the thirty-seven
nests. The queen was present in only 52% of the excavated reproductive brood nests
but she produced the vast majority of the brood (98%) while living. Workers took over
reproduction upon being orphaned. The brood was heavily female biased when
produced by a queen but heavily male biased when worker-produced resulting in a
significant difference in sex ratio between queenright and queenless nests (t = -3.72, p =
0.003), leading to split sex ratios. Together these results generally agree with the
predictions of inclusive fitness theory and support the view that haplodiploidy is
important in the evolution of eusociality.
Introduction
Eusocial behaviour, as exhibited in the Hymenoptera, is highly altruistic requiring
subordinate worker offspring to reduce or eliminate personal reproductive options to
instead help their parent to reproduce (Wilson, 1971). Inclusive fitness (Hamilton,
1964a) is the most widely accepted theory describing how eusociality could have
evolved. Hamilton's rule of inclusive fitness allows individuals to maximize their own
fitness indirectly by helping their close relatives to survive and reproduce. It states that
if the product of the measure of relatedness between two individuals and the beneficial
action one performs to help the other is greater than the cost to the acting individual
(rb>c) then costly traits can be selected for. Thus the more closely related individuals are
the easier it is for altruistic traits such as eusociality to be selected (Hamilton, 1964a).
Recent challenges to inclusive fitness theory as it applies to the evolution of eusociality
have been brought forth in part by the founder of sociobiology (Wilson and Holldobler,
2005; Wilson and Wilson, 2007). Mathematical models have been presented that
dispute the assumption that close genetic relatedness is important for the evolution of
eusociality favoring instead selection at the group level (Nowak et al., 2010). Despite
this, monogamy, which promotes very close genetic relatedness among full sisters, has
been shown to be particularly important in the evolution of various levels of
cooperation including eusociality (Hughes et al., 2008; Boomsma, 2009; Cornwalis et al.,
2010). The prevalence of eusociality in the Hymenoptera may in part be explained by
their genetic system of haplodiploidy (male haploidy) which causes female offspring of a
singly mated queen to be more closely related to their sisters than their daughters, but
less closely related to their brothers than their sons (Hamilton, 1964b; Bourke and
Franks 1995; Crazier and Pamilo 1996; Boomsma, 2009). If relatedness and
haplodiploidy are important factors in maintenance of eusociality, worker females are
52
predicted to lay male destined eggs themselves and to prefer that the queen produce
reproductive sisters (Trivers and Hare, 1976; Pamilo, 1991).
It is difficult to understand the origins and early evolution of eusocial behaviour
when studying highly derived eusocial forms, such as the corbiculate apids and most
ants in which distinct anatomical differences exist between castes from early
development (Michener, 1974; Bourke and Franks, 1995). Members of the Halictidae
exhibit diverse social behaviours that are much more flexible than those of highly
eusocial clades (Schwarz et al., 2007). There have been at least three evolutionary
origins of eusociality within the subfamily Halictinae (Danforth, 2002) as well as multiple
transitions from eusocial to solitary behaviour (Packer, 1997; Danforth et al., 2003). The
existence of socially polymorphic species in the Halictinae (Sakagami and Munakata,
1972; Eickwort et al., 1996; Richards et al, 2003; reviewed by Packer, 1997) and the
relatively recent evolutionary origin of eusociality (Brady et al., 2006) compared to
highly eusocial bees (Michener and Grimaldi, 1988; Cardinal and Danforth, 2011) further
strengthens the case for studying this group as a model for the origin of primitive
eusociality.
53
Studies of Halictusfarinosus in Utah and California (Nye, 1980; Eickwort, 1985;
Sellars, 2004; Chapter 2) indicate that it is a social ground nesting bee with a colony
cycle comprising two distinct seasonal phases; first the overwintered foundress
independently produces worker females; and second the workers forage to produce
the reproductive brood. Small colony size, little size dimorphism between castes as well
as significant ovary development and frequent mating in workers point to a system of
weak eusociality in H.farinosus (Sellars, 2004; Chapter 2) making it an ideal system in
which to study the origins and early maintenance of eusociality. It is not known however
if H.farinosus workers contribute directly to brood production through oviposition that
leads to adult reproductives. Studies in other social halictines based on allozymes, DNA
fingerprinting and microsatellites generally show that nestmates are closely related
(Crozier et al., 1987; Mueller et al., 1994; Packer and Owen, 1994; Soro et al., 2009) and
that reproductive skew, which measures the degree to which reproductive output is
dominated by an individual, is typically high and in favour of the queen (Mueller et al.,
1994; Paxton et al., 2002; Soro et al., 2009; Ulrich et al., 2009). However, simple kin
selection theory is not sufficient to explain all the complexities of behaviour in these
species: multiple paternities (Richards et al., 1995; Paxton et al., 2002), worker
production of female brood (Richards et al., 1995; Paxton et al., 2002) and nest
switching (Paxton et al., 2002; Soro et al., 2009; Ulrich et al., 2009) are not predicted by
inclusive fitness theory and yet they appear to be common. In this study we use
54
microsatellites to determine the breeding system and kin structure in nests of Halictus
farinosus and compare this to studies of related species of sweat bee.
Methods
Field work and nest excavations
For a detailed description of field site, field methods and dissection methods see
chapter 2.
Genotyping of microsatellite loci
A total of 10 first brood nests and 27 second brood nests containing a total of
572 individuals were genotyped at six variable microsatellite loci developed for study of
the closely related species H. rubicundus (Soro and Paxton, 2009, Table 3-1). DNA was
extracted using a high salt extraction protocol (Paxton et al., 1996) from half of the
thorax of adult bees and a small amount of tissue taken from juvenile stages. A nested
three primer PCR adapted from Schuelke (2000) was used to isolate and label the
microsatellite loci. The M13 sequence (Table 3-1) was added to the 5' region of the
forward primer at each locus that matched a third fluorescently labelled M13 primer
(Table 3-1). Products were labelled with either WELLRED D2 (black), IRDye700 (green) or
TYE665 (blue). The PCR conditions used followed those outlined for the nested PCR by
55
Schuelke (2000) using ideal annealing temperatures found by gradient PCR (Table 3-1).
PCR products were genotyped following the manufacturer's instructions for fragment
analysis using a CEQ8000 molecular sequencer from Beckman Coulter Inc.. In cases
where the results were ambiguous or unexpected, the PCR and genotyping were
repeated.
Analyses
A population sample of 42 first brood females was used to calculate observed
heterozygosity, Nei's unbiased (expected) heterozygosity (Nei, 1978) and the inbreeding
coefficient (Fis= 1- H0/HE) by hand. The same sample of females was used to calculate
deviation from Hardy-Weinberg equilibrium and the frequency of alleles using GENEPOP
4.1(Rousset, 2008). Linkage disequilibrium between loci was calculated with GENEPOP
4.1using the same subset of females and a similar subset using 28 males, with at most
one per nest.
Sexing the brood was done initially by inspection of an individual's alleles at all loci:
individuals that were heterozygous at one or more loci were scored as female. It is
possible that diploid males could have been mistaken for females (Cook and Crozier,
1995). However, physical sexing of juvenile brood is possible after they are past the
larval stages and no males were found among bees determined to be diploid. In
56
addition, inbreeding in this population was relatively low (Table 3-1), thus diploid males
are not expected to influence the results presented here. The probability of a female
being scored as a male was calculated as the probability that she would be homozygous
at all loci using the population allele frequencies:
]
(1)
Where p,2 is the probability that the allele (i) is homozygous and j is the locus and the
product is taken over all available loci. If a bee was past the larval stages sexing was
confirmed by inspection of the specimen. Sex ratio was calculated as the number of
male offspring divided by the total number of offspring weighted equally over nests or
over individuals. Investment ratios were calculated using wet weight of pupae weighted
equally over nests or over individuals. Since fathers could not be captured, their
genotypes were deduced using Matesoft 1.2 (Moilanen et al., 2004) from female
offspring and a single paternity was assumed when all daughters in a nest carried the
same paternal alleles. The chance that multiple mating went undetected was calculated
as the probability of two males being identical over all loci:
j
(2) (Seppa et al., 2011)
57
Bees identified as queens in nests labelled with a suffix of C, D, E, F or G were
marked early in the season so they could be identified during excavation. They were
later identified by their markings and confirmed as the queen by their multilocus
genotype. In aggregations B and N, some females in nests that were discovered late in
the season were determined to be queens by their genotype. Colony 2.0 (Wang, 2004;
Jones and Wang, 2010) was able to reliably assign marked queens to individual offspring
within their nests (p > 0.977), thus permitting confidence in queen assignments in
unmarked nests. The program was able to do this in all cases for both marked and
unmarked queens except for 4 offspring in 3 nests where poor genetic information was
available (offspring had missing allele data for at least four loci). In five nests where the
queen was not present (N0811-1, B313, C208, D303, G402) her genotype was
unambiguously deduced from the nest's eldest offspring with the help of Matesoft 1.2
(Moilanen et al., 2004). In two additional orphaned nests the queen's multilocus
genotype was one of two possible genotypes because homozygosity at one locus made
it impossible to distinguish between maternal and paternal alleles at that locus in
heterozygous daughters. In an additional six nests too few of the queen's offspring were
present to reliably deduce her genotype. Unambiguously deduced queens were added
to the dataset for relatedness and sibship calculations.
58
Genetic relatedness (r) was estimated using Queller and Goodknight's (1989)
regression relatedness algorithm and was calculated using Relatedness 5.0.8. Individuals
found to be drifters (alien workers) and one locus in one nest found to have a null
paternal allele (Rub73, B304) were excluded from the relatedness analysis. Relatedness
estimates were done for the population as a whole and for a subset of nests that were
found to be monogamous. Standard errors were obtained by jackknifingover loci.
Multilocus genotypes were manually inspected for all offspring in each nest and
individuals were assigned first to matrilines, then patrilines within the matrilines,
allowing us to group nestmates into full and half sibships and to deduce parentage and
mating frequency (as in Seppa et al., 2011). Sibships were confirmed using Colony 2.0
(Wang, 2004; Jones and Wang, 2010). Relatedness data, sibship data and manual
inspection of alleles allowed us to reliably deduce kin structure in most nests.
Reproductive skew was calculated following the general formula of Crozier and
Pamilo, (1996).
Where NT is the number of potentially reproductive females (workers and queens) and
Pi is the actual reproductive output of the rth individual. Skew may have been
overestimated in some cases as males carrying only maternal alleles were assumed to
have been produced by the queen. Effective mating frequency was calculated using the
following formula:
me = =r"2
(4) (Starr, 1984; Soro et al., 2009)
Where y. is the proportion of the queen's daughters fathered by male / and the
summation is over all fathers. Sex ratios in queenright and queenless nests were
analysed by a two sample, two tailed T-Test using Minitab* Statistical Software after arcsin
transformation of sex ratio proportions.
Results
Microsatellites
Microsatellite loci were highly variable with many alleles per locus (x = 11.8, range 4
-15) and high observed and expected heterozygosities (Table 3-1). Loci were not in
linkage disequilibrium: p > 0.11 for all loci in both male and female datasets. There was
no significant deviation from Hardy-Weinberg equilibrium in the population subset (p >
0.05 for all loci and over all loci). The inbreeding coefficient in the population sample
was not significantly different from zero (Table 3-1, t = 0.510, p = 0.621). At least one
60
locus (Rub 73) had null alleles that were unreliably amplified; however, this locus was
useful for assigning parentage in most cases, particularly with the aid of Colony 2.0
which can account for the presence of null alleles.
The probability that an additional paternity was present when none were detected
was very small in both worker and queen-produced females (p < 0.0007). The
probability that an individual scored as a male by genotype alone was actually a female
was even smaller (p < 0.0002).
Worker brood
On average only 3.4 workers were produced per nest and an average of 1.7
workers were found in those nests when excavated in late summer. Sex ratio in the
worker brood was strongly in favour of females with an average ratio of 0.149 (SD =
0.31, N=16) over nests and an overall ratio of 0.159. All but one of the worker brood
males came from two nests (five from N0708-2 and four from N0709-4). All individuals
in both broods that were found to be males by inspection were haploid. Only one
female was produced in either of these two (N0709-4.07), and she was the youngest
member of the nest. It could not be determined if the queen in N0708-2 was mated
because of damage to her abdomen during nest excavation. The single worker brood
male produced outside of these two nests was the oldest of his nestmates. Workers
were significantly smaller than their queen (t = 6.49, p < 0.0001) with a size differential
of 8.79% for head width and 5.12% for wing length. Dissections revealed that 71.4% of
workers had developed ovaries (N = 43) and 77.5% were mated (N = 40) (Chapter 2).
Queen's had significantly more ovary development than workers whether or not a
queen was present in the worker's nest at the time of excavation (see Chapter 2).
Overall, workers from queenless colonies did not differ significantly in their level of
ovary development from workers in queenright colonies (Chapter 2).
Relatedness
The average genetic relatedness between all nestmates, including the queen,
was not significantly different from 0.5 (r = 0.508, SE = 0.024, CI = 0.062), and between
all female brood (workers and gynes) in each nest was r = 0.641(SE = 0.026, CI = 0.066),
which is significantly higher than 0.5 and significantly lower than 0.75 (Figure 3-1).
Relatedness between females (excluding the queen) in monogamous nests was not
significantly different than 0.75 (r = 0.730, SE = 0.0134, CI = 0.035). Overall, workers
were not more closely related to the reproductive brood than queens were: r = 0.646
(SE = 0.029, CI = 0.075), compared to r = 0.534 (SE = 0.024, CI = 0.061) for queens. In
addition workers were not more closely related to gynes than queens were overall (r =
0.591, SE = 0.041, CI = 0.107 for workers; r = 0.470 SE = 0.026, CI = 0.066 for queens).
However, in monogamous nests the relatedness value between workers and gynes was
the expected 0.75 (r = 0.749, SE = 0.024, CI = 0.062). Worker to worker (0.538) and gyne
62
to gyne (0.632) relatedness values were significantly different from the predicted value
of 0.75 in the population overall but not in monogamous nests (see Table 3-2). A list of
relatedness values over nests in the population can be found in Table 3-2. There were
no differences in relatedness predictions when deduced queen genotypes from absent
queens were left out.
Social structure reproductive brood
Four general types of social structure were found among summer nests (Table 33). The queen was generally present in type 1nests; she and one or two male mates
could account for all offspring within the nest and reproductive skew was complete (S =
1). In type 2 nests the queen was present and some of the offspring had paternal alleles
at one or more loci indicating that they were worker-produced. Reproductive skew in
these nests was still very high (S > 0.957, Table 3-4). In type 3 nests the queen was not
present at the time of excavation and the nesfs workers were responsible for producing
some or all of the offspring. The eldest offspring, if any, were daughters and sons of the
queen and siblings to the workers while the youngest were laid by the workers,
suggesting that the workers only reproduced successfully after the queen had died.
Reproductive skew in type 3 nests was variable (from 0.02 to 1.00, with an average of
0.77, Table 3-3,3-4) and dependent on when the nest was orphaned. In three of the
type 3 nests (C208, B101, N0811-1) it was determined that at least two workers were
63
responsible for producing offspring. In the remaining six type 3 nests the worker-laid
offspring, all of which were male, could have all been produced by the same individual
but because workers were so closely related this could not be verified reliably with only
six loci. Type 4 nests were those in which kin structure could not be determined, either
because the relationships between nest members were too complicated or some
nestmates appeared to be unrelated to others (see discussion for details).
Type 1nests were the most common (N = 12), closely followed by type 3 (N = 9).
Workers laying eggs while the queen was present was uncommon (type 2, N = 3) as
were situations where genealogies could not be determined (type 4, N = 3). Most
reproductive brood males were produced in the type 3 nests and the majority of all
males (60.8%) were produced by workers. Worker-produced brood was overwhelmingly
male (93.8%) while queen-produced brood was predominately female (74.2%). The
queen produced 96.6% of the female reproductives in the population.
Polyandry
A second patriline was identified in two spring and two summer nests (see Table
3-2). No more than two patrilines were present in any nest and the second patriline was
responsible for less than 33% of the offspring in all cases. No worker polyandry was
confirmed but the vast majority of offspring confirmed to be worker-produced were
64
male (Table 3-4). The effective mating frequency for queens was 1.07 males overall and
1.59 in polyandrous nests.
Sex and investment ratios
Reproductive brood nests had, overall, a balanced sex ratio: 0.465 or 1:1.2 (SD =
0.349, N = 27) with nests weighted equally or 0.455 (1:1.2) with individuals weighted
equally. Monogynous queenright reproductive brood nests (Type 1, Table 3-3) tended to
have more females than males with a ratio of 0.255 or 1:2.9 (SD = 0.228, N = 12)
averaged over nests. The sex ratio in all nests where the queen was present (type 1and
type 2 nests combined) was 0.284 or 1:2.5 (SD = 0.253, N = 15). The sex ratio where the
queen was present but workers produced some brood (type 2 nests) was 0.400 (SD =
0.369, N = 3) or 1:1.5. Queenless nests (Type 3) tended to have more males than
females with a ratio of 0.742 or 2.9:1 (SD = 0.304, N= 9) over nests.
Female pupae were heavier, weighing an average of 102.0 mg (SD = 21.8, N = 59)
compared to 62.4 mg (SD = 14.0, N = 45) for male pupae. This makes the population
wide cost ratio for females over males 1.63:1. The sex investment ratios are then 1:2.0
overall, 1:4.7 in queenright monogynous (type 1) nests, 1:2.5 in queenright polygynous
(type 2) nests, 1:4.1in queenright nests overall (type 1and type 2) and 1.8:1 in
65
queenless (type 3) nests. The sex ratio in queenright nests was significantly more female
biased than the sex ratio in queenless nests (t = -3.72, p = 0.003).
Drifting workers
One of the summer nests (B108) contained a small and slightly worn female that
was unrelated to the nest's queen or to the offspring produced in the nest at the time of
excavation and had no ovary development. A second nest (B101) had three worker
brood females present who were unrelated to its five native workers, two of these
drifters were likely full siblings. It was unlikely that this was due to errors in nest
excavation because all worker brood females other than the replacement queen were
found together near the nest entrance. This nest was incredibly productive as it had
twice the number of workers and 45% more offspring than the next most productive
nest. The unrelated workers had some ovary development but did not contribute
genetically to the brood.
Discussion
Relatedness estimates
Relatedness estimates (Table 3-2) were significantly different from expected
under conditions where all nests are headed by a singly mated monogynous queen in
66
four comparisons. First worker to worker relatednesses in first brood nests were lower
than expected because in two of the ten there was an additional patriline. Workerworker relatedness in brood one nests was not significantly different than expected
when these polyandrous nests were excluded (r = 0.73, SE = 0.02, CI = 0.05). Second,
queen to male comparisons in reproductive brood nests were lower than expected
because of frequent oviposition by workers. This value could have been lower still since
workers produced most of the males only when the queen was not present and
comparisons could not be made when the queen's genotype was not available. Third,
worker to gyne and gyne to gyne relatednesses in the reproductive brood were
significantly lower than expected because of multiple mating and worker-laid gynedestined eggs. Finally, three of the second brood nests were type 4 nests, meaning that
the pedigree could not be reconstructed from the available allelic data. One of these
(B311-2) had high relatedness but parentage was not straightforward, two or three
related individuals may have been laying eggs but no adults were present upon
excavation. Two type 4 (B301, B311) nests had low relatedness between nestmate
females, with their confidence limits reaching into the negatives (Figure 3-1). Nest B311
had a worker that was the most likely parent of the young males and two female
offspring present in the nest may have been her half siblings. Nest B301 had a group of
eight males and one female that were likely laid by the same individual, the remaining
two nestmates, one worn worker and one uneclosed adult female, appeared to be
unrelated to the other nest inhabitants but were likely full siblings of one another.
67
Parasitism (nest usurpation) or excavation error may have accounted for the genetic
patterns these three type 4 nests which all came from a very densely populated area. All
relatedness estimates were as expected for a monogamous haplodiploid organism when
polygynous, polyandrous and type 4 nests were excluded from the relatedness analyses
(Table 3-2).
Polyandry
Polyandry occurred occasionally in this population with a second patriline
detected in four (10.8%) of the nests studied. Even when the queen had mated with
more than one male the majority of the offspring were fathered by only one leading to
low effective mating frequencies. Polyandry effectively lowers relatedness between
female nestmates making it less rewarding for them to help in the nest according to
inclusive fitness theory. The small number of patrilines, small effective mating frequency
and low occurrence of multiple mating in this species agrees well with the results of
similar studies in other primitively eusocial halictines (Crozier et al. 1987; Packer and
Owen 1994; Mueller et al., 1994; Richards et al., 1995; Paxton et al., 2002). This also
matches the predictions of inclusive fitness theory and the finding that monogamy is
prevalent in the early evolution of eusociality (Hughes et al., 2008).
68
Worker Drifting
Worker drifting, where conspecifics from unrelated, nests will join a nest and
behave as a worker, occurred in 7.4% of the second brood nests studied. Curiously,
drifting has been reported in a number of other eusocial halictines (Mueller et al., 1994;
Soro et al., 2009; Ulrich, 2009) indicating that it is a common occurrence. It is
understandable from the results in this study why drifting workers may be accepted into
the nest: drifters come into the nest at no cost since they were not found to produce
any of the nest's offspring. Evidence of oophagy was found in dissected crops of the
dominant egg layer in each of the two nests that contained alien workers (B101, B108)
and in the crop of one of the workers in alien-containing nest B108. Oophagy was only
suspected in one additional worker outside of these two nests indicating that eggs laid
by alien workers may be recognized and removed or that the presence of aliens causes
nest residents to increase their policing behaviour. Why workers would switch nests in
this population is not obvious from our results. Ulrich et al. (2009) suggest that workers
in a population of Halictus scabiosae move from overpopulated nests to neighbouring
ones near the end of the season for hibernation and by joining smaller nests they
increase their chance of becoming the dominant egg layer in that nest in the next
season (Ulrich, 2009). This is unlikely to be the reason in H.farinosus in Green Canyon as
they do not overwinter beneath the natal nest (Nye, 1980) and unlike H. scabiosae they
are not pleometrotic. In addition, drifters in the Green Canyon population appeared to
join the most active nests since the nests with alien workers in this study were the two
69
largest in the population. As occurs in some honey bee and bumble bee species
(Birmingham et al. 2004; Lopez-Vaamonde et al. 2004; Nanork et al. 2005), drifting
worker brood females may aim to increase their chances of opposition by moving to a
larger nest where the dominant would have less control. However, while drifters in
some Bombus species have a greater number mature eggs than native workers
(Birmingham et al. 2004), aliens in H.farinosus had the same amount of ovarian
development as the nests' natives (t = 0.40, p = 0.70, N = 11).
Replacement queens and succession
In 56% (N = 9) of the queenless type 3 nests the worker-produced offspring could
all have belonged to a single worker. In three of the other four nests the worker who
laid the youngest offspring was present but the mother of the older worker-laid progeny
was missing. This indicates that a worker takes over whenever the dominant member of
the nest dies and becomes her replacement, a phenomenon known as serial polygyny
(Bourke and Franks 1995; Paxton et al., 2002). Dissections show that the nine
replacement queens in type 3 nests, deduced by their genotype as being the likely
mother of the youngest offspring, do not differ from queens in terms of ovary
development (t = 0.023, p = 0.819, df = 48) but have significantly more developed
ovaries compared to all other workers (t = 3.44, p = 0.001, df = 38) and compared to
other orphaned workers (t = 2.56, p = 0.018, df = 22). This pattern resembles that found
in Augochlorella aurata (as A. striata Mueller et al., 1994) and Lasioglossum laevissimum
(Packer and Owen, 1994} where one worker replaced the queen and subsequently
dominated reproduction.
It is not readily apparent how the replacement queen is determined. In other
social halictids size (Michener et al., 1971) and especially age (Michener et al., 1971;
Plateaux-Quenu, 1978; Plateaux-Quenu, 1985) have been shown to be important factors
in determining which bee dominates oviposition if the nest is orphaned. The line of
succession in H.farinosus is not determined by size since replacement queens were
smaller than each of their worker nestmates in two of the three nests where we could
compare sizes (Figure 3-2). In addition, replacement queens did not appear to be older
than their fellow workers based upon wing and mandible wear scores. In fact,
replacement queens found in nests with other workers (n = 5) always had the lowest
wing wear among their sisters and four of the nine replacement queens had virtually no
wing wear (score of 0 or 1) indicating that they had done little, if any, foraging.
Replacement queens might simply arise based on which bee displays the most queen­
like and least worker-like behaviour at the time of orphaning (Michener, 1990).
Sex ratio and sex allocation
71
The overall sex ratio in the reproductive brood in this population is very close to
50% but the sex ratio is split with significant differences between queenright and
orphaned nests (p = 0.0004, t = 4.218, df = 22). Queens produced the majority of
females (96.6%) and the workers produced most of the males in this population (60.8%).
All replacement queens (N = 9) were mated and their spermathecae after mating
resembled that of the queens', so inability to produce diploid gynes is an unlikely
explanation for male biased sex ratios in orphaned nests. Similarly, Mueller (1991) found
that queenright colonies of Augochlorella aurata (=striata) were significantly more
female biased than queenless colonies. Split sex ratios are also known from Halictus
rubicundus where eusocial (queen containing) nests produced mostly females and
parasocial (orphaned) nests produced mostly males (Yanega, 1989). However, in
Halictus ligatus queens produced most of the males while abandoned colonies headed
by replacement queens produced mostly gynes (Richards et al., 1995). According to
Richards et al. (1995), queens in H. ligatus lay as many male destined eggs as possible
before workers are able to coerce them into producing gynes, and orphaned workers
continue to produce females presumably to balance the overall sex ratio. While the
reproductive brood of H.farinosus tended to be protandrous, its queens produce few
males before switching to gyne production when workers are present. Theory predicts
extreme differences in sex ratios between nests where the cost to produce one sex or
the other differs (Grafen, 1986). This is predicted to occur to the point where different
nest types specialize in producing one sex or the other where their sex investment
72
strategy will depend on the social structure of the family (Grafen, 1986; Boomsma,
1991). This is likely the cause of the extreme sex and investment ratios in queenright
nests in this population which are highly female biased with an average investment ratio
of nearly 1:5 (males: females) in monogynous queenright nests. No relatedness
asymmetry is present when replacement queens produce daughters and sons (or nieces
and nephews in the case of subordinate workers). Thus, when given the choice, workers
in orphaned nests should prefer to produce whichever sex has the best chance of
reproduction (Fisher, 1930; Trivers and Hare, 1976; Queller and Strassman, 1998) and
given the sex ratio asymmetry of the queen's brood, workers in orphaned nests should
prefer to produce males. This pattern is precisely what we see in this population with
queenright colonies (type 1and 2) specializing in producing females and replacement
queen colonies (type 3) specializing in producing males.
Conclusion
The results of this study agree with the predictions of inclusive fitness theory:
monandry and high relatedness among nestmates in a primitively eusocial organism.
Nestmates were close kin as a result of a high incidence of monogamous foundresses
and low effective mating frequency. Despite comparatively small queen to worker size
disparity (Chapter 2) and high levels of worker ovary development and matedness, H.
farinosus queens still produced the vast majority of offspring while they were alive. Only
73
when a nest had been orphaned did first brood female workers make a significant
contribution to the reproductive brood. Overall the workers were as closely related to
the reproductive brood as the queen. Workers may capitalize on the relatedness
asymmetries presented by haplodiploidy by coercing the queen into producing females
while producing males themselves. The fact that queenright nests specialized in
producing females and orphaned nests headed by replacement queens produced mostly
males indicates that haplodiploidy may be playing a role in the evolutionary
maintenance of the helping behaviour by ensuring that working females are maximally
related to the brood in all nest types.
74
Tables and Figures
Rub02
Primer Sequence (5'3')
f: TGTAAAACGACGGCCAGT
CCAGCCGGCCAACGTTGC
o
Z
Name
He
or
Table 3-1: Primers for microsatellite loci (developed by Soro and Paxton, 2009) and variability in
all individuals of H.farinosus. Including observed and expected heterozygosities, the inbreeding
coefficient (Fis) and hardy-weinberg estimations for all loci. M13 tails were added to the forward
primer for incorporation of a fluorescent dye during PCR.
0.893
0.881
0.0133
0.082
4
0.576
0.619
-0.0754
0.068
144-172
14
0.834
0.852
0.0464
0.407
65
168-197
14
0.846
0.921
-0.0885
0.121
60
203-232
10
0.785
0.790
-0.0042
0.458
60
191-244
14
0.811
0.781
0.0377
0.662
Anneali
ng
temp.f
C)
58
Length
Range
(bp)
n
183-211
15
58
207-215
58
alleles
H-W
test p
value
R:CGGAGCTGAAAACTCAATTACA
G
Rub04
F: TGTAAAACGACGGCCAGT
CGGACGTTTTTCAATGTTTTTC
R: CGTCCGACTGCATTCTCTTTG
Rub55
F: TGTAAAACGACGGCCAGT
GCTATAAAAGGCGAAACGGGTG
R:CTCCTATCCGGTTGACATTGCC
Rub30
F: TGTAAAACGACGGCCAGT
GATCCGCnTCAACCGTCCG
R: 6TGAGCTGGGTCCGGCGAG
Rub 59
F : TGTAAAACGACGGCCAGT
GT6ACCAGGTGCGCTCGTTAC
R: CCGTGTCCCCAGCTCCGTTrC
Rub73
F: TGTAAAACGACGGCCAGT
GCTTTGTTTCTCACTATCGTCCC
R: CGCGCAAAGTTCCCAGGGGTG
75
Table 3-2: Estimates of relatedness between different categories of offspring in the worker and
reproductive broods, given separately for monogamous nests. Expected relatedness is the
predicted value is for a nest where there is a sole, singly mated, foundress who produces all
offspring.
Relationship (y, x)
Observed
relatedness
Expected
relatednes
s
SE
95% CI
Worker Brood
0.523
0.50
Queen to worker
0.043
0.110
1.000
0.000
1.00
0.000
Queen to male
0.022
Worker to worker
0.644*
0.75
0.056
0.745
0.137
Worker to male
0.50
0.353
Reproductive brood
0.446
0.026
Queen to worker
0.50
0.067
0.023
0.794*
1.00
0.060
Queen to male
0.025
Queen to gyne
0.474
0.50
0.064
0.628*
0.032
Worker to worker
0.75
0.082
0.550
0.50
0.029
Worker to male
0.076
0.040
Worker to gyne
0.594*
0.75
0.104
0.652*
0.75
Gyne to gyne
0.024
0.061
Monogamous nests only
0.530
0.50
0.035
0.090
Queen to worker
A
1.000
1.00
0.000
Queen to male
0.000
0.495
0.026
Queen to gyne
0.50
0.067
0.720
Worker to worker
0.75
0.014
0.037
A
0.578
0.75
0.039
Worker to male
0.099
0.749
0.024
Worker to gyne
0.75
0.062
•Significant difference rom expected by confidence interval limits
includes reproductive brood individuals only
N
47
14
74
10
53
134
246
68
240
263
496
54
49
137
74
54
111
76
Table 3-3: Summary of kin structure in the population's reproductive brood nests where nests
are divided into types.
Reproductiv
Type e type
1 Monogynous
2 Polygynous
3 Polygynous
Queen
presen
t
Non queenproduced
brood
Origin of non queenproduced brood
Reproductiv
eskew
Y»
n/a
n/a
Full (S = 1.0)
Y
Males
Males and
females
Males and
females
worker produced
High (S > 0.76)
3
worker produced
unknown/cannot be
determined
Highly variable
9
N
N
nests
12
4 Polygynous
N
Unknown
* While it is generally true that the queen is present, she was missing in two nests found to be
completely monogynous
3
77
Table 3-4: Sociobiological details in all nests based on genotype, sibship patterns and genetic
relatedness data.
Worker brood Nests
Que
en
Pres
ent
Mon
ogyn
Proporti
on of
offspring
laid by
queen
Reproducti
ve skew *
Sex Ratio
(males/
total)
Propor
tiori of
female
sin
worker
offspri
ng
n/a
1.000
1.000
0.00
n/a
Queen'
s
effecti
ve
mating
freque
ncy
1
N070
2-1
Y
N070
2-2
n/a
n/a
1.000
1.000
0.00
n/a
1.39
Y
n/a
n/a
1.000
1.000
0.00
n/a
1
Y
Y
n/a
n/a
1.000
1.000
0.00
n/a
1
Y
Y
n/a
n/a
n/a
1.000
1.000
1.00
n/a
n/a
»»
N070
9-4
Y
Y
n/a
n/a
n/a
1.000
1.000
0.83
n/a
n/a
**
N071
2-1
Y
Y
Y
n/a
n/a
1.000
1.000
0.25
n/a
1
N071
2-3
Y
Y
N
n/a
n/a
1.000
1.000
0.00
n/a
1.80
N071
Y
Y
Y
n/a
n/a
1.000
1.000
0.00
n/a
1
Y
Y
Y
n/a
n/a
1.000
1.000
0.00
n/a
1
Queen
worker
Allen
worker
Proportio
nof
offspring
laid by
queen
Sex Ratio
(males/t
otal)
Propor
tk>n of
female
sin
worker
offspri
ng
Queen'
s
effect)
ve
mating
freque
ncy
Nest
Monan
dry
N
queens
worker
N alien
worker
Y
Y
n/a
Y
Y
N
N070
4-3
Y
Y
N070
6-1
Y
N070
8-2
V
Notes
5-1
N072
5-1
Reproductive brood nests
Nest
Type,
Notes
Mon
ogyn
N081
1-1
NA
N
Y
3
0
0.800
0.837
0.50
0.000
1
3,"*
N081
1-3
Y
N
Y
2
0
0.769
0.759
0.82
0.000
1
2
OrlOb
Y
Y
Y
1
0
1.000
1.000
0.00
n/a
1
1
Or4b
Y
Y
Y
1
0
1.000
1.000
0.33
n/a
1
1
B101
N
N
Y
5
3
0.000
0.915
0.98
0.021
1
3,***
B106
Y
N
Y
1
0
0.957
0.909
0.26
0.000
1
2
B107
Y
N
Y
5
0
0.969
0.984
0.12
1.000
1
2
B108
Y
Y
Y
4
1
1.000
1.000
0.22
n/a
1
1
B211
Y
Y
Y
1
0
1.000
1.000
0.11
n/a
1
1
B301
N
N
n/a
1
0
n/a
4,#
*
Monan
dry
(queen
)
Reproducti
ve skew*
Que
en
Pres
ent
0.80
78
B302
NA
N
Y
1
0
0.400
0.273
0.56
0.167
1
3, ***
B304
Y
Y
Y
2
0
1.000
1.000
0.32
n/a
1
1
B306
Y
Y
Y
2
0
1.000
1.000
0.14
n/a
1
1
B311
N
N
n/a
1
0
0.75
n/a
4,#
B3112
B313
N
N
n/a
1
0
0.67
n/a
4,#
NA
Y
Y
2
0
C208
NA
N
Y
2
0
0.875
0.903
0.13
1.000
1
D303
NA
Y
Y
3
0
1.000
1.000
0.25
n/a
1
1
D401
N
N
n/a
1
0
0.000
1.000
l.OO
0.000
n/a
3, **
E401
N
N
Y
2
0
0.000
1.000
0.82
0.182
n/a
3,**
E407
Y
Y
Y
0
0
1.000
1.000
0.29
n/a
1
1
E4H
Y
Y
N
1
0
1.000
1.000
0.16
n/a
1.80
1
F203
Y
Y
Y
2
0
1.000
1.000
0.40
n/a
1
1
F205
N
N
n/a
1
0
0.000
1.000
1.00
0.000
n/a
3,**
F211
Y
Y
Y
0
0
1.000
1.000
0.00
n/a
1
1
G402
NA
N
N
1
0
0.545
0.016
0.70
0.000
1.39
3
G408
NA
N
Y
2
0
0.000
1.000
1.00
0.000
1
3
1.000
1.000
0.86
n/a
1
1
3
*
From equation S3 in Crozier and Pamilo, 1996
••
All males or only one female - n patrilines cannot be determined
*•*
More than one worker has produced offspring. In 0811-1, C208 and B302 the first
replacement queen was not found, one worker (second replacement queen) was the
mother of the youngest offspring.
#
Kin structure cannot be determined
A
Queen's alleles reliably deduced
79
1.25
*0.75
J—I-
T3
0>
m
w 0.5
u
S
s0.25
(9
-0.25
O OWO W
O OU
OW
C l CW
DW
OUO)I ^
i m^m^- ^n
Z Z Z Z Z Z Z Z Z O O CO CO CO CO CD
N
ooooooooo^7t;st;s;N U
O
Q
O
l
'
>
»
l
>
O
o
O
O
'-'P
K)5o>h>l-'U)00U)t-'vlMW
O
w 4O»OcM
n rMo PMKUJi M
u iP
p pOOC' T M O ^ J O O H
(Nj
MMWHHWHI-'HW
Nest
7! O O
ti
£i o
•-*
NJ o
00
Figure 3-1: Genetic relatedness among H. farinosus females (excluding the queen) in each nest
with 95% confidence intervals, calculated by Relatedness 5.0.8 (Queller and Goodknight, 1989).
80
0.25
c £ 0.2
£1
|o
I
*
_ T3
I
w
0.15
01I
0.1
= s
0.05
41
• Head width
• Wing length
oi aj
e E
§
Ol fl)8
it Q.
5 2 -0.05
BlOl
E401
fl
-0.1
Nest
Figure 3-2: Size differences (mm) between replacement queens and the average size of the
nests remaining workers. Only three nests (BlOl, E401, G408) had an identifiable replacement
queen who was present with other workers. The replacement queen was generally smaller than
the remaining workers.
81
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This is to advise you that as the representative of the Dean of Graduate Studies of York
and after having reviewed the text and verified the insertion of the last corrections
suggested by the members of the thesis committee during the thesis examination, I
authorize Naba Al Najjar to file her thesis entitled "LE VICE MONSTRUEUX, LE
MONSTRE VERTUEUX: une analyse narrative et sociale de Justine, ou les malheurs
de/a vertude Donatien Alphonse Frangois de Sade (1740-1814)".
I am forwarding this message to the two thesis supervisors as well as to the student,
whom I congratulate for this very impressive thesis.
Marie-Christine Pioffet