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SUPPLEMENTARY METHODS
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ESM M1 – Observation nests
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Semi-natural observation nests were constructed by sandwiching a 1 cm x 3 cm x 13 cm
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piece of ultra-light density balsa wood between 2 pieces of clear acrylic sheeting. A 9 cm long
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shallow groove was cut from one end of the wood to serve as an entrance and tunnel. The
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acrylic sheeting was then covered with opaque fabric or plastic, secured with binder clips, and
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hung under a plastic roof in the BCI forest (ESM F1).
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9
Each observation nest was seeded with a single newly emerged female, which we collected
from natural nests at the larval or pupal stage and reared in tissue culture trays in ambient
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conditions until the day after they eclosed. Upon introducing adult females to observation nests,
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we plugged the nest entrance with a piece of cotton for a few hours to prevent them from
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immediately flying away. After that initial adjustment period, however, these females could
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leave and enter the nest freely. Behaviour in these nests appeared similar to behaviour of bees in
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natural and modified natural nests.
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We censused observation nests every 4 days to record newly constructed cells, newly closed
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cells, newly emerged offspring, and the presence or absence of each adult. We could predict
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offspring emergence dates based on the date each cell was closed, and censused daily around this
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time in order to know precise emergence dates. During the next full census after a new bee
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emerged, we measured head width across the widest part of the eyes with callipers and used a
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white Decolor paint pen to mark the thorax with individually distinct patterns.
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One solitary female included in the analysis was collected just a few days prior to her first
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offspring emerging. We could be certain that this was a solitary nest, however, because she had
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laid only two eggs, both were males, the last of which was laid one month prior to collection.
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This female was thus clearly following the solitary trajectory of laying only males in the first
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brood, and was in the inter-brood phase of her life cycle. This is further described in [1].
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Female offspring that emerged from eggs laid in observation nests were assigned to the
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worker caste if they remained in their natal nest and foraged. Foraging behaviour was recorded
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under infrared light with a Sony miniDV camcorder positioned near the entrance of each nest
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during peak foraging times, approximately 90 minutes before sunrise and 60 minutes after sunset
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[2]. Workers were the daughters of their queens in all nests except one, in which the worker was
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unrelated [1].
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ESM M2 – New natural nests
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Dispersers were collected from newly initiated natural nests. We marked and hung sticks of
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similar size and density to those used for nesting by M. genalis throughout the BCI forest. We
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censused these sticks every 3 days to identify newly initiated nests. Three dispersers were
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collected from nests that were 6-9 d old, and 18 from nests that were 1-3 d old. All the
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dispersers had constructed an entrance collar, which is one of the first stages of nest initiation.
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Some had begun constructing and provisioning brood cells, but we included only those
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foundresses that had not yet oviposited (i.e. had no closed brood cells).
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ESM M3 – Socially isolated females
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Female pupae were collected from natural nests and reared in cages made out of clear plastic
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round containers (9.3 cm h x 8.5 cm d) with lids. Each bee had passed the larval feeding stage
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when they were introduced to our study, and do not eat during the pupal growth phase. The
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larval diets were likely to have varied among females [3]. Each cage had 8 small air holes poked
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in the lid and a feeding station on one side, which was a 1.8 ml microcentrifuge tube, with the
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bottom cut off and the open end packed with cotton guaze, and filled with a 50% solution of
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honey water (2008) or a 45% honey, 10% soy protein, 45% water solution (2009). Soy protein is
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known to contain adequate quantities of all amino acids required by honeybees [4], and amino
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acid requirements tend to be similar across species [5]. Soy protein has been used as a complete
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protein source in studies with other halictid bees [6, 7]. We began providing food for each
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female ad libitum upon eclosion. We replaced the cotton every 2 days to avoid bacterial growth.
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Each cage also had an artificial refuge, created by cutting the bottom portion off of another 1.8
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ml microcentrifuge tube, removing the lid, and attaching it to the cage floor. The cages were
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kept under ambient forest conditions in complete darkness (while still developing) or very dim
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light (after eclosion) to replicate light conditions experienced in natural nests. Head width was
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not significantly different between socially isolated females in each year (Z = -1.53, p = 0.13, n =
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141).
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ESM M4 – Collection protocol
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All collections were made at the same time of year, within a 5 week time period (4/26/2008 –
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5/31/2008 and 4/23/2009 – 5/24/2009) to minimize seasonal effects. Collections were also done
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at the same time each day (1330-1700) to minimize circadian effects and to ensure that all bees
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would be present in the nest. Nests entrances were plugged with cotton, placed in a dark box,
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and brought to a holding room with ambient temperature outside the laboratory between 1200-
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1230, where they were kept in the dark and allowed to acclimate for at least 1 hour. Socially
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isolated bees were treated the same way, but were transferred to the collection box in their cages.
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Nests/cages were taken one at a time from the holding box, taking care not to disturb the others.
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Bees in cages and nests were chilled at 4o C for 3 minutes and -18o C for 5 minutes, respectively,
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then immediately cross-pinned to a wax lined petri dish. Heads were removed and flash frozen
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in liquid nitrogen prior to pinning for a separate study. Hemolymph was collected under a
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dissecting stereoscope by puncturing the exoskeleton between the 4th and 5th tergites with a 1 μl
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capillary tube. Hemolymph was deposited in 10 μl of 1x protein buffer (Complete mini-tabs,
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Roche Applied Sciences, Bath, UK) and stored at -80o C. The metasoma (abdomen) was
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removed and stored in insect Ringer’s solution at 4o C.
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ESM M5 – Morphological and anatomical measurements
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Head width was measured as the distance between the most distal portion of each eye
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perpendicular to the frontal midline with callipers while bees remained frozen on dry ice after
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collection. All 2008 caged bee measurements were done by KMK and all 2009 bee
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measurements were done by Ting-Yan Chan.
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Metasoma were dissected within 36 hours (most within 24 hours) of collection to measure
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ovary development. Tergites were removed to expose the ovaries, and a digital photograph was
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taken through the microscope.
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ESM M6 – Vitellogenin assays
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Vitellogenin titers were assayed relative to β-galactosidase standard curves with SDS-PAGE,
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as in Nelson et al. [8]. Hemolymph in protein buffer (3-8 μl) was added to 10 μl of 2x loading
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buffer (0.065 M bis/Tris pH 6.8 with HCl, 20% sucrose, 2% SDS, 2% DTT, 0.01% bromophenol
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blue) and loaded into a 7.5% polyacrylamide gel with a 4% stacking gel (Bio-Rad, Hercules,
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CA) along with a dual colour size ladder (Precision Plus Protein Standards, Bio-Rad, Hercules,
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CA), and 3 or 4 lanes of known quantities of β-galactosidase protein in 10 μl of loading buffer.
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Gels ran in SDS running buffer (10% SDS, 30.3% Trizma Base, + 144.1% glycine) at 100-110V
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for approximately 90 – 180 minutes. Protein bands were stained with Coomassie Brilliant Blue,
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and protein concentration was quantified with Quantity One imaging software (Bio-Rad,
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Hercules, CA).
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Western blots were used to confirm Vg band identity (~180 kDa) against a Vg antibody for 8
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individuals from a separate study (ESM F2). Proteins were transferred to a nitrocellulose
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membrane after segregating with SDS-PAGE similar to that described above. Non-specific
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protein binding was blocked using a 3% solution of instant non-fat milk in 1X PBST and
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incubated overnight at 4C. Vg antibody (1:25,000 in blocking solution) was then hybridized to
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the membrane. The membranes were washed in 1X PBST, and membrane-bound antigen-
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antibody complexes were then visualized with horseradish peroxidase-conjugated goat anti-
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rabbit IgG (GE healthcare) at a dilution of 1:1,000 and developed with Western Lightning
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Chemiluminescence reagent (PerkinElmer) on a Versa-Doc imaging system (Bio-Rad, Hercules,
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CA).
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SUPPLEMENTARY DISCUSSION
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ESM D1 – Queens and their solitary counterparts
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The best classifiers of social phenotype were body size and a size-scaled ovarian index.
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Smith et al. [9] also found significant differences in ovary size between queens of social nests
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and solitary reproductive females. Differences in ovarian traits may or may not have existed
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prior to the onset of egg-laying and offspring emergence, and our data cannot distinguish
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between cause and consequence of the social strategies. Increased ovary activation may be the
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result of socially-mediated endocrine activity in social nests, where competition for dominance
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and reproductive opportunity is expected to be high [10-13]. Aggression from queens to workers
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is common in social nests [14], and M. genalis queens have significantly higher JH titers than
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solitary females (unpublished data). The maintenance of dominance behaviour triggers
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morphological changes in the development of brain regions associated with sensory integration
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and learning [15], and social dominance could trigger other changes as well. However, the
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increase in ovary development may also be a consequence of behavioural specialization. Social
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nests were likely to produce more offspring broods, which increases the probability of collecting
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a queen when she has a fully developed oocyte [1].
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Head width was also a significant predictor of a foundress’ social trajectory, whereby solitary
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females were significantly smaller than queens. Body size as the basis of alternative
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reproductive strategies, independent of fecundity, has also been suggested for other halictid bees
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[16, 17]. Variation in body size among nest foundresses may influence social strategy if it
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reflects relative ability of queens to manipulate daughters into working, either through post-
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emergence aggression or differential provisioning. Trophallaxis is often accompanied by
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aggressive solicitation from queens to workers [14], so it is possible that small queens may not
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be able to obtain food from workers. A small queen could theoretically overcome this problem
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by making an even smaller worker, but minimum size constraints may limit this option in at least
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two ways. Females and female larval provisions are significantly larger than males and male
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provisions [3], which indicates a threshold of larval food below which laying a male egg is a
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more optimal strategy. Secondly, smaller females have smaller compound eyes and smaller
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facets (ommatidia) [2], which would decrease photon capture [18], and hence more severely
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constrain foraging under dim light conditions for small females relative to larger ones [19].
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In contrast with our findings, a previous study of nests with unknown histories, which likely
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included foundresses born at different times of year, found that queens and solitary females were
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the same size [13]. It is possible that seasonal variation in body size, variation in nest quality, or
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contingent events like the death of a worker may have provided too much noise to detect a
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difference between solitary and social foundresses. It is unlikely that our study is biased by
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small foundresses who would not have initiated their own nest in a natural setting because the
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distribution of head widths, ovary development, and reproductive behaviour observed in our
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observation nests mirrors natural nests [1, 9, 13, 20, 21].
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ESM T1 Mean values (± sd) of morphological and physiological measurements among natural
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dispersers, workers, queens, solitary reproductives, and socially isolated females.
head width (mm)
4.39 ± 0.38
n = 21
5.41 ± 1.02
n = 21
1.23 ± .20
n = 21
0.29 ± 0.24
n = 20
workers
3.77 ± 0.22
n = 23
2.33 ± 1.08
n = 23
0.61 ± 0.26
n = 23
0.25 ± 0.32
n = 21
queens
4.08 ± 0.33
n = 23
7.51 ± 1.90
n = 23
1.86 ± 0.51
n = 23
0.52 ± 0.43
n = 22
solitary
females
3.77 ± 0.16
n = 15
5.00 ± 1.35
n = 15
1.33 ± 0.35
n = 15
0.32 ± 0.27
n = 14
3.99 ± 0.40
n = 48
4.10 ± 0.40
n = 93
1.56 ± 0.56
n = 12
2.31 ± 0.90
n = 92
0.39 ± 0.11
n = 12
0.56 ± 0.19
n = 90
0.05 ± 0.07
n = 50
0.05 ± 0.08
n = 76
2009
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Vg titer
(μg/μl)
dispersers
isolated
females
2008
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ovary size
(mm2)
size-scaled
ovarian index
(ovary / head)
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ESM F 1 Observation nests used to study queens, solitary females, and workers. (a) Each
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nest was constructed with a standardized piece of balsa wood. (b) The wood was fixed
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between two pieces of transparent acrylic, covered with opaque fabric, and hung under a
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plastic roof in the BCI forest. (c) New foundresses build a sawdust collar just inside the nest
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entrance, and this is easily recognizable as a sign of new nest activity.
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ESM F 2 SDS–PAGE gel with Coomassie brilliant blue staining (a) and Western blot (b)
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with the Vg antibody sampled from M. genalis. m: dual colour molecular mass marker (Bio-
158
Rad); c: cage-reared isolated females; q: queens from observation nests; w: workers from
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observation nests; d: dispersers from natural nests; β- galactosidase standards not pictured.
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The marker in lane 1 of (b) is MagicMark XP Western Standard (Invitrogen) . Arrows
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indicate Vg.
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REFERENCES
1
Kapheim, K. M. Maternal effects and the evolution of sociality in a facultatively eusocial
165
sweat bee, Megalopta genalis [Ph.D. dissertation]. Los Angeles: University of California;
166
2010.
167
2
Kelber, A., Warrant E. J., Pfaff M., Wallen R., Theobald J. C., Wcislo W. T., et al. 2006
168
Light intensity limits foraging activity in nocturnal and crepuscular bees. Behav Ecol 17, 63-
169
72.
170
3
Kapheim, K., Bernal S., Smith A., Nonacs P., Wcislo W. 2011 Support for maternal
171
manipulation of developmental nutrition in a facultatively eusocial bee, Megalopta genalis
172
(Halictidae). Behav Ecol Sociobiol 65, 1179-1190.
173
4
174
175
physiology and the toxicity of plant proteinase inhibitors. J Insect Physiol 34, 1111-1117.
5
176
177
6
182
Roulston, T. H., Cane J. H. 2000 The effect of diet breadth and nesting ecology on body size
variation in bees (Apiformes). J Kansas Entomol Soc 73, 129-142.
7
180
181
De Groot, A. P. 1953 Protein and amino acid requirements of the honeybee (Apis mellifica
L.) Physiologica Comp. Oecol. 3, 197-285.
178
179
Broadway, R. M., Duffey S. S. 1988 The effect of plant protein quality on insect digestive
Roulston, T. H., Cane J. H. 2002 The effect of pollen protein concentration on body size in
the sweat bee Lasioglossum zephyrum (Hymenoptera : Apiformes). Evol Ecol 16, 49-65.
8
Nelson, C. M., Ihle K. E., Fondrk M. K., Page R. E., Amdam G. V. 2007 The gene
vitellogenin has multiple coordinating effects on social organization. PLoS Biology 5, e62.
183
9
Smith, A., Wcislo W., O’Donnell S. 2008 Body size shapes caste expression, and
184
cleptoparasitism reduces body size in the facultatively eusocial bees Megalopta
185
(Hymenoptera: Halictidae). J Insect Behav 21, 394-406.
186
10 Röseler, P.-F., Röseler I., Strambi A. 1985 Role of ovaries and ecdysteroids in dominance
187
hierarchy establishment among foundresses of the primitively social wasp, Polistes gallicus.
188
Behav Ecol Sociobiol 18, 9-13.
189
11 Röseler, P.-F., Röseler I., Strambi A., Augier R. 1984 Influence of insect hormones on the
190
establishment of dominance hierarchies among foundresses of the paper wasp, Polistes
191
gallicus. Behav Ecol Sociobiol 15, 133-142.
192
12 Bloch, G., Shpigler H., Wheeler D. E., Robinson G. E. 2002 Endocrine influences on the
193
organization of insect societies. In Hormones, Brain and Behavior (ed. D. Pfaff), pp. 195-
194
235. San Diego: Academic Press.
195
13 Smith, A. R., Kapheim K. M., O’Donnell S., Wcislo W. T. 2009 Social competition but not
196
subfertility leads to a division of labour in the facultatively social sweat bee Megalopta
197
genalis (Hymenoptera: Halictidae). Anim Behav 78, 1043-1050.
198
14 Wcislo, W. T., Gonzalez V. H. 2006 Social and ecological contexts of trophallaxis in
199
facultatively social sweat bees, Megalopta genalis and M. ecuadoria (Hymenoptera,
200
Halictidae). Insectes Sociaux 53, 220-225.
201
15 Smith, A. R., Seid M. A., Jiménez L. C., Wcislo W. T. 2010 Socially induced brain
202
development in a facultatively eusocial sweat bee Megalopta genalis (Halictidae). Proc Roy
203
Soc B-Biol Sci 277, 2157-2163.
204
16 Zobel, M. U., Paxton R. J. 2007 Is big the best? Queen size, usurpation and nest closure in a
205
primitively eusocial sweat bee (Lasioglossum malachurum). Behav Ecol Sociobiol 61, 435-
206
447.
207
208
209
210
211
212
213
17 Packer, L., Jessome V., Lockerbie C., Sampson B. 1989 The phenology and social biology of
four sweat bees in a marginal environment - Cape Breton Island. Can J Zool 67, 2871-2877.
18 Theobald, J. C., Greiner B., Wcislo W. T., Warrant E. J. 2006 Visual summation in nightflying sweat bees: a theoretical study. Vison Res 46, 2298-2309.
19 Theobald, J. C., Coates M. M., Wcislo W. T., Warrant E. J. 2007 Flight performance in
night-flying sweat bees suffers at low light levels. J Exp Biol 210, 4034-4042.
20 Smith, A. R., Wcislo W. T., O'Donnell S. 2007 Survival and productivity benefits to social
214
nesting in the sweat bee Megalopta genalis (Hymenoptera : Halictidae). Behav Ecol
215
Sociobiol 61, 1111-1120.
216
21 Wcislo, W. T., Arneson L., Roesch K., Gonzalez V., Smith A., Fernandez H. 2004 The
217
evolution of nocturnal behaviour in sweat bees, Megalopta genalis and M. ecuadoria
218
(Hymenoptera : Halictidae): An escape from competitors and enemies? Biol J Linn Soc 83,
219
377-387.