Abstract Kin selection theory is foundational in helping to explain the evolution of sociality; however, the degree to which indirect fitness benefits may underlie helping behavior in species of early stage sociality has received relatively little empirical attention. Facultatively social bees, which demonstrate multiple forms of social organization, provide prime systems in which to empirically test hypotheses regarding the evolutionary origins of sociality. The subsocial small carpenter bee, Ceratina calcarata, may establish a social nest by manipulating brood provisions to rear a worker daughter, which then assists in critical late-season alloparental care. Here, we combine nest demographic and behavioral data with genetic relatedness estimates to calculate the relative inclusive fitness of both subsocial and social reproductive strategies in C. calcarata. Social mothers benefit from improved likelihood of brood survivorship and have higher fitness than subsocial mothers. Worker daughters have low indirect fitness on average, and will not produce their own offspring. Among-sibling relatedness is significantly higher in social nests than subsocial nests, though mothers of either reproductive strategy may mate multiply. Though this study corroborates the ultimate role of indirect fitness and assured fitness returns in the evolution of social traits, it also offers additional support for maternal manipulation as the proximate mechanism underlying evolutionary transitions in early stage insect societies. INTRODUCTION Eusociality is one of the most complex forms of social organization in nature (Wilson 1971). Although eusocial organisms are represented by a diverse suite of taxa (e.g., naked mole rats, Jarvis 1981; thrips, Crespi 1992; shrimp, Duffy 1996; termites, Thorne 1997), Hymenoptera collectively contain more eusocial species than any other group (Wilson 1971). Obligately eusocial bees (e.g., Apis mellifera) demonstrate complex reproductive division of labor. Each individual’s role within the colony is irreversibly determined during development, and a reproductive queen’s lifetime fitness depends on the collective effort of thousands of sterile workers (Wilson 1971; Wilson and Hölldobler 2005; Michener 2007). Despite their established ecological dominance, however, eusocial bees represent relatively few species. Most of the more than 20,000 bee species worldwide are solitary (Michener 2007) and the remainder demonstrate forms of noneusocial organization (Rehan and Toth 2015). Solitary nesting is ancestral in bees, but evidence suggests that lineages may undergo continuous evolutionary gains or losses in their social complexity (Szathmáry and Smith 1995; Danforth 2002; Rehan and Toth 2015). The evolutionary origins of an obligate and sterile worker caste thus appear paradoxical: why would an individual sacrifice its direct fitness to assist in rearing another’s offspring? Further, how might such a seemingly altruistic behavioral phenotype be selectively reinforced? Inclusive fitness theory suggests that indirect fitness benefits to the altruist may be enough to account for the origin and elaboration of the advanced eusocial worker caste (Hamilton 1964; West-Eberhard 1975; Trivers and Hare 1976; Foster et al. 2006). As formalized by Hamilton (1964), if an altruist’s helping behavior were to contribute to the direct fitness of close genetic relatives, its indirect fitness gains could outweigh the incurred costs of forgoing some or all of its own reproduction. Kin selection is thus considered a plausible explanation for the evolution of sociality in Hymenoptera, in which female siblings are expected to share significantly more of their genetic identity with each other compared to their mother or brothers (Hamilton 1972; Lin and Michener 1972). Though the multiple mating of advanced eusocial species appears to confound these expectations by reducing among-sibling relatedness (Palmer and Oldroyd 2000), monandry is thought to be ancestral to Hymenoptera and suggests indirect fitness could have facilitated the emergence of early stage social traits (Hughes et al. 2008). The biological applicability of kin selection has been the subject of heated debate within the field of social evolution (West-Eberhard 1975; Wilson 2005; Gadagkar 2010; Nowak et al. 2010; Marshall 2011). While the maintenance and elaboration of social traits by inclusive fitness remains a widely accepted theory (Foster et al. 2006; Hughes et al. 2008; Bourke 2011; Quiñones and Pen 2017), relevant examinations within early stage social systems remain limited (Leadbeater et al. 2011; Rehan, Richards, et al. 2014; Kapheim et al. 2015). Similarly, though many insights into the evolution of sociality have been gained through the study of advanced eusocial taxa, reproductive divisions of labor are often obligate in such species, and provide only inferential insights regarding their evolutionary origins (Queller and Strassmann 1998; Wilson and Hölldobler 2005; Toth et al. 2007). Facultatively social species, by contrast, demonstrate a capacity for multiple degrees of social organization, and exhibit at least 2 distinct nesting phenotypes (e.g., solitary and eusocial nesting). Accordingly, such species provide a unique opportunity to empirically investigate whether theoretically predicted mechanisms of evolution have a biologically realized influence on gains or losses in social complexity across the social spectrum (Rehan and Toth 2015; Shell and Rehan 2017a; Toth and Rehan 2017; Quiñones and Pen 2017). Divisions of labor among Hymenoptera are frequently based on differences in age or body size. For instance, larger and older daughters in Polistes paper wasp colonies are more likely to assert reproductive dominance than their smaller or younger siblings if given a viable opportunity to do so (Hughes and Strassmann 1988; reviewed in Jandt et al. 2014). Among facultatively eusocial bees, foundresses are the eldest in the family unit, and often able to manipulate the sex, size, and behavior of their offspring through selective fertilization, provision investment, and physical coercion, respectively (Sakagami and Maeta 1984; Yanega 1989; Aneson and Wcislo 2003; Smith et al. 2003; Rehan and Richards 2010b; Kapheim et al. 2011, 2015). In this way, mothers maximize their reproductive investment while minimizing potential conflict or competition from their offspring. Maternal manipulation of brood has consequently been proposed as a proximate mechanism of early divisions of labor across multiple lineages (Alexander 1974; Craig 1979; Ratnieks and Wenseleers 2008), and evidence shows that, in combination with high intracolonial relatedness, it likely reinforces early stage social organization (Crespi and Ragsdale 2000; Richards et al. 2005; Kapheim et al. 2015). Cost-benefit analyses are an effective means of evaluating different life histories (Hamilton 1964; Trivers 1971) and have been applied in facultatively social bees to compare the relative fitness of alternative reproductive strategies (Augusto and Garófalo 2004; Pech et al. 2008; Rehan, Richards, et al. 2014; Kapheim et al. 2015). Where brood loss to predation or parasitism may represent a significant natural problem for solitary nests, social nesting may provide a means of increasing fitness through improved resistance to these pressures (Rehan et al. 2011; Yagi and Hasegawa 2012; Rehan, Richards, et al. 2014). For example, social nests of the facultatively eusocial sweat bee, Lasioglossum baleicum, were better able to preserve developing larvae compared to solitary nests when under pressure from ant predation (Yagi and Hasegawa 2012). Consequently, higher fitness for social reproductive females and their sterile first-brood workers compared to solitary conspecifics supported the role of inclusive fitness in maintaining social traits (Yagi and Hasegawa 2012). Inclusive fitness benefits also likely contribute to the maintenance of division of labor in facultatively social orchid bees (Pech et al. 2008). However, persistent agonistic interactions among siblings or between a mother and her brood indicate that social behavior may be prompted by physical aggression rather than sibling relatedness in this group (Augusto and Garófalo 2004; Pech et al. 2008). Taken together, studies suggest that while ecological pressures and genetic identity among family groups likely help to maintain and even reinforce social traits through inclusive fitness, 1) social nesting may not be an advantageous strategy for all individuals involved; and 2) physical and/or aggressive interactions among nestmates may be required to elicit and maintain sib-social care behaviors (Ratnieks and Wenseleers 2008). Cost-benefit analyses of facultatively social species are few, yet critical to understanding the underlying mechanisms for social organization in early stage societies. The small carpenter bee, Ceratina calcarata demonstrates a form of facultative incipient sociality across its range in eastern North America (Rehan and Sheffield 2011; Shell and Rehan 2016a) where it produces one brood per year (Johnson 1988; Rehan and Richards 2010b). All reproductively active female C. calcarata nest subsocially by providing extended parental care for their maturing brood. Some mothers, however, establish social nests by producing a worker daughter to assist with late-season brood feeding and defense (Figure 1; Rehan, Berens et al. 2014; Lawson et al. 2016). Maternal manipulation is thought to play an important role in C. calcarata’s nesting biology (Rehan and Richards 2013). Specifically, a reproductive female can choose whether to fertilize her eggs and, with carefully controlled pollen provisioning, can determine both the sex and body size of her developing brood (Rehan, Richards, et al. 2014; Lawson et al. 2016). Socially nesting mothers initiate their nest by provisioning a fertilized egg with a relatively small amount of pollen (Lawson et al. 2016), which subsequently develops into a dwarf eldest daughter (Rehan and Richards 2010b). As differences in body size govern dominance hierarchies in C. calcarata, this particular daughter’s small stature at adulthood is thought to facilitate her mother’s ability to coerce her into a worker role (Rehan and Richards 2013; Rehan, Berens, et al. 2014; Withee and Rehan 2016). Figure 1 View largeDownload slide Annual life cycle and reproductive strategies of Ceratina calcarata. In late spring, each female disperses, mates, and establishes a new nest either subsocially (light gray) or socially (dark gray). During the late summer adult brood feeding period, social mothers and/or their worker daughters forage. A worker daughter may save her orphaned nest from failure in the event of maternal mortality. Sufficiently fed adult brood of either nest type survive a lengthy winter hibernation to disperse the following spring. Figure 1 View largeDownload slide Annual life cycle and reproductive strategies of Ceratina calcarata. In late spring, each female disperses, mates, and establishes a new nest either subsocially (light gray) or socially (dark gray). During the late summer adult brood feeding period, social mothers and/or their worker daughters forage. A worker daughter may save her orphaned nest from failure in the event of maternal mortality. Sufficiently fed adult brood of either nest type survive a lengthy winter hibernation to disperse the following spring. Adult C. calcarata offspring must be fed in late summer to ensure their overwintering survival (Durant et al. 2016; Lewis and Richards 2017; Mikát et al. 2017). It is during this late summer feeding that the worker daughter may contribute to her siblings’ survivorship by acting as a secondary forager: either gathering pollen and nectar alongside her mother, or acting as the sole forager in orphaned nests (Figure 1; Rehan, Berens, et al. 2014). Outside of its social context, the worker daughter’s diminutive size effectively negates her chances of surviving the winter season to reproduce in the following year (Rehan and Richards 2010b; Mikát et al. 2017). Thus, though the decision to establish a brood with a dwarf daughter may initially appear to represent a needlessly high maternal cost, worker daughters are thought to represent an investment in late season brood insurance for some social mothers (Mikát et al. 2017). By contrast, though subsocial mothers rear only reproductively viable brood, their nests may fail when left without feeding services in the likely event of late-season maternal mortality. C. calcarata nests thus provide a natural system in which to empirically assess the inclusive fitness of an incipient form of social nesting. In this study, we estimate genetic relatedness within and between sympatric subsocial and social C. calcarata colonies. Next, we assess the potential influences of mate frequency and maternal body size on social phenotype. We then combine genetic and nest demographic data to calculate the relative costs and benefits of subsocial and social reproduction in a species capable of both reproductive strategies in sympatry. METHODS Nest collections and assessment A total of 167 C. calcarata nests were collected from Durham, New Hampshire during the 2014 through 2016 summer field seasons. Ceratina nests were identified in the field using burrow entrance holes in dead broken stems of staghorn sumac (Rhus typhina) and berry brambles (Rubus spp.). Nests were gathered at dawn, while adults were still dormant, as this ensured collection of foraging individuals (i.e., mother and worker daughter) and prevented offspring escape. Nests were refrigerated to sedate individuals, and were then dissected lengthwise to reveal nest architecture and brood composition. Total brood cells, pollen provisions, brood parasitism, and mortality were recorded, along with the developmental stage, brood cell position, and sex of each offspring (assessable at the pupal developmental stage or later); mothers were then measured for overall body size using head width as an accurate proxy (Rehan and Richards 2010b). As worker daughters are reared in the first brood cell, social nests were defined as those which contained a female offspring in the first brood cell; and subsocial nests were defined as those with a male offspring in the first position (Johnson 1988; Rehan and Richards 2010b; Lawson et al. 2016; Lewis and Richards 2017). Sex ratio (male and female brood count), social category (subsocial vs. social), total clutch size (all provisioned brood cells), live brood (offspring alive at time of measurement), and maternal body size were assessed in all nests for which relevant data was available. DNA extraction, amplification, and allelic profiles Nests used for genotyping were collected during the full brood stage, wherein the reproductive female has finished laying eggs and has assumed a brood guarding and cleaning role. Gathering nests at this stage thus ensures that both the reproductive female and her complete brood are collected. Twenty-seven full brood nests of mixed-sex brood, containing 257 individuals in total, were selected for DNA extraction and genotyping. Of the 27 nests, 19 were social and the remaining 8 were subsocial. A modified Phenol-Chloroform Isolation protocol (Kirby 1956) was used to extract DNA from the abdomen and 3 legs of each adult, and from the full body of each late-stage pupa. Each individual was then screened at 8 polymorphic microsatellite loci (Shell and Rehan 2016b) using the fluorescent M13-tail methodology described in Schuelke (2000). PCR reactions were mixed to a volume of 11 μL as follows: 5.45 μL ddiH20; 2.0 μL 5× HF Buffer (Thermo Scientific); 0.2 μL [10 mM] dNTPs; 0.1 μL Phusion HF Taq Polymerase (Thermo Scientific); 0.25 μL [10 mM] forward primer; 0.5 μL Fluorescent M13 oligo [10 mM], 0.5μL [10 mM] reverse primer; 2.0 μL DNA template. Thermocycler programs were run following primer annealing specifications from Shell and Rehan (2016b). After amplification was confirmed via gel electrophoresis (1% agarose gel) PCR product was mixed with Hi-Di Formamide (Applied Biosystems, Foster City, CA) and submitted to the DNA Analysis Facility at Yale University for fragment analysis on a 3730xl Analyzer (Applied Biosystems). Individual allelic profiles were called via manual inspection of peaks in Peak Scanner 2 (Applied Biosystems). Intra- and intercolony maximum likelihood relatedness scores between mothers and their offspring, as well as among all siblings and non-nestmates, were then assessed using ML-Relate (Kalinowski et al. 2006). Pairwise statistical tests of relatedness were then performed using one randomly sampled female per nest to account for variation in brood counts among nests (Rehan, Richards, et al. 2014). Variation among female sibling allelic profiles also allowed for assignment of an estimated sire count for each genotyped nest (as in Richards et al. 2005). Statistical analyses of demography and relatedness were then performed in JMP Pro 13 (SAS Institute Inc., Cary, NC). Fitness calculations Mature brood which are not fed before the end of the blooming season do not survive the winter (Durant et al. 2016), and orphaned nests with no additional foraging activity will fail (Lewis and Richards 2017). Foraging activity by a mother or worker daughter indicates successful brood feeding and can therefore be used as a proxy for expected brood overwintering survivorship. Empirical frequencies of nest orphanage, and foraging activity by mothers and worker daughters, were made available by a comprehensive study of foraging behavior (Mikát et al. 2017). These values were used to calculate average expected survivorship for subsocial and social foundresses, which were then combined with relatedness and demography datasets to calculate the relative fitness of subsocial and social nesting strategies. On average, C. calcarata nests have a 70% chance of being orphaned during the mature brood feeding stage (Mikát et al. 2017). Therefore, likelihood of survivorship in a subsocial nest was calculated as one minus the average probability of a mother dying or abandoning her brood (Ssub=1-0.70). A worker daughter was observed to adopt foraging responsibilities in 18% of orphaned social nests (Mikát et al. 2017). Thus, likelihood of survivorship in social nests was calculated as one minus the probability that a nest is orphaned and not then fed by a worker daughter (Ssoc=1-(0.70*0.82)). Average subsocial and social maternal fitness values were then calculated as a mother’s relatedness to her offspring (rm) multiplied by the average number of live brood for her nesting type (subsocial=Nsub;social=Nsoc), then multiplied by the probability of offspring survival for that nesting strategy ( Ssub or Ssoc). The average live brood count for social mothers was penalized by 1 to represent the cost of rearing a nonreproductive daughter: Equation 1. Maternal inclusive fitness (IF): subsocial IFsub=[(rm)*(Nsub)]*(Ssub) Equation 2. Maternal inclusive fitness (IF): social IFsoc=[(rm)*(Nsoc-1)]*(Ssoc) A worker daughter is active in 29% of social nests, either foraging alongside her mother (11%) or operating as the sole remaining forager in an orphaned nest (18%) (worker daughters do little to no foraging in the remaining social nests, Mikát et al. 2017). Mothers that survive to the end of the blooming season are expected to be able to provide sufficient late-season feeding for their brood’s overwintering survival. Interestingly, in the 11% of cases where a worker daughter aids her mother in foraging, both individuals forage at half the rate of a lone forager (Mikát et al. 2017). As such, a worker daughter may be expected to receive a halved return on her total potential indirect fitness when she aids her mother in a nonorphaned nest (wDcoop=0.30*0.11), and her full indirect fitness in the 18% of cases where she assumes the role of sole forager for her siblings in the event of nest orphanage (wDsolo=0.70*0.18). The average inclusive fitness for the worker daughter (IFwD) is thus calculated as 1) her total expected average indirect fitness (IndFwD) multiplied by the likelihood of her contributing as the sole remaining forager (wDsolo), plus 2) half her expected average indirect fitness (IndFwD*0.5), multiplied by the likelihood of her foraging alongside her mother (wDcoop). Equation 3. Worker daughter inclusive fitness (IFwD) IFwD=[(IndFwD)*(wDsolo)]+[(IndFwD*0.5)*(wDcoop)] RESULTS Demography Genotyped nests ranged in clutch size from 4 to 15 individuals, with female offspring comprising between 18 and 89% of the total brood. In the population-wide dataset of 167 nests there were 122 social and 45 subsocial nests. Social nests contained significantly larger clutch sizes on average (t-test, t = 2.10, df = 80, P = 0.04), however, the percentage of live brood was not significantly different between nest types (t = −0.068, df = 71, P = 0.95; Figure 2), nor was the frequency of brood parasitism (social = 0.11 ± 0.54; subsocial = 0.27 ± 1.03; t = −1.29, df = 53, P = 0.33). Brood sex ratios were significantly more female biased in social than subsocial nests (mean female %Social = 57 ± 0.02; %subsocial = 36 ± 0.038; t = −4.47, df = 31, P < 0.0001). There was a significant positive correlation between maternal body size and clutch size in social nests (social; F1, 95 = 3.93, r2 = 0.04, P = 0.05), but not for subsocial nests (subsocial; F1, 28 = 0.001, r2 < 0.001, P = 0.99). Similarly, a significant positive correlation between maternal body size and percent female sex investment was found across social nests (F1, 94 = 5.3492, r2 = 0.05, P = 0.02), but not subsocial nests (F1, 28 = 0.0222, r2 < 0.001, P = 0.88). The frequency of social nest formation was found to increase with maternal body size (Logistic regression, χ2=4.04, df = 1; P = 0.04) such that for each 0.1 mm increase in maternal head width the likelihood of forming a social nest increased by 4%. Figure 2 View largeDownload slide Mean clutch size and live brood in social and subsocial nests of Ceratina calcarata. Though social mothers produce significantly more brood (t = 2.10, df = 80, P = 0.04), average brood survivorship to adulthood is nearly identical for social and subsocial nest types (t = −0.068, df = 71, P = 0.95). Figure 2 View largeDownload slide Mean clutch size and live brood in social and subsocial nests of Ceratina calcarata. Though social mothers produce significantly more brood (t = 2.10, df = 80, P = 0.04), average brood survivorship to adulthood is nearly identical for social and subsocial nest types (t = −0.068, df = 71, P = 0.95). Relatedness and inclusive fitness estimates Average intercolony relatedness was low (R = 0.07), though average relatedness between mothers and their brood was identical in both social and subsocial nests (R = 0.55; t-test, t = 0.08 df = 10, P = 0.94; Supplementary Table S1). Average relatedness among female siblings in social nests (R = 0.70), however, was significantly higher than subsocial nests (R = 0.48) (t = −3.11, df = 18, P = 0.0061). Within social nests, average worker daughter relatedness to sisters (R = 0.698) was not significantly different from the relatedness among nonworker sisters (R = 0.701) (t = −0.045, df = 34, P = 0.96). Across all genotyped nests, relatedness values between female offspring and their male siblings was 0.25. Genotyping colonies also revealed that 22 of the 27 nests were singly sired. The relatedness among female siblings of singly mated nests (R = 0.71) was significantly higher than multiply mated nests (R = 0.47) (t = −4.158; df = 5.34; P = 0.008). Intracolony allelic profiles indicate that 16% (3/19) of social nest mothers and 25% (2/8) of subsocial nest mothers were multiply mated, though the frequency of multiple mating was not significantly different between nest types (Fisher’s Exact Test, P = 0.62; Supplementary Table S1). Though social mothers provisioned larger clutches than subsocial on average, accounting for the rearing of a nonreproductive worker daughter resulted in similar direct fitness values for both reproductive strategies (subsocial = 4.29, social = 4.51; Wilcoxon, ÷2 = 0.2218, df = 1, P = 0.64). Higher expected offspring survivorship in social colonies (Ssoc=0.426) compared to subsocial nests (Ssub=0.30) resulted in higher inclusive fitness for social mothers (IFsoc = 1.92; vs. IFsub = 1.29). Though worker daughters do not produce offspring, their average indirect fitness was high (IndFwD=4.06). The likelihood that a worker daughter becomes solely responsible for the survival of the brood, however, is relatively low (wDsolo=0.126). Further, her indirect fitness is halved in the few cases where she forages alongside her mother (wDcoop = 0.033; Equation 3). This low overall probability of indirect fitness gains through helping, combined with her lack of direct fitness, results in low average inclusive fitness for worker daughters (IFwD=0.58). Comparing fitness values calculated for the 3 reproductive strategies across the total assessed population, social mothers have significantly higher fitness than subsocial mothers, and subsocial mothers significantly higher fitness than worker daughters on average (Kruskal–Wallis, df = 2, ÷2 = 154.53, P < 0.0001; Figure 3). Figure 3 View largeDownload slide Histogram and box plots of population-wide distribution of estimated inclusive fitness values for social mothers (dark gray; N = 122), subsocial mothers (light gray; N = 45), and worker daughters (white; N = 122). Average inclusive fitness values for worker daughters (A), subsocial mothers (B), and social mothers (C) (Kruskall–Wallis, df = 2, χ2 = 154.53, P < 0.0001; Wilcoxon for Each Pair, social mother versus subsocial mother P < 0.0001, social mother versus worker daughter P < 0.0001, subsocial mother versus worker daughter P < 0.0001). All values were calculated using nest demography data and imputed relatedness scores. Figure 3 View largeDownload slide Histogram and box plots of population-wide distribution of estimated inclusive fitness values for social mothers (dark gray; N = 122), subsocial mothers (light gray; N = 45), and worker daughters (white; N = 122). Average inclusive fitness values for worker daughters (A), subsocial mothers (B), and social mothers (C) (Kruskall–Wallis, df = 2, χ2 = 154.53, P < 0.0001; Wilcoxon for Each Pair, social mother versus subsocial mother P < 0.0001, social mother versus worker daughter P < 0.0001, subsocial mother versus worker daughter P < 0.0001). All values were calculated using nest demography data and imputed relatedness scores. DISCUSSION C. calcarata demonstrates a relatively simple form of facultative social organization, and one that is characteristic of bees in the earliest stages of social evolution (Rehan and Richards 2010b; Rehan, Berens, et al. 2014; Rehan and Toth 2015). This study is the first empirical assessment of inter- and intracolony relatedness in C. calcarata, and a cost-benefit analysis of a bee representative of both subsocial and incipient social reproductive strategies, in which a single worker daughter may be produced. Demographic analyses confirmed positive relationships between maternal body size and brood composition (Johnson 1988; Rehan and Richards 2010b; Rehan, Berens, et al. 2014), and revealed that socially nesting females provision larger clutch sizes and invest in a significantly greater proportion of female offspring (Figure 2). As the probability of social nesting increased with female body size, this study provides further support for the role of maternal body size in determining division of labor in C. calcarata (Rehan and Richards 2010b; Withee and Rehan 2016), other Ceratina small carpenter bees (e.g., C. japonica, Sakagami and Maeta 1984), and among facultatively social bees more broadly (e.g., Halictus rubicundus, Field et al. 2012; Megalopta genalis, Kapheim et al. 2011). Screening at 8 polymorphic microsatellite loci revealed higher average intracolony relatedness among female siblings in social nests compared to subsocial, and indicates that C. calcarata is capable of limited polyandrous mating. Inclusive fitness calculations reveal that while social nesting is advantageous in C. calcarata, worker daughters receive few fitness benefits for their role. Social nests are initiated though careful control of pollen provisions and sib-social care is likely enforced through differences in body size. C. calcarata provides a clear example of how an early division of labor may be initiated by maternal manipulation and consequently maintained through maternal inclusive fitness benefits. Variations in C. calcarata relatedness and implications of multiple mating Microsatellite screening revealed no difference in maternal relatedness to brood between social and subsocial nests. The higher average relatedness among social female siblings compared to subsocial, however, represents a notable asymmetry, and one which has been detected in other facultatively social bees (e.g., Ceratina australensis, Rehan, Richards, et al. 2014; and Megalopta genalis, Kapheim et al. 2015). High intracolony relatedness, particularly within female-biased broods, is thought to play an important role in the emergence of social organization (Trivers and Hare 1976; Hughes et al. 2008). However, the degree to which social nesting may be heritable or influenced by individual condition remains an aim for further investigation (Hamilton 1964; Crozier and Pamilo 1996). Colony relatedness data additionally indicate C. calcarata females are primarily singly mated (22/27 mothers), with a capacity for occasional multiple mating with 2 or more males (5/27 mothers). Most species of bees are thought to be monandrous (Strassmann 2001; Wilson 2005), though many demonstrate a facultative capacity for polyandry (e.g., Bombus species with 2 to 4 sires, Estoup et al. 1995; Lasioglossum malachurum reporting up to 3 sires, Paxton et al. 2002). Polyandry is taken to an extreme degree in some advanced eusocial bees (e.g., Apis mellifera with over 17 sires on average, Laidlaw and Page 1984), but may be disadvantageous in non-Apis bees, in which intermediate levels of colony genetic heterogeneity may incur a fitness cost. For instance, Bombus terrestris females mated to 2 males had reduced reproductive output compared to females mated to either 1 or 4 males (Baer and Schmid-Hempel 2001). Limited polyandry among social nesting C. calcarata suggests that strict monandry may not be necessary for the persistence of early social traits. Additional studies in C. calcarata and other early stage social bees are needed to better understand the degree to which mating frequency may affect the emergence and maintenance of social phenotypes. The costs and benefits of social and subsocial nesting in C. calcarata According to kin selection theory, genes underlying social care behavior are more likely to be passed on when an altruist’s gains in indirect fitness outweigh its direct fitness losses incurred by forgoing reproduction (Hamilton 1964). The low average inclusive fitness of worker daughters compared to both social and subsocial mothers thus seems to contradict theoretical expectation (Figure 3). Although a worker daughter’s foraging behavior is comparable to that of her mother (Mikát et al. 2017), surviving late-season mothers can sufficiently feed their mature brood alone (Lewis and Richards 2017). For this reason, on the occasion a worker daughter provisions alongside her mother, her contributions may represent a supplemental rather than necessary resource for brood survival. Additionally, although sib-social provisioning may be critical to preventing brood mortality, worker daughters assume their role of sole forager with relatively low frequency in orphaned nests (Mikát et al. 2017). Therefore, despite high relatedness between worker daughters and their siblings, reinforcement of sib-social care behavior in C. calcarata does not appear to be fully explained by indirect fitness benefits to the helping individual (Hamilton 1964). Although she may be making the best of a bad situation by foraging for her siblings, in the absence of inclusive fitness benefits to the worker daughter maintenance of division of labor in C. calcarata appears best explained by maternal manipulation (Johnson 1988; Crespi and Ragsdale 2000; Rehan and Richards 2013; Rehan, Berens, et al. 2014; Withee and Rehan 2016; Mikát et al. 2017). Maternal manipulation of brood has long been theorized as a proximate mechanism underlying the emergence of social traits (Alexander 1974; Craig 1979) and has been suggested as an explanation for facultative sociality in other species (e.g., Lasioglossum malachurum, Richards et al. 2005; M. genalis, Kapheim et al. 2016). For instance, M. genalis queens manipulate pollen provisions to reduce the body size of their offspring (Kapheim et al. 2011). As observed in C. calcarata, M. genalis workers have low inclusive fitness, and it is thought that their small adult body size facilitates physical coercion into their worker roles (Arneson and Wcislo 2003; Kapheim et al. 2016). Body size affects behavioral roles in C. calcarata (Withee and Rehan 2016), and appears to predict the probability of social nesting. Larger C. calcarata mothers may thus be better-equipped to manipulate pollen during the mass provisioning period (Rehan and Ricahrds 2010b; Lawson et al. 2016), and may be more capable of establishing a social hierarchy during the adult offspring feeding stage (Withee and Rehan 2016; Mikát et al. 2017). C. calcarata’s social and subsocial nesting polyphenism has gone previously unreported across its range (Rau 1928; Johnson 1988; Rehan and Richards 2010a; Lawson et al. 2016; Lewis and Richards 2017); this could be attributable to the subtle differences between nest types. Both social and subsocial mothers produce similar clutch sizes, and relatively infrequent brood losses to parasitism do not appear to affect one nesting type more than the other (Figure 2). Further, while mothers of either strategy are capable of independently feeding their brood for a successful overwintering period, all mothers are also equally likely to orphan their adult offspring at the end of the brood rearing season (Mikát et al. 2017). In the event of late season orphanage, a social mother’s direct fitness may be insured through sib-social brood provisioning. Similar insurance-based advantages to social nesting have also been detected in the facultatively social hover wasp, Liostenogaster flavolineata, in which the fitness of brood rearing adults is likely to be assured through the collective effort of multiple helpers (Field et al. 2000). Assured fitness returns have been proposed as an important ultimate mechanism for the maintenance of social reproduction in facultatively eusocial bees (Smith et al. 2007), and among social allodapine bees (Schwarz et al. 2010) and wasps (Gadagkar 1990, 2001). The fitness advantage observed in social nesting C. calcarata thus suggests assured fitness returns may operate in selecting for group living and early divisions of labor (Gadagkar 1990). It therefore appears that, while the emergence of social traits likely requires high relatedness and inclusive fitness benefits, selection on physiological and behavioral traits that maximize maternal fitness may represent recurring proximate mechanisms for early evolutionary transitions towards social organization. Additional empirical assessments of colony demography and the relative fitness of alternative reproductive strategies in other facultatively social species are necessary to understand lineage specific and recurrent processes underlying evolutionary gains and losses of social complexity across taxa. SUPPLEMENTARY MATERIAL Supplementary data are available at Behavioral Ecology online. Funding This work was supported by funding from the University of New Hampshire; National Science Foundation – Integrative Organismal Systems: Behavioral Systems (1456296); and National Science Foundation Graduate Research Fellowship (1450271). We thank Cullen Franchino and Michal Mikát for assistance with behavioral observations and nest collections, and members of the Rehan lab for helpful feedback during manuscript preparation. Data accessibility: Analyses reported in this article can be reproduced using the data provided by Shell and Rehan (2017b). REFERENCES Alexander RD. 1974. 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