ESM - Monogamy in Large Bee Societies: a Stingless Paradox

ESM - Monogamy in large bee societies: A stingless paradox

Electronic Supplementary Material

Electronic supplementary material for the manuscript “Monogamy in large bee societies: A stingless paradox” by Rodolfo Jaffé, Fabiana C. Pioker-Hara, Charles F. dos Santos, Leandro R. Santiago, Denise A. Alves, Astrid de M. P. Kleinert, Tiago M. Francoy, Maria C. Arias, and Vera L. Imperatriz-Fonseca. Includes Material and Methods, Tables S1- S4 and References.

Material and Methods

Sampling

To collect pupae, a fireman ladder was employed to reach the nests, usually located on trees above 6m. Using professional climbing and beekeeping equipment we then secured ourselves to a branch at the nest level, and proceeded to drill a hole in the nest crust to open the brood chamber. We extracted between 5 and 10 different brood combs from each nest, placed them in Petri dishes and took them to the laboratory. Pupae were randomly collected from all combs. All nests successfully recovered from the damage done. To collect adult workers we simply struck the tree branches supporting the nests and waited for the bees to attack our bee suits. All bees were collected from the suits before sampling other nests. All samples were preserved in ethanol 95% and frozen shortly after collection. Samples were collected from three Brazilian populations across three different biomes:

Mossoró:Samples were collected during 2012 near the city of Mossoró (Rio Grande do Norte State),at the Experimental Station of Universidade Federal Rural do Semi-Árido (UFERSA). The region belongs to the biome Caatinga((Por et al. 2005)).
São Paulo:Samples were collected during2012 in the city of São Paulo (São Paulo State), atUniversidade de São Paulo (USP). The region belongs to the biome Atlantic Forest((Por et al. 2005)).
Itirapina:Samples were collected between 2008 and 2010, in the Itirapina Ecological Station and the ArrudaBotelho Institute (São Paulo State). The protected area has 2,300 ha and is one of the last reminiscents of the biome Cerrado ((Por et al. 2005)),being surrounded by fragmented cropland and urban landscapes. Before its establishment in 1984, the Itirapina Ecological Station consisted of grassland exposed to yearly burning. After the creation of the Station, burning stopped allowing a process of ecological succession.

Genotyping

DNA was extracted following a Chelex protocol ((Walsh et al. 1991)) and the microsatellite target sequences were amplified bypolymerase chain reactions (PCR), using fluorescent taggedprimers ((Schuelke 2000)).PCR products were then analyzed in anABI 3730 sequencer at Centro de Estudos do GenomaHumano, USP. Allele sizes were checked by eye usingthe software GeneMarker (Softgenetics).

Analyses

Pupae samples showing homozygosity at all loci were classified as males (n = 11), which in all cases showed genotypes consistent with those of queens (Table S2).Genetic diversity measures were computed using FSTAT (Goudet 2001), based on the genotypes of each queen and her mate. Using the reconstructed queen genotypes we then tested for deviations from the Hardy-Weinberg equilibrium and calculated inbreeding coefficients using Genepop((Rousset 2008)).Patriline non-detection errors (the probability of failing to identify a second male because it shares the same allele combination of the main male) were calculated for each colony following (Foster et al. 1999).Patriline non-sampling errors (the probability of not sampling offspring from a second male) were also calculated for each colony assuming that a putative second patriline was represented in 50%, 25%, and 10% of offspring respectively (Foster et al. 1999). Finally, matriline detection probability (for worker offspring produced by a daughter queen) was calculated for each colony according to (Richards et al. 2005).

ESM - Monogamy in large bee societies: A stingless paradox

Table S1: Sample locations, geographic coordinates of nests, sample type, number of successfully genotyped workers (n), number of queens detected, observed paternity, patrilinenon-detection errorper colony (NDE),patrilinenon-sampling error per colony (NSE) for patrilines represented in 50%, 25%, and 10% of offspring, and mean matriline detection probability per colony (MDP).

Colony / Location / Coordinates / Sample type / n / N. Queens / Obs. Paternity / NDE
(dn) / NSE
(50%/25%/10%) / MDP
M1 / Mossoró / -5.062486,-37.397988 / Pupae / 10 / 1 / 1 / 9.53X10-4 / 0.001/0.06/0.35 / 0.61
M2 / Mossoró / -5.060593,-37.4053 / Pupae / 10 / 1 / 1 / 6.00X10-5 / 0.001/0.06/0.35 / 0.72
M3 / Mossoró / -5.056207,-37.399795 / Pupae / 10 / 1 / 1 / 3.92X10-5 / 0.001/0.06/0.35 / 0.67
M4 / Mossoró / -5.061715,-37.401695 / Pupae / 11 / 1 / 1 / 1.80X10-4 / 4.88x10-4/0.04/0.31 / 0.66
M5 / Mossoró / -5.144641,-37.370356 / Pupae / 12 / 1 / 1 / 9.04X10-6 / 2.44x10-4/0.03/0.28 / 0.64
M6 / Mossoró / -5.111797,-37.387265 / Pupae / 14 / 1 / 1 / 3.92X10-5 / 6.10x10-5/0.02/0.23 / 0.69
S1 / São Paulo / -23.562412,-46.723047 / Pupae / 10 / 1 / 1 / 7.26X10-5 / 0.001/0.06/0.35 / 0.72
S2 / São Paulo / -23.564751,-46.731136 / Pupae / 14 / 1 / 1 / 9.42X10-3 / 6.10x10-5/0.02/0.23 / 0.54
S3 / São Paulo / -23.565816,-46.729781 / Pupae / 12 / 1 / 1 / 2.70X10-4 / 2.44x10-4/0.03/0.28 / 0.57
S4 / São Paulo / -23.555071,-46.723796 / Pupae / 10 / 1 / 1 / 2.22X10-5 / 0.001/0.06/0.35 / 0.75
S5a / São Paulo / -23.560959,-46.72072 / Pupae / 14 / 2 / 1 / 1.30X10-4 / 0.01/0.13/0.46 / 0.72
I1 / Itirapina / -22.210955,-47.892226 / Adults / 4 / 1 / 1 / 2.79X10-5 / 0.06/0.32/0.66 / 0.66
I2 / Itirapina / -22.210286,-47.891253 / Adults / 5 / 1 / 1 / 3.89X10-5 / 0.03/0.24/0.59 / 0.65
I3 / Itirapina / -22.215658,-47.913921 / Adults / 5 / 1 / 1 / 5.53X10-7 / 0.03/0.24/0.59 / 0.72
I4 / Itirapina / -22.214678,-47.914734 / Adults / 5 / 1 / 1 / 1.50X10-4 / 0.03/0.24/0.59 / 0.66
I5 / Itirapina / -22.208159,-47.900689 / Adults / 5 / 1 / 1 / 1.28X10-4 / 0.03/0.24/0.59 / 0.60
I6 / Itirapina / -22.210114,-47.923092 / Adults / 5 / 1 / 1 / 1.25X10-5 / 0.03/0.24/0.59 / 0.61
I7 / Itirapina / -22.207898,-47.924355 / Adults / 5 / 1 / 1 / 4.25X10-6 / 0.03/0.24/0.59 / 0.67
I8b / Itirapina / -22.209845,-47.918986 / Adults / 10 / 1 / 1 / 1.57X10-4 / 0.001/0.06/0.35 / 0.56
I9b / Itirapina / -22.214217,-47.922285 / Adults / 10 / 1 / 1 / 2.79X10-5 / 0.001/0.06/0.35 / 0.62
I10 / Itirapina / -22.217008,-47.915655 / Adults / 5 / 1 / 1 / 5.91X10-6 / 0.03/0.24/0.59 / 0.66
I11 / Itirapina / -22.216196,-47.91967 / Adults / 5 / 1 / 1 / 5.92x10-5 / 0.03/0.24/0.59 / 0.63
I12 / Itirapina / -22.205809,-47.920056 / Adults / 5 / 1 / 1 / 2.50X10-6 / 0.03/0.24/0.59 / 0.65
I13 / Itirapina / -22.204003,-47.926106 / Adults / 5 / 1 / 1 / 5.09X10-11 / 0.03/0.24/0.59 / 0.76
I14 / Itirapina / -22.211921,-47.921966 / Adults / 5 / 1 / 1 / 2.84X10-6 / 0.03/0.24/0.59 / 0.62
I15 / Itirapina / -22.195757,-47.917852 / Adults / 5 / 1 / 1 / 1.66X10-5 / 0.03/0.24/0.59 / 0.58
I16 / Itirapina / -22.2214,-47.919875 / Adults / 5 / 1 / 1 / 3.25X10-6 / 0.03/0.24/0.59 / 0.73
I17 / Itirapina / -22.217836,-47.920829 / Adults / 5 / 1 / 1 / 1.93X10-5 / 0.03/0.24/0.59 / 0.67
I18 / Itirapina / -22.172099,-47.870702 / Adults / 5 / 1 / 1 / 1.44X10-5 / 0.03/0.24/0.59 / 0.57
I19 / Itirapina / -22.172705,-47.883932 / Adults / 4 / 1 / 1 / 8.54X10-6 / 0.06/0.32/0.66 / 0.68
I20 / Itirapina / -22.20786,-47.921931 / Adults / 4 / 1 / 1 / 7.28X10-6 / 0.06/0.32/0.66 / 0.68
I21 / Itirapina / -22.208393,-47.923434 / Adults / 5 / 1 / 1 / 1.12X10-4 / 0.03/0.24/0.59 / 0.56
I22 / Itirapina / -22.198247,-47.902812 / Adults / 3 / 1 / 1 / 2.21X10-6 / 0.13/0.42/0.73 / 0.65
I23 / Itirapina / -22.207077,-47.916283 / Adults / 5 / 1 / 1 / 3.80X10-5 / 0.03/0.24/0.59 / 0.68
I24 / Itirapina / -22.210882,-47.914966 / Adults / 5 / 1 / 1 / 1.80X10-4 / 0.03/0.24/0.59 / 0.51
I25 / Itirapina / -22.207137,-47.903324 / Adults / 5 / 1 / 1 / 8.37X10-7 / 0.03/0.24/0.59 / 0.69
I26 / Itirapina / -22.207623,-47.904914 / Adults / 5 / 1 / 1 / 1.06X10-5 / 0.03/0.24/0.59 / 0.62
I27 / Itirapina / -22.203336,-47.90187 / Adults / 5 / 1 / 1 / 3.31X10-4 / 0.03/0.24/0.59 / 0.60
I28 / Itirapina / -22.222354,-47.908465 / Adults / 5 / 1 / 1 / 4.74X10-4 / 0.03/0.24/0.59 / 0.70
I29 / Itirapina / -22.201622,-47.924506 / Adults / 5 / 1 / 1 / 1.01X10-6 / 0.03/0.24/0.59 / 0.71
I30 / Itirapina / -22.200332,-47.920941 / Adults / 5 / 1 / 1 / 1.40X10-5 / 0.03/0.24/0.59 / 0.67
I31 / Itirapina / -22.214013,-47.921099 / Adults / 5 / 1 / 1 / 1.54X10-6 / 0.03/0.24/0.59 / 0.64
I32 / Itirapina / -22.21416,-47.917437 / Adults / 5 / 1 / 1 / 2.19X10-4 / 0.03/0.24/0.59 / 0.60

a Genotypes consistent with the presence of two, singly mated queens. Non-detection and non-sampling errors for this colony were calculated by averaging the estimates from both matrilines. bThese colonieshad two separate entrances and workers were collected from both entrances.

ESM - Monogamy in large bee societies: A stingless paradox

Table S2: Genotypes from all analyzed samples (see Excel file).

Table S3: Genetic diversity per locus and population. Number of alleles (NA), expected heterozygosity (HE) and inbreeding coefficients (FIS) are provided. FIS estimates were tested against zero (*p<0.05; **p<0.01; ***p<0.001). (χ210 = 30.65; p < 0.001)

Population / Locus / NA / HE / FIS
Mossoró / 4D / 2 / 0.21 / -0.11
2F / 8 / 0.90 / -0.13
2A / 2 / 0.50 / 0.71
3G / 2 / 0.11 / N/A
Tang60 / 5 / 0.72 / -0.14
Overall / 3.80± 2.68 / 0.47 ± 0.14 / 0.06
São Paulo / 4D / 4 / 0.77 / 0.15
2F / 5 / 0.71 / -0.14
2A / 10 / 0.91 / 0.11
3G / 3 / 0.46 / -0.05
Tang60 / 5 / 0.72 / -0.54
Overall / 5.40± 2.70 / 0.69 ± 0.07 / -0.07
Itirapinaa / 4D / 8 / 0.68 / 0.13
2F / 9 / 0.73 / 0.15
2ª / 12 / 0.73 / -0.001
3G / 5 / 0.58 / 0.33**
Tang60 / 8 / 0.61 / 0.25*
1B / 8 / 0.75 / 0.04
1D / 14 / 0.90 / -0.02
Overall / 9.14 ± 2.97 / 0.71 ± 0.04 / 0.11**

aA significantdeparture from the Hardy-Weinberg equilibrium was only detected in Itirapina population (χ210 = 30.65; p < 0.001), due to significant FIS estimates in loci 3G and Tang60.

Table S4:Paternity and colony size across 58 species of social bees (only monogynous species included). Only data from studies employing genetic markers are presented. Paternity frequency is presented as observed paternity (Kobs) and effective paternity (me). The number of analyzed colonies (n) and the reported colony size for each species (mean number of workers) are also provided. Arithmetic means are given for Kobswhile harmonic meansare given for me.

Tribe / Species / Kobs / me / n / Colony size / References
Apini / Apisandreniformis / 13.50 / 10.50 / 60 / 4,900 / 1;2
Apini / Apiscerana / 18.80 / 14.10 / 74 / 6,884 / 3;4
Apini / Apisdorsata / 54.90 / 44.20 / 140 / 36,630 / 5;4
Apini / Apis florea / 11.60 / 7.90 / 81 / 6,271 / 6;4
Apini / Apiskoschevnikovi / 16.20 / 13.30 / 74 / 7,000 / 7;8
Apini / Apislaboriosa / 34.40 / 18.28 / 135 / 36,630 / 9; B.Oldroyd pers. comm.
Apini / Apis mellifera / 12.00 / 11.60 / 61 / 19,524 / 10;11; 12
Apini / Apisnigrocincta / 54.00 / 40.30 / 159 / 6,884 / 13; B.Oldroyd pers. comm.
Augochlorini / Augochlorellastriata / 1.00 / 1.00 / 24 / 7 / 14
Bombini / Bombusaffinis / 1.00 / 1.00 / 1 / 176 / 15;11
Bombini / Bombusardens / 1.00 / 1.00 / 5 / 26 / 16;17
Bombini / Bombusauricomus / 1.00 / 1.00 / 1 / 35 / 15;18
Bombini / Bombusbimaculatus / 1.25 / 1.05 / 8 / 60 / 15;11
Bombini / Bombuscitrinus / 2.50 / 1.76 / 10 / 50 / 15; B. Baer pers. comm.
Bombini / Bombusfervidus / 1.00 / 1.00 / 1 / 88 / 15; 11
Bombini / Bombusgriseocollis / 1.00 / 1.00 / 1 / 46 / 15;19
Bombini / Bombushonshuensis / 1.00 / 1.00 / 1 / 200 / 16; 20
Bombini / Bombushortorum / 1.00 / 1.00 / 5 / 100 / 21; 11
Bombini / Bombushypnorum / 1.87 / 1.18 / 23 / 29 / 22;21; 4
Bombini / Bombus impatiens / 1.55 / 1.04 / 10 / 450 / 15;23; 11
Bombini / Bombuslapidarius / 1.00 / 1.00 / 11 / 200 / 21; 24
Bombini / Bombuslucorum / 1.00 / 1.00 / 12 / 200 / 21;11
Bombini / Bombusmixtus / 4.00 / 3.57 / 5 / 50 / 15; B. Baer pers. comm.
Bombini / Bombusoccidentalis / 1.00 / 1.00 / 23 / 55 / 25; 26
Bombini / Bombuspascuorum / 1.00 / 1.00 / 6 / 120 / 21; 24
Bombini / Bombuspratorum / 1.00 / 1.00 / 5 / 100 / 21; 24
Bombini / Bombusternarius / 2.00 / 2.04 / 12 / 100 / 15; 11
Bombini / Bombusterrestris / 1.00 / 1.00 / 17 / 350 / 21;24
Bombini / Bombusvagans / 1.00 / 1.00 / 4 / 70 / 15;11
Halictini / Lasioglossummalachurum / 1.36 / 1.15 / 30 / 33 / 27; 28; 29
Halictini / Lasioglossumzephyrum / 1.00 / 1.00 / 8 / 20 / 30; 31
Meliponini / Austroplebeiaaustralis / 1.00 / 1.00 / 2 / 2,000 / 32;4
Meliponini / Austroplebeiasymei / 1.00 / 1.00 / 4 / 2,000 / 33;4
Meliponini / Heterotrigonahockingsia / 1.00 / 1.00 / 4 / 7,000 / 33; 34
Meliponini / Heterotrigonamellipesb / 1.00 / 1.00 / 4 / 2,000 / 33
Meliponini / Lestrimellitalimao / 1.23 / 2 / 900 / 35;36
Meliponini / Meliponabeecheii / 2.20 / 1.13 / 10 / 1,192 / 37;34; R. Paxton unpubl. data
Meliponini / Meliponamarginata / 1.00 / 1.00 / 5 / 202 / 38;34
Meliponini / Meliponapanamica / 1.00 / 1.00 / 9 / 550 / 35;34
Meliponini / Meliponaquadrifasciata / 1.00 / 1.00 / 7 / 350 / 35; 38;34
Meliponini / Melipona scutellaris / 1.00 / 1.00 / 54 / 992 / 38;39;34;40
Meliponini / Nannotrigonaperilampoides / 1.19 / 7 / 1,125 / 35;41
Meliponini / Paratrigonasubnuda / 1.16 / 11 / 3,750 / 35; 42
Meliponini / Partamonaaff.cupira / 1.00 / 0.91 / 12 / 3,390 / 35; 43
Meliponini / Plebeiaaff. minima / 1.42 / 5 / 125 / 35; 36
Meliponini / Plebeiadroryana / 1.00 / 1.00 / 2 / 2,960 / 38; 34
Meliponini / Plebeiaremota / 1.00 / 1.00 / 7 / 3,500 / 38;34
Meliponini / Plebeiasaiqui / 1.00 / 1.00 / 4 / 1,500 / 38;4
Meliponini / Scaptotrigonaaff.depilisc / 1.20 / 1.02 / 19 / 10,375 / 44;34
Meliponini / Scaptotrigonabarrocoloradensis / 1.00 / 0.85 / 4 / 5,000 / 35;34
Meliponini / Scaptotrigonamexicana / 1.00 / 1.00 / 5 / 2,000 / 33; 45
Meliponini / Scaptotrigonapectoralis / 1.00 / 1.00 / 7 / 4,600 / 33;43
Meliponini / Schwarzianaquadripunctata / 1.00 / 1.00 / 22 / 1,650 / 35; 46;34
Meliponini / Tetragonaclavipes / 1.00 / 1.00 / 17 / 7,000 / 35; 38;34
Meliponini / Tetragonulacarbonariad / 1.00 / 1.00 / 5 / 2,750 / 47
Meliponini / Tetragonulaclypearise / 1.00 / 1.00 / 4 / 500 / 33;4
Meliponini / Trigonafulviventris / 1.15 / 7 / 8,500 / 35;43
Meliponini / Trigonaspinipesf / 1.00 / 1.00 / 44 / 92,500 / This study; 48

Notes: aPreviousTrigonahockingsi, bPreviousTrigonamellipes, cPreviousScaptotrigona postica, dPreviousTrigona carbonaria, ePreviousTrigonaclypeari, f PreviousTrigonaruficrus.

References:1(Oldroyd et al. 1997); 2(Koeniger et al. 2011); 3(Oldroyd et al. 1998); 4(Hammond and Keller 2004); 5(Wattanaachaiyingcharoen et al. 2003); 6(Palmer and Oldroyd 2001); 7(Rinderer et al. 1998); 8(Koeniger et al. 2011); 9(Paar et al. 2004);10(Estoup et al. 1994); 11(Michener 1974); 12(Schneider and Blyther 1988); 13(Palmer et al. 2001); 14(Mueller et al. 1994); 15(Payne et al. 2003); 16(Kokuvo et al. 2009); 17(Katayma 1997); 18(Katayma 1997); 19(Cameron 1989); 20(Ochiai and Katayama 1982); 21(Schmid-Hempel and Schmid-Hempel 2000); 22(Paxton et al. 2001); 23(Cnaani et al. 2002); 24(Westphal et al. 2006); 25(Owen and Whidden 2013); 26(Whittington and Winston 2003);27(Paxton et al. 2002); 28(Richards et al. 1995); 29(Soro et al. 2009); 30(Barrows 1975); 31(Crozier et al. 1987); 32(Drumond et al. 2000); 33(Palmer et al. 2002); 34(Tóth et al. 2004); 35(Peters et al. 1999); 36(Roubik 1983); 37(Paxton et al. 1999); 38(Tóth et al. 2002b); 39(Alves et al. 2009); 40(Wenseleers et al. 2011); 41(Cauich et al. 2004); 42(Tóth et al. 2002a); 43(Slaa et al. 2003); 44(Paxton et al. 2003); 45(Sánchez et al. 2004); 46(Tóth et al. 2003); 47(Green and Oldroyd 2002); 48(Lindauer and Kerr 1960).

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