Evolution of queen cuticular hydrocarbons and worker reproduction in stingless bees

Transcription

1 In the format provided by the authors and unedited. SUPPLEMENTARY INFORMATION VOLUME: 1 ARTICLE NUMBER: 0185 Evolution of queen cuticular hydrocarbons and worker reproduction in stingless bees Túlio M Nunes 1,2, Benjamin Oldroyd 2, Larissa G. Elias 3, Sidnei Mateus 3, Izabel C. Turatti 1, Norberto P. Lopes 1. 1 NPPNS, Departamento de Física e Química, FCFRP, Universidade de São Paulo, Brazil. 2 Behaviour and Genetics of Social Insects Laboratory, School of Life, Earth and Environmental Sciences A12, University of Sydney, NSW 2006, Australia. 3 Departamento de Biologia, FFCLRP, Universidade de São Paulo, Brazil. NATURE ECOLOGY & EVOLUTION DOI: /s

2 Supplementary Methods Behavioural analysis In order to determine the frequency of worker ovary activation for Lestrimelitta limao and Plebeia minima, three colonies of each species were observed. Queens were kept in their colonies for three months, and throughout this period the process of provision of food and oviposition was regularly observed to determine whether workers lay eggs. The advancing front of the broodnest (where new cells are constructed and oviposition occurs) was observed daily for one hour per colony for 20 consecutive days. Following these observations, queens were removed and the colonies kept queenless for the following three months. The behaviour of workers engaged in cell construction was observed daily for one hour for each colony for 20 consecutive days and instances of worker oviposition recorded. Additionally, 20 workers working on brood cell construction were collected from each hive and dissected under 20x magnification in order to determine the frequency of ovary activation under queenright and queenless conditions. Chemical analysis Queens were individually collected in 2.0 ml vials (Sigma-Aldrich) and killed in by freezing. Cuticular compounds were extracted from the entire insect by adding 1.0 ml of hexane to the tube and resting for 5 minutes at room temperature. The queens body was then removed, the extract dried under a stream of nitrogen and resuspended in 50µL of hexane. Chemical analysis was carried on a gas chromatograph coupled with mass spectrometer (Shimadzu model GCMS-QP2010). The temperature profile used for the analysis was: rising from 120º C- to 300 C at 8 C/min, then hold for 10 min. The identity of the compounds was obtained through the analysis of the mass fragments, comparison with synthetic compounds, and commercial mass spectra libraries. The area under the peaks was used to calculate the relative proportion of each compound in the cuticular profile. Relationship between queen chemical profile and worker reproductive behaviour In order to determine the correlation between queen chemical profiles and worker reproductive behaviour we used a phylogenetic generalised linear model for binomial response 1 using the package "phylolm" 2, as implemented in R environment 3. In the analysis, the dependent variable is a binary trait coded according to queen influence on worker reproductive behaviour (State 0 Queen presence does not interfere on worker reproduction; State 1 Queen presence influences worker reproductive behaviour as in category B in the introduction). The independent variables are represented by the relative concentration of each class of queen cuticular compound, which were analysed individually in different models. To ensure that effects of individual compounds were not neglected, we also carried out phylogenetic generalised linear model analyses using individual compounds. We used a model comparison approach 4 using AIC (Akaike Information Criterion) to rank competing models. NATURE ECOLOGY & EVOLUTION DOI: /s

3 Evolution of queen cuticular hydrocarbon profile In order to infer how queen cuticular profile evolved in Meliponini, we performed ancestral state reconstruction using relative abundance of each class of compound as continuous data. Analyses were performed under a likelihood framework using the R package phytools 5. Values were colour-coded so that the variation in colour correspond to the variation in relative abundance. Evolution of worker reproductive behaviour Ancestral state reconstruction analyses were performed to assess how worker reproductive behaviour evolved in stingless bees. The influence of queen presence on the reproductive status of workers was categorically coded with three possible states: workers always lay eggs, even in queenright colonies; workers lay eggs only in queenless colonies; or workers are sterile and never lay eggs. Reconstructions were based on Bayesian posterior probability distribution. Stochastic reconstructions were used to estimate a Bayesian posterior probability distribution. Our analyses used the results of 1000 stochastic character maps to calculate probabilities at each node for each of the traits 6. NATURE ECOLOGY & EVOLUTION DOI: /s

4 Table S1 List of stingless bee species used in the study categorized by the reproductive behavior of workers under queenright and queenless conditions. Species Worker ovary Classification References activation for this study Frieseomelitta silvestri ST A da Cruz-Landim (2000) 7 Frieseomelitta varia ST A Boleli et al. (1999) 8 Tetragonula carbonaria ST A Gloag et al. (2007) 9, Nunes et al. (2015) 10 Tetragonula hockingsi ST A Palmer et al. (2002) 11 SUPPLEMENTARY INFORMATION Friesella schrottkyi QL B Sakagami et al. (1973) 12, Nunes et al. (2014) 13 Leurotrigona muelleri QL B Terada (1974) 14, Sakagami and Zucchi (1974) 15, da Cruz-Landim (2000)7 Plebeia lucii QL B Teixeira (2007) 16 Melipona scutellaris QR C Tóth et al. (2002) 17 Melipona quadrifasciata QR C Tóth et al. (2002) 17, da Cruz-Landim (2000) 7 Lestrimelitta limao QR C This study Nannotrigona testaceicornis QR C Sakagami et al. (1993) 18 Plebeia minima QR C This study Plebeia remota QR C Van Benthem et al. (1995) 19, Tóth et al. (2004) 20 Plebeia droryana QR C Machado et al. (1984) 21, Tóth et al. (2004) 20, da Cruz-Landim (2000) 7 Scaura tenuis QR C Mateus et al. (1999) 22 Tetragonisca angustula QR C Grosso et al. (2000) 23, Tóth et al. (2004) 20 Tetragona clavipes QR C Sakagami and Zucchi (1967) 24, Tóth et al. (2002) 17 Scaptotrigona aff depilis QR C Beig (1972) 25, Paxton et al. (2003) 26 Partamona vicina QR C Azevedo (2001) 27 Trigonisca nataliae QR C Mateus et al. (2006) 28 Austroplebeia australis QR C Drumond et al. (1999) 29, Drumond et al. (2000) 30 QR, workers have activated ovaries in queenright colonies; QL, workers have activated ovaries under queenless conditions only; ST, permanently sterile workers. NATURE ECOLOGY & EVOLUTION DOI: /s

5 Table S2 Queen cuticular compounds and relative concentration for each species analyzed by GC-MS. Cells are coloured according to increasing relative concentration. The last column is the p value for phylogenetic regression analyses carried out for each compound (see methods). Compound Austroplebeia australis Tetragonula carbonaria Tetragonula hockingsi Leurotrigona muelleri Trigonisca nataliae Partamona vicina Scaptotrigona depilis Tetragona clavipes Scaura tenuis Tetragonisca angustula C C C C C C C C C C C C C C C C C C C C C C C C MeC MeC MeC MeC MeC MeC MeC MeC MeC MeC MeC MeC Frieseomelitta silvestri NATURE ECOLOGY & EVOLUTION DOI: /s Frieseomelitta varia Nannotrigona testaceicornis Lestrimelitta limao Friesella schrottkyi Plebeia droryana Plebeia minima Plebeia remota Plebeia lucii Melipona quadrifasciata Melipona scutellaris P value (phylogenetic regression )

6 MeC MeC MeC MeC MeC MeC MeC MeC MeC MeC MeC MeC MeC DimeC DimeC DimeC DimeC DimeC DimeC DimeC DimeC DimeC DimeC DimeC DimeC C15: C17: C18: C19: C20: C21: C23: C24: C25: C26: C27: C28: C29: C30: C31: C32: C33: C34: C35: C37: C21: NATURE ECOLOGY & EVOLUTION DOI: /s

7 C23: C25: C27: C29: C30: C31: C32: C33: C34: C35: C36: C37: C33: C35: C12Es C14Es C16Es C18Es C20:2Es - C20:1Es C20Es C22:1Es C22Es C24:1Es C24Es C26:1Es C26Es C28:1Es C28Es C30Es C32:1Es C32Es C12:2Al C12:1Al - C12Al C14:1Al C15Al C16:1Al C18:1Al C20:1Al C20Al C22:1Al C24:1Al C16Ac C18:2Ac C18:1Ac NATURE ECOLOGY & EVOLUTION DOI: /s

8 Sum % NI 7,8 3,4 1, ,3 0 1,1 0 0,2 0,2 0 0, NATURE ECOLOGY & EVOLUTION DOI: /s

9 Table S3 Shannon diversity index based on relative concentration of each compound per species. Study species Shannon diversity index Austroplebeia_australis Tetragonula_carbonaria Tetragonula_hockingsi Leurotrigona_muelleri Trigonisca_nataliae 0 Partamona_vicina Scaptotrigona_depilis Tetragona_clavipes Scaura_tenuis Tetragonisca_angustula Frieseomelitta_silvestri Frieseomelitta_varia 0 Nannotrigona_testaceicornis Lestrimelitta_limao Friesella_schrottkyi Plebeia_droryana Plebeia_minima Plebeia_remota 0 Plebeia_lucii Melipona_quadrifasciata Melipona_scutellaris NATURE ECOLOGY & EVOLUTION DOI: /s

10 Table S4 - Ancestral state reconstruction of worker reproductive behaviour. Posterior probabilities for each state represented in piecharts in Figure 1. Numbers in brackets correspond to node number in Figure S2. Node State 0 State 1 State 2 Root (22) Tetragonula clade + Austroplebeia australis (23) Tetragonula clade (24) Clade 26 + clade 27 (25) Trigonisca natalie + Leurotrigona muelleri (26) Clade 28 + Clade 41 (27) Clade 29 + Partamona vicina (28) Clade 30 + clade 31 (29) Tetragona clavipes + S. depilis (30) Clade 32 + Scaura tenuis (31) Clade 33 + clade 35 (32) T. angustula + Frieseomelitta clade (33) Frieseomelitta clade (34) N. testaceicornis +Plebeia clades + F. schrottkyi + L. limao (35) Plebeia clades + F. schrottkyi + L. limao (36) L. limao + F. schrottkyi +P. remota + P. droryana (37) F. schrottkyi +P. remota + P. droryana (38) Plebeia remota + Plebeia droryana (39) Plebeia lucii + Plebeia minima (40) Melipona clade (41) State 0, workers always lay eggs, even in queenright colonies; State 1, workers lay eggs only in queenless colonies; State 2, workers are sterile and never lay eggs. Table S5 - Ancestral state reconstruction of queen cuticle composition. Relative concentration shown in colour code in Figure 2. Numbers in brackets correspond to node number in Figure S2. Node Alkanes Methyl Oxigenated Olefins Alkanes compounds Root (22) Tetragonula clade + Austroplebeia australis (23) Tetragonula clade (24) Clade 26 + clade 27 (25) Trigonisca natalie + Leurotrigona muelleri (26) Clade 28 + Clade 41 (27) Clade 29 + Partamona vicina (28) Clade 30 + clade 31 (29) Tetragona clavipes + S. depilis (30) Clade 32 + Scaura tenuis (31) Clade 33 + clade 35 (32) T. angustula + Frieseomelitta clade (33) Frieseomelitta clade (34) N. testaceicornis +Plebeia clades + F. schrottkyi + L. limao (35) Plebeia clades + F. schrottkyi + L. limao (36) L. limao + F. schrottkyi +P. remota + P. droryana (37) F. schrottkyi +P. remota + P. droryana (38) Plebeia remota + Plebeia droryana (39) Plebeia lucii + Plebeia minima (40) Melipona clade (41) NATURE ECOLOGY & EVOLUTION DOI: /s

11 Figure S2 - Node numbers in the phylogenetic tree used for analyses. Figure S3 Principal Component Analysis of stingless bee queen cuticle profile. Friesella schrottkyi (black); Scaura tenuis (red) and Scaptotrigona depilis (green). NATURE ECOLOGY & EVOLUTION DOI: /s

12 References 1 Ives, A. R. & Garland, T. J. Syst. Biol. 59, 9-26, doi: /sysbio/syp074 (2010). 2 Ho, L. s. T. & Ané, C. Syst. Biol. 63, , doi: /sysbio/syu005 (2014) R Development Core Team. R: A Language and Environment for Statistical Computing (R Foundation for Statistical Computing, 2015). 4 Burnham, K. P. & Anderson, D. R. Model selection and multimodel inference. 2nd edn, 488 (Springer, 2002). 5 Revell, L. Methods in Ecology and Evolution 3, , doi: /j x x (2012). 6 Revell, L. Methods in Ecology and Evolution 4, (2013). 7 da Cruz-Landim, C. Genetics & Molecular Biology 23, (2000). 8 Boleli, I. C., Paulino-Simões, Z. L.; Gentile Bitondi, M. M. Journal of Morphology 242, , ISSN (1999). 9 Gloag, R. S. et al. Insectes Sociaux 54, (2007). 10 Nunes, T. M. et al. Austral Entomology 54, 5, ISSN (2015). 11 Palmer, K. A. et al. Molecular Ecology 11, (2002). 12 Sakagami, S. F., Camillo, C., Zucchi, R. Journal of the Faculty of Science, Hokkaido University. Zoology Series 19, (1973). 13 Nunes, T. M. et al. Scientific Reports 4, 7449 (2014). 14 Terada, Y. PhD thesis Universidade de São Paulo, Ribeirão Preto, Brazil (1974). 15 Sakagami, S. F.; Zucchi, R. The Journal of the Faculty of Science, Hokkaido University, Series VI 19, (1974). 16 Teixeira, L. Master dissertation Universidade Federal de Viçosa, Viçosa, Brasil (2007) 17 Tóth, E. et al. Molecular Ecology 11, (2002). 18 Sakagami, S. et al. 昆蟲 61, , ISSN (1993). 19 van Benthem, F., Imperatriz-Fonseca, V., Velthuis, H. Insectes Sociaux 42, 71-87, ISSN (1995). 20 Tóth, E. et al. Insectes Sociaux 51, 1-11 (2004). 21 Machado, M. F. P. S., Contel, E. P. B., Kerr, W. E. Genetica 65, (1984). 22 Mateus, S., Nogueira-Neto, P., Zucchi, R. XVII Encontro Brasileiro Anual de Etologia, Botucatu Brazil (1999) 23 Grosso, A. F., Bego, L. R., Martinez, A. S. Sociobiology 35, (2000). 24 Sakagami, S. F., Zucchi, R. Journal of the Faculty of Science, Hokkaido Univer 16, (1967). 25 Beig, D. Journal of Apicultural Research 11, (1972). 26 Paxton, R. J. et al. Behavioral Ecology and Sociobiology 53, (2003). 27 Azevedo, G. PhD thesis Universidade de São Paulo, Ribeirão Preto, Brazil (2001). 28 Mateus, S.; Matos, E.; Zucchi, R. VII Encontro Sobre Abelhas, Ribeirão Preto Brazil (2006). 29 Drumond, P. M. et al. Australian Journal of Entomology 38, (1999). 30 Drumond, P. M., Oldroyd, B. P.; Osborne, K. E. Insectes Sociaux 47, (2000). NATURE ECOLOGY & EVOLUTION DOI: /s