Additional material may be found in the online version of this article.
Figure S1: PCA analysis based on microsatellite markers confirming that the nests sampled belong to distinct colonies.
Figure S2: Survival Kaplan-Meier curves for each pairing during the 14 first days of colony establishment (i.e., short-term survival). Incipient colonies were monitored every two days. Hazard ratio for cox-proportional hazard model is reported in front of each pairing. At each census date, bar charts indicate colony of origin of the dead alate(s) in failed incipient colonies. Color bars denote dead alates originating from the studied colony, dark grey bars indicate the death of both alates (i.e., studied colony and partner), and light grey bars represent death of the partner.
Figure S3: Hazard ratio for each pairing in the first 14 days after colony establishment. Additional matrices provide values of every variable tested for each pair of colonies (maximum pathogen load, cumulative pathogen load, relatedness, unweighted Unifrac bacterial difference, weighted Unifrac bacterial difference, unweighted Unifrac fungal difference and weighted Unifrac fungal difference. These values are used to test for the correlations presented in Figure 3).
Figure S4 : (a) Principal Coordinate Analyses (PCoA) of individuals based on their bacterial or fungal difference (weighted and unweighted Unifrac values). Each individual is colored according to its colony of origin, alates are indicated with circles and workers with squares. (b) Pairwise distance matrices between each pair of alates from the same or different colonies. Each pair is colored according to its microbial similarity (i.e., bacterial and fungal similarity from weighted and unweighted Unifrac) obtained from Euclidean distances between the two individuals on the two first PCs of the PCoA. Darker values indicate low microbial differentiation between a pair of individuals (i.e., close on the PCoA analysis). (c) Violin plots of bacterial and fungal differentiation (weighted and unweighted Unifrac) among individuals within and between colonies. Box plots represent median and 1st and 3rd quartile; whiskers include 95% of all observations; dots indicate individual values.
Figure S5: Survival Kaplan-Meier curves for each pairing during the overall length of the long-term survival experiment (450 days). Incipient colonies were monitored every two days. Hazard ratio for cox-proportional hazard model is reported in front of each pairing.
Figure S6: Correlation between hazard ratios of each pairing during the short-term experiment (14 days) and those of the long-term experiment (450 days). Hazard ratios of inbred pairings are highlighted in color.
Figure S7: Number of eggs, workers and soldiers present within inbred and outbred incipient colonies each month for 450 days (15 months) after pairing.
Figure S8: Microbial load (mean number of colony forming units, CFUs) for each pairing investigated.
Table S1: Linear and logarithmic correlations between the susceptibility of a pairing (i.e., hazard ratio) and (i ) the cumulative microbial load and (ii ) the maximum microbial load of the constituent colonies. AIC model selection was used to assess whether linear or logarithmic regression better explains the data in both analyses.
Supporting Information S1: DNA extraction and PCR protocols for termite DNA, as well as bacterial and fungal DNA.
Supporting Information S2: Potential presence of internal parasites, not counted in the microbial load.
FIGURE CAPTIONS
Figure 1 : Kaplan-Meier survival distributions of inbred and outbred incipient colonies during the first 14 days after pairing (a) and along the overall length of the experiment (450 days; b).
Figure 2 : (a) Colony of origin of the 202 surviving alates 14 days after colony establishment (inner circle). For each colony of origin, pie charts represent the distribution of surviving inbred and outbred pairings; outbred pairings are divided and light-colored according to the colony of origin of the partner, inbred pairings are represented by bright colors. (b) Radar plot represents the hazard ratio of each inbred and outbred pairings in the first 14 days after colony establishment. Outbred pairings are marked with a circle, while outbred pairings are represented with a square
Figure 3 : Correlation between hazard ratio of a pairing and the maximum pathogen load (a), cumulative pathogen load (b), relatedness (c), unweighted Unifrac bacterial difference (d), weighted Unifrac bacterial difference (e), unweighted Unifrac fungal difference (f) and weighted Unifrac fungal difference (g). Trendlines represent logarithmic correlations for plots a and b, and denote linear correlations for all the other plots. In each plot, inbred pairings are colored according to their colony of origin.
Figure 4 : (a) Principal Coordinate Analyses (PCoA) of individuals based on their unweighted Unifrac values for bacterial similarity and weighted Unifrac values for fungal similarity. Each individual is colored according to its colony of origin, alates are indicated with circles and workers with squares. (b) Violin plots of bacterial (unweighted Unifrac) and fungal differentiation (weighted Unifrac) among individuals within and between colonies. Box plots represent median and 1st and 3rd quartile; whiskers include 95% of all observations; dots indicate individual values. Results for weighted Unifrac bacterial similarity and unweighted Unifrac fungal similarity are provided in Supplementary Figure S4.
Figure 5 : (a) Graphical representation of the productivity of incipient colonies over the overall duration of the experiment (450 days, 15 months). Productivity is measured as the number of workers (outer circle), soldiers (middle circle) and eggs (inner circle) for each pairing. Productivity of inbred pairings is reported on the upper half-circle, while the productivity of outbred pairings is reported on the bottom half-circle. Box plots represent median and 1st and 3rd quartile; whiskers include 95% of all observations; individual dots indicate outlier values. P values indicate significant effect of the type of pairing on the number of workers and soldiers in a colony over time, with an increased production in inbred colonies (see also Supplementary Figure S6). (b) Kaplan-Meier survival distributions of offspring from inbred and outbred colonies when challenged toward entomopathogens. (c) Violin plot of microbial loads (mean number of CFU) of offspring from inbred and outbred colonies. Box plots represent median and 1st and 3rd quartile; whiskers include 95% of all observations; dots indicate individual values.
Figure 6 : Schematic illustration of the inbreeding depression termite colonies face over the different stages of their lifespan. The black line represents the level of inbreeding depression. Inbreeding depression is low during colony foundation and offspring production, but is higher during colony development, when small colonies face pathogen pressure (this study; DeHeer & Vargo, 2006). The dotted lines represent colony size (i.e., number of workers per colony). The red curve represents the efficiency of social immunity, which increases with colony size until it is expected to slightly decrease due to inbreeding from neotenic reproduction. The high efficiency of social immunity in large mature colonies releases inbreeding depression, allowing the development of inbred neotenic reproductives without suffering costs associated with pathogen pressure (Aguero et al. 2021).
REFERENCES
Abbott D. (1993). Social conflict and reproductive suppression in marmoset and tamarin monkeys. In:Primate Social Conflict (eds. Mason WA & Mendoza SP). State University of New York Press New York, pp. 331-372.
Aguero C., Eyer P.A. & Vargo E.L. (2020). Increased genetic diversity from colony merging in termites does not improve survival against a fungal pathogen. Sci. Rep. 10, 4212.
Aguero C.M., Eyer P.-A., Crippen T.L. & Vargo E.L. (2021a). Reduced environmental microbial diversity on the cuticle and in the galleries of a subterranean termite compared to surrounding soil. Microb. Ecol. 81, 1054-1063.
Aguero C.M., Eyer P.-A., Martin J.S., Bulmer M.S. & Vargo E.L. (2021b). Natural variation in colony inbreeding does not influence susceptibility to a fungal pathogen in a termite. Ecol. Evol. 11, 3072-3083.
Aguilera-Olivares D., Flores-Prado L., Véliz D. & Niemeyer H. (2015). Mechanisms of inbreeding avoidance in the one-piece drywood termite Neotermes chilensis. Insect. Soc. 62, 237-245.
Amos B., Schlotterer C. & Tautz D. (1993). Social structure of pilot whales revealed by analytical DNA proftling. Science , 260, 670-672.
Barribeau S.M., Sadd B.M., du Plessis L., Brown M.J.F., Buechel S.D., Cappelle K., et al. (2015). A depauperate immune repertoire precedes evolution of sociality in bees.Gen. Biol. 16, 83.
Bates D., Mächler M., Bolker B. & Walker S. (2015). Fitting linear mixed-effects models using lme4.J Stat. Soft. 67, 1-48.
Beani L., Bagnères A.G., Elia M., Petrocelli I., Cappa F. & Lorenzi M.C. (2019). Cuticular hydrocarbons as cues of sex and health condition in Polistes dominula wasps.Insect. Soc. 66, 543-553.
Bengtsson B.O. (1978). Avoiding inbreeding: at what cost? J Theor. Biol. 73, 439-444.
Blouin S.F. & Blouin M. (1988). Inbreeding avoidance behaviors. Trends Ecol. Evol. 3, 230-233.
Bolyen E., Rideout J.R., Dillon M.R., Bokulich N.A., Abnet C.C., Al-Ghalith G.A., et al. (2019). Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat. Biotech. 37, 852-857.
Boomsma J.J. (2013). Beyond promiscuity: mate-choice commitments in social breeding. Philos. Trans. Roy. Soc. B. 368.
Brooked M.G., Rowley I., Adams M. & Baverstock P.R. (1990). Promiscuity: an inbreeding avoidance mechanism in a socially monogamous species? Behav. Ecol. Sociobiol. 26, 191-199.
Callahan B.J., McMurdie P.J., Rosen M.J., Han A.W., Johnson A.J.A. & Holmes S.P. (2016). DADA2: High-resolution sample inference from Illumina amplicon data. Nat. Meth. 13, 581-583.
Calleri D., II, Rosengaus R. & Traniello J.A. (2005). Disease and colony foundation in the dampwood termite Zootermopsis angusticollis: The survival advantage of nestmate pairs. Naturwissenschaften , 92, 300-304.
Calleri D.V., McGrail Reid E., Rosengaus R.B., Vargo E.L. & Traniello J.F.A. (2006). Inbreeding and disease resistance in a social insect: effects of heterozygosity on immunocompetence in the termite Zootermopsis angusticollis .Proc. Roy. Soc. B. 273, 2633-2640.
Cassidy S.T., Chapa J., Tran T.-A., Dolezal N., Gerena C., Johnson G., et al. (2021). Disease defences across levels of biological organization: individual and social immunity in acorn ants. Anim. Behav. 179, 73-81.
Chouvenc T. (2019). The relative importance of queen and king initial weights in termite colony foundation success. Insect. Soc. 66, 177-184.
Chouvenc T. & Su N.Y. (2012). When subterranean termites challenge the rules of fungal epizootics.Plos One , 7, e34484.
Clutton-Brock T.H. (1989). Female transfer and inbreeding avoidance in social mammals. Nature , 337, 70-72.
Cole E.L., Bayne H. & Rosengaus R.B. (2020). Young but not defenceless: antifungal activity during embryonic development of a social insect. Roy. Soc. Open Sci. 7, 191418-191418.
Cole E.L., Ilieş I. & Rosengaus R.B. (2018). Competing physiological demands during incipient colony foundation in a social insect: consequences of pathogenic stress.Front. Ecol. Evol. 6.
Cole E.L. & Rosengaus R.B. (2019). Pathogenic dynamics during colony ontogeny reinforce potential drivers of termite eusociality: mate assistance and biparental care.Front. Ecol. Evol. 7.
Cotter S.C. & Kilner R.M. (2010). Personal immunity versus social immunity. Behav. Ecol. 21, 663-668.
Cremer S., Armitage S.A.O. & Schmid-Hempel P. (2007). Social immunity. Curr. Biol. 17, R693-R702.
Cremer S., Pull C.D. & Fürst M.A. (2018). Social immunity: Emergence and evolution of colony-level disease protection. Ann. Rev. Entomol. 63, 105-123.
Davis H.E., Meconcelli S., Radek R. & McMahon D.P. (2018). Termites shape their collective behavioural response based on stage of infection. Sci. Rep. 8, 14433-14433.
de Boer R.A., Vega-Trejo R., Kotrschal A. & Fitzpatrick J.L. (2021). Meta-analytic evidence that animals rarely avoid inbreeding. Nat. Ecol. Evol. 5, 949-964.
DeHeer C.J. & Vargo E.L. (2006). An indirect test of inbreeding depression in the termites Reticulitermes flavipes and Reticulitermes virginicus. Behav. Ecol. Sociobiol. 59, 753-761.
Eyer P.-A., Blumenfeld A.J., Johnson L.N.L., Perdereau E., Shults P., Wang S., et al. (2021a). Extensive human-mediated jump dispersal within and across the native and introduced ranges of the invasive termite Reticulitermes flavipes . Mol. Ecol. 30, 3948-3964.
Eyer P.-A., Salin J., Helms A.M. & Vargo E.L. (2021b). Distinct chemical blends produced by different reproductive castes in the subterranean termite Reticulitermes flavipes . Sci. Rep. 11, 4471.
Fei H.X. & Henderson G. (2003). Comparative study of incipient colony development in the Formosan subterranean termite, Coptotermes formosanus Shiraki (Isoptera,Rhinotermitidae). Insect. Soc. 50, 226-233.
Fougeyrollas R., Dolejšová K., Křivánek J., Sillam-Dussès D., Roisin Y., Hanus R. et al. (2018). Dispersal and mating strategies in two neotropical soil-feeding termites, Embiratermes neotenicus and Silvestritermes minutus (Termitidae, Syntermitinae). Insect . Soc. 65, 251-262.
Fox C.W. & Reed D.H. (2011). Inbreeding depression increases with environmental stress: An experimental study and meta-analysis. Evolution , 65, 246-258.
Gerlach G. & Lysiak N. (2006). Kin recognition and inbreeding avoidance in zebrafish, Danio rerio , is based on phenotype matching. Anim. Behav. 71, 1371-1377.
Hamady M., Lozupone C. & Knight R. (2010). Fast UniFrac: facilitating high-throughput phylogenetic analyses of microbial communities including analysis of pyrosequencing and PhyloChip data. ISME J. 4, 17-27.
Hanby J.P. & Bygott J.D. (1987). Emigration of subadult lions. Anim. Behav. 35, 161-169.
He S., Sieksmeyer T., Che Y., Mora M.A.E., Stiblik P., Banasiak R., et al . (2021). Evidence for reduced immune gene diversity and activity during the evolution of termites. Proc. Roy. Soc. B. 288, 20203168.
Hurst J.L., Payne C.E., Nevison C.M., Marie A.D., Humphries R.E., Robertson D.H.L., et al. (2001). Individual recognition in mice mediated by major urinary proteins.Nature , 414, 631-634.
Hussain A., Tian M.-Y., He Y.-R., Bland J.M. & Gu W.-X. (2010). Behavioral and electrophysiological responses of Coptotermes formosanus Shiraki towards entomopathogenic fungal volatiles. Biol. Cont. 55, 166-173.
Husseneder C., Simms D.M. & Ring D.R. (2006). Genetic diversity and genotypic differentiation between the sexes in swarm aggregations decrease inbreeding in the Formosan subterranean termite. Insect. Soc. 53, 212-219.
Jombart T. (2008). adegenet: a R package for the multivariate analysis of genetic markers.Bioinformatics , 24, 1403-1405.
Kearse M., Moir R., Wilson A., Stones-Havas S., Cheung M., Sturrock S., et al . (2012). Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics , 28, 1647-1649.
Koenig WD, Haydock J & MT. S. (1998). Reproductive roles in the cooperatively breeding acorn woodpecker: incest avoidance versus reproductive competition. Am. Nat. 151, 243-255.
Kokko H., Ots I. & Tregenza T. (2006). When not to avoid inbreeding. Evolution , 60, 467-475.
Kozich J.J., Westcott S.L., Baxter N.T., Highlander S.K. & Schloss P.D. (2013). Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform.Applied Environ. Microbiol. 79, 5112-5120.
Lehmann L. & Perrin N. (2003). Inbreeding avoidance through kin recognition: Choosy females boost male dispersal. Am. Nat. 162, 638-652.
Li G., Gao Y., Sun P., Lei C. & Huang Q. (2013). Factors affecting mate choice in the subterranean termite Reticulitermes chinensis (Isoptera: Rhinotermitidae).J. Ethol. 31, 159-164.
Liu L., Zhao X.-Y., Tang Q.-B., Lei C.-L. & Huang Q.-Y. (2019). The mechanisms of social immunity against fungal infections in eusocial insects. Toxins , 11, 244.
López-Uribe M.M., Sconiers W.B., Frank S.D., Dunn R.R. & Tarpy D.R. (2016). Reduced cellular immune response in social insect lineages. Biol. Lett. 12, 20150984.
Matsuura K. & Kobayashi N. (2010). Termite queens adjust egg size according to colony development.Behav. Ecol. 21, 1018-1023.
Meusemann K., Korb J., Schughart M. & Staubach F. (2020). No evidence for single-copy immune-gene specific signals of selection in termites. Front. Ecol. Evol. 8.
Miyaguni Y., Agarie A., Sugio K., Tsuji K. & Kobayashi K. (2021). Caste development and sex ratio of the Ryukyu drywood termite Neotermes sugioi and its potential mechanisms.Sci. Rep. 11, 15037.
Mullins A.J., Messenger M.T., Hochmair H.H., Tonini F., Su N.-Y. & Riegel C. (2015). Dispersal flights of the Formosan subterranean termite (Isoptera: Rhinotermitidae). J Econ. Entomol. 108, 707-719.
Nichols H.J. (2017). The causes and consequences of inbreeding avoidance and tolerance in cooperatively breeding vertebrates. J. Zool. 303, 1-14.
Otani S., Bos N. & Yek S.H. (2016). Transitional complexity of social insect immunity. Front. Ecol. Evol. 4.
Perdereau E., Bagnères A.G., Bankhead-Dronnet S., Dupont S., Zimmermann M., Vargo E.L. et al.(2013). Global genetic analysis reveals the putative native source of the invasive termite, Reticulitermes flavipes , in France.Mol. Ecol. 22, 1105-1119.
Pusey A. & Wolf M. (1996). Inbreeding avoidance in animals. Trends Ecol. Evol. 11, 201-206.
Queller D.C. & Goodnight K.F. (1989). Estimating relatedness using genetic markers. Evolution , 43, 258-275.
R Development Core Team (2016). Language and environment for statistical computing, R Foundation for Statistical Computing. In: Vienna.
Rosengaus R.B., Cornelisse T., Guschanski K. & Traniello J.F.A. (2007). Inducible immune proteins in the dampwood termite Zootermopsis angusticollis .Naturwissenschaften , 94, 25-33.
Rosengaus R.B., James L.-T., Hartke T.R. & Brent C.S. (2011a). Mate preference and disease risk inZootermopsis angusticollis (Isoptera: Termopsidae).Environ. Entomol. 40, 1554-1565.
Rosengaus R.B., Moustakas J.E., Calleri D.V. & Traniello J.F.A. (2003). Nesting ecology and cuticular microbial loads in dampwood (Zootermopsis angusticollis ) and drywood termites (Incisitermes minor, I. schwarzi, Cryptotermes cavifrons ). J. Insect. Sci. 3.
Rosengaus R.B. & Traniello J.F. (1993). Disease risk as a cost of outbreeding in the termite Zootermopsis angusticollis. Proc. Nat. Acad. Sci. 90, 6641-6645.
Rosengaus R.B. & Traniello J.F. (2001). Disease susceptibility and the adaptive nature of colony demography in the dampwood termite Zootermopsis angusticollis .Behav. Ecol. Sociobiol. 50, 546-556.
Rosengaus R.B., Traniello J.F.A. & Bulmer M. (2011b). Ecology, behavior and evolution of disease resistance in termites. In: biology of termites: a modern synthesis (eds. Bignell D, Roisin Y & Lo N). Springer Dordrecht, The Netherlands, pp. 165-191.
Rosengaus R.B., Traniello J.F.A., Chen T., Brown J.J. & Karp R.D. (1999). Immunity in a social insect.Naturwissenschaften , 86, 588-591.
Ross K.G. & Fletcher D.J.C. (1986). Diploid male production — a significant colony mortality factor in the fire ant Solenopsis invicta (Hymenoptera: Formicidae).Behav. Ecol. Sociobiol. 19, 283-291.
Schwenke R.A., Lazzaro B.P. & Wolfner M.F. (2016). Reproduction–Immunity trade-offs in insects.Ann. Rev. Entomol. 61, 239-256.
Shellman-Reeve J.S. (1990). Dynamics of biparental care in the dampwood termite, Zootermopsis nevadensis (Hagen): response to nitrogen availability. Behav. Ecol. Sociobiol. 26, 389-397.
Shellman-Reeve J.S. (1999). Courting strategies and conflicts in a monogamous, biparental termite.Proc. Roy. Soc. B. 266, 137-144.
Shellman-Reeve J.S. (2001). Genetic relatedness and partner preference in a monogamous, wood-dwelling termite. Anim. Behav. 61, 869-876.
Sillero-Zubiri C., Gottelli D. & Macdonald D.W. (1996). Male philopatry, extra-pack copulations and inbreeding avoidance in Ethiopian wolves (Canis simensis ).Behav. Ecol. Sociobiol. 38, 331-340.
Sinotte V.M., Conlon B.H., Seibel E., Schwitalla J.W., de Beer Z.W., Poulsen M. et al . (2021). Female-biased sex allocation and lack of inbreeding avoidance inCubitermes termites . Ecol. Evol. 11, 5598-5605.
Szulkin M., Stopher K.V., Pemberton J.M. & Reid J.M. (2013). Inbreeding avoidance, tolerance, or preference in animals? Trends Ecol. Evol. 28, 205-211.
Therneau T. & Grambsch P. (2000).Modeling Survival Data: Extending the Cox Model . Springer, New York.
Thompson G.J., Lenz M., Crozier R.H. & Crespi B.J. (2007). Molecular-genetic analyses of dispersal and breeding behaviour in the Australian termite Coptotermes lacteus : evidence for non-random mating in a swarm-dispersal mating system.Austr. J. Zool. 55, 219-227.
Traniello J.F.A., Rosengaus R.B. & Savoie K. (2002). The development of immunity in a social insect: Evidence for the group facilitation of disease resistance. Proc. Nat. Acad. Sci. 99, 6838-6842.
Tranter C., LeFevre L., Evison S.E.F. & Hughes W.O.H. (2014). Threat detection: contextual recognition and response to parasites by ants. Behav. Ecol. 26, 396-405.
Van Meyel S., Körner M. & Meunier J. (2018). Social immunity: why we should study its nature, evolution and functions across all social systems. Curr. Opin. Insect. Sci. 28, 1-7.
Vargo E.L. (2019). Diversity of termite breeding systems. Insects . 10, 52.
Vargo E.L. & Husseneder C. (2011). Genetic structure of termite colonies and populations. In: Biology of termites: A modern synthesis (eds. Bignell D, Roisin Y & Lo N). Springer Dordrecht, pp. 133-164.
Viljakainen L., Evans J.D., Hasselmann M., Rueppell O., Tingek S. & Pamilo P. (2009). Rapid evolution of immune proteins in social insects. Mol. Biol. Evol.26, 1791-1801.
Wang J. (2011). Coancestry: a program for simulating, estimating and analysing relatedness and inbreeding coefficients. Mol. Ecol. Res. 11, 141-145.
Waser P.M., Austad S.N. & Keane B. (1986). When should animals tolerate inbreeding? Am. Nat. 128, 529-537.
White T.J., Burns T., Lee S. & Taylor J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols: A Guide to Methods and Applications (eds. Innis MA, Gelfand DH, Snisky JJ & White TJ). Academic Press San Diego, pp. 315-322.
Wolff J.O. (1992). Parents suppress reproduction and stimulate dispersal in opposite-sex juvenile white-footed mice. Nature , 359, 409-410.
Yanagawa A., Imai T., Akino T., Toh Y. & Yoshimura T. (2015). Olfactory cues from pathogenic fungus affect the direction of motion of termites, Coptotermes formosanus .J. Chem. Ecol . 41, 1118-1126.
Zayed A. & Packer L. (2005). Complementary sex determination substantially increases extinction proneness of haplodiploid populations. Proc. Nat. Acad. Sci. 102, 10742-10746.
Zhang Z.-Y., Ren J., Chu F., Guan J.-X., Yang G.-Y., Liu Y.-T., et al. (2021). Biochemical, molecular, and morphological variations of flight muscles before and after dispersal flight in a eusocial termite, Reticulitermes chinensis . Insect. Sci. 28, 77-92.