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The Genetic Background of Chemical Communication and Chemosensory Gene Evolution in Ants

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The genetic background of chemical communication and chemosensory gene evolution in ants. Master's thesis project.
Darwinian selection can be measured and investigated from gene sequences. A certain gene form favored by positive selection will become more common in the population. Detecting strong positive selection is rare, but it has been found to affect genes involved in immune defense and perception of odorants. Genes under positive selection have a possible role in speciation or adaptation. This is why chemical communication, being based on the sense of smell, is an interesting topic for measuring natural selection and positive selection in particular. Social insects, such as ants, are model organisms for chemical communication. They use chemical communication not only for finding nutrition and detecting intruders, but also in coordinating the activities of several thousands of colony members.

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The Genetic Background of Chemical Communication and Chemosensory Gene Evolution in Ants

  1. 1. The Genetic Background of Chemical Communication and Chemosensory Gene Evolution in Ants Katri Ketola Master’s thesis Supervisor: Jonna Kulmuni Antzz group Department of Biociences University of Helsinki Centre of Excellence in Biological Interactions
  2. 2. Contents 1. Introduction 2. Study questions 3. Materials and methods 4. Results 5. Conclusions 6. References
  3. 3. Measuring selection in sequence data  Natural selection can be positive, purifying or balancing  Positive and purifying selection are detected as a lower amount of variation than expected based on the neutral theory  Balancing selection maintains variation and preserves polymorphisms for a longer time than expected  Natural selection can be detected from:  Allele frequency spectrum (Tajima’s D, Fu and Li’s test)  Ratio of synonymous and non-synonymous mutations (McDonald- Kreitman test)  Tree topology (MDFM test)  Signs of natural selection can be confused with signs of unstable demography (changes in population size)  Strong signals of positive selection are rare but have been found to drive immune defense and perception genes
  4. 4. Chemical communication  Genes related to the sense of smell are expected to evolve during speciation  Sense of smell is a key part in chemical communication  Social insects, such as ants, are model organisms for chemical communication  They need communication for  Finding nutrition  Recognizing predators, nest mates and different castes  Organizing activities of the colony (social communication adds an extra layer of complexity compared to other animals) Photo source: http://www.icr.org/article/talking-ants-are-evidence-for-creation/
  5. 5. Sense of smell: Odorant binding, release and inactivation Source: Leal 2013
  6. 6. Gene families underlying chemical communication  OBPs (odorant binding proteins)  CSPs (chemosensory proteins)  OBP and CSP genes include both conserved and species specific genes  OBP genes have more variation than CSP genes  Species specific genes are under positive selection and are expected to have a role in speciation and adaptation  However, for the most part only conserved OBP and CSP genes are expressed specifically in the antennae  Conserved proteins can still vary between species in their ligand binding abilities  This work is focused on conserved OBP and CSP genes
  7. 7. Functions of studied genes OBP1  Strongly expressed in the antennae in ants and honeybee OBP10  Expressed in several tissues, but strongest expression in the head in ants  Expressed in pupae and in the brains of new adult honeybees CSP1 CSP7  Strongly expressed in the antennae in ants and honeybee  Binds queen pheromone in honeybee  Strongly expressed in the antennae in ants  Binds cuticular hydrocarbons → function in nest mate recognition
  8. 8. Do OBPs affect social organization?  Two social forms: monogyne (one queen) and polygyne (multiple queens)  OBP gene Gp9 differs between social forms: monogyne colonies have allele B, polygyne colonies have both alleles B and b  It was later found out that Gp9 is part of a supergene with over 600 genes  The supergene was caused by an inversion → there’s no recombination between the two alleles  Positive selection in b allele, partly in the binding pocket → natural selection has driven changes in the binding pocket affecting ligand binding abilities?  However, Gp9 is not expressed specifically in the antennae
  9. 9. Study Questions 1. How much sequence variation exists between closely related species that have diverged within the last 500 000 years? Is there within species variation in genes related to chemical communication? 2. Which evolution forces, natural selection or random drift, have caused this variation? Is CSP7, which is known to function in nest mate recognition, under positive selection? 3. Are there systematic differences detected between the two social forms (monogynous and polygynous) that would imply that these genes affect the social structure of an ant colony?
  10. 10. Materials and methods: Sequence data  7 Formica ant species  278 individuals  5-10 primers per gene Original samples Succesful samples Species Number of individuals Location CSP1 CSP7 OBP1 OBP10 F. aquilonia 22 Ru, Ir, Skot, 19 15 21 19 Fi, Skan F. cinerea 97 Fi (mono / poly) 54 57 70 48 F. exsecta 64 Fi (mono / poly), 14 34 53 49 En, Ro, Ru, Swe F. lugubris 18 Ir, Skan, Ru 13 9 14 11 F. polyctena 26 Ge, Skan, Ru 25 24 25 22 F. pratensis 4 Skan, Fi, Ru 2 0 0 0 F. rufa 23 Ge, Skan, Ru 21 16 21 5 F. truncorum 24 Fi (mono / poly) 11 24 24 24
  11. 11. Workflow  Raw sequence data given  Editing sequence data: assembling consensus sequences (CodonCode Aligner), MSA (MAFFT), annotation, phasing (PHASE)  Visualization of variation in the data: Phylogenetic tree (MEGA), PCA - Principal coordinate analysis (GenAlEx), FST - Fixation index (Arlequin), Nucleotide diversity (DnaSP), Fixed differences (DnaSP)  Evolutionary forces analyses: McDonald-Kreitman test (DnaSP), Tajima’s D (DnaSP), Fu and Li test (DnaSP), MFDM  Recombination analysis (HyPhy)  Transcription factor binding site prediction (PROMO)
  12. 12. Main Results: Fixed Differences (DnaSP)  Fixed difference = a site where all of one species’ nucleotides differ from those of the other species  Introns included  Gaps not included OBP10 Aq Cin Ex Lug Pol Ruf Trun F. aquilonia 0 F. cinerea 10 0 F. exsecta 8 10 0 F. lugubris 0 10 8 0 F. polyctena 0 9 7 0 0 F. rufa 0 10 8 0 0 0 F. truncorum 0 8 6 0 0 0 0 OBP1 Aq Cin Ex Lug Pol Ruf Trun F. aquilonia 0 F. cinerea 4 0 F. exsecta 6 5 0 F. lugubris 0 3 6 0 F. polyctena 0 3 5 0 0 F. rufa 0 2 4 0 0 0 F. truncorum 1 4 6 1 1 1 0
  13. 13. Fixed Differences (DnaSP) CSP1 Aq Cin Ex Lug Pol Pra Ruf Trun F. aquilonia 0 F. cinerea 5 0 F. exsecta 1 6 0 F. lugubris 0 5 1 0 F. polyctena 0 5 1 0 0 F. pratensis 1 6 1 0 0 0 F. rufa 0 6 4 0 0 1 0 F. truncorum 0 7 3 0 0 0 1 0  Main conclusion: F. cinerea and F. exsecta differ from other species, but there aren’t necessarily any fixed differences between the rufa group species CSP7 Aq Cin Ex Lug Pol Ruf Trun F. aquilonia 0 F. cinerea 7 0 F. exsecta 13 10 0 F. lugubris 0 7 13 0 F. polyctena 0 7 13 0 0 F. rufa 2 9 14 1 0 0 F. truncorum 1 8 13 0 0 1 0
  14. 14. Phylogenetic tree (MEGA)  Neighbor-joining method, pairwise deletion of gaps F. rufa group
  15. 15. Principal coordinate analysis  All pairwise distances between individuals (MEGA)  Principal coordinate analysis (GenAlEx) Coord.2 Coord. 1 Principal Coordinates (PCoA) F. aquilonia F. cinerea F. exsecta F. lugubris F. polyctena F. rufa F. truncorum Coord.2 Coord. 1 Principal Coordinates (PCoA): Rufa group F. aquilonia F. lugubris F. polyctena F. rufa F. truncorum
  16. 16. FST - Fixation Index (Arlequin): Populations Population CinKSK (mono) CinKU (poly) CinLI (mono) CinTA (poly) CinKSK (mono) 0.00000 CinKU (poly) 0.02057 0.00000 CinLI (mono) 0.08251 * 0.08090 * 0.00000 CinTA (poly) 0.03057 0.07729 ** 0.16077 ** 0.00000 Significance: * P<0.05, ** P<0.01, *** P<0.001 Population FTTv_mono FTTv_poly FTKop FTTv_mono 0,00000 FTTv_poly 0,20742 * 0,00000 FTKop 0,14737 0,22294 * 0,00000 Population Ex_mono Ex_mono 0,00000 Ex_poly 0,02876 F. cinerea F. truncorum F. exsecta
  17. 17. Evolution Analyses: CSP7 Species Number of Seqs. Alfa NI Fisher's exact test (p value) G test (p value) F. aquilonia 28 - - - - F. cinerea 106 -5,133 6,133 0,030521 * 0,01308 * F. exsecta 32 - - - - F. lugubris 17 - - - - F. polyctena 48 1,000 0,000 0,548263 - F. rufa 32 0,212 0,788 1,000000 0,84128 F. truncorum 22 1,000 0,000 1,000000 - McDonald-Kreitman test Significance: * 0.01<P<0.05; ** 0.001<P<0.01; *** P<0.001 NI > 1 purifying selection NI < 1 positive selection
  18. 18. Evolution analyses: CSP7 Species/Population Number of Seqs. Tajima’s D Fu & Li D Fu & Li F F. cinerea KSK 22 -1,54163 0,28059 0,30516 F. cinerea KU 24 -0,10769 0,85933 0,89423 F. cinerea LI 22 -0,28351 0,71813 0,39052 F. cinerea TA 22 -1,19166 0,52765 0,19472 F. exsecta Oulu 24 -1,9913 * -2,44664 * -2,73414 * F. truncorum mono 14 1,74339 # 0,67726 1,01617 F. truncorum poly 2 - - - Species Number of Seqs. Region P value Rm Sig. limit F. cinerea 81 intron 0,075# 30 0,003226 F. exsecta 24 intron 0,086957# 7 0,0125 F. truncorum 18 - - - 0,05 Tajima’s D and Fu and Li’s test MFDM Significances: # (P<0.10) * (P<0.05) ** (P<0.02) Purifying selection Positive selection Balancing selection
  19. 19. Summary of evolution tests Gene MK test Tajima’s D Fu and Li test MFDM CSP1 F. cinerea KU * F. exsecta + CSP7 F. cinerea - F. exsecta +/- F. exsecta +/- F. cinerea + F. truncorum * F. exsecta + OBP1 F. aquilonia + F. exsecta * F. lugubris + OBP10 F. exsecta +/- Significant results are marked with the species name and – for purifying selection, + for positive selection and * for balancing selection. MFDM results are not significant if recombination correction is taken into account.
  20. 20. Conclusions  Variation between species  Most differences were between F. cinerea, F. exsecta and the rufa group  Variation between the rufa group species was the same as variation within F. cinerea or F. exsecta  There are very few differences between the rufa group species → they are very closely related and cannot be separated into different species based on these genes  Possible reasons:  Speciation happened recently and differences haven’t accumulated in these gene yet  There’s still crossing between the species and the data included hybrids  Social forms  No fixed differences between mono and poly samples  Only one or a few SNPs present in some sequences of one social form and not in the other, most of them located in introns  However, pairwise FST values show that mono and poly populations of F. cinerea and F. truncorum differ significantly from each other
  21. 21. Conclusions  Evolutionary forces:  Test results are not necessarily consistent  Possible reasons:  Positive and purifying selection can affect different parts of the gene  Small amount of data for Tajima’s D and Fu and Li’s test  MK test considers only exons  Different time scales  Outgroups used in some tests  Tajima’s D and Fu and Li’s test are sensitive to population structure  Different tests detect different selection forces  Possible selection was detected in each gene  CSP7 has strongest indication of selection:  Purifying (MK test) and positive (MFDM) selection for F. cinerea  Purifying/positive (Tajima, Fu and Li) and positive (MFDM) selection for F. exsecta  Balancing selection (Tajima’s D) for F. truncorum  Positive selection would tie in with the nest mate recognition function of CSP7
  22. 22. References  Danty, E., Briand, L., Michard-Vanhee, C., Perez, V., Arnold, G., Gaudemer, O., Huet, D., Huet, J.C., Ouali, C., Masson, C. & Pernollet, J.C. 1999, "Cloning and expression of a queen pheromone-binding protein in the honeybee: an olfactory-specific, developmentally regulated protein", The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 19, no. 17, pp. 7468-7475.  Leal, W.S. 2013, "Odorant reception in insects: roles of receptors, binding proteins, and degrading enzymes", Annual Review of Entomology, vol. 58, pp. 373-391.  Li, H. 2011, "A new test for detecting recent positive selection that is free from the confounding impacts of demography", Molecular biology and evolution, vol. 28, no. 1, pp. 365-375.  Krieger, M.J. & Ross, K.G. 2002, "Identification of a major gene regulating complex social behavior", Science (New York, N.Y.), vol. 295, no. 5553, pp. 328-332.  Kulmuni, J., Wurm, Y. & Pamilo, P. 2013, "Comparative genomics of chemosensory protein genes reveals rapid evolution and positive selection in ant-specific duplicates", Heredity, vol. 110, no. 6, pp. 538-547.  McKenzie, S.K., Oxley, P.R. & Kronauer, D.J. 2014, "Comparative genomics and transcriptomics in ants provide new insights into the evolution and function of odorant binding and chemosensory proteins", BMC genomics, vol. 15, pp. 718-2164-15-718.  Smadja, C. & Butlin, R.K. 2009, "On the scent of speciation: the chemosensory system and its role in premating isolation", Heredity, vol. 102, no. 1, pp. 77-97.  Ozaki, M., Wada-Katsumata, A., Fujikawa, K., Iwasaki, M., Yokohari, F., Satoji, Y., Nisimura, T. & Yamaoka, R. 2005, "Ant nestmate and non-nestmate discrimination by a chemosensory sensillum", Science (New York, N.Y.), vol. 309, no. 5732, pp. 311-314.  Pelosi, P., Calvello, M. & Ban, L. 2005, "Diversity of odorant-binding proteins and chemosensory proteins in insects", Chemical senses, vol. 30 Suppl 1, pp. i291-2.

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