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.
The Genetic Background of Chemical Communication and Chemosensory Gene Evolution in Ants
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
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. 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. Sense of smell: Odorant binding,
release and inactivation
Source: Leal 2013
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. 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. 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. 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. 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. 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. 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. 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
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
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. 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. 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. 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. 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. 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.
