The Genetic Background of Chemical Communication and Chemosensory Gene

Evolution in AntsKatri Ketola

Master’s thesis

Supervisor: Jonna KulmuniAntzz groupDepartment of BiociencesUniversity of HelsinkiCentre of Excellence in Biological Interactions

Contents

1. Introduction2. Study questions3. Materials and methods4. Results5. Conclusions6. References

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

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/

Sense of smell: Odorant binding, release and inactivation

Source: Leal 2013

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

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

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

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?

Materials and methods: Sequence data

7 Formica ant species 278 individuals 5-10 primers per gene

Original samples Succesful samples

SpeciesNumber 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

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)

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

Fixed Differences (DnaSP)

CSP1 Aq Cin Ex Lug Pol Pra Ruf TrunF. 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 TrunF. 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

Phylogenetic tree (MEGA) Neighbor-joining method, pairwise deletion of gaps

F. rufa group

Principal coordinate analysis All pairwise

distances between individuals (MEGA)

Principal coordinate analysis (GenAlEx)

Principal Coordinates (PCoA)

F. aquiloniaF. cinereaF. exsectaF. lugubrisF. polyctenaF. rufaF. truncorum

Coord. 1

Coor

d. 2

Principal Coordinates (PCoA): Rufa group

F. aquiloniaF. lugubrisF. polyctenaF. rufaF. truncorum

Coord. 1

Coor

d. 2

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

Evolution Analyses: CSP7

SpeciesNumber of Seqs.

Alfa NIFisher'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 selectionNI < 1 positive selection

Evolution analyses: CSP7

Species/PopulationNumber of Seqs. Tajima’s D Fu & Li D Fu & Li F

F. cinerea KSK 22 -1,54163 0,28059 0,30516F. cinerea KU 24 -0,10769 0,85933 0,89423F. cinerea LI 22 -0,28351 0,71813 0,39052F. cinerea TA 22 -1,19166 0,52765 0,19472F. exsecta Oulu 24 -1,9913 * -2,44664 * -2,73414 *F. truncorum mono 14 1,74339 # 0,67726 1,01617F. truncorum poly 2 - - -

SpeciesNumber of Seqs. Region P value Rm Sig. limit

F. cinerea 81 intron 0,075# 30 0,003226

F. exsecta 24 intron 0,086957# 7 0,0125F. 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

Summary of evolution tests

Gene MK test Tajima’s D Fu and Li test MFDMCSP1   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.

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

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

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