Impact of the Diversification of Anopheles Mosquitoes in

Southeast Asia on Malaria:the Leucosphyrus Group as a Case

Study

Cathy WaltonUniversity of Manchester, UK

Malaria transmission (R0>1) depends on a complex interplay of interactions

between parasite, human, mosquito and the environment

Southeast Asia has greatest complexity of mosquito vectors

Sinka et al 2012

Distributed across mainland and insular Southeast Asia

Study taxon: Anopheles leucosphyrus Group

Includes human

and monkey feeding species

Forest dependent

Potential factors involved in diversification of the Anopheles

leucosphyrus group

• Pleistocene environmental change- The Refuge hypothesis (Haffner & Prance 2001;

Amazoniania)• Long-term environmental stability

- Tropics as an ‘ark’or a ‘cradle’• Host choice

Pleistocene environmental change

During interglacial periods:• High sea levels

• High levels of precipitation• Decreased aridity

• Spread of tropical forest throughout the smaller landmass

During glacial periods:• Low sea levels

• Reduced levels of precipitation• Increased aridity

• Replacement of tropical forest by grassland and savannah

Reconstruction of major

biomes for the last glacial

maximum

(Hope et al. 2004)

mtDNA two nuclear markers

Balabacensis

clade

Molecular Phylogeny of theLeucosphyrus Group

Chromogram dating origin of host switch

Balabacensis

clade

Summary scenario

An. baimaiiAn. dirus

An. scanloni

Morgan et al. (2010) Mol Ecol

The Isolation-with-Migration model

The IM model enables the estimation of divergence time between two populations, whilst allowing for, and quantifying, levels of gene flow between the populations.

Traditional methods for estimating divergence time

generally assume no gene flow has occurred since divergence.

Traditional methods do not take ancestral polymorphism into

account.

The IM model incorporates gene flow and separates it from the

confounding influence of ancestral polymorphism.

Hey & Nielsen 2004; Genetics

Estimated divergence times

Diverge times estimated using nuclear sequence data:

Divergence time (mya x u)

P

An. baimaii / An. dirus

An. baimaii / An. scanloni

An. dirus / An. scanloni

Divergence times estimated using mtDNA sequence data:

P

Divergence time (kya)

An. baimaii / An. dirus

An. baimaii N Thailand / India

An. baimaii N Thailand / S Thai

An. dirus N Thailand / Cambodia

Morgan et al. (2010) Mol Ecol

• Speciation events date to the Pleistocene.• Recent mtDNA divergence (100,000 ya) of sympatric An. dirus and An. baimaii is due to

mtDNA introgression • Lack of older mtDNA introgression indicates the prior isolation of these species supporting a role for allopatric fragmentation in the speciation of

An. dirus and An. baimaii

Comparative phylogeography: is Pleistocene environmental change

important generally in shaping genetic diversity?

Prediction of Refuge Hypothesis 1:congruent patterns of genetic diversity across

species

Simulate genetic data under specific models of evolutionary history and determine how well the observed data match the

simulated data using SPLATCHE (Spatial and Temporal

Coalescences in Heterogeneous Environments; Curat et al. 2004).

Used 1032 mtDNA sequences from 9 species.

Used altitude as a proxy for forest cover in 21,679 demes across

Southeast Asia

Morgan et al. 2011 (Mol Ecol)

Carrying capacities for forest-dependent species

GLACIAL

GLACIAL

INTER Morgan et al. 2011

(Mol Ecol)

Best fitting demographic models

•Red lines: 2 allopatric northern

refugia•Blue lines: 2

allopatric southern refugia.

Black lines: Null model – no refugia.

Models best fitting all species: post-glacial expansion from 2 allopatric northern

refugia.Morgan et al. 2011 (Mol Ecol)

Prediction of Refuge Hypothesis 2:simultaneous vicariance of geographic lineages across

species

Simultaneous divergence across several species inferred.

Divergence date estimated at 900,000 years ago.

•Tested using MsBayes (Hickerson et al 2006; BMC Bioinformatics)•Uses hierarchical ABC approach to estimated the number of divergence

events across multiple species

Morgan et al. 2011 (Mol Ecol)

Summary scenario

Restriction of populations to northern allopatric refugia during Pleistocene glacial periods

Divergence of geographic lineages due to isolation in allopatric refugia

Expansion of populations from allopatric refugia during the warmer and wetter interglacial periods

Formation of a suture zone, as lineages from allopatric refugia meet

Conclusions so far…•Pleistocene environmental change (forest fragmentation) has played an important role in the generation of genetic diversity.

•In most cases, recurrent gene flow has probably prevented speciation.

Do the distinct genetic communities in east and west of mainland Southeast Asia differ in factors relating to malaria

transmission?

•Dating species divergence indicates that speciation is generally older and related to ecological differences: host choice (Leucosphyrus Group); larval habitat (Maculatus Group), seasonal abundance (Maculatus Group)

Is there genetic heterogeneity within species within east or west Southeast Asia?

Are inversions in Anopheles baimaii involved in local adaptation?

X 2R2L 3R 3L

X 2R2L 3R 3L

X 3Rb2R 2L 3L

A. dirus A. cracens

3Rb Xa 2La2Ra 3Ra 3La

A. scanloni A. baimaii

• Anopheles baimaii Anopheles baimaii is a major malaria vector in NE India, Myanmar is a major malaria vector in NE India, Myanmar

and Thailand and Thailand • An. baimaiiAn. baimaii is polymorphic for 5 inversions (2Ra, 3Ra, 3La - up to is polymorphic for 5 inversions (2Ra, 3Ra, 3La - up to

50% freq.) 50% freq.) • An. baimaii An. baimaii is able to inhabit a wide latitudinal range from is able to inhabit a wide latitudinal range from

northern Myanmar down to peninsular Thailand. Some populations northern Myanmar down to peninsular Thailand. Some populations

also breed in village wells.also breed in village wells.•Are inversions in involved in local adaptation?Are inversions in involved in local adaptation?

Polymorphic inversions

Inversions inhibit recombinationInversion loop in an inversion

heterozygoteSingle recombination =>

unbalanced gametes and meiotic malsegregation

Double recombination and recombination outside inversion

still possible

Net effect: restricted recombination particularly

around inversion breakpoints

Inversions likely play a role in local adaptation and speciation

Linkage disequilibrium (LD) and inversions

A

B

D

C

E

• Allelic combinations kept together in alternative karyotypes i.e. high linkage disequilibrium (LD)

• Differences accumulate in alternative arrangements.

• High divergence between standard and inverted arrangements

• High heterozygosity in these genomic regions

A landscape-genomics approach to study inversions

• 79 unique collection sites

•147 samples and 3197 RAD

(restriction-site associated DNA)

loci (~90 bp in length) with < 20%

data missing

•Use pair-wise measures of linkage disequilibrium (LD) from RAD loci and network analysis to find ‘high LD clusters’ - loci from genomic regions of low recombination

Locus1 Locus2 rr22

L415 L664 0.9887731L200 L4092 .9815159L1029 L2909 0.9737809L5019 L9769 0.9689778L5083 L5456 0.9627664L6864 L7662 0.9621028L5456 L6514 0.9564581L5019 L5083 0.9547414L5083 L9769 0.954263L3279 L9769 0.9524455

LD network analysis (LDna)

Measure of LD = r r22 (~2.5 million pairwise values)Edges = values above the LD cut off, vertices= loci

Locus1 Locus2 r2

L415 L664 0.9887731L200 L4092 0.9815159L1029 L29090.9737809L5019 L97690.9689778L5083 L54560.9627664

LD network analysis (LDna)

Results LD cluster analysis,landscape genetics data

LDC rr22 cut off Number of loci*

1 0.33 33

2 0.44 32

3 0.33 23

4 0.35 15

5 0.29 12

* some filtering done based on rr2 2 and and number of edgesnumber of edges

LD cut off = 0.45, 1352 rr22 values

LDC1

LDC2

LDC3

LDC4

LDC5

Maping of LD clusters to the An.

dirus genome scaffolds and An. baimaii linkage

map

Evidence for Selection

Geographical structuring is stronger for LDC loci than for other, putatively neutral, loci

Acknowledgements

• Petri Kemppainen, Devojit Sarma, Anil Prakash, Pradya Somboon, Yan Naung Maung Maung, Thaung Hlaing, DR Battacharyya, Jagadish Mahanta

• Katy Morgan, Sam O’Loughlin

• Chang Moh Seng, Jeffery Hii, Ralph Harbach

Estimated migrationNuclear sequence DNA migration rates:

Migration rate (m x u)

P

An. dirus to baimaii

An. scanloni to dirusAn. baimaii to dirus

Microsatellite migration rates:

P

An. dirus to scanloniAn. scanloni to baimaiiAn. baimaii to scanloni

Migration rate (m x u)

An. dirus to baimaii

An. scanloni to dirusAn. baimaii to dirus

An. dirus to scanloniAn. scanloni to baimaiiAn. baimaii to scanloni

Morgan et al. (2010) Mol Ecol

• Uni directional gene

flow from An. scanloni to An.

dirus

• This is consistent with

other data indicating the

ecological divergence and

speciation of An. scanloni

from An. dirus

• LD clusters 1-3 form 3 distinct groups

• LD clusters 4 and 5 form only 2 distinct groups

Discriminant Analysis of Principle Components

An. annularis

An. philippinensis

An. minimus

An. splendidusMorgan et al. 2011 (Mol Ecol);

Chen et al. 2011(Heredity)

LD network analysis (LDna)

LD based on 147 wild caught individuals throughout the distribution range

Inversions are thought to play a major role in

adaptationInversion polymorphism in Anopheles baimaii using

Restriction-site Associated DNA (RAD) sequence Data

• Higher heterozygosity in the heterokaryotypes compared to k1 and k2

• The LD present in the total sample is removed within k1 and k2

P1f P1m

F1fx

F1m

F1fx

F1m

F1fx

F1m

F1fx

F1m

49 F2 individuals18 F2 individuals, rr22 cut off =

0.62

LDC1LDC1

Linkage map for putative chromosome 2 (where LDC1 Linkage map for putative chromosome 2 (where LDC1

loci are found)loci are found)

LD clusters mapped to

linkage groups

• The geographical structuring varies between LDC loci

• LDC loci differ in their geographical structuring from non-LDC loci

Conclusions• Network analysis of linkage disequilibrium can be used to identify loci involved in inversions in de novo population genomic data sets

• Three major inversions were detected by RADs in An. baimaii, consistent with earlier polytene chromosome work

• The inversions are maintained by natural selection in relation to some aspect of the environment

Cox-Singh & Singh 2008