gene family sizes at the tips of the species tree
due to annotation or assembly errors and is not
sensitive to inclusion or exclusion of taxa affecting the root age of the tree nor to the exclusion
of taxa with the poorest assemblies and gene
sets (fig. S10 and tables S22 and S23). Examples
include expansions of cuticular proteins in An.
arabiensis and neurotransmitter-gated ion channels in An. albimanus (table S24).
The evolutionary dynamism of Anopheles genes
extends to their architecture. Comparisons of
single-copy orthologs at deeper phylogenetic
depths showed losses of introns at the root of
the true fly order Diptera and revealed continued losses as the group diversified into the
lineages leading to fruit flies and mosquitoes.
However, anopheline orthologs have sustained
greater intron loss than drosophilids, leading
to a relative paucity of introns in the genes of
extant anophelines (fig. S11 and table S25). Com-
parative analysis also revealed that gene fu-
sion and fission played a substantial role in the
evolution of mosquito genes, with apparent re-
arrangements affecting an average of 10.1% of
all genes in the genomes of the 10 species
with the most contiguous assemblies (fig. S12).
Furthermore, gene boundaries can be flexible;
whole genome alignments identified 325 can-
didates for stop-codon readthrough (fig. S13 and
Because molecular evolution of protein-coding
sequences is a well-known source of phenotypic
change, we compared evolutionary rates among
different functional categories of anopheline orthologs. We quantified evolutionary divergence in
terms of protein sequence identity of aligned orthologs and the dN/dS statistic (ratio of nonsynonymous to synonymous substitutions) computed
using PAML ( 12, 20). Among curated sets of genes
linked to vectorial capacity or species-specific traits
against a background of functional categories defined by Gene Ontology or InterPro annotations,
odorant and gustatory receptors show high evolutionary rates and male accessory gland proteins
exhibit exceptionally high dN/dS ratios (Fig. 3, figs.
S14 and S15, and tables S27 to S29). Rapid diver-
gence in functional categories related to malaria
transmission and/or mosquito control strategies
led us to examine the genomic basis of several
facets of anopheline biology in closer detail.
Insights into mosquito biology and
Mosquito reproductive biology evolves rapidly
and presents a compelling target for vector control. This is exemplified by the An. gambiae male
accessory gland protein (Acp) cluster on chromosome 3R ( 21, 22), where conservation is mostly
lost outside the An. gambiae species complex
(fig. S16). In Drosophila, male-biased genes such
as Acps tend to evolve faster than loci without
male-biased expression ( 23–25). We looked for
a similar pattern in anophelines after assessing
each gene for sex-biased expression using micro-array and RNAseq data sets for An. gambiae ( 12).
In contrast to Drosophila, female-biased genes
show dramatically faster rates of evolution across
the genus than male-biased genes (Wilcoxon rank
sum test, P = 5 × 10− 4) (fig. S17).
Differences in reproductive genes among
anophelines may provide insight into the origin and function of sex-related traits. During
10,000 20,000 0
Number of Genes:
0.5 0 1
Fig. 1. Geography, vector status, molecular phylogeny, gene orthology,
and genome alignability of the 16 newly sequenced anopheline mosqui-
toes and selected other dipterans. (A) Global geographic distributions of
the 16 sampled anophelines and the previously sequenced An. gambiae and
An. darlingi. Ranges are colored for each species or group of species as shown
in (B), e.g., light blue for An. farauti. (B) The maximum likelihood molecular
phylogeny of all sequenced anophelines and selected dipteran outgroups.
Shapes between branch termini and species names indicate vector status
(rectangles, major vectors; ellipses, minor vectors, triangles, nonvectors) and
are colored according to geographic ranges shown in (A). (C) Bar plots show
total gene counts for each species partitioned according to their orthology
profiles, from ancient genes found across insects to lineage-restricted and
species-specific genes. (D) Heat map illustrating the density (in 2-kb sliding
windows) of whole-genome alignments along the lengths of An. gambiae
chromosomal arms: from white where An. gambiae aligns to no other species
to red where An. gambiae aligns to all the other anophelines.