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synonymous sites, and yet show an excess of between-species

Vol 437|20 October 2005|doi:10.1038/nature04107
LETTERS
Adaptive evolution of non-coding DNA in Drosophila
Peter Andolfatto1
A big fraction of eukaryotic genomes consists of DNA that isn’t
translated into protein sequence, and little is thought about its
practical significance. Right here I show that a number of courses of noncoding DNA in Drosophila are evolving significantly slower than
synonymous websites, and yet show an excess of between-species
divergence relative to polymorphism compared with
synonymous websites. The previous is a trademark of selective constraint,
however the latter is a signature of adaptive evolution, resembling
common patterns of protein evolution in Drosophila1,2. I estimate
that about 40–70% of nucleotides in intergenic areas, untranslated parts of mature mRNAs (UTRs) and most intronic DNA
are evolutionarily constrained relative to synonymous websites. Nevertheless, I additionally use an extension to the McDonald–Kreitman test3 to
show that a substantial fraction of the nucleotide divergence in
these areas was pushed to fixation by optimistic choice (about
20% for many intronic and intergenic DNA, and 60% for UTRs).
On the idea of these observations, I recommend that a big fraction of
the non-translated genome is functionally necessary and topic
to each purifying choice and adaptive evolution. These outcomes
indicate that, though optimistic choice is clearly an necessary aspect
of protein evolution, adaptive adjustments to non-coding DNA would possibly
have been significantly extra widespread within the evolution of
D. melanogaster.
The excessive diploma of protein sequence similarity between phenotypically diverged species has led some to suggest that regulatory
evolution could also be of significantly extra significance than protein
evolution4,5. Though most of the everyday eukaryotic genome is
comprised of non-coding DNA, comparatively little is thought
concerning the evolutionary forces performing on it. Some unknown fraction
of the non-translated genome is presumed to be essential for the
regulation of gene expression. Most of our direct information concerning the evolution of regulatory parts comes from a handful of
direct practical studies5,6. A second, oblique method relies on
comparative genomics7
. The rationale for this second method is
that if newly arising mutations are sometimes detrimental to gene
perform, functionally necessary elements of the genome are anticipated to
evolve extra slowly than these missing function8–11.
There are some limitations to the comparative genomics
method. First, a given genomic area could be conserved owing
merely to a decrease mutation price12. Second, recognized regulatory
parts don’t appear to be notably properly conserved as a category,
not less than in Drosophila10. This fnding means that taking an method
based mostly on sequence conservation alone could result in a biased view of
regulatory evolution. Performance of DNA sequences implies that
they are often topic to each damaging and optimistic choice. If a
signifcant fraction of divergence between species noticed in noncoding DNA is positively chosen moderately than selectively impartial or
constrained, this might result in underestimates of the practical
significance of non-coding DNA and trigger researchers to miss
the contribution of arguably essentially the most fascinating class of mutations
in genome evolution—these refecting adaptive variations between
populations and species.
These limitations could be overcome by combining comparative
genomic analyses with population-level variability data1–three,13. To
assess the mode of choice performing on non-coding DNA, I’ve
analysed new and beforehand printed polymorphism information for 35
coding fragments (common size 667 base pairs (bp)) and 153 noncoding fragments (common size 426 bp) scattered throughout the
X chromosome of D. melanogaster (see Supplementary Supplies 1).
To estimate ranges of between-species divergence, I’ve in contrast
D. melanogaster with its intently associated sibling species, D. simulans.
On the idea of the present Drosophila genome annotation (launch
four), I separated the surveyed fragments into a number of classes which are
prone to differ within the depth and mode of choice performing on them
(see Desk 1). It’s obvious that the majority non-coding DNA evolves
significantly slower than synonymous websites (that’s, websites in proteincoding sequences at which mutations don’t lead to amino acid
substitutions; Desk 1). That is the case for introns and UTRs (see additionally
refs 14–16), in addition to intergenic DNA, a lot of which is way from the
closest recognized gene (see Supplementary Supplies 1). I estimate ranges
of constraint in Drosophila non-coding DNA to be 40% for introns,
50% for intergenic areas (IGRs), and 60% for UTRs (Desk 2).
These are all significantly greater than earlier estimates from a
selection of species comparisons11,15–18 . The non-coding DNA
surveyed can be usually much less polymorphic than synonymous websites
in D. melanogaster (Desk 1; p , 10210, Wilcoxon two-sample take a look at for
UTRs and intronsþIGRs versus synonymous websites). Thus, each
polymorphism and divergence in non-coding DNA are signifcantly
lowered relative to synonymous websites in D. melanogaster.
Decreased ranges of polymorphism and divergence in non-coding
DNA resemble common patterns of protein evolution19 and recommend
that non-coding DNA is both functionally constrained or is topic
to a decrease mutation price than synonymous websites. One method to
distinguish between these two fashions is to contemplate the distribution
of polymorphism frequencies. Damaging choice performing on polymorphic variants will maintain them at decrease frequencies in a inhabitants
than anticipated in the event that they had been impartial20. In step with this prediction,
the distribution of polymorphism frequencies at each non-coding
DNA and amino acid websites is skewed in direction of uncommon frequencies relative
to synonymous polymorphisms (as indicated by a extra damaging
Tajima’s D worth20, Fig. 1). The distribution of Tajima’s D values for
non-synonymous websites amongst loci is negatively skewed relative to
synonymous websites, suggesting that amino acid polymorphisms are
topic to purifying choice (Fig. 1; p ¼ zero.002, Wilcoxon twosample take a look at versus synonymous websites). Right here I show that this
identical sample extends to polymorphisms in non-coding DNA
(Fig. 1; Wilcoxon take a look at versus synonymous websites: pooled non-coding,
p ¼ zero.0001; UTRs, p , zero.0001; introns, p ¼ zero.zero01; IGRs, p ¼ zero.005).
This fnding, along with the noticed discount in polymorphism
and divergence, implies that mutations in non-coding DNA are
topic, on common, to stronger damaging choice than synonymous
websites (see additionally Supplementary Supplies 2).
Does selective constraint alone account for patterns of non-coding
DNA evolution? McDonald and Kreitman3 have proposed a frame1
Part of Ecology, Conduct and Evolution, Division of Organic Sciences, College of California San Diego, La Jolla, California 92093, USA.
1149
© 2005 Nature Publishing Group
LETTERS NATURE|Vol 437|20 October 2005
Desk 1 | Polymorphism and divergence in coding and non-coding DNA of D. melanogaster
Mutation class No. of areas Imply p* Imply Dxy† D‡ P§ pk P’{ p#
Synonymous 35 2.87 13.59 604 502 2 323 2
Non-synonymous 35 zero.18 1.72 260 115 ,1026 52 ,1029
Non-coding 153 1.06 5.94 three,168 2,386 zero.14 1,295 ,1023
UTRs 31 zero.54 four.54 471 246 ,1025 107 ,10211
50
UTRs 18 zero.61 5.41 328 160 ,1025 71 ,1029
30
UTRs 13 zero.45 three.35 143 86 zero.034 36 ,1024
Introns 72 1.25 6.71 1,564 1,221 zero.39 675 zero.zero10
IGRs 50 1.11 5.72 1,133 919 .zero.5 513 zero.zero59
pIGRs 20 1.29 6.58 500 400 .zero.5 237 zero.25
dIGRs 30 zero.99 5.18 633 519 .zero.5 276 zero.041
IntronsþIGR 122 1.19 6.25 2,697 2,140 zero.50 1,188 zero.013
Mutation courses: synonymous websites, non-synonymous websites, untranslated transcribed areas (UTRs), intergenic areas inside 2 kb of a gene (pIGRs), intergenic areas greater than four kb away
from a gene (dIGRs).
*p is the weighted common within-species pairwise variety per 100 websites.
†Dxy is the weighted common pairwise divergence per 100 websites between D. melanogaster and D. simulans, corrected for a number of hits (Jukes–Cantor). Dxy at fourfold degenerate synonymous
websites is 12.zero%.
‡D is the estimated quantity of fxed variations between species utilizing a Jukes–Cantor correction for a number of hits (see Strategies).
§P is the quantity of intraspecifc polymorphisms.
kMcDonald–Kreitman take a look at of likelihood utilizing all polymorphisms.
{P’ is the quantity of intraspecifc polymorphisms excluding singletons.
#McDonald–Kreitman take a look at of likelihood excluding singleton polymorphisms. Chances are from two-tailed Fisher’s actual assessments and assume websites are impartial. These are prone to be solely
slight underestimates given possible ranges of intragenic recombination (see Supplementary Supplies 2).
work to tell apart neutrality (and variation in mutation price) from
damaging and optimistic choice within the genome. Their method
compares ranges of polymorphism inside and divergence between
species for a putatively chosen class of websites within the genome to a
impartial customary. If lowered ranges of polymorphism and divergence
in non-coding DNA could be defined by a decrease mutation price, the
ratio of polymorphism to divergence must be much like that for
synonymous websites. Constructive choice will improve divergence relative
to polymorphism at chosen websites, whereas damaging choice is
anticipated to end result within the reverse sample21. Though this framework
was initially designed to detect choice inside protein-coding
genes, it may be generalized to contemplate arbitrary courses of putatively
chosen websites sampled from a number of genomic areas, together with
non-coding DNA (see Supplementary Supplies 2). Utilizing all polymorphisms, there’s a signifcant excess of divergence for amino
acid substitute websites (p ¼ 5 £ 1027
) and for UTRs (p ¼ three £ 1026
,
two-tailed Fisher’s actual take a look at) however not at different subclasses of noncoding DNA (Desk 1). This preliminary evaluation means that,
much like the sample noticed for amino acid substitutions1,2,a
signifcant proportion of nucleotide divergence at UTRs was additionally
pushed to fxation by optimistic choice.
The presence of weakly negatively chosen variants in polymorphism can masks the signature of adaptive evolution within the
genome1,22, making the McDonald–Kreitman take a look at very conservative.
As I’ve proven above that polymorphic variants in non-coding
DNA are topic to stronger selective constraint than synonymous
websites (Desk 1 and Fig. 1), negatively chosen variants contributing to
polymorphism in non-coding DNA are prone to be an element limiting
Desk 2 | Functionally related nucleotides in non-coding DNA
Class C (%)* a(%)† p (a # zero)‡ FRN (%)§
UTRs 60.four 57.5 ,1023 83.2
50
UTRs 52.9 60.eight ,1023 80.9
30
UTRs 70.7 52.9 ,1023 86.2
Introns 39.5 19.three zero.007 51.2
IGRs 49.three 15.three zero.036 57.1
pIGRs 40.6 11.four zero.165 47.four
dIGRs 54.6 18.5 zero.zero19 63.zero
Introns þ IGR 44.2 17.6 zero.013 54.zero
*Constraint (C) is estimated relative to fourfold degenerate synonymous websites.
†a is the estimated fraction of divergence pushed by optimistic choice.
‡Chances (a # zero) have been adjusted for results of linkage inside loci (see
Supplementary Supplies 2.5).
§FRN is the inferred fraction of functionally related nucleotides given ranges of constraint
and a (that’s, FRN < C þ (1 2 C)a).
1150
energy to detect optimistic choice. This downside could be partially
overcome by contemplating solely these mutations that aren’t uncommon
in a pattern from each the impartial and putatively chosen courses
(see ref. 23 and Supplementary Supplies 2). Making use of this method
reveals a signifcant excess of divergence in UTRs and in most
different courses of non-coding DNA relative to synonymous websites
(Desk 1; UTRs, p ¼ 5 £ 10212; introns, p ¼ zero.01; dIGRs, p ¼ zero.04;
intronsþIGRs, p ¼ zero.01). A Hudson–Kreitman–Aguade´ (HKA)
take a look at24 additionally gives statistical help for a lowered ratio of polymorphism to divergence for non-coding DNA relative to synonymous websites (UTRs, p , 1023
; pooled introns and IGRs, p ¼ zero.02; see
Supplementary Supplies 2). Collectively, these outcomes show that a
signifcant fraction of the divergence in UTRs, introns and intergenic
DNA was in all probability pushed to fxation by optimistic choice.
To quantify the depth and the relative significance of optimistic
choice in shaping the evolution of non-coding DNA, I apply two
extensions of the McDonald–Kreitman approach2,13. First I estimate
a, defned because the proportion of the divergence between species that
was pushed by optimistic selection2
. I estimate that about 20% of the
nucleotide divergence in introns and intergenic DNA was pushed to
fxation by optimistic choice, and about 60% for UTRs (Fig. 2a and
Desk 2). Utilizing a hierarchical bayesian framework13, I estimate the

Determine 1 | Imply Tajima’s D values for coding and non-coding DNA. Means
throughout loci are given with bars indicating two customary errors. The
expectation of D underneath the impartial mannequin is proven as a dotted line. Syn,
synonymous websites; NonSyn, non-synonymous websites; NonCod, pooled
non-coding DNA.
© 2005 Nature Publishing Group
NATURE|Vol 437|20 October 2005 LETTERS
choice depth on non-coding DNA (together with UTRs, introns
and IGRs) to be optimistic and signifcantly completely different from zero in most
circumstances (Fig. 2b; Supplementary Supplies three). As this bayesian method
assumes that segregating and fxed variants are topic to the
identical course and depth of choice, it’s prone to underestimate
the magnitude of 2Nes (the depth of choice) for nucleotide
substitutions fxed by optimistic choice (see Supplementary
Supplies 2).
Proof that a signifcant fraction of non-coding DNA is functionally necessary is rising from a range of comparative
genomic research. Nevertheless, my fnding of a big fraction of positively
chosen divergence implies that ‘evolutionary constraint’ will considerably underestimate the fraction of functionally related nucleotides as a result of it ignores the contribution of positively chosen
mutations to divergence. For the instance of UTRs, I estimate
evolutionary constraint to be 60%. Nevertheless, as 58% of the noticed
divergence was positively chosen, this suggests that 83% of nucleotides in UTRs are the truth is functionally related. Likewise, the fraction
of functionally related nucleotides in introns and IGRs is prone to be
about 10–20% greater than recommended by ranges of constraint alone
(Desk 2).
How frequent is adaptation within the Drosophila genome? Tough
calculations (see Supplementary Supplies four) recommend that there has
been about one adaptive amino acid substitution each 20 years since
the cut up of D. melanogaster and D. simulans (see additionally ref. 2). Though
that is substantial, take into account that the full quantity of websites contained in

Determine 2 | Quantifying adaptive divergence and choice depth.
a, Estimates of a, the fraction of nucleotide divergence pushed by optimistic
choice. Error bars point out 90% confidence limits decided by a nonparametric bootstrapping. Estimated chances that a $ zero corrected for
partial linkage are given in Desk 2. b, Estimates of the depth of choice
(2Nes) performing on non-synonymous and non-coding DNA websites. Error bars
point out 90% confidence limits decided by simulation (see Strategies).
Singleton polymorphisms had been excluded in estimates of a and 2Nes (see
Supplementary Supplies three). Abbreviations as in Fig. 1.
introns, intergenic areas and UTRs far outweighs the quantity of
codons within the Drosophila genome25. I estimate that UTRs alone
contribute as a lot to adaptive divergence between species as do
amino acid adjustments, and the summed contribution of non-coding
DNA to adaptive divergence may simply be an order of magnitude
bigger. These fndings help earlier intuitions4,5 concerning the nice
significance of regulatory adjustments in evolution.
METHODS
Information. All loci used on this examine, beforehand printed or newly collected, are
X-linked genomic fragments, with a pattern measurement of 12 D. melanogaster alleles
sampled from a inhabitants in Zimbabwe, and a single D. simulans sequence.
For coding DNA (synonymous and non-synonymous websites), I collected polymorphism and divergence in 31 coding areas chosen randomly with respect
to gene perform, and 51 non-coding areas (27 intergenic and 24 untranslated
transcribed areas). Details about these 82 loci and primers used could be
present in Supplementary Supplies 1. I used polymerase chain response (PCR) to
amplify 700–800-bp areas from genomic DNA extracted from single male fies,
eliminated primers and nucleotides utilizing exonuclease I and shrimp alkaline
phosphatase, and sequenced the cleaned product on each strands utilizing Large-Dye
(Model three, Utilized Biosystems). Sequences had been collected on an ABI 3730
capillary sequencer and had been aligned and edited utilizing this system Sequencher
(Gene Codes).
To the 82 areas surveyed above, I added beforehand printed information for
loci that had the identical pattern measurement (n ¼ 12 fies) and had been surveyed in related
samples from Zimbabwe26,27. A quantity of the beforehand printed loci26 had
to be functionally reassigned when in comparison with Launch four of the annotated
D. melanogaster genome (http://fybase.bio.indiana.edu/annot/dmel-release4.
html). I excluded any loci in areas of lowered recombination (see under).
Beforehand printed loci ftting these necessities had been processed into 106
fragments (four coding, 7 UTR, 23 intergenic and 72 intron). Thus, the full
quantity of areas surveyed on this evaluation is 188. Alignments for every locus
can be found upon request. A reciprocal best-hit BLAST protocol was used to
confrm that the areas in contrast between D. melanogaster and D. simulans
are certainly orthologous. Additional gaps had been launched into some alignments in
areas that had been notably diffcult to align. This process is prone to
upwardly bias estimates of constraint, however is conservative with respect to
detecting optimistic choice.
Analyses. The estimated quantity of synonymous websites, non-synonymous websites,
common pairwise variety (p), common pairwise divergence (Dxy), in addition to
counts of the quantity of polymorphic websites (P) had been carried out utilizing DnaSP
software program (model four; http://www.ub.es/dnasp/) and Perl code written by P.A. The
quantity of divergent websites (D) was estimated as Dxy 2 p utilizing a Jukes–Cantor
correction for a number of hits. Multiply hit websites had been included within the evaluation however
insertion–deletion polymorphisms and mutations overlapping alignment gaps
had been excluded. Derived mutations had been polarized utilizing a single D. simulans
sequence and assuming customary parsimony standards. Tajima’s D worth20 was
estimated from the quantity of polymorphisms and p.
On this examine, I assume that synonymous websites are extra impartial than putatively
chosen courses of websites (see Supplementary Supplies 2.2). I separated noncoding DNA into subclasses that I anticipated a priori to expertise completely different
choice pressures: 50 and 30
untranslated transcribed areas (UTRs), introns,
intergenic areas inside 2 kilobases (kb) of a gene (proximal intergenic areas,
pIGRs), and intergenic areas additional than four kb from the closest gene (distal
intergenic areas, dIGRs). My pattern of intron fragments is biased in direction of
introns bigger than the median intron measurement (86 bp) (ref. 28), making estimates of
constraint greater than anticipated with a random pattern of introns14. Nevertheless,
95% of intronic DNA is contained inside introns longer than the median measurement28,
and thus my estimate refects ranges of constraint for many intronic DNA within the
genome.
For comparisons of polymorphism and divergence between synonymous
websites and non-coding DNA, it was essential to pool websites in every class. I estimate
evolutionary constraint relative to fourfold degenerate synonymous websites utilizing
the method in ref. 15, besides that I pooled courses of websites and used a Jukes–
Cantor correction for a number of hits19. Given variations in base composition
between coding and non-coding areas, I investigated doable variations in
mutations charges owing to the 16 doable adjacent-base contexts of nucleotides
(recommended by A. Kondrashov). There was no signifcant impact of adjacent-base
context on charges of divergence (see Supplementary Supplies 5).
I estimate the proportion of divergence pushed by optimistic selection1,2 as
a ¼ 1–(DSPX/DXPS), the place S denotes synonymous (that’s, putatively impartial)
P P n n websites, X denotes putatively chosen websites, and D ¼ i¼1 and P ¼ Pi D , i i¼1
1151
© 2005 Nature Publishing Group

LETTERS NATURE|Vol 437|20 October 2005
the place Di and Pi are the quantity of divergent and polymorphic variants at locus i,
respectively, and n is the quantity of loci of class S or X. Confdence limits on a
had been estimated utilizing a typical non-parametric bootstrapping process,
assuming websites are impartial. The problem of non-independence of websites inside
surveyed fragments is addressed in Supplementary Supplies 2.5. For consistency, a was estimated for non-synonymous websites in the identical means. The depth
of choice (2Nes) was estimated on putatively chosen courses (pooling websites as
above) utilizing a hierarchical bayesian methodology (http://cbsuapps.tc.cornell.edu)13.
To keep away from issues related to large-scale variation in recombination charges, I
restricted my survey of loci to areas of the X chromosome which have the very best
charges of recombination29 (see Supplementary Fig. 1.1).
Obtained 23 Might; accepted 2 August 2005.
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Supplementary Data is linked to the net model of the paper at
www.nature.com/nature.
Acknowledgements The writer thanks D. Bachtrog for in depth feedback on
the manuscript and assist with information high quality points, C. Bustamante and Ok. Thornton
for offering code, and B. Ballard for Zimbabwe fy strains. P. Haddrill and
Ok. Thornton assisted in designing primers for distal intergenic and coding
areas, respectively. Because of B. Fischman for technical assist, A. Betancourt,
A. Kondrashov, A. Poon, D. Presgraves, M. Przeworski and S. Wright for important
feedback on the manuscript, and L. Chao and J. Huelsenbeck for recommendation.
Thanks additionally to the Washington College Genome Sequencing Heart for
offering unpublished D. simulans sequences. This work was funded partly by a
analysis grant from the Biotechnology and Organic Sciences Analysis Council
(UK) to P.A. The writer is supported by an Alfred P. Sloan Fellowship in
Molecular and Computational Biology.
Writer Data Reprints and permissions data is accessible at
npg.nature.com/reprintsandpermissions. The writer declares no competing
fnancial pursuits. Correspondence and requests for supplies must be
addressed to P.A. (pandolfatto@ucsd.edu).
1152
© 2005 Nature Publishing Group

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