Pervasive Adaptive Evolution among Interactors of the Drosophila Hybrid
Inviability Gene, Nup96
Daven C. Presgraves* and Wolfgang Stephan*
*Section of Evolutionary Biology, Biocenter, University of Munich, Planegg-Martinsried, Germany; and
Department of Biology, University of Rochester
Nup96 is involved in a lethal hybrid incompatibility between 2 fruit fly species, Drosophila melanogaster and Drosophila
simulans. Recurrent adaptive evolution drove the rapid functional divergence of Nup96 in both the D. melanogaster and
the D. simulans lineages. Functional divergence of Nup96 between these 2 species is unexpected as Nup96 encodes part of
the Nup107 subcomplex, an architectural component of nuclear pore complexes, the macromolecular channels in nuclear
envelopes that mediate nucleocytoplasmic traffic in all eukaryotes. Here we study the evolutionary histories of 5 of
Nup96’s protein interactors—3 stable Nup107 subcomplex proteins (Nup75, Nup107, and Nup133) and 2 mobile nucleoporins (Nup98 and Nup153)—and show that all 5 have experienced recurrent adaptive evolution. These results are consistent with selection-driven coevolution among molecular interactors within species causing the incidental evolution of
incompatible interactions seen in hybrids between species. We suggest that genetic conflict–driven processes may have
contributed to the rapid molecular evolution of Nup107 subcomplex genes.
Introduction
New species arise through the evolution of reproductive isolation (Dobzhansky 1937; Coyne and Orr 2004).
Among diverging allopatric Drosophila species, prezygotic
isolation (e.g., sexual isolation) and intrinsic postzygotic
isolation (e.g., hybrid sterility or inviability) evolve gradually and do so at roughly equal rates (Coyne and Orr 1989,
1997). The evolution of intrinsic postzygotic isolation corresponds to the accumulation of alleles that have deleterious
epistatic interactions in species hybrids—so-called hybrid
incompatibilities (Dobzhansky 1937; Muller 1940, 1942;
Orr 1995). Many classical genetic analyses have confirmed
that alleles fixed in 1 species are often functionally incompatible with the genetic backgrounds of closely related species (Coyne and Orr 2004). However, the normal functions
and evolutionary histories of hybrid incompatibility genes
have been determined in only a few cases, in part because
the particular loci involved have proven difficult to identify
(Orr et al. 2004; Wu and Ting 2004; Orr 2005).
In Drosophila, just 4 hybrid incompatibility genes have
been identified. Ods is a duplicated homeobox transcription factor that is misexpressed in the testes of Drosophila
simulans–Drosophila mauritiana hybrids, causing male
sterility (Ting et al. 1998; Barbash and Ashburner 2003;
Barbash et al. 2003; Ting et al. 2004). Hmr is a single-copy
gene encoding a Myb-related protein with putative DNAbinding properties, which causes lethality and female sterility in F1 Drosophila melanogaster–D. simulans hybrids
(Barbash and Ashburner 2003; Barbash et al. 2003). The
D. simulans allele of Nup96, a single-copy gene, encodes
a nuclear pore protein that causes recessive lethality when
combined with a hemizygous D. melanogaster X chromosome (Presgraves et al. 2003). And, JYAlpha, which encodes
a male fertility–essential Na1/K1 ATPase, was recently
found to cause sterility due to its change in genetic map position: JYAlpha is on the fourth chromosome in D. melanogaster but on the third chromosome in D. simulans; hybrids
Key words: Drosophila, hybrid inviability, hybrid incompatibility,
speciation.
E-mail: [email protected].
Mol. Biol. Evol. 24(1):306–314. 2007
doi:10.1093/molbev/msl157
Advance Access publication October 20, 2006
Ó The Author 2006. Published by Oxford University Press on behalf of
the Society for Molecular Biology and Evolution. All rights reserved.
For permissions, please e-mail: [email protected]
homozygous D. melanogaster at all major chromosomes
except the fourth are therefore sterile as they lack JYalpha
(Masly et al. 2006).
It is unclear what role, if any, these particular genes
played in reproductive isolation in nature, but they nevertheless serve as important models whose molecular properties and evolutionary histories are informative about the
evolution and genetics of hybrid incompatibilities in general. For example, a striking, but still tentative, pattern
has already emerged from 3 of these genes: each has a history of recurrent positive selection at its protein-coding sequence (Ting et al. 1998; Presgraves et al. 2003; Barbash
et al. 2004). (The remaining gene, JYAlpha, causes hybrid
sterility not by its functional divergence but by a change in
map position [Lynch and Force 2000]). Adaptive divergence at Ods is confined to the D. mauritiana lineage (Ting
et al. 1998), whereas Hmr and Nup96 evolved adaptively
in both the D. melanogaster and the D. simulans lineages
(Presgraves et al. 2003; Barbash et al. 2004).
Hybrid incompatibility genes are not unconditionally
deleterious but instead depend on alleles at interacting loci.
By identifying specific loci, we can make inferences about
the interactions that lead to hybrid sterility and inviability.
For example, the D. simulans allele of Nup96 causes lethality in hybrids that are hemizygous for the D. melanogaster X
chromosome but not in hybrids hemizygous for the D. simulans X (Presgraves 2003; Presgraves et al. 2003). This is
not surprising as the D. simulans allele of Nup96 is not expected to be incompatible with loci on the D. simulans X
chromosome. On the contrary, the functional divergence
of Nup96 in D. simulans must have coevolved with functional divergence at 1 (or more) of its interacting genes. Because Nup96 does not encode a transcription factor and
because the relevant divergence in Nup96 seems to be concentrated in its protein-coding region (Presgraves et al.
2003), its hybrid inviability likely involves a protein–
protein incompatibility rather than a protein–DNA incompatibility (Landry et al. 2005).
Fortunately, a good deal is known about Nup96’s function and potential protein interactors. Nup96 is 1 of ;30 different nucleoporins that together form a single nuclear pore
complex (NPC) (Allen et al. 2000, 2002; Rout et al. 2000;
Tran and Wente 2006). Thousands of NPCs perforate the
nuclear envelopes of eukaryotic cells, mediating molecular
Adaptive Evolution of Nucleoporins 307
traffic into and out of the nucleus. The NPC is largely conserved throughout eukaryotic evolution in its modular
architecture, complement of nucleoporins, and interactions
among particular subsets of nucleoporins (Allen et al. 2000,
2002; Rout et al. 2000; Vasu and Forbes 2001; Bapteste et al.
2005; Devos et al. 2006; Tran and Wente 2006). Nup96 is 1
of 8 known proteins that form the Nup107 subcomplex
(known as the Nup84 subcomplex in yeast), a stable structural component of the NPC, along with Nup75, Nup107,
Nup133, Nup160, Seh1, Nup37, and Nup43 (Siniossoglou
et al. 1996; Rappsilber et al. 2000; Siniossoglou et al. 2000;
Vasu et al. 2001; Allen et al. 2002; Lutzmann et al. 2002;
Walther et al. 2003; Devos et al. 2004). From what is now
known, Nup96 occupies a central position in the Nup107
subcomplex and interacts directly with Nup75 and
Nup107 (Lutzmann et al. 2002). In addition to these stable
Nup107 subcomplex proteins, 3 dynamic nucleoporins
(Nup98, Nup153, and Sec13) are known to shuttle between
the nuclear interior and the NPC, where they physically dock
at the Nup107 subcomplex (Griffis et al. 2002). All 3 of these
dynamic nucleoporins interact directly with Nup96 (Vasu
et al. 2001; Hodel et al. 2002; Enninga et al. 2003);
Nup153 and Nup98 are also known to bind with Nup107,
Nup133, and Nup160 (Vasu et al. 2001).
Because Nup96 is a hub for multiple interactions in the
Nup107 subcomplex—and because Nup96 has experienced
repeated bouts of adaptive evolution in the D. melanogaster
and the D. simulans lineages—we investigated the possibility of adaptive coevolution between Nup96 and some of its
interacting proteins. We surveyed DNA sequence divergence and polymorphism in D. melanogaster and D. simulans at 5 additional nucleoporins: Nup75, Nup107, Nup133,
Nup98, and Nup153. Here we report that divergence at every
1 of these genes was driven by adaptive evolution.
Materials and Methods
Genes and Fly Stocks
We identified Nup75 (CG5733), Nup107 (CG6743),
Nup133 (CG6958), Nup98 (CG10198), and Nup153
(CG4453) using Blast searches of the D. melanogaster
and D. simulans whole-genome sequences using mouse,
human, Xenopus, and yeast homologs as queries. All of
these nucleoporins appear to be encoded by single-copy
genes in the 2 Drosophila genomes. For the 2 large shuttling
nucleoporins, we sequenced 2.4 kb from the 5# end Nup153
(positions 27–2387 of 9246 bp) and 1.3 kb from 3# end of
Nup98 (positions 1906–3223 of 3223 bp) as these are
known to encode the protein domains that mediate interactions with the Nup107 subcomplex (Enarson et al. 1998;
Vasu et al. 2001; Hodel et al. 2002). For the 3 Nup107
subcomplex nucleoporins, we sequenced their entire protein-coding regions. For completeness, we also included
the previously sequenced entire protein-coding region of
Nup96 in our analyses below (Presgraves et al. 2003).
For each gene, we sampled 11–15 chromosomes from
Zimbabwean populations of both D. melanogaster and D.
simulans. The lines were kindly provided by Dr Charles
Aquadro (Cornell University). We studied sequences from
Zimbabwe to minimize the confounding effects of the recent demographic expansion associated with the out-of-
Africa events of both species (Glinka et al. 2003; Baudry
et al. 2004; Ometto et al. 2005; Thornton and Andolfatto
2006). Toextract and isogenize autosomesinD.melanogaster,
we used standard crosses involving balancer chromosomes.
For those lines for which autosomes could not be made homozygous (e.g., due to the presence of balanced recessive
lethals), we extracted genomic DNA from deficiency heterozygotes in which the deficiency deletes a small chromosomal region including the relevant nucleoporin (i.e., from
flies hemizygous for a Zimbabwe chromosome at the locus
of interest). Details about the particular stocks and crosses
used for each nucleoporin are available upon request.
We obtained Drosophila sechellia and Drosophila yakuba sequences for each gene using Blast searches of the
complete genome sequences available from the Broad Institute and the Washington University Genome Sequencing
Center, respectively.
Sequencing and Analysis
We extracted genomic DNA from ;20 females from
each line using Puregene DNA isolation kits (Gentra Systems, Minneapolis, MN). Polymerase chain reaction and internal sequencing primers were designed using the
available D. melanogaster (Adams et al. 2000) and D. simulans (Washington University Genome Sequencing Center)
genome sequences as guides. We purified PCR products
with EXOSAP-IT (USB, Cleveland, OH) and sequenced
both strands. Some sequencing was done at the University
of Munich, using the DYEnamic ET terminator cycle sequencing kit (Amersham Biosciences, Buckinghamshire,
UK), and some at the University of Rochester, using
ABI BigDye terminator chemistry (PerkinElmer, Wellesley, MA). Sequences were assembled and edited using
Sequencher v. 4.5 (Gene Codes, Ann Arbor, MI) and
manually aligned with Se-Al v. 2.0 (Rambaut 1996). All
sequences have been deposited in GenBank, under accession numbers EF057923–EF058045.
We used DnaSP v. 4.0 for most population genetic
analyses (Rozas et al. 2003). To estimate probabilities for
Tajima’s D (Tajima 1989) and Fay and Wu’s H (Fay and
Wu 2000) statistics (with and without recombination), we
simulated 5,000 neutral genealogies, conditioning on the
observed hw (Watterson 1975), using J. Fay’s web application (http://www.genetics.wustl.edu/jflab/htest.html). The
population recombination rate, q 5 4Nr (Hudson 1987),
used in these simulations was estimated from the data.
Results
Adaptive Evolution of the Nup107 Subcomplex
We used MK tests (McDonald and Kreitman 1991) to
determine if patterns of protein polymorphism and divergence at 6 nucleoporins, Nup75, Nup107, Nup133,
Nup98, Nup153, and, for completeness, Nup96 (Presgraves
et al. 2003), are consistent with histories of neutral mutation
and drift. Under a neutral model of molecular evolution, in
which only neutral mutations contribute to variation within
and divergence between species, the ratio of replacement to
synonymous polymorphisms will equal the ratio of replacement to synonymous fixed differences (Kimura 1983;
308 Presgraves and Stephan
Table 1
MK Tests for Nucleoporins in Drosophila melanogaster and Drosophila simulans
Polymorphisms
Gene
Nup75
Nup96
Nup107
Nup133
Nup98
Nup153
Pooled
Fixed Differences
R
S
R/S Ratio
R
S
R/S Ratio
G
P Value
24
27
29
29
14
10
133
95
108
105
127
44
32
511
0.253
0.250
0.276
0.228
0.318
0.313
0.260
27
27
35
34
14
49
186
37
34
41
33
17
49
211
0.730
0.794
0.854
1.030
0.824
1.000
0.882
9.764
11.888
13.365
22.813
4.054
8.647
78.192
0.0018
0.0006
0.0003
,0.0001
0.0441
0.0033
,10 15
McDonald and Kreitman 1991). Table 1 shows that this null
neutral model can be rejected for all 6 genes. Each—the
4 stable Nup107 subcomplex proteins (Nup75, Nup96,
Nup107, and Nup133) and the 2 dynamic shuttling nucleoporins (Nup98 and Nup153)—shows a significant excess
of amino acid replacement fixations between species, consistent with recurrent bouts of adaptive protein evolution.
Sliding window analyses of replacement/synonymous
(R/S) polymorphism and divergence are provided as supplementary figures, Supplementary Material online.
Lineage-Specific Adaptive Evolution
To determine whether the adaptive evolution of any
particular nucleoporin gene was restricted to the D. melanogaster lineage or to the D. simulans lineage, or occurred
in both lineages, we performed lineage-specific MK tests.
Table 2 shows the results of 3 of these tests for each gene.
For the first test, D. simulans and D. yakuba sequences were
used as near and distant outgroups, respectively, to polarize
mutations in the D. melanogaster lineage. For the second
test, D. melanogaster and D. yakuba were used as near and
distant outgroups, respectively, to polarize mutations in the
D. simulans lineage. These 2 tests include fixations that occurred over the 3–5 Myr of independent evolution in D.
melanogaster and in D. simulans, respectively (Hey and
Kliman 1993; Li 1997; Tamura et al. 2004). For the third
test, D. sechellia and D. melanogaster sequences were used
as near and distant outgroups, respectively, to polarize substitutions in the most recent ;400,000 years of evolution in
the D. simulans lineage (Kliman et al. 2000). The latter test,
therefore, asks about patterns of substitution in the more
recent history of D. simulans. (Speciation between D. simulans and D. sechellia was sufficiently recent that some of
the history of the alleles in our sample could predate the
species split [Kliman et al. 2000], possibly confounding polymorphism and divergence. The D. sechellia–D. simulans
MK tests should therefore be interpreted with caution.) It
is important to note that lineage-specific MK tests suffer reduced statistical power, for 2 reasons. First, mapping mutations onto each lineage reduces the number of observations
in each cell of the MK table. Second, mutations that cannot
be accurately polarized, due to, for example, multiple substitutions or alignment gaps that make inference of ancestral and derived states ambiguous, are excluded from the
analysis.
Table 2
Lineage-Specific MK Tests for Nucleoporins in Drosophila melanogaster and Drosophila simulans
Polymorphisms
Gene
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Nup75
Nup96
Nup107
Nup133
Nup98
Nup153
Pooled
Contrast
D.
D.
D.
D.
D.
D.
D.
D.
D.
D.
D.
D.
D.
D.
D.
D.
D.
D.
D.
D.
D.
melanogaster lineage
simulans lineage
simulans lineage (recent)
melanogaster lineage
simulans lineage
simulans lineage (recent)
melanogaster lineage
simulans lineage
simulans lineage (recent)
melanogaster lineage
simulans lineage
simulans lineage (recent)
melanogaster lineage
simulans lineage
simulans lineage (recent)
melanogaster lineage
simulans lineage
simulans lineage (recent)
melanogaster lineage
simulans lineage
simulans lineage (recent)
* Fisher’s exact probability.
Fixed Differences
R
S
R/S Ratio
R
S
R/S Ratio
G
P Value
2
17
15
4
17
16
16
10
8
12
9
8
6
7
7
6
2
2
46
62
56
21
59
51
35
58
56
37
60
59
49
53
51
18
18
18
12
16
18
172
264
253
0.095
0.288
0.294
0.114
0.293
0.286
0.432
0.167
0.136
0.245
0.170
0.157
0.333
0.389
0.389
0.500
0.125
0.111
0.267
0.235
0.221
9
10
1
12
10
0
6
19
4
10
13
2
4
4
3
12
28
7
53
84
17
19
14
1
19
7
1
27
12
0
15
16
4
9
9
0
26
19
1
115
77
7
0.474
0.714
1.000
0.632
1.429
0.000
0.222
1.583
—
0.667
0.813
0.500
0.444
0.444
—
0.462
1.474
7.000
0.461
1.091
2.429
4.427
3.258
0.682
8.082
8.046
—
1.159
22.314
—
3.664
9.409
1.342
0.141
0.032
—
0.017
13.744
16.133
5.397
54.732
28.816
0.0354
0.0711
0.4090
0.0045
0.0046
1.0000*
0.2071
,0.0001
0.0005*
0.0556
0.0022
0.2467
0.7076
0.8587
0.0366*
0.8957
0.0002
,0.0001
0.0202
,0.0001
,0.0001
Adaptive Evolution of Nucleoporins 309
Table 3
DNA Polymorphism and Divergence at Nucleoporins in Drosophila melanogaster and Drosophila simulans Populations
D. melanogaster
Nup75
Nup96
Nup107
Nup133
Nup98
Nup153
Mean
Map Positiona
bp
n
pb
55B (2R)
95B (3R)
32A (2L)
94C (3R)
95B (3R)
14F (X)
2286
3398
2560
4185
1308
2680
2736
12
15
12
11
15
11
12.7
0.0151
0.0145
0.0163
0.0258
0.0266
0.0104
0.0181
D. simulans
Dc
0.202
0.638
1.189
0.291
0.471
0.994
0.022
n
pb
12
15
11
13
15
11
12.8
0.0426
0.0236
0.0374
0.0221
0.0207
0.0132
0.0266
Dc
0.547
0.746
0.079
0.350
0.615
0.460
0.466
Kad
KSd
Ka/Ks
0.022
0.017
0.024
0.016
0.018
0.030
0.021
0.156
0.101
0.141
0.084
0.108
0.107
0.116
0.138
0.168
0.169
0.191
0.170
0.281
0.186
a
Cytogical position.
p for silent sites, with X-linked loci corrected by multiplying by 4/3.
c
Tajima’s D for silent sites.
d
Ka and Ks are Jukes–Cantor corrected.
b
In the D. melanogaster lineage, 4 of 6 nucleoporins
have experienced an excess of replacement fixations (i.e.,
R/S for fixations is greater than R/S for polymorphism).
Two of 4, Nup75 and Nup96, show individually significant
excesses of replacement fixations consistent with adaptive
evolution (table 2). A third gene, Nup133, shows a marginally nonsignificant excess of replacement fixations.
In the D. simulans lineage, 4 of 6 nucleoporins show individually significant evidence for adaptive evolution
(Nup96, Nup107, Nup133, and Nup153; table 2). Similar histories of adaptive evolution cannot be excluded for the remaining 2 genes because Nup75 shows excess amino acid
divergence that is marginally nonsignificant (table 2, line
15); and Nup98 shows a significant excess of replacement
fixations since the split from D. sechellia (table 2, line 15).
Thus, at least 5 (nearly 6) of 6 nucleoporins show some evidence of recurrent adaptive protein evolution in D. simulans.
Nup107 and Nup153 have experienced recurrent bouts
of positive selection strictly limited to the D. simulans lineage (table 2, lines 8 and 17), whereas none of the genes
show D. melanogaster–limited bouts of positive selection.
Only 1 gene, Nup96, shows strong evidence for parallel
bouts of positive selection in both species (table 2, lines
4 and 5). The data for Nup75 and Nup133 are suggestive
of similar parallel bouts of positive selection, but in 1
of the species (D. simulans and D. melanogaster, respectively), the lineage-specific tests are marginally nonsignificant. Overall, the Nup107 subcomplex appears to have
diverged more in D. simulans than in the D. melanogaster
(table 2, lines 19–21).
inferred larger effective population size (Aquadro et al.
1988; Akashi 1996), D. simulans shows ;50% higher
levels of silent diversity, on average, than D. melanogaster
(table 3). None of the nucleoporin genes in either species
exhibits the dramatically reduced variability expected after
a recent, strong selective sweep.
The frequency spectra of silent polymorphisms, summarized by Tajima’s D statistic, are more skewed toward
rare alleles in D. simulans than in D. melanogaster for 5
of 6 genes. A comparison of the distribution of all silent
polymorphisms pooled across loci differs significantly between species (Kolmogorov–Smirnov test, P 5 0.0002).
This species difference is consistent with either a stronger
signal of population expansion or stronger selective constraints on silent sites in D. simulans than in D. melanogaster (Akashi 1995). The sole exception to this pattern is
Nup107: in D. melanogaster, silent variability at Nup107
shows a negative Tajima’s D (table 3; without and with recombination, P 5 0.092 and P 5 0.013, respectively) and
a negative Fay and Wu’s H (without and with recombination, P 5 0.114 and P 5 0.057, respectively). These patterns of variability are consistent with a fairly old selective
sweep at Nup107 in D. melanogaster (relative to the approximate detection limit of ;0.1Ne generations in Drosophila), but it is difficult to rule out alternative (e.g.,
demographic) explanations. Thus, neither the levels nor
the frequency spectra of silent variability at Nup107 subcomplex genes shows particularly compelling evidence
for recent, strong selective sweeps in either species.
Discussion
Little Evidence for Recent Selective Sweeps
Recent selective sweeps caused by the fixation of new,
favorable mutations can leave characteristic signatures in
the level and distribution of linked neutral nucleotide variability (Maynard Smith and Haigh 1974; Kaplan et al.
1989; Nielsen 2005), for example, by depressing silent variability and causing an excess of rare variants. Table 3
shows summary statistics of the levels of silent (synonymous and noncoding) variability and of the frequency spectra of silent mutations for each of the 6 nucleoporins. Levels
of silent polymorphism at each locus are not unusual for
African populations of these species (Andolfatto 2001;
Mousset and Derome 2004). Consistent with its previously
The structure and function of NPCs are largely conserved among eukaryotes. It is therefore surprising that
all 4 Nup107 subcomplex nucleoporins and both interacting
shuttling nucleoporins studied have histories of recurrent
adaptive evolution during the last 3–5 Myr of divergence
between D. melanogaster and D. simulans. Several recent
analyses of polymorphism and divergence data across loci
in Drosophila have revealed an overall excess of amino
acid divergence consistent widespread adaptive evolution
(Fay et al. 2002; Smith and Eyre-Walker 2002; Sawyer
et al. 2003). However, even in such multilocus surveys,
only 5–10% of genes show individually significant evidence
for adaptive evolution (Smith and Eyre-Walker 2002;
310 Presgraves and Stephan
Presgraves 2005). The fact that each of 6 genes in our survey
shows individually significant evidence for adaptive evolution is therefore striking. This finding—along with the fact
that these proteins are known interactors—suggests that the
bouts of adaptation at these 6 genes are not independent but
rather reflect histories of molecular coevolution. We note,
however, that our evidence is circumstantial as only functional tests can confirm coevolution. These tests require that
the particular functional domains of these nucleoporins become better characterized.
What might cause such a large fraction of the proteins
of the otherwise conserved NPC to diverge so dramatically?
The pattern of divergence observed here suggests that a
simple scenario of ecological adaptation to the extrinsic
environment—say, adaptation to a new host plant—is unlikely. D. melanogaster and D. simulans are thought to have
evolved in allopatry, in southeastern sub-Saharan Africa and
in Madagascar, respectively (Lachaise et al. 1988; Dean and
Ballard 2004; Lachaise and Silvain 2004; Lachaise et al.
2004). For an ecological scenario to account for the divergence seen here, these 2 historically geographically isolated
species must have independently experienced an ecological
challenge—at about the same time in their evolutionary histories—and responded to that challenge with multiple beneficial substitutions at the same (or related) sets of genes.
Although we cannot rule out such a scenario, an alternative
class of explanation seems more plausible.
In particular, the parallel bouts of adaptive evolution at
nucleoporins in both D. melanogaster and D. simulans suggest that these 2 species may have inherited an unresolved
conflict involving NPCs from their common ancestor. Under this scenario, targeted nucleoporins in these 2 species
would have evolved in response to a conflict using different
sets of substitutions, with interacting nucleoporins coevolving. This adaptive divergence would then incidentally lead
to a hybrid incompatibility. There are at least 3 types of
genetic conflicts in which NPCs—the ‘‘gatekeepers of
the nucleus’’ (Wente 2000)—might plausibly be involved.
actions between flies and viruses (but see Dostert et al. 2005;
Wang et al. 2006), it is easy to imagine that fly pathogens
have evolved similar strategies, leading to conflicts that
drive the divergence of Drosophila nucleoporins.
Genetic Conflict over Centromeric Drive
Meiotic drive of selfish centromeres during oogenesis
represents a second genetic conflict potentially fueling the
divergence of the Nup107 subcomplex. Of the 4 meiotic
products of oogenesis, only 1 becomes the primary oocyte
and the other 3 are discarded as polar bodies. This inherent
asymmetry in oogenesis thus creates an opportunity for centromeres, the sites of kinetochore assembly and spindle attachment, to compete for access to the primary oocyte
(Zwick et al. 1999; Henikoff et al. 2001). There is direct
evidence for such centromeric drive in plants and animals
(Pardo-Manuel de Villena and Sapienza 2001; Fishman and
Willis 2005), as well as some indirect evidence suggested
by the rapid evolution of both centromeric heterochromatin
and centromere-binding proteins in plants and animals, including Drosophila (Malik and Henikoff 2001; Malik et al.
2002; Talbert et al. 2002). Centromeric drive is predicted to
have deleterious side effects for both female and male fertility, including nondisjunction (Zwick et al. 1999) and
other meiotic defects (Henikoff and Malik 2002; Malik
2005). Centromere-binding proteins may therefore evolve
rapidly in order to suppress selfish centromeres. How might
the Nup107 subcomplex be involved in centromeric drive?
One of the surprises to emerge from recent work on the
Nup107 subcomplex is its localization during cell division:
during mitosis the nuclear envelope breaks down and NPCs
disassemble; however, the Nup107 subcomplex remains intact, specifically localizes to the kinetochores of chromosomes, and promotes spindle assembly (Belgareh et al.
2001; Loiodice et al. 2004). The members of the Nup107
subcomplex therefore represent another class of rapidly
evolving, centromere-associated proteins potentially involved in suppressing centromeric drive.
Genetic Conflict between Host and Pathogens
Genetic Conflict over Segregation Distortion
First, there is ample evidence that nucleoporins are involved in antagonistic interactions with viruses and viruslike particles of retrotransposons (Cronshaw and Matunis
2004; Smith and Helenius 2004; Fontoura et al. 2005; Irwin
et al. 2005). Some viruses (e.g., adenovirus and herpes simplex virus) are too large to pass through the nuclear pore and
instead dock on the cytoplasmic face of NPCs to inject their
genomes into the nucleus (Cronshaw and Matunis 2004;
Smith and Helenius 2004; Fontoura et al. 2005). Other
viruses directly target particular nucleoporins for disruption.
In human cells, the matrix protein of the vesicular stomatitus
virus inhibits mRNA export by specifically disrupting
Nup98 (Enninga et al. 2002). In response, host cells upregulate expression of Nup98 and Nup96 via an interferonmediated immune response. Similarly, poliovirus (Gustin
and Sarnow 2001) and rhinovirus (Gustin and Sarnow
2002) impair specific nuclear import pathways by proteolyzing Nup153. Furthermore, HIV-1 and Tf1 retrotransposons
also interact with Nup153 (Varadarajan et al. 2005). Although relatively little is known about the molecular inter-
Segregation distortion represents a third genetic conflict that might involve the NPC. In D. melanogaster,
a male-specific meiotic drive complex called Segregation
Distorter (SD) resides on 1–5% of second chromosomes
in natural populations (Hartl and Hiraizumi 1976; Kusano
et al. 2003). During spermatogenesis in SD/SD1 males,
SD1-bearing sperm are incapacitated so that virtually all
progeny inherit SD instead of the 50% expected under Mendelian inheritance. The 3 major loci of the SD complex are
1) the Segregation distorter locus (Sd), the main driver that
encodes a truncated duplicate of RanGAP (Merrill et al.
1999); 2) Enhancer of SD [E(SD)], an upward modifier
of the strength of distortion; and 3) Responder (Rsp), the
target of distortion. Rsp is an array of AT-rich 120-bp
repeats for which repeat copy number is correlated with sensitivity to distortion—sensitive Rsp alleles comprise 700–
2500 repeats, whereas insensitive alleles comprise a few
dozen to ;200 (Wu et al. 1988). Wildtype RanGAP localizes
to the cytoplasm where it functions as a major regulator of
nuclear transport; in contrast, truncated Sd-RanGAP was
Adaptive Evolution of Nucleoporins 311
recently shown to mislocalize to the nuclear interior, where it
causes distortion by disrupting nuclear transport during spermatogenesis (Kusano et al. 2001). Genes able to influence
nuclear transport—including nucleoporins—could in principle suppress or compensate for the deleterious effects of distortion and might therefore evolve in response to this genetic
conflict. We have argued elsewhere that RanGAP-mediated
distortion may be an ancient genetic conflict, predating the
D. melanogaster–D. simulans split, which has driven rapid
divergence of several nuclear transport genes, including RanGAP (Presgraves DC, unpublished data).
Thus, the rapid molecular evolution of the Nup107
subcomplex and its interactors could plausibly reflect histories of 1) host–pathogen conflict, 2) conflict over centromeric drive, or 3) conflict over nuclear transport–related
segregation distortion. Although admittedly speculative,
such genetic conflict–driven scenarios seem likely to explain the simultaneous bouts of recurrent adaptive substitutions concentrated in the Nup107 subcomplex genes of
D. melanogaster and D. simulans.
Coevolutionary Consequences for Hybrid
Incompatibilities
Population genetic models of the evolution of hybrid
incompatibilities usually assume that substitutions occur independently (Orr 1995). For instance, in the simple 2-locus,
2-allele Dobzhansky–Muller model, an ancestral aabb genotype first becomes AAbb in 1 of 2 lineages; then, with equal
probability, a B substitution can occur either in the same lineage yielding AABB (scenario 1) or in the sister lineage yielding aaBB (scenario 2). The pattern of molecular evolution
among the Nup107 subcomplex genes implies, however,
that the histories of interacting genes are not independent—the Nup107 subcomplex genes appear to coevolve.
Such nonindependence among coevolving interactors has
2 consequences for the evolution of hybrid incompatibilities.
First, coevolution accelerates the rate of evolution of hybrid
incompatibilities relative to a model with independent substitutions because substitutions at 1-locus increase the probability of substitutions at interacting loci—within a lineage
an a/A substitution can drive a b/B substitution.
Second, coevolution decreases the probability of
evolving incompatibilities between 2 derived alleles
(derived–derived) versus incompatibilities between 1 derived and once ancestral allele (derived–ancestral). Under
scenario 1 in the model above, the AABB 3 aabb species
cross will produce AaBb hybrids exposed to a possible
incompatibility between the derived B allele and the ancestral a allele (as ‘‘B’’ has never been tested by natural selection on an ‘‘a’’ genetic background). Similarly, under
scenario 2, the AAbb 3 aaBB species cross will produce
AaBb hybrids exposed to incompatibilities between the
derived A and derived B alleles. Coevolution among interacting loci will cause substitutions to be concentrated
in 1 lineage (scenario 1), increasing the probability of
derived–ancestral incompatibilities relative to a model with
independent substitutions (Orr 1995). Thus, the nonindependence of substitutions in coevolving genes should increase the rate of evolution of derived–ancestral hybrid
incompatibilities.
Coevolution between Nup96 and a Partner on the X
Chromosome?
Genetic analysis shows that the D. simulans allele of
Nup96 causes hybrid inviability via an epistatic interaction
with an incompatible ‘‘partner’’ gene (or genes) on the X
chromosome of D. melanogaster (Presgraves 2003; Presgraves et al. 2003). Of the 8 known members of the
Nup107 subcomplex and the three shuttling nucleoporins
that dock with it, only 1 is encoded by a gene on the X chromosome: the shuttling nucleoporin, Nup153. Interestingly,
Nup153 shows evidence of adaptive evolution only in the
D. simulans lineage, suggesting that the D. simulans allele
of Nup96 is functional in the context of the newly evolved
allele of Nup153 in D. simulans. If true, then this putative hybrid incompatibility could represent a derived–
ancestral incompatibility—the derived allele of Nup96
(from D. simulans) is incompatible with the ancestral allele
of Nup153 (from D. melanogaster). It is important to note,
however, that Nup153 remains only a candidate partner
gene until functional tests demonstrate its role in the
Nup96 incompatibility.
Supplementary Material
Supplementary figures and Accession Numbers
EF057923–EF058045 are available at Molecular Biology
and Evolution online (http://www.mbe.oxfordjournals.org/).
Acknowledgments
We thank Andrea Betancourt, Victoria Cattani, J.P.
Masly, Allen Orr, and Shanwu Tang for comments on
the manuscript. This work was supported by an Alexander
von Humboldt Research Fellowship and funding from the
University of Rochester to D.C.P. and by a grant from the
VW-Foundation (I/78815) to W.S.
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Accepted October 17, 2006