Ancient Male Recombination Shaped Genetic Diversity of
Neo-Y Chromosome in Drosophila albomicans
Kazuhiro Satomura1 and Koichiro Tamura*,1,2
1Department of Biological Sciences, Tokyo Metropolitan University, Tokyo, Japan
Research Center for Genomics and Bioinformatics, Tokyo Metropolitan University, Tokyo, Japan*Corresponding author: E-mail: ktamura@tmu.ac.jp.
Associate editor: Yoko Satta
2

Abstract

Key words: meiotic recombination, achiasmy, sex chromosome, founder effect.

Introduction

which meiotic recombination is completely suppressed inmales. In this case, meiotic recombination is shut out immediately after fusion due to the switch of inheritance mode
from biparental to paternal, resulting in a situation where a
newly generated neo-Y chromosome has no chance toacquire preexisting genetic variations from its gametologous
chromosome by meiotic recombination and remains monomorphic until newly occurring mutations accumulate overtime. This is a big difference from typical Y chromosome
evolution, where meiotic recombination is suppressed gradually so that young Y chromosome has a chance to acquirepreexisting genetic variations from the original autosome
during the initial stage of evolution. Therefore, the observed
low genetic diversity of the young neo-Y chromosome may belargely attributable to the founder effect caused by a single
chromosomal fusion event. Additionally, the smaller effective
population size may be partly responsible for the lower
genetic diversity of neo-Y chromosome. Because a pair ofparents has one Y chromosome, three X chromosomes andfour individual autosomes, the genetic diversity of Y chromosome is expected to eventually become one-third of that of X
chromosome and one-fourth of that of autosome. On theother hand, this smaller population size may relax synonymous codon usage bias to increase synonymous substitutions.
Furthermore, if Y chromosome evolves faster than X chromosome and autosomes due to differences in germline cell
production between males and females as known as

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Mol. Biol. Evol. 33(2):367–374 doi:10.1093/molbev/msv221 Advance Access publication October 22, 2015

367

ArticleThe effect of meiotic recombination on genetic diversity has
been well characterized by a number of studies on Y chromosome evolution, as meiotic recombination is suppressed
between X and Y chromosomes to maintain their functional
diversification on sex determination (Nei 1969). These studies
have shown that genetic diversity of Y chromosome is much
lower than that of X chromosome in various species(Whitfield et al. 1995, Berset-Br€andli et al. 2007, Rozen et al.
2009, Muir et al. 2011, Larracuente and Clark 2013, Wilson
Sayres et al. 2014). A variety of evolutionary scenarios involving natural selection under the suppression of meiotic recombination have been invoked to explain the low genetic
diversity of Y chromosome (e.g., Bachtrog 2013 for review).
Among these studies, an extremely low genetic diversity was
explored in the neo-Y chromosome of Drosophila miranda,
suggesting recent positive selection acting on the nonrecombining genome (Bachtrog 2004). The neo-Y chromosome wasoriginated by a fusion of ancestral Y chromosome and an
autosome, resulting in a situation where a huge number ofgenes from the fused autosome are shared by its gametologous neo-X chromosome. This situation gives us an excellent
opportunity to compare evolutionary changes between neoX and neo-Y chromosomes in the same genes.
However, we should notice that neo-Y chromosome evolution does not necessarily mirror Y chromosome evolutionin achiasmatic species, for example, the Drosophila species, inDownloaded from http://mbe.oxfordjournals.org/ by guest on January 24, 2016

Researchers studying Y chromosome evolution have drawn attention to neo-Y chromosomes in Drosophila species due totheir resembling the initial stage of Y chromosome evolution. In the studies of neo-Y chromosome of Drosophila miranda,
the extremely low genetic diversity observed suggested various modes of natural selection acting on the nonrecombining
genome. However, alternative possibility may come from its peculiar origin from a single chromosomal fusion event withmale achiasmy, which potentially caused and maintained the low genetic diversity of the neo-Y chromosome. Here, wereport a real case where a neo-Y chromosome is in transition from an autosome to a typical Y chromosome. The neo-Y
chromosome of Drosophila albomicans harbored a rich genetic diversity comparable to its gametologous neo-X chromosome and an autosome in the same genome. Analyzing sequence variations in 53 genes and measuring recombination
rates between pairs of loci by cross experiments, we elucidated the evolutionary scenario of the neo-Y chromosome of D.
albomicans having high genetic diversity without assuming selective force, i.e., it originated from a single chromosomal
fusion event, experienced meiotic recombination during the initial stage of evolution and diverged from neo-X chromosome by the suppression of recombination tens or a few hundreds of thousand years ago. Consequently, the observedhigh genetic diversity on the neo-Y chromosome suggested a strong effect of meiotic recombination to introduce genetic
variations into the newly arisen sex chromosome.

Satomura and Tamura . doi:10.1093/molbev/msv221

Results and Discussion
Genetic Diversity of Neo-Y Chromosome in
D. albomicans
The neo-Y chromosome of D. albomicans is known to bemuch younger than that of D. miranda (Bachtrog andCharlesworth 2002, Bachtrog 2006). This allows us to expectthat the D. albomicans neo-Y chromosome harbors lessgenetic diversity than the D. miranda neo-Y chromosome
under the assumptions that the neo-Y chromosomes inboth species had a single origin and have not recombined.
In this condition, only a way of neo-Y chromosome’s acquiring genetic diversity is mutation and the younger neo-Y chromosome has had shorter time to accumulate mutations. To
examine this expectation, we determined the nucleotide
sequences of 27 genes on the neo-sex chromosomes and 26
genes on the second chromosome for 16 wild strains of
D. albomicans as well as for 16 wild strains of D. nasuta, asibling species of D. albomicans having no neo-sex chromosome but the canonical sex chromosomes and the third chromosome separately. The result showed an unexpectedly highgenetic diversity of the neo-Y linked genes in D. albomicans(table 1, see supplementary tables S1–S5, Supplementary
Material online, for details). The level of genetic diversities
by means of nucleotide diversity,  (Nei and Tajima 1981)
and Watterson’s  (Watterson 1975), were very similar between the homologous genes on the neo-X and neo-Y chromosomes in D. albomicans as well as between those and thegenes on the second chromosome at either synonymous or
intronic sites. The differences are statistically nonsignificant,
except for the difference in  for intronic sites between
368

FIG. 1. Schematic representation of male karyotypes of D. nasuta (A)and D. albomicans (B).

second and neo-Y chromosomes (P < 0.05). The situation
is virtually the same in D. nasuta, in which the genetic diversities of the genes on the unfused third chromosome werevery similar to those on the second chromosome and thedifferences are all statistically nonsignificant.
Given that genes on Y-chromosome are expected to be
degraded during the course of evolution, the observed highgenetic diversity on the neo-Y chromosome may be explainedby relaxed functional constraints on nonfunctionalized genes.
However, this is implausible in the present case because of thefollowing two reasons. First, there are more synonymous segregating sites (nS) than nonsynonymous segregating sites(nN) in spite of much fewer synonymous sites than nonsynonymous sites in all the 27 genes on the neo-Y chromosome(supplementary table S4, Supplementary Material online).
The nN/nS ratio for the neo-Y linked genes is no greaterthan that for the neo-X linked genes in 16 out of 27 genes(supplementary tables S3 and S4, Supplementary Material
online). The result does not change if we use the ratio between synonymous and nonsynonymous ’s instead of nS
and nN. Second, analysis of variance (ANOVA) for the effective number of codons (ENCs) (Wright 1990) indicates nosignificant difference in synonymous codon usage bias
between the neo-X and neo-Y chromosomes, whereas thevariation among genes is highly significant with P < 0.001(table 2, see supplementary tables S6 and S7,
Supplementary Material online, for details). These results suggest that there is no difference in the functional constraints
between the neo-X and neo-Y linked genes, which is consistent with the inference based on a transcriptome analysis that
only 1–2% of genes on the neo-Y chromosome of D. albomicans have been nonfunctionalized (Zhou and Bachtrog 2012).
Another candidate to explain the high genetic diversity is ahigher rate of point mutation on the neo-Y chromosome
than on the neo-X chromosome. To test this hypothesis,
we compared the average number of synonymous substitutions per site between the neo-X and neo-Y linked genes with
their homologous genes on the D. nasuta third chromosome(table 3). The average values are 0.0337  0.0035 and0.0356  0.0042 for the neo-X and neo-Y genes, respectively,
where the value for the neo-Y chromosome is higher thanthat for the neo-X chromosome in 17 out of 27 genes.
Although the neo-Y linked genes show slightly faster

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“male-driven evolution” (Miyata et al. 1987), this works forhigher genetic diversity of neo-Y chromosome. Therefore, weshould take all these factors into account to examine theeffect of natural selection on the genetic diversity of neo-Y
chromosome.
To examine how natural selection acts on genetic diversity
during Y chromosome evolution, it is necessary to study aneo-Y chromosome that was recombining initially but suppressed recombination gradually during the course of evolution. In this article, we report the case of neo-Y chromosome
evolution in Drosophila albomicans as such an example.
D. albomicans is a member of the Drosophila nasuta subgroupof the Drosophila immigrans species group in the subgenus
Drosophila. The ancestor of D. albomicans is thought to havecanonical X and Y sex chromosomes and three pairs of autosomes (second, third, and fourth chromosomes) as do D.
nasuta and other species in the D. nasuta subgroup at present(fig. 1A). However, the neo-X and neo-Y chromosomes thatcurrent D. albomicans has are thought to have originated by
two independent fusions of X and third chromosomes and of
Y and third chromosomes, respectively (fig. 1B, Wilson et al.
1969, Wakahama et al. 1983). Although the male recombination is perfectly suppressed at present in D. albomicans, weshow that it likely occurred in the ancestral population, which
resulted in high genetic diversity of the neo-Y chromosome inthis species.

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Neo-Y Chromosome Evolution in D. albomicans . doi:10.1093/molbev/msv221

Table 1. Nucleotide Diversity (p), Watterson’s h, and Tajima’s D at Synonymous Sites and Intronic Sites in 53 Nuclear Genesa of D. albomicans
and D. nasuta.
Chromosome
D. albomicans
SecondNeo-X
Neo-Y
D. nasuta
SecondThird

No. of Genes

No. of Sites

No. of Segregating Sites

p  SE (100)h  SE (100)

Tajima’s D  SE

Synonymous sites
intronSynonymous sites
intronSynonymous sites
intron

26
23
27
15
27
15

4,169
3,680
4,511
1,819
4,512
1,855

361
203
280
125
233
85

2.21  0.33
2.24  0.39
1.77  0.25
1.59  0.33
1.80  0.25
1.40  0.34

2.51  0.34
2.34  0.33
1.87  0.25
1.98  0.45
1.54  0.21
1.36  0.33

0.47  0.15**
0.38  0.20
0.17  0.20
0.50  0.22*
0.63  0.22**
0.26  0.29

Synonymous sites
intronSynonymous sites
intron

26
23
27
15

4,167
3,678
4,513
1,850

294
178
324
127

1.91  0.32
1.87  0.33
2.13  0.34
1.69  0.56

1.96  0.30
1.85  0.29
2.14  0.33
1.86  0.65

0.21  0.18
0.11  0.18
0.08  0.19
0.23  0.24aAdh, Apf, Arr1, bur, CaBP1, Crc, Ddc, Droj2, eIF-3, p40, fbp, Fdh, Gpdh, Oscp, porin, Rga, RpL4, Set, SPE, Spn88Ea, srp, Surf4, Tps1, und, CG3609, CG5028, CG6255 on the second
chromosome and aay, Amyrel, blw, ERp60, GstS1, Idh, Jheh3, Mpcp, Mys45A, NHP2, Nurf-38, Pdh, Pdi, Pgm, rept, RnrS, Rpn1, SdhB, Srp68, Tango7, Thiolase, Uch-L3, wal, CG5068,
CG7430, CG8446, CG33138 on the neo-sex (D. albomicans) or the third (D. nasuta) chromosomes. *The value is different from zero with a statistical significance (P < 0.05).
**The value is different from zero with a statistical significance (P < 0.01).

Source of Variation DF
SS
MS
F
P
neo-X vs. neo-Y
1
0.0266
0.02660.0472 0.828
Among genes
26 35527.97
1366.460 2422.445 <0.001
Among strains
15
4.773
0.318
0.564
0.903
Residual
821
463.112
0.564
Total
863 35995.880
41.710evolutionary rate than the neo-X linked genes, the difference
is not significant (P < 0.31), indicating that the higher rate ofpoint mutation is not a major factor of the high genetic
diversity on the neo-Y chromosome.
The observed high genetic diversity on the neo-Y chromosome of D. albomicans is in clear contrast to the case of
D. miranda, where the level of nucleotide variability on the
neo-Y chromosome is 30 times lower than that in highly
recombining portions of the genome (Bachtrog 2004). The
observed high genetic diversity of the neo-Y linked genes
suggests that at least either the assumptions of a single
origin or no recombination does not hold for the neo-Y chromosome evolution in D. albomicans. If we assume multiple
origins without recombination to explain the observed neo-Y
diversity at a very similar level to the autosome diversity, weneed to assume that most of the neo-Y chromosomes were
produced by independent chromosome fusions. This is unrealistic because chromosomal fusions have been obviously rare
events in the evolutionary history of Drosophila species, assuggested by well-conserved karyotypes among closely
related species (Ashburner 1989, chapter 5-1).

Meiotic Recombination in D. albomicans andD. nasuta
The possibility of meiotic recombination in males was examined by cross experiments. The recombination was examined
between Arr1 and Fdh and between Surf4 and CaBP1 on theAncestral Male Recombination in D. albomicans
Although male recombination was not detected in D. albomicans, whether or not male recombination occurred in theancestral D. albomicans population and/or the most recent
common ancestor of D. albomicans and D. nasuta remains
369

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Table 2. Results of ANOVA for ENC among 27 Neo-X and Neo-Y
Linked Genes from 16 D. albomicans Strains.

second chromosome and between GstS1 and Pgm on the
neo-sex (D. albomicnas) or third (D. nasuta) chromosomes,
because the genotypes of these genes were distinguishable bysingle nucleotide polymorphisms (SNPs) used as a genotype
marker. The occurrence of meiotic recombination in the malegermline was examined by presence or absence of recombinants at the backcross first generation (BC1) in three D. albomicans and three D. nasuta chromosomal inversion-freestrain pairs, respectively.
The results showed that male recombination did not occur
in D. albomicans but did in D. nasuta either in the second or
neo-sex/third chromosome (table 4). Between Arr1 and Fdh
on the second chromosome, there was no recombinant frommale F1 hybrids in D. albomicans irrespective of the combination of parental strain pairs. Because there were only a few
recombinants from the female F1 hybrids, we additionally
examined the recombination between Surf4 and CaBP1.
The result was the same: there were recombinants from thefemale F1 hybrids but none from the male F1 hybrids. In
contrast, there were many recombinants between Arr1 andFdh from the male F1 hybrids in D. nasuta, although therecombination rate was lower than that from the female F1
hybrids. The male recombination in D. nasuta was recognized
between GstS1 and Pgm on the third chromosome as well.
Again, there was no recombinant by male recombination in
D. albomicans. These results consistently suggest that male
meiotic recombination occurs in D. nasuta but not in
D. albomicans. It should be noted that the observed differences in the recombination rate among different strain pairs
may arise from the differences in gene arrangement among
different chromosomal inversion types (see Materials andMethods for details).

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Table 3. Number of Synonymous Substitutions Per Site and dA Distance for 27 Genes among Neo-X and Neo-Y Chromosomes in D. albomicans
and Third Chromosome in D. nasuta.
dXY

Gene

dA
Neo-Y nas0.04103
0.01398
0.01456
0.008280.02906
0.048430.048390.00754
0.007110.02260
0.04377
0.09267
0.02605
0.024990.030700.00803
0.04806
0.05462
0.05306
0.026960.040190.08363
0.061220.03538
0.02829
0.01650
0.04652
0.03562  0.00423

Neo-X nas0.000080.017930.00387
0.00135
0.006910.01533
0.01558
0.003590.001830.00059
0.033650.001100.007030.00001
0.01551
0.005310.03100
0.013230.017420.032210.00452
0.027160.00889
0.02386
0.01230
0.00592
0.00269
0.01102  0.00211

Neo-Y nas0.006120.005700.003540.00130
0.00446
0.01858
0.02023
0.00300
0.001200.00474
0.036270.01723
0.006450.004250.01526
0.00433
0.031100.02218
0.031730.001900.01852
0.04326
0.016020.02251
0.01158
0.000940.001170.01301  0.00234

Neo-X neo-Y
0.004140.02766
0.008740.001200.003360.000810.001540.00001
0.000140.000610.00001
0.028270.00095
0.005900.000490.000070.001270.00094
0.00915
0.02230
0.005240.030070.002790.000690.000430.00686
0.00692
0.00525  0.00192

Table 4. Numbers of Recombinants and Nonrecombinants and Recombination Rates (%) Observed at Backcross First Generation in
D. albomicans and D. nasuta.
Numbers of Recombinants and Nonrecombinants and % Recombination Rates
Second$D. albomicans
HRS402
F1(NG3  HRS402)
TSN7
F1(SZ43  TSN7)KM7F1(HNZ12  KM7)
D. nasuta
VNS23
F1(TNR35  VNS23)
C3
F1(K33  C3)
MS77
F1(BZV1  MS77)

370

Neo-sex/Third

Arr1–Fdh

Parent strains

Surf4–CaBP1

GstS1–Pgm#

recnon-rec%recnon-rec%recnon-rec%

F1(NG3  HRS402)
HRS402F1(SZ43  TSN7)TSN7F1(HNZ12  KM7)
KM7

0
3
0
2
0
0

32
29
32
30
32
32

0.0
9.4
0.0
6.3
0.0
0.0

0
5
0
1
0
5

32
27
32
31
32
27

0.0
15.6
0.0
3.2
0.0
15.6

0
17
0
16
0
15

32
15
32
16
32
17

0.0
53.1
0.0
50.0
0.0
46.9

F1(TNR35  VNS23)
VNS23
F1(K33  C3)
C3
F1(BZV1  MS77)
MS77

5
10
6
11
3
8

27
22
26
21
29
24

15.6
31.3
18.8
34.4
9.4
25.0

—
—
—
—
—
—

—
—
—
—
—
—

—
—
—
—
—
—

12
9
3
10
1
12

20
23
29
22
31
20

37.5
28.1
9.4
31.3
3.1
37.5

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Neo-X nas0.034440.024530.01958
0.00830
0.03505
0.04369
0.04804
0.00895
0.00698
0.019280.043310.06347
0.024810.02337
0.032100.008440.046850.050400.04168
0.04134
0.019110.08157
0.05834
0.03966
0.030080.015810.04137
0.03372  0.00352

aay
Amyrel
blw
ERp60
GstS1
Idh
Jheh3
Mpcp
Mys45A
NHP2
Nurf-38
Pdh
Pdi
Pgm
rept
RnrS
Rpn1
SdhB
Srp68
Tango7
Thiolase
Uch-L3wal
CG5068CG7430CG8446CG33138
Average  SE

Neo-Y Chromosome Evolution in D. albomicans . doi:10.1093/molbev/msv221

FIG. 2. Venn diagrams showing overlap of synonymous SNP sites between the D. albomicans and D. nasuta second chromosomes (A) andamong the D. albomicans neo-X and neo-Y chromosomes and the D.
nasuta third chromosome (B).

population structuring. To control this effect, we computed
Tajima’s D as well as  and  for the strains from China andSoutheast Asia and those from Japan and Taiwan separately(supplementary tables S8–S14, Supplementary Material
online) and tested the difference in the Tajima’s D values
by ANOVA. Although the statistical significance was declinedby the smaller sample size due to the population splitting, thetendency of Tajima’s D value obtained from the entire population (negative for the neo-X and second and positive forthe neo-Y) did not change. The ANOVA clearly shows thatthe difference between the neo-X and neo-Y linked genes isstatistically significant as well as the difference among genes is,whereas the difference between the China–Southeast Asian
population and the Japan–Taiwan population is nonsignificant (table 5). The significant difference among genes is
attributed to the differences in functional constraints
among genes as observed in the significant difference in
ENC among genes (table 2). In contrast, the significant difference between the neo-X and neo-Y is not attributable to thedifference in functional constraints, because there is no difference in functional constrains between the neo-X and neoY linked genes as discussed earlier. Therefore, it is concluded
that the obtained positive values of Tajima’s D for the neo-Y
linked genes indicates a population size reduction of the neoY chromosome during the course of evolution most probably
due to the suppression of male recombination.
To examine the sequence divergence between the neo-X
and neo-Y chromosomes occurred after the suppression ofmale recombination, we computed the net number of substitutions per site (dA, Nei 1987, pp. 276–277) for the 27 genes
between the neo-X and neo-Y chromosomes from 16
D. albomicans strains and those and the third chromosome
from 16 D. nasuta strains (table 3). As a result, the average net
sequence divergence between the neo-X and neo-Y chromosomes was computed to be 0.00525  0.00192, which is statistically significant from zero (P < 0.01), suggesting sequence
divergence accumulated between these chromosomes after
the suppression of male recombination. Assuming that the371

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unknown. To consider the possibility of the ancestral malerecombination in D. albomicans, we examined the nucleotide
sequence data in the following two ways.
First, we analyzed the shared SNP sites between the neo-X
and neo-Y chromosomes of D. albomicans as well as between
those and the third chromosome of D. nasuta. If the variations
on the neo-Y chromosome were transmitted by meiotic
recombination, many SNP sites are expected to be sharedby the neo-Y, neo-X, and third chromosomes at present.
Indeed, this is the case: 139 out of 280 and 233 synonymous
SNP sites on the neo-X and neo-Y chromosomes, respectively,
were common, which was much more than the amount
expected from random point mutations occurred independently on both chromosomes, that is, 2  280  233/
(4,511 + 4,512) = 14.5 (fig. 2). In addition, the neo-Y chromosome shares as many SNP sites with the D. nasuta third chromosome as does the neo-X chromosome (107/233 vs. 117/
280). This suggests that the neo-Y chromosome as well as theneo-X chromosome recombined with the unfused third chromosome before it reached fixation. It should be noted thatthe observed shared SNP sites did not come from the ancestral polymorphism without recombination, given that each of
the current neo-X and neo-Y chromosome populations wasdescended from a single fused chromosome.
The second approach is based on the following expectation. If the neo-Y chromosome recombined with the neo-X
and/or ancestral unfused third chromosomes in the initialstage of evolution, the effective population size of the neoY chromosome (together with its gametologous chromosomes) was four for a male and female pair of flies, but it
has been reduced to be one by the male recombination suppression that isolated the population of neo-Y chromosomesfrom the population of neo-X chromosomes. In contrast, if
male recombination has not occurred during the entire
course of the neo-Y evolution, the effective population size
should be increased as the neo-Y chromosome increased
from a single (or at least few) origin to eventually reach fixation. To infer the change in the effective population size, we
computed Tajima’s D for the synonymous sites and the intronic sites (table 1). When population size is reduced, Tajima’s
D is expected to be positive and vice versa in the absence of
the influence of natural selection (Tajima 1989). In D. nasuta,
the value of Tajima’s D is not significantly different from zero
either for synonymous or intronic sites (table 1). In D. albomicans, the genes on the neo-X chromosome and the second
chromosome showed negative values, in which the value
from synonymous sites on the second chromosome was statistically significant from zero (P < 0.01). In contrast, thegenes on the neo-Y chromosome showed positive values, inwhich the value from synonymous sites was statistically significant (P < 0.01). This positive value of Tajima’s D is hardly
explained by the effect of natural selection, because theirhomologous genes on the neo-X chromosome under thesame functional constrains gave negative values. However,since Ohsako et al. (1994) reported a substantial population
subdivision between the China-Southeast Asia populationand the Japan–Taiwan population in D. albomicans, theobtained Tajima’s D values might be largely influenced by

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Table 5. Results of ANOVA for Tajima’s D.
Source of Variation
neo-X vs. neo-Y
CS vs. JT
Among genes
Residual
Total

DF
1
1
26
79
107

SS
4.588
1.768
34.71044.16985.236

MS
4.588
1.768
1.335
0.559
0.797

F
8.206
3.163
2.388

P
0.005
0.079
0.002

CS, China-Southeast Asia; JT, Japan-Taiwan

Conclusion
All the results presented in this article support the scenariothat the neo-Y chromosome of D. albomicans originated froma fusion of an ancestral Y chromosome and a third chromosome, recombined with its gametologous chromosome
during the initial stage of evolution and then ceased meiotic
recombination tens or a few hundreds of thousand years ago(fig. 3). The currently observed high genetic diversity on the
neo-Y chromosome suggests that the diversity was introduced by the ancient meiotic recombination effectively andhas not been altered significantly after the cessation of meiotic recombination. This is a clear contrast to the case of
D. miranda, where the neo-Y chromosome showed an
extremely low genetic diversity. Although Bachtrog (2004)
suggested a selective sweep causing the low genetic diversity,
we alternatively propose a founder effect due to the peculiar
generation by a chromosomal fusion event and the subsequent immediate cessation of meiotic recombination byachiasmy. In this study, we did not find any evidence of
selective force acting on the genetic diversity on the neo-Y
chromosome in D. albomicans. In contrast, the observed highgenetic diversity clearly indicates a strong effect of meiotic
recombination to introduce genetic diversity into the newly
generated neo-Y chromosome. Yet, we still do not know
about the driving force for the fixation of the neo-X andneo-Y chromosomes. We speculate that the fused
372

FIG. 3. Schematic diagram of the evolution of neo-X and neo-Y chromosomes in D. albomicans. The width of branches represents the population size and the gray color represents the meiotic recombination inmales. After the speciation of D. albomicans and D. nasuta occurred 0.55
(0.34–0.75) million years ago (Ma), in D. albomicans the neo-X and neoY chromosomes emerged via chromosomal fusion events between the
canonical third chromosome and the canonical X and Y chromosomes,
respectively. Then, the neo-X and neo-Y chromosomes have gradually
substituted for the preexisting third chromosome with recombining
each other and with the third chromosome (indicated by crescentshaped arrows) to share the polymorphisms at the sequence level.
Finally, the male recombination was ceased 0.24 (0.07-0.41) Ma, resulting
in the divergence of neo-X and neo-Y chromosomes and the reductionin the effective population size especially for neo-Y chromosome.

chromosomes had some selective advantage from the facts
that both neo-X and neo-Y have reached fixation within ashort evolutionary time and that fusions of Muller’s elements
have occurred independently many times during the evolution of Drosophila species (Ashburner 1989, chapter 5-1). If
this is the case, there must be a footprint of selective sweep
detectable among the genomic nucleotide sequences of
D. albomicans, which remains open to further genome-wide
studies. Especially, data for the complete genome sequence of
D. albomicans are indispensable to investigate the stratum
structure accompanying gradual suppression of meiotic
recombination during Y chromosome evolution.

Materials and Methods
Drosophila Strains
Sixteen Drosophila albomicans and sixteen D. nasuta isofemale strains were used for the comparative sequence analyses. The D. albomicans strains were collected in six geographic
locations in Japan, three in China, three in Taiwan, and four inSoutheast Asia. The D. nasuta strains were collected in fivegeographic locations in India, two in Sri Lanka, two in
Seychelles, two in Mauritius islands, two in Reunion islands,
and three in Africa. The collection locality and year of thesestrains were listed in supplementary table S15, Supplementary
Material online. These strains were cultured in the standard
cornmeal medium at 20  C.

DNA Sequences
We selected 53 genes (aay, Adh, Amyrel, Apf, Arr1, blw, bur,
CaBP1, CG3609, CG5028, CG5068, CG6255, CG7430, CG8446,
CG33138, Crc, Ddc, Droj2, eIF-3p40, ERp60, fbp, Fdh, Gpdh,

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rate of neutral substitution to be 1.1  10 8 per year among
Drosophila species (Tamura et al. 2004), we estimate thedivergence time of the neo-X and neo-Y chromosomes as
the time of recombination suppression to be ~240,000
years (with 95% confidence interval ranging from 70,000 to
410,000 years). Similarly, we estimate the time of D. albomicans and D. nasuta speciation to be about 546,000 years (with
95% confidence interval ranging from 344,000 to 748,000
years), which is significantly older than 120,000 years (with
95% confidence intervals ranging from 15,000 to 290,000
years) estimated by Bachtrog (2006). However, because thevariation of time estimates among genes is considerably large(from 0 to 1.5 My) in both studies, the obtained difference inthe average is attributed to the difference in the genes used.
At any rate, the time of recombination suppression between
the neo-X and neo-Y chromosomes was most probably muchlater than the speciation of D. albomicans and D. nasuta,
supporting the scenario that the neo-Y chromosome recombined with the neo-X chromosome for a certain period oftime and then ceased the recombination tens or a few hundreds of thousand years ago.

Neo-Y Chromosome Evolution in D. albomicans . doi:10.1093/molbev/msv221

Chromosomal Assignment of Genes
Whether each gene is located on the neo-sex or second chromosome was determined by the correspondence of chromosomal arms between D. albomicans and Drosophilamelanogaster. According to Chang et al. (2008), the second
chromosome of D. albomicans corresponds to Muller’s elements B and E and the neo-Y chromosome corresponds to
Muller’s elements C and D. Therefore, genes on Muller’s element B, that is, chromosomal arm 2L in D. melanogaster, wereassumed to be on the second chromosome in D. albomicans.
In this way, the chromosomal assignment was made for all the53 genes. To confirm the chromosomal assignment, we
further examined whether the pattern of inheritance was
sex-linked or autosomal in 32 F2 hybrids of X108 males andNG3 females for 22 genes using SNP makers obtained fromthe nucleotide sequences determined. As the result, we confirmed that ten genes (Amyrel, blw, GstS1, NHP2, Pdi, Pgm,
SdhB, CG5068, CG7430, CG33138) on the third chromosome
in D. nasuta or its corresponding part of the neo-X and neo-Y
chromosomes in D. albomicans and 12 genes on the second
chromosome (Adh, Arr1, CaBP1, Crc, eIF-3p40, Fdh, Gpdh,
RpL4, Surf4, Tps1, CG3609, CG5028) (supplementary table
S17, Supplementary Material online). For all the 22 genes,
their chromosomal assignments were consistent with those
expected from the correspondence to the chromosomal arms
in D. melanogaster. Therefore, we concluded that theassignments for the rest of the genes based on Muller’s element were valid.

Measurement of Recombination Rates
To measure the recombination rate in D. albomicans andD. nasuta, we counted the number of recombinants resulted
from cross experiments. These two fly species were known tohave chromosomal inversion polymorphisms (Ranganathand Krishnamurthy 1975, Lin et al. 1977). Because crossing
over is suppressed in inversion heterozygotes (Sturtevant1926), it is critical to use a strain pair that have the same
inversion type as the parents. Therefore, we observed salivary
grand chromosomes from 25 or more individuals of the F1
hybrid for 112 and 41 combinations of strain pairs in
D. albomicans and D. nasuta, respectively, to confirm theabsence of inversion loop indicating inversion heterozygotes(supplementary fig. S1, Supplementary Material online). On
the basis of the observation, we selected three strain pairs of
D. albomicans (NG3 female and HRS402 male, SZ43 female
and TSN7 male, and HNZ12 female and KM7 male) and threestrain pairs of D. nasuta (TNR35 female and VNS23 male, K33
female and C3 male, and BZV1 female and MS77 male) as theparent strains for the cross experiments (supplementarytables S18 and S19, Supplementary Material online).
To count the recombinants, we used the pairs of genesthat can distinguish between the parental strains in all the six
strain pairs. Two pairs of genes on the second chromosome(Arr1 and Fdh, and Surf4 and CaBP1) and a pair of genes(GstS1 and Pgm) on the third chromosome (D. nasuta) or
the neo-sex chromosomes (D. albomicans) met this condition. Although the strains in the same strain pair are homosequential, there are chromosomal inversions among
different species pairs (supplementary tables S18 and S19,
Supplementary Material online). Therefore, the chromosomal
position of marker genes and the distance between them may
not be the same among strain pairs.
For each cross, we conducted a pair mating on the standard cornmeal medium at 25  C. Then, we backcrossed asingle female F1 hybrid obtained to a single male of thepaternal strain and counted the recombinants detected by
the SNP markers among 16 males and 16 females in the
backcross first generation (BC1) to measure the female
recombination rate. Similarly, to measure the male recombination rate, we backcrossed a single male F1 hybrid to a single
female of the paternal strain and counted the number ofrecombinants in the BC1 generation.

Comparative Sequence Analyses
Nucleotide sequence alignments for the 55 individual genes in
D. albomicans and D. nasuta were obtained by using Clustal
W (Thompson et al. 1994) with local manual adjustments.
The genetic diversity was quantified by means of Watterson’s (Watterson 1975) and nucleotide diversity,  (Nei andTajima 1981) under the condition of complete gap site deletion by using DnaSP5.1 (Librado and Rozas 2009).
The number of synonymous substitutions per sites wasestimated by the Nei–Gojobori method (Nei and Gojobori
373

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GstS1, Idh, Jheh3, Mpcp, Mys45A, NHP2, Nurf-38, Oscp, Pdh,
Pdi, Pgm, porin, rept, Rga, RnrS, RpL4, Rpn1, SdhB, Set, SPE,
Spn88Ea, srp, Srp68, Surf4, Tango7, Thiolase, Tps1, Uch-L3, und,wal), which were single copy and orthologous in the 12
Drosophila genomes (Drosophila 12 Genomes Consortium
2007), to determine the nucleotide sequences for the 16
D. albomicans strains and the 16 D. nasuta strains. Because
many of these strains were heterozygous for many of thegenes, we crossed a single male from each strain to a single
female of an inbred D. albomicans strain, NG3, for which
nucleotide sequences of all the genes were predetermined,
and determined the nucleotide sequences of the neo-X andneo-Y linked genes from a single female and a single male of
the F1 hybrid, respectively.
The DNA fragments of each target gene were amplified by
PCR with Ex Taq polymerase (Takara Co. Ltd.) from total
DNA extracted from a whole body by a silica-gel based
method following Boom et al. (1990). We newly designed
101 PCR primers and used them together with two (Gpdh
F and Gpdh R) and seven (Amyrel F, Amyrel R, Apf F, Apf R,
NHP2 F, NHP2 R and Pdh F) sets designed by Kwiatowski et al.
(1997) and Chang et al. (2008), respectively, to amplify the
DNA fragments for the 53 genes in total (supplementary table
S16, Supplementary Material online). The resultant PCR fragments were purified by the silica-gel based method and thenucleotide sequences were determined by using BigDye
Terminator Ready Reaction Cycle Sequencing Kit v1.1 orv3.1 and a 3130xl Genetic Analyzer (Applied Biosystems).
The nucleotide sequences obtained have been depositedto GenBank (accession numbers LC057726–LC059853).

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Satomura and Tamura . doi:10.1093/molbev/msv221

1986) with Jukes–Cantor multiple-hit correction (Jukes andCantor 1969) by using MEGA5 software (Tamura et al. 2011).
Using the synonymous distances obtained, we computed thenumber of nucleotide substitutions per site (dXY) and the
net number of nucleotide substitutions per site (dA) between
the neo-X and neo-Y chromosomes in D. albomicans and thethird chromosome in D. nasuta.

Supplementary Material
Supplementary figure S1 and tables S1–S19 are available at
Molecular Biology and Evolution online (http://www.mbe.
oxfordjournals.org/).

Acknowledgments

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The authors thank Aya Takahashi for stimulating discussion
and valuable comments. Further thanks to the associate
editor and two anonymous reviewers for their valuable comments and suggestions on earlier versions of this article. This
work was partially supported by Japan Society for the
Promotion of Science Grants-in-Aid for Scientific Research
Project (23370096) to K.T.

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