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Impacts of Sex Ratio Meiotic Drive on Genome Structure
and Function in a Stalk-Eyed Fly
Josephine A. Reinhardt
1,
*, Richard H. Baker
2
, Aleksey V. Zimin
3
, Chloe Ladias
1
, Kimberly A. Paczolt
4
,
John H. Werren
5
, Cheryl Y. Hayashi
2
, and Gerald S. Wilkinson
4
1
Biology Department, State University of New York at Geneseo, Geneseo, New York, USA
2
Sackler Institute for Comparative Genomics, American Museum of Natural History, New York, New York, USA
3
Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland, USA
4
Department of Biology, University of Maryland, College Park, Maryland, USA
5
Department of Biology, University of Rochester, Rochester, New York, USA
*Corresponding author: E-mail: reinhardt@geneseo.edu.
Accepted: 15 June 2023
Abstract
Stalk-eyed flies in the genus Teleopsis carry selfish genetic elements that induce sex ratio (SR) meiotic drive and impact the
fitness of male and female carriers. Here, we assemble and describe a chromosome-level genome assembly of the stalk-eyed
fly, Teleopsis dalmanni, to elucidate patterns of divergence associated with SR. The genome contains tens of thousands of
transposable element (TE) insertions and hundreds of transcriptionally and insertionally active TE families. By resequencing
pools of SR and ST males using short and long reads, we find widespread differentiation and divergence between X
SR
and
X
ST
associated with multiple nested inversions involving most of the SR haplotype. Examination of genomic coverage and
gene expression data revealed seven X-linked genes with elevated expression and coverage in SR males. The most extreme
and likely drive candidate involves an XSR-specific expansion of an array of partial copies of JASPer, a gene necessary for main-
tenance of euchromatin and associated with regulation of TE expression. In addition, we find evidence for rapid protein evo-
lution between X
SR
and X
ST
for testis expressed and novel genes, that is, either recent duplicates or lacking a Dipteran
ortholog, including an X-linked duplicate of maelstrom, which is also involved in TE silencing. Overall, the evidence suggests
that this ancient X
SR
polymorphism has had a variety of impacts on repetitive DNA and its regulation in this species.
Key words: selfish genetic elements, transposable elements, Diptera, JASPer.
Significance
Sex ratio meiotic drivers can have powerful impacts on evolution, impacting the genome structure and putting species in
danger of extinction, but the genomic causes and consequences of drive are poorly understood. By assembling and ana-
lyzing the genomes of two stalk-eyed flies, we are able to document the dramatic consequences of meiotic drive, includ-
ing a promising candidate involved in chromatin regulation, adding to a growing body of work connecting disruption of
genome packaging to these selfish genetic elements.
© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits
non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
Introduction
The genome was once thought to be little more than a
blueprint needed to accomplish biological functions and re-
production of an organism. Yet research in the past few
decades has demonstrated that genomes of most organ-
isms are heavily colonized by selfish genetic elements
(SGEs) with their own evolutionary interests (Werren et al.
1988; Burt and Trivers 2006; Werren 2011; McLaughlin
GBE
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and Malik 2017). Meiotic drivers are a well-studied cat-
egory of SGE, also known as segregation distorters. These
elements spread by manipulating gametogenesis in their
favor, leading to greater than 50% representation of the
driver in mature gametes (Lyttle 1991). Gamete killers are
a common type of meiotic driver (sperm and spore killers),
which cause gametes not inheriting the driver to fail to de-
velop. If drivers are on a sex chromosome, a skew in the sex
ratio (SR) of offspring will result. Such SR distortion may
cause population collapse or even extinction (Hamilton
1967) but can be maintained stably (reviewed in
Curtsinger and Feldman 1980; Jaenike 2001; Lindholm
et al. 2016), and production of excess females has been
theorized to contribute to success in interspecific competi-
tion (Unckless and Clark 2014; Mackintosh et al. 2021).
The molecular mechanisms underlying meiotic drive have
been elucidated in an increasing number of species in recent
years (Grognet et al. 2014; Hu et al. 2017; Nuckolls et al.
2017), (t locus), and signs of common mechanisms have be-
gun to emerge in Drosophila systems (Wu et al. 1988;
Houtchens and Lyttle 2003; Montchamp-Moreau et al.
2006; Nagao et al. 2010; Gell and Reenan 2013;
Larracuente 2014; Helleu et al. 2016; Lin et al. 2018;
Courret et al. 2019; Muirhead and Presgraves 2021;
Vedanayagam et al. 2021), including a role for repetitive
DNA either in the driver or in the target of drive, disruptions
in DNA packaging (histones or protamines), and a role for
genome defense pathways (Courret et al. 2019). However,
meiotic drivers are often associated with chromosomal inver-
sions (Jaenike 2001), making them resistant to standard gen-
etic analysis (Wu and Beckenbach 1983; Dyer et al. 2007;
Paczolt et al. 2017; Fuller et al. 2020). Like any inversion,
drive-associated inversions may exchange alleles within in-
version type via recombination when homozygous, but this
will be relatively rare if drive is at a low frequency. A reduc-
tion in recombination slows or prevents purging of new dele-
terious mutations (Muller 1964) and may also reduce
nucleotide diversity (Smith and Haigh 1974; Charlesworth
et al. 1993), presumably leading to reduced fitness to carriers
over time. The role of such linked variation in the evolution-
ary trajectory of drive systems remains poorly understood.
Here, we analyze the impacts of SGEs within the genome
of a stalk-eyed fly (Teleopsis dalmanni). In this species, 10–
30% of males possess X-linked elements that prevent prop-
er development of Y-bearing sperm and result in carrier
males producing 90% or more daughters (Presgraves
et al. 1997). This SR X chromosome has multiple impacts
on individual fitness (Wilkinson et al. 2006; Finnegan
et al. 2019; Meade et al. 2019) including reduced sexual or-
nament (eyespan) size in SR males (Wilkinson et al. 1998;
Johns et al. 2005; Cotton et al. 2014). The SR X chromo-
some (X
SR
) appears to have originated approximately
500 Kya (Paczolt et al. 2017), and hundreds of mostly
X-linked genes are differentially expressed in the testes of
SR males (Reinhardt et al. 2014). X
SR
contains at least one
large chromosomal inversion compared with the standard
(ST) arrangement (X
ST
) and likely more, as recombination
has not been detected in X
SR
/X
ST
females (Johns et al.
2005; Paczolt et al. 2017). Recombination occurs between
X
SR
haplotypes in homozygous females, but the rate of re-
combination is about half of that in X
ST
females (Paczolt
et al. 2017). Reduced recombination and effective popula-
tion size have likely contributed to drastically reduced poly-
morphism on X
SR
(Christianson et al. 2011).
But how much differentiation has occurred between X
SR
and X
ST
in this species? How many inversions are on the X?
What impacts has long-term association with a meiotic
drive element had on the landscape of genetic variation
on X
SR
? Can we identify likely candidate genes involved in
establishing the meiotic drive phenotype? To answer these
questions, we created a chromosome-level genome assem-
bly for T. dalmanni, annotated transposable elements and
genes, and then combined RNA sequencing (RNAseq),
pooled short-read and long-read resequencing data from
males exhibiting SR to identify sequence, copy number,
and expression differences between the two types of X
chromosomes.
Results
A Chromosome Length Assembly of the T. dalmanni
Genome
A primary assembly of the genome of T. dalmanni
(NLCU01000000), a stalk-eyed fly from southeast Asia,
was created using MaSuRCA from hybrid sequencing
data containing long-read and short-read sequences
(supplementary table S1, Supplementary Material online).
All data used in this assembly were generated using females
from a standard SR inbred line (see methods). After haplo-
tig filtering, the assembly was scaffolded using chromatin
conformation information, producing three chromosome-
length scaffolds with a total size of 438.2 Mbp
(NLCU04000000) comprising 95.7% of the filtered
MaSuRCA assembly. We validated the assembly by com-
parison with an independently generated linkage map pro-
duced using a backcross family from a prior QTL study
(Wilkinson et al. 2014) (supplementary fig. S1,
Supplementary Material online). Although the maps were
largely concordant, a 13.3-Mbp region (61–74 Mbp) is in-
verted between the assembly and the linkage map, consist-
ent with an inversion difference between the two
populations used in the QTL study. BUSCO analysis con-
firmed the presence of 96.7% of 3,285 conserved
Dipteran genes (supplementary table S2, Supplementary
Material online), with 2.0% of BUSCO genes duplicated.
Overall, 89.8% of 1-to-1 Drosophila melanogaster ortho-
logs are located on the same Muller element in these two
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Schizophoran fly species (fig. 1A). As previously reported
(Baker and Wilkinson 2010), the 97.2-Mbp Teleopsis X
chromosome is orthologous to chromosome 2L in D. mela-
nogaster (Muller element B). Stalk-eyed flies are among
only a few Dipterans in which Muller B is the X (Vicoso
and Bachtrog 2015). The two autosomes, previously
(Baker and Wilkinson 2010) referred to as “C1” and
“C2” are similarly sized (176 and 165 Mbp). We also pro-
duced a draft assembly of a closely related cryptic species
(Christianson et al. 2005; Paczolt et al. 2017), T. dalmanni
sp 2 (Td2) to polarize molecular evolutionary changes.
This assembly contains 50,545 scaffolds and is less com-
plete (Diptera BUSCO = 90.0%, N50 = 35,545) than the
T. dalmanni sensu stricto assembly. Also as expected, gen-
omic coverage in females was approximately twice that in
males across the X, but not the autosomes (fig. 1B).
Compared with D. melanogaster (dm6 RepeatMasker
open-4.0.6), repetitive sequences cover more of the T. dal-
manni genome (∼13.5% vs. ∼35.8%). About 10.0% of the
genome is comprised of unclassified interspersed elements,
whereas the rest includes 716 classified families from 38
superfamilies of Class I (DNA) elements (4.8% of genome)
and Class II elements including LINE (10.7%) and LTR
(5.9%) elements, but no SINE elements (supplementary
fig. S2A, Supplementary Material online). LINE elements
were significantly (χ
2
= 3401, P < 0.001) overrepresented
in T. dalmanni compared with the other two species. The
most abundant transposable element (TE) superfamilies in
T. dalmanni include R1-LOA, Jockey, and RTE-BovB
non-LTR (LINE) Class II elements (24,798, 15,719, and
11,712 copies, respectively), Gypsy LTR Class-II elements
(14,484 copies), and TcMar-Mariner Class I (DNA) elements
(13,210 copies). Overall, transposable elements are more
abundant on the T. dalmanni X than on the two autosomes
(293.9 TEs/Mbp on the X vs. 204.5 TEs/Mbp on the two
autosomes, χ
2
= 364.46, P < 0.0001) (fig. 1C). TE distribu-
tion also varies by element type, with more X-linked DNA
elements (26.6%, χ
2
= 364, P < 0.0001) and LINE elements
(25.3%, χ
2
= 372, P < 0.0001) but fewer X-linked LTR ele-
ments than expected (14.9%, χ
2
= 532, P < 0.0001) given
the X comprises 22.2% of the genome. The assembly is
from female tissue, so we do not have a Y-chromosome
FIG. 1.—A chromosome length assembly of the stalk-eyed fly genome reveals gene synteny and movement compared with D. melanogaster, and unique
patterns of sequence variation and differentiation on the X chromosome influenced by meiotic drive. (A) Slopegraph indicating the locations of 7,634 T. dal-
manni—D. melanogaster 1-to-1 orthologs in each genome. Genes are ordered left to right by their Muller element (A–F) in D. melanogaster, with 89.8%
found on the same Muller element in the two species. The X chromosome in T. dalmanni is Muller element B (chromosome 2L in D. melanogaster). C1 consists
of Muller D and A (chromosomes 3L and X in D. melanogaster), and C2 contains Muller C, F, and E in that order (chromosomes 2R, 4, and 3R in D. mela-
nogaster). (B) As expected, the X chromosome has reduced WGS coverage (reads per bp) in a male genomic DNA library compared with a female library. (C)
Transposable element copies are more abundant on the T. dalmanni X than on the two autosomes. (D) New transposable element insertions are less common
on the X.
Comparative Genomics of Meiotic Drive X GBE
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assembly; however, we found several transposable ele-
ments showing excess of male coverage (supplementary
table S3, Supplementary Material online), suggesting an
excess of insertions on the Y chromosome. For example,
one Penelope element was present on male reads 8-fold
more than on female reads. Many TE families are transcrip-
tionally active (supplementary fig. S2B, Supplementary
Material online) and are producing new insertions in the
genome (fig. 1D).
The SR X Is Diverging from the ST X due to Multiple
Overlapping Inversions
Using long reads generated from SR male siblings aligned
to the reference genome, we identified six large
X-chromosomal inversions that differ between SR males
and the reference genome (fig. 2A), which we further vali-
dated by examining short-read pool-seq data from SR and
ST males at breakpoint regions (supplementary table S4
and fig. S3, Supplementary Material online). These inver-
sions spanned the entire 97-Mbp chromosome except for
a small region at the proximal end (0–2.04 Mbp) and a re-
gion between 60.4 Mbp and 82.7 Mb. Many of the inver-
sions overlap, particularly near the proximal end. Of the
four inversions with outgroup data available from the Td2
draft genome, three are derived in the SR lineage and
one (inversion 2) is derived in the ST lineage
(supplementary table S4, Supplementary Material online).
We compared patterns of genetic variation on the auto-
somes and the X
SR
and X
ST
chromosomes using the pool-
seq short-read resequencing data from two pools of SR
and four pools of ST males derived from two field sites
(Gombak Field Studies Center and Kanching Forest Park)
near Kuala Lumpur, Malaysia (fig. 2B and Cand
supplementary fig. S4, Supplementary Material online).
As expected, the nucleotide diversity on autosomes in SR
(0.020134 ± 0.0000839) and ST (0.021375 ± 0.001432)
pools does not differ significantly (t-test SR vs. ST for C1,
P = 0.309, for C2, P = 0.3332) from each other but we
find that X
SR
has significantly reduced diversity
(0.0057492 ± 0.001775) compared with X
ST
(0.015670 ±
0.001410 CI, t-test P = 0.00122, fig. 2B). All three chro-
mosomes contain regions with reduced nucleotide diver-
sity in all pools (supplementary fig. S4A, Supplementary
Material online), which are presumably centromeric re-
gions (Begun and Aquadro 1992; Begun et al. 2007),
as they are either near the center of the metacentric
autosomes or the end of the X chromosome. Genetic
differentiation (F
ST
) is strongly elevated across the X
chromosome between SR types (X
SR
vs. X
ST
) and to a less-
er degree was elevated between collection sites but with-
in the SR type (fig. 2C) but is not elevated on the autosomes
(supplementary fig. S4B, Supplementary Material online).
Patterns of nucleotide diversity and differentiation appear
to be influenced by proximity to the inversions on the
X chromosome (fig. 2). F
ST
was elevated, and poly-
morphism reduced where there is a higher density of
overlapping inversions. In particular, the region between
17 Mbp and 20 Mb, where five of the six inversions over-
lap, has high F
ST
between X
SR
and X
ST
and reduced diver-
sity within all pools.
Gene Expression and Copy Number Variation Reveal
Drive Candidates on the SR X
Using the pool-seq reads from X
SR
and X
ST
males above in
combination with X
SR
and X
ST
RNAseq reads previously ob-
tained from pools of mature testes (Reinhardt et al. 2014),
we jointly evaluated differences in gene copy number and
expression between X
SR
and X
ST
. We identified 596 DE
genes (37.6% had higher expression in X
SR
) and 120 genes
with differential genomic coverage (DC genes, 62.5% with
higher coverage on X
SR
). Among the 48 genes that were
both DE and DC, there was a highly significant and positive
association in the direction of DE and differential genomic
coverage (FET P < 0.0001) with only four genes having
higher X
SR
coverage but higher X
ST
expression. This could
be due to a direct effect of the copy number on expression
but is not definitive of a causal relationship. As expected
from prior work (Reinhardt et al. 2014; Paczolt et al.
2017), most SR–ST differences in gene expression
(75.6%) and genomic coverage (89.2%) were confined
to the X. Seven annotated protein-coding genes (JASPer,
Pbp95, Tetraspanin 29Fb, Minichromosome maintenance
10, isopeptidase-T-3, Cyclin K, and santa-maria) exhibited
both differential expression (DE) and differential genomic
coverage (DC) between SR and ST males (supplementary
table S5, Supplementary Material online). Strikingly, all se-
ven were X linked and had a higher level of both expression
and coverage in SR males. We also identified 11 transpos-
able element families that differ in either expression or
copy number between SR and ST, with roughly an equal
number of families differentially expressed in each
(supplementary table S3 and fig. S2C, Supplementary
Material online). Maverick, Gypsy, and R1-LOA elements
are overexpressed in SR males, whereas TcMariner,
PIF-Harbinger, and two types of RTE elements have excess
coverage on the SR X chromosome.
The highest level of differential expression and coverage
was found for an X-linked paralog of JASPer (Jil-1
Anchoring and Stabilizing Protein), a gene which normally
regulates the maintenance of euchromatin and is involved
in female fertility and X-chromosome dosage compensa-
tion (Albig et al. 2019; Dou et al. 2020). Examination of
genomic coverage near the JASPer region (X:18.18 Mbp)
shows a 20-fold increase in coverage over a 1.5-Kbp region
containing a 1.1-Kbp gene (fig. 3A). Long- and short-read
sequences from SR males showed supplemental alignment
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to nearby regions of the X at 18.19 and 18.27 Mbp. These
two regions also contain a ∼1.5-Kbp region with 20-fold in-
creased raw short-read coverage. At 18.19 Mbp, the gene
exhibits the same two-exon structure and length as the
copy at 18.16 Mbp but is positioned in the opposite orien-
tation. At 18.27 Mbp, a smaller (∼600 bp) single-exon
region is transcribed. Translations of the transcripts pro-
duced by these three partial JASPer genes contain only
one of the two major functional domains of D. melanoga-
ster JASPer, PWWP, which normally interacts with activat-
ing chromatin marks (H3K36me3). There are also three
full-length paralogs of JASPer on the C2 autosome and
two additional X-linked copies that contain only the other
functional domain, LEDGF, known to interact with
JASPer’s functional partner, JIL-1 in Drosophila (fig. 3B).
Although eight other D. melanogaster proteins contain
PWWP domains, a maximum likelihood phylogeny shows
that the T. dalmanni JASPer PWWP domains are closer
orthologs to the PWWP domain of D. melanogaster
JASPer than to other Dipteran PWWP domains (fig. 3C),
confirming these are partial JASPer paralogs. Neither of
the LEDGF-only X-linked copies exhibit differential ex-
pression or coverage between SR and ST males (fig. 3A).
The JASPer paralogs are all single copy in X
ST
but are amp-
lified in copy number in X
SR
. Using only uniquely mapping
SR and ST short reads, we estimated the increase in copy
number of each JASPer copy on X
SR
. The fold increase
rates in genomic coverage of SR libraries across the amp-
lified regions are ∼58.9-fold (at 18.181:Mbp), 3.4-fold (at
18.194:Mbp) and 6.5-fold (at 18.270:Mbp) compared
with ST libraries (fig. 3A, “unique”). For the copy at
18.181:Mbp, we were also able to identify long reads
which aligned uniquely to sequence matching both the
left and right flanking regions and extended into the
gene copy and these contained five or six tandem copies
of the PWWP-only JASPer paralog (supplementary fig. S5,
Supplementary Material online). We also searched for
supplemental alignments of PWWP-JASPer aligning reads
to other places on the X chromosome, as these could in-
dicate additional places that a PWWP-JASPer might have
FIG. 2.—Comparison of the SR X chromosome (X
SR
) with the ST X chromosome (X
ST
) using long-read and short-read resequencing. (A) Six inversions were
identified using alignment of PacBio long-read sequencing of males carrying the X
SR
chromosome to the reference genome. The shaded area indicates a
7-Mbp region where five of the six inversions overlap. Two replicate WGS Illumina sequencing pools were constructed from screened males exhibiting stand-
ard SRs from each of two collection sites near Kuala Lumpur: Gombak Research station (GST) and Kanching Forest park (KST). One pool of males exhibiting
female-biased SRs was also sequenced from each site. (B) Nucleotide diversity (pi) estimated from pool-seq libraries is lower across the X for the two SR pools
compared with the four ST pools. (C) Compared with between-replicate comparisons (KST and GST), differentiation (F
ST
) is only slightly elevated between
populations with the same SR type (between populations) but F
ST
is notably elevated chromosome wide in pairwise comparisons between ST and SR pool-seq
(between SR types). (D) Locations of named genes that were differentially expressed (DE) in testes between SR and ST RNAseq samples or have differential
coverage (DC) between SR and ST WGS pools or both (DCDE) are also shown.
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inserted, providing an explanation of the excess coverage
observed at 10.181 Mbp. We found a few reads mapping
partly elsewhere on the X chromosome. However, none
of these locations were represented by more than a single
sequencing read.
Rapid Protein Evolution between X
ST
and X
SR
Given most of the X chromosome shows elevated genetic
differentiation (FST) between X
ST
and X
SR
, we calculated
dN/dS to detect protein evolution occurring between these
two chromosomal haplotypes, and also between
FIG. 3.—JASPer-PWWP has amplified in expression and coverage on the SR X. (A) Examination of mapped reads in X-linked gene regions with homology
to JASPer shows elevated expression (RNAseq) and coverage in pool-seq WGS libraries (pool-seq raw) from SR males, but not ST males from the same collec-
tions (GST and KST). Only those copies of JASPer including the PWWP domain showed increases in expression and coverage. Examination of uniquely mapping
pool-seq reads (pool-seq Unique) demonstrates that the excess SR coverage is largely limited to the JASPer copy at 18.181 Mbp. (B) T. dalmanni contains eight
transcripts with detectable homology to D. melanogaster JASPer. Copies on the second autosome (C2) contain both canonical domains (PWWP and LEDGF)
whereas the five X-linked copies contain only one of the two domains. Amino acid percent identity compared with D. melanogaster JASPer is shown for each
domain. (C) T. dalmanni JASPer copies are orthologous to PWWP from D. melanogaster JASPer as confirmed by a maximum likelihood phylogeny of all PWWP
domains in D. melanogaster and all PWWP domains from T. dalmanni JASPer copies. The three X linked JASPer PWWP domains from ∼18 Mbp are identical in
amino acid sequence.
FIG. 4.—Rapid protein evolution on the SR X chromosome. dN/dS between T. dalmanni and T. dalmanni sp 2 (Td2) was assessed for 9,525 protein-coding
genes, and dN/dS between the X
SR
and X
ST
chromosomes was assessed for 2,642 X-linked genes. Significance of comparisons was assessed using linear mod-
els on log-transformed dN/dS values and ANOVA (supplementary tables S5–S8, Supplementary Material online). The expression pattern of genes was anno-
tated based on tissue-specific RNAseq in T. dalmanni (Reinhardt et al. 2014), and the age of genes was based on a prior comparative analysis (Baker et al.
2016). (A) dN/dS was significantly correlated in the two comparisons. (B) dN/dS between X
SR
and X
ST
copies of genes was elevated in testis-specific genes
and newer categories of genes, with no significant interaction. (C) The same was true for the interspecific comparison (T2dN/dS).
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T. dalmanni and a cryptic sister species we described pre-
viously, T. dalmanni sp 2 (Paczolt et al. 2017). For X-linked
genes observed in both comparisons, dN/dS was positive-
ly correlated (Pearson’s product–moment correlation =
0.57, P < 2.2e
−16
, fig. 4A). Using prior annotations of
gene novelty and expression pattern (Reinhardt et al.
2014; Baker et al. 2016) and a linear model to predict
dN/dS, we find that novel protein-coding genes (dupli-
cated genes and genes without an identifiable ortholog
outside of Diopsidae) and testis-expressed genes have sig-
nificantly higher dN/dS in both the interspecific and X
SR
–
X
ST
comparisons (fig. 4B and Cand supplementary tables
S6–S9, Supplementary Material online). In the interspecif-
ic comparison, there is also a significant interaction effect
between gene age and expression.
In the X
SR
–X
ST
comparison, 25 genes were putatively
evolving under positive selection (dN/dS > 1, table 1). Of
those with a dN/dS estimate in the interspecific compari-
son, most (11/13) were not under positive selection be-
tween species (dN/dS < 1). Among the positively selected
genes, nine of 25 were also differentially expressed be-
tween SR and ST. These nine included three genes with
Drosophila orthologs, Pol32 (a component of DNA poly-
merase involved in double-strand break repair), an unchar-
acterized transmembrane protein (Tetraspanin 29Fb), and
Cyclin K, which phosphorylates RNA polymerase II and con-
tributes to pre-mRNA processing, transcription, and chro-
matin structure.
Discussion
The presence of meiotic drivers can lead to dramatic gen-
omic changes due to their conflicting interests with their
hosts (Burt and Trivers 2006). Here, we analyzed the
impacts on a stalk-eyed fly (T. dalmanni) genome of a long-
term association with such an element. Prior work sug-
gested widespread genetic differentiation between X
SR
and X
ST
chromosomes (Reinhardt et al. 2014) and identified
at least one inversion distinguishing the two (Paczolt et al.
2017). By assembling the genome into three chromosome-
length scaffolds, we find dramatic differentiation between
X
SR
and X
ST
that extends across the entire X chromosome, a
sign that genetic recombination between the two types of
X chromosomes has been severely limited for a long time
(figs. 1 and 2). By combining pool-seq short-read and long-
read resequencing data, we located the breakpoints of six
overlapping inversions that span most of the X (fig. 2 and
supplementary table S3, Supplementary Material online)
and have permitted expression, copy number, and se-
quence divergence to accumulate between the inversion
haplotypes.
We identified several genes that have diverged dramat-
ically in copy number, and expression between X
SR
and
X
ST
including a paralog of JASPer (JIL-1 Anchoring and
Stabilizing Protein, also known as dP75). JASPer has mul-
tiple new paralogs in the genus Teleopsis, and these have
amplified in expression and copy number on X
SR
relative
to X
ST
(fig. 3 and supplementary table S4, Supplementary
Material online). In D. melanogaster, JASPer positively reg-
ulates the transition of heterochromatin to euchromatin
with its partner JIL-1, is involved in X-chromosome dosage
compensation (Albig et al. 2019), and is essential during
oogenesis (Dou et al. 2020). A partial paralog of JASPer
appears to have formed tandem arrays on X
SR
, with
only part of the gene—the chromatin-binding PWWP
domain—being duplicated and upregulated in expression
in X
SR
(fig. 3). This amplified partial paralog (Td-JASPer:
X:18.181Mbp) is found in a region overlapped by five of
the six inversions we identified as distinguishing X
SR
and
X
ST
(fig. 2). Interestingly, the excess coverage detected
in the Illumina sequencing (∼X) is much more than can
be explained by the spanning PacBio reads at the copy
inserted at18.181 Mbp and we were unable to identify
additional copies inserted elsewhere on X
SR
. It may be
that additional copies are present on X
SR
, but that the
flanking sequences are also X
SR
specific and/or were
not assembled completely in the reference genome.
Td-JASPer:X:18.181Mbp is also testis specific, whereas
the full-length paralog closest in sequence to the D. mel-
anogaster ortholog (Td-JASPer:C2:156Mbp) is primarily
expressed in the ovary (fig. 3B).
PWWP domains, like those found in JASPer, primarily
bind to H3K36me3 chromatin marks (Albig et al. 2019;
Dou et al. 2020), which are associated with regions of ac-
tive gene expression. Although we do not know the target
of any JASPer paralog in T. dalmanni, it would seem likely
this function is conserved given it occurs across metazoa.
Plausibly, the PWWP-only JASPer duplicates expressed
from X
SR
might bind to their targets but are unable to re-
cruit JIL-1 and therefore fail to properly activate target
genes, similar to a dominant negative allele. JIL-1/JASPer
binding is also implicated in positive regulation of expres-
sion of Gypsy5 retroelements, which are found in arrays
in Drosophila telomeres (Albig et al. 2019), providing a
plausible connection between this drive candidate and dif-
ferences in expression of certain TEs. We found that Gypsy
elements are generally less common on the X chromosome
and several Gypsy families show a male bias in genomic
coverage suggesting bias towards insertion on the Y
(supplementary table S3, Supplementary Material online).
The identification of a partial duplicate of a chromatin-
binding protein bears a striking resemblance to another
case of Dipteran meiotic drive, the so-called “Paris” SR drive
system in D. simulans (Montchamp-Moreau et al. 2006).
As in the present case, the SR copy of the causal gene—
HP1D2—retains one domain—the histone-binding chro-
modomain, but not the protein binding chromoshadow
(CSD) domain (Helleu et al. 2016). Knocking out the
Comparative Genomics of Meiotic Drive X GBE
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standard copy of HP1D2 or deleting its CSD partly recapitu-
lates drive, and HP1D2 binds specifically to Y-chromosomal
heterochromatin. If the chromatin targets of PWWP-JASPer
are similarly Y specific, development of Y-bearing, but not
X
SR
-bearing, sperm could be similarly disrupted.
The widespread divergence of X-linked sequences be-
tween X
SR
and X
ST
has had impacts beyond meiotic drive it-
self. Not only are hundreds of genes differentially expressed
or varying in copy number, we further find that many
X-linked genes are diverging rapidly (dN/dS > 1) between
the chromosome types (fig. 4). Unsurprisingly, new genes
and testis-specific genes evolved more quickly both be-
tween species and between the SR types, though the pat-
terns of evolution are loosely correlated between the
comparisons (r = 0.56). The model built to predict X
SR
–X
ST
dN/dS explains less (19%) of the total variation in dN/dS
than the interspecific comparison (30%), indicating
that drive-associated protein evolution may be more idio-
syncratic and less driven by “typical” trends in molecular
evolution (e.g., gene expression level and age). Whereas
some of this difference is presumably due to stronger gen-
etic drift occurring on X
SR
due to a lack of recombination
(Muller 1964), some genes are evolving under positive se-
lection (table 1) and these are rarely (2 of 13 cases) the
same genes as those evolving adaptively between species.
Among those diverging adaptively between X
SR
and X
ST
but not between species are a number of novel and unchar-
acterized genes, the genome defense gene maelstrom,
Pol32, a DNA polymerase subunit involved in DNA repair at
chromosome fragile sites (Ji et al. 2019), and RNA-binding
protein La autoantigen like. Given JASPer’s putative role in
chromatin regulation, the identification of maelstrom and
Pol32 is intriguing. Maelstrom acts to silence transposable
elements by promoting the spread of heterochromatic states
to nearby genes (Sienski et al. 2012), and chromatin states
have been found to be tightly integrated with genome integ-
rity via multiple DNA repair pathways (reviewed in
Papamichos-Chronakis and Peterson 2013), including at
chromosome fragile sites.
Overall, these results extend the work on the genomic
impacts of SR meiotic drive (reviewed in Courret et al.
2019) to a charismatic organism—Teleopsis stalk-eyed
flies—and suggest that although the specific mechanisms
of drive are idiosyncratic, drivers may be converging repeat-
edly on similar genomic vulnerabilities.
Material and Methods
Genome Assembly of T. dalmanni s.s.
A draft genome assembly for T. dalmanni, NLCU01000000,
was created with a combination of Roche 454, Illumina,
Table 1
Genes Diverging under Positive Selection between SR and ST X Chromosomes
Name Gene Age Differential Coverage Differential Expression X
SR
–X
ST
dN/dS Interspecific dN/dS
ORF2072 Single copy No No 2.632 NA
Barren Duplicate No No 2.273 NA
ORF702 Orphan No No 2 NA
ORF1962 Orphan No No 1.887 0.73529412
ORF1691.1 Orphan No Up in X
SR
1.667 1.28205128
Maelstrom Duplicate No No 1.587 0.34602076
La autoantigen-like Single copy No No 1.471 0.36363636
ORF2361 Orphan No No 1.471 NA
Calmodulin Single copy No No 1.429 NA
ORF2114 Orphan No Up in X
ST
1.37 NA
CG10195 Single copy No No 1.351 0.135318
ORF2326 Orphan No No 1.333 NA
ORF705 Orphan No Up in X
ST
1.299 0.70921986
CG34109 Single copy No No 1.205 0.31446541
ORF1530 Orphan Up in X
ST
Up in X
ST
1.19 NA
ORF1691.2 Orphan No Up in X
SR
1.19 1.31578947
La autoantigen-like Single copy No No 1.163 0.1980198
Pol32 Single copy No Up in X
ST
1.111 0.42194093
Heat shock protein 60 Duplicate No No 1.099 NA
Tetraspanin 29Fb NA Up in X
SR
Up in X
SR
1.087 NA
ORF739 Orphan No Up in X
ST
1.064 NA
ORF944 Orphan No No 1.064 No
Cyclin K Duplicate Up in X
SR
Up in X
SR
1.053 0.70921986
CG15435 Single copy No No 1.042 0.24630542
ORF1457 Orphan No No 1.02 0.36630037
Reinhardt et al. GBE
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and Pacific Biosciences sequence data (supplementary table
S1, Supplementary Material online) using MaSuRCa (Zimin
et al. 2013) and is available on Ensembl metazoa,
GCA002237135v2 (Kersey et al. 2018). A chromosome-
level assembly (NLCU04000000) was then created by
incorporating chromatin conformation information and
validated with a high-density linkage map.
All DNA sequences used in the assembly were obtained
from an inbred population (line “2A”) of T. dalmanni. This
population is derived from flies that were first collected
near the Gombak River in peninsular Malaysia (3 12′N,
101 42′E) in 1989 and then maintained as a control line
for an artificial selection study on relative eyespan
(Wilkinson 1993; Wolfenbarger and Wilkinson 2001).
After 50 generations of selection, full-sib mating was con-
ducted for seven generations to establish the line, which
has subsequently been maintained without additional in-
breeding. This population has been used in several prior
studies (Christianson et al. 2005; Wilkinson et al. 2014)
and does not carry any drive-associated genetic markers.
Contaminating bacterial scaffolds were identified and re-
moved prior to submission using a modification of the
Wheeler et al. (2013) DNA-based homology pipeline
(Poynton et al. 2018). Male and female genomic short-read
resequencing data were aligned to each scaffold using
NextGenMap v0.5.5 (Sedlazeck et al. 2013) with default
parameters, and relative coverage of male and female reads
was used to identify X-linked scaffolds (cf. Vicoso and
Bachtrog 2015), with the expectation that the normalized
ratio of female to male reads should be approximately 1
to 1 for autosomal and 2 to 1 for X-linked scaffolds. The
NLCU01 assembly was then filtered for haplotigs and
other redundant sequences using Purge Haplotigs v1.1.1
(Roach et al. 2018) prior to scaffolding to create the
NLCU04000000 assembly. A prior scaffolding attempt was
also done without first haplotig filtering resulting in an as-
sembly not used in the present analysis (NLCU02000000).
For scaffolding, chromatin conformation capture data
were generated using a Phase Genomics (Seattle, WA)
Proximo Hi-C Plant Kit, which is a commercially available
version of the Hi-C protocol (Lieberman-Aiden et al.
2009). Following the manufacturer’s instructions, intact
cells from unsexed pupae from the 2A inbred line were
crosslinked using a formaldehyde solution, digested using
the Sau3AI restriction enzyme, and proximity ligated with
biotinylated nucleotides to create chimeric molecules com-
posed of fragments from different regions of the genome
that were physically proximal in vivo, but not necessarily
genomically proximal. Continuing with the manufacturer’s
protocol, molecules were pulled down with streptavidin
beads and processed into an Illumina-compatible sequen-
cing library. Sequencing was performed on an Illumina
HiSeq 4000, generating a total of 202,608,856 100-bp
read pairs. Reads were aligned to the draft assembly
(NLCU01.30_45_breaks.fasta) following the manufac-
turer’s recommendations. Briefly, reads were aligned using
BWA-MEM v. 0.7.15-r1144-dirty (Li and Durbin 2009) with
the -5SP and -t 8 options specified, and all other options de-
fault. SAMBLASTER (commit 37142b37e4f0026e1b83-
ca3f1545d1807ef77617, Faust and Hall 2014) was used
to flag PCR duplicates, which were later excluded from ana-
lysis. Alignments were then filtered with samtools (Li et al.
2009) using the -F 2304 filtering flag to remove nonprimary
and secondary alignments. Putative misjoined contigs were
broken using Juicebox v1.11.08 (Rao et al. 2014; Durand
et al. 2016) based on the Hi-C alignments. A total of 113
breaks in 105 contigs were introduced, and the same align-
ment procedure was repeated from the beginning on the
resulting corrected assembly. The Phase Genomics’
Proximo Hi-C genome scaffolding platform (commit
aa3382b6e63f7b99e92a9d95c553ef1c6a5a6a38) was
used to create chromosome-scale scaffolds from the cor-
rected assembly as described (Bickhart et al. 2017). As in
the LACHESIS method (Burton et al. 2013), this process
computes a contact frequency matrix from the aligned
Hi-C read pairs, normalized by the number of Sau3AI re-
striction sites (GATC) on each contig, and constructs scaf-
folds in such a way as to optimize expected contact
frequency and other statistical patterns in Hi-C data. In add-
ition to Hi-C data, chromosomal linkage information (see
below) was used as input to the scaffolding process.
Linkage groups from a linkage map were used to constrain
chromosome assignment during the clustering phase of
Proximo by discarding any suggested clustering steps that
would incorporate contigs from different linkage groups
onto the same chromosome, but linkage map data were
not used during subsequent ordering and orientation ana-
lyses in Proximo. Approximately 528,000 separate Proximo
runs were performed to optimize the number of scaffolds
and scaffold construction to make the scaffolds as concord-
ant with the observed Hi-C data as possible. This process re-
sulted in a set of three chromosome-scale scaffolds
containing a total of 438.2 Mb, comprising 95.7% of the
filtered MaSuRCa assembly. Finally, Juicebox was again
used to correct the remaining scaffolding errors.
Linkage groups used to constrain and later validate the
gene order in the Hi-C assembly were created by mating
a female hybrid offspring obtained from a cross between
a male from the 2A inbred strain and a female from a non-
inbred population of T. dalmanni collected near Bukit
Lawang, Sumatra (3 35′N, 98 6′E), to a male from the 2A
strain. This backcross produced 249 (131 female and 118
male) individuals that were individually genotyped using
multiplex shotgun sequencing (Andolfatto et al. 2011)
and multiple STR loci (Wilkinson et al. 2014). Genotypes
were determined as either heterozygous or homozygous
for each scaffold by combining all loci present on a scaffold
into a single “super locus.” Reads were aligned using BWA
Comparative Genomics of Meiotic Drive X GBE
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v0.7.17 (Li and Durbin 2009), and genotypes were assessed
as either homozygous or heterozygous using samtools
v.1.9 (Li et al. 2009) (mpileup -v). Because this was a back-
cross, for autosomal loci, those individuals with the back-
cross allele (pure “2A”) should be homozygous at
informative markers whereas individuals with the nonback-
cross allele should be heterozygous, with an expectation of
a 1 to 1 ratio of these genotypes. This results in an overall 3
to 1 ratio of the backcross to the nonbackcross allele for
autosomal markers and X-linked markers in females, and
an overall 1 to 1 ratio for X-linked markers in hemizygous
males. Markers were retained as potentially informative if
at least one individual was found to carry the nonbackcross
(Wilkinson et al. 2014) allele, which we defined as the less
common allele across all female individuals. Markers were
removed if they violated expected allele ratios for a back-
cross using a binomial test against the expectations de-
scribed above, or if more than 20% of female individuals
were found to carry only the nonbackcross allele. Finally,
within each individual, all markers from a given scaffold
were pooled to give an overall number of reads supporting
each genotype and requiring a minimum coverage of five
reads per marker. Individuals were assigned in the final ma-
trices as “a” (for 2A/backcross genotypes) or “b” (for Bukit
Lawang/foreign genotypes).
Separate genotype matrices were then created for the
X-linked scaffolds (as determined by male and female
coverage) (Vicoso and Bachtrog 2015) and autosomal scaf-
folds and rank ordered by the number of individuals geno-
typed. We then used JOINMAP v4.1 (Stam 1993) to assign
the top 1,000 autosomal scaffolds into one of two linkage
groups (chromosomes). Only scaffolds with a LOD score > 5
were assigned to a chromosome. We used a similar process
for the top 250 X-linked scaffolds but used a LOD score >
10 to assign scaffolds to the chromosome. These linkage
groups were used to constrain the Hi-C genome assembly
as noted above. Then, independent from the Hi-C scaffold-
ing process, we ordered scaffolds within each linkage
group by regression mapping using a Haldane mapping
function. We used regression mapping, rather than max-
imum likelihood, because it is less sensitive to missing geno-
type data (Van Ooijen 2006) as is typical for multiplex
shotgun sequencing data sets. We removed markers from
the final map if there was evidence for significant (after
Bonferroni correction) lack of fit to their nearest neighbors.
The resulting linkage map included 762 scaffolds spanning
147.1 Mbp. Collinearity between the Hi-C and linkage
maps was assessed by comparing the relative position of
scaffolds which were found in both maps. Chromosomal
synteny of the assembly scaffolds with D. melanogaster
chromosome arms was assessed by alignment of a set of
7,634 previously annotated (Baker et al. 2016) 1-to-1
Drosophila orthologs to the genome assembly
using GMAP v2019-12-01 (Wu and Watanabe 2005)
(–npaths=1 –format=gff3_gene –min-identity=0.9).
Assemblies were assessed for completeness using
BUSCOv5.0.0 (Seppey et al. 2019) comparing to pan-
Dipteran (diptera_odb10 2020-08-05) and pan-eukaryotic
(eukaryota_odb10 2020-09-10) genesets.
Draft Genome Assembly of T. dalmanni Species 2
We previously described a cryptic species of stalk-eyed fly
(Paczolt et al. 2017), which we refer to as T. dalmanni sp
2 (Td2) and which corresponds to prior collections of T. dal-
manni from several sites in peninsular Malaysia, such as
Cameron Highlands (Christianson et al. 2005; Swallow
et al. 2005). To produce a draft genome of this species,
we extracted HMW DNA from a single male from a labora-
tory population of Td2 using the Gentra Puregene tissue kit
(Qiagen #158667). 1 µg of DNA was sent to the New York
Genome Center (NYGC) where it was prepped with the
Chromium Genome-linked read kit (10× Genomics) and se-
quenced on a half lane of an Illumina HiSeqX machine, pro-
ducing a total of 416 million reads. These reads were
assembled at NYGC using Supernova v2.0.1 (Weisenfeld
et al. 2017). The resultant draft genome contained
10,290 scaffolds greater than 10 kb with a N50 of
45.2 kb and total genome size of 355 Mb. Although in-
complete, this genome was sufficient to determine inver-
sion history and polarize molecular divergence for some
genes.
Short-Read Resequencing of SR and ST Males
To identify sequence and structural variations specific to
X
SR
, we sequenced DNA from replicate pools of SR males
(males with female-biased offspring SRs), or ST males either
collected in the field from two different sites in peninsular
Malaysia or representing the first three generations of
sons descended from field-collected females. One SR and
two ST sample pools were created from the DNA of males
from each of two collection sites (Gombak and Kanching)
that were previously phenotyped for offspring SR (Paczolt
et al. 2017). When an excess of individuals was available
from a collection and SR category, genotype data from
nine X-linked STR loci from the same analysis were used
to avoid oversampling closely related individuals (e.g.,
male full siblings from the same brood). Haplotype diversity
within pools was not significantly different to haplotype di-
versity among all candidate males for that pool (following
Christianson et al. 2011, Dunnett’s t-test, P > 0.05 for all
comparisons, supplementary table S8, Supplementary
Material online). DNA was extracted using the DNeasy
Blood and Tissue Kit (Qiagen #69504) and quantified using
the PicoGreen Quant-IT dsDNA quantification kit
(Thermofisher Q33130). Pools were then assembled using
an equimolar amount of DNA from each sample. Sample
size for each pool ranged from 15 to 18 individuals
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(supplementary table S8, Supplementary Material online).
Six barcoded libraries were prepared and multiplexed on
two lanes of a HiSeq1500 set to a RapidRun mode to gen-
erate 150-bp paired-end sequences. Bam-formatted align-
ments of these libraries to the genome were produced
using NextGenMap v0.5.5 (Sedlazeck et al. 2013) with de-
fault parameters and used in subsequent analyses. Pairwise
genetic diversity for each pool and F
ST
between each pair-
wise combination of pool-seq data was calculated from
the pooled resequencing alignments in 5-kb nonsliding
windows using popoolation2 (Kofler et al. 2011). After
pool-seq had been completed, it was determined that
four pooled ST individuals were actually Td2 males.
Genetic markers distinguishing these species had not
been identified until after pooling; see supplementary
table S8, Supplementary Material online, (Paczolt et al.
2017), and a previous work (Christianson et al. 2005;
Swallow et al. 2005) had suggested that Td2 would not
be present in the collection sites we visited. Both SR pools
and one ST pool (Kanching ST2) were entirely composed
of T. dalmanni s.s. (supplementary Table S8, Supplementary
Material online) so all analyses where excess polymorphism
(potentially caused by species divergence from the Td2 indivi-
duals) within a pool could impact the results of analysis were
repeated using only these three samples, and results of the re-
duced analysis were found to be qualitatively similar to the full
analysis (supplementary table S9, Supplementary Material
online).
Long-Read Resequencing of a SR Haplotype
To identify inversion breakpoints between X
SR
and X
ST
, we
used long-read sequencing (Pacific Biosciences). A pool of
full-sib males bearing a single identical-by-descent (IBD)
X
SR
haplotype was created by mating an SR/SR female to
a male from the 2A strain and then backcrossing the female
progeny to males from the same strain. We then genotyped
107 sons from this backcross at three X-linked STR loci
(ms125, ms395, and CRC) to distinguish X
SR
and X
ST
sons
(Paczolt et al. 2017). DNA from 46 X
SR
sons was then ex-
tracted using the Gentra PureGene Tissue Kit (Qiagen
#158667) and pooled, followed by a phenol–chloroform
extraction and ethanol precipitation. A PacBio long insert
(15 Kb) library was then prepared and run on three
PacBio Sequel SMRT cells. These runs yielded a total of
15.1 Gb of sequence, with a mean read length of 4.9 Kb
and maximum read length of 92.8 Kb. Raw long reads
were aligned to the genome using ngmlr v0.2.7 with de-
fault parameters, and structural variants were called using
Sniffles v1.0.12 (Sedlazeck et al. 2018), requiring at least
two reads to support each variant call. The resulting output
was filtered to find inversions that were fixed within the SR
PacBio long reads relative to the reference genome. Each
putative inversion was then validated as a fixed
SR-specific inversion by comparison with the read-pair
orientation in the SR and ST pool-seq data at the breakpoint
using IGV (Thorvaldsdottir et al. 2013). An inversion was
considered validated if reads from the SR pool-seq samples
but none of the ST pool-seq samples agreed with the snif-
fles call at that position (supplementary table S3,
Supplementary Material online). Finally, to polarize the dir-
ection of the inversion mutation, an alignment of the T. dal-
manni sp 2 genome assembly was performed using blat
(Kent 2002) and scaffold alignments near the breakpoints
were examined to determine if they 1) support the standard
arrangement (span the breakpoint), 2) support the SR ar-
rangement (scaffold breaks and aligns to other end of
breakpoint), 3) support another arrangement (scaffold pre-
sent near breakpoint), or 4) are uninformative (no scaffolds
map near the breakpoint) (supplementary table S3,
Supplementary Material online).
Transposable Element Annotation
Transposable elements were annotated in the T. dalmanni
s.s. assembly using RepeatModeler v. 1.0.4 (Smit and
Hubley 2008) with default parameters and the
NLCU04000000 assembly as input. RepeatModeler seed
alignments and consensus sequences were submitted to
dfam. Resulting consensus fasta formatted TE sequences
were input into RepeatMasker open4.0.9 (Smit et al.
2013) with the assembly as the reference, producing a
repeat-masked reference genome and repeat annotations.
The tool One code to find them all v2014 (Bailly-Bechet
et al. 2014) was used with the RepeatMasker output
(.out) to count the numbers and locations of each type of
insertion in the T. dalmanni genome. These were compared
with the RepeatMasker annotations for two other Dipterans
(D. melanogaster dm6 RepeatMasker open-4.0.6 and
Anopheles gambiae anoGam1 RepeatMasker open-4.0.5)
analyzed using the same procedure. Further classification
of element superfamilies followed prior universal classifica-
tion schemes (Wicker et al. 2007; Makałowski et al. 2019).
X (or autosomal) chromosome bias in the distribution of ele-
ments of each annotated family was determined by compar-
ing the observed number of intact elements of each element
type on the X to the number expected assuming the X com-
prises 22.5% of the genome using a chi-squared
goodness-of-fit test. To correct for multiple testing, we ap-
plied a Benjamini–Hochberg (BH) 5% false discovery rate
(FDR).
Novel, polymorphic insertions of TE’s were called in each
of the six Illumina resequencing pools using
PopoolationTE2 (Kofler et al. 2016) running the “separate”
analysis mode on the repeat masked assembly and TE con-
sensus sequences. Sites were subsampled to 20× coverage
and were discarded if coverage was less than 20× in any
sample. To compare the rate of insertions of TE’s between
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the samples, insertions were inferred to be orthologous if
they were an insertion of the same element within
500 bp in multiple pools.
The expression of RepeatModeler TE families in SR and ST
male testes was assessed using TEtools copyright (C) 2015
Laurent Modolo (Lerat et al. 2016), using the default settings
and including alignment with bowtie2 v2.2.4 (Langmead and
Salzberg 2012) using the RepeatModeler TE library and
RNAseq reads from two pools of SR male testis and two pools
of ST male testis (BioProject PRJNA240197). Unannotated
repetitive elements (“Unknown” interspersed and simple
repeats) were removed after normalization but prior to differ-
ential expression analysis with DESeq2 v1.32.0 (Love et al.
2014). Differential TE expression between the SR and ST pools
was assessed using the negative binomial Wald test on the
DESeq-normalized counts for each TE family. TEtools was
also used to estimate the TE family copy number within the
SR and ST genomic resequencing pools and in male and fe-
male genomic libraries (SRS2309195–SRS2309198). We
identified potentially Y-inserted elements by qualitative com-
parison DESeq2 normalized genomic read counts from one
male and one female HiSeq library.
Annotation of Gene Duplication and Expression and
Differential Coverage
A set of protein-coding genes annotated from a transcrip-
tome assembly (BioProject PRJNA240197) was aligned to
the three largest scaffolds using GMAP v2019-12-01 allow-
ing for up to ten gene alignments (“paths”) per gene
(–npaths=10 –format=gff3_gene). Annotations were re-
moved as potential TEs misannotated as genes if they had
>50% alignment overlap with any TE annotation from
RepeatMasker. BEDTools (Quinlan and Hall 2010) (intersect
-wao) was used to determine the number of bases of over-
lap for each exon; then, the proportion of overlapping
bases was calculated across the entire length of each
gene alignment (“path” in GMAP terminology). RNAseq
data from SR and ST male testis (Reinhardt et al. 2014)
were aligned to the genome using HISAT2 (Kim et al.
2019) v2.0.1-beta (–dta -X 800). Genomic pool-seq data
were aligned to the genome using NextGenMap v0.5.5
(Sedlazeck et al. 2013). Genomic expression and coverage
in each library were estimated using Featurecounts v2.0.0
(Liao et al. 2014). Differential coverage and differential ex-
pression were each assessed from the Featurecounts read
count matrix on a by-feature basis using DESeq2 (Love
et al. 2014) with the default Wald test on the negative bi-
nomial distribution.
Molecular Evolutionary Analyses
Divergence was assessed in comparison with the T. dalman-
ni sp 2 (Paczolt et al. 2017) genome described above. The
Td2 draft genome scaffolds were aligned to the three
largest (chromosomal) scaffolds in the Hi-C assembly for
T. dalmanni s.s. using GMAP v2019-12-01 (Wu and
Watanabe 2005) (–nosplicing –format=samse). A Td2 con-
sensus was called from the GMAP alignment of Td2 scaf-
folds to the T. dalmanni genome by sorting and indexing
with samtools (Li et al. 2009) v1.3.1, then calling the con-
sensus with bcftools v 1.9 (bcftools call –ploidy 1 -mA) (Li
2011). Regions which did not have an aligned scaffold or
align as gaps show up as stretches of “N’s” in the consen-
sus when these parameters are used.
For molecular evolutionary comparisons, the bam-
formatted pool-seq library alignments were used with
bcftools (bcftools call –ploidy 1 -c ; vcfutils.pl vcf2fq),
to create a majority-rule X
SR
consensus sequence for
the large X-chromosomal scaffold (PGA_scaffold1) from
a bam file combining both X
SR
pools into a single bam
file. In addition, to have a comparable (similar sequencing
and allelic coverage) X
ST
consensus, an X
ST
alignment
(bam) file was produced using one replicate from each
collection site (Gombak ST1 and Kanching ST2) and con-
sensus called as above. The best alignment of the coding
regions of genes previously (Baker et al. 2016) assembled
and annotated using data from a multitissue RNAseq
experiment (BioProject PRJNA240197) was localized to
the genome via alignment with GMAP v2019-12-01
(–npaths=1 –format=gff3_gene –min-identity=0.9).
Gene sequences were extracted from the X
SR
and X
ST
consensus X chromosomes described above using
GffRead v0.12.7 (Pertea and Pertea 2020) and the
mRNA gff annotations from GMAP. Some genes con-
tained in-frame stop codons in one or more libraries
(e.g., stop codons were polymorphic). These genes
were trimmed to the longest open reading frame present
in all libraries, and if what remained was longer than 50
amino acids, they were retained. Genes were also ex-
cluded if they contained only ambiguity sequence (“N”)
in one or more of the consensus genomes or were less
than 50 aa in length. After exclusions, we counted
nonsynonymous divergent sites and calculated pairwise
dN/dS of X
SR
versus X
ST
for 2,642 X-linked genes and
for T. dalmanni versus Td2 for 9,525 genes using the
SNAP utility v6/15/98 (Korber et al. 2000). For the lin-
ear models and ANOVA predicting ln(dN/dS), genes
were identified as being testis specific based on prior
analyses of a multitissue transcriptome (Reinhardt
et al. 2014; Baker et al. 2016). Following Baker et al.
2016, gene duplication events occurring within
Diopsidae were assigned based on a four-species tran-
scriptomic analysis and protein coding genes without
identifiable Dipteran orthologs were designated as or-
phan genes. In addition, genes that were previously de-
signated as single copy were assessed as duplicates if
they mapped (GMAP, see above) to multiple locations
(paths) in the genome.
Reinhardt et al. GBE
12 Genome Biol. Evol. 15(7) https://doi.org/10.1093/gbe/evad118 Advance Access publication 26 June 2023
Downloaded from https://academic.oup.com/gbe/article/15/7/evad118/7207967 by guest on 08 July 2023
Supplementary Material
Supplementary data are available at Genome Biology and
Evolution online (http://www.gbe.oxfordjournals.org/).
Acknowledgments
The authors thank Melanie Kirk, Nathaniel Lowe, Wyatt
Shell, George Ru, and Gabriel Welsh for the assistance
with analysis, sample preparation, and fly rearing; Philip
Johns and Max Brown for fly collections; Shawn Sullivan
and Hayley Mangelson for HiC analysis; Najib El-Sayed
and Suwei Zhao for HiSeq library prep and sequencing as-
sistance; Ellen Martinson for bacterial contamination
screening; and Molly Schumer, Peter Andolffato, and Wei
Wang for reagents and advice on multiplexed shotgun
genotyping (MSG). We thank two anonymous reviewers
for suggestions that improved the manuscript. Funding
for this work was provided by the National Science
Foundation grants DEB-0951816 to R.H.B., DEB-0952260
to G.S.W., and DEB1257053 and DEB1950078 to J.H.W;
by the USDA National Institute of Food and Agriculture
grant 2018-67015-28199 to A.V.Z; by the University of
Maryland by the Research Foundation for the State
University of New York; and by the Geneseo Foundation.
Author Contributions
J.A.R., R.H.B., and G.S.W. prepared the manuscript. J.A.R.,
A.V.Z., K.A.P., C.L., J.H.W., G.S.W., and R.H.B. analyzed
the data. C.Y.H. and G.S.W. provided sequencing data.
Data Availability
Raw data and genome assemblies used in this project are
available on NCBI BioProjects PRJNA655584 (sex ratio rese-
quencing), PRJNA391339 (Teleopsis dalmanni s.s. genome
assembly), PRJNA662429 (multiplexed shotgun genotyp-
ing), and PRJNA659474 (Teleopsis dalmanni sp2 aka iso-
late:KP12SP2M1 genome assembly). The Whole Genome
Shotgun project for Teleopsis dalmanni s.s. has been de-
posited at GenBank under the accession NLCU00000000.
The version described in this paper is version
NLCU04000000. The following additional data sets are
available on the digital repository at University of
Maryland (DRUM): transposable element family consensus
fasta sequences for Teleopsis dalmanni (ID 1903/26380),
the MSG genotype matrix (ID fgxn-tuaf), and gff3 format-
ted gene annotations (ID gfqi-iktk).
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Associate editor: Rebecca Zufall
Comparative Genomics of Meiotic Drive X GBE
Genome Biol. Evol. 15(7) https://doi.org/10.1093/gbe/evad118 Advance Access publication 26 June 2023 15
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