bioRxiv preprint doi: https://doi.org/10.1101/2020.06.28.176735; this version posted June 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
1 Distinct genetic architectures underlie divergent thorax, leg, and wing pigmentation
2 between Drosophila elegans and D. gunungcola
3 Jonathan H. Massey1,2, Jun Li1,3, David L. Stern2, Patricia J. Wittkopp1,4*
4
5 1Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI
6 48109, USA
7 2Janelia Research Campus of the Howard Hughes Medical Institute, Ashburn, VA 20147, USA
8 3Institute of Evolution and Ecology, School of Life Sciences, Central China Normal University,
9 Wuhan 430079, China
10 4Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann
11 Arbor, MI 48109, USA
12
13
14 *Corresponding author
16
17 Running head: Pigmentation divergence between D. elegans and D. gunungcola
18 Keywords: yellow, ebony, optomotor-blind
19 Word count: 4,553 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.28.176735; this version posted June 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
20 Abstract
21 Understanding the genetic basis of species differences is a major goal in evolutionary biology.
22 Pigmentation divergence between Drosophila species often involves genetic changes in
23 pigmentation candidate genes that pattern the body and wings, but it remains unclear how these
24 changes affect pigmentation evolution in multiple body parts between the same diverging
25 species. Drosophila elegans and D. gunungcola show pigmentation differences in the thorax,
26 legs, and wings, with D. elegans exhibiting male-specific wing spots and D. gunungcola lacking
27 wing spots with intensely dark thoraces and legs. Here, we performed QTL mapping to identify
28 the genetic architecture of these differences. We find a large effect QTL on the X chromosome
29 for all three body parts. QTL on Muller Element E were found for thorax pigmentation in both
30 backcrosses but were only marginally significant in one backcross for the legs and wings.
31 Consistent with this observation, we isolated the effects of the Muller Element E QTL by
32 introgressing D. gunungcola alleles into a D. elegans genetic background and found that D.
33 gunungcola alleles linked near the pigmentation candidate gene ebony caused intense darkening
34 of the thorax, minimal darkening of legs, and minimal shrinking of wing spots. D. elegans ebony
35 mutants showed changes in pigmentation consistent with Ebony having different effects on
36 pigmentation in different tissues. Our results suggest that multiple genes have evolved
37 differential effects on pigmentation levels in different body regions.
38
39
40
41
42 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.28.176735; this version posted June 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
43 Introduction
44 Pigmentation differences within and between species illustrate some of the most striking
45 examples of phenotypic evolution in nature. In insects, pigmentation intensity in the body, legs,
46 and wings varies widely, often distinguishing sexes, populations, and species (True, 2003;
47 Wittkopp et al., 2003). The genetic and developmental processes determining pigment patterning
48 are well understood, which has facilitated the use of insect pigmentation as a model system to
49 investigate the genetic basis of phenotypic evolution (Wittkopp et al., 2003; Kopp, 2009).
50 Multiple studies have revealed that the same genes have often evolved independently to cause
51 pigmentation variation; that genetic variation within these genes often explains the majority of
52 pigmentation differences; and that mutations affecting the expression of enzymes and
53 transcription factors cause pigmentation to evolve (Massey and Wittkopp, 2016). These results
54 support the hypothesis that the genetic architecture of phenotypic evolution is, at least to some
55 degree, predictable (Stern and Orgogozo, 2008).
56
57 The insect pigmentation synthesis pathway (Figure 1) therefore provides a roadmap for
58 predicting which genes likely contribute to pigmentation differences within and between insect
59 species. Differences in how dopamine is metabolized in this pathway ultimately lead to
60 combinations of black and brown melanins as well as yellow, tan, and colorless sclerotins that
61 diffuse into the developing cuticle in sex-, population-, and species-specific ways (reviewed in
62 True, 2003). The genes yellow, tan, and ebony in this pathway have repeatedly evolved to
63 contribute to these differences. These include both within and between species differences in
64 abdominal, thorax, and wing pigmentation (reviewed in Massey and Wittkopp, 2016). In each bioRxiv preprint doi: https://doi.org/10.1101/2020.06.28.176735; this version posted June 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
65 case, mutations in cis-regulatory DNA underlie these phenotypic changes, emphasizing the
66 importance of gene regulatory evolution in generating animal pigmentation diversity.
67
68 Most studies identifying the genetic basis of divergent pigmentation have focused on the
69 divergence of one particular element of the pigment pattern within each species pair. It thus
70 remains unclear how often the same genes and/or mutations are responsible for divergent
71 pigmentation seen in multiple body parts. In some instances, closely related species show
72 striking pigmentation differences across most body regions. Pigmentation divergence between
73 Drosophila americana and D. novamexicana, for example, involves differences in thorax and
74 abdominal pigmentation intensity that are partly explained by evolution of the ebony gene (Lamb
75 et al., 2020). Divergence in pupal case coloration between D. americana and D. virilis, however,
76 is caused by evolution at the dopamine N-acetyltransferase (Dat) gene (Ahmed-Braimah and
77 Sweigart, 2015). In other instances, species differ not only in the pigmentation intensity across
78 multiple body parts but also in pigmentation patterning. Pigmentation evolution in redheaded
79 pine sawfly larvae, for example, involves changes in body coloration and spot patterning that is
80 partially explained by overlapping quantitative trait loci (QTL) (Linnen et al., 2018).
81
82 Species differences between D. elegans and D. gunungcola involve dramatic changes in
83 pigmentation intensity across multiple body parts, although body pigmentation is also
84 polymorphic in D. elegans (Bock and Wheeler, 1972; Hirai and Kimura, 1997). Populations of
85 D. elegans from Hong Kong and Indonesia have light brown thoraxes and legs, and males
86 possess dark black spots at the tips of their wings (Hirai and Kimura, 1997; Figure 2). In Japan
87 and Taiwan, populations have dark black thoraxes while still possessing male-specific wing bioRxiv preprint doi: https://doi.org/10.1101/2020.06.28.176735; this version posted June 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
88 spots (Hirai and Kimura, 1997). In D. gunungcola, pigmentation is less variable than in D.
89 elegans (Sultana et al., 1999; Massey et al., 2020). Males and females have dark black thoraxes
90 like the D. elegans Japanese and Taiwanese populations but also show dark black-pigmented
91 legs, and males do not have wing spots (Yeh et al., 2006; Massey et al., 2020; Figure 2).
92 Although these two species diverged 2-2.8 million years ago (Prud'Homme et al., 2006), they
93 can produce fertile, female F1 hybrids in the laboratory (Yeh et al., 2006), allowing us to
94 investigate the genetic basis of pigmentation evolution across multiple body regions in the same
95 mapping population.
96
97 Here, we use quantitative trait locus (QTL) mapping in two backcross populations between the
98 light D. elegans morph from Hong Kong (HK) and D. gunungcola from Sukarami, Indonesia
99 (SK) to identify gene regions associated with thorax, leg, and wing pigmentation divergence. For
100 all three traits, we identified a major effect QTL on the X chromosome, where pigmentation
101 candidate genes omb and yellow are located. We also find evidence for QTL linked to Muller
102 Element E for all three traits, but these effects depend on genetic background. We attempted to
103 isolate these effects by crossing D. gunungcola alleles into a D. elegans genetic background and
104 by introgressing a ~1.5 Mb region containing the ebony locus, which is found on Muller Element
105 E. We find that homozygous D. gunungcola alleles linked to this introgression darkened thorax
106 pigmentation intensity to D. gunungcola levels, but only minimally affected leg and wing
107 pigmentation, consistent with the differences observed in QTL mapping data for the three traits.
108 These data suggest that during the evolution of pigmentation across multiple body parts, epistasis
109 influences the extent to which individual pigmentation loci affect pigmentation divergence in
110 each tissue. In the case of D. elegans and D. gunungcola, evolution of at least two loci is bioRxiv preprint doi: https://doi.org/10.1101/2020.06.28.176735; this version posted June 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
111 necessary to cause species differences in leg and wing pigmentation, but evolution at a single
112 locus is sufficient for divergence in thorax pigmentation.
113
114
115 Materials and Methods
116 Fly stocks
117 Drosophila elegans HK (Hong Kong) and D. gunungcola SK (Sukarami) species stocks were a
118 gift from John True (Stony Brook University). Stock maintenance and food recipes are described
119 in Massey et al. (2020). In brief, stocks were maintained at 23ºC on a 12 h light-dark cycle. At
120 the third instar larval stage of development, adults were transferred onto new food, and
121 Fisherbrand filter paper (cat# 09-790-2A) was added to the larval vials to facilitate pupariation.
122
123 Generating hybrid progeny
124 Male and female D. elegans HK flies will reproduce with male and female D. gunungcola SK in
125 the laboratory to produce fertile F1 hybrid female and sterile F1 hybrid male offspring (Yeh et al.,
126 2006). Creating these F1 hybrids in populations large enough for genetic analysis, however, is
127 difficult. Detailed methods are described in Massey et al. (2020). In brief, both D. elegans HK
128 and D. gunungcola SK species stocks were expanded to establish populations of more than
129 10,000 flies. Next, virgin males and females from each species were placed in heterospecific
130 crosses (D. elegans HK males with D. gunungcola SK females and vice versa) in groups of ten
131 males and females to generate fertile F1 hybrid female offspring. Dozens of these crosses were
132 set to create ~120 F1 hybrid female offspring. Since F1 hybrid males are sterile (Yeh et al., 2006;
133 Yeh and True, 2014), the F1 hybrid females were used to generate two backcross populations for bioRxiv preprint doi: https://doi.org/10.1101/2020.06.28.176735; this version posted June 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
134 QTL mapping. Briefly, for the D. elegans HK backcross population, ~60 F1 hybrid females were
135 crossed in the same vial with ~60 D. elegans HK males and transferred onto new food every two
136 weeks for ~2.5 months, resulting in 724 recombinant individuals. For the D. gunungcola SK
137 backcross population, ~60 F1 hybrid females were crossed in the same vial with ~60 D.
138 gunungcola SK males and transferred onto new food every two weeks for ~2.5 months, resulting
139 in 241 recombinant individuals.
140
141 Pigmentation quantification
142 For thorax pigmentation QTL analysis, male recombinants from each backcross population were
143 organized into three thorax pigmentation classes. The lightest recombinants showed thorax
144 pigmentation intensities similar to D. elegans HK and were given a score of 0; recombinants with
145 intermediate thorax pigmentation intensities were given a score of 1; and recombinants with dark
146 thorax pigmentation intensities similar to D. gunungcola SK were given a score of 2 (Figure 3A).
147 To quantify the effects of the Muller Element E introgression region on thorax pigmentation
148 (Figure 4B,C), individuals were placed thorax-side up on Scotch double sided sticky tape on
149 glass microscope slides (Fisherbrand) (cat# 12-550-15) and imaged at the same exposure using a
150 Canon EOS Rebel T6 camera mounted to a Canon MP-E 65 mm macro lens equipped with a ring
151 light. The images were then imported into ImageJ software (version 1.50i) (Wayne Rasband,
152 National Institutes of Health, USA; http://rsbweb.nih.gov/ij/) and converted to 32 bit grayscale.
153 Using the “straight line segment” tool, a line was drawn between the anterior scutellar bristles
154 (Figure 4B white dashed box) to measure the mean grayscale value of the cuticle. Data shown in
155 Figure 4C were inverted so that higher values indicated darker pigmentation. Raw data for
156 Figure 3 and 4 are deposited on Dryad. bioRxiv preprint doi: https://doi.org/10.1101/2020.06.28.176735; this version posted June 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
157
158 For leg pigmentation QTL analysis, methods were identical to the thorax procedures above
159 except recombinants were organized into light (0), intermediate (1), and dark (2) classes based
160 on the pigmentation intensity of the medial side of their right hindleg femur (Figure 3B). This
161 region of the leg was chosen because it contains few cuticular bristles that could obscure
162 pigmentation. To quantify the effects of the Muller Element E introgression region on leg
163 pigmentation (Figure 4B,E), the same methods as above were used except right medial hindleg
164 images were captured. In ImageJ, the “polygon selections” tool was used to draw a polygon
165 selection around the medial side of the right hindleg femur (Figure 4B red transparency
166 selection) to measure the mean grayscale value of the cuticle. Data shown in Figure 4E were
167 inverted so that higher values indicated darker pigmentation. Raw data for Figure 3 and 4 are
168 deposited on Dryad.
169
170 For wing spot pigmentation QTL analysis, methods are described in Massey et al., (2020).
171 Briefly, right wings from male recombinants from each backcross population were imaged, and
172 spots were quantified in ImageJ using the “polygon selections” tool to quantify wing spot size
173 relative to wing size (Figure 4B red transparency selection). The same procedure was used to
174 quantify the effects of the Muller Element E introgression region (Figure 4D). Raw data for
175 Figure 3 and 4 are deposited on Dryad.
176
177 Library preparation, sequencing, and genome assembly
178 Detailed methods for preparing and sequencing the genomic DNA (gDNA) libraries for the D.
179 elegans HK and D. gunungcola SK backcross populations and advanced introgression line were bioRxiv preprint doi: https://doi.org/10.1101/2020.06.28.176735; this version posted June 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
180 described in Massey et al., (2020). Briefly, gDNA was extracted from male recombinants by
181 homogenizing individuals singly in each well of a 96-well plate (Corning, cat# 3879). Each
182 recombinant gDNA sample was then barcoded with unique adaptors, pooled into a single
183 multiplexed sequencing library, size selected, and sequenced in a single lane of Illumina HiSeq
184 by the Janelia Quantitative Genomics Team. Methods for assembling the D. elegans HK and D.
185 gunungcola SK genomes to facilitate marker generation were described in Massey et al. (2020).
186
187 Marker generation with Multiplexed Shotgun Genotyping
188 Chromosome ancestry “genotypes” for the backcross progeny and introgression line were
189 estimated with two Multiplexed Shotgun Genotyping (MSG) (Andolfatto et al., 2011) libraries,
190 following methods described in Cande et al., (2012). Briefly, reads generated from the Illumina
191 backcross sequencing library were mapped to the assembled D. elegans HK and D. gunungcola
192 SK parental genomes to estimate chromosome ancestry for each backcross individual. We
193 generated 3,425 and 3,121 markers for the D. elegans HK and D. gunungcola SK backcrosses,
194 respectively, for QTL analysis [markers, phenotypes, and procedures for QTL mapping are
195 deposited on Dryad (doi:10.5061/dryad.gb5mkkwm5)]. PDFs of chromosomal breakpoints for
196 each recombinant are available here:
197 https://deepblue.lib.umich.edu/data/concern/data_sets/j098zb17n?locale=en.
198
199 QTL analysis
200 QTL analysis was performed using R/qtl (Broman and Sen, 2009) in R for Mac version 3.3.3. (R
201 Core Team 2018). We imported ancestry data for both backcross populations into R/qtl using a
202 custom script (https://github.com/dstern/read_cross_msg). This script directly imports the bioRxiv preprint doi: https://doi.org/10.1101/2020.06.28.176735; this version posted June 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
203 conditional probability estimates produced by the Hidden Markov Model (HMM) of MSG
204 (described in detail in Andolfatto et al., 2011). We performed genome scans with a single QTL
205 model using the “scanone” function of R/qtl and Haley-Knott regression (Haley and Knott, 1992)
206 for thorax, leg, and wing pigmentation. For QTL mapping using the D. elegans HK backcross
207 population, we excluded 18 and 20 individuals for thorax and leg pigmentation, respectively,
208 because fly samples were either too poor to image or sequencing reads were too shallow to map.
209 For the D. gunungcola SK backcross population, we excluded 12 and 9 individuals for thorax
210 and leg pigmentation, respectively, for the same reasons. For wing pigmentation QTL analysis,
211 we previously (Massey et al., 2020) reported on an ~400 kb fine-mapped region on the X
212 chromosome explaining the majority of variation for wing spot size. We also reported on QTL
213 underlying variation in wing spot size with spotless recombinants removed from the analysis
214 (Massey et al., 2020). Here, in Figure 3D, we show QTL underlying this variation to emphasize
215 the role of Muller Element E in spot size divergence. Significance of QTL peaks at α = 0.01 was
216 determined by performing 1000 permutations of the data.
217
218 Introgression of black body color alleles from D. gunungcola SK into D. elegans HK
219 To isolate individual QTL underlying body color differences between D. elegans HK and D.
220 gunungcola SK, F1 hybrid females and D. elegans HK backcross recombinants were generated
221 using the methods described above. Next, dark black/brown female recombinants were
222 repeatedly backcrossed with D. elegans HK males for four generations (BC3-BC6). Finally, we
223 generated a single BC6 dark black homozygous introgression line that we then genotyped using
224 MSG (Figure 4A,B)
225 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.28.176735; this version posted June 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
226 Creating an ebony null allele in Drosophila elegans HK via CRISPR-Cas9 genome editing
227 Using methods described in Bassett et al. 2013, we in vitro transcribed (MEGAscript T7
228 Transcription Kit, Invitrogen) three single guide RNAs (sgRNAs) (Supplemental File S1) with
229 target sequences designed based on conserved sites between D. elegans HK and D. gunungcola
230 SK in exon 2 of the ebony gene. After transcription, sgRNAs were purified using an RNA Clean
231 and Concentrator 5 kit (Zymo Research), eluted with nuclease-free water, and quantified using a
232 Qubit RNA BR Assay Kit (Thermo Fisher Scientific). Next, mature (>2 weeks old) D. elegans
233 HK males and females were transferred to 60 mm embryo lay cages (GENESEE Part Number:
234 59-100) on top of 60 mm grape plates (3% agar + 25% grape juice + 0.3% sucrose) at high
235 densities (>300 flies) after brief CO2 anesthesia. After CO2 anesthesia, mature, mated D. elegans
236 HK females will often dispel an embryo from their abdomen where it sticks briefly to the anus.
237 Tapping the grape plate + embryo cage down on a hard surface 10 times causes the embryos to
238 stick to the grape agar. Flies were then transferred back into food vials and embryos were lined
239 up on glass cover slips taped to a glass microscope slide. For CRISPR/Cas9 injections into D.
240 elegans HK embryos, Cas9 protein (PNA Bio #CP01), phenol red, and all three sgRNAs were
241 mixed together at 400 ng/µl, 0.05%, and 100 ng/µl final injection concentrations, respectively.
242 All CRISPR injections were performed in-house, using previously described methods (Miller et
243 al, 2002). Finally, we screened for germline mutants based on body pigmentation and confirmed
244 loss of Ebony protein by western blot (Supplemental Figure S1). To the best of our knowledge,
245 these are the first gene editing experiments to succeed in D. elegans. We attempted the same
246 experiments in D. gunungcola SK, but failed to recover any mutants.
247
248 Western blotting bioRxiv preprint doi: https://doi.org/10.1101/2020.06.28.176735; this version posted June 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
249 Western blot methods were followed similar to Wittkopp et al. (2002). In brief, for each replicate
250 per genotype, four newly eclosed (within 60 min) male flies were homogenized in 100 µl of 125
251 mM Tris pH 6.8, 6% SDS and centrifuged for 15 min. The supernatant was then transferred to a
252 new protein low-bind Eppendorf tube with an equal amount of 2X Laemmli sample buffer [4%
253 SDS, 20% glycerol, 120 mM Tris-Cl (pH 6.8)], boiled for 10 min, and stored at -80ºC. Before
254 gel electrophoresis, samples were thawed at room temperature for 30 min, and 20 µl of each
255 sample was loaded into individual wells of an Invitrogen NuPAGE 4-12% Bis-Tris Gel. The gel
256 was run at 175 V for 60 min, washed, and transferred to an Invitrogen iBlot 2 PVDF Mini Stack
257 Kit. The mini stack was run on an iBlot 2 (ThermoFisher, Catalog Number: IB21001) to perform
258 western blotting transfer, and blocked using an Invitrogen Western Breeze anti-rabbit kit
259 (Catalog Number: WB7106). After two washes with diH2O, samples were incubated in 1:400
260 rabbit anti-Ebony (Wittkopp et al., 2002) overnight at 4ºC. Finally, samples were washed using
261 the Western Breeze wash solution, incubated in secondary antibody solution alk-phos. (Catalog
262 Number: WP20007) with conjugated anti-rabbit for 30 min, washed for 2 min with diH2O, and
263 prepared for chromogenic detection.
264
265 Statistics
266 Statistical tests were performed in R for Mac version 3.3.3 (R Core Team 2018). ANOVAs were
267 performed with post hoc Tukey HSD for pairwise comparisons adjusted for multiple
268 comparisons. See “QTL analysis” methods for statistical tests used during QTL mapping.
269
270
271 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.28.176735; this version posted June 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
272 Results
273
274 X-linked divergence contributed to pigmentation divergence of all three traits
275 We examined the genetic basis for divergence of thorax, leg, and wing pigmentation between D.
276 elegans HK and D. gunungcola SK (Figure 2A). First, to assess the contribution of the X
277 chromosome in pigmentation divergence, we compared F1 hybrid males from reciprocal crosses
278 between D. elegans HK and D. gunungcola SK, which inherited their X chromosome from either
279 their D. elegans HK (F1E) or D. gunungcola SK (F1G) mother, respectively. We observed
280 striking pigmentation differences in the thorax, legs, and wings between the hybrids (Figure 2B).
281 F1 hybrid males inheriting their X chromosome from D. elegans HK mothers possessed wing
282 spots (quantified in Massey et al., 2020) and had thorax and leg pigmentation similar to D.
283 elegans HK (Figure 2A,B). In contrast, F1 hybrids from D. gunungcola SK mothers lacked wing
284 spots and had thorax and leg pigmentation similar to D. gunungcola SK (Figure 2A,B). These
285 observations suggest that X-linked loci contributed to pigmentation divergence in multiple body
286 parts.
287
288 QTL on the X chromosome underlie pigmentation divergence
289 To assess the genome-wide distribution of genes contributing to pigmentation divergence for
290 each body part, we performed quantitative trait locus (QTL) mapping. We generated two
291 backcross populations by crossing F1 hybrid females to males from each parental species and
292 quantified pigmentation variation for thorax, leg, and wing spots (see Methods) (Figure 3). For
293 each trait in both backcrosses, we identified major effect QTL on the X chromosome (Figure 3;
294 Table 1). The X-linked QTL peaks for thorax and leg pigmentation appear broader than the wing bioRxiv preprint doi: https://doi.org/10.1101/2020.06.28.176735; this version posted June 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
295 spot pigmentation QTL (Figure 3), which is also reflected in their support intervals (Table 1).
296 For the wing spot, this QTL was previously refined to a ~400 kb region containing the
297 pigmentation candidate gene optomotor-blind (omb) (Massey et al., 2020; Table 1), consistent
298 with the single, narrow peak for wing spot variation (Figure 3C). The broader QTL peaks for
299 variation in thorax and leg pigmentation may result from multiple X-linked genes affecting these
300 traits.
301
302 QTL on Muller Element E underlie pigmentation divergence
303 We detected major effect QTL for thorax and leg, but not wing, pigmentation on Muller Element
304 E (Figure 3A,B), with the strongest effect linked to the left end of the chromosome (Table 1). For
305 these traits, the QTL peaks cover the entire Muller element, which might indicate low
306 recombination rate or the presence of multiple genes on Muller Element E contributing to thorax
307 and leg pigmentation divergence. Nonetheless, this result indicates that at least one gene on
308 Muller Element E affects thorax and leg pigmentation divergence differently than wing spot
309 pigmentation. Prior work has also shown, however, that autosomal QTL on Muller Elements C
310 and E affect the size of wing spots even though the X chromosome is sufficient to control their
311 presence or absence (Massey et al, 2020; Figure 3D).
312
313 To investigate further whether autosomal variation might affect wing spot divergence, and
314 whether this variation might be hidden by a large epistatic effect of the X chromosome, we
315 repeated QTL analysis on a subset of backcross recombinants that displayed wing spots of any
316 size and excluded offspring that completely lacked spots (Figure 3D). This analysis revealed
317 small effect QTL linked to Muller Elements C and E that affect wing spot size (Figure 3D). Like bioRxiv preprint doi: https://doi.org/10.1101/2020.06.28.176735; this version posted June 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
318 thorax and leg pigmentation QTL, the largest effect was linked to the left end of Muller Element
319 E.
320
321 ebony is a candidate gene for the Muller Element E QTL
322 The pigmentation gene ebony is located near the left end of Muller Element E, where we
323 observed linkage to thorax, leg, and wing spot size pigmentation differences in the backcross
324 populations, making it an excellent candidate for the locus harboring the genetic variant(s)
325 affecting these traits (Figure 3). To test this hypothesis, we attempted to create stable
326 introgression lines by repeatedly backcrossing D. gunungcola SK alleles into a D. elegans HK
327 genetic background. For each backcross, we selected for dark black female recombinants to cross
328 with virgin male D. elegans HK for six generations (see Methods). Our goal was to isolate
329 multiple, independent introgression lines to fine map QTL involved in pigmentation divergence;
330 however, the majority of introgressions failed to propagate. We were not able to maintain any
331 introgression lines containing D. gunungcola SK alleles linked to the X chromosome in a D.
332 elegans HK genetic background (Massey et al., 2020), suggesting that divergence on the X
333 chromosome also contributed to hybrid incompatibilities. We did, however, recover a single
334 introgression line with a dark black thorax that resembled D. gunungcola SK (Figure 4A,B).
335 Sequencing this line revealed that it was homozygous for an approximately 1.5 Mb region of the
336 left end of D. gunungcola SK Muller Element E loci that included the ebony gene (Figure 4A).
337 These data provide independent confirmation that variation in genes on the left end of Muller
338 Element E contribute to thorax pigmentation divergence and support the hypothesis that ebony
339 contributes to thorax pigmentation evolution.
340 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.28.176735; this version posted June 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
341 We next quantified pigmentation variation of the Muller Element E introgression line to
342 determine whether genetic variation in this region is sufficient to explain the Muller Element E
343 QTL effects. We found that this introgression completely recapitulated the species difference in
344 thorax pigmentation, but it had only weak effects on leg pigmentation and wing spot size (Figure
345 4C-E). This result allowed us to partially separate the genetic effects on Muller Element E,
346 further implicating ebony as a candidate gene with a large effect on thorax pigmentation
347 divergence and a small effect on leg and wing spot pigmentation.
348
349 To determine whether ebony contributes to thorax pigmentation divergence, we attempted to
350 knock out the ebony gene in both species using CRISPR-Cas9 genome editing to perform a
351 reciprocal hemizygosisty test (Stern, 2014). Unfortunately, we were able to generate only a
352 single ebony knockout line in D. elegans HK (Figure 4F; Supplemental Figure S1). We found
353 that the D. elegans HK ebony line displayed a dark black thorax (Figure 4F) from loss of Ebony
354 activity, similar to ebony mutant effects in D. melanogaster (Wittkopp et al., 2002). This result
355 further supports the hypothesis that lower Ebony activity and/or expression in D. gunungcola SK
356 underlies its dark thorax color. Interestingly, loss of Ebony activity did not darken leg
357 pigmentation or expand the size of wing spots in D. elegans HK (Figure 4F). One explanation for
358 this observation is that ebony is not normally expressed in these tissues in D. elegans HK, so loss
359 of Ebony function is not reflected phenotypically in these regions. Alternatively, because
360 pigmentation reflects the balance of expression between ebony, tan, and yellow (Wittkopp et al.,
361 2002; Wittkopp et al., 2009), differences in expression of these other genes might explain why
362 we did not observe the accumulation of dark black melanins in these tissues of D. elegans HK
363 ebony mutants (Figure 1). This observation could explain why genetic variation linked near the bioRxiv preprint doi: https://doi.org/10.1101/2020.06.28.176735; this version posted June 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
364 ebony locus seems to affect thorax pigmentation divergence more than leg and wing
365 pigmentation divergence.
366
367
368 Discussion
369 We found that pigmentation divergence in the thorax, legs, and wings between D. elegans HK
370 and D. gunungcola SK is primarily influenced by genes on the X chromosome and Muller
371 Element E. Despite the fact that QTL regions overlap for these three body regions, several
372 observations suggest that pigmentation divergence results from evolution of multiple genes that
373 act in different ways on the three body regions examined. First, the X-linked QTLs that influence
374 thorax and leg pigmentation are broader than the QTL for wing spots. Second, QTL on Muller
375 Element E have a strong effect on thorax and leg pigmentation but minimal effect on wing spots.
376 Third, a 1.5 Mbp introgression of Muller Element E had a strong effect on thorax pigmentation
377 but minimal effects on leg pigmentation and wing spots. Thus, there is evidence for independent
378 genetic variation influencing pigmentation in each of these three body regions.
379
380 Evidence from previous studies is similar to our results. In D. melanogaster, for example, genetic
381 variants in the tan gene affect pigmentation in multiple abdominal segments, but genetic variants
382 in other genes affect each segment independently (Dembeck et al., 2015). Similarly, a single,
383 large effect QTL changes bristle number across multiple body regions in D. melanogaster, but
384 QTL on different chromosomes have independent effects on bristle number in the thorax versus
385 the abdomen (Mackay, 1995). Genetic variation in individual genes, therefore, can have bioRxiv preprint doi: https://doi.org/10.1101/2020.06.28.176735; this version posted June 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
386 pleiotropic effects on phenotypic differences in multiple body parts, whereas genetic variation in
387 less pleiotropic genes can have minimal effects in specific body parts.
388
389 Multicellular organisms develop distinct body parts through the action of gene regulatory
390 networks (Davidson, 2010). Differences in which (and how) genes are used during phenotypic
391 evolution likely reflect the activity of these networks (Stern and Orgogozo, 2008). If a gene is
392 not expressed in a given tissue, for example, then genetic variation at this gene will not be
393 reflected phenotypically. In our study, QTL on the X chromosome influenced pigmentation
394 differences in all three body parts (Figure 3), but QTL linked to the left end of Muller Element E
395 mainly affected thorax pigmentation (Figure 4). We predict, therefore, that species differences in
396 the spatial expression pattern of genes with distinct functions in the insect sclerotization and
397 pigmentation synthesis pathway (Figure 1) underlie these results.
398
399 In Drosophila, pigmentation coloring and patterning is determined by the action of multiple
400 enzymes that are regulated by multiple transcription factors (reviewed in Massey and Wittkopp,
401 2016). The enzymes Yellow and Ebony both require dopamine to synthesize dark black or light
402 yellow pigments, respectively, and transcription factors determine where these enzymes are
403 expressed (Figure 1; Massey and Wittkopp, 2016). Mutations that disrupt Yellow activity cause
404 flies to develop light yellow pigments, because Ebony converts excess dopamine into lightly
405 colored sclerotins. Reciprocally, mutations that disrupt Ebony activity cause flies to develop dark
406 black pigments, because Yellow converts excess dopamine into dark black melanins (Figure 1).
407 The phenotypic effect of mutations in one enzyme, therefore, require the reciprocal activity of
408 the other (Wittkopp et al., 2002). When we introgressed D. gunungcola SK Muller Element E bioRxiv preprint doi: https://doi.org/10.1101/2020.06.28.176735; this version posted June 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
409 alleles containing the ebony gene into a D. elegans HK genetic background, we observed
410 darkening of the thorax but not the leg or wing (Figure 4B). This likely resulted from the
411 combined effects of low D. gunungcola SK ebony expression with high D. elegans HK yellow
412 expression. We hypothesize, then, that the D. elegans HK yellow allele is not normally expressed
413 in the legs or in regions outside the wing spot. Instead, the D. gunungcola SK yellow allele likely
414 evolved an expanded expression pattern in the legs and a restricted expression pattern in the
415 wings (Prud’homme et al., 2006). Dissecting these mechanisms in detail will shed light on how
416 the evolution of gene regulatory networks shape phenotypic divergence and vice versa.
417
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431 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.28.176735; this version posted June 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
432 Acknowledgements
433 We thank members of the Wittkopp, Stern, and Rebeiz labs for helpful discussions. For fly
434 strains, we thank J. True (Stony Brook University). For guidance with CRISPR/Cas9 genome
435 editing and embryo injections, we thank Abigail Lamb. For helpful advice on creating F1
436 hybrids, we thank Shu-Dan Yeh (National Central University). Funding was provided by
437 University of Michigan, Department of Ecology and Evolutionary Biology, Peter Olaus
438 Okkelberg Research Award, National Institutes of Health (NIH) training grant T32GM007544,
439 and Howard Hughes Medical Institute Janelia Graduate Research Fellowship to JHM; NIH R01
440 GM089736 and 1R35GM118073 to PJW.
441
442 Competing Interests
443 The authors declare no competing interests.
444
445 Data Archiving
446 All supporting data can be accessed at University of Michigan Deep Blue
447 (https://deepblue.lib.umich.edu/data/concern/data_sets/j098zb17n? locale=en) and Dryad.
448
449
450
451
452
453
454 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.28.176735; this version posted June 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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566 Figure Legends
567 568 Figure 1 Insect sclerotization and pigmentation synthesis pathway
569 Pigmentation enzymes (shown in red) convert substrates (shown in black) into pigmentation
570 precursors that polymerize into darkly colored melanins or lightly colored sclerotins. Synthesis
571 of black melanin depends on Yellow function, whereas Ebony converts dopamine into lightly
572 colored sclerotins. Tan catalyzes the reverse reaction of Ebony, converting NBAD molecules
573 back into dopamine.
574
575 Figure 2 Differences in reciprocal F1 male hybrids suggest X-linked sequence divergence in
576 pigmentation evolution between Drosophila elegans HK and D. gunungcola SK
577 (A) D. elegans HK (Hong Kong) and D. gunungcola SK (Sukarami) show divergent thorax, leg,
578 and wing pigmentation. (B) F1 male hybrids inheriting their X chromosome from D. elegans HK
579 mothers (F1E, bottom left) possess wing spots and have thorax and leg pigmentation similar to D.
580 elegans HK. F1 male hybrids inheriting their X chromosome from D. gunungcola SK mothers
581 (F1G, bottom right) lack wing spots and have thorax and leg pigmentation similar to D.
582 gunungcola SK.
583
584
585 Figure 3 Quantitative trait locus (QTL) mapping of thorax, leg, and wing pigmentation
586 divergence
587 (A) Thorax pigmentation was organized into three classes (light, intermediate, and dark). QTL
588 map for thorax pigmentation is shown for D. elegans HK backcross (red) and D. gunungcola SK
589 backcross (blue). (B) Leg pigmentation was organized into three classes (light, intermediate, and bioRxiv preprint doi: https://doi.org/10.1101/2020.06.28.176735; this version posted June 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
590 dark). QTL map for leg pigmentation is shown for D. elegans HK backcross (red) and D.
591 gunungcola SK backcross (blue). (C) Wing spot pigmentation was quantified relative to wing
592 size (see Methods). QTL map for wing spot pigmentation is shown for D. elegans HK backcross
593 (red) and D. gunungcola SK backcross (blue) [Reprinted from Massey et al. (2020), Copyright
594 (2020), with permission from John Wiley and Sons]. (D) Similar to (C), wing spot pigmentation
595 was quantified relative to wing size, however, recombinants completely lacking wing spots were
596 excluded (red “X”) from the QTL analysis. QTL map for wing spot size is shown for D. elegans
597 HK backcross (red) and D. gunungcola SK backcross (blue) [Reprinted from Massey et al.
598 (2020), Copyright (2020), with permission from John Wiley and Sons]. Transparent yellow bars
599 indicate the chromosomal location of pigmentation candidate genes. LOD (logarithm of the
600 odds) is indicated on the y-axis. The x-axis represents the physical map of Muller Elements X, B,
601 C, D, E, and F based on the D. elegans HK assembled genome (see Methods). D. elegans HK
602 and D. gunungcola SK have six chromosomes (Yeh et al. 2006; Yeh and True 2014) that
603 correspond to D. melanogaster chromosomes as follows: X = X, B = 2L, C = 2R, D = 3L, E =
604 3R, and F = 4. Individual SNP markers are indicated with black tick marks along the x-axis.
605 Horizontal red and blue lines mark P = 0.01 for the D. elegans HK and D. gunungcola SK
606 backcross, respectively.
607
608 Figure 4 D. gunungcola SK alleles linked to ebony on Muller Element E have varied effects
609 on thorax, leg, and wing pigmentation divergence
610 (A) Multiplexed Shotgun Genotyping (MSG) (Andolfatto et al., 2011) was used to estimate
611 genome-wide ancestry assignments for a single introgression line generated by repeated
612 backcrossing of dark D. gunungcola SK recombinant females with D. elegans HK males (see bioRxiv preprint doi: https://doi.org/10.1101/2020.06.28.176735; this version posted June 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
613 Methods). The posterior probability that a region is homozygous for D. elegans HK (red) or D.
614 gunungcola SK (blue) ancestry is plotted along the y-axis. (B) Images highlighting pigmentation
615 and chromosome differences between species and the Muller Element E introgression line. The
616 white dashed box highlights the scutellar region of the thorax used to quantify thorax
617 pigmentation in (C) (see Methods). Dashed red transparencies highlight regions of the wing and
618 leg used to quantify wing spot and leg pigmentation in (D) and (E), respectively (see Methods).
619 The black arrowhead emphasizes the wing spot region that has shrunk in the introgression line.
620 (C) Quantification of thorax pigmentation differences between species and the Muller Element E
-10 621 introgression line (One-way ANOVA F3,35 = 33.9; P = 1.84 x 10 ; groups not sharing the same
622 letter are significantly different based on post-hoc Tukey HSD at P < 0.00001). (D)
623 Quantification of wing spot pigmentation differences between species and the Muller Element E
-16 624 introgression line (One-way ANOVA F2,88 = 78.6; P < 2.0 x 10 ; groups not sharing the same
625 letter are significantly different based on post-hoc Tukey HSD at P = 0.02) [Reprinted from
626 Massey et al. (2020), Copyright (2020), with permission from John Wiley and Sons]. (E)
627 Quantification of leg pigmentation differences between species and the Muller Element E
-16 628 introgression line. (One-way ANOVA F3,33 = 481; P < 2.0 x 10 ; groups not sharing the same
629 letter are significantly different based on post-hoc Tukey HSD at P < 5 x 10-7). Gray triangles
630 represent individual replicates for (C-E). (F) Images highlighting effects of knocking out the
631 pigmentation candidate gene ebony in D. elegans HK.
632
633 Supplementary Figure S1 Quantification of Ebony band intensity in western blot
634 (A) Western blot using anti-Ebony antibody to detect Ebony protein levels (black arrowhead) in
635 newly eclosed D. elegans HK, the Muller Element E introgression line, and the D. elegans HK bioRxiv preprint doi: https://doi.org/10.1101/2020.06.28.176735; this version posted June 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
636 ebony knockout line. Each number denotes a unique biological replicate involving four
637 homogenized flies. A second band just below the ~94 kDa Ebony band appeared in both the
638 wild-type and ebony knockout extracts (as observed in Wittkopp et al., 2002). (B) Using this
639 secondary band as a loading control, we quantified Ebony protein levels for each genotype and
640 observed no statistically significant differences. The western blot image was imported into
641 ImageJ software (version 1.50i) (Wayne Rasband, National Institutes of Health, USA;
642 http://rsbweb.nih.gov/ij/) and converted to 32 bit grayscale. Using the “straight line segment”
643 tool, the mean grayscale value of each band was quantified and inverted so that higher values
644 indicated darker band intensity (Student’s t-test; t = -0.495; df = 2.75; P = 0.646; two-tailed).
645 Gray triangles represent individual replicates, ns = not significant.
646
647
648
649
650 651 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.28.176735; this version posted June 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Dark pigment (brown) Dark pigment (black) Dopamine Dopamine melanin melanin PO Pale DDC Yellow Tyrosine DOPA Dopamine Ebony Tan N-acetyl dopamine N-β-alanyl dopamine (NADA) (NBAD) PO PO
NADA sclerotin NADA sclerotin Colorless pigment Light pigment (yellow-tan) bioRxiv preprint doi: https://doi.org/10.1101/2020.06.28.176735; this version posted June 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A D. elegans HK D. gunungcola SK
X B C D E F X B C D E F
B F1E hybrid F1G hybrid (♀ D. ele x ♂ D. gun) (♀ D. gun x ♂ D. ele)
X B C D E F X B C D E F bioRxiv preprint doi: https://doi.org/10.1101/2020.06.28.176735; this version posted June 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
omb, D. elegans backcross (N = 706) tan yellow ebony A 40 D. gunungcola backcross (N = 229) 30 20 LOD LOD 10
Thorax 0
XA B C D E F B 40 D. elegans backcross (N = 704) Chromosome 30 D. gunungcola backcross (N = 232) 20 LOD LOD Leg 10 0 C XA B C D E F 200 D. elegans backcross (N = 656) Chromosome 150 D. gunungcola backcross (N = 199) 100 LOD LOD 50
Wing Spot 0
AX B C D E F D 7 7 6 6 50 D. elegans backcross (N =5 362) 5 Chromosome4 4 LOD LOD 3 3 40 D. gunungcola backcross2 (N = 128) 2 1 1 30 0 0 A B CA DB EC FD E F LOD LOD 20 X Chromosome Chromosome 10
Wing Spot 0
XA B C D E F Chromosome Chromosome ebony A Prob. Homozygouspar1 D. elegans HK Prob. Homozygous D. gunungcola SK par2 0 X 0 B 0 C 0 D 0 E F0 6.25 Mb A B C D E F
B D. elegans HK D. gunungcola SK Introgression D. elegans HK Introgression
X B C D E F X B C D E F X B C D E F X B C D E F
C 200 b b F D. ele HKebonyKO 180 a a 160
(mean grayscale) (mean 140
Thorax pigmentation pigmentation Thorax HK SK F6 F1_het D 0.16 a c 0.14 ) 2 0.12 b 0.10 (mm 0.08 Wing spot X B C D E F
Wing spot size size spot Wing absent * 0.06 * E HK SK F6_dark HET 200 b 180 c 160 a 140 a 120 (mean grayscale) (mean Leg pigmentation HKHK SKSK Intro.F6 F1_hetHK Intro. Table 1 QTLs detected for thorax, leg, and wing pigmentation divergence
Trait Backcross Chr. QTL interval QTL peak (bp) LOD Candidate (bp)a (bp) Thorax D. elegans HK X 8,996,888- 9,309,667 34.2 omb: pigmentation 10,120,567 10,700,000 yellow: 11,412,900
Thorax D. elegans HK E 244,349- 409,133 27.8 ebony: pigmentation 6,527,255 1,580,200
Leg D. elegans HK X 8,727,567- 9,456,223 34.0 omb: pigmentation 14,805,348 10,700,000 yellow: 11,412,900
Leg D. elegans HK C 14,797- 27,017 5.27 ? pigmentation 7,500,000
Leg D. elegans HK E 7,600- 606,610 27.9 ebony: pigmentation 6,588,062 1,580,200
Wing spot D. elegans HK X 10,297,836- 10,304,581 220 omb: 10,744,020b 10,700,000
Wing spotc D. elegans HK X 10,117,675- 10,303,766 49.1 omb: 10,748,235b 10,700,000
Thorax D. gunungcola SK X 6,969,794- 7,704,294 20.0 omb: pigmentation 9,711,406 10,700,000 yellow: 11,412,900
Thorax D. gunungcola SK E 2,083,292- 3,766,760 30.3 ebony: pigmentation 3,843,775 1,580,200
Leg D. gunungcola SK X 10,029,176- 10,117,133 39.5 omb: pigmentation 11,595,407 10,700,000 yellow: 11,412,900
Leg D. gunungcola SK E 517,241- 11,251,902 6.41 ebony: pigmentation 27,451,006 1,580,200
Wing spot D. gunungcola SK X 10,474,499- 11,223,359 38.9 omb: 11,584,862b 10,700,000
Wing spotc D. gunungcola SK C 6,655,757 - 8,420,192 4.37 Dll: 12,279,025b 5,221,911
Wing spotc D. gunungcola SK E 10,907- 12,292 6.85 ebony: 4,009,870b 1,580,200
a LOD drop 1.5 support interval (the region where the LOD score is within 1.5 of its maximum; Broman et al., 2003).
b Previously reported in Massey, J. H. et al. (2020). Co-evolving wing spots and mating displays are genetically separable traits in Drosophila. Evolution doi: 10.1111/evo.13990.
c Variation in wing spot size with spotless recombinant wings excluded.
A HK HK HK Intro. Intro. Intro. eKO (1) (2) (3) (1) (2) (3)
B ns 1.00 0.95 0.90 0.85 0.80 No band
(mean grayscale) (mean 0.75 present
Ebony band intensity intensity band Ebony HK F6 E - HK HK e ele D. ele Introgression D.