Pleiotropy promotes male exaggerated weapon and its associated fighting behaviour in a water strider

Exaggerated sexually selected traits, often carried by males, are characterized by the evolution of hyperallometry, resulting in their disproportionate growth relative to the rest of the body 1–3. While the evolution of allometry has attracted much attention for centuries, our understanding of the developmental genetic mechanisms underlying its emergence remains fragmented 4,5. Here we show that the hyperallometric legs in the males of the water strider Microvelia longipes are associated with a specific signature of gene expression during development. Using RNAi knockdown, we demonstrate that a broadly expressed growth factor, Bone Morphogenetic Protein 11 (BMP11, also known as Growth Differentiation Factor 11), regulates leg allometries through increasing the allometric coefficient and mean body size in males. In contrast, BMP11 RNAi reduced mean body size but did not affect slope in females. Furthermore, our data show that a tissue specific factor, Ultrabithorax (Ubx), increases intercept without affecting mean body size. This indicates a genetic correlation between mean body size and variation in allometric slope, but not intercept. Strikingly, males treated with BMP11 RNAi exhibited a severe reduction in fighting frequency compared to both controls and Ubx RNAi-treated males. Overall, we demonstrate a genetic correlation between male body size, the exaggerated weapon, and the intense fighting behaviour associated with it in M. longipes. Our results provide evidence for a role of pleiotropy in the evolution of allometric slope.

( Figure 2A). This is reflected in the first major axis of variation, which separated the legs with similar contribution (about 35% of the total variation) in both males and females ( Figure 2A). This result 1 0 8 suggests that morphological divergence in serial homologs between the sexes may not result from a 1 0 9 global change in gene expression but rather from specific sets of genes.
To identify the genes underlying the growth differences in serial homologs, we examined the 1 1 1 differentially expressed genes between the first and the third leg (leg-biased genes), and compared the 1 1 2 divergence in expression between these two legs in males and females. We found that the third legs 1 1 3 diverged more from the first legs in males than in females, both in terms of number of differentially  Similar results were observed for the second legs in males, which are moderately exaggerated females. Similar results were also observed for biased genes in female third legs ( Figure 2B), indicating that a large proportion of up-regulated genes in the third legs are sex-specific. In the first 1 2 0 legs, which have more similar scaling relationship between males and females, we found that about 1 2 1 4 63% of leg-biased genes were common between the sexes ( Figure 2C). Moreover, hierarchical 1 2 2 clustering analyses revealed that genes which were up-regulated in the first legs specifically in males showed similar expressions with the first legs of females ( Figure 2C). Likewise, female-restricted genes showed similar expressions with the first legs of males ( Figure 2C). Finally, leg-biased genes 1 2 5 shared among sexes do not show any average differences in expression between males and females 1 2 6 (Supplementary figure 5). Altogether, these data suggest that the morphological divergence of serial 1 2 7 homologs is associated with up-regulation of a unique set of genes in the third legs of both sexes, with 1 2 8 a higher magnitude, both in number and degree of differential expression, in male exaggerated third 1 2 9 legs. We note also that the third legs express a considerable set of common genes between males and 1 3 0 females, possibly underlying more global regulators of leg growth and allometry.   companion paper for sex-biased genes).

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To test the role of these genes in trait exaggeration, we conducted an RNA interference (RNAi) screen targeting about 30 candidates representing transcripts that were leg-biased, sex-biased, broadly 1 4 8 expressed or tissue-specific (Supplementary table 5). This functional screen identified two genes, Bone Morphogenetic Protein 11 (BMP11, also known as Growth Differentiation Factor 11) and whereas Ubx is a tissue-specific Hox protein known to be confined to the second and third thoracic 1 5 3 segments in water striders 29 . In our dataset, BMP11 is expressed in all legs but was up-regulated in the  Importantly, RNAi against BMP11 also significantly reduced the allometric coefficient of male adult males were also lower compared to their corresponding controls, but to a lower degree than in the third Contrary to BMP11, Ubx knockdown did not affect the slope but rather shifted the intercept due to a Supplementary table 6). We also detected a small effect in the second legs and no effect in the first Second, this tissue-specific gene is associated with a change in intercept but not in allometric 1 8 4 coefficient. We have shown that the evolution of exaggerated third legs in M. longipes was accompanied with sneaking behaviour by focusing on the female rather than trying to chase rival males away, or by 1 9 6 abandoning the site altogether without a fight (Supplementary video 2). We quantified this "docile" 1 9 7 behaviour by calculating the frequency of fights on floaters and found that BMP11 males fought on 1 9 8 average twelve times less than control males ( Figure 4A; t-test: t = 3.6134, df = 3.0559, p-value < 1 9 9 0.05). BMP11 females were attracted by male's signals on floaters, but we failed to observe any 2 0 0 mating events during our trials (Supplementary video 2). We further observed that Ubx RNAi-treated We have shown that leg exaggeration in M. longipes males is associated with a specific Exaggerated traits represent striking cases of differential growth between various organs, and production of the growth factor occurs locally rather than through heightened sensitivity to a systemic 2 2 7 factor. Whether and how this increased tissue-specific expression of growth factors is connected to systemic growth pathways remains to be tested 11 .

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Our experiments describe a common effect of BMP11 on mean body length in both sexes. By 2 3 0 contrast to males, the effect of BMP11 on body length is decoupled from its effect on leg length in 2 3 1 7 females ( Figure 5C). It is possible that increased body size has been favoured in both sexes, through 2 3 2 increased competitiveness in males and increased fertility in females 33,34 . Another explanation could 2 3 3 be that smaller females were disfavoured due to reproductive incompatibility as body size increased in 2 3 4 males. On the other hand, the lack of correlation in leg length between the sexes may be a 2 3 5 consequence of sexual conflict resolution through dimorphism 35 .

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Studies of scaling relationships reported a strong stasis of the allometric coefficient at the 2 3 7 microevolutionary time scale [12][13][14] . Even at the macro-scale, slope is known to evolve slower compared 2 3 8 to other scaling parameters such as intercept 12,13 . Several hypotheses were formulated to explain such 2 3 9 evolutionary stasis, including a lack of genetic variation, strong stabilizing selection acting on slope, more pleiotropic effect than that of intercept, possibly explaining the observed differences in 2 4 5 evolutionary stasis between these two scaling parameters.

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An important question is how trait exaggeration evolves despite the evolutionary stasis of 2 4 7 slope. We have shown that the evolution of hyperallometry in M. longipes is associated with increased 2 4 8 mean body size and male aggressiveness compared to its sister species M. pulchella 21 . These changes 2 4 9