尼泊爾埋葬蟲的社會演化與生殖適應的基因體研究

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(1)國立台灣師範大學生命科學系中央研究院生物多樣性國際研究生學程 Department of Life Science, National Taiwan Normal University Taiwan International Graduate Program on Biodiversity, Academia Sinica. 博士論文 Doctoral Dissertation. 尼泊爾埋葬蟲的社會演化與生殖適應的基因體研究 Social evolution and genomic investigation of breeding adaptation in burying beetles. 研究生：陳伯飛 Bo-Fei Chen 指導教授：沈聖峰博士 Sheng-Feng Shen, PhD 蔡怡陞博士 Isheng Jason Tsai, PhD. 中華民國 108 年八月 August 2019.

(2) Acknowledgement This dissertation can not be done without many people’s comments, assistance, and support. First of all, I would like to thank my two supervisors and also mentors, Sheng-Feng Shen (沈聖峰) and Isheng Jason Tasi (蔡怡陞). From them, I learned not only knowledge and technic of scientific researches, but more importantly, the attitude of pursuing scientific truth. In addition to my two mentors, I would also like to thank the two postdoctoral fellows Meng-Shin Shiao (蕭孟昕) and Mark Liu (劉彥廷) who led me into the field of molecular biology. They let me realize the power of molecular and genomic tools in the field of behavioral ecology. In terms of experiments and logistics, I would like to thank the current and former members of Dr. Shen's lab, Shih-Fan Chan (詹仕凡), Tzu-Neng Yuan (袁子能), Yancheng Lin (林彥成), Hsiang-Yu Tsai (蔡祥瑜), Yi Shin Jang (張譯心), Yu-Heng Lin (林宇恆), Yan Lin (林燕), Pai-Yu Ariel Cheng (鄭百佑), Catherine Lan (藍永懿), Jingfu Lin (林敬富), An-yu Chang (張安瑜), Syuan-Jyun Sun (孫烜駿), E-Ping Rau (饒益品), Yuanwen Zheng (鄭詠文), Yi-chen Lee (李宜蓁), Jian-Nan Liu (劉建男), Hsueh-Chen Chen (陳雪溱 ), Tsung-Wei Wnag (汪琮瑋 ), Gary Sing, Chang-Yu Chang (張昌佑), Tong-Chang Ni (倪東正), Wei-Kai Shih (石維楷), Sih Chen Lin (林思辰), Chinhsien Tsai (蔡瑾嫻), Yun-Jia Yeh (葉 i.

(3) 芸嘉), Yuki Haba (羽場優紀), Jessie Ku (辜昱嘉), Yu-Meng Fan (范郁盟), and Chien Tsun Chen (陳建存); the current and former members of Dr. Tsai’s lab, Huei-Mien Ke (柯惠棉), Tracy Jiaye Lee (李佳燁), Yu-fei Lin (林渝非), Cheng-Kuo Lai (賴政國), Hsinhan Li (李昕翰), Tzuhao Kuo (郭子灝), Dang Liu (劉當), and Chan-Yi Ivy Lin (林展伊); two Research Specilists of Biodiversity Research Center, Dr. Meiyeh Lu (呂美曄) and Dr. Tzi-Yuan Wang (王子元); NGS High Throughput Genomics Core at BRCAS; the staff of TIGP BIODIV (生物多樣性國際研究生學程), Jane Wu (吳莉珍), Sage Gong (龔政哲), and Vanessa Chen (陳捃教), without their comments and help in experiments or logistic support, I could not survive in the past few years. Finally, I appreciate my parents (陳永茂和吳玉梅), thank them for supporting me to be a scientist as my career. Thanks to my brother (陳文軒) for helping me to accompany my parents. I would also like to thank my wife, Yu-Ching Liu (劉育菁), for helping me in both reseach and daily life.. ii.

(4) Abstract Competition shapes the evolution of life and determines how organisms live now. Carcasses are nutritious but unpredictable and transient resources that drive intense competition among scavengers and microbiomes. Burying beetles (Coleoptera: Silphidae: Nicrophorus) are one of the unique scavenging insects which use small vertebrate carcasses as the sole resources to reproduce. To compete against the major interspecific competitor, blowflies, they formed cooperative groups on carcasses. This study used a series of field and laboratory experiments to clarify the mechanism of group formation in burying beetles and found that interspecific competition drove Nicrophorus nepalensis to use a sulfurcontaining organic volatile compound, dimethyl disulfide (DMDS) as the cue to indicate interspecific competition and form social groups on carcasses. On the other hand, the interspecific competition also drove N. nepalensis to evolve two breeding types, i.e., continuous breeding (CB) and seasonal breeding (SB), among populations. To understand the transition in the molecular mechanism between two breeding types, I performed the genomic comparison among N. nepalensis and 14 Hexapoda species and the transcriptomic comparisons between two N. nepalensis populations. The results showed the insects of two breeding types had convergent evolution at gene levels, respectively, and N. nepalensis shared breeding-type specific genes with both breeding types. These two studies provide evidence to demonstrate how N. nepalensis adapt to interspecific competition in terms of cooperative behavior and also adjusting breeding seasons. When the pressure of interspecific competition increases, N. iii.

(5) nepalensis shifts from intraspecific competition to cooperation to compete with interspecific competitors. Because N. nepalensis has both breedingtype specific gene features in its genome, N. nepalensis can adjust the breeding season to avoid interspecific competitors when the competitive pressure is too high. Differentiation in the length of breeding seasons implies N. nepalensis has local adaptation among populations. In future work, the study would be focused on the molecular evolution among populations using genomic data in order to further understand the local adaptation and its driving forces in N. nepalensis. Keywords: social evolution, cooperative behavior, comparative genomics, seasonal breeder, continuous breeder, burying beetle. iv.

(6) Table of contents Acknowledgement ..................................................................................... i Abstract .................................................................................................... iii Chapter 1 – Introduction..........................................................................1 Evolution driven by competition .............................................................1 Behavioral mechanisms of group formation ...........................................2 Genomic and transcriptomic comparisons in breeding regulations ........3 References ................................................................................................5 Chapter 2 – A chemically-triggered transition from conflict to cooperation in burying beetles .................................................................9 Abstract ....................................................................................................9 Introduction............................................................................................11 Results ....................................................................................................13 Group size, social investment and conflict along the environmental gradient ...............................................................................................13 Cooperation triggered by interspecific competition ...........................14 A sulfur-containing organic compound as the cue of interspecific competition .........................................................................................15 Dominance hierarchy and body temperature among group members17 Discussion ..............................................................................................20 Materials and methods ...........................................................................22 References ..............................................................................................29 Chapter 3 – The genomic and transcriptomic investigations of seasonal and continuous breeding adaptations in burying beetles ...................46 Abstract ..................................................................................................46 v.

(7) Introduction............................................................................................47 Results ....................................................................................................49 Assembly, gene annotation, and genome characterization of the N. nepalensis genome..............................................................................49 Convergent evolution at gene levels within CB and SB insects ........50 N. nepalensis shared genetic features with both breeding types ........52 Gene-expression changes between seasonal and continuous breeding N. nepalensis populations ...................................................................53 Genes and their function correlated with sexual maturation and developmental stages ..........................................................................55 Breeding-type-specific orthologues or PDEGs and circadian clock genes in the regulation of continuous and seasonal breeding ............57 Discussion ..............................................................................................59 Materials and methods ...........................................................................61 References ..............................................................................................67 Chapter 4 – Conclusion ........................................................................135 References ............................................................................................137. vi.

(8) Chapter 1 – Introduction Evolution driven by competition Competition is an interaction between individuals in which both are harmed. Resource competition is one of major types of competition. When resource, usually food, is limited, individuals using the same resource always need to compete for it. Defeated individuals not only lose the chance to access resources but most likely lose the chance to pass their genes to the next generation. Therefore, competition is a strong selective force determining how organisms live now. The carcass is a nutritious but unpredictable and transient resource. The vertebrate carcass contains 7-11% nitrogen per gram of dry weight, which releases 18 to 24 calories while decompoding1,2. It also contains essential elements such as potassium, sulfur, sodium, calcium, magnesium, and phosphorus1,2. Because of the rich nutrients, a large number of scavengers and microbial decomposers compete for a carcass rapidly when it shows up. When a carcass appears on the soil surface, the soft tissue takes a few days to a few weeks to be completely consumed, leaving only the hard tissue that is difficult to decompose1-3. In order to compete for such precious and nutrient-rich resources, many scavengers have evolved unique behaviors to preserve carcasses and exclude other competitors from using carcasses. The burying beetle is one of these unique scavengers. The burying beetle belongs to Coleoptera: Silphidae: Nicrophorus. The genus Nicrophorus contains about 70 to 80 species, mainly distributing in the 1.

(9) northern hemisphere4. Only a few species are distributed to South Asia or Latin America, and all of them are montane rather than lowland species4. The phylogenetic tree shows that the burying beetle originated from the temperate zone of the old world5-7. Burying beetles use small vertebrate carcasses as the sole breeding resource. Their unique way of preserving a carcass is to bury a small vertebrate carcass into the soil and then roll it into a meatball to prevent other animal competitors from discovering and approaching the carcass4. During processing a carcass, they also smear their gut microbiota to the surface of the carcass to prevent other microbiota from corrupting the carcass8. When burying beetles reproduce, they form a cooperative group of more than one pair of males and females9. It is generally believed that cooperative breeding is also evolved to resist competitors within and between species10-16. This thesis mainly reports how competition affects the evolution of burying beetles from the perspective of behavioral mechanisms and comparative genomics.. Behavioral mechanisms of group formation Understanding the mechanism of forming a cooperative group has always been the core of social evolution studies. Previous studies have shown that many ecological factors, such as ecological constraint and environmental fluctuation, have led to the evolution of cooperative groups17-20. What proximate promoters can trigger animals to form cooperative groups is still little known. In preliminary observations, it was found that Nicrophorus nepalensis in Taiwan did not form a cooperative group in the simple condition of laboratories, only one pair of burying beetles could use a rat carcass. However, N. nepalensis formed cooperative groups in fields. I 2.

(10) speculated that the behavioral differences between laboratories and fields could be related to inter-species competitions. In the second chapter of this thesis, I used a series of experiments to show that N. nepalensis formed cooperative groups on the carcasses in the present their primary competitors, maggots of blowflies. The formation of cooperative groups is triggered by a volatile organic compound emitted by the decomposing carcasses. I also used behavioral and thermal videos to identify the hierarchy ranks of N. nepalensis individuals. The individuals with higher ranks spent more time dealing with carcasses and have more conflicting behaviors toward lower-ranking individuals, and also maintained a higher body temperature to keep themselves in an active condition.. Genomic and transcriptomic comparisons in breeding regulations Being capable of reproduction is one of the definitions of life. All animals must reproduce offspring to pass their genes to the next generation. Depending on the length of the breeding season, animals can be divided into two major breeding types, the continuous breeder (CB) and seasonal breeder (SB)21-29. CB can breed throughout a year, but SB only breeds in the most favorable season. So far, no studies have compared the transition in the molecular mechanisms between these two breeding types. Previous studies showed that different N. nepalensis populations belonged to different breeding types, and the main cause of such differences in breeding seasons is interspecific competition. In the third chapter of this thesis, I compared the de novo assembled N. nepalensis genome with the genomes 3.

(11) of 6 CB and 6 SB insects. The results show that there are convergent evolutions at the gene level in the CB or SB insects, respectively. Both breeding types of insects share breeding-type-specific orthologues or enriched protein families, respectively. Moreover, N. nepalensis shared a partial of breeding-type-specific genes with both breeding types. Analyses of transcriptomes between two breeding types of N. nepalensis show that a high proportion of the genes which shared with either breeding types have differentially expressed patterns between two breeding types. It implies that these shared genes are involved in the molecular regulation of two breeding types in N. nepalensis. In addition to confirming the importance of shared genes, the transcriptomic comparison also reveals the top 20 genes most involved in gene regulation of both breeding types.. 4.

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(15) (Diptera: Tephritidae) in Heraklion, Crete, Southern Greece. Environmental Entomology 29, 1180-1187, 1188 (2000). 27. Somoano, A., Ventura, J. & Miñarro, M. Continuous breeding of fossorial water voles in northwestern Spain: potential impact on apple orchards. Folia Zoologica 66, 29-36, 28 (2017).. 28. Verhulst, S. & Nilsson, J.-Å. The timing of birds' breeding seasons: a review of experiments that manipulated timing of breeding. Philosophical Transactions of the Royal Society B: Biological Sciences 363, 399-410 (2008).. 29. Visser, M. E., Caro, S. P., Oers, K. v., Schaper, S. V. & Helm, B. Phenology, seasonal timing and circannual rhythms: towards a unified framework. Philosophical Transactions of the Royal Society B: Biological Sciences 365, 3113-3127 (2010).. 8.

(16) Chapter 2 – A chemically-triggered transition from conflict to cooperation in burying beetles Abstract Although interspecific competition has long been recognized as a major driver of trait divergence and adaptive evolution, relatively little effort has focused on how it influences the evolution of intraspecific cooperation. Here we identify the mechanism by which the perceived pressure of interspecific competition influences the transition from intraspecific conflict to cooperation in a facultative cooperatively breeding species, the Asian burying beetle Nicrophorus nepalensis. In their natural environment in central Taiwan, N. nepalensis is typically aggressive to conspecifics and only cooperate with others of their own species at critical carcass resources in the presence of blowflies, their primary competitors. We demonstrate that beetles form larger groups and are more cooperative in carcass preparation in warmer environments where the pressure of interspecific competition with blowflies is highest. To test the hypothesis that the presence of blowflies promotes beetle cooperation and to identify the mechanism by which this occurs, we manipulated blowfly larvae on carcasses in the lab. We not only found that beetles are more cooperative at carcasses when blowfly maggots have begun to digest the tissue, but that this social cooperation appears to be triggered by a single chemical cue, dimethyl disulfide (DMDS), emitted from carcasses consumed by blowflies but not from control carcasses lacking blowflies. Our results provide experimental evidence that interspecific competition promotes the 9.

(17) transition from intraspecific conflict to cooperation in N. nepalensis via a surprisingly simple social chemical cue that is a reliable indicator of interspecific competition.. 10.

(18) Introduction Unraveling the mechanisms that shift individuals from being competitive to cooperative is critical for understanding not only the evolution of sociality but also of biological organization at many scales1. Interspecific competition has long been recognized as a major driver of trait divergence and adaptive evolution2-4. However, the proximate mechanisms affecting how individuals perceive the pressure of interspecific competition, which in turn shapes their cooperative and competitive strategies, remain poorly understood. Burying beetles (Nicrophorus spp.) use small vertebrate carcasses as their sole resource for reproduction and often face intense intra- and interspecific competition for access to these precious but limiting resources5-7. Previous work has suggested that the key benefit of cooperation in the Asian burying beetle N. nepalensis is that cooperative carcass preparation-including carcass cleaning, shaping, and burial, as well as the elimination of competing species5-8—enables beetles to outcompete their primary competitor, blowflies (family Calliphoridae), particularly in warmer environments where blowflies are most abundant. By experimentally manipulating burying beetle group size along an elevational gradient, we showed that in cooler environments where the pressure of interspecific competition is low, beetles in large groups are more aggressive toward same-sex conspecifics and often engage in intense and even lethal fights that result in a single individual monopolizing the carcass and having a higher probability of breeding successfully than those in large groups9. In contrast, in warmer environments where blowflies are more common, burying beetles cooperate with conspecifics to more 11.

(19) quickly bury carcasses and escape blowfly competition10, ultimately gaining greater reproductive success9. Although the presence of blowflies at carcasses appears to facilitate a shift from competitive to cooperative behavior in N. nepalensis, it remains unclear what drives this transition in beetle social behaviour and how individuals know to reduce conflict and tolerate conspecifics.. 12.

(20) Results Group size, social investment and conflict along the environmental gradient To determine how ecology influences inter- and intraspecific social interactions in natural burying beetle populations, we first quantified beetle social behavior and dynamics by video recording their breeding behaviors at 25 sites along two elevational gradients in eastern and western Taiwan, each spanning more than 1000 m in elevation. We calculated the time that beetles spent on cooperative carcass preparation (hereafter cooperative investment) both in terms of total investment (i.e. the cumulative time of the social group) and on a per capita basis for large (groups larger than the median size) and small groups (groups smaller than the median size), as well as in cool (<14.5°C) and warm environments (>14.5°C). We found that group size peaked at moderate temperatures (χ21 = 5.52, P = 0.019, n = 245; Fig. 1a) and that per capita cooperative investment along the temperature gradient varied with group size (group size × temperature interaction, χ21 = 11.20, P = 0.001, n = 89). Specifically, per capita cooperative investment increased with daily minimum temperature in large groups (χ21 = 5.39, P = 0.02, n = 33), but not in small groups (χ21 = 0.05, P = 0.83, n = 56; Fig 1b). Similarly, total cooperative investment increased with daily minimum temperature in large groups (χ21 = 4.88, P = 0.03, n = 33), but not in small groups (χ21 = 0.24, P = 0.60, n = 56; Fig. 1c). In contrast, per capita social conflict along the temperature gradient, measured as the number of intraspecific conflict events for each individual, varied with group size (mean group size × temperature interaction, χ21 = 13.

(21) 6.64, P < 0.01, n = 82, Fig. 1d), such that conflict increased with group size in cool environments (χ21 = 11.24, P < 0.001, n = 40; Fig. 1d), but not in warm environments (χ21 = 1.59, P = 0.2, n = 42; Fig. 1d). To confirm that these patterns of social conflict and cooperation were the result of changes in social behavior and not simply changes in activity associated with differences in ambient temperature, we further separated cooperative investment into (i) time spent simply walking on the carcass and (ii) more complex carcass-preparation behaviors, which are presumably more costly—including maggot and rotten tissue removal, as well as carcass dragging, depilation, and burial. We found that time spent on more complex carcass preparation behaviors increased with increasing daily minimum temperature in large groups (χ21 = 5.39, P = 0.02, n = 33; Fig. 2a), but not in small groups (χ21 = 0.17, P = 0.68, n = 56; Fig. 2a). However, there was no significant relationship between walking time and daily minimum temperature in large (χ21 = 0.24, P = 0.60, n = 33; Fig. 2b) or small groups (χ21 = 0.79, P = 0.37, n = 56; Fig. 2b), suggesting that the increase in total cooperative investment in warmer environments was not simply the result of increased activity at warmer temperatures. Cooperation triggered by interspecific competition Our field results demonstrate that N. nepalensis exhibits remarkably flexible social behaviors along elevational and thermal gradients: beetles are normally asocial and aggressive towards conspecifics in cooler environments but become social and cooperate with conspecifics in warmer environments where the competition for critical resources with other species is intense10. However, to demonstrate experimentally that 14.

(22) blowfly competition for carcasses drives the transition from intraspecific competition to intraspecific cooperation, we performed a series of laboratory experiments to manipulate the presence or absence of blowflies at carcasses directly. Our first experiment introduced blowfly competition to burying beetles by exposing carcasses to adult blowflies in an incubator at 26°C for two days, conditions that match those in the field and are optimal for blowflies to lay eggs and for their maggots to consume the carcass partially. We then allowed six beetles (three males and three females) to breed on the carcass. We found that more beetles cooperated (t = 5.26, P < 0.001; Fig. 3a), and that each individual beetle spent significantly more time cooperating, in the blowfly treatment than in the control treatment containing carcasses but no blowflies (t = 3.27, P = 0.002; Fig. 3b). As a consequence, the total cooperative investment was higher in the blowfly treatment than in the control treatment (t = 5.37, P < 0.001; Fig. 3c). Although there was no difference in per capita social conflict between the blowfly and control treatments (t = -0.33, P = 0.75; Fig. 3d), after controlling for total investment time by dividing per capita social conflict by the total cooperative investment, the adjusted per capita number of social conflicts per unit time was significantly lower in the blowfly treatment than in the control treatment (t = -2.58, P = 0.013; Fig. S1). Thus, social conflict in burying beetles was lower and cooperation higher when blowflies were present on carcasses. A sulfur-containing organic compound as the cue of interspecific. 15.

(23) competition What is the mechanism driving the transition from intraspecific competition to intraspecific cooperation? Since blowfly species are diurnal but N. nepalensis is nocturnal, it is unlikely that the physical presence of blowflies influences N. nepalensis behavior. Previous studies have demonstrated that sulfur-containing volatile organic compounds (S-VOCs) emitted from decomposing carcasses attract burying beetles to this key resource11,12. Because GC-MS analysis showed that dimethyl disulfide (DMDS) appeared earlier and was more abundant in the blowfly treatment than in the control (Fig. 4a), we hypothesized that DMDS is the key infochemical13—indicating not only the presence of a decaying carcass but also the degree of interspecific competition at that carcass—that mediates the transition between cooperative and competitive strategies in N. nepalensis. To experimentally test this hypothesis, we injected DMDS into the body cavity of mouse carcasses. We found that more individuals cooperated (t = -3.76, P < 0.001; Fig. 4b), and that each individual spent more time cooperating, in DMDS treated carcasses relative to controls (t = -2.55, P = 0.014; Fig. 4c). Thus, there was a higher total cooperative investment in the DMDS treatment than in the control (t = -3.8, P < 0.001; Fig. 4d). These results were similar to those observed in the blowfly treatment from the initial experiment. The only difference between the DMDS and blowfly treatments was that there was marginally more social conflict in the DMDS treatment than in the hexane control (t = -1.97, P = 0.054; Fig. 4e), whereas this trend-while in the same direction-was not 16.

(24) significant in the blowfly treatment (t = -0.33, P = 0.75), presumably because there were no real competitors that beetles need to remove in the DMDS treatment. Dominance hierarchy and body temperature among group members To further examine the individual investments and conflicts between the treatments and control. We identified a clear dominance hierarchy among group members. Again, the patterns of cooperative investment and conflict between the blowfly treatments and control and between DMDS and hexane control were similar (Fig. 5). Individuals of each rank in the blowfly and DMDS treatments invested more in cooperative carcass preparation than the same rank individual in their controls (Fig. 5a and 5b, respectively). However, Alpha individuals invest significantly more in carcass preparation than Beta and Gamma individuals, regardless the sex of individuals, in all treatments and controls (Fig. 5a and 5b). Alpha individuals also initiated more conflict events than Beta and Gamma individuals in all four treatments and controls (Fig. 5c and 5d). Alpha and Beta but not Gamma individuals had significantly more conflict events in the DMDS treatments than the hexane control, whereas there was no significant difference in levels of social conflict level between the blowfly treatment and control treatments in for each rank (Fig. 5c and 5d). Finally, patterns of injuries were different between blowfly treatment and control and between DMDS and hexane control: lower-ranking individuals sustained more injuries than higher-ranking individuals in the control but not in blowfly treatment. There is no significant difference in levels of injuries in each rank between the control and blowfly treatment, though 17.

(25) (Fig. 6a). On the other hand, Gamma individuals had the highest levels of injuries in the hexane control but not in the DMDS treatments. Individuals in each rank had a higher level of injuries in the DMDS treatment than hexane control, corresponding to the higher level of conflict in the DMDS treatment than in hexane control (Fig. 6b). In addition to measuring behavioral responses, we also compared the physiological responses (i.e., changes in surface body temperature) for all treatments and controls using infrared thermography, which allows noninvasive, precise (0.1 ℃ precision) and continuous body temperature recordings (see Method). Interestingly, higher-ranking individuals generally had higher surface body temperature, except in the blowfly treatment (Fig. 7a and 7b). Higher surface body temperature leads to both higher investment time (Fig. 7c) and higher levels of conflict (Fig. 7d). Thus, the higher surface body temperature of Gamma individuals in the blowfly treatment than the control suggests that Gamma individuals were physiologically prepared for higher investment levels. Similarly, the higher surface body temperature of individuals in the DMDS treatment than the hexane control corresponds well with the higher investment and conflict levels observed in the DMDS treatment. Note that the results were the same when using the relative body temperature—i.e., the difference between absolute body temperatures and environmental temperatures (Fig. S2)— and individuals’ body temperatures maintained a particular range that was consistently higher than the environmental temperature (Fig. S3). This suggests that active thermoregulation was involved in influencing the body temperature of beetles. Together, these results demonstrate that inter18.

(26) specific competition and the DMDS cue not only change social behavior but also cause underlying physiological changes in burying beetles.. 19.

(27) Discussion Our study shows that burying beetles transition from competitive to more cooperative interactions as the pressure of interspecific competition increases. Accumulating empirical evidence from other animals suggests that social conflict in cooperative societies is often lower in adverse environments with strong interspecific competition14. This pattern of reduced social conflict under strong interspecific competition has largely been explained by the fact that the cost of engaging in competitive interactions increases under adverse conditions15. Yet, there is little empirical evidence demonstrating that social animals increase their investment in cooperation under the threat of interspecific competition. One exception comes from cooperatively breeding superb fairy-wrens (Malurus cyaneus) that cooperate more in nest defense when exposed to a greater threat of interspecific brood parasitism16. However, it remains unclear how intraspecific conflict in fairy-wrens is influenced by the threat of interspecific competition. Our study helps fill this knowledge gap by showing that cooperative carcass preparation to reduce blowfly competition in warmer environments is critical for predicting both the cooperative and competitive interactions among individuals of the same species. Furthermore, we show that the conditional cooperative and competitive strategies17,18 used by N. nepalensis to maximize their utility of carcasses are mediated by a surprisingly simple chemical mechanism. DMDS is produced during the decomposition process and acts as an indicator of the level of competition from blowflies. Although interspecific 20.

(28) competition has long been recognized as a major ecological force that drives adaptive evolution2-4, relatively little effort has focused on how it influences intraspecific cooperation14,19,20. Our discovery of a novel social chemical cue provides unambiguous evidence that interspecific competition has shaped social evolution in N. nepalensis. DMDS acts as a kairomone because it is produced by heterospecifics (i.e., blowfly digestion), but benefits the receiver13, and not as a pheromone produced by conspecifics21,22. Pheromones are often used for kin discrimination, and studying the olfactory sensory system and its genes have greatly advanced our understandings of the role that chemically-driven kin recognition has played in social evolution, especially in ants23,24. Here we demonstrate that interspecific chemical communication is also important to insect social evolution. By showing that chemically-mediated interspecific competition is a key driver of intraspecific cooperation and of social evolution more generally, our work demonstrates the value of integrating ultimate and proximate levels to study the evolution of cooperation25. Ultimately, our findings suggest that the role of between-species competitive interactions driving within-species competitive and cooperative interactions are likely to have been important for the evolution of social behavior in a number of animal species, perhaps even in our own species’ social evolution at a time when we were not the only hominid inhabiting the planet.. 21.

(29) Materials and methods Field Study. The field study was conducted in Taiwan from 2012 to 2015 along elevational gradients composed primarily of uncultivated forest in Nantou county (spanning 1688 m to 2650 m above sea level) and Hualien county (spanning 1193 m to 2720 m above sea level). A variety of social behaviors, including per capita social conflict and investment in cooperative carcass preparation (i.e., cooperative investment), were scored on the first night of video observation (from 19:00 to 05:00) using the Observer® XT 14 (Noldus). To quantify total cooperative investment, we estimated the cumulative time that each beetle spent depilating rat hair, cleaning rat carcasses by removing maggots, or dragging carcasses during carcass burial and preparation. We measured per capita cooperative investment as the total cooperative investment divided by the mean group size, defined as the maximum number of beetles that stayed on or under carcasses averaged over three time periods (22:30 to 22:40; 01:30 to 01:40 and 04:30 to 04:40) during each video observation. Investment in cooperation was quantified as the duration of cumulative time sampled for a 10 mins observation period in each hour (i.e., 100 mins for each breeding experiment). In total, there were 89 breeding experiments (resulting in 8900 mins of video recordings) from which we were able to quantify total cooperative investment. Aggressive interactions were defined as a social conflict if a beetle attacked, wrestled, chased, or escaped from another same-sexed individual (see below for definitions of each behavior). We measured total social conflict as the total number of aggressive interactions over the 240 mins observation period. We measured per capita social 22.

(30) conflict as the total number of aggressive interactions divided by the mean group size for each observation period. Conflict was quantified as the total number of aggressive interactions sampled for two 120 mins observation periods (from 19:30 to 21:30 and from 23:30 to 1:30). In total, there were 82 breeding experiments (resulting in 19,680 mins of video recordings) from which we were able to quantify conflict behavior. We determined the mean group size on the first night of each beetle’s arrival in 245 breeding experiments (resulting in 7350 mins of video recordings). Collection and maintenance. Lab experiments were carried out using N. nepalensis individuals from laboratory-reared strains that originated from Meifeng, Nantou County, Taiwan (24°5' N, 121°10'). Burying beetles were collected using hanging pitfall traps baited with 100 g rotten chicken breasts. Collected beetles were randomly paired and supplied with frozen and re-thawed 75 ± 5 g dead rats (Rattus norvegicus) in 23 × 15.5 × 16 cm plastic boxes filled with 10 cm moist peat for reproduction. The emerged beetles were housed individually in 7.3 × 7.3 × 3.5 cm plastic boxes filled with 2 cm moist peat and fed with dead superworms (Zophobas morio) once a week. All individuals were kept in environmental chambers at 13.2 ~ 19.7 °C (to resemble the natural daily temperature fluctuation in their natural habitat) on a 14L : 10D photoperiod. Experimental beetles were between 40 and 80 days of age, which is their optimal age for reproduction (individuals can live for over three months in the laboratory). Experimental design and procedure. For each experimental replicate, three unrelated males and three unrelated females were randomly chosen from different families to avoid relatedness affecting their behaviors. Each 23.

(31) individual was weighed to the nearest 0.1 mg and marked with a Uni POSCA paint marker on the elytra and coated with Scorch® Super GlueGel for individual identification in videos. The marking and weighing of beetles were done 2 hrs prior to beginning an experiment to ensure that all beetles would return to normal activity levels. All six marked beetles were placed into the experimental boxes in random order at the beginning of each experiment. Experimental boxes consisted of a smaller plastic container (23 × 15.5 × 13.5 cm filled with 13.5 cm moist peat) located inside a larger plastic container (45 × 34.5 × 25 cm filled with 13.5 cm moist peat). A 4 cm high iron net with 2 cm2 mesh was placed around the small container to prevent beetles moving carcasses outside the field of view of the digital cameras, but beetles could still move freely between the inner and outer areas. A digital camera and a FLIR SC300 thermal camera were fitted on the top of a 25 × 20 × 55 cm acrylic box, which was fixed on the cap of the large container. To equalize the temperature of the experimental apparatus, boxes were filled with moist peat and put into the environmental chambers one day before the experiments began. The blowfly treatment was conducted by exposing a 75 ± 5 g rat thawed carcass to blowflies, oriental latrine flies (Chrysomya megacephala), in 32 × 32 × 32 fly cages for 50 hrs before the start of each experiment. Fly cages contained oriental latrine flies that had emerged from 10 g pupa and been kept in environmental chambers at 26°C on a 14L : 10D photoperiod. Except for maggot-digested carcasses, all other carcasses in the same weight range were thawed at 4°C for 24 hrs before experiments began. Carcasses used in all treatments were moved into the environmental 24.

(32) chambers 8 hrs prior to the start of experiments to equalize their temperatures. The hexane control and DMDS treatment used thawed-only carcasses injected with 2 ml hexane or 0.01 M DMDS solution, respectively, into abdominal cavities through the anus using 3 ml Terumo® Syringes and needles 1 hr prior to the start of the experiment. The thawedonly carcasses served as controls. The carcasses in controls and all treatments were moved into the experimental boxes and put on the surface of peat in smaller containers 1 hr before experiments began. Behavioral videos were recorded either from 7 PM until the day and time at which a carcass was completely buried into peat or for 72 hrs if the beetles did not completely bury the carcass (under natural conditions, a carcass would be completely consumed by blowflies if beetles did not completely bury it within 72 hrs). In total, 1020 hrs of videos were analyzed from 23 blowfly control replicates, 23 blowfly treatment replicates, 32 hexane control replicates, and 24 DMDS replicates. Social conflict and cooperative investment behaviors were recorded in the first 10 hrs (7 PM to 5 AM) of each experimental treatment using The Observer® XT 14 (Noldus). Behavioral analyses. In total, 1020 hours of videos were analyzed from 23 blowfly control replicates, 23 blowfly treatment replicates, 32 hexane control replicates, and 24 DMDS replicates. A variety of social behaviors, including social conflicts and investments in preparing carcasses, were recorded in the first 10 hours (7 pm to 5 am) after experiments began using The Observer® XT 14 (Noldus). Four individual interactions, such as wrestles, attack, chases, and escapes, between individuals of the same gender, were defined as social conflicts. Two individuals grasping and 25.

(33) biting each other was defined as a wrestle; one individual biting another was defined as an attack; a chase was defined as one individual rapidly run after another after any other interaction, such as a wrestle, attack, or other body contacts; and one individual rapidly running away from another after any other interaction described above was defined as an escape (also see supplementary videos). Social conflicts occurred randomly during observing periods, so they were recorded for the entire 10 hours (7 pm to 5 am). We calculated per capita conflicts as the total number of social conflicts divided by the maximal group size for each observation period. We quantified the total time each individual spent processing the carcass cooperatively by evaluating the cumulative time that each beetle spent on inspecting carcasses, depilating rat hair, removing maggots and blowfly eggs, moving carcasses, and digging the peat surrounding carcasses. The investment behaviors were sampled for 10 minutes in the middle period in each hour. Therefore, the maximal investment time of each beetle would be 100 minutes. The group size of each period was quantified as the maximum number of beetles that stayed on or under carcasses during the sampled 10 minutes. We calculated per capita investment time as the total investment time divided by the maximal group size for each observation period. Determination of dominance hierarchies. We determined the hierarchical organization of dominance relations among beetles using the social network transitivity analysis27. Both the number of attacks and chases and the roles of behavioral actors or receivers were used to construct dominance hierarchies. There were instances in which an individual 26.

(34) attacked others, but ran away from the carcass and did not return if others fought back; in these situations, the social network transitivity analysis could miscalculate dominance hierarchies. Therefore, all constructed dominance relations were manually checked and corrected. Gas chromatography-mass spectrometry (GC-MS) analysis. To determine the composition of volatile organic compounds (VOCs) emitted by the control and blowfly treated carcasses, two control and two blowfly treated carcasses were used to collect the VOCs (all carcasses were prepared using the same procedure described in materials and methods). The prepared carcasses were put on the peat surface in glass vacuum desiccators (15 cm diameter × 22 cm tall) filled with 5 cm of moist peat. The ground-glass rim of the desiccator lid and the stopcock of the lid were greased with a thin layer of petroleum jelly to prevent a leak of emitted VOCs or contamination from the atmosphere. The VOCs were sampled using the solid-phase micro-extraction26. The SPME holder with CAR/PDMS fiber (Supelco, previously desorbed for 5 min in GC injection port heated to 200°C) was inserted through the hold of the stopcock (stopcock was removed) into the atmosphere surrounding the rat carcass, and the fiber was exposed for 15 min; immediately after the 15 min, it was GC-MS-analysed using 6890N Network Gas Chromatograph (Agilent Technologies) equipped with an HP-5ms column (Agilent J&W) and 5975 Mass Selective Detector (Agilent Technologies). The GC oven was operated at an initial temperature 40°C for 1 min and then ramped at a rate 10°C/min to 250°C (with 10-min hold). The temperatures of the GC inlet and detector were set to 200°C and 260°C, respectively. The SPME 27.

(35) samples were GC analyzed split-less. Helium (1 mL/min) was used as a carrier gas. The GC-MS results showed DMDS was the major VOC emitted by the blowfly-treated carcasses. Therefore, DMDS was injected into carcasses in further experiments. Two DMDS-injected carcasses (also prepared using the same procedure described in Methods) were used in GC-MS-analyses (following the procedure described above) to determine the composition of volatile organic compounds (VOCs) they emitted. Statistical analyses. Multivariate analyses were performed using generalized linear models (GLMs) to determine statistical significance for differences between blowfly controls and treatments or hexane controls and DMDS treatments in mean group size, per capita investment, total investment, total conflict number, and adjusted per capita conflict number. Due to the random effects of 6 individuals in each replicate, generalized linear mixed models (GLMMs) were used in the multivariate analyses of all individual comparison between blowfly controls and treatments or hexane controls and DMDS treatments. The conditional R2 values in the linear regressions between mean body temperature and investment time or conflict numbers were calculated using the protocol proposed by Nakagawa and Schielzeth28. All statistical analyses were performed in R using R packages, such as stats, lme4, car, multcomp (http://cran.rproject.org/), and glmmADMB (http://glmmadmb.r-forge.r-project.org/).. 28.

(36) References 1. Bourke AFG (2011) Principles of Social Evolution (Oxford University Press, Oxford).. 2. Bolnick DI, et al. (2010) Ecological release from interspecific competition leads to decoupled changes in population and individual niche width. Proc. R. Soc. Lond., Ser. B: Biol. Sci. 277(1689):17891797.. 3. Pianka ER (1974) Niche overlap and diffuse competition. Proc. Natl. Acad. Sci. USA 71(5):2141-2145.. 4. Hardin G (1960) The competitive exclusion principle. Science 131(3409):1292-1297.. 5. Pukowski E (1933) Ökologische Untersuchungen an Necrophorus F. Zeitschrift fur Morphologie und Ökologie der Tiere 27:518 – 586.. 6. Scott MP (1998) The ecology and behavior of burying beetles. Annu. Rev. Entomol. 43(1):595-618.. 7. Rozen D, Engelmoer D, & Smiseth P (2008) Antimicrobial strategies in burying beetles breeding on carrion. Proc. Natl. Acad. Sci. USA 105(46):17890-17895.. 8. Cotter SC & Kilner RM (2010) Sexual division of antibacterial resource defence in breeding burying beetles, Nicrophorus vespilloides. J. Anim. Ecol. 79(1):35-43.. 9. Sun S-J, et al. (2014) Climate-mediated cooperation promotes niche expansion in burying beetles. eLife 3:e02440.. 10. Liu M, et al. (in review) Ecological transitions in grouping benefits explain the paradox of environmental quality and sociality. 29.

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(39) Figure 1 | Changes in N. nepalensis group size and social behaviors during carcass preparation along a temperature gradient. The relationship between daily minimum air temperature and (a) mean group size, (b) per capita cooperative investment, (c) total cooperative investment in large (closed circles) and small groups (open circles). Group size peaked at moderate temperatures, whereas per capita and total cooperative investment increased with daily minimum temperature in large but not small groups. Solid lines denote predicted relationships from GLMs, whereas dashed lines denote non-significant relationships. (d) Per capita 32.

(40) social conflict increased with group size in cool environments (closed circles), but not in warm environments (open circles). Lines represent leastsquared means (solid lines denote significant relationships and dotted lines non-significant relationships), and blue shaded areas represent 95% confidence intervals expected from GLMMs.. 33.

(41) Figure 2 | Complex carcass preparation and simple walking behaviors during cooperative carcass preparation along the temperature gradient. The time that beetles spent on (a) complex carcass preparation behaviors and (b) walking on the carcass in relation to daily minimum air temperature in large and small groups. Compared to small groups (open circles), large groups (closed circles) spent more time on complex carcass preparation but not on walking as daily minimum air temperature increased, suggesting that the increase in total cooperative investment in warmer environments was not simply the result of increased activity at warmer temperatures. Lines represent least-squared means (solid lines denote significant relationships and dotted lines non-significant relationships), and the blue shaded area represents 95% confidence intervals expected from GLMMs.. 34.

(42) Figure 3 | N. nepalensis social behaviors in control and blowfly treatments. (a) Mean group size, (b) per capita cooperative investment, (c) total cooperative investment, and (d) per capita social conflict of burying beetles on carcasses. Beetles formed larger groups and had greater per capital and total cooperative investment in carcass preparation in the presence of blowflies than in control treatments where blowflies were absent. ** P ≤ 0.01; *** P ≤ 0.001.. 35.

(43) Figure 4 | Results of gas chromatography-mass spectrometry (GC-MS) analyses and N. nepalensis social behaviors in hexane and DMDS treatments. (a) GC-MS analyses showed an abundance of sulfide compounds, including dimethyl sulfide (DMS), dimethyl disulfide (DMDS) and dimethyl trisulfide (DMTS) in control, blowfly, and DMDS treatments during the first 10 hrs. DMDS was the major sulfide compound emitted by maggot-digested carcasses. Shaded areas represent 95% confidence intervals expected from GLMMs. (b) Mean group sizes, (c) per capita cooperative investment, (d) total cooperative investment, and (e) per capita 36.

(44) social conflict of burying beetles on carcasses in DMDS and hexane control treatments. Beetles formed larger groups and had greater per capital and total cooperative investment on carcasses in the DMDS treatment compared to the hexane control treatment. * P ≤ 0.05; *** P ≤ 0.001.. 37.

(45) Figure 5 | Average investment time per individual in each group in (a) the. blowfly treatment and control and (b) the hexane control and. DMDS treatment; Individual conflict numbers for individuals of all hierarchies in (c) the blowfly treatment and control and (d) the hexane control and DMDS treatment. Individuals of all hierarchies in the blowfly and the DMDS treatments spent more time on preparing carcasses than the same ranking individuals in the blowfly control (Alpha, GLMM: z = 2.705, P = 0.007; Beta, GLMM: z = 3.945, P < 0.001; Gamma: GLMM: z = 3.459, P < 0.001) and hexane control (Alpha, GLMM: z = -3.502, P < 0.001; Beta, GLMM: z = -2.024, P = 0.043; Gamma: GLMM: z = -2.39, P 38.

(46) = 0.017), respectively. Moreover, Alpha individuals, regardless of sex, in all treatments and controls invested significantly more than Beta (control, GLMM: z = -8.194, P < 0.001; blowfly treatment, GLMM: z = -7.733, P < 0.001; hexane control, GLMM: z = -11.455, P < 0.001; DMDS treatment, GLMM: z = -9.321, P < 0.001) and Gamma individuals (control, GLMM: z = -7.425, P < 0.001; blowfly treatment, GLMM: z = -7.465, P < 0.001; hexane control, GLMM: z = -10.907, P < 0.001; DMDS treatment, GLMM: z = -8.809, P < 0.001). On the other hand, Alpha individuals in all treatments and controls launched more conflict events than Beta (control, GLMM: z = -5.458, P < 0.001; blowfly treatment, GLMM: z = -3.158, P = 0.005; hexane control, GLMM: z = -3.436, P = 0.002; DMDS treatment, GLMM: z = -6.2, P < 0.001) and Gamma individuals (control, GLMM: z = -8.304, P < 0.001; blowfly treatment, GLMM: z = -5.929, P < 0.001; hexane control, GLMM: z = -6.382, P < 0.001; DMDS treatment, GLMM: z = -11.039, P < 0.001). Beta individuals also initiated more conflict events than Gamma individuals (control, GLMM: z = -3.404, p = 0.002; blowfly treatment, GLMM: z = -2.927, P = 0.01; hexane control, GLMM: z = 3.398, P = 0.02; DMDS treatment, GLMM: z = -5.605, P < 0.001) in all treatments and controls. Furthermore, Alpha and Beta but not Gamma individuals had more conflict behaviors in the DMDS treatment than in the hexane control (Alpha, GLMM: z = -3.415, P < 0.001; Beta, GLMM: z = -2.469, P = 0.01), whereas there was no significant difference in conflict levels for any hierarchies between the control and the blowfly treatment. * P < 0.05; ** P ≤ 0.01; *** P ≤ 0.001.. 39.

(47) Figure 6 | The individual injury levels in (a) the blowfly treatment and control and (b) the DMDS treatment and hexane control. Beta (GLMM: z = 2.914, P = 0.01) and Gamma individuals (GLMM: z = 3.762, P < 0.001) sustained more injuries than Alpha individuals in the control but not in the blowfly treatments. However, there was no significant difference in the injury levels for each rank between the control and the blowfly treatments. On the other hand, Gamma individuals had higher injury levels than Alpha individuals (GLMM: z = 2.346, P = 0.049) in the hexane control but not in the DMDS treatments. But individuals in each rank sustained more injuries in the DMDS treatments than in the hexane control (Alpha, GLMM: z = 2.666, P = 0.008; Beta, GLMM: z = -1.922, P = 0.055; Gamma, GLMM: z = -2.031, P = 0.042). In each figure, ⋅ P < 0.1; * P < 0.05; ** P ≤ 0.01; *** P ≤ 0.001.. 40.

(48) Figure 7 | The individual mean body temperatures during the first two hours of (a) the blowfly treatment and control and (b) the hexane control and DMDS treatment. Alpha (control, GLMM: z = -4.927, P < 0.001; hexane control, GLMM: z = 3.538, P = 0.001; DMDS treatment, GLMM: z = 3.029, P = 0.007) and Beta individuals (control, GLMM: z = -3, P = 0.007; hexane control, GLMM: z = 2.861, P = 0.012; DMDS treatment, GLMM: z = 2.615, P = 0.024) had higher mean body temperatures than Gamma individuals in the control, the hexane control, and the DMDS treatment. In the blowfly treatment, Alpha individuals also had higher body temperatures than Gamma individuals (GLMM: z = 2.412, 41.

(49) P = 0.042). Furthermore, Gamma individuals in the blowfly treatment exhibited higher body temperatures than in the control (GLMM: z = 2.58, P = 0.01). But individuals of each rank had higher body temperatures in the DMDS treatment than in the hexane control (Alpha, GLMM: z = 3.729, P < 0.001; Beta, GLMM: z = -3.4, P < 0.001; Gamma, GLMM: z = 4.147, P < 0.001). The linear regressions between the mean body temperature and (c) investment time and (d) conflict numbers. The mean body temperatures were positively correlated with either individual investment time (GLMM: χ2 = 8.091, P = 0.004, R2 = 0.054) or individual conflict numbers (GLMM: χ2 = 7.054, P = 0.008, R2 = 0.478). In each figure,* P < 0.05; ** P ≤ 0.01; *** P ≤ 0.001.. 42.

(50) Figure S1 | Adjusted per capita social conflict in different experimental treatments. Adjusted per capita social conflict (i.e., per capita social conflict divided by the total cooperative investment time) was lower in the blowfly than control treatments. There was no difference in adjusted per capita social conflict between the DMDS and hexane control treatments. * P < 0.05.. 43.

(51) Figure S2 | The linear regressions between the relative mean body temperature (the difference between absolute body temperatures and environmental temperatures) and (a) investment time and (b) conflict numbers. The relative mean body temperatures were positively correlated with either individual investment time (GLMM: χ2 = 5.663, P = 0.017, R2 = 0.054) or individual conflict numbers (GLMM: χ2 = 7.852, P = 0.005, R2 = 0.458).. 44.

(52) Figure S3 | The frequency of environmental temperature and individual body temperature across the three ranks. The beetle body temperature is higher than the environmental temperature. Higher-ranking individuals actively generated more heat than lower-ranking individuals.. 45.

(53) Chapter 3 – The genomic and transcriptomic investigations of seasonal and continuous breeding adaptations in burying beetles Abstract Seasonal change shapes the evolution of organisms and determines how Earth looks around us. Seasonal breeding is one of ubiquitous adaptation triggered by photoperiods. In contrast, continuous breeders can reproduce year-round regulated by their internal biological rhythms. Investigating the genomic differentiation behind breeding types is crucial to understanding the evolutionary transitions. Here we present the reference-quality genome of the burying beetle Nicrophorus nepalensis which exhibits different breeding regulations between populations in Taiwan. Comparing genomes among Hexapoda, we find continuous breeding is likely the ancestral trait in insects and two breeding types of insects both show convergent evolution at gene levels. N. nepalensis surprisingly shares genetic features with both reproductive types in its genome. Transcriptomic comparison suggests these genes were strongly associated with the polymorphic breeding regulations in N. nepalensis. Our results provide the first comparative study between two breeding types and give the insight to clarify the regulation of circannual rhythms.. 46.

(54) Introduction To reproduce successfully, breeding at appropriate timing is crucial for almost all animals that inhabit in regions displaying distinct seasons. The energetic costs of reproduction are enormous; birth must occur at the most favorable season in which food abundance is at the peak of annual variations to maximize benefits in both parents and offspring1-3. Organisms use predictive cues such as photoperiod, temperature, and rainfall to adjust morphological traits, physiological conditions, and behaviors to prepare for breeding in the coming favorable environments4,5. Different from the seasonal breeders (SBs), the continuous breeders (CBs) can reproduce allyear-round by regulating neuroendocrine systems with internal biological rhythms6-8. Hence, identifying the underlying molecular mechanisms between different breeding behaviors are key to understanding these evolutionary transitions but also to providing the perspectives on the regulation of circannual rhythms3. The genomes and transcriptomes from a species displaying both SB and CB would allow us to extensively study the selection on this breeding differentiations. However, a paucity of genomic data fitted for this criterion has impeded such investigations. The burying beetles, Nicrophorus nepalensis have shown to display remarkable polymorphisms in breeding behaviors among populations in Taiwan9. Most individuals of the north population only breed from late autumn to early spring using the short photoperiod as the cue. In contrast, the central population breeds year-round along the altitude (Supplementary Fig. 1). The two populations of beetles evolved two breeding strategies, respectively, due to the combination of interspecific competitions and 47.

(55) topographies9. The beetles choose the areas with temperature favoring them but not the interspecific competitors, blowflies9. Also, the over-high or -low temperature limited the physiological performance of N. nepalensis in their survivorship and development. Unlike the polymorphic breeding behaviors in N. nepalensis, most of the temperate Nicrophorus species are seasonal breeders reproducing in the favorable seasons, varied from late spring to early autumn10. Nicrophorus incorporate phenotypic diversity within and among species and therefore constitute excellent candidates for evolutionary studies in the regulation of breeding behaviors. Here we report a chromosome-level genome assembly of N. nepalensis. Comparative analyses of the N. nepalensis genome with those of continuous and seasonal breeding Hexapoda species allow us to relize the similarity and differentiation between two breeding types in gene levels. We find both breeding types of insects showed convergent evolution at gene levels and N. nepalensis shared breeding-type specific orthologues and gene expansions with either continuous or seasonal breeding types. Transcriptome profiling between two breeding types in N. nepalensis reveals differential genetic regulation in sexual maturity between two populations. Combining the genome and transcriptomes, we then identify a set of genes strongly correlated to the continuous and seasonal breeding in N. nepalensis.. 48.

(56) Results Assembly, gene annotation, and genome characterization of the N. nepalensis genome We first generated a draft assembly of N. nepalensis using Illumina reads, then corrected misjoins and scaffolded using in situ Hi-C reads11 (Supplementary Table 1). The final assembly was a total of 173.87 Mb from 1,595 scaffolds, which was 96.6% of its predicted size (Supplementary Fig 2). The genome consisted of 8 chromosome-length scaffolds (lengths from 7.8 to 32.2 Mb and N50 length of 23.47 Mb) containing 90% of the total sequence (Fig. 1 and Supplementary Table 2). Previous karyotyping studies have shown two Nicrophorus species were diploid with six pairs of autosomes and one X chromosome (2n = 13)12,13. The N. nepalensis chromosomes could be mainly represented by these eight chromosome-length scaffolds. The N. nepalensis genome had a high degree of continuity and completeness when compared with the published genome of other insect species (Supplementary Table 2). The sex chromosome of N. nepalensis was determined by the orthologous genes, paired-end read coverages and single nucleotide polymorphisms (SNPs) densities (Fig. 1 and Supplementary Fig. 3). The overall within-individual SNPs density was 11.19 SNPs/Kb (Fig. 1b and Supplementary Fig. 3b and d), which was high in insects (1.60 SNPs/Kb in the malaria mosquito, Anopheles gambiae14; 0.48 SNPs/Kb in the mountain pine beetle, Dendroctonus ponderosae15). The highly heterozygous genome suggested high genetic variation within and between N. nepalensis populations. We annotated the N. nepalensis gene set based on the MAKER216 49.

(57) pipeline combining reference insect protein homology support, transcriptome sequencing from head tissues of 54 individuals of two populations (Supplementary Table 3), and ab initio gene prediction. A total of 11,263 protein-coding gene models were annotated (Supplementary Table 2). Of these, 90.6% were expressed in the head tissues, and 95.5% could be assigned Gene Ontology (GO) terms using Argot217. Unlike other model insect genomes18,19, the N. nepalensis genome contained only 9.74% of the genome composed of transposable elements and interspersed repeats (Supplementary Table 4). Therefore, there was no obvious complementary distribution between genes and repeats at each scaffold (Supplementary Fig. 4). Convergent evolution at gene levels within CB and SB insects Our first step was to construct a phylogenetic tree to check evolutionary patterns of two breeding types in insects using single-copy orthologues. Orthology of N. nepalensis proteome were assigned against gene models from six CB and six SB insects and two Collembola (springtails) species. Daphnia pulex was used as the outgroup. N. nepalensis uses day length as the cue to indicate breeding seasons, so SBs and CBs were defined accordingly (see Methods for species details). A total of 16,498 orthologous groups (OGs) were assigned (Supplementary Fig. 5 and Supplementary Table 5), of which 910 single copy orthologues were used to construct the phylogenetic tree of the 16 species. The phylogeny showed CB was likely the ancestral trait, and SB would be the derived trait in insects (Fig. 2a). Also, both breeding types evolved multiple times, suggesting the reproductive regulation is a flexible trait in insects. 50.

(58) Since the two traits had changed multiple times in insects, we speculated the genetic regulation of two breeding types should be quite divergent in insects. To clarify our hypothesis, we investigated if there were orthologues and enriched gene families conserving within two breeding types by comparing conserved OGs and numbers of protein family (Pfam) domains across 15 Hexapoda species. Surprisingly, we identified 109 OGs only presented in at least half of each insect species group of breeding types, and 49 Pfam domains only enriched in either CB or SB insects (Fig. 2). It suggested each breeding types of insects do share general genetic features. Of 109 breeding-type specific OGs, 76 OGs were specific to CB insects, and 33 OGs were specific to SB insects although 51.4% of these OGs were uncharacterized or hypothetical proteins (see Fig. 2b for gene details). Two springtails overall shared more OGs with CB insects (16/76 OGs) than SB insects (3/33 OGs). Comparison of expanded gene families also showed a similar pattern. The principal component analysis (PCA) of Pfam domains did not cluster the insects by their phylogeny, suggesting significant functional diversification since the lineage evolved (Fig. 2a and c). Two springtails were clustered with CB insects rather than SB insects (Supplementary Fig. 6). This result showed two springtails had more analogous genes with CB insects. Within 49 enriched Pfam, 20 were observed in SB insects, and 29 were observed in SB insects. Two springtails overall shared more gene expansions with CB insects (18/29) than SB insects (12/20). Two springtails shared more OGs and enriched Pfam with CB insects, which were consistent with the phylogenetic tree suggesting the CB was likely the ancestral trait for insects. 51.

(59) Although the derived SB independently evolved multiple times in insects, we still identified 29 OGs, and 7 enriched Pfam presented in SB insects but not in two springtails and CB insects, also, 16 OGs and 13 enriched Pfam presented in CB insects and two springtails but SB insects (Fig 2b and d). It implied SB insects gained or lost these genes after these lineages evolved. This was the evidence to show SB insects had convergent evolution at gene levels in SB insects. On the other hand, convergent evolution also was observed in CB insects. A total of 60 OGs and 5 enriched Pfam were identified in CB insects but not in two springtails and SB insects (Fig 2b and d) implying CB insects gained and kept these genetic features after insects evolved. N. nepalensis shared genetic features with both breeding types Comparisons of OGs and Pfam also revealed N. nepalensis shared OGs and enriched Pfam with SB and CB insects, respectively. Of 16,498 OGs, 11,027 N. nepalensis genes (97.9%) were included in OGs containing orthologues from at least one other species (Fig. 5 and Supplementary Table 5). 219 genes were not assigned to any orthologous groups, and only 17 genes are parts of the five N. nepalensis-specific orthologous groups. Of the breeding-type-specific OGs, N. nepalensis shared seven OGs with CB insects and 10 OGs with SB insects, suggesting N. nepalensis had both genetic adaptations with two breeding types of insects (Fig. 2b and Supplementary Fig. 7a). The Pfam domain analyses also showed the same patterns. N. nepalensis clustered with two breeding types in the PCA of Pfam domain (Fig. 2c) and shared seven enriched Pfam with each breeding type of insects (Fig. 2d and Supplementary Fig. 7b). N. nepalensis had both 52.

(60) gene features in its genome explained why N. nepalensis can shift CB and SB among populations. N. nepalensis shared two gene sets, i.e., conserved orthologues and enriched Pfam genes (EPGs), with both breeding types. However, enrichment of GO terms revealed two gene sets had similar gene functions. CB-specific genes were mostly associated with regulation in the cellular level, e.g., ion transmembrane transport, cytoskeleton, and endocytosis (Supplementary Table 6 and 7). On the contrary, SB-specific genes were mainly related to development, metabolism, and response to stimulus (Supplementary Table 8 and 9). To be able to reproduce throughout a year, CBs needed to produce germ cells regularly and frequently in a year. We speculated this is the reason why CB-specific genes were mostly involved in the cellular process. SB only activated reproductive organs and produced secondary sex characteristics in certain seasons; therefore SB-specific genes were mostly related to development, metabolism, and response to stimulus. Gene-expression changes between seasonal and continuous breeding N. nepalensis populations To investigate if the CB- and SB-specific genes were differentially expressed between two breeding types of N. nepalensis, we generated transcriptomes from individuals of SB (north) and CB (central) populations in Taiwan (Supplementary Table 3). Previous studies showed the CB individuals would become sexually mature two weeks after emerging, but the ovaries of SB individuals would stay undeveloped two weeks after emerging while reared in the long day9. Therefore, we compared the 53.