物种：红带袖蝶(寡食性)&青衫黄袖蝶(多食性)
Interspecies mating between H. melpomene and H. cydno (while rare on a per individual basis) does occur and is generally observed to occur between H. melpomene males and H. cydno females. These crosses result in sterile females, but fertile F1 males that can back-cross with either species.
H. cydno is a host plant generalist and, in Central America, H. melpomene is a host plant specialist. These differences in mating and oviposition may cause broad patterns of differential expression between H. cydno and H. melpomene.
棉铃虫雌性(S)和烟青虫雄性(R)杂交只产生可育雄性， 但杂交成功率比较高， 且产后代数较多。
烟青虫雌性和棉铃虫雄性可杂交并产生可育后代, 二者性信息素组分比率差异的遗传学研巧提供了很好的实验材料， 但杂交成功率极低， 且产后代数量少。
Three sensory tissue types (antenna, legs, and mouthparts) and three biological groups (males, unmated females, and mated females).
With this design we were able to describe species-, sex-, and life-stage(mated vs. unmated) specific expression profiles that reflect important differences in mate and host plant choice for H. melpomene and H. cydno.
Chemosensory candidates are not in physical proximity to known wing-color pattern genes or other genomic regions associated with visual mate preference.
ORs and OBPs largely comprised antenna-specific genes, whereas CSPs, GRs, and OBPs generally comprised mouthpartand leg-specific genes.
Sex-based expression differences observed between males and females may stem from their different evolutionary needs to recognize odorant volatiles during mate choice and host plant selection.
Moreover, genes showing conserved sex-based differences may precede the speciation of H. melpomene and H. cydno, making them more likely to correspond to broad differences in sex-specific traits.
Differences in chemosensory gene expression between unmated and mated females might reflect a change in sensory priorities from ==mate==
==choice (in unmated females)== to ==host plant choice for oviposition (in mated females)==.
It is possible that this increased chemosensory expression corresponds to both ==host plant generalist behavior== and ==strict conspecific mate choice preferences== in H.cydno, behaviors that might require increasingly specialized and sensitive chemosensory discrimination genes.
Only one chemosensory gene, however, showed species level expression patterns consistent across biological groups and tissue type: HmOBP20 (q-value ≤ 0.001 for all comparisons). HmOBP20 was found to be overexpressed in H. melpomene antennae relative to H. cydno, and this pattern was observed in males, mated females, and unmated females (Fig. 5). As such, ==HmOBP20 presents a compelling candidate for species-specific recognition in H. melpomene.==
The most pronounced difference in expression is observed for HmGR56 in H. cydno male mouthparts. HmGR56 is a Heliconius-specific GR putatively involved in synephrine (a common plant alkaloid) recognition and may present an interesting candidate for host plant recognition.
We found that chemosensory genes occur as small clusters, were present on every chromosome, and were spread across the genome. These clustering patterns were in line with expectations for genes originating from duplications as per the birth–death model of gene family evolution.
While close proximity (within 1 Mb) could indicate that the chemosensory genes are physically linked to the wing-pattern genes (in linkage disequi librium, LD), none of these chemosensory genes were identified as candidates based on expression and admixture data.
Due to the association of color pattern with mate-preference behavior in H. melpomene and H. cydno, we specifically explored the levels of LD between our chemosensory genes (n = 252) and the optix color pattern interval.
连锁不平衡指的是在某一群体中，两个基因同时遗传的频率大于随机组合的频率。下面通过一个例子来说明。
基因A的两个allel 分别用A和a 表示，基因B的allel分别用B和b表示，如果这这两个基因完全独立遗传，也就是说其allel 完全随机组合，那么后代中会出现4种单倍型，AB， Ab, aB， ab, 而且出现的概率都是相同的，都是0.25；如果这两个基因在遗传时不是独立的，意味着后代中单倍型出现的概率不是完全随机的了，我们就可以说两个基因是存在连锁关系的，基因在遗传时出现连锁的现象就叫做连锁不平衡。 https://www.jianshu.com/p/477bdb7f57fa
Multiple studies support the importance of chemically mediated species boundaries, which indicate that species can differ in both the type and relative abundance of chemosensory genes that facilitate the detection of speciesspecific chemical cues, generally volatile odorants.
This increased chemosensory expression in H. cydno might, for example, confer males and females of this species a greater ability to distinguish conspecific mates or recognize a wider range of host plants.
This is particularly true for females, who select ovipositing sites in part by tasting the putative host plant surface with their forelegs, a behavior known as “drumming”. In agreement with these behavioral observations, we find that in H. cydno (a plant generalist) HmGR22, HmGR3, HmGR56, and HmGR5 are overexpressed in the legs and mouthparts of females.
We propose that HmOBP20 represents an independently evolved strategy for discrimination and reinforcement of species barriers in Heliconius. In addition to presenting strong species specific genomic signals, HmOBP20 is not physically linked with any of the classic color pattern genes (optix [chr18], WntA [chr10], and cortex [chr15]), nor is it linked to any of the previously identified male preference QTLs (chr1, chr17, chr18). Instead, it is located on a different chromosome (chr19).
Targeted resequencing is a variation of re-sequencing where only a small subset of the genome is sequenced, such as the exome, a particular chromosome, a set of genes or a region of interest. Focusing on a subset of the genome is generally done to reduce costs, although there may be certain cases, such as in a clinical setting, where it is important to sequence only particular regions. Also, by focusing all of the sequencing on a small region of the genome, it is possible to detect low levels of variation that might have otherwise been missed. Some researchers are starting to use targeted resequencing for genome-wide association studies (GWAS) instead of arrays as it is better suited for measuring rare alleles.
==Linkage Disequilibrium==
Chemosensory genes at the intercept of these two estimates are likely to be involved in species-specific processes. Finally, because the color pattern locus optix has been shown to be associated with mate preference behavior in H. melpomene and H. cydno, and linkage between chemosensory and color pattern genes could facilitate the forming of smell-based prezygotic barriers, we tested for the nonrandom association of chemosensory loci (50 kb around transcription start site) at and around the optix gene (1,000 kb region) in 50 kb windows. Pairwise comparisons of SNPs within each 50 kb window were averaged to produce a mean r2.
==Admixture Values (fd)==
The admixture values (f
d) are based on the ABBA-BABA test which measures an excess of derived allele sharing between the sympatric non-sister taxa compared to an allopatric population of H. melpomene from French Guiana. We included H. numata as an outgroup. Negative fdvalues indicate low admixture or sharing of derived alleles between the sympatric Panama populations of H. melpomene and H. cydno whereas positive fdvalues indicate high admixture. The fdvalues were calculated for 100 kb windows with a step size of 20 kb. For chemosensory genes overlapping windows were averaged.
[1]：van Schooten, B., Meléndez-Rosa, J., Van Belleghem, S.M., Jiggins, C.D., Tan, J.D., McMillan, W.O., Papa, R., 2020. Divergence of chemosensing during the early stages of speciation. Proc Natl Acad Sci USA 201921318. https://doi.org/10/gg4m6g.
[2]：https://www.jianshu.com/p/477bdb7f57fa
[3]：王琛柱. 从棉铃虫和烟青虫的种间杂交理解生物学物种概念[J]. Chinese Science Bulletin, 2006, 51: 2573–2575. DOI:10/gg4m6d.