Transcriptomic comparisons and allele specific expression reveals the electric signal duration divergence in radiative mormyrid electric fish evolution

Electric fishes have independently evolved six times. Most of these fish are weakly electric and they use their discharge mainly for orientation and communication. In the African weakly electric fish genus Campylomormyrus, electric organ discharge (EOD) signals are strikingly different in shape and duration among closely related species, they contribute to pre-zygotic isolation and may have triggered an adaptive radiation. We performed mRNA sequencing on electric organs (EOs) and skeletal muscle (SMs; from which the EOs derive) from three species with short (0.4 ms), medium (5 ms), and long (40 ms) EODs and two different cross-species hybrids. Using pairwise comparison of differential gene expression between EOs and SMs, we identified 1,444 up regulated genes in EO shared by all five species/hybrids cohorts, rendering them candidate genes for EO-specific properties in Campylomormyrus. To understand how gene expression contributes to the variation in EOD duration, we made cross species comparisons among species and tissue. We identified three types of EOD-duration-related expression patterns and several candidate genes, including KCNJ2, KLF5 and SLC24a2, their upregulation may contribute to increased EOD duration, along with a down-regulated gene KCNK6. Hybrids between a short (C. compressirostris) and a long (C. rhynchophorus) discharging species exhibit EODs of intermediate duration and showed imbalanced expression of KCNJ2 alleles. The preferential expression of the C. rhynchophorus allele is in line with a higher expression level in that parental species and points towards a cis-regulatory difference at this locus, relative to EOD duration. A further candidate gene, KLF5, is a transcription factor potentially balancing potassium channel gene expression, a crucial process for the formation of an EOD. Unraveling the genetic basis of the species-specific modulation of the EOD in Campylomormyrus is crucial for understanding the adaptive radiation of this emerging model taxon of ecological (perhaps even sympatric) speciation. Overall design: C. tshokwe samples were collected from Brazzaville, Congo river in 2012. Other samples were artifically bred and raised at the Univerrsity of Potsdam. Those fish were anesthetized by a lethal dose of clove oil, and dissected on cold 99% ethanol. Electric organ and skeletal muscle tissue from each specimen were flash frozen in liquid nitrogen, and further preserved in -80°C.电鱼（electric fishes）独立演化了六次。这类鱼类大多为弱电鱼（weakly electric fish），其放电主要用于定位与通讯。在非洲弱电鱼属弯吻电鱼属（Campylomormyrus）中，近缘物种间的电器官放电（electric organ discharge，简称EOD）信号在波形与时长上存在显著差异，这类信号参与合子前生殖隔离，甚至可能驱动了适应性辐射。我们对三种分别具有短时长（0.4毫秒）、中时长（5毫秒）和长时长（40毫秒）EOD的物种，以及两种不同的跨物种杂交个体的电器官（electric organs，简称EOs）和其同源组织骨骼肌（skeletal muscle，简称SMs）开展了mRNA测序（mRNA sequencing）。通过对EOs与SMs之间的差异基因表达进行两两比较，我们在全部5个物种/杂交组的EOs中鉴定出1444个上调基因，这些基因可作为弯吻电鱼属中EOs特异性性状的候选基因。 为探究基因表达如何影响EOD时长的变异，我们进行了物种间与组织间的跨物种比较分析。共鉴定出三类与EOD时长相关的表达模式，以及多个候选基因，包括KCNJ2、KLF5与SLC24a2，这些基因的上调可能会延长EOD时长；同时还发现一个下调基因KCNK6。短放电物种（C. compressirostris）与长放电物种（C. rhynchophorus）的杂交个体的EOD时长呈中间型，且其KCNJ2等位基因的表达存在失衡。C. rhynchophorus等位基因的优先表达与其亲本物种中更高的表达水平相符，这表明相对于EOD时长而言，该位点存在顺式调控差异。另一个候选基因KLF5属于转录因子，可能参与平衡钾通道基因的表达，而这一过程对EOD的形成至关重要。阐明弯吻电鱼属中物种特异性EOD调控的遗传基础，对于理解这一新兴的生态物种形成（甚至可能是同域物种形成）模式类群的适应性辐射具有关键意义。 实验整体设计：2012年，研究人员在刚果河流域的布拉柴维尔采集了C. tshokwe的样本；其余样本均在波茨坦大学人工繁育饲养。实验鱼经致死剂量的丁香油麻醉后，在预冷的99%乙醇中进行解剖。每只个体的电器官与骨骼肌组织均经液氮快速冷冻，随后保存于-80℃冰箱中。