1. Introduction
Ever since the book “On the Origin of Species by Means of Natural Selection” was published by Charles Darwin 160 years ago (Darwin, 1859), adaptive evolution has become an important research question (Tian et al., 2017). Understanding the driving forces of speciation processes is a primary goal of evolutionary biology (Feldmeyer et al., 2015; Marie Curie et al., 2012). And inferring the patterns and processes that accompany the generation of phenotypic diversity and new species is a primary goal of evolutionary biology (Mason and Taylor, 2015). The development of high-throughput sequencing technology provided more genetic information of evolutionary history (Lemmon and Lemmon, 2013; Mccormack et al., 2013), adaptive traits (Ma et al., 2023; Sun et al., 2023; Yu et al., 2023), and the evolution of phenotypic diversity and speciation (Mason and Taylor, 2015). Thereinto transcriptome analysis has been widely used in the study to reveal the species evolutionary mechanisms (Dong et al., 2013; Hao et al., 2019; Koenig et al., 2013; Naumenko et al., 2017; Shao et al., 2015). Comparative evolutionary approaches can address whether interspecific differences are the result of neutral divergence over evolutionary history or whether they are the result of nonrandom processes, such as adaptation to different environmental conditions (Marra et al., 2014). Thus comparative transcriptomics was usually used to study the genetic basis of evolutionary differences among species.
Bats belong to the order Chiroptera. Due to the constant changes in external and internal factors during bat evolution, different bat species have evolved different adaptive mechanisms to adapt to the environment in the process of natural selection. Evolution is typically thought to proceed through the divergence of genes, proteins, and ultimately phenotypes (Clark et al., 2007; Hughes et al., 2010; Soskine and Tawfik, 2010). The changes in phenotype can reflect the changes in genes. The new phenotype or biological character of bats reflected the changes in the molecular level of genetic material under selective pressure fundamentally. Echolocation is an important phenotype in bats, and it is a complex phenotypic trait, which involves the production, reception, and auditory processing of ultrasonic pulses for obstacle avoidance, orientation, and hunting (Au and Simmons, 2007; Marianne et al., 2004). Rhinolophid bats possess the most sophisticated echolocation systems(Jones and Teeling, 2006), and detected the positive selection of auditory genes in the study of genome data (Dong et al., 2016). It revealed the adaptation of auditory sensory perception in the rhinolophid bat lineages (Dong et al., 2016; Wang et al., 2019; Zhao et al., 2019). Current studies into the evolution of bat acoustic have been conducted at the population level or in bats with different echolocation types, the molecular mechanisms underlying acoustic differences between closely related bat species are rare.
The Rhinolophus macrotis group, belonging to the family Rhinolophidea, is a specific species group among the genusRhinolophus . The macrotis group currently includes six species, namely R. episcopus ; R. siamensis ; R. rex ;R. osgoodi ; R. marshalli ; and R. schnitzleri(Liu et al., 2019; Zhang et al., 2018). These species are closely related species, and experienced recent and rapid diversification during the Pleistocene (Zhang et al., 2018). Compared with otherRhinolophus species, the echolocation frequencies for the species within the macrotis group exhibits differences except R. osgoodi , which were lower relative to their body size (Zhang et al., 2009). Although some acoustic related genes showed sequence convergence in echolocation bats (Davies et al., 2012; Li et al., 2008; Li et al., 2010; Liu et al., 2012; Liu et al., 2011), different echolocation characters among closely related species may indicated they have undergone different evolutionary processes, which may reflected at the genome level. Previous studies on morphology and behavior for this special phenomenon showed that both pinna and nasal capsule size were better predictors of echolocation call frequencies than forearm length (Wu et al., 2015). Studies using transcriptomics for R. episcopus and R. siamensis found differentially expressed genes relevant to the variation in echolocation frequency (Li et al., 2022a), and the expression variation related to acoustic signals (resting frequency) and body size (forearm length) was widely governed by natural selection (Li et al., 2022b). However, little is known about the molecular basis on gene sequences for this specific phenomenon, which will help us to understand the evolution mode for the auditory system in bats.
The species within the macrotis group are closely related withR. pusillus . Species in the macrotis group (exceptR. osgoodi ) have low frequencies relative to body size (specieslow), whereas R. osgoodi and R. pusillus emit echolocation with normal frequencies (speciesnormal). The difference in echolocation frequency provides the basis for understanding adaptations in their evolutionary history. The whole-genome sequence of these species is not available yet. Transcriptome analysis will help us to determine the molecular genetics for the adaptation of the macrotis group. In this study, we performed a comparative transcriptome analysis and obtained the sequence for multi-tissues of specieslowand speciesnormal for the first time, to (1) identify genes under positive selection and GO categories significantly enriched involved in the adaptation; (2) characterize the sequence differences between specieslow and speciesnormal; and (3) uncover the potential genetic mechanism underlying the adaptation for specieslow.