Background - The majority of DNA contained within vertebrate genomes is non-coding, with a certain proportion of this thought to play regulatory roles during development. Conserved Non-coding Elements (CNEs) are an abundant group of putative regulatory sequences that are highly conserved across divergent groups and are thus assumed to be under strong selective constraint. Many CNEs may contain regulatory factor binding sites, and their frequent spatial association with key developmental genes – such as those regulating the development of the sensory systems – suggests crucial roles in regulating gene expression and hence cellular patterning. Yet surprisingly little is known about the molecular evolution of CNEs across diverse mammalian taxa or their role in specific phenotypic adaptations. We examined 3,110 vertebrate-specific and ~82,000 mammalian-specific CNEs across 19 and 9 mammalian orders respectively, and tested for changes in the rate of evolution of CNEs located in the proximity of genes underlying the development or functioning of auditory systems. As we focused on CNEs putatively associated with genes underlying the development/functioning of the auditory systems, we incorporated several echolocating taxa in our dataset because of their highly specialised and derived auditory systems. Results - Phylogenetic reconstructions of concatenated CNE sequences broadly recovered the accepted mammal relationships despite high levels of sequence conservation. We found that CNE substitution rates were highest in rodents and lowest in primates, consistent with previous findings. Comparisons of substitution rates in CNEs from several genomic regions containing genes linked to auditory system development and hearing genes revealed differences between echolocating and non-echolocating taxa. Wider taxonomic sampling of four CNEs associated with the homeobox genes Hmx2 and Hmx3 ¬ – which are required for mammalian inner ear development – revealed family-wise variation across diverse bat species. Specifically within one family of echolocating bats, known to utilise frequency-modulated echolocation calls varying widely in frequency and intensity, high levels of sequence divergence were found. Conclusions - Levels of selective constraint acting on CNEs differed both across genomic locations and taxa, with observed variation in substitution rates of CNEs among bat species. More work in needed to determine whether this variation can be linked to echolocation, and wider taxonomic sampling is necessary to fully document levels of conservation in CNEs across diverse taxa.

Concatenated alignments of vertebrate-specific CNEs for 89 genomic regions based on data from 26 mammalian genomesVert_specCNEs.zipAlignments of four conserved non-coding elements located in the same genomic region as HMX2/3Alignments of four conserved non-coding elements located in the same genomic region as HMX2/3 in divergent mammalian species. Sequences were either taken from online sources, such as Ensembl, or sequenced directly using PCR and Sanger sequencing.HMX2_3.zip