Recent findings of sequence convergence in the Prestin gene among some bats and cetaceans suggest that parallel adaptations for high-frequency hearing have taken place during the evolution of echolocation. To determine if this gene is an exception, or instead similar processes have occurred in other hearing genes, we have examined Tmc1 and Pjvk, both of which are associated with non-syndromic hearing loss in mammals. These genes were amplified and sequenced from a number of mammalian species, including echolocating and non-echolocating bats and whales, and were analysed together with published sequences. Sections of both genes showed phylogenetic signals that conflicted with accepted species relationships, with coding regions uniting laryngeal echolocating bats in a monophyletic clade. Bayesian estimates of posterior probabilities of convergent and divergent substitutions provided more direct evidence of sequence convergence between the two groups of laryngeal echolocating bats as well as between echolocating bats and dolphins. We found strong evidence of positive selection acting on some echolocating bat species and echolocating cetaceans, contrasting with purifying selection on non-echolocating bats. Signatures of sequence convergence and molecular adaptation in two additional hearing genes suggest that the acquisition of high-frequency hearing has involved multiple loci.

The increased accessibility of soft-tissue data through diffusible iodine-based contrast-enhanced computed tomography (diceCT) enables comparative biologists to increase the taxonomic breadth of their studies with museum specimens. However, it is still unclear how soft-tissue measurements from preserved specimens reflect values from freshly collected specimens and whether diceCT preparation may affect these measurements. Here, we document and evaluate the accuracy of diceCT in museum specimens based on the soft-tissue reconstructions of brains and eyes of five bats. Based on proxies, both brains and eyes were roughly 60% of the estimated original sizes when first imaged. However, these structures did not further shrink significantly over a 4-week staining interval, and 1 week in 2.5% iodine-based solution yielded sufficient contrast for differentiating among soft-tissues. Compared to six "fresh" bat specimens imaged shortly after field collection (not fixed in ethanol), the museum specimens had significantly lower relative volumes of the eyes and brains. Variation in field preparation techniques and conditions, and long-term storage in ethanol may be the primary causes of shrinkage in museum specimens rather than diceCT staining methodology. Identifying reliable tissue-specific correction factors to adjust for the shrinkage now documented in museum specimens requires future work with larger samples.

Diversification and adaptive radiations are tied to evolvability, which in turn is linked to morphological integration. Tightly integrated structures typically evolve in unison, whereas loosely integrated structures evolve separately. Highly integrated structures are therefore thought to constrain evolutionary change by limiting morphological disparity. Mounting evidence suggests that high integration may facilitate evolutionary change along a single trajectory. We used geometric morphometrics to compare cranial disparity and integration among phyllostomid bats-which exhibit the greatest dietary diversity of any mammalian family-and their sister taxa within the superfamily Noctilionoidea. Our results reveal that phyllostomids are more tightly integrated and have less disparity in cranial shape than their outgroups, despite exhibiting tenfold higher species richness and significantly increased rates of speciation. Phyllostomid cranial morphology appears to have diverged from that of other noctilionoids by evolving along a single axis of morphological variation that describes the relative length of the rostrum. We propose that phyllostomids were able to evolve to occupy a wide range of dietary niches by varying rostrum length, possibly along a line of least evolutionary resistance. This study provides a compelling empirical example of how increased integration can lead to adaptation, implying that both high and low integration can underlie diverse phenotypes in adaptive radiation.

During their evolutionary radiation, mammals have colonized diverse habitats. Arguably the subterranean niche is the most inhospitable of these, characterized by reduced oxygen, elevated carbon dioxide, absence of light, scarcity of food, and a substrate that is energetically costly to burrow through. Of all lineages to have transitioned to a subterranean niche, African mole-rats are one of the most successful. Much of their ecological success can be attributed to a diet of plant storage organs, which has allowed them to colonize climatically varied habitats across sub-Saharan Africa, and has probably contributed to the evolution of their diverse social systems. Yet despite their many remarkable phenotypic specializations, little is known about molecular adaptations underlying these traits. To address this, we sequenced the transcriptomes of seven mole-rat taxa, including three solitary species, and combined new sequences with existing genomic data sets. Alignments of more than 13,000 protein-coding genes encompassed, for the first time, all six genera and the full spectrum of ecological and social variation in the clade. We detected positive selection within the mole-rat clade and along ancestral branches in approximately 700 genes including loci associated with tumorigenesis, aging, morphological development, and sociality. By combining these results with gene ontology annotation and protein–protein networks, we identified several clusters of functionally related genes. This family wide analysis of molecular evolution in mole-rats has identified a suite of positively selected genes, deepening our understanding of the extreme phenotypic traits exhibited by this group.

The loss of previously adaptive traits is typically linked to relaxation in selection, yet the molecular steps leading to such repeated losses are rarely known. Molecular studies of loss have tended to focus on gene sequences alone, but overlooking other aspects of protein expression might underestimate phenotypic diversity. Insights based almost solely on opsin gene evolution, for instance, have made mammalian color vision a textbook example of phenotypic loss. We address this gap by investigating retention and loss of opsin genes, transcripts, and proteins across ecologically diverse noctilionoid bats. We find multiple, independent losses of short-wave-sensitive opsins. Mismatches between putatively functional DNA sequences, mRNA transcripts, and proteins implicate transcriptional and post-transcriptional processes in the ongoing loss of S-opsins in some noctilionoid bats. Our results provide a snapshot of evolution in progress during phenotypic trait loss, and suggest vertebrate visual phenotypes cannot always be predicted from genotypes alone.

IntroductionMany mammals have evolved highly adapted hearing associated with ecological specialisation. Of these, bats possess the widest frequency range of vocalisations and associated hearing sensitivities, with frequencies of above 200 kHz in some lineages that use laryngeal echolocation. High frequency hearing in bats appears to have evolved via structural modifications of the inner ear, however, studying these minute features presents considerable challenges and hitherto few such attempts have been made. To understand these adaptations more fully, as well as gain insights into the evolutionary origins of ultrasonic hearing and echolocation in bats, we undertook micro-computed tomography (μCT) scans of the cochleae of representative bat species from 16 families, encompassing their broad range of ecological diversity. To characterise cochlear gross morphology, we measured the relative basilar membrane length and number of turns, and compared these values between echolocating and non-echolocating bats, as well as other mammals.ResultsWe found that hearing and echolocation call frequencies in bats correlated with both measures of cochlear morphology. In particular, relative basilar membrane length was typically longer in echolocating species, and also correlated positively with the number of cochlear turns. Ancestral reconstructions of these parameters suggested that the common ancestor of all extant bats was probably capable of ultrasonic hearing; however, we also found evidence of a significant decrease in the rate of morphological evolution of the basilar membrane in multiple ancestral branches within the Yangochiroptera suborder. Within the echolocating Yinpterochiroptera, there was some evidence of an increase in the rate of basilar membrane evolution in some tips of the tree, possibly associated with reported shifts in call frequency associated with recent speciation events.ConclusionsThe two main groups of echolocating bat were found to display highly variable inner ear morphologies. Ancestral reconstructions and rate shift analyses of ear morphology point to a complex evolutionary history, with the former supporting ultrasonic hearing in the common bat ancestor but the latter suggesting that morphological changes associated with echolocation might have occurred later. These findings are consistent with theories that sophisticated laryngeal echolocation, as seen in modern lineages, evolved following the divergence of the two main suborders.

Among mammals, several lineages have independently adapted to a subterranean niche and possess similar phenotypic traits for burrowing (e.g., cylindrical bodies, short limbs, and absent pinnae). Previous research on mole-rats has revealed molecular adaptations for coping with reduced oxygen, elevated carbon dioxide, and the absence of light. In contrast, almost nothing is known regarding molecular adaptations in other subterranean lineages (e.g., true moles and golden moles). Therefore, the extent to which the recurrent phenotypic adaptations of divergent subterranean taxa have arisen via parallel routes of molecular evolution remains untested. To address these issues, we analyzed ∼8,000 loci in 15 representative subterranean taxa of four independent transitions to an underground niche for signatures of positive selection and convergent amino acid substitutions. Complementary analyses were performed in nonsubterranean “control” taxa to assess the biological significance of results. We found comparable numbers of positively selected genes in each of the four subterranean groups; however, correspondence in terms of gene identity between gene sets was low. Furthermore, we did not detect evidence of more convergent amino acids among subterranean species pairs compared with levels found between nonsubterranean controls. Comparisons with nonsubterranean taxa also revealed loci either under positive selection or with convergent substitutions, with similar functional enrichment (e.g., cell adhesion, immune response, and coagulation). Given the limited indication that positive selection and convergence occurred in the same loci, we conclude that selection may have acted on different loci across subterranean mammal lineages to produce similar phenotypes.

The vestibular system maintains the body’s sense of balance and, therefore, was probably subject to strong selection during evolutionary transitions in locomotion. Among mammals, bats possess unique traits that place unusual demands on their vestibular systems. First, bats are capable of powered flight, which in birds is associated with enlarged semicircular canals. Second, many bats have enlarged cochleae associated with echolocation, and both cochleae and semicircular canals share a space within the petrosal bone. To determine how bat vestibular systems have evolved in the face of these pressures, we used micro-CT scans to compare canal morphology across species with contrasting flight and echolocation capabilities. We found no increase in canal radius in bats associated with the acquisition of powered flight, but canal radius did correlate with body mass in bat species from the suborder Yangochiroptera, and also in non-echolocating Old World fruit bats from the suborder Yinpterochiroptera. No such trend was seen in members of the Yinpterochiroptera that use laryngeal echolocation, although canal radius was associated with wing-tip roundedness in this group. We also found that the vestibular system scaled with cochlea size, although the relationship differed in species that use constant frequency echolocation. Across all bats, the shape of the anterior and lateral canals was associated with large cochlea size and small body size respectively, suggesting differential spatial constraints on each canal depending on its orientation within the skull. Thus in many echolocating bats, it seems that the combination of small body size and enlarged cochlea together act as a principal force on the vestibular system. The two main groups of echolocating bats displayed different canal morphologies, in terms of size and shape in relation to body mass and cochlear size, thus suggesting independent evolutionary pathways and offering tentative support for multiple acquisitions of echolocation.