Comparative analyses of bat genomes identify distinct evolution of immunity in Old World fruit bats.

Bats have been identified as natural reservoir hosts of several zoonotic viruses, prompting suggestions that they have unique immunological adaptations. Among bats, Old World fruit bats (Pteropodidae) have been linked to multiple spillovers. To test for lineage-specific molecular adaptations in these bats, we developed a new assembly pipeline to generate a reference-quality genome of the fruit bat Cynopterus sphinx and used this in comparative analyses of 12 bat species, including six pteropodids. Our results reveal that immunity-related genes have higher evolutionary rates in pteropodids than in other bats. Several lineage-specific genetic changes were shared across pteropodids, including the loss of NLRP1, duplications of PGLYRP1 and C5AR2, and amino acid replacements in MyD88. We introduced MyD88 transgenes containing Pteropodidae-specific residues into bat and human cell lines and found evidence of dampened inflammatory responses. By uncovering distinct immune adaptations, our results could help explain why pteropodids are frequently identified as viral hosts.

Evolution of bitter receptor genes and ontogenetic dietary shift in a frog.

Vertebrate Tas2r taste receptors detect bitter compounds that are potentially poisonous. Previous studies found substantial variation in the number of Tas2r genes across vertebrates, with some frog species carrying the largest number. Peculiar among vertebrates, frogs undergo metamorphosis, often associated with a dietary shift between tadpoles and adults. A possible explanation for the large size of frog Tas2r families could be that distinct sets of Tas2r genes are required for tadpoles and adults, suggesting differential expression of Tas2r genes between tadpoles and adults. To test this hypothesis, we first examined 20 amphibian genomes and found that amphibians generally possess more Tas2r genes than do other vertebrate clades. We next focused on the American bullfrog (Lithobates catesbeianus) to examine the expression of its Tas2r genes in herbivorous tadpoles and insectivorous adult frogs. We report that close to one fifth of its 180 Tas2r genes are differentially expressed (22 genes enriched in adults and 11 in tadpoles). Tuning properties were determined for a subset of differentially expressed genes by a cell-based functional assay, with the adult-enriched Tas2r gene set covering a larger range of ligands compared to the tadpole-enriched subset. These results suggest a role of Tas2r genes in the ontogenetic dietary shift of frogs and potentially initiate a new avenue of ontogenetic analysis of diet-related genes in the animal kingdom.

Local Adaptation of Bitter Taste and Ecological Speciation in a Wild Mammal.

Sensory systems are attractive evolutionary models to address how organisms adapt to local environments that can cause ecological speciation. However, tests of these evolutionary models have focused on visual, auditory, and olfactory senses. Here, we show local adaptation of bitter taste receptor genes in two neighboring populations of a wild mammal-the blind mole rat Spalax galili-that show ecological speciation in divergent soil environments. We found that basalt-type bitter receptors showed higher response intensity and sensitivity compared with chalk-type ones using both genetic and cell-based functional analyses. Such functional changes could help animals adapted to basalt soil select plants with less bitterness from diverse local foods, whereas a weaker reception to bitter taste may allow consumption of a greater range of plants for animals inhabiting chalk soil with a scarcity of food supply. Our study shows divergent selection on food resources through local adaptation of bitter receptors, and suggests that taste plays an important yet underappreciated role in speciation.

ACE2 receptor usage reveals variation in susceptibility to SARS-CoV and SARS-CoV-2 infection among bat species.

Bats are the suggested natural hosts for severe acute respiratory syndrome coronavirus (SARS-CoV) and the causal agent of the coronavirus disease 2019 (COVID-19) pandemic, SARS-CoV-2. The interaction of viral spike proteins with their host receptor angiotensin-converting enzyme 2 (ACE2) is a critical determinant of potential hosts and cross-species transmission. Here we use virus-host receptor binding and infection assays to examine 46 ACE2 orthologues from phylogenetically diverse bat species, including those in close and distant contact with humans. We found that 24, 21 and 16 of them failed to support infection by SARS-CoV, SARS-CoV-2 or both viruses, respectively. Furthermore, we confirmed that infection assays in human cells were consistent with those in two bat cell lines. Additionally, we used genetic and functional analyses to identify critical residues in bat ACE2 receptors associated with viral entry restrictions. Our results suggest that many bat species may not be the potential hosts of one or both viruses and that no correlation was identified between proximity to humans and probability of being natural hosts of SARS-CoV or SARS-CoV-2. This study demonstrates dramatic variation in susceptibility to SARS-CoV and SARS-CoV-2 infection among bat species and adds knowledge towards a better understanding of coronavirus-bat interaction.

Loss of sweet taste despite the conservation of sweet receptor genes in insectivorous bats.

The evolution of taste perception is usually associated with the ecology and dietary changes of organisms. However, the association between feeding ecology and taste receptor evolution is unclear in some lineages of vertebrate animals. One example is the sweet taste receptor gene Tas1r2 Previous analysis of partial sequences has revealed that Tas1r2 has undergone equally strong purifying selection between insectivorous and frugivorous bats. To test whether the sweet taste function is also important in bats with contrasting diets, we examined the complete coding sequences of both sweet taste receptor genes (Tas1r2 and Tas1r3) in 34 representative bat species. Although these two genes are highly conserved between frugivorous and insectivorous bats at the sequence level, our behavioral experiments revealed that an insectivorous bat (Myotis ricketti) showed no preference for natural sugars, whereas the frugivorous species (Rousettus leschenaultii) showed strong preferences for sucrose and fructose. Furthermore, while both sweet taste receptor genes are expressed in the taste tissue of insectivorous and frugivorous bats, our cell-based assays revealed striking functional divergence: the sweet taste receptors of frugivorous bats are able to respond to natural sugars whereas those of insectivorous bats are not, which is consistent with the behavioral preference tests, suggesting that functional evolution of sweet taste receptors is closely related to diet. This comprehensive study suggests that using sequence conservation alone could be misleading in inferring protein and physiological function and highlights the power of combining behavioral experiments, expression analysis, and functional assays in molecular evolutionary studies.

Molecular evolution and deorphanization of bitter taste receptors in a vampire bat.

Bats represent the largest dietary radiation in a single mammalian order, and have become an emerging model group for studying dietary evolution. Taste receptor genes have proven to be molecular signatures of dietary diversification in bats. For example, all 3 extant species of vampire bats have lost many bitter taste receptor genes (Tas2rs) in association with their dietary shift from insectivory to sanguivory. Indeed, only 8 full-length Tas2rs were identified from the high-quality genome of the common vampire bat (Desmodus rotundus). However, it is presently unknown whether these bitter receptors are functional, since the sense of taste is less important in vampire bats, which have an extremely narrow diet and rely on other senses for acquiring food. Here, we applied a molecular evolutionary analysis of Tas2rs in the common vampire bat compared with non-vampire bats. Furthermore, we provided the first attempt to deorphanize all bitter receptors of the vampire bat using a cell-based assay. We found that all Tas2r genes in the vampire bat have a level of selective pressure similar to that in non-vampire bats, suggesting that this species must have retained some bitter taste functions. We demonstrated that 5 of the 8 bitter receptors in the vampire bat can be activated by some bitter compounds, and observed that the vampire bat generally can not detect naturally occurring bitter compounds examined in this study. Our study demonstrates functional retention of bitter taste in vampire bats as suggested by cell-based functional assays, calling for an in-depth study of extra-oral functions of bitter taste receptors.

Three STIGMA AND STYLE STYLISTs Pattern the Fine Architectures of Apical Gynoecium and Are Critical for Male Gametophyte-Pistil Interaction.

The gynoecium is derived from the fusion of carpels and is considered to have evolved from a simple setup followed by adaptive adjustment in cell type and tissue distribution to facilitate efficient sexual reproduction [1, 2]. As a sequence of the adjustment, the apical gynoecium differentiates into a stigma and a style. Both the structural patterning and functional specification of the apical gynoecium are critical for plant fertility [3, 4]. However, how the fine structures of the apical gynoecium are established at the interface interacting with pollen and pollen tubes remain to be elucidated. Here, we report a novel angiosperm-specific gene family, STIGMA AND STYLE STYLIST 1-3 (SSS1, SSS2, and SSS3). The SSS1 expresses predominately in the transmitting tract tissue of style, SSS2 expresses intensively in stigma, and SSS3 expresses mainly in stylar peripheral region round the transmitting tract. SSSs coregulate the patterning of the apical gynoecium via controlling cell expansion or elongation. Both the architecture and function of apical gynoecium can be affected by the alteration of SSS expression, indicating their critical roles in the establishment of a proper female interface for communication with pollen tubes. The NGATHA3 (NGA3) transcription factor [5, 6] can directly bind to SSSs promoter and control SSSs expression. Overexpression of SSSs could rescue the stylar defect of nga1nga3 double mutant, indicating their context in the same regulatory pathway. Our findings reveal a novel molecular mechanism responsible for patterning the fine architecture of apical gynoecium and establishing a proper interface for pollen tube growth, which is therefore crucial for plant sexual reproduction.

Competitive oxidation and ubiquitylation on the evolutionarily conserved cysteine confer tissue-specific stabilization of Insig-2.

Insig-2 is an ER membrane protein negatively controlling lipid biosynthesis. Here, we find that Insig-2 is increased in the tissues, including liver, but unaltered in the muscle of gp78-deficient mice. In hepatocytes and undifferentiated C2C12 myoblasts, Insig-2 is ubiquitylated on Cys215 by gp78 and degraded. However, the C215 residue is oxidized by elevated reactive oxygen species (ROS) during C2C12 myoblasts differentiating into myotubes, preventing Insig-2 from ubiquitylation and degradation. The stabilized Insig-2 downregulates lipogenesis through inhibiting the SREBP pathway, helping to channel the carbon flux to ATP generation and protecting myotubes from lipid over-accumulation. Evolutionary analysis shows that the YECK (in which C represents Cys215 in human Insig-2) tetrapeptide sequence in Insig-2 is highly conserved in amniotes but not in aquatic amphibians and fishes, suggesting it may have been shaped by differential selection. Together, this study suggests that competitive oxidation-ubiquitylation on Cys215 of Insig-2 senses ROS and prevents muscle cells from lipid accumulation.

Convergent reduction of V1R genes in subterranean rodents.

BACKGROUND: Vomeronasal type 1 receptor genes (V1Rs) are expected to detect intraspecific pheromones. It is believed that rodents rely heavily on pheromonal communication mediated by V1Rs, but pheromonal signals are thought to be confined in subterranean rodents that live in underground burrows. Thus, subterranean rodents may show a contrasting mode of V1R evolution compared with their superterranean relatives. RESULTS: We examined the V1R evolution in subterranean rodents by analyzing currently available genomes of 24 rodents, including 19 superterranean and 5 subterranean species from three independent lineages. We identified a lower number of putatively functional V1R genes in each subterranean rodent (a range of 22-40) compared with superterranean species (a range of 63-221). After correcting phylogenetic inertia, the positive correlation remains significant between the small V1R repertoire size and the subterranean lifestyle. To test whether V1Rs have been relaxed from functional constraints in subterranean rodents, we sequenced 22 intact V1Rs in 29 individuals of one subterranean rodent (Spalax galili) from two soil populations, which have been proposed to undergo incipient speciation. We found 12 of the 22 V1Rs to show significant genetic differentiations between the two natural populations, indicative of diversifying selection. CONCLUSION: Our study demonstrates convergent reduction of V1Rs in subterranean rodents from three independent lineages. Meanwhile, it is noteworthy that most V1Rs in the two Spalax populations are under diversifying selection rather than relaxed selection, suggesting that functional constraints on these genes may have retained in some subterranean species.

Functional divergence of bitter taste receptors in a nectar-feeding bird.

Nectar may contain many secondary metabolites that are commonly toxic and bitter-tasting. It has been hypothesized that such bitter-tasting secondary metabolites might keep the nectar exclusive to only a few pollinators. To test this hypothesis, we examined functional changes of bitter taste receptor genes (Tas2rs) in a species of nectar-feeding bird (Anna's hummingbird) by comparing these genes with those from two closely related insect-feeding species (chimney swift and chuck-will's widow). We previously identified a larger number of Tas2rs in the hummingbird than in its close insectivorous relatives. In the present study, we demonstrate higher sensitivity and new functions in the hummingbird Tas2r gene copies generated by a lineage-specific duplication, which has been shaped by positive selection. These results suggest that the bitter taste may lead to increased sensitivities and specialized abilities of the hummingbird to detect bitter-tasting nectar. Moreover, this study potentially supports the hypothesis that bitter-tasting nectar may have been specialized for some pollinators, thus enforcing plant-pollinator mutualism.

Trehalase Gene as a Molecular Signature of Dietary Diversification in Mammals.

Diet is a key factor in determining and structuring animal diversity and adaptive radiations. The mammalian fossil record preserves phenotypic evidence of many dietary shifts, whereas genetic changes followed by dietary diversification in mammals remain largely unknown. To test whether living mammals preserve molecular evidence of dietary shifts, we examined the trehalase gene (Treh), which encodes an enzyme capable of digesting trehalose from insect blood, in bats and other mammals with diverse diets. Bats represent the largest dietary radiation among all mammalian orders, with independent origins of frugivory, nectarivory, carnivory, omnivory, and even sanguivory in an otherwise insectivorous clade. We found that Treh has been inactivated in unrelated bat lineages that independently radiated into noninsectivorous niches. Consistently, purifying selection has been markedly relaxed in noninsectivorous bats compared with their insectivorous relatives. Enzymatic assays of intestinal trehalase in bats suggest that trehalase activity tends to be lost or markedly reduced in noninsectivorous bats compared with their insectivorous relatives. Furthermore, our survey of Treh in 119 mammal species, which represent a deeper evolutionary timeframe, additionally identified a number of other independent losses of Treh in noninsectivorous species, recapitulating the evolutionary pattern that we found in bats. These results document a molecular record of dietary diversification in mammals, and suggest that such molecular signatures of dietary shifts would help us understand both historical and modern changes of animal diets.

Lineage-specific duplication and adaptive evolution of bitter taste receptor genes in bats.

By generating raw genetic material and diverse biological functions, gene duplication represents a major evolutionary mechanism that is of fundamental importance in ecological adaptation. The lineage-specific duplication events of bitter taste receptor genes (Tas2rs) have been identified in a number of vertebrates, but functional evolution of new Tas2r copies after duplication remains largely unknown. Here, we present the largest data set of bat Tas2rs to date, identified from existing genome sequences of 15 bat species and newly sequenced from 17 bat species, and demonstrate lineage-specific duplications of Tas2r16, Tas2r18 and Tas2r41 that only occurred in Myotis bats. Myotis bats are highly speciose and represent the only mammalian genus that is naturally distributed on every continent except Antarctica. The occupation of such diverse habitats might have driven the Tas2r gene expansion. New copies of Tas2rs in Myotis bats have shown molecular adaptation and functional divergence. For example, three copies of Tas2r16 in Myotis davidii showed differential sensitivities to arbutin and salicin that may occur in their insect prey, as suggested by cell-based functional assays. We hypothesize that functional differences among Tas2r copies in Myotis bats would increase their survival rate through preventing the ingestion of an elevated number of bitter-tasting dietary toxins from their insect prey, which may have facilitated their adaptation to diverse habitats. Our study demonstrates functional changes of new Tas2r copies after lineage-specific duplications in Myotis bats and highlights the potential role of taste perception in exploiting new environments.

Testing the sensory trade-off hypothesis in New World bats.

Detection of evolutionary shifts in sensory systems is challenging. By adopting a molecular approach, our earlier study proposed a sensory trade-off hypothesis between a loss of colour vision and an origin of high-duty-cycle (HDC) echolocation in Old World bats. Here, we test the hypothesis in New World bats, which include HDC echolocators that are distantly related to Old World HDC echolocators, as well as vampire bats, which have an infrared sensory system apparently unique among bats. Through sequencing the short-wavelength opsin gene (SWS1) in 16 species (29 individuals) of New World bats, we identified a novel SWS1 polymorphism in an HDC echolocator: one allele is pseudogenized but the other is intact, while both alleles are either intact or pseudogenized in other individuals. Strikingly, both alleles were found to be pseudogenized in all three vampire bats. Since pseudogenization, transcriptional or translational changes could separately result in functional loss of a gene, a pseudogenized SWS1 indicates a loss of dichromatic colour vision in bats. Thus, the same sensory trade-off appears to have repeatedly occurred in the two divergent lineages of HDC echolocators, and colour vision may have also been traded off against the infrared sense in vampire bats.

Transcriptome sequencing and phylogenetic analysis of four species of luminescent beetles.

The evolution of bioluminescence has prompted scientific attention to illuminate phylogenetic relationships of luminescent beetles. However, genomic resources are virtually lacking in rhagophthalmids (Rhagophthalmidae) and their related firefly beetles lampyrids (Lampyridae). Here, we employed the Illumina Hiseq 2000 platform and sequenced the whole-body transcriptomes of the four luminescent beetles: one rhagophthalmid (Rhagophthalmus sp.) and three fireflies (Asymmetricata circumdata, Aquatica ficta, and Pyrocoelia pectoralis). We obtained 55.4, 43.4, 38.6, and 36.7 million clean reads for the four species, respectively. All reads were assembled into contigs from which unigenes were derived. All unigenes were annotated by publicly available databases, and a total of 4325 orthologous genes were identified. Using multiple phylogenetic approaches, our transcriptome data confirmed the distinctiveness of Rhagophthalmidae from Lampyridae, which was also supported by our mitogenome analysis using three newly determined mitogenome sequences and 12 previously published ones. Together, this study is the first report of whole transcriptome sequencing data in Rhagophthalmidae and Lampyridae species, representing a valuable genomic resource for studying the origin and evolution of some remarkable traits in these beetles such as bioluminescence. Moreover, our transcriptome and mitogenome data provide useful phylogenetic information that could be of importance in future studies of phylogenetic inference.

Two-Step Functional Innovation of the Stem-Cell Factors WUS/WOX5 during Plant Evolution.

WUS and WOX5, which are expressed, respectively, in the organizing center (OC) and the quiescent center (QC), are essential for shoot/root apical stem-cell maintenance in flowering plants. However, little is known about how these stem-cell factors evolved their functions in flowering plants. Here, we show that the WUS/WOX5 proteins acquired two distinct capabilities by a two-step functional innovation process in the course of plant evolution. The first-step is the apical stem-cell maintenance activity of WUS/WOX5, which originated in the common ancestor of ferns and seed plants, as evidenced by the interspecies complementation experiments, showing that ectopic expression of fern Ceratopteris richardii WUS-like (CrWUL) surrounding OC/QC, or exclusive OC-/QC-expressed gymnosperms/angiosperms WUS/WOX5 in Arabidopsis wus-1 and wox5-1 mutants, could rescue their phenotypes. The second-step is the intercellular mobility that emerged in the common ancestor of seed plants after divergence from the ferns. Evidence for this includes confocal imaging of GFP fusion proteins, showing that WUS/WOX5 from seed plants, rather than from the fern CrWUL, can migrate into cells adjacent to the OC/QC. Evolutionary analysis showed that the WUS-like gene was duplicated into two copies prior to the divergence of gymnosperms/angiosperms. Then the two gene copies (WUS and WOX5) have undergone similar levels of purifying selection, which is consistent with their conserved functions in angiosperm shoot/root stem-cell maintenance and floral organ formation. Our results highlight the critical roles and the essential prerequisites that the two-step functional innovation of these genes performs and represents in the origin of flowering plants.

Sympatric speciation revealed by genome-wide divergence in the blind mole rat Spalax.

Sympatric speciation (SS), i.e., speciation within a freely breeding population or in contiguous populations, was first proposed by Darwin [Darwin C (1859) On the Origins of Species by Means of Natural Selection] and is still controversial despite theoretical support [Gavrilets S (2004) Fitness Landscapes and the Origin of Species (MPB-41)] and mounting empirical evidence. Speciation of subterranean mammals generally, including the genus Spalax, was considered hitherto allopatric, whereby new species arise primarily through geographic isolation. Here we show in Spalax a case of genome-wide divergence analysis in mammals, demonstrating that SS in continuous populations, with gene flow, encompasses multiple widespread genomic adaptive complexes, associated with the sharply divergent ecologies. The two abutting soil populations of S. galili in northern Israel habituate the ancestral Senonian chalk population and abutting derivative Plio-Pleistocene basalt population. Population divergence originated ∼0.2-0.4 Mya based on both nuclear and mitochondrial genome analyses. Population structure analysis displayed two distinctly divergent clusters of chalk and basalt populations. Natural selection has acted on 300+ genes across the genome, diverging Spalax chalk and basalt soil populations. Gene ontology enrichment analysis highlights strong but differential soil population adaptive complexes: in basalt, sensory perception, musculature, metabolism, and energetics, and in chalk, nutrition and neurogenetics are outstanding. Population differentiation of chemoreceptor genes suggests intersoil population's mate and habitat choice substantiating SS. Importantly, distinctions in protein degradation may also contribute to SS. Natural selection and natural genetic engineering [Shapiro JA (2011) Evolution: A View From the 21st Century] overrule gene flow, evolving divergent ecological adaptive complexes. Sharp ecological divergences abound in nature; therefore, SS appears to be an important mode of speciation as first envisaged by Darwin [Darwin C (1859) On the Origins of Species by Means of Natural Selection].

Sequence and organization of complete mitochondrial genome of the firefly, Aquatica leii (Coleoptera: Lampyridae).

The firefly Aquatica leii (Coleoptera: Lampyridae) is widely distributed in China. In this study, we sequenced and characterized the first complete mitochondrial genome of the firefly from the subfamily Luciolinae. The circular genome of 16,856 bp in length contains 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and a non-coding AT-rich region. Overall base composition of the genome is 42.28% A, 34.80% T, 13.91% C and 9.01% G, with an AT bias of 77.08%. All protein-coding genes start with an ATN codon, and terminate with the typical stop codon TAA, TAG or a single T. The non-coding AT-rich region is unusually long (2239 bp), containing six 113 bp tandem repeats and a microsatellite-like (TA)7 element. The genome sequence is useful for studying the evolution of sexual signaling and many ecological specializations in fireflies.