Adaptive Evolution of the Myo6 Gene in Old World Fruit Bats (Family: Pteropodidae)

Myosin VI (encoded by the Myo6 gene) is highly expressed in the inner and outer hair cells of the ear, retina, and polarized epithelial cells such as kidney proximal tubule cells and intestinal enterocytes. The Myo6 gene is thought to be involved in a wide range of physiological functions such as hearing, vision, and clathrin-mediated endocytosis. Bats (Chiroptera) represent one of the most fascinating mammal groups for molecular evolutionary studies of the Myo6 gene. A diversity of specialized adaptations occur among different bat lineages, such as echolocation and associated high-frequency hearing in laryngeal echolocating bats, large eyes and a strong dependence on vision in Old World fruit bats (Pteropodidae), and specialized high-carbohydrate but low-nitrogen diets in both Old World and New World fruit bats (Phyllostomidae). To investigate what role(s) the Myo6 gene might fulfill in bats, we sequenced the coding region of the Myo6 gene in 15 bat species and used molecular evolutionary analyses to detect evidence of positive selection in different bat lineages. We also conducted real-time PCR assays to explore the expression levels of Myo6 in a range of tissues from three representative bat species. Molecular evolutionary analyses revealed that the Myo6 gene, which was widely considered as a hearing gene, has undergone adaptive evolution in the Old World fruit bats which lack laryngeal echolocation and associated high-frequency hearing. Real-time PCR showed the highest expression level of the Myo6 gene in the kidney among ten tissues examined in three bat species, indicating an important role for this gene in kidney function. We suggest that Myo6 has undergone adaptive evolution in Old World fruit bats in relation to receptor-mediated endocytosis for the preservation of protein and essential nutrients.     

Cloning and Molecular Evolution of the Aldehyde Dehydrogenase 2 Gene (Aldh2) in Bats (Chiroptera)

Old World fruit bats (Pteropodidae) and New World fruit bats (Phyllostomidae) ingest significant quantities of ethanol while foraging. Mitochondrial aldehyde dehydrogenase (ALDH2, encoded by the Aldh2 gene) plays an important role in ethanol metabolism. To test whether the Aldh2 gene has undergone adaptive evolution in frugivorous and nectarivorous bats in relation to ethanol elimination, we sequenced part of the coding region of the gene (1,143 bp, ~73 % coverage) in 14 bat species, including three Old World fruit bats and two New World fruit bats. Our results showed that the Aldh2 coding sequences are highly conserved across all bat species we examined, and no evidence of positive selection was detected in the ancestral branches leading to Old World fruit bats and New World fruit bats. Further research is needed to determine whether other genes involved in ethanol metabolism have been the targets of positive selection in frugivorous and nectarivorous bats.     

Parallel Evolution of the Glycogen Synthase 1 (Muscle) Gene Gys1 Between Old World and New World Fruit Bats (Order: Chiroptera)

Glycogen synthase, which catalyzes the synthesis of glycogen, is especially important for Old World (Pteropodidae) and New World (Phyllostomidae) fruit bats that ingest high-carbohydrate diets. Glycogen synthase 1, encoded by the Gys1 gene, is the glycogen synthase isozyme that functions in muscles. To determine whether Gys1 has undergone adaptive evolution in bats with carbohydrate-rich diets, in comparison to insect-eating sister bat taxa, we sequenced the coding region of the Gys1 gene from 10 species of bats, including two Old World fruit bats (Pteropodidae) and a New World fruit bat (Phyllostomidae). Our results show no evidence for positive selection in the Gys1 coding sequence on the ancestral Old World and the New World Artibeus lituratus branches. Tests for convergent evolution indicated convergence of the sequences and one parallel amino acid substitution (T395A) was detected on these branches, which was likely driven by natural selection.     

Phosphoenolpyruvate Carboxykinase 1 Gene (Pck1) Displays Parallel Evolution between Old World and New World Fruit Bats

Bats are an ideal mammalian group for exploring adaptations to fasting due to their large variety of diets and because fasting is a regular part of their life cycle. Mammals fed on a carbohydrate-rich diet experience a rapid decrease in blood glucose levels during a fast, thus, the development of mechanisms to resist the consequences of regular fasts, experienced on a daily basis, must have been crucial in the evolution of frugivorous bats. Phosphoenolpyruvate carboxykinase 1 (PEPCK1, encoded by the Pck1 gene) is the rate-limiting enzyme in gluconeogenesis and is largely responsible for the maintenance of glucose homeostasis during fasting in fruit-eating bats. To test whether Pck1 has experienced adaptive evolution in frugivorous bats, we obtained Pck1 coding sequence from 20 species of bats, including five Old World fruit bats (OWFBs) (Pteropodidae) and two New World fruit bats (NWFBs) (Phyllostomidae). Our molecular evolutionary analyses of these sequences revealed that Pck1 was under purifying selection in both Old World and New World fruit bats with no evidence of positive selection detected in either ancestral branch leading to fruit bats. Interestingly, however, six specific amino acid substitutions were detected on the ancestral lineage of OWFBs. In addition, we found considerable evidence for parallel evolution, at the amino acid level, between the PEPCK1 sequences of Old World fruit bats and New World fruit bats. Test for parallel evolution showed that four parallel substitutions (Q276R, R503H, I558V and Q593R) were driven by natural selection. Our study provides evidence that Pck1 underwent parallel evolution between Old World and New World fruit bats, two lineages of mammals that feed on a carbohydrate-rich diet and experience regular periods of fasting as part of their life cycle.     

Adaptive evolution in the glucose transporter 4 gene Slc2a4 in Old World fruit bats (family: Pteropodidae)

Frugivorous and nectarivorous bats are able to ingest large quantities of sugar in a short time span while avoiding the potentially adverse side-effects of elevated blood glucose. The glucose transporter 4 protein (GLUT4) encoded by the Slc2a4 gene plays a critical role in transmembrane skeletal muscle glucose uptake and thus glucose homeostasis. To test whether the Slc2a4 gene has undergone adaptive evolution in bats with carbohydrate-rich diets in relation to their insect-eating sister taxa, we sequenced the coding region of the Slc2a4 gene in a number of bat species, including four Old World fruit bats (Pteropodidae) and three New World fruit bats (Phyllostomidae). Our molecular evolutionary analyses revealed evidence that Slc2a4 has undergone a change in selection pressure in Old World fruit bats with 11 amino acid substitutions detected on the ancestral branch, whereas, no positive selection was detected in the New World fruit bats. We noted that in the former group, amino acid replacements were biased towards either Serine or Isoleucine, and, of the 11 changes, six were specific to Old World fruit bats (A133S, A164S, V377F, V386I, V441I and G459S). Our study presents preliminary evidence that the Slc2a4 gene has undergone adaptive changes in Old World fruit bats in relation to their ability to meet the demands of a high sugar diet.     

The Glycogen Synthase 2 Gene (Gys2) Displays Parallel Evolution Between Old World and New World Fruit Bats

Frugivorous and nectarivorous bats rely largely on hepatic glycogenesis and glycogenolysis for postprandial blood glucose disposal and maintenance of glucose homeostasis during short time starvation, respectively. The glycogen synthase 2 encoded by the Gys2 gene plays a critical role in liver glycogen synthesis. To test whether the Gys2 gene has undergone adaptive evolution in bats with carbohydrate-rich diets in relation to their insect-eating sister taxa, we sequenced the coding region of the Gys2 gene in a number of bat species, including three Old World fruit bats (OWFBs) (Pteropodidae) and two New World fruit bats (NWFBs) (Phyllostomidae). Our results showed that the Gys2 coding sequences are highly conserved across all bat species we examined, and no evidence of positive selection was detected in the ancestral branches leading to OWFBs and NWFBs. Our explicit convergence test showed that posterior probabilities of convergence between several branches of OWFBs, and the NWFBs were markedly higher than that of divergence. Three parallel amino acid substitutions (Q72H, K371Q, and E666D) were detected among branches of OWFBs and NWFBs. Tests for parallel evolution showed that two parallel substitutions (Q72H and E666D) were driven by natural selection, while the K371Q was more likely to be fixed randomly. Thus, our results suggested that the Gys2 gene has undergone parallel evolution on amino acid level between OWFBs and NWFBs in relation to their carbohydrate metabolism.     

Old World fruit bats can be long-distance seed dispersers through extended retention of viable seeds in the gut

Seed dispersal and pollination by animals play a crucial role in the maintenance of forest ecosystems worldwide. Frugivorous bats are important pollen and seed dispersers in both the Palaeo- and Neotropics, and at least 300 plant species are known to rely on Old World fruit bats (Megachiroptera, Pteropodidae) for their propagation. However, rapid food transit times (generally less than 30 minutes) in frugivorous bats have been thought to limit their ability to disperse seeds to just a few tens of kilometres. Here we demonstrate regular daytime (greater than 12 hours) retention of food and viable fig seeds (Ficus, Moraceae) in the gut of the Old World fruit bat Cynopterus sphinx: a behaviour not previously reported for any frugivorous bat. Field observations indicate that this behaviour also occurs in other genera. Old World fruit bats are highly mobile and many species undertake considerable foraging and migration flights. Our findings indicate that Old World fruit bats have the potential to disperse small seeds hundreds of kilometres. This necessitates a reappraisal of their importance in transporting zoochorous seeds to remote areas and facilitating gene flow between isolated populations of plants, both within mainlands and across ocean barriers.     

Relation of fig fruit characteristics to fruit-eating bats in the New and Old World tropics

Abstract. Long-term studies of a Panamanian fig community have revealed that the figs separate into two major groups based on distinct patterns in fruit characteristics including fruit size, colour, scent and synchrony of ripening. Furthermore, these differences can be linked to sensory, morphological and behavioural capabilities of the figs' primary dispersers. One group of figs attracts primarily bats; the other group is visited mainly by birds. Whereas fruits of 'bat' figs span a wide range of size classes, ripen synchronously and remain green(ish) when ripe, all fruits of 'bird' figs have small fruit which ripen asynchronously and turn red when ripe. Among 'bat' figs, fruit size is correlated with body size of the bats that prefer them. Based on the consistent differences between 'bat' and 'bird' fig fruits in Panama we expect similar patterns in Old World figs. Furthermore, since fig-eating bats of the Old World differ in morphology, behaviour and sensory capabilities from fig-eating bats of the New World we speculate that these differences should be reflected in differences in fruit characteristics of Old and New World 'bat' figs. Personal observations and literature reports of Old World bats and figs are consistent with our predictions.     

Evolution of the ABPA Subunit of Androgen-Binding Protein Expressed in the Submaxillary Glands in New and Old World Rodent Taxa

The salivary androgen-binding proteins (ABPs) are members of the secretoglobin gene family present in mammals. Each ABP is a heterodimer assembled as an ABPA subunit encoded by an Abpa gene and linked by disulfide bridges to an ABPBG subunit encoded by an Abpbg gene. The ABP dimers are secreted into the saliva of mice and then transferred to the pelage after grooming and subsequently to the environment allowing an animal to mark territory with a biochemical signal. The putative role of the mouse salivary ABPs is that of pheromones mediating mate selection resulting in assortative mating in the Mus musculus species complex. We focused on comparing patterns of molecular evolution between the Abpa genes expressed in the submaxillary glands of species of New World and Old World muroids. We found that in both sets of rodents the Abpa genes expressed in the submaxillary glands appear to be evolving under a similar evolutionary regime, with relatively high nonsynonymous substitution rates, suggesting that ABP might play a similar biological role in both systems. Thus, ABP could be involved with mate recognition and species isolation in New World as well as Old World muroids.     

Sources of Assimilated Proteins in Old and New World Phytophagous Bats

Dietary proteins are considered crucial for growth and maintenance in mammals, but many fruit‐eating mammals feed largely on a protein‐poor diet. In the Chiroptera, frugivory has evolved twice, in the Old World Pteropodidae and in the New World Phyllostomidae, especially the Stenodermatinae. Recent studies based on the analysis of nitrogen isotope ratios (δ¹⁵N) suggest that phyllostomids feed to varying degrees on arthropods to meet their nitrogen (N) requirements. Arthropod feeding has rarely been observed in Pteropodidae. Thus, we asked whether pteropodids meet their N requirements by feeding exclusively on plant matter. We predicted that tissue from pteropodid wing membranes should be depleted in ¹⁵N relative to those of obligate insectivorous rhinolophoid, vespertilionid or emballonurid bats, if pteropodids acquire proteins exclusively from fruits, leaves or nectar. We found that δ¹⁵N in pteropodids were significantly lower than in obligate insectivorous bats. In addition, mean δ¹⁵N of Old World pteropodids was similar to that of obligate frugivorous stenodermatines of the New World tropics. We infer from these data that pteropodids are predominantly phytophagous bats. From a nutritional perspective, pteropodids and stenodermatines are very similar, suggesting that they share convergent physiological adaptations to compensate for the lack of dietary nitrogen.     

Bioinformatic Analysis Reveals Genome Size Reduction and the Emergence of Tyrosine Phosphorylation Site in the Movement Protein of New World Bipartite Begomoviruses

Begomovirus (genus Begomovirus, family Geminiviridae) infection is devastating to a wide variety of agricultural crops including tomato, squash, and cassava. Thus, understanding the replication and adaptation of begomoviruses has important translational value in alleviating substantial economic loss, particularly in developing countries. The bipartite genome of begomoviruses prevalent in the New World and their counterparts in the Old World share a high degree of genome homology except for a partially overlapping reading frame encoding the pre-coat protein (PCP, or AV2). PCP contributes to the essential functions of intercellular movement and suppression of host RNA silencing, but it is only present in the Old World viruses. In this study, we analyzed a set of non-redundant bipartite begomovirus genomes originating from the Old World (N = 28) and the New World (N = 65). Our bioinformatic analysis suggests ∼120 nucleotides were deleted from PCP’s proximal promoter region that may have contributed to its loss in the New World viruses. Consequently, genomes of the New World viruses are smaller than the Old World counterparts, possibly compensating for the loss of the intercellular movement functions of PCP. Additionally, we detected substantial purifying selection on a portion of the New World DNA-B movement protein (MP, or BC1). Further analysis of the New World MP gene revealed the emergence of a putative tyrosine phosphorylation site, which likely explains the increased purifying selection in that region. These findings provide important information about the strategies adopted by bipartite begomoviruses in adapting to new environment and suggest future in planta experiments.     

Multiple episodes of convergence in genes of the dim light vision pathway in bats

The molecular basis of the evolution of phenotypic characters is very complex and is poorly understood with few examples documenting the roles of multiple genes. Considering that a single gene cannot fully explain the convergence of phenotypic characters, we choose to study the convergent evolution of rod vision in two divergent bats from a network perspective. The Old World fruit bats (Pteropodidae) are non-echolocating and have binocular vision, whereas the sheath-tailed bats (Emballonuridae) are echolocating and have monocular vision; however, they both have relatively large eyes and rely more on rod vision to find food and navigate in the night. We found that the genes CRX, which plays an essential role in the differentiation of photoreceptor cells, SAG, which is involved in the desensitization of the photoactivated transduction cascade, and the photoreceptor gene RH, which is directly responsible for the perception of dim light, have undergone parallel sequence evolution in two divergent lineages of bats with larger eyes (Pteropodidae and Emballonuroidea). The multiple convergent events in the network of genes essential for rod vision is a rare phenomenon that illustrates the importance of investigating pathways and networks in the evolution of the molecular basis of phenotypic convergence.     

The evolution of echolocation in bats

Recent molecular phylogenies have changed our perspective on the evolution of echolocation in bats. These phylogenies suggest that certain bats with sophisticated echolocation (e.g. horseshoe bats) share a common ancestry with non-echolocating bats (e.g. Old World fruit bats). One interpretation of these trees presumes that laryngeal echolocation (calls produced in the larynx) probably evolved in the ancestor of all extant bats. Echolocation might have subsequently been lost in Old World fruit bats, only to evolve secondarily (by tongue clicking) in this family. Remarkable acoustic features such as Doppler shift compensation, whispering echolocation and nasal emission of sound each show multiple convergent origins in bats. The extensive adaptive radiation in echolocation call design is shaped largely by ecology, showing how perceptual challenges imposed by the environment can often override phylogenetic constraints.     

The role of frugivorous bats in tropical forest succession

Discussion of successional change has traditionally focused on plants. The role of animals in producing and responding to successional change has received far less attention. Dispersal of plant propagules by animals is a fundamental part of successional change in the tropics. Here we review the role played by frugivorous bats in successional change in tropical forests. We explore the similarities and differences of this ecological service provided by New and Old World seed-dispersing bats and conclude with a discussion of their current economic and conservation implications. Our review suggests that frugivorous New World phyllostomid bats play a more important role in early plant succession than their Old World pteropodid counterparts. We propose that phyllostomid bats have shared a long evolutionary history with small-seeded early successional shrubs and treelets while pteropodid bats are principally dispersers of the seeds of later successional canopy fruits. When species of figs (Ficus) are involved in the early stages of primary succession (e.g. in the river meander system in Amazonia and on Krakatau, Indonesia), both groups of bats are important contributors of propagules. Because they disperse and sometimes pollinate canopy trees, pteropodid bats have a considerable impact on the economic value of Old World tropical forests; phyllostomid bats appear to make a more modest direct contribution to the economic value of New World tropical forests. Nonetheless, because they critically influence forest regeneration, phyllostomid bats make an important indirect contribution to the economic value of these forests. Overall, fruit-eating bats play important roles in forest regeneration throughout the tropics, making their conservation highly desirable.     

Nectar-feeding bird and bat niches in two worlds: pantropical comparisons of vertebrate pollination systems

Aim We review several aspects of the structure of regional and local assemblages of nectar‐feeding birds and bats and their relationships with food plants to determine the extent to which evolutionary convergence has or has not occurred in the New and Old World tropics. Location Our review is pantropical in extent and also includes the subtropics of South Africa and eastern Australia. Within the tropics, it deals mostly with lowland forest habitats. Methods An extensive literature review was conducted to compile data bases on the regional and local species richness of nectar‐feeding birds and bats, pollinator sizes, morphology, and diets. Coefficients of variation (CVs) were used to quantify the morphospace occupied by the various families of pollinators. The extent to which plants have become evolutionarily specialized for vertebrate pollination was explored using several criteria: number and diversity of growth forms of plant families providing food for all the considered pollinator families; the most common flower morphologies visited by all the considered pollinator families; and the number of plant families that contain genera with both bird‐ and bat‐specialized species. Results Vertebrate pollinator assemblages in the New World tropics differ from those in the Old World in terms of their greater species richness, the greater morphological diversity of their most specialized taxa, and the greater degree of taxonomic and ecological diversity and morphological specialization of their food plants. Within the Old World tropics, Africa contains more specialized nectar‐feeding birds than Asia and Australasia; Old World nectar‐feeding bats are everywhere less specialized than their New World counterparts. Main conclusions We propose that two factors – phylogenetic history and spatio‐temporal predictability (STP) of flower resources – largely account for hemispheric and regional differences in the structure of vertebrate pollinator assemblages. Greater resource diversity and resource STP in the New World have favoured the radiation of small, hovering nectar‐feeding birds and bats into a variety of relatively specialized feeding niches. In contrast, reduced resource diversity and STP in aseasonal parts of Asia as well as in Australasia have favoured the evolution of larger, non‐hovering birds and bats with relatively generalized feeding niches. Tropical Africa more closely resembles the Neotropics than Southeast Asia and Australasia in terms of resource STP and in the niche structure of its nectar‐feeding birds but not its flower‐visiting bats.     

New World Bats Harbor Diverse Influenza A Viruses

Aquatic birds harbor diverse influenza A viruses and are a major viral reservoir in nature. The recent discovery of influenza viruses of a new H17N10 subtype in Central American fruit bats suggests that other New World species may similarly carry divergent influenza viruses. Using consensus degenerate RT-PCR, we identified a novel influenza A virus, designated as H18N11, in a flat-faced fruit bat (Artibeus planirostris) from Peru. Serologic studies with the recombinant H18 protein indicated that several Peruvian bat species were infected by this virus. Phylogenetic analyses demonstrate that, in some gene segments, New World bats harbor more influenza virus genetic diversity than all other mammalian and avian species combined, indicative of a long-standing host-virus association. Structural and functional analyses of the hemagglutinin and neuraminidase indicate that sialic acid is not a ligand for virus attachment nor a substrate for release, suggesting a unique mode of influenza A virus attachment and activation of membrane fusion for entry into host cells. Taken together, these findings indicate that bats constitute a potentially important and likely ancient reservoir for a diverse pool of influenza viruses.     

Molecular Evidence for an Old World Origin of Galapagos and Caribbean Band-Winged Grasshoppers (Acrididae: Oedipodinae: Sphingonotus)

Patterns of colonization and diversification on islands provide valuable insights into evolutionary processes. Due to their unique geographic position and well known history, the Galapagos Islands are an important model system for evolutionary studies. Here we investigate the evolutionary history of a winged grasshopper genus to infer its origin and pattern of colonization in the Galapagos archipelago. The grasshopper genus Sphingonotus has radiated extensively in the Palaearctic and many species are endemic to islands. In the New World, the genus is largely replaced by the genus Trimerotropis. Oddly, in the Caribbean and on the Galapagos archipelago, two species of Sphingonotus are found, which has led to the suggestion that these might be the result of anthropogenic translocations from Europe. Here, we test this hypothesis using mitochondrial and nuclear DNA sequences from a broad sample of Sphingonotini and Trimerotropini species from the Old World and New World. The genetic data show two distinct genetic clusters representing the New World Trimerotropini and the Old World Sphingonotini. However, the Sphingonotus species from Galapagos and the Caribbean split basally within the Old World Sphingonotini lineage. The Galapagos and Caribbean species appear to be related to Old World taxa, but are not the result of recent anthropogenic translocations as revealed by divergence time estimates. Distinct genetic lineages occur on the four investigated Galapagos Islands, with deep splits among them compared to their relatives from the Palaearctic. A scenario of a past wider distribution of Sphingonotus in the New World with subsequent extinction on the mainland and replacement by Trimerotropis might explain the disjunct distribution.     

A Novel Endogenous Betaretrovirus in the Common Vampire Bat (Desmodus rotundus) Suggests Multiple Independent Infection and Cross-Species Transmission Events

The Desmodus rotundus endogenous betaretrovirus (DrERV) is fixed in the vampire bat D. rotundus population and in other phyllostomid bats but is not present in all species from this family. DrERV is not phylogenetically related to Old World bat betaretroviruses but to betaretroviruses from rodents and New World primates, suggesting recent cross-species transmission. A recent integration age estimation of the provirus in some taxa indicates that an exogenous counterpart might have been in recent circulation.     

A Novel Hepacivirus with an Unusually Long and Intrinsically Disordered NS5A Protein in a Wild Old World Primate

GB virus B (GBV-B; family Flaviviridae, genus Hepacivirus) has been studied in New World primates as a model for human hepatitis C virus infection, but the distribution of GBV-B and its relatives in nature has remained obscure. Here, we report the discovery of a novel and highly divergent GBV-B-like virus in an Old World monkey, the black-and-white colobus (Colobus guereza), in Uganda. The new virus, guereza hepacivirus (GHV), clusters phylogenetically with GBV-B and recently described hepaciviruses infecting African bats and North American rodents, and it shows evidence of ancient recombination with these other hepaciviruses. Direct sequencing of reverse-transcribed RNA from blood plasma from three of nine colobus monkeys yielded near-complete GHV genomes, comprising two distinct viral variants. The viruses contain an exceptionally long nonstructural 5A (NS5A) gene, approximately half of which codes for a protein with no discernible homology to known proteins. Computational structure-based analyses indicate that the amino terminus of the GHV NS5A protein may serve a zinc-binding function, similar to the NS5A of other viruses within the family Flaviviridae. However, the 521-amino-acid carboxy terminus is intrinsically disordered, reflecting an unusual degree of structural plasticity and polyfunctionality. These findings shed new light on the natural history and evolution of the hepaciviruses and on the extent of structural variation within the Flaviviridae.