tissue-collection-of-bats-for-omics-analyses-and-primary-cell-culture

Tissue Collection of Bats for -Omics Analyses and Primary Cell Culture

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of ...

Yale University

Stony Brook University

Biodiversity patterns in Asia are poorly understood due to inferences drawn from incorrect taxonomy and limited survey effort. The Streak-breasted Scimitar Babblers (Pomatorhinus ruficollis complex) have a wide distribution across southern Asia and exhibit a high degree of plumage variability within and among populations. Continued use of traditional subspecies designations over revised species-limits based on plumage and DNA sequence data furthers confusion in this group and obscures complex biogeographic patterns. In this study, we combined previously published DNA sequences with newly sampled populations to produce a comprehensive dataset for the ruficollis complex. Phylogenetic analysis of these data confirms that traditional subspecies based on plumage alone are paraphyletic and therefore not good descriptors of evolutionary history. With increased sampling, our study supported previous delimitations of phylogenetic species as distinct units, refined the range limits of two taxa - P. reconditus (throughout central China) and P. nigrostellatus (Hainan, Guangxi, N Vietnam), showed two additional clades that may be distinct species, and uncovered a 'suture' zone where populations of multiple species occur in the same localities. Diversification within the ruficollis complex indicates a clade of Sino-Himalayan and SE Asian species sister to a clade distributed in central and southern China species. The 'suture' zone where different ruficollis species are in contact coincides with the meeting of these four major geographic areas in a highly geomorphologically complex region. 

Untangling taxonomic confusion and diversification patterns of the Streak-breasted Scimitar Babblers (Timaliidae: Pomatorhinus ruficollis complex) in southern Asia.

The earliest record of plant visiting in bats dates to the Middle Miocene of La Venta, the world's most diverse tropical palaeocommunity. Palynephyllum antimaster is known from molars that indicate nectarivory. Skull length, an important indicator of key traits such as body size, bite force and trophic specialization, remains unknown. We developed Bayesian models to infer skull length based on dental measurements. These models account for variation within and between species, variation between clades, and phylogenetic error structure. Models relating skull length to trophic level for nectarivorous bats were then used to infer the diet of the fossil. The skull length estimate for Palynephyllum places it among the larger lonchophylline bats. The inferred diet suggests Palynephyllum fed on nectar and insects, similar to its living relatives. Omnivory has persisted since the mid-Miocene. This is the first study to corroborate with fossil data that highly specialized nectarivory in bats requires an omnivorous transition. 

Bayesian hierarchical models suggest oldest known plant-visiting bat was omnivorous.

All behaviors of an organism are rooted in sensory processing of signals from its environment, and natural selection shapes sensory adaptations to ensure successful detection of cues that maximize fitness. Sensory drive, or divergent selection for efficient signal transmission among heterogeneous environments, has been a useful hypothesis for describing sensory adaptations, but its current scope has primarily focused on visual and acoustic sensory modalities. Chemosensation, the most widespread sensory modality in animals that includes the senses of smell and taste, is characterized by rapid evolution and has been linked to sensory adaptations to new environments in numerous lineages. Yet, olfaction and gustation have been largely underappreciated in light of the sensory drive hypothesis. Here, we examine why chemosensory systems have been overlooked and discuss the potential of chemosensation to shed new insight on the sensory drive hypothesis and vice versa. We provide suggestions for developing a framework to better incorporate studies of chemosensory adaptation that have the potential to shape a more complete, coherent, and holistic interpretation of the sensory drive.

Evolutionary ecology of chemosensation and its role in sensory drive.

Gene duplication is an important process in the evolution of gene content in eukaryotic genomes. Understanding when gene duplicates contribute new molecular functions to genomes through molecular adaptation is one important goal in comparative genomics. In large gene families, however, characterizing adaptation and neofunctionalization across species is challenging, as models have traditionally quantified the timing of duplications without considering underlying gene trees. This protocol combines multiple approaches to detect adaptation in protein duplicates at a phylogenetic scale. We include a description of models for gene tree-species tree reconciliation that enable different types of inference, as well as a practical guide to their use. Although simulation-based approaches successfully detect shifts in the rate of duplication/retention, the conflation between the duplication and retention processes, the distinct trajectories of duplicates under non-, sub-, and neofunctionalization, as well as dosage effects offer hitherto unexplored analytical avenues. We introduce mathematical descriptions of these probabilities and offer a road map to computational implementation whose starting point is parsimony reconciliation. Sequence evolution information based on the ratio of nonsynonymous to synonymous nucleotide substitution rates (dN/dS) can be combined with duplicate survival probabilities to better predict the emergence of new molecular functions in retained duplicates. Together, these methods enable characterization of potentially adaptive candidate duplicates whose neofunctionalization may contribute to phenotypic divergence across species.

Protocols for the Molecular Evolutionary Analysis of Membrane Protein Gene Duplicates.

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.

Multifactorial processes underlie parallel opsin loss in neotropical bats.

In mammals, social and reproductive behaviors are mediated by chemical cues encoded by hyperdiverse families of receptors expressed in the vomeronasal organ. Between species, the number of intact receptors can vary by orders of magnitude. However, the evolutionary processes behind variation in receptor number, and its link to fitness-related behaviors are not well understood. From vomeronasal transcriptomes, we discovered the first evidence of intact vomeronasal type-1 receptor (V1r) genes in bats, and we tested whether putatively functional bat receptors were orthologous to those of related taxa, or whether bats have evolved novel receptors. Instead of lineage-specific duplications, we found that bat V1rs show high levels of orthology to those of their relatives, and receptors are under comparative levels of purifying selection as non-bats. Despite widespread vomeronasal organ loss in bats, V1r copies have been retained for >65 million years. The highly conserved nature of bat V1rs challenges our current understanding of mammalian V1r function and suggests roles other than conspecific recognition or mating initiation in social behavior.

Expressed Vomeronasal Type-1 Receptors (V1rs) in Bats Uncover Conserved Sequences Underlying Social Chemical Signaling.

Multigene families evolve from single-copy ancestral genes via duplication, and typically encode proteins critical to key biological processes. Molecular analyses of these gene families require high-confidence sequences, but the high sequence similarity of the members can create challenges for sequencing and downstream analyses. Focusing on the common vampire bat, Desmodus rotundus, we evaluated how different sequencing approaches performed in recovering the largest mammalian protein-coding multigene family: olfactory receptors (OR). Using the genome as a reference, we determined the proportion of intact protein-coding receptors recovered by: (a) amplicons from degenerate primers sequenced via Sanger technology, (b) RNA-Seq of the main olfactory epithelium, and (c) those genes captured with probes designed from transcriptomes of closely-related species. Our initial re-annotation of the high-quality vampire bat genome resulted in >400 intact OR genes, more than doubling the original estimate. Sanger-sequenced amplicons performed the poorest among the three approaches, detecting <33% of receptors in the genome. In contrast, the transcriptome reliably recovered >50% of the annotated genomic ORs, and targeted sequence capture recovered nearly 75% of annotated genes. Each sequencing approach assembled high-quality sequences, even if it did not recover all receptors in the genome. While some variation may be due to limitations of the study design (e.g., different individuals), variation among approaches was mostly caused by low coverage of some receptors rather than high rates of assembly error. Given this variability, we caution against using the counts of intact receptors per species to model the birth-death process of multigene families. Instead, our results support the use of orthologous sequences to explore and model the evolutionary processes shaping these genes.

Evaluating the performance of targeted sequence capture, RNA-Seq, and degenerate-primer PCR cloning for sequencing the largest mammalian multigene family.

Laurel R. Yohe

Department of Geology & Geophysics

Yale University

Tissue Collection of Bats for -Omics Analyses and Primary Cell Culture