Exon capture optimization in large-genome amphibians
Evan McCartney-Melstad, Genevieve G. Mount, H. Bradley Shaffer
Background Gathering genomic-scale data efficiently is challenging for non-model species with large, complex genomes. Transcriptome sequencing is accessible for even large-genome organisms, and sequence capture probes can be designed from such mRNA sequences to enrich and sequence exonic regions. Maximizing enrichment efficiency is important to reduce sequencing costs, but, relatively little data exist for exon capture experiments in large-genome non-model organisms. Here, we conducted a replicated factorial experiment to explore the effects of several modifications to standard protocols that might increase sequence capture efficiency for large-genome amphibians. Methods We enriched 53 genomic libraries from salamanders for a custom set of 8,706 exons under differing conditions. Libraries were prepared using pools of DNA from 3 different salamanders with approximately 30 gigabase genomes: California tiger salamander (Ambystoma californiense), barred tiger salamander (Ambystoma mavortium), and an F1 hybrid between the two. We enriched libraries using different amounts of c0t-1 blocker, individual input DNA, and total reaction DNA. Enriched libraries were sequenced with 150 bp paired-end reads on an Illumina HiSeq 2500, and the efficiency of target enrichment was quantified using unique read mapping rates and average depth across targets. The different enrichment treatments were evaluated to determine if c0t-1 and input DNA significantly impact enrichment efficiency in large-genome amphibians. Results Increasing the amounts of c0t-1 and individual input DNA both reduce the rates of PCR duplication. This reduction led to an increase in the percentage of unique reads mapping to target sequences, essentially doubling overall efficiency of the target capture from 10.4% to nearly 19.9%. We also found that post-enrichment DNA concentrations and qPCR enrichment verification were useful for predicting the success of enrichment. Conclusions Increasing the amount of individual sample input DNA and the amount of c0t-1 blocker both increased the efficiency of target capture in large-genome salamanders. By reducing PCR duplication rates, the number of unique reads mapping to targets increased, making target capture experiments more efficient and affordable. Our results indicate that target capture protocols can be modified to efficiently screen large-genome vertebrate taxa including amphibians.
Model Inadequacy and Mistaken Inferences of Trait-Dependent Speciation
Daniel L. Rabosky, Emma E. Goldberg
(Submitted on 22 Dec 2014)
Species richness varies widely across the tree of life, and there is great interest in identifying ecological, geographic, and other factors that affect rates of species proliferation. Recent methods for explicitly modeling the relationships among character states, speciation rates, and extinction rates on phylogenetic trees- BiSSE, QuaSSE, GeoSSE, and related models – have been widely used to test hypotheses about character state-dependent diversification rates. Here, we document the disconcerting ease with which neutral traits are inferred to have statistically significant associations with speciation rate. We first demonstrate this unfortunate effect for a known model assumption violation: shifts in speciation rate associated with a character not included in the model. We further show that for many empirical phylogenies, characters simulated in the absence of state-dependent diversification exhibit an even higher Type I error rate, indicating that the method is susceptible to additional, unknown model inadequacies. For traits that evolve slowly, the root cause appears to be a statistical framework that does not require replicated shifts in character state and diversification. However, spurious associations between character state and speciation rate arise even for traits that lack phylogenetic signal, suggesting that phylogenetic pseudoreplication alone cannot fully explain the problem. The surprising severity of this phenomenon suggests that many trait-diversification relationships reported in the literature may not be real. More generally, we highlight the need for diagnosing and understanding the consequences of model inadequacy in phylogenetic comparative methods.
Fitness costs in spatially structured environments
The clustering of individuals that results from limited dispersal is a double-edged sword: while it allows for local interactions to be mostly among related individuals, it also results in increased local competition. Here I show that, because they mitigate local competition, fitness costs such as reduced fecundity or reduced survival are less costly in spatially structured environments than in non spatial settings. I first present a simple demographic example to illustrate how spatial structure weakens selection against fitness costs. Then, I illustrate the importance of disentangling the evolution of a trait from the evolution of potential associated costs, using an example taken from a recent study investigating the effect of spatial structure on the evolution of host defence. In this example indeed, the differences between spatial and non-spatial selection gradients are entirely due to differences in the fitness costs, thereby undermining interpretations of the results made in terms of the trait only. This illustrates the need to consider fitness costs as proper traits in both theoretical and empirical studies.
An experimental test of the relationship between melanism and desiccation survival in insects
Subhash Rajpurohit, Lisa Marie Peterson, Andrew Orr, Anthony J. Marlon, Allen G Gibbs
We used experimental evolution to test the ?melanism-desiccation? hypothesis, which proposes that dark cuticle in several Drosophila species is an adaptation for increased desiccation tolerance. We selected for dark and light body pigmentation in replicated populations of D. melanogaster and assayed traits related to water balance. We also scored pigmentation and desiccation tolerance in populations selected for desiccation survival. Populations in both selection regimes showed large differences in the traits directly under selection. However, after over 40 generations of pigmentation selection, dark-selected populations were not more desiccation-tolerant than light-selected and control populations, nor did we find significant changes in carbohydrate amounts that could affect desiccation resistance. Body pigmentation of desiccation-selected populations did not differ from control populations after over 140 generations of selection. Our results do not support an important role for melanization in Drosophila water balance.
Spider web DNA: a new spin on noninvasive genetics of predator and prey
Charles Cong Yang Xu, Ivy J Yen, Dean Bowman, Cameron R. Turner
Noninvasive genetic approaches enable biomonitoring without the need to directly observe or disturb target organisms. Environmental DNA (eDNA) methods have recently extended this approach by assaying genetic material within bulk environmental samples without a priori knowledge about the presence of target biological material. This paper describes a novel and promising source of noninvasive spider DNA and insect eDNA from spider webs. Using black widow spiders (Latrodectus spp.) fed with house crickets (Acheta domesticus), we successfully extracted and amplified mitochondrial DNA sequences of both spider and prey from spider web. Detectability of spider DNA did not differ between assays with amplicon sizes from 135 to 497 base pairs. Spider DNA and prey eDNA remained detectable at least 88 days after living organisms were no longer present on the web. Spider web DNA may be an important tool in conservation research, pest management, biogeography studies, and biodiversity assessments.
Tools and Methods from the Anopheles 16 Genome Project
Aaron Steele, Michael C. Fontaine, Andres Martin, Scott J Emrich
The dramatic reduction in sequencing costs has resulted in many initiatives to sequence certain organisms and populations. These initiatives aim to not only sequence and assemble genomes but also to perform a more broader analysis of the population structure. As part of the Anopheline Genome Consortium, which has a vested interest in studying anpopheline mosquitoes, we developed novel methods and tools to further the communities goals. We provide a brief description of these methods and tools as well as assess the contributions that each offers to the broader study of comparative genomics.
Heterochrony explains convergent testis evolution in primates
Ekin Saglican, Ezgi Ozkurt, Haiyang Hu, Babur Erdem, Philipp Khaitovich, Mehmet Somel
The testis displays striking anatomical divergence among primates. Multi-male species, such as chimpanzees, have recurrently evolved large testicles relative to single-male species, such as humans. However, the developmental mechanisms behind testis divergence and whether they involve convergent molecular changes, have remained unknown. Through comparative analysis of transcriptomic data, we show that a species’ testis expression profile, like testis size, can be a reliable indicator of mating type among primates, and possibly murids. Differential expression, in turn, largely reflects changes in the relative proportions of somatic/pre-meiotic versus meiotic/post-meiotic cell types. By studying mouse and macaque testis development, we find that single-male species’ testis expression profiles are paedomorphic relative to multi-male species’ profiles. For instance, human and gorilla testis profiles resemble those of adolescent mice. Our results suggest that heterochronic shifts involving conserved transcription regulators have been repeatedly employed in primate evolution, leading to rapid, convergent changes in testis size and histology.
Protein folding and binding can emerge as evolutionary spandrels through structural coupling
Michael Manhart, Alexandre V Morozov
Binding interactions between proteins and other molecules mediate numerous cellular processes, including metabolism, signaling, and regulation of gene expression. These interactions evolve in response to changes in the protein’s chemical or physical environment (such as the addition of an antibiotic), or when genes duplicate and diverge. Several recent studies have shown the importance of folding stability in constraining protein evolution. Here we investigate how structural coupling between protein folding and binding — the fact that most proteins can only bind their targets when folded — gives rise to evolutionary coupling between the traits of folding stability and binding strength. Using biophysical and evolutionary modeling, we show how these protein traits can emerge as evolutionary “spandrels” even if they do not confer an intrinsic fitness advantage. In particular, proteins can evolve strong binding interactions that have no functional role but merely serve to stabilize the protein if misfolding is deleterious. Furthermore, such proteins may have divergent fates, evolving to bind or not bind their targets depending on random mutation events. These observations may explain the abundance of apparently nonfunctional interactions among proteins observed in high-throughput assays. In contrast, for proteins with both functional binding and deleterious misfolding, evolution may be highly predictable at the level of biophysical traits: adaptive paths are tightly constrained to first gain extra folding stability and then partially lose it as the new binding function is developed. These findings have important consequences for our understanding of fundamental evolutionary principles of both natural and engineered proteins.
Postmating reproductive barriers contribute to the incipient sexual isolation of US and Caribbean Drosophila melanogaster
Joyce Y Kao, Seana Lymer, Sea H Hwang, Albert Sung, Sergey V Nuzhdin
The nascent stages of speciation start with the emergence of sexual isolation. Understanding how reproductive barriers influence this evolutionary process is an ongoing effort. We present here a study of Drosophila melanogaster populations from the southeast United States and Caribbean islands undergoing incipient sexual isolation. The existence of premating reproductive barriers have been previously established, but they do not fully account for the degree of isolation present. To assess the influence of postmating barriers, we investigated putative postmating barriers of female remating and egg laying behavior, as well as hatchability of eggs laid and female longevity after mating. While we did not find any effects in female remating or egg laying, we did observe lower hatchability in the central region of our geographical spread as well as shorten female life spans after mating to genetically different males in females originating from the northern- and southernmost locations of those surveyed. These results serve as evidence that long-term consequences after mating such as the fitness of offspring and shortened lifespan have a stronger effect than short-term postmating behaviors.
Sex-biased expression in the Drosophila melanogaster group
Rebekah L. Rogers, Ling Shao, Jaleal S. Sanjak, Peter Andolfatto, Kevin R. Thornton
(Submitted on 1 Aug 2014)
Here, we provide revised gene models for D. ananassae, D. yakuba, and D. simulans, which include UTRs and empirically verified intron-exon boundaries, as well as ortholog groups identified using a fuzzy reciprocal-best-hit blast comparison. Using these revised annotations, we perform differential expression testing using the cufflinks suite to provide a broad overview of differential expression between reproductive tissues and the carcass. We identify thousands of genes that are differentially expressed across tissues in D. yakuba and D. simulans, with roughly 60% agreement in expression patterns of orthologs in D. yakuba and D. simulans. We identify several cases of putative polycistronic transcripts, pointing to a combination of transcriptional read-through in the genome as well as putative gene fusion and fission events across taxa. We furthermore identify hundreds of lineage specific genes in each species with no blast hits among transcripts of any other Drosophila species, which are candidates for neofunctionalized proteins and a potential source of genetic novelty.