Evolution Of Novel Phenotypes Research Articles

OrthoPhyl - Streamlining large scale, orthology-based phylogenomic studies of bacteria at broad evolutionary scales.

There are a staggering number of publicly available bacterial genome sequences (at writing, 2.0 million assemblies in NCBI's GenBank alone), and the deposition rate continues to increase. This wealth of data begs for phylogenetic analyses to place these sequences within an evolutionary context. A phylogenetic placement not only aids in taxonomic classification, but informs the evolution of novel phenotypes, targets of selection, and horizontal gene transfer. Building trees from multi-gene codon alignments is a laborious task that requires bioinformatic expertise, rigorous curation of orthologs, and heavy computation. Compounding the problem is the lack of tools that can streamline these processes for building trees from large scale genomic data. Here we present OrthoPhyl, which takes bacterial genome assemblies and reconstructs trees from whole genome codon alignments. The analysis pipeline can analyze an arbitrarily large number of input genomes (>1200 tested here) by identifying a diversity spanning subset of assemblies and using these genomes to build gene models to infer orthologs in the full dataset. To illustrate the versatility of OrthoPhyl, we show three use-cases: E. coli/Shigella, Brucella/Ochrobactrum, and the order Rickettsiales. We compare trees generated with OrthoPhyl to trees generated with kSNP3 and GToTree along with published trees using alternative methods. We show that OrthoPhyl trees are consistent with other methods while incorporating more data, allowing for greater numbers of input genomes, and more flexibility of analysis.

Decision-making and challenges within the evolving treatment algorithm in spinal muscular atrophy: a clinical perspective

ABSTRACT Introduction The clinical application of disease modifying therapies has dramatically changed the paradigm of the management of people with spinal muscular atrophy (SMA), from sole reliance on symptomatic care directed toward the downstream consequences of muscle weakness, to proactive intervention and even preventative care. Areas covered In this perspective, the authors evaluate the contemporary therapeutic landscape of SMA and discuss the evolution of novel phenotypes and the treatment algorithm, including the key factors that define individual treatment choice and treatment response. The benefits achieved by early diagnosis and treatment through newborn screening are highlighted, alongside an appraisal of emerging prognostic methods and classification frameworks to inform clinicians, patients, and families about disease course, manage expectations, and improve care planning. A future perspective of unmet needs and challenges is provided, emphasizing the key role of research. Expert opinion SMN-augmenting therapies have improved health outcomes for people with SMA and powered the practice of personalized medicine. Within this new proactive diagnostic and treatment paradigm, new phenotypes and different disease trajectories are emerging. Ongoing collaborative research efforts to understand the biology of SMA and define optimal response are critical to refining future approaches.

Conserved Molecular Players Involved in Human Nose Morphogenesis Underlie Evolution of the Exaggerated Snout Phenotype in Cichlids.

Instances of repeated evolution of novel phenotypes can shed light on the conserved molecular mechanisms underlying morphological diversity. A rare example of an exaggerated soft tissue phenotype is the formation of a snout flap in fishes. This tissue flap develops from the upper lip and has evolved in one cichlid genus from Lake Malawi and one genus from Lake Tanganyika. To investigate the molecular basis of snout flap convergence, we used mRNA sequencing to compare two species with snout flap to their close relatives without snout flaps from each lake. Our analysis identified 201 genes that were repeatedly differentially expressed between species with and without snout flap in both lakes, suggesting shared pathways, even though the flaps serve different functions. Shared expressed genes are involved in proline and hydroxyproline metabolism, which have been linked to human skin and facial deformities. Additionally, we found enrichment for transcription factor binding sites at upstream regulatory sequences of differentially expressed genes. Among the enriched transcription factors were members of the FOX transcription factor family, especially foxf1 and foxa2, which showed an increased expression in the flapped snout. Both of these factors are linked to nose morphogenesis in mammals. We also found ap4 (tfap4), a transcription factor showing reduced expression in the flapped snout with an unknown role in craniofacial soft tissue development. As genes involved in cichlid snout flap development are associated with human midline facial dysmorphologies, our findings hint at the conservation of genes involved in midline patterning across distant evolutionary lineages of vertebrates, although further functional studies are required to confirm this.

My road to the ants: A model clade for eco-evo-devo.

This chapter is the story of how I pioneered ants as a system for studying eco-evo-devo, a field that integrates developmental biology with ecology and evolutionary biology. One aim of eco-evo-devo is to understand how the interactions between genes and their environments during development facilitates the origin and evolution of novel phenotypes. In a series of six parts, I review some of the key discoveries from my lab on how novel worker caste systems in ants--soldiers and supersoldiers--originated and evolved. I also discuss some of the ideas that emerged from these discoveries, including the role that polyphenisms, hidden developmental potentials, and rudimentary organs play in facilitating developmental and evolutionary change. As superorganisms, I argue that ants are uniquely positioned to reveal types of variation that are often difficult to observe in nature. In doing so, they have the potential to transform our view of biology and provide new perspectives in medicine, agriculture, and biodiversity conservation. With my story I hope to inspire the next generation of biologists to continue exploring the unknown regions of phenotypic space to solve some of our most pressing societal challenges.

Rapid Genomic Evolution Drives the Diversification of Male Reproductive Genes in Dung Beetles.

The molecular basis for the evolution of novel phenotypes is a central question in evolutionary biology. In recent years, dung beetles have emerged as models for novel trait evolution as they possess distinct precopulatory traits such as sexually dimorphic horns on their head and thorax. Here, we use functional and evolutionary genomics to investigate the origins and the evolution of postcopulatory reproductive traits in male dung beetles. Male ejaculates that underlie postcopulatory sexual selection are excellent candidates to study novel trait evolution as they are complex, fast evolving, and often highly divergent in insects. We assemble de novo transcriptomes of male accessory glands and testes of a widespread dung beetle, Catharsius molossus, and we perform an evolutionary analysis of closely and distantly related insect genomes. Our results show there is rapid innovation at the genomic level even among closely related dung beetles. Genomic expansion and contraction drive the divergence of male reproductive traits and their functions. The birth of scores of completely novel reproductive genes is reinforced by the recruitment of these genes for high expression in male reproductive tissues, especially in the accessory glands. We find that male accessory glands of C. molossus are specialized for secretory function and express female, egg, and embryo-related genes as well as serine protease inhibitors, whilst the testes are specialized for spermatogenesis and sperm function. Finally, we touch upon putative functions of these evolutionary novelties using structure-function analysis as these proteins bear no homology to any other known proteins.

Enhanced Outcrossing, Directional Selection and Transgressive Segregation Drive Evolution of Novel Phenotypes in Hybrid Swarms of the Dutch Elm Disease Pathogen Ophiostoma novo-ulmi.

In the 1970s, clones of the two subspecies of Ophiostoma novo-ulmi, subsp. americana (SSAM) and subsp. novo-ulmi (SSNU) began to overlap in Europe, resulting in hybrid swarms. By 1983–1986, hybrids with high, SSAM-like growth and pathogenic fitness comprised ~75% of popula-tions at Limburg, Netherlands and Orvieto, Italy. We resampled these populations in 2008 to examine trends in hybrid fitness traits. Since preliminary sampling in 1979–1980, MAT-1 locus frequency had increased from ~0% to ~32% at Orvieto and 5% to ~43% at Limburg, and vegeta-tive incompatibility type frequency had changed from near clonal to extremely diverse at both sites. This represents an enormous increase in outcrossing and recombination potential, due in part to selective acquisition (under virus pressure) of MAT-1 and vic loci from the resident O. ulmi and in part to SSAM × SSNU hybridisation. Overt virus infection in the 2008 samples was low (~4%), diagnostic SSAM and SSNU cu and col1 loci were recombinant, and no isolates exhib-ited a parental SSAM or SSNU colony pattern. At both sites, mean growth rate and mean patho-genicity to 3–5 m clonal elm were high SSAM-like, indicating sustained directional selection for these characters, though at Orvieto growth rate was slower. The once frequent SSNU-specific up-mut colony dimorphism was largely eliminated at both sites. Perithecia formed by Limburg isolates were mainly an extreme, long-necked SSNU-like form, consistent with transgressive segregation resulting from mismatch of SSAM and SSNU developmental loci. Orvieto isolates produced more parental-like perithecia, suggesting the extreme phenotypes may have been se-lected against. The novel phenotypes in the swarms are remodelling O. novo-ulmi in Europe. Locally adapted genotypes may emerge.

A two-step PCR assembly for construction of gene variants across large mutational distances.

Construction of empirical fitness landscapes has transformed our understanding of genotype–phenotype relationships across genes. However, most empirical fitness landscapes have been constrained to the local genotype neighbourhood of a gene primarily due to our limited ability to systematically construct genotypes that differ by a large number of mutations. Although a few methods have been proposed in the literature, these techniques are complex owing to several steps of construction or contain a large number of amplification cycles that increase chances of non-specific mutations. A few other described methods require amplification of the whole vector, thereby increasing the chances of vector backbone mutations that can have unintended consequences for study of fitness landscapes. Thus, this has substantially constrained us from traversing large mutational distances in the genotype network, thereby limiting our understanding of the interactions between multiple mutations and the role these interactions play in evolution of novel phenotypes. In the current work, we present a simple but powerful approach that allows us to systematically and accurately construct gene variants at large mutational distances. Our approach relies on building-up small fragments containing targeted mutations in the first step followed by assembly of these fragments into the complete gene fragment by polymerase chain reaction (PCR). We demonstrate the utility of our approach by constructing variants that differ by up to 11 mutations in a model gene. Our work thus provides an accurate method for construction of multi-mutant variants of genes and therefore will transform the studies of empirical fitness landscapes by enabling exploration of genotypes that are far away from a starting genotype.

Genetic assimilation and accommodation: Models and mechanisms.

Genetic assimilation and genetic accommodation are mechanisms by which novel phenotypes are produced and become established in a population. Novel characters may be fixed and canalized so they are insensitive to environmental variation, or can be plastic and adaptively responsive to environmental variation. In this review we explore the various theories that have been proposed to explain the developmental origin and evolution of novel phenotypes and the mechanisms by which canalization and phenotypic plasticity evolve. These theories and models range from conceptual to mathematical and have taken different views of how genes and environment contribute to the development and evolution of the properties of phenotypes. We will argue that a deeper and more nuanced understanding of genetic accommodation requires a recognition that phenotypes are not static entities but are dynamic system properties with no fixed deterministic relationship between genotype and phenotype. We suggest a mechanistic systems-view of development that allows one to incorporate both genes and environment in a common model, and that enables both quantitative analysis and visualization of the evolution of canalization and phenotypic plasticity.

Can gene-inactivating mutations lead to evolutionary novelty?

Evolutionary novelty is difficult to define. It typically involves shifts in organismal or biochemical phenotypes that can be seen as qualitative as well as quantitative changes. In laboratory-based experimental evolution of novel phenotypes and the human domestication of crops, the majority of the mutations that lead to adaptation are loss-of-function mutations that impair or eliminate the function of genes rather than gain-of-function mutations that increase or qualitatively alter the function of proteins. Here, I speculate that easier access to loss-of-function mutations has led them to play a major role in the adaptive radiations that occur when populations have access to many unoccupied ecological niches. I discuss five possible objections to this claim: that genes can only survive if they confer benefits to the organisms that bear them, antagonistic pleiotropy, the importance of pre-existing genetic variation in populations, the danger that adaptation by breaking genes will, over long times, cause organisms to run out of genes, and the recessive nature of most loss-of-function mutations.

Caste-specific morphological modularity in the ant tribe Camponotini (Hymenoptera, Formicidae)

BackgroundThe morphological structures of organisms form tightly integrated but mutually independent character complexes (modules) linked through common development and function. Even though their abundance, diversity, and complex caste systems make camponotine ants ideal subjects to research developmental modularity and phenotypic integration, no studies investigating these phenomena have been conducted in this taxonomic group. This study attempts to identify and visualize integrated character complexes in 14 taxa from the genera Camponotus and Colobopsis using statistical analyses of morphometry.ResultsThe identified modules differ between castes: Minor workers have small heads and long extremities, while major workers have enlarged heads modified for defence, and short, thick appendages; extremities (legs and antennae) are strongly correlated in both worker castes. Gynes show weaker integration of extremities, but a strong correlation of mesosoma and eyes, and highly variable median ocellus size. Gynes infested by mermithid nematodes exhibit reduction of gyne-specific characters and altered patterns of phenotypic integration.ConclusionThe integrated character complexes described herein can largely be interpreted as functional, caste-specific modules related to behavioural ecology and task allocation within ant colonies. This modular nature of the body plan is hypothesized to facilitate the evolution of novel phenotypes and thus contributes to the tremendous evolutionary success of ants. The study of these modules can help to further elucidate the evolution and ontogeny of castes in camponotine ants, as well as the effects of parasite infestation on the phenotype.

Selection for novel metabolic capabilities in Salmonella enterica.

Bacteria are known to display extensive metabolic diversity and many studies have shown that they can use an extensive repertoire of small molecules as carbon‐ and energy sources. However, it is less clear to what extent a bacterium can expand its existing metabolic capabilities by acquiring mutations that, for example, rewire its metabolic pathways. To investigate this capability and potential for evolution of novel phenotypes, we sampled large populations of mutagenized Salmonella enterica to select very rare mutants that can grow on minimal media containing 124 low molecular weight compounds as sole carbon sources. We found mutants growing on 18 of these novel carbon sources, and identified the causal mutations that allowed growth for four of them. Mutations that relieve physiological constraints or increase expression of existing pathways were found to be important contributors to the novel phenotypes. For the remaining 14 novel phenotypes, whole genome sequencing of independent mutants and genetic analysis suggested that these novel metabolic phenotypes result from a combination of multiple mutations. This work, by virtue of identifying the genetic and mechanistic basis for new metabolic capabilities, sheds light on the properties of adaptive landscapes underlying the evolution of novel phenotypes.

Evolutionary shifts in anti-predator responses of invasive cane toads (Rhinella marina)

A potential prey item’s response to encountering a predator depends on aspects of the predator (e.g., its locomotor capacity), the local environment (e.g., proximity to shelter) and the physiological state of the prey item, but in addition, anti-predator tactics also vary geographically among different populations within wide-ranging species. Using standardised trials, we tested responses of cane toads (Rhinella marina) to being placed on a laboratory runway and encouraged to flee. Overall, the toads least capable of rapid locomotion were the ones most likely to respond to simulated predator attack by exuding toxins rather than attempting to escape. A toad’s willingness to move down the runway and its propensity to exude toxin from the parotoid glands rather than fleeing were repeatable in successive trials and influenced by the animal’s location of origin, morphology, previous experience, and parentage. We found that specimens from Australia were more willing to flee than were those from the native range (French Guiana) or Hawai’i; larger toads, and those with relatively longer legs, were more willing to flee; captive-raised toads were less willing to flee and more willing to exude toxin. Captive-raised offspring resembled their wild-caught parents both in propensity to run and in propensity to exude toxin. Thus, geographic divergence among cane toad populations in anti-predator responses reflects a complex combination of processes, including both developmental plasticity and heritability. The process of colonising novel environments exposes an organism to a host of different predators that exert selection on morphology, behaviour and physiology. Over time, this can lead to the evolution of novel phenotypes that are adapted to local conditions. Anthropogenically facilitated biological invasions provide a unique opportunity to study how species respond to colonisation of new environments. Here, we studied geographic variation in cane toad (Rhinella marina) anti-predator behaviours from a range of locations (including invasive and native populations) using standardised trials. Our results suggest that invasive Australian cane toads have undergone a shift in their anti-predator response, becoming more willing to flee than are conspecifics from other areas. Location of origin, morphology, prior experience and parentage all played a role in shaping a toad’s anti-predator response, suggesting that the behaviour represents a combination of plastic and heritable variation.

Viviparity stimulates diversification in an order of fish.

Species richness is distributed unevenly across the tree of life and this may be influenced by the evolution of novel phenotypes that promote diversification. Viviparity has originated ∼150 times in vertebrates and is considered to be an adaptation to highly variable environments. Likewise, possessing an annual life cycle is common in plants and insects, where it enables the colonization of seasonal environments, but rare in vertebrates. The extent to which these reproductive life-history traits have enhanced diversification and their relative importance in the process remains unknown. We show that convergent evolution of viviparity causes bursts of diversification in fish. We built a phylogenetic tree for Cyprinodontiformes, an order in which both annualism and viviparity have arisen, and reveal that while both traits have evolved multiple times, only viviparity played a major role in shaping the patterns of diversity. These results demonstrate that changes in reproductive life-history strategy can stimulate diversification.

Large-Scale Coding Sequence Change Underlies the Evolution of Postdevelopmental Novelty in Honey Bees.

Whether coding or regulatory sequence change is more important to the evolution of phenotypic novelty is one of biology’s major unresolved questions. The field of evo–devo has shown that in early development changes to regulatory regions are the dominant mode of genetic change, but whether this extends to the evolution of novel phenotypes in the adult organism is unclear. Here, we conduct ten RNA-Seq experiments across both novel and conserved tissues in the honey bee to determine to what extent postdevelopmental novelty is based on changes to the coding regions of genes. We make several discoveries. First, we show that with respect to novel physiological functions in the adult animal, positively selected tissue-specific genes of high expression underlie novelty by conferring specialized cellular functions. Such genes are often, but not always taxonomically restricted genes (TRGs). We further show that positively selected genes, whether TRGs or conserved genes, are the least connected genes within gene expression networks. Overall, this work suggests that the evo–devo paradigm is limited, and that the evolution of novelty, postdevelopment, follows additional rules. Specifically, evo–devo stresses that high network connectedness (repeated use of the same gene in many contexts) constrains coding sequence change as it would lead to negative pleiotropic effects. Here, we show that in the adult animal, the converse is true: Genes with low network connectedness (TRGs and tissue-specific conserved genes) underlie novel phenotypes by rapidly changing coding sequence to perform new-specialized functions.

Genetic variability in reaction norms in fishes

The ability of populations to adapt to environmental change and the spatial scale at which this adaptation occurs are fundamentally important issues in evolutionary biology, and ones that may benefit greatly from the study of genetic variability in reaction norms, which represent the plasticity of phenotypic traits across an environmental gradient. Therefore variable reaction norms can reflect genetic differences in the ability of individuals, families, populations, and species to respond to natural and anthropogenic environmental change. Fishes are ideal organisms in which to study plasticity because of their remarkable intraspecific morphological, physiological, behavioural, and life history variation. Here, we review studies demonstrating genetic variability in reaction norms in fishes. Genetic variability in plasticity among full- and half-sib families suggests potential for some populations to develop an adaptive norm of reaction (recalling that plasticity need not be adaptive). Reaction norm variability among populations suggests that adaptive genetic divergence can occur rapidly when selection pressures are strong and that the spatial scale of adaptation is much smaller than previously believed for some species with high dispersal capabilities. These studies demonstrate the potential of using reaction norms to study the evolution of novel phenotypes and the influence of temporal environmental variability and gene flow on the evolution of phenotypic plasticity, which can then be used to predict how populations will respond to directional environmental change. To promote future research into genetic variability in reaction norms, we propose questions that would benefit from such an approach and discuss some important considerations for designing experiments to investigate questions related to genetic variation in plasticity and phenotypic evolution.

Large-scale coding sequence change underlies the evolution of postdevelopmental novelty in honey bees.

Whether coding or regulatory sequence change is more important to the evolution of phenotypic novelty is one of biology's major unresolved questions. The field of evo-devo has shown that in early development changes to regulatory regions are the dominant mode of genetic change, but whether this extends to the evolution of novel phenotypes in the adult organism is unclear. Here, we conduct ten RNA-Seq experiments across both novel and conserved tissues in the honey bee to determine to what extent postdevelopmental novelty is based on changes to the coding regions of genes. We make several discoveries. First, we show that with respect to novel physiological functions in the adult animal, positively selected tissue-specific genes of high expression underlie novelty by conferring specialized cellular functions. Such genes are often, but not always taxonomically restricted genes (TRGs). We further show that positively selected genes, whether TRGs or conserved genes, are the least connected genes within gene expression networks. Overall, this work suggests that the evo-devo paradigm is limited, and that the evolution of novelty, postdevelopment, follows additional rules. Specifically, evo-devo stresses that high network connectedness (repeated use of the same gene in many contexts) constrains coding sequence change as it would lead to negative pleiotropic effects. Here, we show that in the adult animal, the converse is true: Genes with low network connectedness (TRGs and tissue-specific conserved genes) underlie novel phenotypes by rapidly changing coding sequence to perform new-specialized functions.

Peripheral Isolate Speciation of a Lake Malawi Cichlid Fish from Shallow Muddy Habitats

Much of the exceptional diversity of cichlid fishes in the African Great Lakes can be explained by geographic variation among isolated populations of species specialised to live on patchily distributed rocky habitat. However, there are also many endemic species that are not specialised for rocky shores. These appear to experience weaker geographic isolation. Major decreases in lake volume may have segregated such populations in isolated refugia in the distant past, but subsequent range changes have likely eliminated most of the phylogeographic signal of these events. Divergence in currently isolated peripheral water bodies may be more amenable to the study of recent processes of allopatric divergence. We investigate a haplochromine cichlid fish, here referred to as Lethrinops sp. ‘chilingali’, isolated in a small satellite lake near Lake Malawi, and the candidate sister taxon, Lethrinops lethrinus, which inhabits shallow muddy habitats in the main lake and associated water bodies. The satellite lake form from Lake Chilingali showed significant morphological differentiation, with a less ventrally-placed mouth and shorter snout, associated with a shift in diet from a diverse range of benthic invertebrates towards specialisation on mid-water chaoborid larvae and pupae. The Lake Chilingali population showed substantially reduced mitochondrial DNA diversity and no haplotype sharing was observed with populations from the main lake system. In laboratory experiments, putative species showed a high degree of assortative mating and territorial males were significantly more aggressive towards intruders of their own population. This study adds to the evidence that rapid evolution of novel phenotypes in peripheral habitats can add to the diversity of lacustrine cichlids through the evolution of at least partial reproductive isolation in allopatry.

Extensive Translational Regulation of Gene Expression in an Allopolyploid (Glycine dolichocarpa)

All flowering plants have experienced repeated rounds of polyploidy (whole-genome duplication), which has in turn driven the evolution of novel phenotypes and ecological tolerances and been a major driver of speciation. The effects of polyploidy on gene expression have been studied extensively at the level of transcription and, to a much lesser extent, at the level of the steady state proteome, but not at the level of translation. We used polysome profiling by RNA-Seq to quantify translational regulation of gene expression in a recently formed (∼100,000 years ago) allotetraploid (Glycine dolichocarpa) closely related to the cultivated soybean (Glycine max). We show that there is a high level of concordance between the allopolyploid transcriptome and translatome overall but that at least one-quarter of the transcriptome is translationally regulated. We further show that translational regulation preferentially targets genes involved in transcription, translation, and photosynthesis, causes regional and possibly whole-chromosome shifts in expression bias between duplicated genes (homoeologs), and reduces transcriptional differences between the polyploid and its diploid progenitors, possibly attenuating misregulation resulting from genome merger and/or doubling. Finally, translational regulation correlates positively with long-term retention of homoeologs from a paleopolyploidy event, suggesting that it plays a significant role in polyploid evolution.

Partial Complementarity of the Mimetic Yellow Bar Phenotype in Heliconius Butterflies

Heliconius butterflies are an excellent system for understanding the genetic basis of phenotypic change. Here we document surprising diversity in the genetic control of a common phenotype. Two disjunct H. erato populations have each recruited the Cr and/or Sd loci that control similar yellow hindwing patterns, but the alleles involved partially complement one another indicating either multiple origins for the patterning alleles or developmental drift in genetic control of similar patterns. We show that in these H. erato populations cr and sd are epistatically interacting and that the parental origin of alleles can explain phenotypes of backcross individuals. In contrast, mimetic H. melpomene populations with identical phenotypes (H. m. rosina and H. m. amaryllis) do not show genetic complementation (F1s and F2s are phenotypically identical to parentals). Finally, we report hybrid female inviability in H. m. melpomene × H. m. rosina crosses (previously only female infertility had been reported) and presence of standing genetic variation for alternative color alleles at the Yb locus in true breeding H. melpomene melpomene populations (expressed when in a different genomic background) that could be an important source of variation for the evolution of novel phenotypes or a result of developmental drift. Although recent work has emphasized the simple genetic control of wing pattern in Heliconius, we show there is underlying complexity in the allelic variation and epistatic interactions between major patterning loci.

Identification of transcriptome SNPs between Xiphophorus lines and species for assessing allele specific gene expression within F 1 interspecies hybrids

Variations in gene expression are essential for the evolution of novel phenotypes and for speciation. Studying allelic specific gene expression (ASGE) within interspecies hybrids provides a unique opportunity to reveal underlying mechanisms of genetic variation. Using Xiphophorus interspecies hybrid fishes and high-throughput next generation sequencing technology, we were able to assess variations between two closely related vertebrate species, Xiphophorus maculatus and Xiphophorus couchianus, and their F 1 interspecies hybrids. We constructed transcriptome-wide SNP polymorphism sets between two highly inbred X. maculatus lines (JP 163 A and B), and between X. maculatus and a second species, X. couchianus. The X. maculatus JP 163 A and B parental lines have been separated in the laboratory for ≈ 70 years and we were able to identify SNPs at a resolution of 1 SNP per 49 kb of transcriptome. In contrast, SNP polymorphisms between X. couchianus and X. maculatus species, which diverged ≈ 5–10 million years ago, were identified about every 700 bp. Using 6524 transcripts with identified SNPs between the two parental species ( X. maculatus and X. couchianus), we mapped RNA-seq reads to determine ASGE within F 1 interspecies hybrids. We developed an in silico X. couchianus transcriptome by replacing 90,788 SNP bases for X. maculatus transcriptome with the consensus X. couchianus SNP bases and provide evidence that this procedure overcomes read mapping biases. Employment of the in silico reference transcriptome and tolerating 5 mismatches during read mapping allow direct assessment of ASGE in the F 1 interspecies hybrids. Overall, these results show that Xiphophorus is a tractable vertebrate experimental model to investigate how genetic variations that occur during speciation may affect gene interactions and the regulation of gene expression.