Search

Amphibians inhabiting montane riparian zones in the Neotropics are particularly vulnerable to decline, but the reasons are poorly understood. Because environmental contaminants, endocrine disruption, and pathogens often figure prominently in amphibian declines it is imperative that we understand how these factors are potentially interrelated to affect montane populations. One possibility is that increased precipitation associated with global warming promotes the deposition of contaminants in montane regions. Increased exposure to contaminants, in turn, potentially elicits chronic elevations in circulating stress hormones that could contribute to montane population declines by compromising resistance to pathogens and/or production of sex steroids regulating reproduction. Here, we test this hypothesis by examining contaminant levels, stress and sex steroid levels, and nematode abundances in male drab treefrogs, Smilisca sordida, from lowland and montane populations in Costa Rica. We found no evidence that montane populations were more likely to possess contaminants (i.e., organochlorine, organophosphate and carbamate pesticides or benzidine and chlorophenoxy herbicides) than lowland populations. We also found no evidence of elevational differences in circulating levels of the stress hormone corticosterone, estradiol or progesterone. However, montane populations possessed lower androgen levels, hosted more nematode species, and had higher nematode abundances than lowland populations. Although these results suggested that nematodes contributed to lower androgens in montane populations, we were unable to detect a significant inverse relationship between nematode abundance and androgen level. Our results suggest that montane populations of this species are not at greater risk of exposure to contaminants or chronic stress, but implicate nematodes and compromised sex steroid levels as potential threats to montane populations.

Studies of the transcriptome have shown that a substantial fraction of interspecific differences in gene expression is the result of sex-biased gene expression. These results suggest that sex-dependent selection may be an important force in generating differences between species but to date all studies have focused on Drosophila. We examined a sample of the transcriptome in the gonads of two species of Xenopus to provide an additional test of how sex-biased gene expression may contribute to differences in gene expression between species. In contrast to Drosophila, Xenopus provides an example of a ZW system with morphologically indistinguishable sex chromosomes. About 81% of the transcriptome was differentially expressed between X. laevis and X. muelleri and there were more genes that were male-biased compared to the number of genes that were female-biased or non-sex-biased. While there were more genes up-regulated in males of Xenopus, the largest magnitude difference in expression between species occurred in female-biased genes, and male-biased genes were proportionally more abundant for the smallest changes in expression between species. Our results suggest that more genes have a role in the function of the testis compared to the ovary and female-biased genes play a principle role in expression divergence between species. These results differ from those in the Drosophila XY system in that more female-biased genes had >4-fold difference of expression between species than male-biased genes, suggesting that ZW sex chromosomes may facilitate enhanced gene expression divergence between species.

Accurate gene model annotation of reference genomes is critical for making them useful. The modENCODE project has improved the D. melanogaster genome annotation by using deep and diverse high-throughput data. Since transcriptional activity that has been evolutionarily conserved is likely to have an advantageous function, we have performed large-scale interspecific comparisons to increase confidence in predicted annotations. To support comparative genomics, we filled in divergence gaps in the Drosophila phylogeny by generating draft genomes for eight new species. For comparative transcriptome analysis, we generated mRNA expression profiles on 81 samples from multiple tissues and developmental stages of 15 Drosophila species, and we performed cap analysis of gene expression in D. melanogaster and D. pseudoobscura. We also describe conservation of four distinct core promoter structures composed of combinations of elements at three positions. Overall, each type of genomic feature shows a characteristic divergence rate relative to neutral models, highlighting the value of multispecies alignment in annotating a target genome that should prove useful in the annotation of other high priority genomes, especially human and other mammalian genomes that are rich in noncoding sequences. We report that the vast majority of elements in the annotation are evolutionarily conserved, indicating that the annotation will be an important springboard for functional genetic testing by the Drosophila community., National Institutes of Health (U.S.) (NIDDK (DK015600-18)), National Institutes of Health (U.S.) (Extramural program (1ROIGM082843)), National Institutes of Health (U.S.) (Extramural program (U01HB004271))

Ecologists recognize the balance of nature as a complex system that arises from interacting and connected parts; disturbing the balance can affect other species in nonlinear and dynamic ways. Like ecosystems, the cell and genome are extensively connected by an abundance of molecular interactions. Upsetting the molecular balance, through changes in gene regulation, abnormal copy number variants, and changes in cell size can create consequences for genome and organismal function (1, 2). In PNAS, Gibbons et al. (3) advance the idea of genome balance further by identifying a new source of genetic variation that results in balancing transcriptional subunits of the ribosome. Using data from human and mouse, the authors characterized copy number variation (CNV) in tandemly repeated ribosomal DNA (rDNA) arrays and found that subunits of the array vary together in what they define as concerted copy number variation (cCNV). cCNV raises new questions about how CNV can function to restore balance in eukaryotic genomes, especially on the assembly and function of the ribosome.

Biotic and abiotic factors have been proposed to explain patterns of reproductive character displacement, but which factor is most important to character displacement of acoustic signals is not clear. Male vocalizations of the frog Pseudacris feriarum are known to undergo reproductive character displacement in areas of sympatry with P. brimleyi and P. nigrita. Despite evidence for reinforcement as an important mechanism, local adaptation via sensory drive might explain this pattern because Pseudacris breed in different habitat types and mating signals are exposed to a variety of environments. We tested the sensory drive hypothesis by playing synthesized vocalizations representing the spectrum of variation in P. feriarum at 12 different study sites. If sensory drive has occurred, then vocalizations should transmit better in the site of origin or at ecologically similar sites. We found that variation in acoustic signals did not produce better transmission in particular sites, the effect of site was uniform, and acoustic signals often transmitted better in habitats external to their origin. Ecological variation among habitats did not explain signal degradation. Our playback experiments, ecological analyses, and comparisons of different habitat types provide no support for sensory drive as a process promoting reproductive character displacement in this system. Reinforcement is the more likely primary mechanism.

The production of multiple transcript isoforms from one gene is a major source of transcriptome complexity. RNA-Seq experiments, in which transcripts are converted to cDNA and sequenced, allow the resolution and quantification of alternative transcript isoforms. However, methods to analyze splicing are underdeveloped and errors resulting in incorrect splicing calls occur in every experiment. We used RNA-Seq data to develop sequencing and aligner error models. By applying these error models to known input from simulations, we found that errors result from false alignment to minor splice motifs and antisense stands, shifted junction positions, paralog joining, and repeat induced gaps. By using a series of quantitative and qualitative filters, we eliminated diagnosed errors in the simulation, and applied this to RNA-Seq data from Drosophila melanogaster heads. We used high-confidence junction detections to specifically interrogate local splicing differences between transcripts. This method out-performed commonly used RNA-seq methods to identify known alternative splicing events in the Drosophila sex determination pathway. We describe a flexible software package to perform these tasks called Splicing Analysis Kit (Spanki), available at http://www.cbcb.umd.edu/software/spanki . Splice-junction centric analysis of RNA-Seq data provides advantages in specificity for detection of alternative splicing. Our software provides tools to better understand error profiles in RNA-Seq data and improve inference from this new technology. The splice-junction centric approach that this software enables will provide more accurate estimates of differentially regulated splicing than current tools.

X chromosomes are preferentially transmitted through females, which may favor the accumulation of X-linked alleles/genes with female-beneficial effects. Numerous studies have shown that genes with sex-biased expression are under- or over-represented on the X chromosomes of a wide variety of organisms. The patterns, however, vary between different animal species, and the causes of these differences are unresolved. Additionally, genes with sex-biased expression tend to be narrowly expressed in a limited number of tissues, and narrowly expressed genes are also non-randomly X-linked in a taxon-specific manner. It is therefore unclear whether the unique gene content of the X chromosome is the result of selection on genes with sex-biased expression, narrowly expressed genes, or some combination of the two. To address this problem, we measured sex-biased expression in multiple Drosophila species and at different developmental time points. These data were combined with available expression measurements from Drosophila melanogaster and mouse to reconcile the inconsistencies in X-chromosome content among taxa. Our results suggest that most of the differences between Drosophila and mammals are confounded by disparate data collection/analysis approaches as well as the correlation between sex bias and expression breadth. Both the Drosophila and mouse X chromosomes harbor an excess of genes with female-biased expression after controlling for the confounding factors, suggesting that the asymmetrical transmission of the X chromosome favors the accumulation of female-beneficial mutations in X-linked genes. However, some taxon-specific patterns remain, and we provide evidence that these are in part a consequence of constraints imposed by the dosage compensation mechanism in Drosophila.

Analysis of the relationship between gene copy number and gene expression in aneuploid male Drosophila cells reveals a global compensation mechanism in addition to X chromosome-specific dosage compensation., Extensive departures from balanced gene dose in aneuploids are highly deleterious. However, we know very little about the relationship between gene copy number and expression in aneuploid cells. We determined copy number and transcript abundance (expression) genome-wide in Drosophila S2 cells by DNA-Seq and RNA-Seq. We found that S2 cells are aneuploid for >43 Mb of the genome, primarily in the range of one to five copies, and show a male genotype (∼ two X chromosomes and four sets of autosomes, or 2X;4A). Both X chromosomes and autosomes showed expression dosage compensation. X chromosome expression was elevated in a fixed-fold manner regardless of actual gene dose. In engineering terms, the system “anticipates” the perturbation caused by X dose, rather than responding to an error caused by the perturbation. This feed-forward regulation resulted in precise dosage compensation only when X dose was half of the autosome dose. Insufficient compensation occurred at lower X chromosome dose and excessive expression occurred at higher doses. RNAi knockdown of the Male Specific Lethal complex abolished feed-forward regulation. Both autosome and X chromosome genes show Male Specific Lethal–independent compensation that fits a first order dose-response curve. Our data indicate that expression dosage compensation dampens the effect of altered DNA copy number genome-wide. For the X chromosome, compensation includes fixed and dose-dependent components., Author Summary While it is widely recognized that mutations in protein coding genes can have harmful consequences, one can also have too much or too little of a good thing. Except for the sex chromosomes, genes come in sets of two in diploid organisms. Extra or missing copies of genes or chromosomes result in an imbalance that can lead to cancers, miscarriages, and disease susceptibility. We have examined what happens to gene expression in Drosophila cells with the types of gross copy number changes that are typical of cancers. We have compared the response of autosomes and sex chromosomes and show that there is some compensation for copy number change in both cases. One response is universal and acts to correct copy number changes by changing transcript abundance. The other is specific to the X chromosome and acts to increase expression regardless of gene dose. Our data highlight how important gene expression balance is for cell function.

Chromosomes that harbor dominant sex determination loci are predicted to erode over time—losing genes, accumulating transposable elements, degenerating into a functional wasteland and ultimately becoming extinct. The Drosophila melanogaster Y chromosome is fairly far along this path to oblivion. The few genes on largely heterochromatic Y chromosome are required for spermatocyte-specific functions, but have no role in other tissues. Surprisingly, a recent paper shows that divergent Y chromosomes can substantially influence gene expression throughout the D. melanogaster genome.1 These results show that variation on Y has an important influence on the deployment of the genome. BioEssays 30:409–411, 2008. © 2008 Wiley Periodicals, Inc.

Background Interspecific hybrids of frogs of the genus Xenopus result in sterile hybrid males and fertile hybrid females. Previous work has demonstrated a dramatic asymmetrical pattern of misexpression in hybrid males compared to the two parental species with relatively few genes misexpressed in comparisons of hybrids and the maternal species (X. laevis) and dramatically more genes misexpressed in hybrids compared to the paternal species (X. muelleri). In this work, we examine the gene expression pattern in hybrid females of X. laevis × X. muelleri to determine if this asymmetrical pattern of expression also occurs in hybrid females. Results We find a similar pattern of asymmetry in expression compared to males in that there were more genes differentially expressed between hybrids and X. muelleri compared to hybrids and X. laevis. We also found a dramatic increase in the number of misexpressed genes with hybrid females having about 20 times more genes misexpressed in ovaries compared to testes of hybrid males and therefore the match between phenotype and expression pattern is not supported. Conclusion We discuss these intriguing findings in the context of reproductive isolation and suggest that divergence in female expression may be involved in sterility of hybrid males due to the inherent sensitivity of spermatogenesis as defined by the faster male evolution hypothesis for Haldane's rule.

Reproductive isolation between biological species is characterized by Haldane's rule, which states that the heterogametic sex (XY or ZW) suffers the most dysfunctional effects of hybridization. It has been hypothesized that, in addition to X-linked recessive genes, Haldane's rule also reflects the faster evolution of genes related to male reproduction (faster-male evolution). We used sex-reversed hybrid Xenopus to test faster-male evolution by examining the fertility of sex-reversed individuals with the genotype of the inverse phenotypic sex. Hybrid males are sterile and hybrid females are fertile irrespective of their genotypic sex. Gene expression profiles match these adult phenotypes, and our results demonstrate that faster-male evolution is the most likely mechanism producing hybrid male sterility.

Understanding the general features of speciation is an important goal in evolutionary biology, and despite significant progress, several unresolved questions remain. We analyzed an extensive comparative dataset consisting of more than 1900 crosses between 92 species of toads to infer patterns of reproductive isolation. This unique dataset provides an opportunity to examine the strength of reproductive isolation, the development and sex ratios of hybrid offspring, patterns of fertility and infertility, and polyploidization in hybrids all in the context of genetic divergence between parental species. We found that the strength of intrinsic postzygotic isolation increases with genetic divergence, but relatively high levels of divergence are necessary before reproductive isolation is complete in toads. Fertilization rates were not correlated to genetic divergence, but hatching success, the number of larvae produced, and the percentage of tadpoles reaching metamorphosis were all inversely related with genetic divergence. Hybrids between species with lower levels of divergence developed to metamorphosis, while hybrids with higher levels of divergence stopped developing in gastrula and larval stages. Sex ratios of hybrid offspring were biased towards males in 70% of crosses and biased towards females in 30% of crosses. Hybrid females from crosses between closely related species were completely fertile, while approximately half (53%) of hybrid males were sterile, with sterility predicted by genetic divergence. The degree of abnormal ploidy in hybrids was positively related to genetic divergence between parental species, but surprisingly, polyploidization had no effect on patterns of asymmetrical inviability. We discuss explanations for these patterns, including the role of Haldane's rule in toads and anurans in general, and suggest mechanisms generating patterns of reproductive isolation in anurans.

DNA sequences on X chromosomes often have a faster rate of evolution when compared to similar loci on the autosomes, and well articulated models provide reasons why the X-linked mode of inheritance may be responsible for the faster evolution of X-linked genes. We analyzed microarray and RNA–seq data collected from females and males of six Drosophila species and found that the expression levels of X-linked genes also diverge faster than autosomal gene expression, similar to the “faster-X” effect often observed in DNA sequence evolution. Faster-X evolution of gene expression was recently described in mammals, but it was limited to the evolutionary lineages shortly following the creation of the therian X chromosome. In contrast, we detect a faster-X effect along both deep lineages and those on the tips of the Drosophila phylogeny. In Drosophila males, the dosage compensation complex (DCC) binds the X chromosome, creating a unique chromatin environment that promotes the hyper-expression of X-linked genes. We find that DCC binding, chromatin environment, and breadth of expression are all predictive of the rate of gene expression evolution. In addition, estimates of the intraspecific genetic polymorphism underlying gene expression variation suggest that X-linked expression levels are not under relaxed selective constraints. We therefore hypothesize that the faster-X evolution of gene expression is the result of the adaptive fixation of beneficial mutations at X-linked loci that change expression level in cis. This adaptive faster-X evolution of gene expression is limited to genes that are narrowly expressed in a single tissue, suggesting that relaxed pleiotropic constraints permit a faster response to selection. Finally, we present a conceptional framework to explain faster-X expression evolution, and we use this framework to examine differences in the faster-X effect between Drosophila and mammals., Author Summary As species diverge over evolutionary time, they accumulate differences in the sequences of their genes and how those genes are expressed. We show that gene expression changes accumulate faster for genes on the X chromosome than for genes on the other chromosomes (autosomes) in Drosophila (the “faster-X” effect). The X chromosome is only found in a single copy in males, whereas the autosomes are found in two copies in both sexes. To compensate for the reduced dosage of X-linked genes in males, a molecular complex binds the Drosophila X chromosome to upregulate gene expression in males. We demonstrate that genes that escape this dosage compensation process have faster evolving expression levels. X-linked genes are inherited in a unique manner, and we hypothesize that this permits a faster rate of adaptive evolution, thereby driving the faster-X evolution of gene expression. We compare these observations with the recently described faster-X evolution of gene expression in mammals, and we explain how differences in dosage compensation, mutation rate, and population size could affect the extent of the faster-X effect.

Background Drosophila females commit tremendous resources to egg production and males produce some of the longest sperm in the animal kingdom. We know little about the coordinated regulation of gene expression patterns in distant somatic tissues that support the developmental cost of gamete production. Results We determined the non-gonadal gene expression patterns of Drosophila females and males with or without a germline. Our results show that germline-dependent expression in the non-gonadal soma is extensive. Interestingly, gene expression patterns and hormone titers are consistent with a hormone axis between the gonads and non-gonadal soma. Conclusions The germline has a long-range influence on gene expression in the Drosophila sexes. We suggest that this is the result of a germline/soma hormonal axis.

BACKGROUND: Reproductive isolation is a defining characteristic of populations that represent unique biological species, yet we know very little about the gene expression basis for reproductive isolation. The advent of powerful molecular biology tools provides the ability to identify genes involved in reproductive isolation and focuses attention on the molecular mechanisms that separate biological species. Herein we quantify the sterility pattern of hybrid males in African Clawed Frogs (Xenopus) and apply microarray analysis of the expression pattern found in testes to identify genes that are misexpressed in hybrid males relative to their two parental species (Xenopus laevis and X. muelleri). METHODOLOGY/PRINCIPAL FINDINGS: Phenotypic characteristics of spermatogenesis in sterile male hybrids (X. laevis x X. muelleri) were examined using a novel sperm assay that allowed quantification of live, dead, and undifferentiated sperm cells, the number of motile vs. immotile sperm, and sperm morphology. Hybrids exhibited a dramatically lower abundance of mature sperm relative to the parental species. Hybrid spermatozoa were larger in size and accompanied by numerous undifferentiated sperm cells. Microarray analysis of gene expression in testes was combined with a correction for sequence divergence derived from genomic hybridizations to identify candidate genes involved in the sterility phenotype. Analysis of the transcriptome revealed a striking asymmetric pattern of misexpression. There were only about 140 genes misexpressed in hybrids compared to X. laevis but nearly 4,000 genes misexpressed in hybrids compared to X. muelleri. CONCLUSIONS/SIGNIFICANCE: Our results provide an important correlation between phenotypic characteristics of sperm and gene expression in sterile hybrid males. The broad pattern of gene misexpression suggests intriguing mechanisms creating the dominance pattern of the X. laevis genome in hybrids. These findings significantly contribute to growing evidence for allelic dominance in hybrids and have implications for the mechanism of species differentiation at the transcriptome level.

Gene regulation was long predicted to play a vital role in speciation and species divergence. Only recently with the advent of new technologies, however, has it been possible to address the question of the relative contributions of different mechanisms of gene expression to the evolution of phenotypic diversity. Here we broaden the question and ask whether microRNAs, a large class of small regulatory RNAs, play a role in reproductive isolation between species by contributing to hybrid male sterility. MicroRNAs from the testes of clawed frogs (Xenopus) were extracted and the expression profiles of sterile hybrids were compared with males of a parental species. Hybrid testes were largely microRNA-depleted relative to those of nonhybrids, and this pattern was validated with quantitative RT-PCR. A number of candidate differential microRNAs from this study have previously been described as testis-specific in the mouse, suggesting that microRNA structural conservation may be associated with functional retention.

Background Gene dosage change is a mild perturbation that is a valuable tool for pathway reconstruction in Drosophila. While it is often assumed that reducing gene dose by half leads to two-fold less expression, there is partial autosomal dosage compensation in Drosophila, which may be mediated by feedback or buffering in expression networks. Results We profiled expression in engineered flies where gene dose was reduced from two to one. While expression of most one-dose genes was reduced, the gene-specific dose responses were heterogeneous. Expression of two-dose genes that are first-degree neighbors of one-dose genes in novel network models also changed, and the directionality of change depended on the response of one-dose genes. Conclusions Our data indicate that expression perturbation propagates in network space. Autosomal compensation, or the lack thereof, is a gene-specific response, largely mediated by interactions with the rest of the transcriptome.