The Loci of Behavioral Evolution: Evidence That Fas2 and tilB Underlie Differences in Pupation Site Choice Behavior between Drosophila melanogaster and D. simulans.

The behaviors of closely related species can be remarkably different, and these differences have important ecological and evolutionary consequences. Although the recent boom in genotype-phenotype studies has led to a greater understanding of the genetic architecture and evolution of a variety of traits, studies identifying the genetic basis of behaviors are, comparatively, still lacking. This is likely because they are complex and environmentally sensitive phenotypes, making them difficult to measure reliably for association studies. The Drosophila species complex holds promise for addressing these challenges, as the behaviors of closely related species can be readily assayed in a common environment. Here, we investigate the genetic basis of an evolved behavioral difference, pupation site choice, between Drosophila melanogaster and D. simulans. In this study, we demonstrate a significant contribution of the X chromosome to the difference in pupation site choice behavior between these species. Using a panel of X-chromosome deficiencies, we screened the majority of the X chromosome for causal loci and identified two regions associated with this X-effect. We then collect gene disruption and RNAi data supporting a single gene that affects pupation behavior within each region: Fas2 and tilB. Finally, we show that differences in tilB expression correlate with the differences in pupation site choice behavior between species. This evidence associating two genes with differences in a complex, environmentally sensitive behavior represents the first step toward a functional and evolutionary understanding of this behavioral divergence.

The complex genetic architecture of male mate choice evolution between Drosophila species.

Mate choice behaviors are among the most important reproductive isolating barriers in many animals. Little is known about the genetic basis of reproductively isolating behaviors, but examples to date provide evidence that they can have a simple genetic basis. However, it is unclear if these results indicate that individual genes with large effects are common, or are instead due to ascertainment biases. Here, we present the results of a QTL mapping study for the most important behavioral isolating barrier between Drosophila simulans and D. sechellia: male mate choice. Our QTL results initially suggested that differences in male mate choice may be due to a couple loci with large effects. However, as we divided the largest-effect QTL using stable introgression strains, we found evidence of multiple interacting loci. We further find that separate regions of the genome control different aspects of male choice. Taken together, our results suggest that the genetic architecture of mate choice behavior, in this case, is more complex than QTL mapping suggested, highlighting potential challenges to future mapping studies. We discuss the implications of these results as they relate to signal-receiver coevolution, mate choice, and reproductive isolation.

Maximum likelihood estimation of frequencies of known haplotypes from pooled sequence data.

DNA samples are often pooled, either by experimental design or because the sample itself is a mixture. For example, when population allele frequencies are of primary interest, individual samples may be pooled together to lower the cost of sequencing. Alternatively, the sample itself may be a mixture of multiple species or strains (e.g., bacterial species comprising a microbiome or pathogen strains in a blood sample). We present an expectation-maximization algorithm for estimating haplotype frequencies in a pooled sample directly from mapped sequence reads, in the case where the possible haplotypes are known. This method is relevant to the analysis of pooled sequencing data from selection experiments, as well as the calculation of proportions of different species within a metagenomics sample. Our method outperforms existing methods based on single-site allele frequencies, as well as simple approaches using sequence read data. We have implemented the method in a freely available open-source software tool.

Combining genome-wide methods to investigate the genetic complexity of courtship song variation in Drosophila melanogaster.

Little is currently known about the genetic complexity of quantitative behavioral variation, the types of genes involved, or their effects on intermediate phenotypes. Here, we conduct a genome-wide association study of Drosophila melanogaster courtship song variation using 168 sequenced inbred lines, and fail to find highly significant associations. However, by combining these data with results from a well-powered Evolve and Resequence (E&R) study on the same trait, we provide statistical evidence that some power to associate genotype and phenotype is available. Genes that are significant in both analyses are enriched for expression in the nervous system, and affect neural development and synaptic growth when perturbed. Quantitative complementation at one of these loci, Syntrophin-like 1, supports a hypothesis that variation at this locus affects variation in the inter-pulse interval of courtship song. These results suggest that experimental evolution may provide an approach for genome-scale replication in Drosophila.

Fine-mapping natural alleles: quantitative complementation to the rescue.

Mapping the genes responsible for natural variation and divergence is a challenging task. Many studies have mapped genes to genomic regions or generated lists of candidates, but few studies have implicated specific genes with a high standard of evidence. I propose that combining recent advances in genomic engineering with a modified version of the quantitative complementation test will help turn candidate genes into causal genes. By creating loss-of-function mutations in natural strains, and using these mutations to quantitatively fail-to-complement natural alleles, fine mapping should be greatly facilitated. As an example, I propose that the CRISPR/Cas9 system could be combined with the FLP/FRT system to fine-map genes in the numerous systems where inversions have frustrated these efforts.

Population-based resequencing of experimentally evolved populations reveals the genetic basis of body size variation in Drosophila melanogaster.

Body size is a classic quantitative trait with evolutionarily significant variation within many species. Locating the alleles responsible for this variation would help understand the maintenance of variation in body size in particular, as well as quantitative traits in general. However, successful genome-wide association of genotype and phenotype may require very large sample sizes if alleles have low population frequencies or modest effects. As a complementary approach, we propose that population-based resequencing of experimentally evolved populations allows for considerable power to map functional variation. Here, we use this technique to investigate the genetic basis of natural variation in body size in Drosophila melanogaster. Significant differentiation of hundreds of loci in replicate selection populations supports the hypothesis that the genetic basis of body size variation is very polygenic in D. melanogaster. Significantly differentiated variants are limited to single genes at some loci, allowing precise hypotheses to be formed regarding causal polymorphisms, while other significant regions are large and contain many genes. By using significantly associated polymorphisms as a priori candidates in follow-up studies, these data are expected to provide considerable power to determine the genetic basis of natural variation in body size.

siRNA-mediated methylation of Arabidopsis telomeres.

Chromosome termini form a specialized type of heterochromatin that is important for chromosome stability. The recent discovery of telomeric RNA transcripts in yeast and vertebrates raised the question of whether RNA-based mechanisms are involved in the formation of telomeric heterochromatin. In this study, we performed detailed analysis of chromatin structure and RNA transcription at chromosome termini in Arabidopsis. Arabidopsis telomeres display features of intermediate heterochromatin that does not extensively spread to subtelomeric regions which encode transcriptionally active genes. We also found telomeric repeat-containing transcripts arising from telomeres and centromeric loci, a portion of which are processed into small interfering RNAs. These telomeric siRNAs contribute to the maintenance of telomeric chromatin through promoting methylation of asymmetric cytosines in telomeric (CCCTAAA)(n) repeats. The formation of telomeric siRNAs and methylation of telomeres relies on the RNA-dependent DNA methylation pathway. The loss of telomeric DNA methylation in rdr2 mutants is accompanied by only a modest effect on histone heterochromatic marks, indicating that maintenance of telomeric heterochromatin in Arabidopsis is reinforced by several independent mechanisms. In conclusion, this study provides evidence for an siRNA-directed mechanism of chromatin maintenance at telomeres in Arabidopsis.

The Sponges of the Carmel Pinnacles Marine Protected Area.

California's network of marine protected areas was created to protect the diversity and abundance of native marine life, but the status of some taxa is very poorly known. Here we describe the sponges (phylum Porifera) from the Carmel Pinnacles State Marine Reserve, as assessed by a SCUBA-based survey in shallow waters. Of the 29 sponge species documented, 12 (41%) of them were previously unknown. Using a combination of underwater photography, DNA sequencing, and morphological taxonomy, we greatly improve our understanding of the status and distribution of previously described species and formally describe the new species as Hymedesmia promina sp. nov., Phorbas nebulosus sp. nov., Clathria unoriginalis sp. nov., Clathria rumsena sp. nov., Megaciella sanctuarium sp. nov., Mycale lobos sp. nov., Xestospongia ursa sp. nov., Haliclona melissae sp. nov., Halichondria loma sp. nov., Hymeniacidon fusiformis sp. nov., Scopalina carmela sp. nov., and Obruta collector gen. nov., sp. nov. An additional species, Lissodendoryx topsenti (de Laubenfels 1930), is moved to Hemimycale, and H. polyboletus comb. nov., nom. nov. is created due to preoccupation by H. topsenti (Burton, 1929). Several of the new species appear to be rare and/or have very restricted distributions, as they were not found at comparative survey sites outside of Carmel Bay. These results illustrate the potential of qualitative presence/absence systematic surveys of understudied taxa to discover and document substantial novel diversity.

The order Axinellida (Porifera: Demospongiae) in California.

Sponges are common and diverse in California, but they have received little study in the region, and the identities of many common species remain unclear. Here we combine fresh collections and museum vouchers to revise the order Axinellida for California. Seven new species are described: Endectyon (Endectyon) hispitumulus, Eurypon curvoclavus, Aulospongus viridans, Aulospongus lajollaensis, Halicnemia litorea, Halicnemia montereyensis, and Halicnemia weltoni. One new combination is also described, and two existing species are reduced to junior synonyms, resulting in a total of 13 species; a dichotomous key to differentiate them is provided. DNA data from 9 of the 13 species is combined with publicly available data to produce updated global phylogenies for the order.

Confirmation of the southern African distribution of the marine sponge Hymeniacidon perlevis (Montagu, 1814) in the context of its global dispersal.

Background: Intertidal rocky shore surveys along the South African coastline (∼3,000 km) have demonstrated the presence and abundance of the encrusting orange sponge Hymeniacidon perlevis (Montagu, 1814), a well-known globally distributed species. After analysing the southern African populations, we gained a better understanding of the genetic structure of this now-accepted global species. Apart from confirming the presence of a single population of H. perlevis, we also determined its distribution in the southern African intertidal rocky shore ecosystem, compared its genetic diversity to congeners, predict its global distribution via environmental niche modelling, and discussed possible underlying mechanisms controlling the species' global distribution. Methods: We surveyed the South African coastline and sampled sponges at 53 rocky shore sites spanning over 3,000 km, from Grosse Bucht south of Lüderitz (Namibia) to Kosi Bay on the east coast of South Africa. DNA sequences of the nuclear rDNA internal transcribed spacer (ITS1) and the COI mitochondrial gene were obtained from 61 samples and compared them to a world-wide sample of other H. perlevis sequences. Using environmental predictor variables from the global dataset BIO-ORACLE, we predicted the probability of global occurrence of the species using an ensemble of eight distribution models. Results: South African specimens were found to be 99-100% identical to other populations of H. perlevis (=H. sinapium) from other world-wide regions. The presence of a single population of H. perlevis in southern Africa is supported by genetic data, extending its distribution to a relatively wide geographical range spanning more than 4,000 km along the temperate southern African coast. The predicted global occurrence by ensemble model matched well with the observed distribution. Surface temperature mean and range were the most important predictor variables. Conclusion: While H. perlevis appears to have been introduced in many parts of the world, its origins in Europe and southern Africa are unclear.

Four new Scopalina from Southern California: the first Scopalinida (Porifera: Demospongiae) from the temperate Eastern Pacific.

Sponges (phylum Porifera) are common inhabitants of kelp forest ecosystems in California, but their diversity and ecological importance are poorly characterized in this biome. Here I use freshly collected samples to describe the diversity of the order Scopalinida in California. Though previously unknown in the region, four new species are described here: Scopalina nausicae sp. nov., S. kuyamu sp. nov., S. goletensis sp. nov., and S. jali sp. nov.. These discoveries illustrate the considerable uncharacterized sponge diversity remaining in California kelp forests, and the utility of SCUBA-based collection to improve our understanding of this diversity.

The order Tethyida (Porifera) in California: taxonomy, systematics, and the first member of the family Hemiasterellidae in the Eastern Pacific.

Sponges (phylum Porifera) are ubiquitous inhabitants of marine ecosystems and have been shown to provide substantial ecosystem services. Despite this ecological importance, the sponge fauna in California has received little study. Here I use a collection of kelp forest sponges to describe the diversity of the order Tethyida in California. This order contains the genus Tethya, which has been included in long-term ecological monitoring projects. I show that Tethya vacua is a junior synonym of Tethya californiana, and thus all Tethya populations known in California belong to a single species. Genetic data from California's only Timeidae, Timea authia, indicate that this species is in fact in the Tethyidae and is the third known member of the genus Tethytimea. I also describe the first member of the family Hemiasterellidae from the Eastern Pacific, Galaxia gaviotensis gen. nov. sp. nov. By combining field photographs, morphological taxonomy, and phylogenetic analysis of these samples, this work will facilitate future efforts to understand the evolution of this order and the ecological role of sponges in the California kelp forest.

Light dependent courtship behavior in Drosophila simulans and D. melanogaster.

Differences in courtship signals and perception are well-known among Drosophila species. One such described difference is the dependency on light, and thus presumably vision, for copulation success. Many studies have described a difference in light-dependent copulation success between D. melanogaster and D. simulans, identifying D. simulans as a light-dependent species, and D. melanogaster as a light-independent one. However, many of these studies use assays of varying design and few strains to represent the entire species. Here, we attempt to better characterize this purported difference using 11 strains of each species, paired by collection location, in behavioral assays conducted at two different exposure times. We show that, while there is a species-wide difference in magnitude of light-dependent copulation success, D. melanogaster copulation success is, on average, still impaired in the dark at both exposure times we measured. Additionally, there is significant variation in strain-specific ability to copulate in the dark in both species across two different exposure times. We find that this variation correlates strongly with longitude in D. melanogaster, but not in D. simulans. We hypothesize that differences in species history and demography may explain behavioral variation. Finally, we use courtship assays to show that light-dependent copulation success in one D. simulans strain is driven in part by both males and females. We discuss potential differences in courtship signals and/or signal importance between these species and potential for further comparative studies for functional characterization.

The Genetics of Male Pheromone Preference Difference Between Drosophila melanogaster and Drosophila simulans.

Species of flies in the genus Drosophila differ dramatically in their preferences for mates, but little is known about the genetic or neurological underpinnings of this evolution. Recent advances have been made to our understanding of one case: pheromone preference evolution between the species D. melanogaster and D. simulans Males of both species are very sensitive to the pheromone 7,11-HD that is present only on the cuticle of female D. melanogaster In one species this cue activates courtship, and in the other it represses it. This change in valence was recently shown to result from the modification of central processing neurons, rather than changes in peripherally expressed receptors, but nothing is known about the genetic changes that are responsible. In the current study, we show that a 1.35 Mb locus on the X chromosome has a major effect on male 7,11-HD preference. Unfortunately, when this locus is divided, the effect is largely lost. We instead attempt to filter the 159 genes within this region using our newfound understanding of the neuronal underpinnings of this phenotype to identify and test candidate genes. We present the results of these tests, and discuss the difficulty of identifying the genetic architecture of behavioral traits and the potential of connecting these genetic changes to the neuronal modifications that elicit different behaviors.

Genomic analysis of adaptive differentiation in Drosophila melanogaster.

Drosophila melanogaster shows clinal variation along latitudinal transects on multiple continents for several phenotypes, allozyme variants, sequence variants, and chromosome inversions. Previous investigation suggests that many such clines are due to spatially varying selection rather than demographic history, but the genomic extent of such selection is unknown. To map differentiation throughout the genome, we hybridized DNA from temperate and subtropical populations to Affymetrix tiling arrays. The dense genomic sampling of variants and low level of linkage disequilibrium in D. melanogaster enabled identification of many small, differentiated regions. Many regions are differentiated in parallel in the United States and Australia, strongly supporting the idea that they are influenced by spatially varying selection. Genomic differentiation is distributed nonrandomly with respect to gene function, even in regions differentiated on only one continent, providing further evidence for the role of selection. These data provide candidate genes for phenotypes known to vary clinally and implicate interesting new processes in genotype-by-environment interactions, including chorion proteins, proteins regulating meiotic recombination and segregation, gustatory and olfactory receptors, and proteins affecting synaptic function and behavior. This portrait of differentiation provides a genomic perspective on adaptation and the maintenance of variation through spatially varying selection.

Extensive intraspecies cryptic variation in an ancient embryonic gene regulatory network.

Innovations in metazoan development arise from evolutionary modification of gene regulatory networks (GRNs). We report widespread cryptic variation in the requirement for two key regulatory inputs, SKN-1/Nrf2 and MOM-2/Wnt, into the C. elegans endoderm GRN. While some natural isolates show a nearly absolute requirement for these two regulators, in others, most embryos differentiate endoderm in their absence. GWAS and analysis of recombinant inbred lines reveal multiple genetic regions underlying this broad phenotypic variation. We observe a reciprocal trend, in which genomic variants, or knockdown of endoderm regulatory genes, that result in a high SKN-1 requirement often show low MOM-2/Wnt requirement and vice-versa, suggesting that cryptic variation in the endoderm GRN may be tuned by opposing requirements for these two key regulatory inputs. These findings reveal that while the downstream components in the endoderm GRN are common across metazoan phylogeny, initiating regulatory inputs are remarkably plastic even within a single species.

Genomic islands of speciation in Anopheles gambiae.

The African malaria mosquito, Anopheles gambiae sensu stricto (A. gambiae), provides a unique opportunity to study the evolution of reproductive isolation because it is divided into two sympatric, partially isolated subtaxa known as M form and S form. With the annotated genome of this species now available, high-throughput techniques can be applied to locate and characterize the genomic regions contributing to reproductive isolation. In order to quantify patterns of differentiation within A. gambiae, we hybridized population samples of genomic DNA from each form to Affymetrix GeneChip microarrays. We found that three regions, together encompassing less than 2.8 Mb, are the only locations where the M and S forms are significantly differentiated. Two of these regions are adjacent to centromeres, on Chromosomes 2L and X, and contain 50 and 12 predicted genes, respectively. Sequenced loci in these regions contain fixed differences between forms and no shared polymorphisms, while no fixed differences were found at nearby control loci. The third region, on Chromosome 2R, contains only five predicted genes; fixed differences in this region were also verified by direct sequencing. These "speciation islands" remain differentiated despite considerable gene flow, and are therefore expected to contain the genes responsible for reproductive isolation. Much effort has recently been applied to locating the genes and genetic changes responsible for reproductive isolation between species. Though much can be inferred about speciation by studying taxa that have diverged for millions of years, studying differentiation between taxa that are in the early stages of isolation will lead to a clearer view of the number and size of regions involved in the genetics of speciation. Despite appreciable levels of gene flow between the M and S forms of A. gambiae, we were able to isolate three small regions of differentiation where genes responsible for ecological and behavioral isolation are likely to be located. We expect reproductive isolation to be due to changes at a small number of loci, as these regions together contain only 67 predicted genes. Concentrating future mapping experiments on these regions should reveal the genes responsible for reproductive isolation between forms.

Male mate choice via cuticular hydrocarbon pheromones drives reproductive isolation between Drosophila species.

Mate discrimination is a key mechanism restricting gene flow between species. While studied extensively with respect to female mate choice, mechanisms of male mate choice between species are far less studied. Thus, we have little knowledge of the relative frequency, importance, or overall contribution of male mate discrimination to reproductive isolation. In the present study, we estimated the relative contributions of male and female choice to reproductive isolation between Drosophila simulans and D. sechellia, and show that male mate discrimination accounts for the majority of the current isolation between these species. We further demonstrate that males discriminate based on female cuticular hydrocarbon pheromones, and collect evidence supporting the hypothesis that male mate discrimination may alleviate the costs associated with heterospecific courtship and mating. Our findings highlight the potentially significant contribution of male mate choice to the formation of reproductive isolating barriers, and thus the speciation process.

Oviposition preferences for ethanol depend on spatial arrangement and differ dramatically among closely related Drosophila species.

Recent work on the model fly Drosophila melanogaster has reported inconsistencies in their preference for laying eggs on intermediate concentrations of ethanol. In this study, we resolve this discrepancy by showing that this species strongly prefers ovipositing on ethanol when it is close to a non-ethanol substrate, but strongly avoids ethanol when options are farther apart. We also show fluidity of these behaviors among other Drosophila species: D. melanogaster is more responsive to ethanol than close relatives in that it prefers ethanol more than other species in the close-proximity case, but avoids ethanol more than other species in the distant case. In the close-proximity scenario, the more ethanol-tolerant species generally prefer ethanol more, with the exception of the island endemic D. santomea This species has the lowest tolerance in the clade, but behaves like D. melanogaster We speculate that this could be an adaptation to protect eggs from parasites or predators such as parasitoid wasps, as larvae migrate to non-toxic substrates after hatching. These natural differences among species are an excellent opportunity to study how genes and brains evolve to alter ethanol preferences, and provide an interesting model for genetic variation in preferences in other organisms, including humans.