Problems with paralogs: the promise and challenges of gene duplicates in evo-devo research.

Gene duplicates, or paralogs, serve as a major source of new genetic material and comprise seeds for evolutionary innovation. While originally thought to be quickly lost or non-functionalized following duplication, now a vast number of paralogs are known to be retained in a functional state. Daughter paralogs can provide robustness through redundancy, specialize via sub-functionalization, or neo-functionalize to play new roles. Indeed, the duplication and divergence of developmental genes have played a monumental role in the evolution of animal forms (e.g. Hox genes). Still, despite their prevalence and evolutionary importance, the precise detection of gene duplicates in newly sequenced genomes remains technically challenging and often overlooked. This presents an especially pertinent problem for evolutionary developmental biology (evo-devo), where hypothesis testing requires accurate detection of changes in gene expression and function, often in non-traditional model species. Frequently, these analyses rely on molecular reagents designed within coding sequences that may be highly similar in recently duplicated paralogs, leading to cross-reactivity and spurious results. Thus, care is needed to avoid erroneously assigning diverged functions of paralogs to a single gene, and potentially misinterpreting evolutionary history. This perspective aims to overview the prevalence and importance of paralogs and to shed light on the difficulty of their detection and analysis while offering potential solutions.

Evolution and molecular mechanisms of wing plasticity in aphids.

Aphids present a fascinating example of phenotypic plasticity, in which a single genotype can produce dramatically different winged and wingless phenotypes that are specialized for dispersal versus reproduction, respectively. Recent work has examined many aspects of this plasticity, including its evolution, molecular control mechanisms, and genetic variation underlying the trait. In particular, exciting discoveries have been made about the signaling pathways that are responsible for controlling the production of winged versus wingless morphs, including ecdysone, dopamine, and insulin signaling, and about how specific genes such as REPTOR2 and vestigial are regulated to control winglessness. Future work will likely focus on the role of epigenetic mechanisms, as well as developing transgenic tools for more thoroughly dissecting the role of candidate plasticity-related genes.

Dopamine mediates the pea aphid wing plasticity.

Many organisms exhibit phenotypic plasticity, in which developmental processes result in different phenotypes depending on their environmental context. Here we focus on the molecular mechanisms underlying that environmental response. Pea aphids (Acyrthosiphon pisum) exhibit a wing dimorphism, in which pea aphid mothers produce winged or wingless daughters when exposed to a crowded or low-density environment, respectively. We investigated the role of dopamine in mediating this wing plasticity, motivated by a previous study that found higher dopamine titres in wingless- versus winged-producing aphid mothers. In this study, we found that manipulating dopamine levels in aphid mothers affected the numbers of winged offspring they produced. Specifically, asexual female adults injected with a dopamine agonist produced a lower percentage of winged offspring, while asexual females injected with a dopamine antagonist produced a higher percentage of winged offspring, matching expectations based on the titre difference. We also found that genes involved in dopamine synthesis, degradation and signalling were not differentially expressed between wingless- and winged-producing aphids. This result indicates that titre regulation possibly happens in a non-transcriptional manner or that sampling of additional timepoints or tissues is necessary. Overall, our work emphasizes that dopamine is an important component of how organisms process information about their environments.

Aphid male wing polymorphisms are transient and have evolved repeatedly.

Polymorphic phenotypes have long been used to examine the maintenance of genetic variation within and between species. Most studies have focused on persistent polymorphisms, which are retained across species boundaries, and their positive effects on speciation rates. Far less is known about the macroevolutionary impacts of more transient polymorphisms, which are also common. Here we investigated male wing polymorphisms in aphids. We estimated the phylogenetic history of wing states across species, along with several other traits that could affect wing evolution. We found that male wing polymorphisms are transient: they are found in only ~4% of extant species, but have likely evolved repeatedly across the phylogeny. We reason that the repeated evolution of transient polymorphisms might be facilitated by the existence of the asexual female wing plasticity, which is common across aphids, and would maintain the wing development program even in species with wingless males. We also discovered that male wingedness correlates positively with host plant alternation and host plant breadth, and that winged morphs and wing polymorphisms may be associated with higher speciation rates. Our results provide new evolutionary insights into this well-studied group and suggest that even transient polymorphisms may impact species diversification rates.

Pea aphid winged and wingless males exhibit reproductive, gene expression, and lipid metabolism differences.

Alternative, intraspecific phenotypes offer an opportunity to identify the mechanistic basis of differences associated with distinctive life history strategies. Wing dimorphic insects, in which both flight-capable and flight-incapable individuals occur in the same population, are particularly well-studied in terms of why and how the morphs trade off flight for reproduction. Yet despite a wealth of studies examining the differences between female morphs, little is known about male differences, which could arise from different causes than those acting on females. Here we examined reproductive, gene expression, and biochemical differences between pea aphid (Acyrthosiphon pisum) winged and wingless males. We find that winged males are competitively superior in one-on-one mating circumstances, but wingless males reach reproductive maturity faster and have larger testes. We suggest that males tradeoff increased local matings with concurrent possible inbreeding for outbreeding and increased ability to find mates. At the mechanistic level, differential gene expression between the morphs revealed a possible role for activin and insulin signaling in morph differences; it also highlighted genes not previously identified as being functionally important in wing polymorphism, such as genes likely involved in sperm production. Further, we find that winged males have higher lipid levels, consistent with their use as flight fuel, but we find no consistent patterns of different levels of activity among five enzymes associated with lipid biosynthesis. Overall, our analyses provide evidence that winged versus wingless males exhibit differences at the reproductive, gene expression, and biochemical levels, expanding the field's understanding of the functional aspects of morph differences.

Male-Biased microRNA Discovery in the Pea Aphid.

Epigenetic mechanisms modulate gene expression levels during development, shaping how a single genome produces a diversity of phenotypes. Here, we begin to explore the epigenetic regulation of sexual dimorphism in pea aphids (Acyrthosiphon pisum) by focusing on microRNAs. Previous analyses of microRNAs in aphids have focused solely on females, so we performed deep sequencing of a sample containing early-stage males. We used this sample, plus samples from Genbank, to find 207 novel pea aphid microRNA coding loci. We localized microRNA loci to a chromosome-level assembly of the pea aphid genome and found that those on the X chromosome have lower overall expression compared to those on autosomes. We then identified a set of 19 putative male-biased microRNAs and found them enriched on the X chromosome. Finally, we performed protein-coding RNA-Seq of first instar female and male pea aphids to identify genes with lower expression in males. 10 of these genes were predicted targets of the 19 male-biased microRNAs. Our study provides the most complete set of microRNAs in the pea aphid to date and serves as foundational work for future studies on the epigenetic control of sexual dimorphism.

Wing plasticity and associated gene expression varies across the pea aphid biotype complex.

Developmental phenotypic plasticity is a widespread phenomenon that allows organisms to produce different adult phenotypes in response to different environments. Investigating the molecular mechanisms underlying plasticity has the potential to reveal the precise changes that lead to the evolution of plasticity as a phenotype. Here, we study wing plasticity in multiple host-plant adapted populations of pea aphids as a model for understanding adaptation to different environments within a single species. We describe the wing plasticity response of different "biotypes" to a crowded environment and find differences within as well as among biotypes. We then use transcriptome profiling to compare a highly plastic pea aphid genotype to one that shows no plasticity and find that the latter exhibits no gene expression differences between environments. We conclude that the loss of plasticity has been accompanied by a loss of differential gene expression and therefore that genetic assimilation has occurred. Our gene expression results generalize previous studies that have shown a correlation between plasticity in morphology and gene expression.

Intraspecific variation in symbiont density in an insect-microbe symbiosis.

Many insects host vertically transmitted microbes, which can confer benefits to their hosts but are costly to maintain and regulate. A key feature of these symbioses is variation: for example, symbiont density can vary among host and symbiont genotypes. However, the evolutionary forces maintaining this variation remain unclear. We studied variation in symbiont density using the pea aphid (Acyrthosiphon pisum) and the bacterium Regiella insecticola, a symbiont that can protect its host against fungal pathogens. We found that relative symbiont density varies both between two Regiella phylogenetic clades and among aphid "biotypes." Higher density symbiont infections are correlated with stronger survival costs, but variation in density has little effect on the protection Regiella provides against fungi. Instead, we found that in some aphid genotypes, a dramatic decline in symbiont density precedes the loss of a symbiont infection. Together, our data suggest that the optimal density of a symbiont infection is likely different from the perspective of aphid and microbial fitness. Regiella might prevent loss by maintaining high within-host densities, but hosts do not appear to benefit from higher symbiont numbers and may be advantaged by losing costly symbionts in certain environments. The standing variation in symbiont density observed in natural populations could therefore be maintained by antagonistic coevolutionary interactions between hosts and their symbiotic microbes.

Extreme developmental instability associated with wing plasticity in pea aphids.

A key focus of evolutionary developmental biology is on how phenotypic diversity is generated. In particular, both plasticity and developmental instability contribute to phenotypic variation among genetically identical individuals, but the interactions between the two phenomena and their general fitness impacts are unclear. We discovered a striking example of asymmetry in pea aphids: the presence of wings on one side and the complete or partial absence of wings on the opposite side. We used this asymmetric phenotype to study the connection between plasticity, developmental instability and fitness. We found that this asymmetric wing development (i) occurred equally on both sides and thus is a developmental instability; (ii) is present in some genetically unique lines but not others, and thus has a genetic basis; and (iii) has intermediate levels of fecundity, and thus does not necessarily have negative fitness consequences. We conclude that this dramatic asymmetry may arise from incomplete switching between developmental targets, linking plasticity and developmental instability. We suspect that what we have observed may be a more widespread phenomenon, occurring across species that routinely produce distinct, alternative phenotypes.

Correction to: The genome sequence of the grape phylloxera provides insights into the evolution, adaptation, and invasion routes of an iconic pest.

An amendment to this paper has been published and can be accessed via the original article.

The genome sequence of the grape phylloxera provides insights into the evolution, adaptation, and invasion routes of an iconic pest.

BACKGROUND: Although native to North America, the invasion of the aphid-like grape phylloxera Daktulosphaira vitifoliae across the globe altered the course of grape cultivation. For the past 150 years, viticulture relied on grafting-resistant North American Vitis species as rootstocks, thereby limiting genetic stocks tolerant to other stressors such as pathogens and climate change. Limited understanding of the insect genetics resulted in successive outbreaks across the globe when rootstocks failed. Here we report the 294-Mb genome of D. vitifoliae as a basic tool to understand host plant manipulation, nutritional endosymbiosis, and enhance global viticulture. RESULTS: Using a combination of genome, RNA, and population resequencing, we found grape phylloxera showed high duplication rates since its common ancestor with aphids, but similarity in most metabolic genes, despite lacking obligate nutritional symbioses and feeding from parenchyma. Similarly, no enrichment occurred in development genes in relation to viviparity. However, phylloxera evolved > 2700 unique genes that resemble putative effectors and are active during feeding. Population sequencing revealed the global invasion began from the upper Mississippi River in North America, spread to Europe and from there to the rest of the world. CONCLUSIONS: The grape phylloxera genome reveals genetic architecture relative to the evolution of nutritional endosymbiosis, viviparity, and herbivory. The extraordinary expansion in effector genes also suggests novel adaptations to plant feeding and how insects induce complex plant phenotypes, for instance galls. Finally, our understanding of the origin of this invasive species and its genome provide genetics resources to alleviate rootstock bottlenecks restricting the advancement of viticulture.

A large genomic insertion containing a duplicated follistatin gene is linked to the pea aphid male wing dimorphism.

Wing dimorphisms have long served as models for examining the ecological and evolutionary tradeoffs associated with alternative phenotypes. Here, we investigated the genetic cause of the pea aphid (Acyrthosiphon pisum) male wing dimorphism, wherein males exhibit one of two morphologies that differ in correlated traits that include the presence or absence of wings. We mapped this trait difference to a single genomic region and, using third generation, long-read sequencing, we identified a 120 kb insertion in the wingless allele. This insertion includes a duplicated follistatin gene, which is a strong candidate gene in the minimal mapped interval to cause the dimorphism. We found that both alleles were present prior to pea aphid biotype lineage diversification, we estimated that the insertion occurred millions of years ago, and we propose that both alleles have been maintained in the species, likely due to balancing selection.

Host and symbiont genetic determinants of nutritional phenotype in a natural population of the pea aphid.

A defining feature of the nutritional ecology of plant sap-feeding insects is that the dietary deficit of essential amino acids (EAAs) in plant sap is supplemented by EAA-provisioning microbial symbionts in the insect. Here, we demonstrated substantial variation in the nutritional phenotype of 208 genotypes of the pea aphid Acyrthosiphon pisum collected from a natural population. Specifically, the genotypes varied in performance (larval growth rates) on four test diets lacking the EAAs arginine, histidine and methionine or aromatic EAAs (phenylalanine and tryptophan), relative to the diet containing all EAAs. These data indicate that EAA supply from the symbiotic bacteria Buchnera can meet total aphid nutritional demand for only a subset of the EAA/aphid genotype combinations. We then correlated single nucleotide polymorphisms (SNPs) identified in the aphid and Buchnera genomes by reduced genome sequencing against aphid performance for each EAA deletion diet. This yielded significant associations between performance on the histidine-free diet and Buchnera SNPs, including metabolism genes predicted to influence histidine biosynthesis. Aphid genetic correlates of performance were obtained for all four deletion diets, with associations on the arginine-free diet and aromatic-free diets dominated by genes functioning in the regulation of metabolic and cellular processes. The specific aphid genes associated with performance on different EAA deletion diets are largely nonoverlapping, indicating some independence in the regulatory circuits determining aphid phenotype for the different EAAs. This study demonstrates how variation in the phenotype of associations collected from natural populations can be applied to elucidate the genetic basis of ecologically important traits in systems intractable to traditional forward/reverse genetic techniques.

Expression profiling of winged- and wingless-destined pea aphid embryos implicates insulin/insulin growth factor signaling in morph differences.

Developmental plasticity allows the matching of adult phenotypes to different environments. Although considerable effort has gone into understanding the evolution and ecology of plasticity, less is known about its developmental genetic basis. We focused on the pea aphid wing polyphenism, in which high- or low-density environments cause viviparous aphid mothers to produce winged or wingless offspring, respectively. Maternally provided ecdysone signals to embryos to be winged or wingless, but it is unknown how embryos respond to that signal. We used transcriptional profiling to investigate the gene expression state of winged-destined (WD) and wingless-destined (WLD) embryos at two developmental stages. We found that embryos differed in a small number of genes, and that gene sets were enriched for the insulin-signaling portion of the FoxO pathway. To look for a global signature of insulin signaling, we examined the size and stage of WD and WLD embryos but found no differences. These data suggest the hypothesis that FoxO signaling is important for morph development in a tissue-specific manner. We posit that maternally supplied ecdysone affects embryonic FoxO signaling, which ultimately plays a role in alternative morph development. Our study is one of an increasing number that implicate insulin signaling in the generation of alternative environmentally induced morphologies.

A Laterally Transferred Viral Gene Modifies Aphid Wing Plasticity.

Organisms often respond to changing environments by altering development of particular traits. These plastic traits exhibit genetic variation; i.e., genotypes respond differently to the same environmental cues. Theoretical studies have demonstrated the importance of this variation, which is targeted by natural selection, in adapting plastic responses to maximize fitness [1, 2]. However, little is known about the underlying genetic mechanisms. We identify two laterally transferred genes that contribute to variation in a classic example of phenotypic plasticity: the pea aphid's ability to produce winged offspring in response to crowding. We discovered that aphid genotypes vary extensively for this trait and that aphid genes of viral origin are upregulated in response to crowding solely in highly inducible genotypes. We knocked down expression of these genes to demonstrate their functional role in wing plasticity. Through phylogenetic analysis, we found that these genes likely originated from a virus that infects rosy apple aphids and causes their hosts to produce winged offspring [3]. The function of these genes has therefore been retained following transfer to pea aphids. Our results uncover a novel role for co-opted viral genes, demonstrating that they are used to modulate ecologically relevant, plastic phenotypes. Our findings also address a critical question about the evolution of environmentally sensitive traits: whether the genes that control the expression of plastic traits also underlie variation in plasticity. The genes we identify originated from outside aphids themselves, and thus, our work shows that genes formerly unrelated to plasticity can fine-tune the strength of plastic responses to the environment.

Molecular Mechanisms of Wing Polymorphism in Insects.

Many insects are capable of developing into either long-winged or short-winged (or wingless) morphs, which enables them to rapidly match heterogeneous environments. Thus, the wing polymorphism is an adaptation at the root of their ecological success. Wing polymorphism is orchestrated at various levels, starting with the insect's perception of environmental cues, then signal transduction and signal execution, and ultimately the transmitting of signals into physiological adaption in accordance with the particular morph produced. Juvenile hormone and ecdysteroid pathways have long been proposed to regulate wing polymorphism in insects, but rigorous experimental evidence is lacking. The breakthrough findings of ecdysone receptor regulation on transgenerational wing dimorphism in the aphid Acyrthosiphon pisum and of insulin signaling in the planthopper Nilaparvata lugens greatly broaden our understanding of wing polymorphism at the molecular level. Recently, the advent of high-throughput sequencing coupled with functional genomics provides powerful genetic tools for future insights into the molecular bases underlying wing polymorphism in insects.

Extensive Differential Splicing Underlies Phenotypically Plastic Aphid Morphs.

Phenotypic plasticity results in a diversity of phenotypes from a single genotype in response to environmental cues. To understand the molecular basis of phenotypic plasticity, studies have focused on differential gene expression levels between environmentally determined phenotypes. The extent of alternative splicing differences among environmentally determined phenotypes has largely been understudied. Here, we study alternative splicing differences among plastically produced morphs of the pea aphid using RNA-sequence data. Pea aphids express two separate polyphenisms (plasticity with discrete phenotypes): a wing polyphenism consisting of winged and wingless females and a reproduction polyphenism consisting of asexual and sexual females. We find that pea aphids alternatively splice 34% of their genes, a high percentage for invertebrates. We also find that there is extensive use of differential spliced events between genetically identical, polyphenic females. These differentially spliced events are enriched for exon skipping and mutually exclusive exon events that maintain the open reading frame, suggesting that polyphenic morphs use alternative splicing to produce phenotype-biased proteins. Many genes that are differentially spliced between polyphenic morphs have putative functions associated with their respective phenotypes. We find that the majority of differentially spliced genes is not differentially expressed genes. Our results provide a rich candidate gene list for future functional studies that would not have been previously considered based solely on gene expression studies, such as ensconsin in the reproductive polyphenism, and CAKI in the wing polyphenism. Overall, this study suggests an important role for alternative splicing in the expression of environmentally determined phenotypes.

Diurnal and developmental differences in gene expression between adult dispersing and flightless morphs of the wing polymorphic cricket, Gryllus firmus: Implications for life-history evolution.

The functional basis of life history adaptation is a key topic of research in life history evolution. Studies of wing-polymorphism in the cricket Gryllus firmus have played a prominent role in this field. However, prior in-depth investigations of morph specialization have primarily focused on a single hormone, juvenile hormone, and a single aspect of intermediary metabolism, the fatty-acid biosynthetic component of lipid metabolism. Moreover, the role of diurnal variation in life history adaptation in G. firmus has been understudied, as is the case for organisms in general. Here, we identify genes whose expression differs consistently between the morphs independent of time-of-day during early adulthood, as well as genes that exhibit a strong pattern of morph-specific diurnal expression. We find strong, consistent, morph-specific differences in the expression of genes involved in endocrine regulation, carbohydrate and lipid metabolism, and immunity - in particular, in the expression of an insulin-like-peptide precursor gene and genes involved in triglyceride production. We also find that the flight-capable morph exhibited a substantially greater number of genes exhibiting diurnal change in gene expression compared with the flightless morph, correlated with the greater circadian change in the hemolymph juvenile titer in the dispersing morph. In fact, diurnal differences in expression within the dispersing morph at different times of the day were significantly greater in magnitude than differences between dispersing and flightless morphs at the same time-of-day. These results provide important baseline information regarding the potential role of variable gene expression on life history specialization in morphs of G. firmus, and the first information on genetically-variable, diurnal change in gene expression, associated with a key life history polymorphism. These results also suggest the existence of prominent morph-specific circadian differences in gene expression in G. firmus, possibly caused by the morph-specific circadian rhythm in the juvenile hormone titer.

Disentangling the Causes for Faster-X Evolution in Aphids.

The faster evolution of X chromosomes has been documented in several species, and results from the increased efficiency of selection on recessive alleles in hemizygous males and/or from increased drift due to the smaller effective population size of X chromosomes. Aphids are excellent models for evaluating the importance of selection in faster-X evolution because their peculiar life cycle and unusual inheritance of sex chromosomes should generally lead to equivalent effective population sizes for X and autosomes. Because we lack a high-density genetic map for the pea aphid, whose complete genome has been sequenced, we first assigned its entire genome to the X or autosomes based on ratios of sequencing depth in males (X0) to females (XX). Then, we computed nonsynonymous to synonymous substitutions ratios (dN/dS) for the pea aphid gene set and found faster evolution of X-linked genes. Our analyses of substitution rates, together with polymorphism and expression data, showed that relaxed selection is likely to be the greatest contributor to faster-X because a large fraction of X-linked genes are expressed at low rates and thus escape selection. Yet, a minor role for positive selection is also suggested by the difference between substitution rates for X and autosomes for male-biased genes (but not for asexual female-biased genes) and by lower Tajima's D for X-linked compared with autosomal genes with highly male-biased expression patterns. This study highlights the relevance of organisms displaying alternative chromosomal inheritance to the understanding of forces shaping genome evolution.