Search for: All records

The evolution of sexual secondary characteristics necessitates regulatory factors that confer sexual identity to differentiating tissues and cells. InColias eurythemebutterflies, males exhibit two specialized wing scale types—ultraviolet-iridescent (UVI) and spatulate scales—which are absent in females and likely integral to male courtship behavior. This study investigates the regulatory mechanisms and single-nucleus transcriptomics underlying these two sexually dimorphic cell types during wing development. We show thatDoublesex(Dsx) expression is itself dimorphic and required to repress the UVI cell state in females, while unexpectedly, UVI activation in males is independent fromDsx. In the melanic marginal band,Dsxis required in each sex to enforce the presence of spatulate scales in males, and their absence in females. Single-nucleus RNAseq reveals that UVI and spatulate scale cell precursors each show distinctive gene expression profiles at 40% of pupal development, with marker genes that include regulators of transcription, cell signaling, cytoskeletal patterning, and chitin secretion. Both male-specific cell types share a low expression of theBric-a-brac(Bab) transcription factor, a key repressor of the UVI fate. Bab ChIP-seq profiling suggests that Bab binds thecis-regulatory regions of gene markers associated to UVI fate, including potential effector genes involved in the regulation of cytoskeletal processes and chitin secretion, and loci showing signatures of recent selective sweeps in a UVI-polymorphic population. These findings open new avenues for exploring wing patterning and scale development, shedding light on the mechanisms driving the specification of sex-specific cell states and the differentiation of specialized cell ultrastructures. 

Long noncoding RNAs (lncRNAs) are transcribed elements increasingly recognized for their roles in regulating gene expression. Thus far, however, we have little understanding of how lncRNAs contribute to evolution and adaptation. Here, we show that a conserved lncRNA,ivory, is an important color patterning gene in the buckeye butterflyJunonia coenia.ivoryoverlaps withcortex, a locus linked to multiple cases of crypsis and mimicry in Lepidoptera. Along with a companion paper by Livraghi et al., we argue thativory, notcortex, is the color pattern gene of interest at this locus. InJ. coenia, a cluster ofcis-regulatory elements (CREs) in the first intron ofivoryare genetically associated with natural variation in seasonal color pattern plasticity, and targeted deletions of these CREs phenocopy seasonal phenotypes. Deletions of differentivoryCREs produce other distinct phenotypes as well, including loss of melanic eyespot rings, and positive and negative changes in overall wing pigmentation. We show that the color pattern transcription factors Spineless, Bric-a-brac, and Ftz-f1 bind to theivorypromoter during wing pattern development, suggesting that they directly regulateivory. This case study demonstrates howcis-regulation of a single noncoding RNA can exert diverse and nuanced effects on the evolution and development of color patterns, including modulating seasonally plastic color patterns. 

Evolutionary variation in the wing pigmentation of butterflies and moths offers striking examples of adaptation by crypsis and mimicry. Thecortexlocus has been independently mapped as the locus controlling color polymorphisms in 15 lepidopteran species, suggesting that it acts as a genomic hotspot for the diversification of wing patterns, but functional validation through protein-coding knockouts has proven difficult to obtain. Our study unveils the role of a long noncoding RNA (lncRNA) which we nameivory, transcribed from thecortexlocus, in modulating color patterning in butterflies. Strikingly,ivoryexpression prefigures most melanic patterns during pupal development, suggesting an early developmental role in specifying scale identity. To test this, we generated CRISPR mosaic knock-outs in five nymphalid butterfly species and show thativorymutagenesis yields transformations of dark pigmented scales into white or light-colored scales. Genotyping ofVanessa carduigermline mutants associates these phenotypes to small on-target deletions at the conserved first exon ofivory. In contrast,cortexgermline mutant butterflies with confirmed null alleles lack any wing phenotype and exclude a color patterning role for this adjacent gene. Overall, these results show that a lncRNA gene acts as a master switch of color pattern specification and played key roles in the adaptive diversification of wing patterns in butterflies. 

Ancient multifunctional regulatory elements underlie the evolution of butterfly wing color patterns. 

Abstract The painted lady butterfly, Vanessa cardui, has the longest migration routes, the widest hostplant diversity, and one of the most complex wing patterns of any insect. Due to minimal culturing requirements, easily characterized wing pattern elements, and technical feasibility of CRISPR/Cas9 genome editing, V. cardui is emerging as a functional genomics model for diverse research programs. Here, we report a high-quality, annotated genome assembly of the V. cardui genome, generated using 84× coverage of PacBio long-read data, which we assembled into 205 contigs with a total length of 425.4 Mb (N50 = 10.3 Mb). The genome was very complete (single-copy complete Benchmarking Universal Single-Copy Orthologs [BUSCO] 97%), with contigs assembled into presumptive chromosomes using synteny analyses. Our annotation used embryonic, larval, and pupal transcriptomes, and 20 transcriptomes across five different wing developmental stages. Gene annotations showed a high level of accuracy and completeness, with 14,437 predicted protein-coding genes. This annotated genome assembly constitutes an important resource for diverse functional genomic studies ranging from the developmental genetic basis of butterfly color pattern, to coevolution with diverse hostplants. 

Characterizing the genetic complexity of adaptation and trait evolution is a major emphasis of evolutionary biology and genetics. Incongruent findings from genetic studies have resulted in conceptual models ranging from a few large-effect loci to massively polygenic architectures. Here, we combine chromatin immunoprecipitation sequencing, Hi-C, RNA sequencing, and 40 whole-genome sequences from Heliconius butterflies to show that red color pattern diversification occurred via many genomic loci. We find that the red wing pattern master regulatory transcription factor Optix binds dozens of loci also under selection, which frequently form three-dimensional adaptive hubs with selection acting on multiple physically interacting genes. Many Optix-bound genes under selection are tied to pigmentation and wing development, and these loci collectively maintain separation between adaptive red color pattern phenotypes in natural populations. We propose a model of trait evolution where functional connections between loci may resolve much of the disparity between large-effect and polygenic evolutionary models. 

Developmental plasticity allows genomes to encode multiple distinct phenotypes that can be differentially manifested in response to environmental cues. Alternative plastic phenotypes can be selected through a process called genetic assimilation, although the mechanisms are still poorly understood. We assimilated a seasonal wing color phenotype in a naturally plastic population of butterflies ( Junonia coenia ) and characterized three responsible genes. Endocrine assays and chromatin accessibility and conformation analyses showed that the transition of wing coloration from an environmentally determined trait to a predominantly genetic trait occurred through selection for regulatory alleles of downstream wing-patterning genes. This mode of genetic evolution is likely favored by selection because it allows tissue- and trait-specific tuning of reaction norms without affecting core cue detection or transduction mechanisms.