Review of the Narrow-Banded Hawkmoth, Neogurelca montana (Rothschild & Jordan, 1915) (Lepidoptera: Sphingidae) in China, with Morphological and Phylogenetic Analysis.

Neogurelca montana (Rothschild & Jordan, 1915) is a species of the genus Neogurelca Hogenes & Treadaway, 1993, that was previously known from Sichuan, Yunnan, and Tibet, China. Recently, however, this species was also found in Beijing and Hebei. These populations differ from those in southwest China in body colour and the shape of the yellow patches of the hindwing-a paler body colour and triangular patches in the former and darker body colour and fan-like patches in the latter. Wing morphology, male and female genitalia, and molecular evidence (DNA barcodes) were analysed for the different localities of this species and three other Neogurelca species-N. hyas, N. himachala, and N. masuriensis. Our molecular data support the Beijing population of montana as a valid subspecies, which we describe as N. montana taihangensisssp. nov. Wing and genital morphology confirm the molecular conclusions. We also collected larvae of the new subspecies in the Beijing suburbs and describe its life history and larval hosts and compare them with those of N. himachala.

High-quality haploid genomes corroborate 29 chromosomes and highly conserved synteny of genes in Hyles hawkmoths (Lepidoptera: Sphingidae).

BACKGROUND: Morphological and traditional genetic studies of the young Pliocene genus Hyles have led to the understanding that despite its importance for taxonomy, phenotypic similarity of wing patterns does not correlate with phylogenetic relationship. To gain insights into various aspects of speciation in the Spurge Hawkmoth (Hyles euphorbiae), we assembled a chromosome-level genome and investigated some of its characteristics. RESULTS: The genome of a male H. euphorbiae was sequenced using PacBio and Hi-C data, yielding a 504 Mb assembly (scaffold N50 of 18.2 Mb) with 99.9% of data represented by the 29 largest scaffolds forming the haploid chromosome set. Consistent with this, FISH analysis of the karyotype revealed n = 29 chromosomes and a WZ/ZZ (female/male) sex chromosome system. Estimates of chromosome length based on the karyotype image provided an additional quality metric of assembled chromosome size. Rescaffolding the published male H. vespertilio genome resulted in a high-quality assembly (651 Mb, scaffold N50 of 22 Mb) with 98% of sequence data in the 29 chromosomes. The larger genome size of H. vespertilio (average 1C DNA value of 562 Mb) was accompanied by a proportional increase in repeats from 45% in H. euphorbiae (measured as 472 Mb) to almost 55% in H. vespertilio. Several wing pattern genes were found on the same chromosomes in the two species, with varying amounts and positions of repetitive elements and inversions possibly corrupting their function. CONCLUSIONS: Our two-fold comparative genomics approach revealed high gene synteny of the Hyles genomes to other Sphingidae and high correspondence to intact Merian elements, the ancestral linkage groups of Lepidoptera, with the exception of three simple fusion events. We propose a standardized approach for genome taxonomy using nucleotide homology via scaffold chaining as the primary tool combined with Oxford plots based on Merian elements to infer and visualize directionality of chromosomal rearrangements. The identification of wing pattern genes promises future understanding of the evolution of forewing patterns in the genus Hyles, although further sequencing data from more individuals are needed. The genomic data obtained provide additional reliable references for further comparative studies in hawkmoths (Sphingidae).

Distal-less and spalt are distal organisers of pierid wing patterns.

Two genes, Distal-less (Dll) and spalt (sal), are known to be involved in establishing nymphalid butterfly wing patterns. They function in several ways: in the differentiation of the eyespot's central signalling cells, or foci; in the differentiation of the surrounding black disc; in overall scale melanisation (Dll); and in elaborating marginal patterns, such as parafocal elements. However, little is known about the functions of these genes in the development of wing patterns in other butterfly families. Here, we study the expression and function of Dll and sal in the development of spots and other melanic wing patterns of the Indian cabbage white, Pieris canidia, a pierid butterfly. In P. canidia, both Dll and Sal proteins are expressed in the scale-building cells at the wing tips, in chevron patterns along the pupal wing margins, and in areas of future scale melanisation. Additionally, Sal alone is expressed in the future black spots. CRISPR knockouts of Dll and sal showed that each gene is required for the development of melanic wing pattern elements, and repressing pteridine granule formation, in the areas where they are expressed. We conclude that both genes likely play ancestral roles in organising distal butterfly wing patterns, across pierid and nymphalid butterflies, but are unlikely to be differentiating signalling centres in pierids black spots. The genetic and developmental mechanisms that set up the location of spots and eyespots are likely distinct in each lineage.

Scale granules and colours: Sexual dimorphism in Trichonis (Lepidoptera: Lycaenidae, Theclinae).

A large fraction of dorsal wing surface ground scales show an unusual granulated nature, composed of material apparently extruded from the scale lumen in male individuals of both Trichonis Hewitson, 1865 species in the tribe Eumaeini, a rare Guyanian-Amazonian genus. Only a few not-granulated male specimens are known, females are not granulated. The granulated scales are investigated by various microscopic (optical, scanning and transmission electron microscopy, focused ion beam lamella cutting) and spectroscopic (optical reflectance, energy-dispersive X-ray (EDS), Raman) techniques. The characteristic blue colour unique in the South American representatives of the tribe is documented and analysed. EDS spectra show that the granules contain additional calcium and oxygen as compared with the un-granulated regions of the same scale. Electron diffraction (inside the TEM) did not reveal any crystalline component in the granules. The granulated wing surfaces of the males exhibit a UV absorption band at 280 nm, characteristic for biogenic CaCO3; therefore, the material of the granules is tentatively identified as CaCO3. It is shown that the granules influence the optical properties of the dorsal wing surface resulting in a characteristic spectrum.

Novel Doublesex Duplication Associated with Sexually Dimorphic Development of Dogface Butterfly Wings.

Sexually dimorphic development is responsible for some of the most remarkable phenotypic variation found in nature. Alternative splicing of the transcription factor gene doublesex (dsx) is a highly conserved developmental switch controlling the expression of sex-specific pathways. Here, we leverage sex-specific differences in butterfly wing color pattern to characterize the genetic basis of sexually dimorphic development. We use RNA-seq, immunolocalization, and motif binding site analysis to test specific predictions about the role of dsx in the development of structurally based ultraviolet (UV) wing patterns in Zerene cesonia (Southern Dogface). Unexpectedly, we discover a novel duplication of dsx that shows a sex-specific burst of expression associated with the sexually dimorphic UV coloration. The derived copy consists of a single exon that encodes a DNA binding but no protein-binding domain and has experienced rapid amino-acid divergence. We propose the novel dsx paralog may suppress UV scale differentiation in females, which is supported by an excess of Dsx-binding sites at cytoskeletal and chitin-related genes with sex-biased expression. These findings illustrate the molecular flexibility of the dsx gene in mediating the differentiation of secondary sexual characteristics.

Contributions to the knowledge of Neotropical Lycaenidae: taxonomy of Johnsonita/ Salazar amp; Constantino, 1995 with descriptions of seven new species (Theclinae: Eumaeini).

The Transandean-Andean genus Johnsonita Salazar Constantino, 1995 is revised on the basis of wing and genitalia morphology. Apart from eight species formerly placed in Johnsonita (Thecla assula Draudt, 1919; Thecla auda Hewitson, 1867; Thecla catadupa Hewitson, 1869; Thecla chaluma Schaus, 1902, Thecla chlamydem Druce, 1907; Thecla pardoa d'Abrera, 1995; Johnsonita johnsoni Salazar Constantino, 1995 and Johnsonita johnbanksi Bálint, 2003), seven new species are described: Johnsonita carpia Bálint, Boyer Pyrcz, sp. n. (Ecuador), Johnsonita iacinta Bálint, Boyer Pyrcz, sp. n. (Peru), Johnsonita ianusca Bálint, Lorenc-Brudecka Pyrcz, sp. n. (Ecuador), Johnsonita oxalida Bálint, Boyer Pyrcz, sp. n. (Peru), Johnsonita subcunicula Bálint, Cerdeña Pyrcz, sp. n. (Peru), Johnsonita turquisca Bálint, Boyer Pyrcz, sp. n. (Ecuador) and Johnsonita zubkova Bálint, Boyer Lorenc-Brudecka, sp. n. (Peru). One species, erroneously recorded from Bolivia under the name Thecla chaluma Schaus, 1902, is not formally described due to insufficient material. An identification key, type material revision, descriptions or redescriptions, and diagnosis for all the taxa recognized are given. Lectotypes for the nominal species Thecla assula Draudt, 1919 and Thecla chlamydem Druce, 1907 are designated. Male secondary wing characters are tabulated. A brief discussion on classification, life-history and male secondary characters of Johnsonita is presented.

Revealing the specific status of Mellicta distans Higgins, 1955, stat. n. (Lepidoptera, Nymphalidae) with morphological and molecular characters.

A detailed comparison of the taxa Mellicta aurelia distans Higgins, 1955, M. aurelia aurelia (Nickerl, 1850), and M. alatauica (Staudinger, 1881) was performed. The geographic isolation of M. aurelia distans, its habitat preferences, as well as clear differences in the female and male genitalia compared with M. aurelia aurelia, indicate that the taxon distans is an independent species. Molecular data based on sequencing of cytochrome oxidase subunit I (COI) of the mitochondrial DNA confirmed our morphological results. Both the morphological and the molecular results showed a high degree of divergence of M. alatauica from the related species.

The enigmatic case of the genus Argyresthia in the Azores Islands (Lepidoptera: Argyresthiidae).

The species composition of the genus Argyresthia Hübner, 1825 in the Azores is examined. Argyresthia brumella, sp. nov., is described and illustrated from Terceira and Flores Islands. Argyresthia minusculella Rebel, 1940, syn. nov. and Tinea poecilella Rebel, 1940, syn. nov. are synonymized with Argyresthia atlanticella Rebel, 1940. The high variability of A. atlanticella is revealed through the polymorphic wing pattern and the intraspecific genetic divergence of the DNA barcode COI in the specimens examined.

Are the Yellow and Red Marked Club-Tail Losaria coon the Same Species?

Losaria coon (Fabricius, 1793) is currently comprised of ten subspecies, which were originally described under two names, Papilio coon and P. doubledayi before 1909, when they were combined as one species. The main difference between them is the colour of abdomen and hindwing subterminal spots-yellow in coon and red in doubledayi. Wing morphology, male and female genitalia, and molecular evidence (DNA barcodes) were analysed for multiple subspecies of L. coon and three other Losaria species-rhodifer, neptunus, and palu. Our molecular data support the separation of L. coon and L. doubledayi stat. rev. as two distinct species, with L. rhodifer positioned between them in phylogenetic analyses. Wing morphology and genitalic structures also confirm the molecular conclusions. Our findings divide L. coon into two species occupying different geographic ranges: with L. coon restricted to southern Sumatra, Java, and Bawean Island, while L. doubledayi occurs widely in regions from North India to northern Sumatra, including Hainan and Nicobar Islands. Hence, future conservation efforts must reassess the status and threat factors of the two species to form updated strategies.

How many scales on the wings? A case study based on Colias crocea (Geoffroy, 1785) (Hexapoda: Lepidoptera, Pieridae).

The covering by scales of the wings of Lepidoptera contributes to multiple functions that are critical for their survival and reproduction. In order to gain a better understanding about their distribution, we have exhaustively studied 4 specimens of Colias crocea (Geoffroy, 1785). We have quantified the sources of variability affecting scale density. The results indicate that the scale covering of butterfly wings may be remarkably heterogeneous, and that the importance of the sources of variability differs between forewings and hindwings. Thus, in forewing the greatest variability occurs between sectors, while in the hindwings it occurs between sides, with a higher density of scales on the underside, considerably higher (almost 19%) than on the upperside. It seems likely that this difference has an adaptive value, as the hindwing underside is more exposed (in resting position) to predators. These results are in contrast with the generally accepted notion that scale covering is uniform and homogeneous. Moreover, the cover scale density is independent of the size of the specimen and therefore an average density of scales can be attributed to this species. According to our measurements C. crocea has 312 scales/mm2 and the total number of scales per individual is about 520,000 on average.

Structural color in Junonia butterflies evolves by tuning scale lamina thickness.

In diverse organisms, nanostructures that coherently scatter light create structural color, but how such structures are built remains mysterious. We investigate the evolution and genetic regulation of butterfly scale laminae, which are simple photonic nanostructures. In a lineage of buckeye butterflies artificially selected for blue wing color, we found that thickened laminae caused a color shift from brown to blue. Deletion of the optix patterning gene also altered color via lamina thickening, revealing shared regulation of pigments and lamina thickness. Finally, we show how lamina thickness variation contributes to the color diversity that distinguishes sexes and species throughout the genus Junonia. Thus, quantitatively tuning one dimension of scale architecture facilitates both the microevolution and macroevolution of a broad spectrum of hues. Because the lamina is an intrinsic component of typical butterfly scales, our findings suggest that tuning lamina thickness is an available mechanism to create structural color across the Lepidoptera.

From iridescent blues to vibrant purples, many butterflies display dazzling 'structural colors' created not by pigments but by microscopic structures that interfere with light. For instance, the scales that coat their wings can contain thin films of chitin, the substance that normally makes the external skeleton of insects. In slim layers, however, chitin can also scatter light to produce color, the way that oil can create iridescence at the surface of water. The thickness of the film, which is encoded by the genes of the butterfly, determines what color will be produced. Yet, little is known about how common thin films are in butterflies, exactly how genetic information codes for them, and how their thickness and the colors they produce can evolve. To investigate, Thayer et al. used a technique called Helium Ion Microscopy and examined the wings of ten related species of butterflies, showing that thin film structures were present across this sample. However, the different species have evolved many different structural colors over the past millions of years by changing the thickness of the films. Next, Thayer et al. showed that this evolution could be reproduced at a faster pace in the laboratory using common buckeye butterflies. These insects mostly have brown wings, but they can have specks of blue created by thin film structures. Individuals with more blue on their wings were mated and over the course of a year, the thickness of the film structures increased by 74%, leading to shiny blue butterflies. Deleting a gene called optix from the insects also led to blue wings. Optix was already known to control the patterns of pigments in butterflies, but it now appears that it controls structural colors as well. From solar panels to new fabrics, microscopic structures that can scatter light are useful in a variety of industries. Understanding how these elements exist and evolve in organisms may help to better design them for human purposes.

Description of a new subspecies of Dodona dipoea Hewitson, [1866] (Lycaenidae: Riodininae) from S.W. Chongqing, China.

Dodona dipoea pseudokaterina subsp. nov. is described from southwestern Chongqing, China. The wing patterns of the type specimens are similar to those of D. katerina Monastyrskii Devyatkin, 2000, but the genitalic characters of both sexes show that they belong to D. dipoea Hewitson, [1866]. Adults and genitalia of both sexes of the new subspecies, and adults of the nominate subspecies are illustrated.

Unravelling the genes forming the wing pattern supergene in the polymorphic butterfly Heliconius numata.

BACKGROUND: Unravelling the genetic basis of polymorphic characters is central to our understanding of the origins and diversification of living organisms. Recently, supergenes have been implicated in a wide range of complex polymorphisms, from adaptive colouration in butterflies and fish to reproductive strategies in birds and plants. The concept of a supergene is now a hot topic in biology, and identification of its functional elements is needed to shed light on the evolution of highly divergent adaptive traits. Here, we apply different gene expression analyses to study the supergene P that controls polymorphism of mimetic wing colour patterns in the neotropical butterfly Heliconius numata. RESULTS: We performed de novo transcriptome assembly and differential expression analyses using high-throughput Illumina RNA sequencing on developing wing discs of different H. numata morphs. Within the P interval, 30 and 17 of the 191 transcripts were expressed differentially in prepupae and day-1 pupae, respectively. Among these is the gene cortex, known to play a role in wing pattern formation in Heliconius and other Lepidoptera. Our in situ hybridization experiments confirmed the relationship between cortex expression and adult wing patterns. CONCLUSIONS: This study found the majority of genes in the P interval to be expressed in the developing wing discs during the critical stages of colour pattern formation, and detect drastic changes in expression patterns in multiple genes associated with structural variants. The patterns of expression of cortex only partially recapitulate the variation in adult phenotype, suggesting that the remaining phenotypic variation could be controlled by other genes within the P interval. Although functional studies on cortex are now needed to determine its exact developmental role, our results are in accordance with the classical supergene hypothesis, whereby several genes inherited together due to tight linkage control a major developmental switch.

Plasticity and divergence in ultraviolet reflecting structures on Dogface butterfly wings.

The vast diversity of animal coloration is generated through a combination of pigment and structural colors. These colors can greatly influence the fitness and life history of an organism. Butterflies and their wing colors are an excellent model to study how these colors can impact the development and success of an organism. In this study, we explore species differences in structurally-based ultraviolet coloration in the Zerene butterfly. We show clear species differences in ultraviolet (UV) pattern and reflectance spectra. By varying larval diet, we show evidence for developmental plasticity in the structure and organization of UV reflecting scales in Zerene cesonia. We further show that feeding the larval host plant of Zerene eurydice to Z. cesonia does not result in greater similarity in scale structure or UV coloration to the sister species. These results not only demonstrate a connection between plasticity in a male ornamentation, UV wing pattern, and larval resource acquisition, but also identify candidate structural and organizational changes in wing scales responsible for the trait variation.

The contribution of nanostructures towards the wing patterning of yellow Catopsilia pomona. How it differs from the lime?

Polyphenism, an adaptation to survive throughout the year, is shown by many butterflies including Catopsilia pomona. With the variation of seasons, different morphs were found. Among all the morphs, lime exists throughout the year whereas the yellow one is available only in the winter season. The current study deciphers the colouration mechanism of yellow morph using various microscopic and spectroscopic techniques. The scanning electron microscopy analysis reveals various types of scales on the dorsal as well as the ventral side. The shape of the cover scale varies from region to region. The fine structural arrangement of the scale like window, ridge, microrib, crossrib and pigments vary throughout the wing. The pigment present in the wing is pterin as evidenced from the shape and its isolation technique. Absorption spectroscopy further confirms the presence of various types of pterin within the wing. Scanning electron microscopy discloses the dense amount of pigments within the wing. The fine structural arrangement of the wing of yellow C. pomona is compared with the yellow region of the lime C. pomona. All together, the current study describes the fine structural arrangement of the wing of yellow C. pomona and the various types of pterin which contribute towards the wing colouration. The advantage of yellow morph over lime is also discussed in this paper.

Male Bicyclus anynana Butterflies Choose Females on the Basis of Their Ventral UV-Reflective Eyespot Centers.

Butterflies often use their dorsal and ventral wing color patterns for distinct signaling functions. Color patterns on hidden dorsal wing surfaces are often used in sexual signaling, while exposed ventral patterns are often used to ward off predator attacks. At rest, however, part of the ventral forewings are often hidden by the hindwings, allowing individuals to also use the patterns on this wing surface for sexual signaling. Here, we test this hypothesis in Bicyclus anynana (Butler, Lepidoptera, Nymphalidae) butterflies by first determining the degree of sexual dimorphism in ventral forewing patterns, focusing on the eyespots, from both wet and dry season forms, and then testing the role of the larger ventral forewing eyespots of dry season females in male mate choice. We also test male investment in reproduction. We show that ventral forewing UV-reflective eyespot centers, in addition to dorsal forewing eyespot centers previously examined in this species, play a role in sexual signaling as males preferentially mated with females with their ventral eyespot centers intact instead of blocked with black paint. This male preference, however, did not translate into a detectable higher reproductive investment via a single mating toward ornamented females. This study provides an example of how ventral forewing patterns, often hidden by hindwings, are used in sexual communication, in this case by females to attract males.

Characterization of Pleurotinae, with review of Pleurota species close to P. aristella (Linnaeus) from Morocco (Lepidoptera: Gelechioidea: Oecophoridae).

Morphological traits characterizing and delimiting Pleurotinae (Oecophoridae) are provided and discussed. The evidence supports the validity of the subfamily as suggested by recent molecular studies. The Pleurota aristella (Linnaeus, 1767) species group is characterized, and six new species belonging to the group from Morocco are described: Pleurota tricolor Tabell, sp. nov., P. pellicolor Tabell, sp. nov., P. lacteella Tabell, sp. nov., P. moroccoensis Tabell, sp. nov., P. ochreopalpella Tabell, sp. nov., and P. atlasensis Tabell, sp. nov. Habitus images and label data are provided for the types of P. goundafella Zerny, 1935; P. insignella Zerny, 1935; P. ochreostrigella Baker, 1885; P. macrosella Rebel, 1900; P. staintoniella Baker, 1888; P. mauretanica Baker, 1888; and P. algeriella Baker, 1885. DNA barcodes of the new species are compared with all available Pleurotinae sequences (BIN n = 117) in BOLD.

Aristaless Controls Butterfly Wing Color Variation Used in Mimicry and Mate Choice.

Neotropical Heliconius butterflies display a diversity of warningly colored wing patterns, which serve roles in both Müllerian mimicry and mate choice behavior. Wing pattern diversity in Heliconius is controlled by a small number of unlinked, Mendelian "switch" loci [1]. One of these, termed the K locus, switches between yellow and white color patterns, important mimicry signals as well as mating cues [2-4]. Furthermore, mate preference behavior is tightly linked to this locus [4]. K controls the distribution of white versus yellow scales on the wing, with a dominant white allele and a recessive yellow allele. Here, we combine fine-scale genetic mapping, genome-wide association studies, gene expression analyses, population and comparative genomics, and genome editing with CRISPR/Cas9 to characterize the molecular basis of the K locus in Heliconius and to infer its evolutionary history. We show that white versus yellow color variation in Heliconius cydno is due to alternate haplotypes at a putative cis-regulatory element (CRE) downstream of a tandem duplication of the homeodomain transcription factor aristaless. Aristaless1 (al1) and aristaless2 (al2) are differentially regulated between white and yellow wings throughout development with elevated expression of al1 in developing white wings, suggesting a role in repressing pigmentation. Consistent with this, knockout of al1 causes white wings to become yellow. The evolution of wing color in this group has been marked by retention of the ancestral yellow color in many lineages, a single origin of white coloration in H. cydno, and subsequent introgression of white color from H. cydno into H. melpomene.

Experimental field tests of Batesian mimicry in the swallowtail butterfly Papilio polytes.

The swallowtail butterfly Papilio polytes is known for its striking resemblance in wing pattern to the toxic butterfly Pachliopta aristolochiae and is a focal system for the study of mimicry evolution. Papilio polytes females are polymorphic in wing pattern, with mimetic and nonmimetic forms, while males are monomorphic and nonmimetic. Past work invokes selection for mimicry as the driving force behind wing pattern evolution in P. polytes. However, the mimetic relationship between P. polytes and P. aristolochiae is not well understood. In order to test the mimicry hypothesis, we constructed paper replicas of mimetic and nonmimetic P. polytes and P. aristolochiae, placed them in their natural habitat, and measured bird predation on replicas. In initial trials with stationary replicas and plasticine bodies, overall predation was low and we found no differences in predation between replica types. In later trials with replicas mounted on springs and with live mealworms standing in for the butterfly's body, we found less predation on mimetic P. polytes replicas compared to nonmimetic P. polytes replicas, consistent with the predator avoidance benefits of mimicry. While our results are mixed, they generally lend support to the mimicry hypothesis as well as the idea that behavioral differences between the sexes contributed to the evolution of sexually dimorphic mimicry.

A distinct species, Dodona formosana, detected in the Dodona eugenes species complex: clarification of the taxonomic status of the Punch butterfly in Taiwan.

The Tailed Punch, Dodona eugenes, is widely distributed in East Asia with seven subspecies currently recognized. However, two of them, namely ssp. formosana and ssp. esakii found in Taiwan, are hard to distinguish from each other due to ambiguous diagnostic characters. In this study, their taxonomic status is clarified by comparing genitalia characters and phylogenetic relationships based on mitochondrial sequences, COI and COII (total 2211 bps). Our results show that there is no reliable feature to separate these two subspecies. Surprisingly we found that Dodona in Taiwan is more closely related to the Orange Punch, D. egeon, than to other subspecies of D. eugenes. Therefore, the following nomenclatural changes are proposed: Dodona eugenes formosana is revised to specific status as Dodona formosana Matsumura, 1919, stat. rev, and ssp. esakii is sunk to a junior synonym of Dodona formosanasyn. n.