A global molecular phylogeny yields insights into the dispersal and invasion history of Junonia, a butterfly genus with remarkable dispersal abilities.

The nymphalid butterfly genus Junonia has remarkable dispersal abilities. Occurring on every continent except Europe and Antarctica, Junonia are often among the only butterflies on remote oceanic islands. The biogeography of Junonia has been controversial, plagued by taxonomic disputes, small phylogenetic datasets, incomplete taxon sampling, and shared interspecific mitochondrial haplotypes. Junonia originated in Africa but its route into the New World remains unknown. Presented here is, to our knowledge, the most comprehensive Junonia phylogeny to date, using full mitogenomes and nuclear ribosomal RNA repeats from 40 of 47 described species. Junonia is monophyletic and the genus Salamis is its probable sister clade. Genetic exchange between Indo-Pacific Junonia villida and New World Junonia vestina is evident, suggesting a trans-Pacific route into the New World. However, in both phylogenies, the sister clades to most New World Junonia contain both African and Asian species. Multiple trans-Atlantic or trans-Pacificinvasions could have contributed to New World diversification. Hybridization and lateral transfer of mitogenomes, already well-documented in New World Junonia, also occurs in at least two Old World lineages (Junonia orithya/Junonia hierta and Junonia iphita/Junonia hedonia). Variation associated with reticulate evolution creates challenges for phylogenetic reconstruction, but also may have contributed to patterns of speciation and diversification in this genus.

Mathematical modeling of the eyespots in butterfly wings.

Butterfly wing color patterns are a representative model system for studying biological pattern formation, due to their two-dimensional simple structural and high inter- and intra-specific variabilities. Moreover, butterfly color patterns have demonstrated roles in mate choice, thermoregulation, and predator avoidance via disruptive coloration, attack deflection, aposematism, mimicry, and masquerade. Because of the importance of color patterns to many aspects of butterfly biology and their apparent tractability for study, color patterns have been the subjects of many attempts to model their development. Early attempts focused on generalized mechanisms of pattern formation such as reaction-diffusion, diffusion gradient, lateral inhibition, and threshold responses, without reference to any specific gene products. As candidate genes with expression patterns that resembled incipient color patterns were identified, genetic regulatory networks were proposed for color pattern formation based on gene functions inferred from other insects with wings, such as Drosophila. Particularly detailed networks incorporating the gene products, Distal-less (Dll), Engrailed (En), Hedgehog (Hh), Cubitus interruptus (Ci), Transforming growth factor-ß (TGF-ß), and Wingless (Wg), have been proposed for butterfly border ocelli (eyespots) which helps the investigation of the formation of these patterns. Thus, in this work, we develop a mathematical model including the gene products En, Hh, Ci, TGF-ß, and Wg to mimic and investigate the eyespot formation in butterflies. Our simulations show that the level of En has peaks in the inner and outer rings and the level of Ci has peaks in the inner and middle rings. The interactions among these peaks activate cells to produce white, black, and yellow pigments in the inner, middle, and outer rings, respectively, which captures the eyespot pattern of wild type Bicyclus anynana butterflies. Additionally, our simulations suggest that lack of En generates a single black spot and lack of Hh or Ci generates a single white spot, and a deficiency of TGF-ß or Wg will cause the loss of the outer yellow ring. These deficient patterns are similar to those observed in the eyespots of Vanessa atalanta, Vanessa altissima, and Chlosyne nycteis. Thus, our model also provides a hypothesis to explain the mechanism of generating the deficient patterns in these species.

Color pattern evolution in Vanessa butterflies (Nymphalidae: Nymphalini): non-eyespot characters.

A phylogenetic approach was used to study color pattern evolution in Vanessa butterflies. Twenty-four color pattern elements from the Nymphalid ground plan were identified on the dorsal and ventral surfaces of the fore- and hind wings. Eyespot characters were excluded and will be examined elsewhere. The evolution of each character was traced over a Bayesian phylogeny of Vanessa reconstructed from 7750 DNA base pairs from 10 genes. Generally, the correspondence between character states on the same surface of the two wings is stronger on the ventral side compared to the dorsal side. The evolution of character states on both sides of a wing correspond with each other in most extant species, but the correspondence between dorsal and ventral character states is much stronger in the forewing than in the hindwing. The dorsal hindwing of many species of Vanessa is covered with an extended Basal Symmetry System and the Discalis I pattern element is highly variable between species, making this wing surface dissimilar to the other wing surfaces. The Basal Symmetry System and Discalis I may contribute to behavioral thermoregulation in Vanessa. Overall, interspecific directional character state evolution of non-eyespot color patterns is relatively rare in Vanessa, with a majority of color pattern elements showing non-variable, non-directional, or ambiguous character state evolution. The ease with which the development of color patterns can be modified, including character state reversals, has likely made important contributions to the production of color pattern diversity in Vanessa and other butterfly groups.

A tale of two haplotype groups: Evaluating the New World Junonia ring species hypothesis using the distribution of divergent COI haplotypes.

The New World Junonia butterflies are a possible ring species with a circum-Caribbean distribution. Previous reports suggest a steady transition between North and South American forms in Mesoamerica, but in Cuba the forms were thought to co-exist without interbreeding representing the overlapping ends of the ring. Three criteria establish the existence of a ring species: a ring-shaped geographic distribution, gene flow among intervening forms, and genetic isolation in the region of range overlap. We evaluated mitochondrial cytochrome oxidase I haplotypes in Junonia from 9 species in the Western Hemisphere to test the Junonia ring species hypothesis. Junonia species are generally not monophyletic with respect to COI haplotypes, which are shared across species. However, two major COI haplotype groups exist. Group A predominates in South America, and Group B predominates in North and Central America. Therefore, COI haplotypes can be used to assess the degree of genetic influence a population receives from each continent. Junonia shows a ring-shaped distribution around the Caribbean, and evidence is consistent with gene flow among forms of Junonia, including those from Mesoamerica. However, we detected no discontinuity in gene flow in Cuba or elsewhere in the Caribbean consistent with genetic isolation in the region of overlap. Though sampling is still very limited in the critical region, the only remaining possiblity for a circum-Caribbean discontinuity in gene flow is at the Isthmus of Panama, where there may be a transition from 98% Group B haplotypes in Costa Rica to 85-100% Group A haplotypes in South America.

Phylogenetic analysis of the complete mitochondrial genome of the Japanese peacock butterfly Aglais io geisha (Stichel 1907) (Insecta: Lepidoptera: Nymphalidae).

The peacock butterfly Aglais io (Linnaeus, 1758) (Nymphalidae: Nymphalinae: Nymphalini) is a colorful and charismatic flagship butterfly species whose range spans from the British Isles and Europe through temperate Asia and the Far East. In Europe, it has been used as a model species for studying the effects of GMO maize pollen on caterpillar growth and survivorship. The Japanese subspecies, Aglais io geisha (Stichel 1907), is not as well studied as its European counterpart. Genome skimming by Illumina sequencing allowed the assembly of a complete circular mitochondrial genome (mitogenome) of 15,252 bp from A. io geisha consisting of 80.6% AT nucleotides, 13 protein-coding genes, 22 tRNAs, two rRNAs, and a control region in the gene order typical of butterfly species. Aglais io geisha COX1 gene features an atypical start codon (CGA) while COX1, COX2, CYTB, ND1, ND3, ND4, and ND5 display incomplete stop codons finished by the addition of 3' A residues to the mRNA. Bayesian phylogenetic reconstruction places A. io geisha within a clade with European A. io mitogenomes in the tribe Nymphalini, which is consistent with previous phylogenetic hypotheses.

Phylogenetic analysis of the complete mitochondrial genome of the Blomfild's Beauty butterfly Smyrna blomfildia (Fabricius 1781) (Insecta: Lepidoptera: Nymphalidae: Nymphalini).

The Blomfild's Beauty butterfly Smyrna blomfildia (Fabricius 1781) (Lepidoptera: Nymphalidae: Nymphalini) is a sexually dimorphic species found in Mexico, Central, and South America. Males are territorial and are more vibrantly colored than females. Genome skimming by Illumina sequencing allowed the assembly of a complete circular mitochondrial genome (mitogenome) of 15,149 bp from S. blomfildia consisting of 83.9% AT nucleotides, 13 protein-coding genes, 22 tRNAs, two rRNAs, and a control region in the typical butterfly gene order. The S. blomfilda COX1 gene features an atypical start codon (CGA) while ATP6, COX1, COX2, CYTB, ND1, ND3, ND4, and ND5 display partial stop codons completed by the addition of 3' A residues to the mRNA. Bayesian phylogenetic reconstruction places Smyrna as a member of the tribe Nymphalini and sister to a clade containing genera Araschnia, Vanessa, Polygonia, and Aglais, which differs from its classic taxonomic placement in tribe Coeini.

The complete mitochondrial genome of the file ramshorn snail Planorbella pilsbryi (Mollusca: Gastropoda: Hygrophila: Planorbidae).

The file ramshorn snail Planorbella pilsbryi Baker, 1926 (Gastropoda: Hygrophila: Planorbidae) is a widespread herbivorous North American freshwater snail found in diverse habitats, including standing and moving water bodies. Genome skimming by Illumina sequencing allowed the assembly of a complete nuclear rRNA repeat sequence and a complete circular mitogenome of 13,720 bp from P. pilsbryi consisting of 75.3% AT nucleotides, 22 tRNAs, 13 protein-coding genes, 2 rRNAs and a control region in the typical order found in panpulmonate snails. Planorbella pilsbryi COXI features a rare TTG start codon while COXII, CYTB, ND2, ND3, and ND5 exhibit incomplete stop codons completed by the addition of 3' A residues to the mRNA. Phylogenetic reconstruction of mitochondrial protein-coding gene and rRNA sequences places P. pilsbryi as sister taxon to Planorbella duryi (Planorbidae) within family Planorbidae, which is consistent with previous phylogenetic hypotheses.

The complete mitochondrial genome of the Indian leafwing butterfly Kallima paralekta (insecta: Lepidoptera: Nymphalidae).

The Indian leafwing butterfly Kallima paralekta (Horsfield, 1829) (Nymphalidae) is an Asian forest-dwelling, leaf-mimic. Genome skimming by Illumina sequencing permitted assembly of a complete circular mitogenome of 15,200 bp from K. paralekta consisting of 79.5% AT nucleotides, 22 tRNAs, 13 protein-coding genes, two rRNAs and a control region in the typical butterfly gene order. Kallima paralekta COX1 features an atypical CGA start codon, while ATP6, COX1, COX2, ND4, ND4L, and ND5 exhibit incomplete stop codons completed by 3' A residues added to the mRNA. Phylogenetic reconstruction places K. paraleckta within the monophyletic genus Kallima, sister to Mallika in the subfamily Nymphalinae. These data support the monophyly of tribe Kallimini and contribute to the evolutionary systematics of the Nymphalidae.

Drosophila transposon insertions as unknowns for structured inquiry recombination mapping exercises in an undergraduate genetics course.

Structured inquiry approaches, in which students receive a Drosophila strain of unknown genotype to analyze and map the constituent mutations, are a common feature of many genetics teaching laboratories. The required crosses frustrate many students because they are aware that they are participating in a fundamentally trivial exercise, as the map locations of the genes are already established and have been recalculated thousands of times by generations of students. We modified the traditional structured inquiry approach to include a novel research experience for the students in our undergraduate genetics laboratories. Students conducted crosses with Drosophila strains carrying P[lacW] transposon insertions in genes without documented recombination map positions, representing a large number of unique, but equivalent genetic unknowns. Using the eye color phenotypes associated with the inserts as visible markers, it is straightforward to calculate recombination map positions for the interrupted loci. Collectively, our students mapped 95 genetic loci on chromosomes 2 and 3. In most cases, the calculated 95% confidence interval for meiotic map location overlapped with the predicted map position based on cytology. The research experience evoked positive student responses and helped students better understand the nature of scientific research for little additional cost or instructor effort.

The complete mitochondrial genome of the Malagasy clouded mother-of-pearl butterfly Protogoniomorpha ancardii duprei (Insecta: Lepidoptera: Nymphalidae).

The Malagasy clouded mother-of-pearl butterfly, Protogoniomorpha ancardii duprei (Nymphalidae), is the Madagascar subspecies of a widespread sub-Saharan leaf-mimic. Genome skimming allowed the assembly of the complete P. ancardii duprei circular mitogenome (15,220 bp) consisting of 80% AT nucleotides, 13 protein-coding genes, 22 tRNAs, two rRNAs, and a control region in typical butterfly gene order. Protogoniomorpha ancardii duprei COX1 has a CGA start codon while COX1, COX2, CYTB, NAD1, and NAD4 exhibit partial stop codons completed by 3' A residues added to the mRNA. Phylogenetic reconstruction places Protogoniomorpha as sister to genus Yoma within monophyletic tribe Junoniini.

The complete mitochondrial genome and phylogenetic analysis of the European map butterfly Araschnia levana (Insecta: Lepidoptera: Nymphalidae).

The European map butterfly Araschnia levana (Linnaeus, 1758) is a species showing extreme seasonal polyphenism. The complete 15,207 bp circular A. levana mitogenome consisting of 81.6% AT nucleotides, was assembled by Illumina genome skimming. It includes 22 tRNAs, 13 protein-coding genes, 2 rRNAs, and a control region in the typical butterfly gene order. Araschnia levana COX1 features an atypical CGA start codon and ATP6, COX1, COX2, ND1, ND3, and ND4 have incomplete stop codons completed by 3'A residues added to the mRNA. Phylogenetic reconstruction places A. levana as a basal lineage within tribe Nymphalini, consistent with previous phylogenetic hypotheses.

The complete mitochondrial genome of the Jackson's leaf butterfly Mallika jacksoni (Insecta: Lepidoptera: Nymphalidae).

The Jackson's leaf butterfly Mallika jacksoni (Sharpe 1896), is a leaf-mimicking species from tropical East Africa. Genome skimming by Illumina sequencing permitted the assembly of the complete circular M. jacksoni 15,183 bp mitogenome. It consists of 79.4% AT nucleotides, 22 tRNAs, 13 protein-coding genes, 2 rRNAs, and a control region in the typical butterfly gene order. Mallika jacksoni COX1 has a CGA start codon while ATP6, COX1, COX2, ND3, ND4, and ND5 exhibit partial stop codons completed by 3'-A residues added to the mRNA. Phylogenetic reconstruction places M. jacksoni as sister to Kallima within nymphalid tribe Kallimini.

Phylogenetic analysis of the complete mitochondrial genome of the white peacock butterfly Anartia jatrophae saturata (Insecta: Lepidoptera: Nymphalidae).

The white peacock butterfly Anartia jatrophae saturata Staudinger, 1884 (Nymphalidae: Nymphalinae: Victorini), lives in the neotropics. Genome skimming with Illumina sequencing of A. jatrophae saturata allowed the assembly of a complete circular mitogenome of 15,297 bp, consisting of 81.4% AT nucleotides, 22 tRNAs, 13 protein-coding genes, two rRNAs, and a control region. Anartia jatrophae COX1 features an atypical start codon (CGA); ATP6, COX1, ND1, ND4, ND4L, ND5, and ND6 exhibit incomplete stop codons completed in the mRNA by the addition of 3' A residues. Contrary to previous phylogenetic hypotheses, phylogenetic reconstruction places A. jatrophae as sister to nymphalid tribe Nymphalini.

It's a moth! It's a butterfly! It's the complete mitochondrial genome of the American moth-butterfly Macrosoma conifera (Warren, 1897) (Insecta: Lepidoptera: Hedylidae)!

The taxonomic placement of the moth-butterfly, Macrosoma conifera (Warren 1897) (Lepidoptera: Hedylidae), has been controversial. The 15,344 bp complete M. conifera circular mitogenome, assembled by genome skimming, consists of 81.7% AT nucleotides, 22 tRNAs, 13 protein-coding genes, 2 rRNAs and a control region in the typical butterfly gene order. Macrosoma conifera COX1 features an atypical CGA start codon while ATP6, COX1, COX2, and ND5 exhibit incomplete stop codons completed by the post-transcriptional addition of 3' A residues. Phylogenetic reconstruction places M. conifera as sister to the skippers (Hesperiidae), which is consistent with several recent phylogenetic analyses.

Reply to 'A refutation to 'A new A-P compartment boundary and organizer in holometabolous insect wings'.

Here we reply to the "Refutation" of Lawrence, Casal, de Cellis, and Morata, who critique our paper presenting evidence for an organizer and compartment boundary subdividing the widely recognized posterior wing compartment of butterflies and moths (Lepidoptera) and Drosophila, that we called the F-P boundary. Lawrence et al. present no data from the Lepidoptera and while the data that they present from Drosophila melanogaster mitotic clones are intriguing and may be informative with respect to the timing of the activity of the A-P and F-P organizers, considerable ambiguity remains regarding how their data should be interpreted with respect to the proposed wing compartment boundaries. Thus, contrary to their claims, Lawrence et al. have failed to falsify the F-P boundary hypothesis. Additional studies employing mitotic clones labeled with easily detectable markers that do not affect cytoskeletal organization or rates of cell division such as GFP and RFP clones produced by G-Trace or Twin Spot Generator (TSG) may further clarify the number of compartment boundaries in Drosophila wings. At the same time, because Drosophila wings are diminutive and highly modified compared to other insects, we also urge great caution in making generalizations about insect wing development based exclusively on studies in Drosophila.Replying to: Lawrence, P.A., Casal, J., de Celis, J., Morata, G. A refutation to 'A new A-P compartment boundary and organizer in holometabolous insect wings'. Sci. Rep. 9 (2019), https://doi.org/10.1038/s41598-019-42668-y .

The complete mitochondrial genome of the brown pansy butterfly, Junonia stygia (Aurivillius, 1894), (Insecta: Lepidoptera: Nymphalidae).

The brown pansy, Junonia stygia (Aurivillius, 1894) (Lepidoptera: Nymphalidae), is a widespread West African forest butterfly. Genome skimming by Illumina sequencing allowed assembly of a complete 15,233 bp circular mitogenome from J. stygia consisting of 79.5% AT nucleotides. Mitochondrial gene order and composition is identical to other butterfly mitogenomes. Junonia stygia COX1 features an atypical CGA start codon, while ATP6, COX1, COX2, ND4, and ND4L exhibit incomplete stop codons. Phylogenetic reconstruction supports a monophyletic Subfamily Nymphalinae, Tribe Junoniini, and genus Junonia. The phylogenetic tree places Junonia iphita and J. stygia as basal mitogenome lineages sister to the remaining Junonia sequences.

A simulation study of mutations in the genetic regulatory hierarchy for butterfly eyespot focus determination.

The color patterns on the wings of butterflies have been an important model system in evolutionary developmental biology. A recent computational model tested genetic regulatory hierarchies hypothesized to underlie the formation of butterfly eyespot foci [Evans, T.M., Marcus, J.M., 2006. A simulation study of the genetic regulatory hierarchy for butterfly eyespot focus determination. Evol. Dev. 8, 273-283]. The computational model demonstrated that one proposed hierarchy was incapable of reproducing the known patterns of gene expression associated with eyespot focus determination in wild-type butterflies, but that two slightly modified alternative hierarchies were capable of reproducing all of the known gene expressions patterns. Here we extend the computational models previously implemented in Delphi 2.0 to two mutants derived from the squinting bush brown butterfly (Bicyclus anynana). These two mutants, comet and Cyclops, have aberrantly shaped eyespot foci that are produced by different mechanisms. The comet mutation appears to produce a modified interaction between the wing margin and the eyespot focus that results in a series of comet-shaped eyespot foci. The Cyclops mutation causes the failure of wing vein formation between two adjacent wing-cells and the fusion of two adjacent eyespot foci to form a single large elongated focus in their place. The computational approach to modeling pattern formation in these mutants allows us to make predictions about patterns of gene expression, which are largely unstudied in butterfly mutants. It also suggests a critical experiment that will allow us to distinguish between two hypothesized genetic regulatory hierarchies that may underlie all butterfly eyespot foci.

Our love-hate relationship with DNA barcodes, the Y2K problem, and the search for next generation barcodes.

DNA barcodes are very useful for species identification especially when identification by traditional morphological characters is difficult. However, the short mitochondrial and chloroplast barcodes currently in use often fail to distinguish between closely related species, are prone to lateral transfer, and provide inadequate phylogenetic resolution, particularly at deeper nodes. The deficiencies of short barcode identifiers are similar to the deficiencies of the short year identifiers that caused the Y2K problem in computer science. The resolution of the Y2K problem was to increase the size of the year identifiers. The performance of conventional mitochondrial COI barcodes for phylogenetics was compared with the performance of complete mitochondrial genomes and nuclear ribosomal RNA repeats obtained by genome skimming for a set of caddisfly taxa (Insect Order Trichoptera). The analysis focused on Trichoptera Family Hydropsychidae, the net-spinning caddisflies, which demonstrates many of the frustrating limitations of current barcodes. To conduct phylogenetic comparisons, complete mitochondrial genomes (15 kb each) and nuclear ribosomal repeats (9 kb each) from six caddisfly species were sequenced, assembled, and are reported for the first time. These sequences were analyzed in comparison with eight previously published trichopteran mitochondrial genomes and two triochopteran rRNA repeats, plus outgroup sequences from sister clade Lepidoptera (butterflies and moths). COI trees were not well-resolved, had low bootstrap support, and differed in topology from prior phylogenetic analyses of the Trichoptera. Phylogenetic trees based on mitochondrial genomes or rRNA repeats were well-resolved with high bootstrap support and were largely congruent with each other. Because they are easily sequenced by genome skimming, provide robust phylogenetic resolution at various phylogenetic depths, can better distinguish between closely related species, and (in the case of mitochondrial genomes), are backwards compatible with existing mitochondrial barcodes, it is proposed that mitochondrial genomes and rRNA repeats be used as next generation DNA barcodes.

RAD7 homologues contribute to Arabidopsis UV tolerance.

Frequent exposure of plants to solar ultraviolet radiation (UV) results in damaged DNA. One mechanism of DNA repair is the light independent pathway Global Genomic Nucleotide Excision Repair (GG-NER), which repairs UV damaged DNA throughout the genome. In mammals, GG-NER DNA damage recognition is performed by the Damaged DNA Binding protein 1 and 2 (DDB1/2) complex which recruits the Xeroderma Pigmentosa group C (XPC) / RAD23D complex. In the yeast Saccharomyces cerevisiae, distinct proteins, Radiation sensitive 7 and 16 (Rad7p and Rad16p), recognize the damaged DNA strand and then recruit the XPC homologue, Rad4p, and Rad23p. The remainder of the proteins involved GG-NER are well conserved. DDB1, DDB2, XPC/RAD4, and RAD23 homologues have been described in the model plant Arabidopsis thaliana. In this study we characterize three Arabidopsis RAD7 homologues, RAD7a, RAD7b, and RAD7c. Loss of function alleles of each of the three RAD7 homologues result in increased UV sensitivity. In addition, RAD7b and RAD7c overexpression lines exhibited increased UV tolerance. Thus RAD7 homologues contribute to UV tolerance in plants as well as in yeast. This is the first time any system has been shown to utilize both the DDB1/2 and RAD7/16 damage recognition complexes.

A new A-P compartment boundary and organizer in holometabolous insect wings.

Decades of research on the highly modified wings of Drosophila melanogaster has suggested that insect wings are divided into two Anterior-Posterior (A-P) compartments separated by an axis of symmetry. This axis of symmetry is created by a developmental organizer that establishes symmetrical patterns of gene expression that in turn pattern the A-P axis of the wing. Butterflies possess more typical insect wings and butterfly wing colour patterns provide many landmarks for studies of wing structure and development. Using eyespot colour pattern variation in Vanessa butterflies, here we show an additional A-P axis of symmetry running between wing sectors 3 and 4. Boundaries of Drosophila mitotic clones suggest the existence of a previously undetected Far-Posterior (F-P) compartment boundary that coincides with this additional A-P axis. A similar compartment boundary is evident in butterfly mosaic gynandromorphs. We suggest that this additional compartment boundary and its associated developmental organizer create an axis of wing colour pattern symmetry and a gene expression-based combinatorial code, permitting each insect wing compartment to acquire a unique identity and allowing for the individuation of butterfly eyespots.