Mitogenomes of Giant-Skipper Butterflies reveal an ancient split between deep and shallow root feeders

Background: Giant-Skipper butterflies from the genus Megathymus are North American endemics. These large and thick-bodied Skippers resemble moths and are unique in their life cycles. Grub-like at the later stages of development, caterpillars of these species feed and live inside yucca roots. Adults do not feed and are mostly local, not straying far from the patches of yucca plants. Methods: Pieces of muscle were dissected from the thorax of specimens and genomic DNA was extracted (also from the abdomen of a specimen collected nearly 60 years ago). Paired-end libraries were prepared and sequenced for 150bp from both ends. The mitogenomes were assembled from the reads followed by a manual gap-closing procedure and a phylogenetic tree was constructed using a maximum likelihood method from an alignment of the mitogenomes. Results: We determined mitogenome sequences of nominal subspecies of all five known species of Megathymus and Agathymus mariae to confidently root the phylogenetic tree. Pairwise sequence identity indicates the high similarity, ranging from 88-96% among coding regions for 13 proteins, 22 tRNAs and 2 rRNA, with a gene order typical for mitogenomes of Lepidoptera. Phylogenetic analysis confirms that Giant-Skippers (Megathymini) originate within the subfamily Hesperiinae and do not warrant a subfamily rank. Genus Megathymus is monophyletic and splits into two species groups. M. streckeri and M. cofaqui caterpillars feed deep in the main root system of yucca plants and deposit frass underground. M. ursus, M. beulahae and M. yuccae feed in the yucca caudex and roots near the ground, and deposit frass outside through a "tent" (a silk tube projecting from the center of yucca plant). M. yuccae and M. beulahae are sister species consistently with morphological similarities between them. Conclusions: We constructed the first DNA-based phylogeny of the genus Megathymus from their mitogenomes. The phylogeny agrees with morphological considerations.


This article is included in the Phylogenetics
Giant-Skippers (Lepidoptera: Hesperiidae: Megathymini) are large, fat-bodied butterflies endemic to the North American continent 1-3 . Their caterpillars adapted to feeding inside large roots and fleshy leaves of Yucca and Agave plants and relatives. Protected from many predators living within their nutrition-rich food sources, Megathymini are larger in size than most other skippers, and don't feed as adults. Genus Megathymus is characterized by root-feeding caterpillars, mostly in Yucca plants, that build a "tent" (a silk tube projecting above the ground) at least prior to pupation. Caterpillars of the genus Agathymus live inside Agave leaves and make a "trapdoor" (a round, hardened disk of silk) to close the entrance to their leaf chamber before pupation.
All specimens, but one, were collected in 2013 and pieces of their muscles cut out of the thorax were preserved in 100% ethanol to ensure best DNA quality. However, M. beulahae paratype specimen was collected in 1957 15 and stored pinned, spread and dry in a museum drawer for 60 years. DNA was extracted from its abdomen prior to genitalia dissection and produced good quality genomic reads resulting in a complete mitogenome assembly. Similarly to the results reported previously 16 , we see that dry insect collections are an invaluable source of specimens for DNA studies; DNA can be extracted from Lepidoptera without damaging specimens beyond standard genitalia dissection procedure; and good quality DNA sequences can be obtained from specimens collected many decades ago. In conclusion, we sequenced mitochondrial genomes of all five known species of Megathymus and one species of Agathymus as an outgroup, and constructed the first DNA-based phylogeny of Megathymus. The phylogeny is fully consistent with morphological and behavioral similarities between species. Our results support phylogenetic placement of Megathymini within the subfamily Hesperiinae and clarify the relationships between Megathymus species. In particular, the major phylogenetic split is between the shallow   analyzed the data and wrote the manuscript.

Competing interests
No competing interests were disclosed. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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3. This work determined mitogenome sequences of nominal subspecies of all five known species of Megathymus and Agathymus mariae and constructed the phylogenetic trees of the main Hesperiinae groups, the results confirms that Giant-Skippers (Megathymini) should not warrant a subfamily rank and that the monophyletic Megathymus splits into two species groups. In addition, the reconstructed mitogenomic phylogeny is fully consistent with morphological and behavioral similarities between the closely related species of the genus Megathymus. Overall this interesting work deserves to be published and indexed.

Open Peer Review
The manuscript's writing is good with an appropriate title and other content; the design, method and analysis is generally correct though some phylogenetic analyses should be more robust.
If the work considers the molecular dating of the Megatheymus divergence by molecular clock method, and incorporates the analysis of relevant earth environmental factors, the results and significance will be more remarkable.

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No competing interests were disclosed. Competing Interests: 23  This paper is an interesting contribution to our understanding of the evolution of Megathymini and the phylogentic placement of the tribe within the Hesperiidae. Using mitogenomes of all recognized species of Megathymus, the authors confirm morphologically-based species and species groups. Moreoverthey identify a deep mitogenomic divergence between lineages that correspond with important life history traits that define species groups.
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