Research Article |
Corresponding author: Nick V. Grishin ( grishin@chop.swmed.edu ) Academic editor: Thomas Simonsen
© 2019 Jing Zhang, Qian Cong, Jinhui Shen, Ernst Brockmann, Nick V. Grishin.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Zhang J, Cong Q, Shen J, Brockmann E, Grishin NV (2019) Three new subfamilies of skipper butterflies (Lepidoptera, Hesperiidae). ZooKeys 861: 91-105. https://doi.org/10.3897/zookeys.861.34686
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We obtained and analyzed whole genome data for more than 160 representatives of skipper butterflies (family Hesperiidae) from all known subfamilies, tribes and most distinctive genera. We found that two genera, Katreus Watson, 1893 and Ortholexis Karsch, 1895, which are sisters, are well-separated from all other major phylogenetic lineages and originate near the base of the Hesperiidae tree, prior to the origin of some subfamilies. Due to this ancient origin compared to other subfamilies, this group is described as Katreinae Grishin, subfam. n. DNA sequencing of primary type specimens reveals that Ortholexis melichroptera Karsch, 1895 is not a female of Ortholexis holocausta Mabille, 1891, but instead a female of Ortholexis dimidia Holland, 1896. This finding establishes O. dimidia as a junior subjective synonym of O. melichroptera. Furthermore, we see that Chamunda Evans, 1949 does not originate within Pyrginae Burmeister, 1878, but, unexpectedly, forms an ancient lineage of its own at the subfamily rank: Chamundinae Grishin, subfam. n. Finally, a group of two sister genera, Barca de Nicéville, 1902 and Apostictopterus Leech, [1893], originates around the time Hesperiinae Latreille, 1809 have split from their sister clade. A new subfamily Barcinae Grishin, subfam. n. sets them apart from all other Hesperiidae.
Africa, Asia, genomics, higher classification, phylogeny
New methods bring new discoveries. While careful expert-driven morphological analysis can be insightful in revealing synapomorphies and predicting evolutionary relationships between animals, DNA sequences offer additional insights. Phylogenetic analysis at the genomic scale is expected to give an unprecedented resolution and clarify many questions, providing a firm basis for the best taxonomic classification. Butterflies are attracting attention with a number of large scale phylogeny studies published recently (
Sequenced specimens from the new Hesperiidae subfamilies. DNA sample numbers are given for each specimen, additional data are in the Suppl. material
Here, we tackle the questions about deep phylogeny of Hesperiidae using whole genome shotgun analysis. We selected 160 representative species of skippers that cover all known subfamilies and tribes, including some genera that we thought would be interesting to analyze at the genomic scale. To make this work taxonomically sound, we used type genera and their type species where possible, and for some species used their primary type specimens. Our genomic methods break the time barrier and allow us to work with specimens more than a century old from museum collections. We find that while the backbone of the current classification of Hesperiidae stands the test of genomic data (
Bodies of freshly collected specimens were stored in RNAlater, and their wings and genitalia dried and kept in envelopes to address possible misidentification issues. DNA was extracted from a piece of tissue of these specimens. For specimens in museum collections, DNA was extracted either from the abdomen or from a leg. The abdomen was gently pushed from above and below (while watching for the legs not to be damaged) until it cracked off, and placed in a DNA extraction buffer. After extraction (see below), the abdomen was transferred to 10% KOH solution and genitalia were dissected in a standard manner. A leg was used for primary type specimens. A leg was removed from a specimen using fine forceps and placed in a plastic tube. The forceps were wiped with clean paper tissue after each sample was taken.
DNA was extracted from legs (and abdomens) non-destructively using Macherey-Nagel (MN) reagents. 70 µl buffer T1 and 10 µl protK were added to the tube without crushing the leg, and the mixture was incubated at 57 °C for 24 hours. Then, 80 µl buffer B3 was added and incubation continued for 2 hours, after which 85 µl of absolute EtOH was added and thoroughly mixed. The resulting liquid was transferred to a different tube and DNA extraction continued according to MN protocol (https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Genomic%20DNA/UM_gDNATissueXS.pdf), leaving the leg intact. Mate-pair libraries were constructed according to our published protocols (
The libraries were sequenced for 150 bp from both ends targeting 4 to 6 Gbp of data (depending on the expected genome size) on Illumina HiSeq x10 at GENEWIZ. The resulting reads were matched using Diamond (
Diagnostic DNA characters were identified in nuclear genomic sequences using our recently published procedure (see SI Appendix to
We obtained whole genome shotgun sequence reads for 160 Hesperiidae specimen of representative species. The lengths of resulting genomic regions were: nuclear total 11,835,126 +/-3,035,464, Z-chromosome 99,237 +/-24,462, mitogenomes 12,144 +/-958. We considered Z-chromosome separately. Butterfly males carry two copies of Z, and females possess Z and W. In Z, recombination is reduced to half of that in autosomes, and sexual selection acts differently on genes encoded by it. Thus, the analysis of genes encoded by the Z-chromosome may provide additional information about species evolution. Phylogenetic trees were constructed from coding regions of nuclear genome, Z-chromosome and mitogenome. The trees were rooted with the genomic sequence of Pterourus glaucus that we obtained previously (
Several conclusions confirmed previous findings (
Unexpected placement of Ortholexis holocausta (Mabille, 1891) (Fig.
Phylogenetic trees. The trees are constructed from protein-coding regions of a nuclear genome b Z-chromosome, and c mitochondrial genome. The trees are rooted with Pterourus glaucus (NVG-1670). Specimen names are not shown in the Z-chromosome tree and can be deduced from the nuclear tree by corresponding dotted lines. Details about specimens are in Suppl. material
Katreus Watson, 1893.
In appearance, most similar to Celaenorrhinus Hübner, [1819] and its relatives (
Katreus with its invalid synonym Choristoneura Mabille, 1889 (junior homonym of Choristoneura Lederer 1859 in Lepidoptera: Tortricidae) and subjective synonyms Loxolexis Karsch, 1895 and Daratus Lindsey, 1925 (replacement name for Choristoneura) (Fig.
Taxonomy of these skippers has been confusing until it was resolved by
We sequenced a syntype of Erionota holocausta Mabille, 1891 (Fig.
The next find was particularly unexpected and was not likely to happen in the absence of DNA sequences. Nearly as ancient as Katreinae subfam. n., is the lineage consisting of a single genus Chamunda Evans, 1949, which is sister to the group collectively known as “grass skippers”: subfamilies Heteropterinae Aurivillius, 1925, Trapezitinae Waterhouse & Lyell, 1914 and Hesperiinae Latreille, 1809 (Fig.
Chamunda Evans, 1949.
Keys to C.10 in
Only Chamunda, a monotypic genus for Plesioneura chamunda Moore, 1866 (Fig.
The subfamily-worthy uniqueness of this butterfly from southwestern Asia, dubbed “Olive” or “Crescent Spotted Flat”, is perhaps the largest surprise of our study. Chamunda is not clearly distinct in appearance, it is similar to Lobocla (Eudaminae) and Celaenorrhinus (Tagiadinae) in the spotting of the forewing. Uniqueness of Chamunda was not noticed before Evans, who established a new monotypic genus for this skipper (
These two genera that are apparently each other’s closest relatives have been enigmatic for decades (
Barca de Nicéville, 1902.
Keys to F.4a in
Barca de Nicéville, 1902 with its invalid synonym Dejeania Oberthür, 1896 (junior homonym of Dejeania Robineau-Desvoidy, 1830 in Diptera) (Fig.
These two genera from southwestern China were (with disclaimers) placed in Heteropterinae by Evans (
While classification relies on phylogeny, it does not require phylogeny to be fully resolved. Good classification only requires a clade itself to be well supported and distinct from other clades of the same rank. However, the exact position of that clade in the tree, which reflects the order in time when these clades originated, does not need to be fully resolved. Thus, accurate classification is a simpler task than phylogenetic inference. These considerations are relevant to our treatment of Chamundinae subfam. n. While in the Z-chromosome tree (Fig.
Nevertheless, the decision to treat Chamundinae as a subfamily is supported by the following reasons. We consider three clades in the trees (Fig.
Genomics analysis has been instrumental in revealing the ancient origins of several groups of Hesperiidae that have not been understood before. Moreover, previous studies based on smaller DNA datasets, such as several genes (
We are grateful to Robert K. Robbins, John M. Burns, and Brian Harris (National Museum of Natural History, Smithsonian Institution, Washington DC), Geoff Martin, David Lees, and Blanca Huertas (Natural History Museum, London, UK), Paul A. Opler and Boris Kondratieff (Colorado State University Collection, Fort Collins, CO, USA), Wolfram Mey and Viola Richter (Museum für Naturkunde, Berlin, Germany), Weiping Xie (Los Angeles County Museum of Natural History, Los Angeles, CA, USA), Rodolphe Rougerie (Muséum National d’Histoire Naturelle, Paris, France), Edward G. Riley, Karen Wright, and John Oswald (Texas A&M University Insect Collection, College Station, TX, USA) for facilitating access to the collections under their care and stimulating discussions, and the late Edward C. Knudson for leg samples of specimens from the Texas Lepidoptera Survey collection, which is now at the McGuire Center for Lepidoptera and Biodiversity, Gainesville, FL, USA. Special thanks to Olaf H. H. Mielke and Carlos G. C. Mielke for sampling specimens for DNA analysis from the collections of O. H. H. Mielke and of Departamento de Zoologia, Universidade Federal do Paraná, Curitiba, Brazil; to Steve Collins (African Butterfly Research Institute) and Bernard Hermier for many enlightening discussions, and numerous suggestions; and anonymous reviewer for helpful comments. We acknowledge the Texas Advanced Computing Center (TACC) at The University of Texas at Austin (http://www.tacc.utexas.edu) for providing invaluable HPC resources that were essential to carry out this study, which has been supported by the grants from the National Institutes of Health GM127390 and the Welch Foundation I-1505.
Table S1. Specimen data and DNA sequences
Data type: table, text and DNA sequences
Explanation note: Table S1 with data for 160 sequenced specimens and diagnostic nucleotide characters of the new subfamilies mapped to the reference genome of Cecropterus lyciades.