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Historical museum specimens are valuable for exploring population genetics and evolutionary questions because they can provide snapshots of morphological and genetic characteristics from populations over space and time. Unfortunately, DNA found in older museum specimens is frequently degraded, so obtaining genotypes from many individual samples necessary for rigorous molecular population genetic studies is challenging. Previous studies have varied greatly in their success at obtaining genotypes from older preserved insect material. Many well-intentioned collection curators have used research results showing poor preservation of DNA preserved in museum specimens to inform curatorial best practices, in some cases choosing not to allow DNA extraction by destructive sampling because, in their estimation, the likelihood of success would be low. Recent methodological advances in DNA extraction, amplification, and genotyping have allowed some researchers to include mid-19th century samples in molecular genetic analyses. Here we present a robust, high-throughput, and low-cost DNA extraction and genotyping protocol for historical insect specimens employing restriction digests of PCR products followed by high sensitivity electrophoresis. Using this technique, we obtained mitochondrial haplotypes for 100% of 48 New World Junonia butterfly specimens (Nymphalidae) ranging in age from pre-1813 to 1909 and show that the haplotype frequencies obtained are statistically indistinguishable from 20th-century and contemporary reference populations of Junonia (1632 specimens) matched by geographic region. As most extant insect specimens were collected after 1813, based on our findings we would expect that many or even most pinned specimens preserved in museum collections contain usable DNA for mitochondrial haplotyping.
Historical museum specimens are valuable for exploring population genetics and evolutionary questions because they can provide snapshots of morphological and genetic characteristics from populations over space and time. Unfortunately, DNA found in older museum specimens is frequently degraded, so obtaining genotypes from many individual samples necessary for rigorous molecular population genetic studies is challenging. Previous studies have varied greatly in their success at obtaining genotypes from older preserved insect material. Many well-intentioned collection curators have used research results showing poor preservation of DNA preserved in museum specimens to inform curatorial best practices, in some cases choosing not to allow DNA extraction by destructive sampling because, in their estimation, the likelihood of success would be low. Recent methodological advances in DNA extraction, amplification, and genotyping have allowed some researchers to include mid-19th century samples in molecular genetic analyses. Here we present a robust, high-throughput, and low-cost DNA extraction and genotyping protocol for historical insect specimens employing restriction digests of PCR products followed by high sensitivity electrophoresis. Using this technique, we obtained mitochondrial haplotypes for 100% of 48 New World Junonia butterfly specimens (Nymphalidae) ranging in age from pre-1813 to 1909 and show that the haplotype frequencies obtained are statistically indistinguishable from 20th-century and contemporary reference populations of Junonia (1632 specimens) matched by geographic region. As most extant insect specimens were collected after 1813, based on our findings we would expect that many or even most pinned specimens preserved in museum collections contain usable DNA for mitochondrial haplotyping.
Delineating species boundaries in phylogenetic groups undergoing recent radiation is a daunting challenge akin to discretizing continuity. Here, we propose a general approach exemplified by American butterflies from the genus Junonia Hübner notorious for the variety of similar phenotypes, ease of hybridization, and the lack of consensus about their classification. We obtain whole‐genome shotgun sequences of about 200 specimens. We reason that discreteness emerges from continuity by means of a small number of key players, and search for the proteins that diverged markedly between sympatric populations of different species, while keeping low polymorphism within these species. Being 0.25% of the total number, these three dozen ‘speciation’ proteins indeed partition pairs of Junonia populations into two clusters with a prominent break in between, while all proteins taken together fail to reveal this discontinuity. Populations with larger divergence from each other, comparable to that between two sympatric species, form the first cluster and correspond to different species. The other cluster is characterized by smaller divergence, similar to that between allopatric populations of the same species and comprise conspecific pairs. Using this method, we conclude that J. genoveva (Cramer), J. litoralis Brévignon, J. evarete (Cramer), and J. divaricata C. & R. Felder are restricted to South America. We find that six species of Junonia are present in the United States, one of which is new: Junonia stemosa Grishin, sp.n. (i), found in south Texas and phenotypically closest to J. nigrosuffusa W. Barnes & McDunnough (ii) in its dark appearance. In the pale nudum of the antennal club, these two species resemble J. zonalis C. & R. Felder (iii) from Florida and the Caribbean Islands. The pair of sister species, J. grisea Austin & J. Emmel (iv) and J. coenia Hübner (v), represent the classic west/east U.S.A. split. The mangrove feeder (as caterpillar), dark nudum J. neildi Brévignon (vi) enters south Texas as a new subspecies Junonia neildi varia Grishin ssp.n. characterized by more extensive hybridization with and introgression from J. coenia, and, as a consequence, more variable wing patterns compared with the nominal J. n. neildi in Florida. Furthermore, a new mangrove‐feeding species from the Pacific Coast of Mexico is described as Junonia pacoma Grishin sp.n. Finally, genomic analysis suggests that J. nigrosuffusa may be a hybrid species formed by the ancestors of J. grisea and J. stemosa sp.n. This published work has been registered on Zoobank, http://zoobank.org/urn:lsid:zoobank.org:act:C6E70C96-463D-4E6A-95CC-B038.
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