Contributions to Zoology 89 (2020) 127-145 CTOZ brill.com/ctoz Evolutionary history of species of the firefly subgenus Hotaria (Coleoptera, Lampyridae, Luciolinae, Luciola) inferred from DNA barcoding data Taeman Han Applied Entomology Division, Department of Agricultural Biology, National Institute of Agricultural Science, Nongsaengmyeong-ro 166, Iseo-myeon, Wanju- gun, Jeollabuk-do 55365, Korea Seung-Hyun Kim Applied Entomology Division, Department of Agricultural Biology, National Institute of Agricultural Science, Nongsaengmyeong-ro 166, Iseo-myeon, Wanju- gun, Jeollabuk-do 55365, Korea Hyung Joo Yoon Applied Entomology Division, Department of Agricultural Biology, National Institute of Agricultural Science, Nongsaengmyeong-ro 166, Iseo-myeon, Wanju- gun, Jeollabuk-do 55365, Korea In Gyun Park Applied Entomology Division, Department of Agricultural Biology, National Institute of Agricultural Science, Nongsaengmyeong-ro 166, Iseo-myeon, Wanju- gun, Jeollabuk-do 55365, Korea Haechul Park Applied Entomology Division, Department of Agricultural Biology, National Institute of Agricultural Science, Nongsaengmyeong-ro 166, Iseo-myeon, Wanju- gun, Jeollabuk-do 55365, Korea [email protected] Abstract The firefly subgenus Hotaria sensu lato of the genus Luciola currently includes four morphospecies: L. (H.) parvula, L. (H.) unmunsana, L (H.) papariensis, and L. (H.) tsushimana. The latter three are taxonomically controversial based on both morphological and molecular data. We examined the phylogenetic relation- ships and evolutionary history of the species and related congeners using partial COI gene sequences (DNA barcoding). Our phylogenetic analyses consistently supported the monophyly of Hotaria sensu lato, but did not resolve the generic rank. The two types of L. (H.) parvula in Japan can be considered distinct © Han et al., 2019 | doi:10.1163/18759866-20191420 This is an open access article distributed under the terms of the cc-by 4.0 License. Downloaded from Brill.com09/27/2021 05:10:11AM via free access <UN> 128 Han et al. species that arose by pseudocryptic speciation during the Miocene, with substantial genetic divergence (15.41%). Three morphospecies, L. (H.) unmunsana, L (H.) papariensis, and L. (H.) tsushimana, split into several polyphyletic or paraphyletic groups, forming entangled species groups. They are considered an incipient group that is distinguishable genetically but not morphologically, with evidence for recent allo- patric speciation events corresponding to geologic events and sea-level changes during the Pliocene and Pleistocene. Group III of L. (H.) unmunsana collected from the Jeolla region is a new taxon. Keywords Fireflies – Luciola – Hotaria – incipient species diversity – molecular clock – allopatric speciation Introduction the morphological differentiation. Previous studies of the distributional patterns of the Fireflies belonging to the family Lampyri- two Korean Luciola species are based on dae may be the most charismatic among all the descriptions by Doi (1931, 1932). Kim & insects owing to their beautiful light signals Nam (1981) reported that L. (H.) papariensis for spectacular courtship displays. Thus, they is dominant in the northern part and L. (H.) have inspired many poems, songs, and stories unmunsana is abundant in the southern part as well as research. Fireflies include more than of South Korea. Sim & Kwon (2000) obtained 2,000 species in 100 genera (Lewis & Cratsely, similar results but found that the two spe- 2008). Six species in five genera and two sub- cies are sympatric at every site surveyed, ex- families are known in Korea (Kang, 2012). In cept for a remote volcanic Island, Jeju, where the genus Luciola of Luciolinae, three species only H. (H.) unmunsana is found. Kim et al. are recognized in Korea. L. (s. str.) lateralis is (2004) suggested that L. (H.) papariensis and morphologically distinct from the other two L. (H.) unmunsana are not different species Korean species, L. (Hotaria) unmunsana and because the remarkable the pronotal semicir- L. (H.) papariensis, based on its black scutel- cular speckle is polymorphic within species lum and large punctures on the pronotum. and sometimes differ between species. Kang However, L. (H.) unmunsana and L. (H.) papa- (2012) also pointed out that L. (H.) paparien- riensis are highly similar to each other. sis may be the same as L. (H.) unmunsana Doi (1931, 1932) described L. unmunsana or at least may not be distributed in South (type locality: Mt. Unmun, Cheongdo-gun, Korea based on topotypical specimens of L. Gyeongsangbuk-do, South Korea) and L. pa- unmunsana collected from the type locality, pariensis (type locality: Pabalri, Pungsan-gun, Mt. Unmun, with the blackish semicircular Hamgyeongnam-do, North Korea). The color speckle on the pronotum. This conclusion pattern of the pronotum, which is orange- was based on the description of Doi (1932), red in L. (H.) unmunsana and yellowish- which did not consider intraspecific varia- brown with blackish semicircular speckle tion in the pronotal speckle. Closely related at the anterior part in L. (H.) papariensis, species, similar to the two Korean species of is a diagnostic character (Doi, 1931, 1932). Luciola, are also found in Japan. L. (H.) tsu- However, the type specimens were lost and shimana Nakane, 1970 is distributed only on accordingly cannot be examined to confirm Tsushima Is. and L. (H.) parvula Kiesenwetter, Downloaded from Brill.com09/27/2021 05:10:11AM via free access <UN> EVOLUTIONARY HISTORY OF HOTARIA SPECIES 129 1874 is widely found on three major islands papariensis and L. (H.) unmunsana. Our find- (Honshu, Shikoku, and Kyushu). ings clarify the species statuses of four conge- These four morphospecies are currently ners in Hotaria and provide insight into intra- classified in the subgenus Hotaria Yuasa, 1937 and interspecific divergence. of the genus Luciola (Sim et al., 1999; Sim & Kwon, 2000; Suzuki, 2001), but the taxonomic position of Hotaria is still unclear. It has been Material and methods treated as a genus (Yuasa, 1937; Nakane & Ohba, 1981; Kurosawa et al., 1985; Hirashima, Taxon sampling and initial morphospecies 1989; Suzuki, 1997; Ohba, 1998; Choi et al., identification 2002), as a subgenus (McDermott, 1966; Bal- In total, 72 specimens regarded as Luciola lantyen, 1968; Suzuki, 2001), and as synony- (Hotaria) papariensis and L. (H.) unmunsana mous with Luciola (Oba et al., 2011). were collected in South Korea. This additional During the past two decades, Korean and sampling was expected to provide more im- Japanese fireflies in Luciola have been investi- proved species identification and genetic gated extensively using molecular approaches structures for the focal taxa by expanding on (see reviewed by Suzuki, 2001; Kim et al., 2004). the locations and sample sizes than the pre- Suzuki et al. (1993) used 13 allozyme loci to an- vious studies. These samples were kept alive alyze populations of Hotaria parvula, which until they could be stored at –80°C for DNA can be distinguished into two types accord- extraction. For morphospecies identification, ing to male body size, the dimorphism of flash the descriptions of Doi (1931, 1932) were used interval patterns in males, and their allopatric owing to the lack of type specimens. The gen- distribution in Japan (Ohba, 1983, 1986, 1987), eral features of the specimens were observed and found two genetically distinct ecological under a stereoscopic microscope (MZ 16A and types. Ohmiya et al. (1995) found that H. par- MZ 6; Leica, Solms, Germany). L. (H.) papari- vula is separated from Luciola lineages, includ- ensis was identified based on the presence of ing L. cruciata and L. lateralis, in an analysis of the black semicircular speckle on the anterior luciferase amino acid sequences. Suzuki (1997) part of the pronotum and L. (H.) unmunsana examined a partial sequence of mitochondrial lacked the speckle on the pronotum. Finally, 16S ribosomal RNA (16S rRNA) and showed 22 specimens were identified as L. (H.) papari- H. parvula and H. tsushimana form a mono- ensis from five localities, mainly in the north- phyletic group that is separated from other Lu- ern part of South Korea, and 50 specimens ciola species. Kim et al. (2004) analyzed lucif- were recognized as L. (H.) unmunsana from erase and mitochondrial DNA sequences and five localities, mainly in the middle to south- found that H. papariensis, H. unmunsana, and ern parts of South Korea (table 1, supplemen- H. tsushimana do not exhibit sufficient diver- tary table S1). Unlike previous studies (Kim & gence to be considered separate species (Kim Nam, 1981; Sim & Kwon, 2000; Kim et al., 2004; et al., 2000; Choi et al., 2002, 2003). However, Kang, 2012), we did not find these two species they used only a few local samples (<five spec- co-occurring at the same collecting sites. All imens). Therefore, the precise species statuses materials were preserved in the insect col- of these species are unclear. lection at the Applied Entomology Division, In this study, we performed a DNA barcod- Department of Agricultural Biology, National ing analysis using COI gene sequences to eval- Institute of Agricultural Science (NIAST), uate larger sample sizes of Luciola (Hotaria) Jeonju, Korea. Downloaded from Brill.com09/27/2021 05:10:11AM via free access <UN> 130 Han et al. TABLE 1 Summary of the firefly species examined in this study Species ID Species Samplesize Collection Source of COI sequences localities Present study NCBI 1 Luciolar (Hotaria) 54 South Korea (54)* ○ ○ unmunsana