204 Interspecific hybrid between Paracercion sieboldii and P. mela no­ tum from Japan (Odonata: Coenagrionidae) Genta Okude1,2 & Ryo Futahashi2 1 Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan; [email protected] 2 Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan; [email protected] Abstract. Interspecific hybrids have been occasionally found in the field. Here were describe a male of the interspecific hybrid betweenParacercion sieboldii and P. melanotum with inter- mediate phenotypes between the two parent species from Japan. Nuclear and mitochondrial DNA analyses indicated that this individual was derived from interspecific mating between a female P. sieboldii and a male P. melanotum. To our knowledge, this is the only report of the hybrid between these two species. Further key words. Damselfly, Zygoptera, hybridisation, heterospecific matings Introduction Interspecific hybrids of Odonata have been occasionally found (Asahina 1974; Tennessen 1982; Corbet 1999; Sánchez-Guillén et al. 2014; Futahashi et al. 2018). Hybrid individuals can be identified by analysing nuclear and mitochondrial DNA, whereas it is often difficult to judge if they are hybrids between closely related species only by their morphological characteristics (Futahashi & Hayashi 2004; Futahashi et al. 2018). Here we describe a male representing an interspecific hy- brid of Paracercion sieboldii and P. melanotum. To our knowledge, this is the only report of a hybrid between these two species. Including this individual, 21 combi- nations of Odonata hybrids have been discovered so far in Japan. Materials and Methods The following specimens were studied: Paracercion sieboldii 4♂1♀, Ami, Ibaraki, Japan, 31-v-2017; leg. RF. P. melanotum 3♂2♀, Ami, Ibaraki, Japan, 08-ix-2017; leg. RF. Hybrid 1♂, Ami, Ibaraki, Japan, 19-v-2017; leg. GO. We extracted DNA from two legs of each insect using a Maxwell 16 LEV Blood DNA kit (Promega). Nuclear ITS2 region was PCR-amplified from the DNA sam- ples using the primers 5.8S-S1 (5’-CGG TGG ATC ACT CGG CTC GT-3’) and 28S- AS1 (5’-TTT CAC TCG CCG TTA CTA AGG GAA TC-3’), whereas mitochondrial 16S rRNA gene was amplified using the primers 16S-F0 (5’-TTA AAC CGG TYT GAA CTC AGA TC-3’) and 16S-R1 (5’-CGC CTG TTT ATC AAA AAC AT-3’) (Futahashi et al. 2018). PCR-amplified products were subjected to DNA sequenc- Notulae odonatologicae 9(5) 2020: 173-228204-208 – DOI:10.5281/zenodo.3823261 205 ing using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and 3130xl Genetic Analyzer (Applied Biosystems). The sequence data of P. sieboldii and P. melanotum were deposited in DDBJ/EMBL/GenBank databases (accession numbers are LC365694-LC365747). Full views of a hybrid adult were taken using a CCD scanner GT-X830 (Epson), and magnified views of adults were taken using a stereoscopic microscope S8APO (Leica Microsystems) with a digital high definition microscope camera MC120HD (Leica). Results Subsequent DNA analyses confirmed that the nuclear DNA sequence was derived from both Paracercion sieboldii and P. melanotum (Fig. 1, left column), whereas ma- ternally inherited mitochondrial DNA sequence was identical to that of P. sieboldii (Fig. 1, right column), indicating that this individual resulted from an interspecific mating between a female P. sieboldii and a male P. melanotum. Full views of the hybrid adult are shown in Fig. 2A. Discussion Paracercion sieboldii and P. melanotum are widely distributed in Japan, and the ex- ternal characteristics of males are very similar. The males of both species can be dis- tinguished based on the marking pattern of the head, the colour of the compound eyes, and the shape of the caudal appendages (Ozono et al. 2017). A crescent- shaped spot appears on dorsum of antefrons in P. melanotum (Fig. 2B, red arrow- head), but not in P. sieboldii. The post-ocular spots are large and pear-like in P. sie­ boldii but small and elongated in P. melanotum (Fig. 2B, blue arrowhead). Therefore, Fig. 1. Results of nuclear (ITS2 region) and mitochondrial (16S rRNA region) DNA analyses of Paracercion sieboldii, P. melanotum, and their hybrid from Japan. Squares indicate species- specific nucleotides. Notulae odonatologicae 9(5) 2020: 173-228 206 in the colour pattern of the head, the hybrid represented intermediate phenotypes (Fig. 2B). The colour of the compound eyes is greenish in P. sieboldii and bluish in P. melanotum. In the hybrid, the colour of the compound eyes was similar to that of P. sieboldii (Fig. 2B). The length of the inferior appendages is longer than superior appendages in male P. sieboldii (Fig. 2C, arrowhead), while they are almost the same in male P. melanotum. In the hybrid, the length of inferior appendages was slightly longer than the superior appendages (Fig. 2C). Fig. 2. Male of the interspecific hybrid between Paracercion sieboldii and P. melanotum. A – Lateral and dorsal views of the hybrid. B – Dorsal views of heads and prothorax of P. sieboldii, P. melanotum, and their hybrid from Japan. Red and blue arrowheads indicate post ocular spot and marking on dorsum of antefrons, respectively. C – Lateral and dorsal views of abdominal segments 8–10. Arrowhead indicates inferior appendage. Notulae odonatologicae 9(5) 2020: 173-228 207 Table 1. Records of interspecific hybrids in Japanese Odonata. Based on Futahashi et al. (2018, 2019), Nishimura et al. (2019), Okazaki et al. (2019), and Okude et al. (2020). Hybrid combination Family Field Rearing Paracercion melanotum × P. sieboldii Coenagrionidae 1♂ - Ischnura elegans × I. senegalensis Coenagrionidae - 30♂31♀ Mnais costalis × M. pruinosa Calopterygidae 1♂8♀ - Anax parthenope × A. nigrofasciatus Aeshnidae 174♂10♀ 52♂64♀ Anax parthenope × A. guttatus Aeshnidae 1♂ - Anax parthenope × A. panybeus Aeshnidae 1♂1♀ - Somatochlora uchidai × S. graeseri Corduliidae 6♂3♀ - Sympetrum darwinianum × S. maculatum Libellulidae 2♂ 2♂ Sympetrum frequens × S. depressiusculum Libellulidae 16♂35♀ 11♂9♀ Sympetrum eroticum× S. baccha Libellulidae 77♂1♀ 2♂1♀ Sympetrum eroticum× S. parvulum Libellulidae 1♂ - Sympetrum eroticum× S. kunckeli Libellulidae 29♂ - Sympetrum eroticum× S. pedemontanum Libellulidae 2♂1♀ 1♀ Sympetrum croceolum × S. speciosum Libellulidae 1♂ - Sympetrum croceolum × S. uniforme Libellulidae 5♂1♀ - Tramea virginia × T. basilaris Libellulidae 1♂ - Tramea virginia × T. transmarina Libellulidae 3♂1♀ - Zyxomma obtusum × Z. petiolatum Libellulidae 9♂ - Orthetrum albistylum × O. japonicum Libellulidae 1♂ - Orthetrum melania × O. pruinosum Libellulidae 4♂ - Libellula quadrimaculata × L. angelina Libellulidae 7♂19♀ 5♂17♀ Black spots on abdominal segment 8 are often developed in P. sieboldii, but there are individual differences, and are undeveloped in P. melanotum (Fig. 2C). The hy- brid had obvious black spots on abdominal segment 8, reminiscent of P. sieboldii (Fig. 2C). Overall, the hybrid mostly showed a phenotype intermediate between the two parent species. Including the individual in this study, DNA analyses have confirmed 21 combi- nations of hybrids among 203 Odonata species in Japan (Table 1, Futahashi et al. 2018). Although only one hybrid between P. sieboldii and P. melanotum has been discovered so far, additional individuals may be found by careful examination of specimens, because it is possible to identify hybrids based on their external charac- teristics. Moreover, since it is possible to analyse DNA even in individuals collected more than 50 years ago (Okude et al. 2018), further discoveries will be made by examining specimens in historic collections. Notulae odonatologicae 9(5) 2020: 173-228 208 Acknowledgements. This work was partially supported by the JSPS KAKENHI Grant Num- bers JP18J21561 to GO and JP18H02491, JP18H04893, JP19H03287 to RF. References Asahina S. 1974. Interspecific hybrids Aeschna 55: 51-53 [In Japanese, English among the Odonata. Japanese Journal of abstract] Zoology 17: 67-75, 2 pls Okude G., Fukatsu T. & Futahashi Corbet P.S. 1999. Dragonflies, Behav- R. 2020. Interspecific crossing between ior and Ecology of Odonata. Cornell blue-tailed damselflies Ischnura el­ University Press, Ithaca egans and I. senegalensis in the labora- Futahashi R., Futahashi H., Shim- tory. Entomological Science. https://doi. bori O., Nakada T. & Iwata T. 2019. org/10.1111/ens.12408 The dragonflies and damselflies of Okude G., Futahashi R. & Udono K. Toyama Prefecture, Central Honshu, 2018. DNA analysis of a hybrid between Japan in 2018. Bulletin of the Toyama Sympetrum eroticum and Sympetrum Science Museum 43: 87-99 [In Japanese, pedemontanum collected in 1962. 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