Biodiversity Data Journal 11: e96800 CO) doi: 10.3897/BDJ.11.e96800 open access Research Article Molecular investigation on diversity of the land snail genus Aegista (Gastropoda, Camaenidae) in South Korea Kazuki Kimura*, Satoshi Chiba*, Jae-Hong Pak$ + Center for Northeast Asian Studies, Tohoku University, Sendai, Japan § Department of Biology, Kyungpook National University, Taegu, South Korea Corresponding author: Kazuki Kimura (k.kimura.0Q00@gmail.com) Academic editor: Dimitris Poursanidis Received: 28 Oct 2022 | Accepted: 04 Jan 2023 | Published: 31 Jan 2023 Citation: Kimura K, Chiba S, Pak J-H (2023) Molecular investigation on diversity of the land snail genus Aegista (Gastropoda, Camaenidae) in South Korea. Biodiversity Data Journal 11: e96800. https://doi.org/10.3897/BDJ.11.e96800 Abstract Aegista Albers, 1850 is a large genus of the land snail family Camaenidae Pilsbry, 1895 and distributed in south, southeast and east Asian countries (from India and Nepal to Korea and Japan). Fourteen species and subspecies of Aegista are known from South Korea. They were described, based only on shell morphology during 1887-1943 and our knowledge on diversity of Korean Aegista has seldom been updated since then. In this study, we provide the report on the first molecular investigation of diversity of Aegista in South Korea, which unmasked some of overlooked diversity of this group. Keywords Aegista, cryptic species, land snails, molecular phylogeny, the Korean Peninsula © Kimura K et al. 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. 2 Kimura K et al Introduction The Korean Peninsula is located at the edge of the eastern part of the Asian continent (Fig. 1). It has had intermittent land-bridge connections with mainland China and its nearby large archipelago (i.e. the Japanese archipelago) through multiple glacial cycles (Kim and Kennett 1998, Kong et al. 2006). Moreover, the Peninsula has never been covered by ice sheets during the Quaternary, which makes it possible for terrestrial organisms to inhabit the Peninsula for a long period (Chung et al. 2017). In East Asia, for example, the Korean Peninsula is the only region where an ancient divided member of the salamander family Plethodontidae Gray, 1850 (Amphibia), whose centre of diversity is North and Middle America, is found (Min et al. 2005). The ancestral group of the Asian plethodontid salamander is estimated to have migrated from the North American continent around 45 Mya and Asian plethodontid populations have probably been extirpated, except in the climatologically stable region, the Korean Peninsula (Shen et al. 2015). These complex geographical characteristics make this region attractive for biodiversity and phylogeographic studies. 1000km 110°E care i 4 Ko rean P «+ Peninsula Asian P a z 30°N continent ssh v3 ona S Japanese archipelago Korean Peninsula Figure 1. EES Map showing the sampling localities. The number corresponds to the locality No. in Table 1. Our analyses also included the sequences of the specimens from Japan, Taiwan and Laos. Molecular investigation on diversity of the land snail genus Aegista (Gastropoda, ... 3 Aegista Albers, 1850 is a large genus of the land snail family Camaenidae Pilsbry, 1895 and distributed in south, southeast and east Asian countries (from India and Nepal to Korea and Japan) (Hirano et al. 2015, Nurinsiyah et al. 2019). Although some researchers treat Landouna Godwin-Austen, 1918 as a distinct genus (Kohler et al. 2018, Nahok et al. 2021), as suggested by Hirano et al. (2014), we treated members of the genus as Aegista in this study because the monophyly of Landouria is still unclear. Fourteen species and subspecies of Aegista are known from South Korea (Kuroda 1958). They were described during 1887-1943 and, unfortunately, our Knowledge on diversity of Korean Aegista has seldom been updated since then. The classification of these Korean Aegista is based only on shell morphology. However, recent molecular phylogenetic studies of land snails have revealed cryptic species within a single morphospecies and demonstrated the unreliability of shell morphology for species identification (e.g. Chiba and Davison (2008), Nantarat et al. (2014)). Hirano et al. (2014) examined a lot of species of Aegista (n >70) from almost all regions of Japan in addition to its surrounding regions such as Taiwan and China and found that multiple phylogenetic clades inhabit the Japanese archipelago, which has a profound biogeographic connection with the Korean Peninsula. Whether the Korean Aegista species include cryptic species and distribution pattern of the phylogenetic lineages in the Korean Peninsula remain to be studied. In the present study, we aim to address this question. Material and methods Samples Individuals of 12 Aegista species and subspecies (Fig. 2) were collected in South Korea from 2018 to 2021 (Table 1). In total, 42 individuals were used in this study and six of them were not identified to the species-level because they were still small juveniles. A small portion of the foot muscle from each snail individual was stored in 99.5% ethanol for DNA extraction. For some species, DNA sampling with body swabbing (Morinha et al. 2014) was used in order to avoid unwanted effects of sample collecting on snail populations. The examined Aegista specimens are preserved in Tohoku University Museum (TUMC) or the authors’ collection (Voucher No. in Table 1). Table 1. List of the species included in the molecular phylogenetic analyses. Asterisks indicate the sequences newly obtained in this study. Species names Locality Locality Voucher # GenBank GenBank No (COl) (ITS) A. aemula (Gude, 1900) Kusama, Niimi KC3103 AB852664 AB852931 A. aemula Tojyo, Shobara KC1472 AB852665 AB852932 A. awajiensis (Gude, 1900) Yashima, Takamatsu H0695 AB852625 AB852892 Species names Kimura K et al A. awajiensis A. caelurea Kuroda & Habe, 1960 A. calcicola (Kuroda, 1989) A. calcicola A. carinata (Gude, 1901) A. carinata A. cavicollis (Pilsbry, 1900) A. certus (Zilch, 1938) A. chejuensis (Pilsbry & Hirase, 1908) A. chejuensis A. chejuensis A. chosenica (Pilsbry, 1927) A. chosenica A. collinsoni casta (Pilsbry, 1901) A. collinsoni casta A. collinsoni collinsoni (Adams, 1868) A. commoda commoda (Adams, 1868) A. commoda nioyaka (Pilsbry & Hirase, 1904) A. cretacea Gude, 1900 A. cretacea A. cretacea A. cretacea A. deflexa Pilsbry, 1902 Locality Locality Voucher # No Naka KC4521 Ishigaki, Okinawa Aeg02 Ishimaki, Toyohasi ismkB1 Inasa, Hamamatsu ismkA3 Monobe, Kami KC2573 Naka H0662 Sakyo, Kyoto KC7121 Mt. Nanren, KC2953 Henchung Pongedong, Cheju KKC137 Hangyeong, Cheju KKC2492 Hangyeong, Cheju TUMC112741 Mt. Soyo, Kyongido KKC1462 Tokchokk Island, KKC4685 Incheon Kamikoshiki, H1026 Kagoshima Mt. Inao, KC3206 Minamiosumi Kushimoto tsnmA2 Yasuda, Shodoshima H0417 Oya, Shizuoka H0398 Yoshii, lbara KC1822 Tojyo, Shobara KC2045 Bessho, Izumo KC6350 Mt. Hakugaku, Kure KC2776 Tobishima Island, KC2990 Sakata GenBank (COl) AB852624 AB852626 AB852712 AB852713 AB852714 AB852715 AB852661 AB852704 LC742391* LC742407* LC742408* LC742403* LC742426* AB852716 AB852717 AB852718 AB852719 AB852720 AB852721 AB852722 AB852723 AB852724 AB852666 GenBank (ITS) AB852891 AB852893 AB852982 AB852983 AB852984 AB852985 AB852928 AB852974 LC742435* LC742451* LC742452* LC742447* LC742470* AB852986 AB852987 AB852988 AB852989 AB852990 AB852991 AB852992 AB852993 AB852994 AB852933 Molecular investigation on diversity of the land snail genus Aegista (Gastropoda, ... Species names A. diversa Kuroda & Miyanaga, 1936 A. diversa A. elegantissima elegantissima (Pfeiffer, 1849) A. eumenes (Westerlund, 1883) A. eumenes A. fausta Kuroda & Habe, 1951 A. friedeliana friedeliana (von Martens, 1864) A. friedeliana humerosa (Pilsbry & Hirase, 1904) A. goodwini (Smith, 1876) A. goodwini A. gottschei (Méllendorff, 1887) A. gottschei gottschei A. gottschei gottschei A. gottschei gottschei A. gottschei gottschei A. gottschei kyobuntonis Kuroda & Miyanaga, 1943 A. gottschei kyobuntonis > . granti (Pfeiffer, 1865) > . hachijoensis (Pilsbry, 1902) > . hachijoensis > . hatakedai Kuroda & Habe, 1951 > . hilgendorfi (Kobelt, 1879) > . hilgendorfi > . horrida (Pilsbry, 1900) Locality Namsan, Chunchon Namsan, Chunchon Mt. Hedo, Kunigami Amidadera, Shimonoseki Goya, Tagawa Mt. Toma, Hatsukaichi Goya, Tagawa Kamikoshiki, Kagoshima Akasaka, Ogaki Sakyo, Kyoto Incheon Mt. Soyo, Kyongido Mt. Soyo, Kyongido Chumk, Incheon Mt. Soyo, Kyongido Kobun Island, Chollanamdo Kobun Island, Chollanamdo Wulai, Taipei Kurio, Yakushima Satamisaki, Minamiosumi Kusama, Niimi Akasaka, Ogaki Ibuki, Maibara Yunohama, Tsuruoka Locality Voucher # No 5 KKC4974 5 TUMC112742 KC9200 KC3209 KC3080 KC7539 KC3072 H1024 otmA1 KC3299 H0707 3 TUMC112743 3 KKC1463 6 KKC1595 3 KKC4194 7 KKC2712 7 KKC2956 KC2965 KC4997 H0415 KC7286 hilA1 hilE1 H0429 GenBank (COl) LC742433* LC742434* AB852667 AB852725 AB852726 AB852627 AB852628 AB852629 AB852727 AB852728 AB852630 LC742402* LC742404* LC742405* LC742422* LC742409* LC742411* AB852631 AB852729 AB852730 AB852668 AB852731 AB852732 AB852669 GenBank (ITS) LC742477* LC742478* AB852935 AB852995 AB852996 AB852894 AB852895 AB852896 AB852997 AB852998 AB852897 LC742446* LC742448* LC742449* LC742466* LC742453* LC742455* AB852898 AB852999 AB853000 AB852936 AB853001 AB853002 AB852937 Species names A. incertus (Pfeiffer, 1866) A. inexpectata Kuroda & Minato, 1977 A. inexpectata A. izuensis (Pilsbry & Hirase, 1904) A. kandai Azuma, 1970 A. kanmuriyamensis Azuma & Azuma, 1982 A. kiusiuensis kiusiuensis (Pilsbry, 1900) A. kiusiuensis tokunovaga (Pilsbry & Hirase, 1905) A. kobensis discus (Pilsbry & Hirase, 1904) A. kobensis kobensis (Schmacker & O. Boettger, 1890) A. kobensis kobensis A. kobensis koshikijimana (Pilsbry & Hirase, 1904) A. kobensis pertenuis (Pilsbry & Hirase, 1904) A. kunimiensis Azuma & Azuma, 1982 A. latizona (Kuroda & Habe, 1949) A. lautsi (Schmacker & O. Boettger, 1890) A. lautsi A. lepidophora (Gude, 1900) A. lepidophora A. mackensii (Adams & Reeve, 1850) A. mackensii Kimura K et al Locality Wulai, Taipei Ishimaki, Toyohashi Nippara, Okutama Yugashima, Izu Yakata Island, Saeki Sakauchi, Ibigawa Onotsu, Kikai Akirigami, Kagoshima Tosayama, Kochi Ashiu, Nantan Yasuda, Shodoshima Kamikoshiki, Kagoshima Hiburi Island, Ehime Kunimi, Shizukuishi Toubara, Kishiwada Mt. Nanren, Henchung Kenting, Henchung Mt. Nekumachiji, Ogimi Mt. Katsuu, Nago Ishigaki, Okinawa lriomote, Okinawa Locality Voucher # No KC2968 KC2536 KC1158 H0423 KC4222 KC2225 KC4280 KC4381 KC1245 KC3355 H0413 H1138 KC1618 KC2242 krobA1 KC2304 KC2299 H1311 H1310 KC2324 kriw3 GenBank (COl) AB852705 AB852670 AB852671 AB852733 AB852662 AB852632 AB852672 AB852673 AB852633 AB852634 AB852635 AB852636 AB852637 AB852638 AB852734 AB852639 AB852640 AB852674 AB852675 AB852676 AB852677 GenBank (ITS) AB852975 AB852938 AB852939 AB853003 AB852929 AB852899 AB852940 AB852941 AB852900 AB852901 AB852902 AB852903 AB852904 AB852905 AB853004 AB852906 AB852907 AB852942 AB852943 AB852944 AB852945 Molecular investigation on diversity of the land snail genus Aegista (Gastropoda, ... Species names A. marginata (Pilsbry & Hirase, 1903) A. mayasana (Azuma, 1969) A. meiacoshimensis (Adams & Reeve, 1850) A. meiacoshimensis A. mesogonia (Pilsbry, 1900) A. nitens (Pilsbry & Hirase, 1904) A. nitens A. oculus (Pfeiffer, 1850) A. okinoshimae (Pilsbry, 1902) A. okinoshimae A. optima (Pilsbry, 1902) A. oshimana (Pilsbry & Hirase, 1903) A. oshimana A. ottoi (Pilsbry, 1927) A. pallens (Jacobi, 1898) A. pannosa (Pilsbry, 1902) A. proba eminens (Pilsbry & Hirase, 1904) A. proba goniosoma (Pilsbry & Hirase, 1904) A. proba goniosoma A. proba goniosoma A. proba goniosoma A. proba mikuriyensis (Pilsbry, 1902) A. proba minula (Pilsbry, 1900) Locality No Mt. Hedo, Kunigami Mt. Maya, Kobe Ishigaki, Okinawa Iriomote, Okinawa Yasuda, Shodoshima Mt. Yuwan, Uken Inutabu, Kagoshima Miyako, Okinawa Moshima, Okinoshima Ashizuri, Tosashimizu Naka En, Tatsugo Setouchi, Kagoshima Kobun Island, 7 Chollanamdo Oda, Uchiko Mt. Aoba, Sendai Atawa, Mihama Inasa, Hamamatsu Toyama, Minamishinano Kaminago, Shibakawa Ten’ei Kuchisakamoto, Shizuoka lbuki, Maibara Locality Voucher # KC2007 H0397 psd02 KC3211 H0403 KC3109 KC4378 H2849 KC5683 KC1600 KC4503 KC2011 KC2682 KKC2729 KC1818 Aeg05 H1234 H1126 KC3302 KC2206 H0910 KC3191 H1005 GenBank (COl) AB852678 AB852641 AB852706 AB852707 AB852735 AB852736 AB852737 AB852642 AB852738 AB852739 AB852740 AB852679 AB852680 LC742410* AB852741 AB852681 AB852643 AB852644 AB852645 AB852646 AB852647 AB852648 AB852649 GenBank (ITS) AB852947 AB852908 AB852976 AB852977 AB853005 AB853006 AB853007 AB852909 AB853008 AB853009 AB853010 AB852949 AB852950 LC742454* AB853011 AB852951 AB852910 AB852911 AB852912 AB852913 AB852914 AB852915 AB852916 Kimura K et al Species names Locality Locality Voucher # GenBank GenBank No (COl) (ITS) . proba minula Hatsuchi, Okazaki H0655 AB852650 + AB852917 . proba proba (Adams, 1868) Kawaradani, KC2268 AB852651 AB852918 Shirahama . proxima (Pilsbry & Hirase, 1909) Mt. Palgon, Taegu 8 KKC514 LC742398* LC742442* . proxima Mageundambong, AS) KKC522 LC742399* LC742443* Taegu . proxima Samchok, 10 TUMC112744 LC742400* LC742444* Kangwondo . proxima Samchok, 11 KKC600 LC742401* LC742445* Kangwondo . proxima Yeongju, 12 KKC3866 LC742418* LC742462* Kyongsanpukdo . proxima Sichon, 13 KKC3985 LC742419* LC742463* Kyongsannamdo . proxima Masan, 14 KKC4001 LC742420* LC742464* Kyongsannamdo . proxima Koje Island, 15 KKC4758 LC742428* LC742472* Kyongsannamdo . proxima Koje Island, 15 KKC4759 LC742429* LC742473* Kyongsannamdo . proxima Koje Island, 15 KKC4785 LC742430* LC742474* Kyongsannamdo . proxima Koje Island, 15 KKC4787 LC742431* LC742475* Kyongsannamdo . pseudopatula (Méllendorff, 1899) Gansu KC9593 AB852652 = AB852919 . pyramidata hebes (Pilsbry, 1927) Seosan, 16 KKC3584 LC742416* LC742460* Chunchonnamdo . pyramidata hebes Seosan, 16 KKC3585 LC742417* LC742461* Chunchonnamdo A. pyramidata pyramidata (Pilsbry, | Kageodo, 17 KKC3308 LC742412* LC742456* 1927) Chollanamdo A. pyramidata pyramidata Kageodo, 17 TUMC112745 LC742413* LC742457* Chollanamdo Molecular investigation on diversity of the land snail genus Aegista (Gastropoda, ... Species names A. pyramidata pyramidata A. pyramidata pyramidata A. quelpartensis (Pilsbry & Hirase, 1908) A. quelpartensis A. quelpartensis A. quelpartensis A. quelpartensis A. scepasma (Reeve, 1854) A. scepasma A. shikokuensis (Pilsbry & Hirase, 1903) A. smithiana (Pilsbry, 1901) A. smithiana A. sp. A. sp. A. sp. A. sp.1 A. sp.10 A. sp.11 A. sp.12 A. sp.13 Locality Kageodo, Chollanamdo Kageodo, Chollanamdo Hallim, Cheju Hallim, Cheju Hangyeong, Cheju Andokk, Seogwipo Hangyeong, Cheju lheya, Okinawa Mt. Nekumachiji, Ogimi Tsurugi Haruno, Kochi Tosayamada, Kami Pongedong, Cheju Talsong, Taegu Chechon, Chunchonpukdo Hon Island, Chollanamdo Hon Island, Chollanamdo UI Island, Incheon Totsui, Yura Takihata, Kawachinagano Kurokawa, Miyata Muang Kham Mt. Yudono, Tsuruoka Locality Voucher # No 17 KKC3371 17 KKC3391 18 KKC222 18 KKC224 19 TUMC112746 20 KKC351 2 KKC2489 KC3627 KC3607 H0657 H0678 KC1224 1 KKC138 21 KKC484 22 KKC4217 23 KKC4623 23 KKC4629 24 KKC4705 awjA3 tkhtA1 KC5621 KC3594 KC3113 GenBank (COl) LC742414* LC742415* LC742393* LC742394* LC742395* LC742396* LC742406* AB852682 AB852683 AB852742 AB852743 AB852744 LC742392* LC742397* LC742423* LC742424* LC742425* LC742427* AB852711 AB852753 AB852754 AB852701 AB852653 GenBank (ITS) LC742458* LC742459* LC742437* LC742438* LC742439* LC742440* LC742450* AB852952 AB852953 AB853012 AB853013 AB853014 LC742436* LC742441* LC742467* LC742468* LC742469* LC742471* AB85298 1 AB853024 AB853025 AB852971 AB852920 10 Species names A. sp.2 A. sp.3 A. sp.4 A. sp.5 A. sp.6 A. sp.7 A. sp.8 A. sp.9 A. squarrosa tokunoshimana (Pilsbry & Hirase, 1904) A. subchinensis (M6llendorff, 1884) A. tenuissima omorii Kuroda, 1936 A. tenuissima tenuissima (Pilsbry & Hirase, 1908) > . tokyoensis Sorita, 1980 > . trochula (Adams, 1868) A. tumida cavata (Pilsbry, 1902) A. tumida cavata A. tumida tumida (Gude, 1901) > . turritus (Gude, 1900) A. vermis (Reeve, 1852) A. vulgivaga lanx (Pilsbry, 1902) A. vulgivaga vulgivaga (Schmacker & Boettger, 1890) A. vulgivaga vulgivaga A. vulgivaga vulgivaga A. vulgivaga vulgivaga A. vulgivaga vulgivaga A. vulgivaga vulgivaga Kimura K et al Locality Kodomari, Nakadomari Akiu, Sendai Ten’ei Ogawa, Wakasa Mt. Myojyo, ltoigawa Dorogawa, Tenkawa Shirasaki, Yura Mijikano, Katsuragi Inutabu, Kagoshima Wulai, Taipei Mt. Soyo, Kyongido Namsan, Chunchon Bunkyo, Tokyo Chikugo, Nagasaki Hikigawa, Shirahama Hongu, Tanabe Takihata, Kawachinagano Onna, Okinawa Iriomote, Okinawa Suii, Anan Mt. Myojyo, ltoigawa Shiramine, Hakusan Mt. Fujiwara, Inabe Taga Sakyo, Kyoto Nariwa, Takahashi Locality Voucher # KC2243 H0972 KC7649 KC1848 H0420 H0424 srskA3 H0407 KC4945 KC2956 KKC4185 TUMC112747 H0652 KC6702 KC4607 KC4576 H0700 H0780 KC2836 HO0666 Aeg03 H0703 KC1148 KC1949 KC2075 KC4916 GenBank (COl) AB852745 AB852746 AB852747 AB852748 AB852749 AB852750 AB852751 AB852752 AB852654 AB852655 LC742462* LC742432* AB852656 AB852663 AB852657 AB852658 AB852659 AB852710 AB852660 AB852684 AB852685 AB852686 AB852687 AB852688 AB852689 AB852690 GenBank (ITS) AB853016 AB853017 AB853018 AB853019 AB853020 AB853021 AB853022 AB853023 AB852921 AB852922 LC742465* LC742476* AB852923 AB852930 AB852924 AB852925 AB852926 AB852980 AB852927 AB852954 AB852955 AB852956 AB852957 AB852958 AB852959 AB852960 Molecular investigation on diversity of the land snail genus Aegista (Gastropoda, ... Species names A. vulgivaga vulgivaga A. vulgivaga vulgivaga A. vulgivaga vulgivaga A. vulgivaga vulgivaga A. vulgivaga vulgivaga A. vulgivaga vulgivaga Euhadra awaensis occidentalis Azuma, Tatewaki & Okamura,1987 Euhadra herklotsi (von Martens, 1861) Euhadra peliomphala (Pfeiffer, 1850) Figure 2. EESl Shells of the Korean Aegista species used in this study. a A. chejuensis; b A. chosenica; c A. diversa; d A. gottschei gottschei; e A. gottschei kyobuntonis; f A. ottol; g A. proxima; h A. pyramidata pyramidata; i A. pyramidata hebes; j A. quelpartensis; k A. tenuissima tenuissima; 1A. tenuissima omorii. Locality Mt. Nachi, Nachikatsuura Kumanogawa, Shingu Takihata, Kawachinagano Ishimaki, Toyohashi Inasa, Hamamatsu Toyama, Minamishinano Locality Voucher # No KC4596 KC7057 H0698 Aeg21 KC2105 KC3303 KC7333 KC3081 KC1149 GenBank (COl) AB852691 AB852692 AB852693 AB852694 AB852695 AB852696 AB852698 AB852699 AB852700 GenBank (ITS) AB85296 1 AB852962 AB852963 AB852964 AB852965 AB852966 AB852968 AB852969 AB852970 11 12 Kimura K et al Phylogenetic analyses Total DNA was isolated from each foot piece and swabbed subsance using the DNeasy Blood & Tissue Kit or QlAamp DNA Micro Kit (Qiagen), according to the manufacturer’s instructions. Fragments of mitochondrial (cytochrome oxidase subunit | [COI] gene) and nuclear (internal transcribed spacer [ITS] 1-2 regions and 5.8S rRNA gene) DNA markers were amplified and sequenced. The PCR conditions and primer sets were decided following Hirano et al. (2014). The PCR products were purified using Exo-SAP-IT (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK). Sequencing was performed using a BigDye™ Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA) and electrophoresed using an ABI 3130xl sequencer (Applied Biosystems, Carlsbad, CA, USA). The resulting COI and ITS sequences have been deposited in the DDBJ/EMBL/GenBank database (Table 1). In addition to these new sequence data for Korean Aegista, already existing datasets of the genus and its closely related genus Euhadra Pilsbry, 1890 were obtained from GenBank in order to conduct the phylogenetic analyses (Table 1). The mitochondrial and nuclear sequences were aligned with MUSCLE v.3.8 (Edgar 2004). In order to eliminate any uncertainty in the ITS alignments, trimAl (v. 1.2) with automated option (Capella-Gutiérrez et al. 2009) was used to exclude ambiguous alignment regions. The phylogenetic trees were obtained for a concatenated dataset (533 sites for COI and 1,147 sites for ITS) using Maximum Likelihood (ML) and Bayesian Inference (BI) methods. Prior to the ML and BI analyses, ModelFinder (Kalyaanamoorthy et al. 2017) was used to select the optimal partition scheme for codon position and the appropriate models for sequence evolution. As a result, the following models were selected: TIM2+F+G4 model for codon 1 of COl, TIM+F+R4 for codon 2 of COI, GIR+F+ASC+G4 for codon 3 of COl, TVM+F+R3 for ITS in the ML analysis; GTR+G for codons 1, 2 and 3 of COI and ITS in the BI analysis. ML analysis was performed with IQ-TREE (v. 1.6.8) using the options of edge- unlinked branch lengths between partitions and a perturbation strength of 1.0 (Nguyen et al. 2014, Chernomor et al. 2016). For the ML analyses, we assessed nodal support by performing ultrafast bootstrap analyses with 10,000 replications in IQ-TREE (Hoang et al. 2018). BI analysis was performed using MrBayes (v. 3.2.7) with two simultaneous runs (Ronquist and Huelsenbeck 2003). Each run consisted of four simultaneous chains for two million generations and sampling of trees every 100 generations. We discarded the first 2,001 trees as burn-in after examining the convergence of runs and their effective sample sizes (ESSs) using Tracer (v. 1.6) and used the remaining samples to estimate tree topology, branch length and substitution parameters. Species delimitation analyses To validate the Korean species of Aegista, three species delimitation analyses were conducted. For topology-based approach, ML Poisson Tree Process model (mIPTP; Zhang et al. (2013)) and the Bayesian Poisson Tree Process model (bPTP; Zhang et al. (2013)) were used and the topology of the obtained ML tree was applied for both models. For the bPTP model, we set default parameters (number of MCMC generation: 100,000; burn-in: Molecular investigation on diversity of the land snail genus Aegista (Gastropoda, ... 13 10%). Both analyses were run using web servers (hittp://species.h-its.org/ptp/). For distance-based approach, Assemble Species by Automatic Partitioning (ASAP) analysis (Puillandre et al. 2020) was performed using the COI dataset. This analysis was run using Kimura (K80) distances with the default setting at the ASAP website (hitps:// bioinfo.mnhn.fr/abi/public/asap/asapweb.html). The partitions with the first and second-best asap-scores were considered according to Puillandre et al. (2020). Results The results of the phylogenetic analyses for the concatenated sequences using ML and BI methods were mostly consistent. Only well-supported clades (the posterior probabilities 2 0.95 or ultrafast bootstrap support values 2 90%) are considered hereafter. The 12 Aegista species in South Korea were separated into two major clades (Fig. 3). Subclade A1 was composed of at least nine species of Korean Aegista and subclade A2 was composed of a Korean and two Japanese species. Subclade B2 included the remaining Korean species and showed a sister relationship with subclade B1 composed of two Japanese species. The monophyly of A. chosenica was supported only in the BI tree. Neither the pairs of the subspecies of A. gottschei (i.e. A. gottschei gottschei and A. gottschei kyobuntonis) nor A. pyramidata (i.e. A. pyramidata pyramidata and A. pyramidata hebes) showed any sister relationship. For the Korean species in subclade A1, A2, and B2 the mIPTP, bPTP and ASAP analyses provided the same pattern of species delimitation (Fig. 3) and delineated 17 species (asap- score for the ASAP: 5.00), while the ASAP also partitioned 26 species with the second-best asap-score (6.50). For all analyses, the number of the species delineated was higher than the number of the Korean Aegista species recognised. None of the six small individuals that we could not identify was treated as a species with the identified snails in the species delimitation analyses. Discussion Our phylogenetic analyses, using the COl and ITS markers, revealed that multiple phylogenetic lineages of Aegista inhabit the Korean Peninsula and they are included in clade A or B. Hirano et al. (2014) showed that the majority of the Aegista species in the mainland of Japan are included in the two clades. Our result provides an example showing a close relationship between terrestrial biotas of Japan and South Korea. In both clades A and B, the lineages of Korean Aegista are not ancestral, which may suggest dispersal events of their progenitors from the Japanese archipelago to the Korean Peninsula. To test this hypothesis, further studies are needed to examine when Aegista has diversified and compare it with the geohistorical background of the Asian continent and the Japanese archipelago. Discovery of fossils of Aegista or big data arising from NGS technologies will help to address the history of diversification of this genus. Of the 14 Aegista species and subspecies known from South Korea, phylogenetic positions of A. pumilio (Pilsbry & 14 Kimura K et al Hirase, 1909) and A. gottschei fusanica (Pilsbry, 1926) are unclear. Although the unidentified small individuals may include these two taxa, further examination using specimens of them is needed to clarify their phylogenetic relationship with the other Korean Aegista. pwagonss (KC4521) A aeragenstes (OES) otinpond casts (KCX206) oantumuiay (KC27T6) Aurted (OXCAB 251130} mein (KCAROT A hredehera humernsa Eunatra povomphaur Figure 3. EE ML tree of the Aegista species, based on the concatenated data (1680 bp) of the COI and ITS DNA markers. Each OTU label represents a species name. Numbers at nodes represent Bayesian posterior probabilities (right) and Maximum Likelihood ultrafast bootstrap support values (left). Only Bayesian posterior probabilities 2 0.95 and bootstrap support values 2 90% are shown. Scale bar indicates 0.07 substitutions per site. Arrow heads indicate clades and subclades. Blue shadows mark the Korean Aegista species. Black bars represent putative species suggested by the species delimitation analyses. Molecular investigation on diversity of the land snail genus Aegista (Gastropoda, ... 15 The species delimitation analyses suggested the presence of cryptic species in A. chosenica (Fig. 3). In addition, each of A. gottschei kyobuntonis and A. pyramidata hebes would need to be treated as a distinct species. Kuroda (1958) proposed the scientific name A. pyramidata hebesioides Kuroda, 1958 for the latter because A. hebes (Pilsbry & Hirase, 1905) is known from Taiwan. Although Kuroda’s proposal based on the comparison between species and subspecies and thus was regarded as unnecessary, it would need to be reconsidered. The analyses also suggested that several unrecognised species of Aegista inhabit South Korea. It seems that at least three species have been overlooked even if the unidentified individuals include A. pumilio and A. gottschei fusanica. Previous studies exhibited that Aegista shows a striking divergence in shell morphology and provides an example of parallel evolution of morphological traits (Hirano et al. 2014, Hirano et al. 2015). Our results also found examples of the independent evolution of shell flatness and hair-like ornamentation of the shell. For example, A. tenuissima omori has a flat shell, while A. chejuensis has a globular shell. Conclusions In this study, we provide the report on first molecular investigation of diversity of Aegista in South Korea, which unmasked some of overlooked diversity of this group. To fully understand diversity of Korean Aegista, further studies using additional specimens including A. pumilio and A. gottschei fusanica, and an additional sampling effort, especially in the central region of the Korean Peninsula, are required. Acknowledgements We express our sincere gratitude to W. Lee, J. Youn and Y. Kim for considerable assistance in our fieldwork and T. Hirano for important information on the study materials. We are also grateful to Parin Jirapatrasilp, Sheikh Sajan and Roberto Eugenio Vogler for improving the earlier manuscript. Funding program This research was supported by JSPS Grant-in-Aid for Scientific Research (No 17H04611; 22K06383) and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2016R1A6A1A05011910). References ° Capella-Gutiérrez S, Silla-Martinez JM, Gabaldon T (2009) trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 25 (15): 1972-1973. https://doi.org/10.1093/bioinformatics/btp348 16 Kimura K et al Chernomor O, von Haeseler A, Minh BQ (2016) Terrace aware data structure for phylogenomic inference from supermatrices. Systematic Biology 65 (6): 997-1008. https://doi.org/10.1093/sysbio/syw037 Chiba S, Davison A (2008) Anatomical and molecular studies reveal several cryptic species of the endemic genus Mandarina (Pulmonata: Helicoidea) in the Ogasawara Islands. 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