Biodiversity Data Journal : Research Article
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Research Article
Molecular investigation on diversity of the land snail genus Aegista (Gastropoda, Camaenidae) in South Korea
expand article infoKazuki 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.000@gmail.com)
Academic editor: Dimitris Poursanidis
Received: 28 Oct 2022 | Accepted: 04 Jan 2023 | Published: 31 Jan 2023
© 2023 Kazuki Kimura, Satoshi Chiba, Jae-Hong Pak
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: 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
 Open Access

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

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.

Figure 1.  

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.

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 Landouria Godwin-Austen, 1918 as a distinct genus (Köhler 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.
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List of the species included in the molecular phylogenetic analyses. Asterisks indicate the sequences newly obtained in this study.

Species names Locality Locality No Voucher # GenBank (COI) GenBank (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
A. awajiensis Naka KC4521 AB852624 AB852891
A. caelurea Kuroda & Habe, 1960 Ishigaki, Okinawa Aeg02 AB852626 AB852893
A. calcicola (Kuroda, 1989) Ishimaki, Toyohasi ismkB1 AB852712 AB852982
A. calcicola Inasa, Hamamatsu ismkA3 AB852713 AB852983
A. carinata (Gude, 1901) Monobe, Kami KC2573 AB852714 AB852984
A. carinata Naka H0662 AB852715 AB852985
A. cavicollis (Pilsbry, 1900) Sakyo, Kyoto KC7121 AB852661 AB852928
A. certus (Zilch, 1938) Mt. Nanren, Henchung KC2953 AB852704 AB852974
A. chejuensis (Pilsbry & Hirase, 1908) Pongedong, Cheju 1 KKC137 LC742391* LC742435*
A. chejuensis Hangyeong, Cheju 2 KKC2492 LC742407* LC742451*
A. chejuensis Hangyeong, Cheju 2 TUMC112741 LC742408* LC742452*
A. chosenica (Pilsbry, 1927) Mt. Soyo, Kyongido 3 KKC1462 LC742403* LC742447*
A. chosenica Tokchokk Island, Incheon 4 KKC4685 LC742426* LC742470*
A. collinsoni casta (Pilsbry, 1901) Kamikoshiki, Kagoshima H1026 AB852716 AB852986
A. collinsoni casta Mt. Inao, Minamiosumi KC3206 AB852717 AB852987
A. collinsoni collinsoni (Adams, 1868) Kushimoto tsnmA2 AB852718 AB852988
A. commoda commoda (Adams, 1868) Yasuda, Shodoshima H0417 AB852719 AB852989
A. commoda nioyaka (Pilsbry & Hirase, 1904) Oya, Shizuoka H0398 AB852720 AB852990
A. cretacea Gude, 1900 Yoshii, Ibara KC1822 AB852721 AB852991
A. cretacea Tojyo, Shobara KC2045 AB852722 AB852992
A. cretacea Bessho, Izumo KC6350 AB852723 AB852993
A. cretacea Mt. Hakugaku, Kure KC2776 AB852724 AB852994
A. deflexa Pilsbry, 1902 Tobishima Island, Sakata KC2990 AB852666 AB852933
A. diversa Kuroda & Miyanaga, 1936 Namsan, Chunchon 5 KKC4974 LC742433* LC742477*
A. diversa Namsan, Chunchon 5 TUMC112742 LC742434* LC742478*
A. elegantissima elegantissima (Pfeiffer, 1849) Mt. Hedo, Kunigami KC9200 AB852667 AB852935
A. eumenes (Westerlund, 1883) Amidadera, Shimonoseki KC3209 AB852725 AB852995
A. eumenes Goya, Tagawa KC3080 AB852726 AB852996
A. fausta Kuroda & Habe, 1951 Mt. Toma, Hatsukaichi KC7539 AB852627 AB852894
A. friedeliana friedeliana (von Martens, 1864) Goya, Tagawa KC3072 AB852628 AB852895
A. friedeliana humerosa (Pilsbry & Hirase, 1904) Kamikoshiki, Kagoshima H1024 AB852629 AB852896
A. goodwini (Smith, 1876) Akasaka, Ogaki otmA1 AB852727 AB852997
A. goodwini Sakyo, Kyoto KC3299 AB852728 AB852998
A. gottschei (Möllendorff, 1887) Incheon H0707 AB852630 AB852897
A. gottschei gottschei Mt. Soyo, Kyongido 3 TUMC112743 LC742402* LC742446*
A. gottschei gottschei Mt. Soyo, Kyongido 3 KKC1463 LC742404* LC742448*
A. gottschei gottschei Chumk, Incheon 6 KKC1595 LC742405* LC742449*
A. gottschei gottschei Mt. Soyo, Kyongido 3 KKC4194 LC742422* LC742466*
A. gottschei kyobuntonis Kuroda & Miyanaga, 1943 Kobun Island, Chollanamdo 7 KKC2712 LC742409* LC742453*
A. gottschei kyobuntonis Kobun Island, Chollanamdo 7 KKC2956 LC742411* LC742455*
A. granti (Pfeiffer, 1865) Wulai, Taipei KC2965 AB852631 AB852898
A. hachijoensis (Pilsbry, 1902) Kurio, Yakushima KC4997 AB852729 AB852999
A. hachijoensis Satamisaki, Minamiosumi H0415 AB852730 AB853000
A. hatakedai Kuroda & Habe, 1951 Kusama, Niimi KC7286 AB852668 AB852936
A. hilgendorfi (Kobelt, 1879) Akasaka, Ogaki hilA1 AB852731 AB853001
A. hilgendorfi Ibuki, Maibara hilE1 AB852732 AB853002
A. horrida (Pilsbry, 1900) Yunohama, Tsuruoka H0429 AB852669 AB852937
A. incertus (Pfeiffer, 1866) Wulai, Taipei KC2968 AB852705 AB852975
A. inexpectata Kuroda & Minato, 1977 Ishimaki, Toyohashi KC2536 AB852670 AB852938
A. inexpectata Nippara, Okutama KC1158 AB852671 AB852939
A. izuensis (Pilsbry & Hirase, 1904) Yugashima, Izu H0423 AB852733 AB853003
A. kandai Azuma, 1970 Yakata Island, Saeki KC4222 AB852662 AB852929
A. kanmuriyamensis Azuma & Azuma, 1982 Sakauchi, Ibigawa KC2225 AB852632 AB852899
A. kiusiuensis kiusiuensis (Pilsbry, 1900) Onotsu, Kikai KC4280 AB852672 AB852940
A. kiusiuensis tokunovaga (Pilsbry & Hirase, 1905) Akirigami, Kagoshima KC4381 AB852673 AB852941
A. kobensis discus (Pilsbry & Hirase, 1904) Tosayama, Kochi KC1245 AB852633 AB852900
A. kobensis kobensis (Schmacker & O. Boettger, 1890) Ashiu, Nantan KC3355 AB852634 AB852901
A. kobensis kobensis Yasuda, Shodoshima H0413 AB852635 AB852902
A. kobensis koshikijimana (Pilsbry & Hirase, 1904) Kamikoshiki, Kagoshima H1138 AB852636 AB852903
A. kobensis pertenuis (Pilsbry & Hirase, 1904) Hiburi Island, Ehime KC1618 AB852637 AB852904
A. kunimiensis Azuma & Azuma, 1982 Kunimi, Shizukuishi KC2242 AB852638 AB852905
A. latizona (Kuroda & Habe, 1949) Toubara, Kishiwada krobA1 AB852734 AB853004
A. lautsi (Schmacker & O. Boettger, 1890) Mt. Nanren, Henchung KC2304 AB852639 AB852906
A. lautsi Kenting, Henchung KC2299 AB852640 AB852907
A. lepidophora (Gude, 1900) Mt. Nekumachiji, Ogimi H1311 AB852674 AB852942
A. lepidophora Mt. Katsuu, Nago H1310 AB852675 AB852943
A. mackensii (Adams & Reeve, 1850) Ishigaki, Okinawa KC2324 AB852676 AB852944
A. mackensii Iriomote, Okinawa kriw3 AB852677 AB852945
A. marginata (Pilsbry & Hirase, 1903) Mt. Hedo, Kunigami KC2007 AB852678 AB852947
A. mayasana (Azuma, 1969) Mt. Maya, Kobe H0397 AB852641 AB852908
A. meiacoshimensis (Adams & Reeve, 1850) Ishigaki, Okinawa psd02 AB852706 AB852976
A. meiacoshimensis Iriomote, Okinawa KC3211 AB852707 AB852977
A. mesogonia (Pilsbry, 1900) Yasuda, Shodoshima H0403 AB852735 AB853005
A. nitens (Pilsbry & Hirase, 1904) Mt. Yuwan, Uken KC3109 AB852736 AB853006
A. nitens Inutabu, Kagoshima KC4378 AB852737 AB853007
A. oculus (Pfeiffer, 1850) Miyako, Okinawa H2849 AB852642 AB852909
A. okinoshimae (Pilsbry, 1902) Moshima, Okinoshima KC5683 AB852738 AB853008
A. okinoshimae Ashizuri, Tosashimizu KC1600 AB852739 AB853009
A. optima (Pilsbry, 1902) Naka KC4503 AB852740 AB853010
A. oshimana (Pilsbry & Hirase, 1903) En, Tatsugo KC2011 AB852679 AB852949
A. oshimana Setouchi, Kagoshima KC2682 AB852680 AB852950
A. ottoi (Pilsbry, 1927) Kobun Island, Chollanamdo 7 KKC2729 LC742410* LC742454*
A. pallens (Jacobi, 1898) Oda, Uchiko KC1818 AB852741 AB853011
A. pannosa (Pilsbry, 1902) Mt. Aoba, Sendai Aeg05 AB852681 AB852951
A. proba eminens (Pilsbry & Hirase, 1904) Atawa, Mihama H1234 AB852643 AB852910
A. proba goniosoma (Pilsbry & Hirase, 1904) Inasa, Hamamatsu H1126 AB852644 AB852911
A. proba goniosoma Toyama, Minamishinano KC3302 AB852645 AB852912
A. proba goniosoma Kaminago, Shibakawa KC2206 AB852646 AB852913
A. proba goniosoma Ten’ei H0910 AB852647 AB852914
A. proba mikuriyensis (Pilsbry, 1902) Kuchisakamoto, Shizuoka KC3191 AB852648 AB852915
A. proba minula (Pilsbry, 1900) Ibuki, Maibara H1005 AB852649 AB852916
A. proba minula Hatsuchi, Okazaki H0655 AB852650 AB852917
A. proba proba (Adams, 1868) Kawaradani, Shirahama KC2268 AB852651 AB852918
A. proxima (Pilsbry & Hirase, 1909) Mt. Palgon, Taegu 8 KKC514 LC742398* LC742442*
A. proxima Mageundambong, Taegu 9 KKC522 LC742399* LC742443*
A. proxima Samchok, Kangwondo 10 TUMC112744 LC742400* LC742444*
A. proxima Samchok, Kangwondo 11 KKC600 LC742401* LC742445*
A. proxima Yeongju, Kyongsanpukdo 12 KKC3866 LC742418* LC742462*
A. proxima Sichon, Kyongsannamdo 13 KKC3985 LC742419* LC742463*
A. proxima Masan, Kyongsannamdo 14 KKC4001 LC742420* LC742464*
A. proxima Koje Island, Kyongsannamdo 15 KKC4758 LC742428* LC742472*
A. proxima Koje Island, Kyongsannamdo 15 KKC4759 LC742429* LC742473*
A. proxima Koje Island, Kyongsannamdo 15 KKC4785 LC742430* LC742474*
A. proxima Koje Island, Kyongsannamdo 15 KKC4787 LC742431* LC742475*
A. pseudopatula (Möllendorff, 1899) Gansu KC9593 AB852652 AB852919
A. pyramidata hebes (Pilsbry, 1927) Seosan, Chunchonnamdo 16 KKC3584 LC742416* LC742460*
A. pyramidata hebes Seosan, Chunchonnamdo 16 KKC3585 LC742417* LC742461*
A. pyramidata pyramidata (Pilsbry, 1927) Kageodo, Chollanamdo 17 KKC3308 LC742412* LC742456*
A. pyramidata pyramidata Kageodo, Chollanamdo 17 TUMC112745 LC742413* LC742457*
A. pyramidata pyramidata Kageodo, Chollanamdo 17 KKC3371 LC742414* LC742458*
A. pyramidata pyramidata Kageodo, Chollanamdo 17 KKC3391 LC742415* LC742459*
A. quelpartensis (Pilsbry & Hirase, 1908) Hallim, Cheju 18 KKC222 LC742393* LC742437*
A. quelpartensis Hallim, Cheju 18 KKC224 LC742394* LC742438*
A. quelpartensis Hangyeong, Cheju 19 TUMC112746 LC742395* LC742439*
A. quelpartensis Andokk, Seogwipo 20 KKC351 LC742396* LC742440*
A. quelpartensis Hangyeong, Cheju 2 KKC2489 LC742406* LC742450*
A. scepasma (Reeve, 1854) Iheya, Okinawa KC3627 AB852682 AB852952
A. scepasma Mt. Nekumachiji, Ogimi KC3607 AB852683 AB852953
A. shikokuensis (Pilsbry & Hirase, 1903) Tsurugi H0657 AB852742 AB853012
A. smithiana (Pilsbry, 1901) Haruno, Kochi H0678 AB852743 AB853013
A. smithiana Tosayamada, Kami KC1224 AB852744 AB853014
A. sp. Pongedong, Cheju 1 KKC138 LC742392* LC742436*
A. sp. Talsong, Taegu 21 KKC484 LC742397* LC742441*
A. sp. Chechon, Chunchonpukdo 22 KKC4217 LC742423* LC742467*
A. sp. Hon Island, Chollanamdo 23 KKC4623 LC742424* LC742468*
A. sp. Hon Island, Chollanamdo 23 KKC4629 LC742425* LC742469*
A. sp. Ul Island, Incheon 24 KKC4705 LC742427* LC742471*
A. sp.1 Totsui, Yura awjA3 AB852711 AB852981
A. sp.10 Takihata, Kawachinagano tkhtA1 AB852753 AB853024
A. sp.11 Kurokawa, Miyata KC5621 AB852754 AB853025
A. sp.12 Muang Kham KC3594 AB852701 AB852971
A. sp.13 Mt. Yudono, Tsuruoka KC3113 AB852653 AB852920
A. sp.2 Kodomari, Nakadomari KC2243 AB852745 AB853016
A. sp.3 Akiu, Sendai H0972 AB852746 AB853017
A. sp.4 Ten’ei KC7649 AB852747 AB853018
A. sp.5 Ogawa, Wakasa KC1848 AB852748 AB853019
A. sp.6 Mt. Myojyo, Itoigawa H0420 AB852749 AB853020
A. sp.7 Dorogawa, Tenkawa H0424 AB852750 AB853021
A. sp.8 Shirasaki, Yura srskA3 AB852751 AB853022
A. sp.9 Mijikano, Katsuragi H0407 AB852752 AB853023
A. squarrosa tokunoshimana (Pilsbry & Hirase, 1904) Inutabu, Kagoshima KC4945 AB852654 AB852921
A. subchinensis (Möllendorff, 1884) Wulai, Taipei KC2956 AB852655 AB852922
A. tenuissima omorii Kuroda, 1936 Mt. Soyo, Kyongido 3 KKC4185 LC742462* LC742465*
A. tenuissima tenuissima (Pilsbry & Hirase, 1908) Namsan, Chunchon 5 TUMC112747 LC742432* LC742476*
A. tokyoensis Sorita, 1980 Bunkyo, Tokyo H0652 AB852656 AB852923
A. trochula (Adams, 1868) Chikugo, Nagasaki KC6702 AB852663 AB852930
A. tumida cavata (Pilsbry, 1902) Hikigawa, Shirahama KC4607 AB852657 AB852924
A. tumida cavata Hongu, Tanabe KC4576 AB852658 AB852925
A. tumida tumida (Gude, 1901) Takihata, Kawachinagano H0700 AB852659 AB852926
A. turritus (Gude, 1900) Onna, Okinawa H0780 AB852710 AB852980
A. vermis (Reeve, 1852) Iriomote, Okinawa KC2836 AB852660 AB852927
A. vulgivaga lanx (Pilsbry, 1902) Suii, Anan H0666 AB852684 AB852954
A. vulgivaga vulgivaga (Schmacker & Boettger, 1890) Mt. Myojyo, Itoigawa Aeg03 AB852685 AB852955
A. vulgivaga vulgivaga Shiramine, Hakusan H0703 AB852686 AB852956
A. vulgivaga vulgivaga Mt. Fujiwara, Inabe KC1148 AB852687 AB852957
A. vulgivaga vulgivaga Taga KC1949 AB852688 AB852958
A. vulgivaga vulgivaga Sakyo, Kyoto KC2075 AB852689 AB852959
A. vulgivaga vulgivaga Nariwa, Takahashi KC4916 AB852690 AB852960
A. vulgivaga vulgivaga Mt. Nachi, Nachikatsuura KC4596 AB852691 AB852961
A. vulgivaga vulgivaga Kumanogawa, Shingu KC7057 AB852692 AB852962
A. vulgivaga vulgivaga Takihata, Kawachinagano H0698 AB852693 AB852963
A. vulgivaga vulgivaga Ishimaki, Toyohashi Aeg21 AB852694 AB852964
A. vulgivaga vulgivaga Inasa, Hamamatsu KC2105 AB852695 AB852965
A. vulgivaga vulgivaga Toyama, Minamishinano KC3303 AB852696 AB852966
Euhadra awaensis occidentalis Azuma,Tatewaki & Okamura,1987 KC7333 AB852698 AB852968
Euhadra herklotsi (von Martens, 1861) KC3081 AB852699 AB852969
Euhadra peliomphala (Pfeiffer, 1850) KC1149 AB852700 AB852970
Figure 2.  

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. ottoi; g A. proxima; h A. pyramidata pyramidata; i A. pyramidata hebes; j A. quelpartensis; k A. tenuissima tenuissima; l A. tenuissima omorii.

Phylogenetic analyses

Total DNA was isolated from each foot piece and swabbed subsance using the DNeasy Blood & Tissue Kit or QIAamp DNA Micro Kit (Qiagen), according to the manufacturer’s instructions. Fragments of mitochondrial (cytochrome oxidase subunit I [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 COI, TIM+F+R4 for codon 2 of COI, GTR+F+ASC+G4 for codon 3 of COI, 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 (mlPTP; 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: 10%). Both analyses were run using web servers (http://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 (https://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 ≥ 0.95 or ultrafast bootstrap support values ≥ 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.

Figure 3.  

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 ≥ 0.95 and bootstrap support values ≥ 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.

For the Korean species in subclade A1, A2, and B2 the mlPTP, 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 COI 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 & 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.

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 omorii 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

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