Abstract

Semisulcospira freshwater snails are a prominent element of the freshwater macrozoobenthos throughout East Asia and have undergone substantial diversification throughout Japan, particularly in ancient Lake Biwa. Up to 19 species are currently recognized in Japan, including a large number of endemics in Lake Biwa that are placed in the subgenus Biwamelania. However, previous workers have encountered pervasive polymorphism in morphological characters, rendering current species hypotheses tentative. Based on extensive sampling of populations from throughout Japan, followed by genetic characterization with one nuclear (28S rDNA) and two mitochondrial gene fragments (cytochrome c oxidase subunit I and 16S rDNA), the phylogenetic lineage differentiation of Semisulcospira in Japan is studied for the first time in an attempt to delineate species boundaries. The phylogenetic analyses revealed insufficient differentiation in 28S to address phylogenetic relationships amongst the Japanese species. While the mtDNA markers revealed substantial lineage differentiation, there was wide-ranging incongruence between the topology of the mtDNA-based trees and morphospecies. Moreover, there was no discernible phylogeographic structure. Haplotypes from the main mtDNA clades were found to be represented in nearly all Japanese regions, with Lake Biwa being a reservoir for every mtDNA clade. I conclude that mtDNA markers are unsuitable to address species limits in Semisulcospira from Japan and discuss polymorphisms in morphology and mtDNA plus introgression as possible causes for the observed incongruence, but without being able to identify its ultimate source.

INTRODUCTION

Freshwater snails of the genus Semisulcospira Boettger, 1886 are a conspicuous and often abundant element of the freshwater macrozoobenthos throughout most of eastern Asia, including Japan, Korea, southeastern China and Taiwan (e.g. von Martens, 1905; Yen, 1939; Davis, 1969). Semisulcospira species have been extensively studied in their role as intermediate hosts of human pathogens (e.g. Abbott, 1948; Komiya, Suzuki & Ito, 1961; Urabe, 2003; Cheng et al., 2011; Lou et al., 2011; Fu et al., 2012), and are increasingly used as biological indicators in the environmental monitoring of lakes and rivers throughout the region (Kurozumi, Ozaki & Ohki, 2004; Karube et al., 2010; Gao, Niu & Hu, 2011; Pan, Wang & He, 2011).

In stark contrast to their significance for public health and in environmental studies, our knowledge of their evolutionary history, phylogenetic systematics and taxonomy has remained remarkably incomplete. Semisulcospira is the type genus of the Semisulcospiridae—a family of freshwater cerithioideans found in eastern Asia and western North America (Strong & Köhler, 2009). The eastern Asian and western North American semisulcospirid lineages are thought to have diverged during the Palaeocene-Eocene Thermal Maximum about 55 Ma as a result of the drowning of the Thulean land bridge that once connected East Asia and North America (Strong & Köhler, 2009). Based on this biogeographical scenario and a time-calibrated mitochondrial phylogeny, Strong & Köhler (2009) proposed that the last common ancestor of Semisulcospira may have lived about 25 Ma. This inferred evolutionary origin of Semisulcospira corresponds well with the earliest appearance of shells attributable to Semisulcospira in the fossil record of Japan in the Lower to early Mid Miocene, about 15–23 Ma (Yasuno, 2010; Matsuoka & Taguchi, 2013).

In addition to the type genus, the family Semisulcospiridae includes the following (nominal) genera (in order of their description): Juga H. & A. Adams, 1854 (from western North America; see Strong & Frest, 2007), SenckenbergiaYen, 1939 (from China), Hua Chen, 1943 (from China and northern Vietnam; see Strong & Köhler, 2009), NamrutuaAbbott, 1948 (from China), KoreoleptoxisBurch & Jung, 1988 and KoreanomelaniaBurch & Jung, 1988 (both from Korea) and Biwamelania Matsuoka & Nakamura, 1981 (as subgenus of Semisulcospira; from Japan). In the taxonomic literature, however, there is little consistency in how these taxa have been delimited (Burch & Jung, 1988; Ko, Lee & Kwon, 2001; Prozorova & Rasshepkina, 2006; Lee et al., 2007) and it remains questionable whether all of them truly represent natural entities (Strong & Köhler, 2009). For example, Biwamelania was originally established as a subgenus of Semisulcospira for a putative independent radiation of snails endemic to Lake Biwa (Matsuoka, 1985; Nishino et al., 2000). However, it has subsequently been questioned whether the two nominal subgenera, Semiculcospira (Semisulcospira) and S. (Biwamelania), are indeed monophyletic sister taxa (Kamiya, Shimamoto & Hashimoto, 2011). Semisulcospira (including Biwamelania) is the only viviparous genus within a family of otherwise oviparous taxa and also the only native semisulcospirid in Japan. Varying numbers of species were recognized by different authors mainly by means of adult and juvenile shell characters, but also by employing protein electrophoretic and karyological data (e.g. Burch & Davis, 1967; Burch, 1968; Davis, 1969; Watanabe, 1984; Watanabe & Nishino, 1995). Despite having been studied extensively in the past, the actual number of Semisulcospira species in Japan has remained controversial. In the most comprehensive taxonomic revision to date, Davis (1969) listed 27 nominal species-group taxa of which only 10 were recognized as ‘true’ biological species and 17 were relegated to synonymy. Subsequently, Watanabe (1984) and Watanabe & Nishino (1995) described an additional nine species endemic to Lake Biwa mainly based on shell characteristics, which they placed in Biwamelania.

With respect to the Japanese species, taxonomic difficulties have arisen from considerable polymorphism in shell and radular characters as revealed by comparative morphological, protein electrophoretic and karyological studies (e.g. Davis, 1972; Urabe, 1992; Nakano, Wada & Izawa, 1996). This polymorphism has been attributed in part to environmental attributes such as substrate structure and water turbidity, i.e. ecophenotypism (Urabe, 1998, 2000). Moreover, protein electrophoretic and karyological studies have highlighted instances of incongruence between morphology and genetic characters that hinted at the likely existence of cryptic species and/or species hybrids (Burch, 1968; Kimura & Oniwa, 1984; Kobayashi, 1986; Oniwa & Kimura, 1986a, b; Urabe, 1993; Kimura, Sakaida & Sano, 1996; Nakano et al., 1996).

While molecular phylogenetic studies have provided insights into the relationships at the family level (Strong & Köhler, 2009; Strong et al., 2011), no DNA-based studies have yet addressed the relationships among the Japanese Semisulcospira species. A phylogenetic study of Korean species revealed widespread nonmonophyly of morphospecies in a mitochondrial phylogeny and lack of phylogenetic resolution (Lee et al., 2007). Lee et al. (2007) recovered a topology that contained two kinds of clades that differed in their phylogenetic structure: a numerically predominant (modal) Korean clade exhibited drainage-level structuring and was characterized by short internal branches. In contrast, a subset of rare, genetically divergent haplotypes formed a deeply branched sister clade to the modal clade and lacked geographic structuring. As a possible explanation for this phenomenon, the retention of ancestral polymorphisms or introgressive hybridization was discussed. However, subsequently it has been shown that the tips of the deeply branched, nonstructured Korean clade has sister lineages in Japan (Miura et al., 2013). Based on this observation it was suggested that the admixture of Japanese and Korean mtDNA lineages in Korean drainages was caused by dispersal from Japan to Korea during the Pleistocene (between 1.43 and 0.76 Ma) when sea levels were much lower than today (Miura et al., 2013).

The present study is based on extensive sampling of Semisulcospira populations from Japan, followed by genetic characterization with one nuclear (28S rDNA [28S]) and two mitochondrial gene fragments (cytochrome c oxidase subunit I gene [COI] and 16S rDNA [16S]). Most of the 16S sequences have previously been published by Miura et al. (2013), while all COI sequences are new (Table 1). In contrast to the work of Miura et al. (2013), however, all samples have been identified to morphospecies based on the most recent taxonomic revisions, in order to address the phylogenetic differentiation of the Japanese Semisulcospira species across a comprehensive taxonomic and geographical range and to evaluate critically the utility of mtDNA markers for resolving the complex taxonomy of these snails.

Table 1.

Materials used in this study with voucher information and GenBank accession numbers

Taxon Voucher Locality Loc. Code [map] Specimen Clade 16S GenBank accession COI GenBank accession 28S genotype 
Juga nigrina ZMB 106257 Albion R. tributary W of Ukiah, Montgomery Woods, California, USA  KU160155 KT820544  
 – USA – –  AY010523 – – 
Juga plicata – USA – –  AF101004 – – 
Juga silicula – USA – –  AF101003 – – 
 ZMB 114630 Capitol State Forest, Thurston Co., Washington, USA [49°55′N, 133°02′W] – – – KT820545 – 
Hua jacqueti ZMB 114163 Lao Cai, Vietnam [22°31.138′N, 103°59.632′E] – – FJ471494 KT820542 GT36 
 ZMB 114165 Lao Cai, Vietnam [22°31.138′N, 103°59.632′E] – – FJ471495 KT820543 – 
Koreoleptoxis amurensis n. comb. ZMB 113248 R. Bolshaya Ussurska, Amur basin, Primorskiy Kray, Russia [45°58.565′N, 134°1.393′E] – a b B B FJ471497 FJ471498 KT820546 KT820547 GT9 
S. tegulata ZMB 111989 Chonnam, Youngam-gun, Seongjin-ri, Korea – FJ471502 KT820551 GT10 
S. forticosta ZMB 111991 Kyungbuk, Munkyung-shi, Gaeun-eup, Korea – FJ471501 KT820548 GT1 
S. gottschei ZMB 111992 Chungbuk, Danyang-gun, Cheokseong-myun, Korea – FJ471500 KT820550 GT1 
 ZMB 112961 Kangwondo, Inje-gun, Naechon-myun, Korea – FJ471499 KT820549 GT1 
S. dolorosa ZMB 102182 M.-Park, Marugama R., Saporro, Hokkaido – HM486088 KT820573 – 
S. decipiens ZMB 114571 Off mouth of Kusatsu R., Lake Biwa, Shiga Pref., Honshu 33 – KT820554 – 
 ZMB 114574 Off mouth of Kusatsu R., Lake Biwa, Shiga Pref., Honshu 33 – KT820556 – 
 ZMB 114579 Takagi Pond, Yasu-shi, Shiga Pref., Honshu – HM486174 KT820559 – 
 ZMB 114580 Okishima Strait, Oumihachiman-shi, Lake Biwa, Shiga Pref., Honshu 32 – KT820560 – 
 ZMB 114587 Near water-quality monitoring station, Lake Biwa, Shiga Pref., Honshu – HM486083 KT820563 GT1 
 ZMB 114718 Bay at northern tip nr Yanokuma, Lake Biwa, Shiga Pref., Honshu [35°28.4′N, 136°7.14′E] 23 a b c G K I HM486089 HM486090 HM486091 KT820568 KT820569 KT820570 – – – 
 ZMB 114724 Bay at northern tip nr Yanokuma, Lake Biwa, Shiga Pref., Honshu [35°28.4′N, 136°7.14′E] 34 HM486145 KT820628 GT31 
 ZMB 114765 W coast in Omi-Imatsu, Lake Biwa, Shiga Pref., Honshu [35°23.22′N, 136°2.03′E] 21 a b c G G K HM486092 HM486093 HM486094 KT820565 KT820566 KT820567 – GT1 GT16 
 S. habei yamaguchi ZMB 114724 Bay at northern tip nr Yanokuma, Lake Biwa, Shiga Pref., Honshu [35°28.4′N, 136°7.14′E] 23 b c G I HM486146 HM486147 KT820627 KT820629 GT31 GT1 
S. kurodai ZMB 114713 River in Sasayama, Hyogo Pref., Honshu [35°4′N, 135°15′E] 17 a b c d K I K G HM486097 HM486098 HM486099 HM486100 KT820574 KT820575 KT820576 KT820577 – GT1 GT1 GT1 
 ZMB 114754 Creek in Sasayama c. 3 km W crossroads 372/173, Hyogo Pref., Honshu [35°4′N, 135°15′E] 17 a b K K HM486101 HM486102 KT820578 KT820579 – GT1 – 
S. libertina ZMB 112963 Chungnam, Asan-shi, Umbong-myun, Korea – – KT820581 GT1 
 ZMB 111988 Jeju, Namjeju-gun, Korea – – KT820580 GT1 
 ZMB 114589 Tributary of Tenjin R., Ootsu-shi, Shiga Pref., Honshu 20 HM486084 KT820582 – 
 ZMB 114591 Irrigation ditch, Ika-gun, Takatsuki-cho, Shiga Pref., Honshu – HM486086 KT820583 – 
 ZMB 114703 Gion District (old quarter), Kyoto, Kamo-gawa, Kyoto Pref., Honshu [35°2.5′N, 135°44.6′E] 18 a b C C HM486149 HM486150 KT820633 KT820634 GT1 GT19 
 ZMB 114705 Creek 15 km NW Ube at road No. 2 to Hiroshima, Yamaguchi Pref., Honshu [34°2.5′N, 131°17′E] a b E K HM486103 HM486104 KT820607 KT820608 GT17 GT1 
 ZMB 114710 Crossroads 192/293 nr Ikeda, Yoshino-gawa, Tokushima Pref., Shikoku [34°0.5′N, 133°47.3′E] 37 a b c C K C HM486107 HM486108 HM486172 KT820584 KT820585 KT820586 – – – 
 ZMB 114711 Creek nr Kake, Hiroshima Pref., Honshu [34°38.86′N, 132°22.1′E] a c E E HM486109 HM486110 KT820587 – – – 
 ZMB 114716 Yotsuya, Shimo-sakamoto 1-chome, Otsu, Lake Biwa, Shiga Pref., Honshu [35°3.2′N, 135°52.5′E] 19 a b C C HM486156 HM486157 KT820640 KT820641 – – 
 ZMB 114717 River in Haga, Hyogo Pref., Honshu [35°9.7′N, 134°32.6′E] 15 HM486111 KT820614 GT1 
 ZMB 114734 Small river, NW Mikamo at road No. 181, Hiroshima Pref., Honshu [35°9.4′N, 133°37.1′E] 12 a b E C HM486118 HM486119 KT820603 KT820604 GT1 – 
 ZMB 114739 Shobara-gawa, road No. 183, Hiroshima Pref., Honshu [34°52.3′N, 133°3.5′E] 11 E G HM486125 KT820595 GT1 
 ZMB 114740 Creek in Mino, Tsuji Station, affluent of Yoshino-gawa, Tokushima Pref., Shikoku [34°2′N, 134°2′E] 36 a b C C HM486127 – KT820599 KT820600 GT11 – 
 ZMB 114746 Larger river in Tsuyama, Okayama Pref., Honshu [35°3.7′N, 134°1.1′E] 14 HM486166 KT820654 – 
 ZMB 114747 Creek nr Muikaichi at road No. 434, affluent of Nishiki-gawa, Shimane Pref., Honshu [34°17′N, 131°58′E] a b G G HM486167 HM486168 KT820652 KT820653 GT1 GT1 
 ZMB 114751 R. in Wasaka, 10 km SW Obama (Kita-gawa drainage), Fukui Pref., Honshu [35°36′N, 135°45′E] 22 a b C C HM486128 HM486129 KT820593 KT820594 GT1 GT15 
 ZMB 114752 creek between Haga and Asayo at road No. 429, Hyogo Pref., Honshu [35°11.05′N, 134°38.02′E] 16 HM486130 KT820588 – 
 ZMB 114760 Yabe-gawa in Yabe, Fukuoka Pref., Kyushu [33°11.02′N, 130°43′E] a b J K – HM486131 KT820591 KT820592 GT1 – 
 ZMB 114776 Mouth of creek discharging into lake, Maibara City, Lake Biwa, Shiga Pref., Honshu [35°20.1′N, 136°16.4′E] 25 HM486170 KT820655 GT1 
 ZMB 114780 Creek Seno-gawa, ca. 30 km E Hiroshima at road No. 2, Hiroshima Pref., Honshu [34°15′N, 132°36′E] 10 a b E E HM486132 – KT820605 KT820606 – – 
S. morii ZMB 114577 Ootsu-shi, Lake Biwa, Shiga Pref., Honshu 19 HM486079 KT820558 GT1 
S. multigranosa ZMB 114584 Kusatsu-shi, South basin off Shimogasa-cho, Lake Biwa, Shiga Pref., Honshu 33 HM486081 KT820562 GT1 
 ZMB 114590 Oumihachiman-shi, off mouth Hino R., Lake Biwa, Shiga Pref., Honshu 20 HM486085 KT820564 GT1 
 ZMB 114728 Katata harbour, Otsu, Lake Biwa, Shiga Pref., Honshu [35°6.97′N, 135°54.5′E] 20 HM486163 KT820648 GT1 
 ZMB 114762 Hikone beach, Lake Biwa, Shiga Pref., Honshu [35°16.8′N, 136°14.5′E] 26 a b c d H G G K HM486133 HM486134 HM486135 HM486136 KT820615 KT820616 KT820617 – GT1 GT12 GT1 
 ZMB 114769 Nagahama Beach, Lake Biwa, Shiga Pref., Honshu [35°23′N, 136°15.2′E] 24 a b I K HM486095 HM486096 KT820571 KT820572 GT13 GT3 
 ZMB 114770 Nagahama beach, Lake Biwa, Shiga Pref., Honshu [35°23′N, 136°15.2′E] 24 a b I J HM486137 HM486138 KT820620 KT820621 GT3 GT13 
 ZMB 114775 Maibara City, mouth of a creek discharging into lake, Lake Biwa, Shiga Pref., Honshu [35°20.1′N, 136°16.4′E] 25 a b G G HM486139 HM486173 KT820618 KT820619 GT11 GT14 
S. nakasokae ZMB 114704 Creek 15 km NW Ube at road No. 2 to Hiroshima, Yamaguchi Pref., Honshu, [34°2.5′N, 131°17′E] a b K E HM486140 HM486141 KT820625 KT820626 GT1 GT1 
 ZMB 114732 Seta-gawa in Uji, Kyoto Pref., Honshu [34°53.2′N, 135°48.39′E] 31 a b c G I G HM486142 HM486143 HM486144 KT820622 KT820623 KT820624 GT1 GT22 GT26 
S. niponica ZMB 114575 Off mouth of Kusatsu R., Lake Biwa, Shiga Pref., Honshu 33 HM486078 KT820557 GT1 
S. reiniana ZMB 114583 Seta-gawa, below Nango flood-control barrage, Ootsu-shi, Shiga Pref., Honshu 19 HM486148 KT820630 – 
 ZMB 114586 Tributary of Tenjin R., Ootsu-shi, Shiga Pref., Honshu 19 HM486082 KT820631 GT1 
 ZMB 114588 Yasu-shi, Yanamune R., Shiga Pref., Honshu 33 – KT820632 – 
 ZMB 114706 Creek nr Oguni at waterfall, Kumamoto Pref., Kyushu [33°7.5′N, 131°2.9′E] a b K I HM486105 HM486106 – KT820609 GT1 – 
 ZMB 114707 A ffluent to Lake Biwa, Higoshiomi City S Hikone, Notogawa City, Noto-gawa, Shiga Pref., Honshu [35°10.6′N, 136°9.5′E] 28 a b K C HM486151 HM486152 KT820644 KT820645 GT1 GT1 
 ZMB 114708 Seta-gawa nr Otsu, Lake Biwa outlet, Shiga Pref., Honshu [34°58.5′N, 135°54.5′E] 29 a b c E F E HM486153 HM486154 HM486155 KT820635 KT820636 KT820637 – – GT1 
 ZMB 114721 Creek nr Notsu, 50 km S Oita, Oita Pref., Kyushu [32°59.7′N, 131°49.4′E] a b J J HM486112 HM486113 KT820597 KT820598 GT2 – 
 ZMB 114722 Creek nr Yame, S Kurume, Fukuoka Pref., Kyushu [33°13.37′N, 130°36.94′E] A b J K HM486114 HM486115 KT820612 KT820613 GT1 GT1 
 ZMB 114726 Creek nr Naokawa, 70 km S Oita at road No. 10, Oita Pref., Kyushu [32°59.7′N, 131°45.2′E] a b I I HM486116 HM486117 KT820589 KT820590 – GT1 
 ZMB 114735 Larger river in Mikamo Hiroshima Pref., Honshu [35°4.9′N, 133°37.1′E] 13 a b K K HM486120 HM486159 KT820642 KT820643 GT1 – 
 ZMB 114736 R. in Mikara, Awaji-Shima I., Hyogo Pref., Honshu [34°18′N, 134°45.3′E] 35 a b K K HM486121 HM486122 KT820610 KT820611 – GT1 
 ZMB 114737 Echi-gawa, S of Hikone, affluent to Lake Biwa, Shiga Pref., Honshu [35°11.6′N, 136°10.9′E] 27 a b K K HM486158 HM486160 KT820646 KT820647 – GT1 
 ZMB 114738 River nr Kintaykio, crossroads 1/2, Yamaguchi Pref., Honshu [34°3′N, 132°1′E] a b E I HM486123 HM486124 KT820601 KT820602 GT1 GT1 
 ZMB 114739 Shobara-gawa, road No. 183, Hiroshima Pref., Honshu [34°52.3′N, 133°3.5′E] 11 HM486126 KT820596 GT1 
 ZMB 114779 Seta-gawa in Uji, Kyoto Pref., Honshu [34°53.2′N, 135°48.39′E] 31 a b J I HM486161 HM486162 KT820638 KT820639 GT1 GT1 
 114782 Seta-gawa between Otsu and Uji, Shiga Pref., Honshu [34°54.26′N, 135°52′E] 30 HM486171 KT820656 GT22 
S. reticulata ZMB 114572 Off mouth of Kusatsu R., Lake Biwa, Shiga Pref., Honshu 33 – KT820555 – 
 ZMB 114576 Ootsu-shi, Lake Biwa, Shiga Pref., Honshu 20 – KT820552 – 
 ZMB 114581 Oumihachiman-shi, off mouth Hino-gawa, Lake Biwa, Shiga Pref., Honshu 32 HM486080 KT820561 GT1 
 ZMB 114592 Near water-quality monitoring station, Lake Biwa, Shiga Pref., Honshu – HM486087 KT820553 GT1 
 ZMB 114728 Katata harbour, Otsu, Lake Biwa, Shiga Pref., Honshu [35°6.97′N, 135°54.5′E] 20 b c K K HM486164 HM486165 KT820649 KT820650 GT1 GT1 
Semisulcospira sp. ZMB 114764 West coast, Omi-Imatsu, Lake Biwa, Shiga Pref., Honshu [35°23.22′N, 136°2.03′E] 21 HM486169 KT820651 – 
Taxon Voucher Locality Loc. Code [map] Specimen Clade 16S GenBank accession COI GenBank accession 28S genotype 
Juga nigrina ZMB 106257 Albion R. tributary W of Ukiah, Montgomery Woods, California, USA  KU160155 KT820544  
 – USA – –  AY010523 – – 
Juga plicata – USA – –  AF101004 – – 
Juga silicula – USA – –  AF101003 – – 
 ZMB 114630 Capitol State Forest, Thurston Co., Washington, USA [49°55′N, 133°02′W] – – – KT820545 – 
Hua jacqueti ZMB 114163 Lao Cai, Vietnam [22°31.138′N, 103°59.632′E] – – FJ471494 KT820542 GT36 
 ZMB 114165 Lao Cai, Vietnam [22°31.138′N, 103°59.632′E] – – FJ471495 KT820543 – 
Koreoleptoxis amurensis n. comb. ZMB 113248 R. Bolshaya Ussurska, Amur basin, Primorskiy Kray, Russia [45°58.565′N, 134°1.393′E] – a b B B FJ471497 FJ471498 KT820546 KT820547 GT9 
S. tegulata ZMB 111989 Chonnam, Youngam-gun, Seongjin-ri, Korea – FJ471502 KT820551 GT10 
S. forticosta ZMB 111991 Kyungbuk, Munkyung-shi, Gaeun-eup, Korea – FJ471501 KT820548 GT1 
S. gottschei ZMB 111992 Chungbuk, Danyang-gun, Cheokseong-myun, Korea – FJ471500 KT820550 GT1 
 ZMB 112961 Kangwondo, Inje-gun, Naechon-myun, Korea – FJ471499 KT820549 GT1 
S. dolorosa ZMB 102182 M.-Park, Marugama R., Saporro, Hokkaido – HM486088 KT820573 – 
S. decipiens ZMB 114571 Off mouth of Kusatsu R., Lake Biwa, Shiga Pref., Honshu 33 – KT820554 – 
 ZMB 114574 Off mouth of Kusatsu R., Lake Biwa, Shiga Pref., Honshu 33 – KT820556 – 
 ZMB 114579 Takagi Pond, Yasu-shi, Shiga Pref., Honshu – HM486174 KT820559 – 
 ZMB 114580 Okishima Strait, Oumihachiman-shi, Lake Biwa, Shiga Pref., Honshu 32 – KT820560 – 
 ZMB 114587 Near water-quality monitoring station, Lake Biwa, Shiga Pref., Honshu – HM486083 KT820563 GT1 
 ZMB 114718 Bay at northern tip nr Yanokuma, Lake Biwa, Shiga Pref., Honshu [35°28.4′N, 136°7.14′E] 23 a b c G K I HM486089 HM486090 HM486091 KT820568 KT820569 KT820570 – – – 
 ZMB 114724 Bay at northern tip nr Yanokuma, Lake Biwa, Shiga Pref., Honshu [35°28.4′N, 136°7.14′E] 34 HM486145 KT820628 GT31 
 ZMB 114765 W coast in Omi-Imatsu, Lake Biwa, Shiga Pref., Honshu [35°23.22′N, 136°2.03′E] 21 a b c G G K HM486092 HM486093 HM486094 KT820565 KT820566 KT820567 – GT1 GT16 
 S. habei yamaguchi ZMB 114724 Bay at northern tip nr Yanokuma, Lake Biwa, Shiga Pref., Honshu [35°28.4′N, 136°7.14′E] 23 b c G I HM486146 HM486147 KT820627 KT820629 GT31 GT1 
S. kurodai ZMB 114713 River in Sasayama, Hyogo Pref., Honshu [35°4′N, 135°15′E] 17 a b c d K I K G HM486097 HM486098 HM486099 HM486100 KT820574 KT820575 KT820576 KT820577 – GT1 GT1 GT1 
 ZMB 114754 Creek in Sasayama c. 3 km W crossroads 372/173, Hyogo Pref., Honshu [35°4′N, 135°15′E] 17 a b K K HM486101 HM486102 KT820578 KT820579 – GT1 – 
S. libertina ZMB 112963 Chungnam, Asan-shi, Umbong-myun, Korea – – KT820581 GT1 
 ZMB 111988 Jeju, Namjeju-gun, Korea – – KT820580 GT1 
 ZMB 114589 Tributary of Tenjin R., Ootsu-shi, Shiga Pref., Honshu 20 HM486084 KT820582 – 
 ZMB 114591 Irrigation ditch, Ika-gun, Takatsuki-cho, Shiga Pref., Honshu – HM486086 KT820583 – 
 ZMB 114703 Gion District (old quarter), Kyoto, Kamo-gawa, Kyoto Pref., Honshu [35°2.5′N, 135°44.6′E] 18 a b C C HM486149 HM486150 KT820633 KT820634 GT1 GT19 
 ZMB 114705 Creek 15 km NW Ube at road No. 2 to Hiroshima, Yamaguchi Pref., Honshu [34°2.5′N, 131°17′E] a b E K HM486103 HM486104 KT820607 KT820608 GT17 GT1 
 ZMB 114710 Crossroads 192/293 nr Ikeda, Yoshino-gawa, Tokushima Pref., Shikoku [34°0.5′N, 133°47.3′E] 37 a b c C K C HM486107 HM486108 HM486172 KT820584 KT820585 KT820586 – – – 
 ZMB 114711 Creek nr Kake, Hiroshima Pref., Honshu [34°38.86′N, 132°22.1′E] a c E E HM486109 HM486110 KT820587 – – – 
 ZMB 114716 Yotsuya, Shimo-sakamoto 1-chome, Otsu, Lake Biwa, Shiga Pref., Honshu [35°3.2′N, 135°52.5′E] 19 a b C C HM486156 HM486157 KT820640 KT820641 – – 
 ZMB 114717 River in Haga, Hyogo Pref., Honshu [35°9.7′N, 134°32.6′E] 15 HM486111 KT820614 GT1 
 ZMB 114734 Small river, NW Mikamo at road No. 181, Hiroshima Pref., Honshu [35°9.4′N, 133°37.1′E] 12 a b E C HM486118 HM486119 KT820603 KT820604 GT1 – 
 ZMB 114739 Shobara-gawa, road No. 183, Hiroshima Pref., Honshu [34°52.3′N, 133°3.5′E] 11 E G HM486125 KT820595 GT1 
 ZMB 114740 Creek in Mino, Tsuji Station, affluent of Yoshino-gawa, Tokushima Pref., Shikoku [34°2′N, 134°2′E] 36 a b C C HM486127 – KT820599 KT820600 GT11 – 
 ZMB 114746 Larger river in Tsuyama, Okayama Pref., Honshu [35°3.7′N, 134°1.1′E] 14 HM486166 KT820654 – 
 ZMB 114747 Creek nr Muikaichi at road No. 434, affluent of Nishiki-gawa, Shimane Pref., Honshu [34°17′N, 131°58′E] a b G G HM486167 HM486168 KT820652 KT820653 GT1 GT1 
 ZMB 114751 R. in Wasaka, 10 km SW Obama (Kita-gawa drainage), Fukui Pref., Honshu [35°36′N, 135°45′E] 22 a b C C HM486128 HM486129 KT820593 KT820594 GT1 GT15 
 ZMB 114752 creek between Haga and Asayo at road No. 429, Hyogo Pref., Honshu [35°11.05′N, 134°38.02′E] 16 HM486130 KT820588 – 
 ZMB 114760 Yabe-gawa in Yabe, Fukuoka Pref., Kyushu [33°11.02′N, 130°43′E] a b J K – HM486131 KT820591 KT820592 GT1 – 
 ZMB 114776 Mouth of creek discharging into lake, Maibara City, Lake Biwa, Shiga Pref., Honshu [35°20.1′N, 136°16.4′E] 25 HM486170 KT820655 GT1 
 ZMB 114780 Creek Seno-gawa, ca. 30 km E Hiroshima at road No. 2, Hiroshima Pref., Honshu [34°15′N, 132°36′E] 10 a b E E HM486132 – KT820605 KT820606 – – 
S. morii ZMB 114577 Ootsu-shi, Lake Biwa, Shiga Pref., Honshu 19 HM486079 KT820558 GT1 
S. multigranosa ZMB 114584 Kusatsu-shi, South basin off Shimogasa-cho, Lake Biwa, Shiga Pref., Honshu 33 HM486081 KT820562 GT1 
 ZMB 114590 Oumihachiman-shi, off mouth Hino R., Lake Biwa, Shiga Pref., Honshu 20 HM486085 KT820564 GT1 
 ZMB 114728 Katata harbour, Otsu, Lake Biwa, Shiga Pref., Honshu [35°6.97′N, 135°54.5′E] 20 HM486163 KT820648 GT1 
 ZMB 114762 Hikone beach, Lake Biwa, Shiga Pref., Honshu [35°16.8′N, 136°14.5′E] 26 a b c d H G G K HM486133 HM486134 HM486135 HM486136 KT820615 KT820616 KT820617 – GT1 GT12 GT1 
 ZMB 114769 Nagahama Beach, Lake Biwa, Shiga Pref., Honshu [35°23′N, 136°15.2′E] 24 a b I K HM486095 HM486096 KT820571 KT820572 GT13 GT3 
 ZMB 114770 Nagahama beach, Lake Biwa, Shiga Pref., Honshu [35°23′N, 136°15.2′E] 24 a b I J HM486137 HM486138 KT820620 KT820621 GT3 GT13 
 ZMB 114775 Maibara City, mouth of a creek discharging into lake, Lake Biwa, Shiga Pref., Honshu [35°20.1′N, 136°16.4′E] 25 a b G G HM486139 HM486173 KT820618 KT820619 GT11 GT14 
S. nakasokae ZMB 114704 Creek 15 km NW Ube at road No. 2 to Hiroshima, Yamaguchi Pref., Honshu, [34°2.5′N, 131°17′E] a b K E HM486140 HM486141 KT820625 KT820626 GT1 GT1 
 ZMB 114732 Seta-gawa in Uji, Kyoto Pref., Honshu [34°53.2′N, 135°48.39′E] 31 a b c G I G HM486142 HM486143 HM486144 KT820622 KT820623 KT820624 GT1 GT22 GT26 
S. niponica ZMB 114575 Off mouth of Kusatsu R., Lake Biwa, Shiga Pref., Honshu 33 HM486078 KT820557 GT1 
S. reiniana ZMB 114583 Seta-gawa, below Nango flood-control barrage, Ootsu-shi, Shiga Pref., Honshu 19 HM486148 KT820630 – 
 ZMB 114586 Tributary of Tenjin R., Ootsu-shi, Shiga Pref., Honshu 19 HM486082 KT820631 GT1 
 ZMB 114588 Yasu-shi, Yanamune R., Shiga Pref., Honshu 33 – KT820632 – 
 ZMB 114706 Creek nr Oguni at waterfall, Kumamoto Pref., Kyushu [33°7.5′N, 131°2.9′E] a b K I HM486105 HM486106 – KT820609 GT1 – 
 ZMB 114707 A ffluent to Lake Biwa, Higoshiomi City S Hikone, Notogawa City, Noto-gawa, Shiga Pref., Honshu [35°10.6′N, 136°9.5′E] 28 a b K C HM486151 HM486152 KT820644 KT820645 GT1 GT1 
 ZMB 114708 Seta-gawa nr Otsu, Lake Biwa outlet, Shiga Pref., Honshu [34°58.5′N, 135°54.5′E] 29 a b c E F E HM486153 HM486154 HM486155 KT820635 KT820636 KT820637 – – GT1 
 ZMB 114721 Creek nr Notsu, 50 km S Oita, Oita Pref., Kyushu [32°59.7′N, 131°49.4′E] a b J J HM486112 HM486113 KT820597 KT820598 GT2 – 
 ZMB 114722 Creek nr Yame, S Kurume, Fukuoka Pref., Kyushu [33°13.37′N, 130°36.94′E] A b J K HM486114 HM486115 KT820612 KT820613 GT1 GT1 
 ZMB 114726 Creek nr Naokawa, 70 km S Oita at road No. 10, Oita Pref., Kyushu [32°59.7′N, 131°45.2′E] a b I I HM486116 HM486117 KT820589 KT820590 – GT1 
 ZMB 114735 Larger river in Mikamo Hiroshima Pref., Honshu [35°4.9′N, 133°37.1′E] 13 a b K K HM486120 HM486159 KT820642 KT820643 GT1 – 
 ZMB 114736 R. in Mikara, Awaji-Shima I., Hyogo Pref., Honshu [34°18′N, 134°45.3′E] 35 a b K K HM486121 HM486122 KT820610 KT820611 – GT1 
 ZMB 114737 Echi-gawa, S of Hikone, affluent to Lake Biwa, Shiga Pref., Honshu [35°11.6′N, 136°10.9′E] 27 a b K K HM486158 HM486160 KT820646 KT820647 – GT1 
 ZMB 114738 River nr Kintaykio, crossroads 1/2, Yamaguchi Pref., Honshu [34°3′N, 132°1′E] a b E I HM486123 HM486124 KT820601 KT820602 GT1 GT1 
 ZMB 114739 Shobara-gawa, road No. 183, Hiroshima Pref., Honshu [34°52.3′N, 133°3.5′E] 11 HM486126 KT820596 GT1 
 ZMB 114779 Seta-gawa in Uji, Kyoto Pref., Honshu [34°53.2′N, 135°48.39′E] 31 a b J I HM486161 HM486162 KT820638 KT820639 GT1 GT1 
 114782 Seta-gawa between Otsu and Uji, Shiga Pref., Honshu [34°54.26′N, 135°52′E] 30 HM486171 KT820656 GT22 
S. reticulata ZMB 114572 Off mouth of Kusatsu R., Lake Biwa, Shiga Pref., Honshu 33 – KT820555 – 
 ZMB 114576 Ootsu-shi, Lake Biwa, Shiga Pref., Honshu 20 – KT820552 – 
 ZMB 114581 Oumihachiman-shi, off mouth Hino-gawa, Lake Biwa, Shiga Pref., Honshu 32 HM486080 KT820561 GT1 
 ZMB 114592 Near water-quality monitoring station, Lake Biwa, Shiga Pref., Honshu – HM486087 KT820553 GT1 
 ZMB 114728 Katata harbour, Otsu, Lake Biwa, Shiga Pref., Honshu [35°6.97′N, 135°54.5′E] 20 b c K K HM486164 HM486165 KT820649 KT820650 GT1 GT1 
Semisulcospira sp. ZMB 114764 West coast, Omi-Imatsu, Lake Biwa, Shiga Pref., Honshu [35°23.22′N, 136°2.03′E] 21 HM486169 KT820651 – 

MATERIAL AND METHODS

Samples

This study is based on samples collected in 2007 by the author at multiple sites on Kyushu, Honshu and Shikoku (Fig. 1). The material has been deposited in the Malacological Collection of the Museum für Naturkunde, Berlin (ZMB) (Table 1). Morphospecies were identified by means of external shell characters, based on figures and descriptions given by Davis (1969, 1972). Specimens that could not be unambiguously allocated to morphospecies were labelled as Semisulcospira sp. Each analysed specimen has a unique identifier consisting of voucher number and a letter (e.g. ZMB 100.000a).

Figure 1.

Map of the study area with location of sampling sites on Kyushu, Shikoku and Honshu. Circle colour indicates membership of one of haplotype lineages A–K as shown in Figure 2 and Supplementary Material Figures S1 and S2. For detailed localities refer to Table 1.

Figure 1.

Map of the study area with location of sampling sites on Kyushu, Shikoku and Honshu. Circle colour indicates membership of one of haplotype lineages A–K as shown in Figure 2 and Supplementary Material Figures S1 and S2. For detailed localities refer to Table 1.

DNA sequences and molecular analyses

Total DNA was extracted from samples preserved in 75–90% ethanol by application of a CTAB extraction protocol for molluscan tissues (Winnepenninckx, Backeljau & De Wachter, 1993). Approximately 1 mm3 of foot muscle was macerated at 60 °C in 300 µl of CTAB buffer (2% CTAB, 1.4 M NaCl, 20 mM EDTA, 100 mM Tris–HCl, pH 8.0, 0.2% β-mercaptoethanol) containing 10 µl of Proteinase K. PCR amplifications were conducted in 25 µl volumes containing 1× PCR buffer, 200 µM each dNTP, 2.0 mM MgCl2, 0.5 µM each Primer, 1.25 units of Taq polymerase and c. 50 ng DNA. After an initial denaturation step of 3 min at 96 °C, 35 cycles of each 60 s at 94 °C, 50–55 and 72 °C were performed, followed by a final extension step of 5 min at 72 °C. Partial sequences of 16S were amplified using the primers 16SL-Juga (Strong & Köhler, 2009) and H3059-Inv (Palumbi et al., 1991); partial sequences of COI were amplified using the primers L1490 and H2198 (Folmer et al., 1994). Partial sequences of 28S were sequenced using the primers D23F and D6R (Park & Ó Foighil, 2000). Both strands of the amplified gene fragments were Sanger sequenced using PCR primers. Sequence electropherograms were corrected manually and assembled using BioEdit v. 7.2.5 (Hall, 1999). All new sequences have been deposited in GenBank (Table 1).

Sequence alignment and phylogenetic analyses

The 16S and 28S sequences were aligned using the online version of MAFFT (available at http://mafft.cbrc.jp/alignment/server/), employing the E-INS-i strategy suitable for thorough alignments of sequences with multiple conserved domains and long gaps (Katoh et al., 2002). COI sequences did not require alignment as they had a consistent length of 658 bp after the primer sites were trimmed. Specimens with identical sequences were represented in the datasets as unique haplotypes/genotypes.

Prior to the phylogenetic analyses, the best-fit model of nucleotide substitution was identified for each gene fragment by employing MrModeltest (Nylander, 2002). Maximum-likelihood phylograms were reconstructed by using raxmlGui (Silvestro & Michalak, 2010) for each fragment separately as well as for a concatenated dataset of both mtDNA fragments. A data partition was applied to the concatenated dataset that allowed parameters to be estimated separately for each gene fragment. Two-hundred thorough ML bootstrap replicates were performed to assess branch support. The nuclear 28S sequences were analysed separately as described above.

RESULTS

The generalized time-reversible model of sequence evolution with invariant sites gamma distribution of rates across sites (GTR + I + Γ; Tavaré, 1986) was identified as the best-fit model for both the 16S and COI datasets by means of the Akaike Information Criterion.

Phylogenetic reconstructions of the mtDNA dataset were based on a concatenated COI and 16S dataset for 121 individuals with a combined length of 1,574 bp (COI: 658 bp; 16S: 916 bp). Five basal semisulcospirids (Juga spp. and Hua jacqueti) were used as outgroups to root the trees (see Strong & Köhler, 2009, for a phylogeny of the Semisulcospiridae). The ingroup was represented by specimens of Koreoleptoxis amurensis (Gerstfeldt, 1859) n. comb. from the Russian Far East, sequences of Korean semisulcospirids, and 110 specimens from Japan representing the 11 nominal species S. libertina, S. dolorosa (Gould, 1859), S. niponica (Smith, 1876), S. reiniana (Brot, 1876), S. decipiens (Westerlund, 1883), S. multigranosa (Boettger, 1886), S. nakasokae Kuroda, 1929, S. kurodai Kajiyama & Habe, 1961, S. reticulata Kajiyama & Habe, 1961), S. habeiDavis, 1969 and S. moriiWatanabe, 1984. The Japanese samples originated from the four main islands of Japan, Hokkaido (1 sample), Honshu (55 samples), including Lake Biwa (23 samples), Shikoku including Awaji Island (3 samples) and Kyushu (5 samples) (Table 1; Fig. 1). Seven species (S. niponica, S. multigranosa, S. decipiens, S. reticulata, S. habei and S. morii) are endemic to Lake Biwa on Honshu. Between one and four specimens from each sample were sequenced. Presumably due to primer mismatch, I was not able to amplify both mtDNA fragments from all specimens. Thus, the 110 Japanese specimens in the concatenated dataset were represented by 96 16S sequences and 103 COI sequences. Missing sequence fragments in the concatenated dataset were coded as unknown. To test the effect of these missing sequences on the tree topology, the 16S and COI sequences also were analysed separately.

Phylogenetic reconstructions based on the concatenated dataset (Fig. 2) as well as on the 16S and COI datasets separately (Supplementary Material Figs S1, S2) produced trees with largely consistent topologies. All trees consistently contained the same divergent mtDNA clades (labelled A-K in Fig. 2 and Supplementary Material Figs S1, S2). Most significantly, all individuals were consistently found to be members of the same main clade. With respect to the effects on the principal branching pattern, the 16S tree differed from the concatenated tree in the following respects: (1) clade A formed the sister group of clades F-K, but was not the most basal offshoot of Semisulcospira; (2) clade D was paraphyletic with respect to clade E; (3) clades G and H were sister clades and (4) clade J was paraphyletic with respect to clade K (Supplementary Material Fig. S1). The topology of the COI phylogeny differed from that of the concatenated tree as follows: (1) clade A formed the sister group of clade B; (2) both together were sister to clades F-K, not occupying the most basal split within Semisulcospira and (3) clades F and G were sisters (Supplementary Material Fig. S2). In summary, the phylogenetic position of clade A appears the least supported. This clade becomes the most basal offshoot within Semisulcospira in the concatenated tree, reflecting the incongruence between the two datasets.

Figure 2.

Best maximum-likelihood phylogram for the concatenated 16S and COI dataset of Semisulcospira. Hua jacqueti and Juga spp. were used as outgroups to root the tree. Numbers on branches indicate bootstrap support. Scale bar indicates inferred 4% sequence divergence. Colours and symbols: shaded boxes, Korean samples; names in green, S. libertina species complex; names in black, S. niponica species complex; red circles, samples from Lake Biwa; blue squares, samples from Shikoku; green triangles, samples from Kyushu; branches without symbol, samples from Honshu outside Lake Biwa. Haplotype lineages A–K indicated on topology. Figured shells exemplify morphology of sequenced specimens, to scale. For localities refer to Table 1.

Figure 2.

Best maximum-likelihood phylogram for the concatenated 16S and COI dataset of Semisulcospira. Hua jacqueti and Juga spp. were used as outgroups to root the tree. Numbers on branches indicate bootstrap support. Scale bar indicates inferred 4% sequence divergence. Colours and symbols: shaded boxes, Korean samples; names in green, S. libertina species complex; names in black, S. niponica species complex; red circles, samples from Lake Biwa; blue squares, samples from Shikoku; green triangles, samples from Kyushu; branches without symbol, samples from Honshu outside Lake Biwa. Haplotype lineages A–K indicated on topology. Figured shells exemplify morphology of sequenced specimens, to scale. For localities refer to Table 1.

The maximum uncorrected pairwise distances among the newly produced sequences of Japanese samples were 18.6% for both mtDNA markers. Two of the deeply-divergent mtDNA lineages (B, D) comprised samples exclusively from outside Japan (B: Koreoleptoxis amurensis from the Russian Far East and S. tegulata from Korea; D: S. libertina, S. gottschei and S. forticosta from Korea); clade E contained one specimen from Korea rendering the Japanese radiation nonmonophyletic (A, C, E–K). Clade D represented the ‘Korean modal mtDNA clade’ recovered by Lee et al. (2007).

In all trees, none of the Japanese species appeared as a monophyletic cluster, while in several instances different species shared identical haplotypes, such as S. libertina and S. reiniana, S. sp. and S. multigranosa, S. nakasokae and S. reiniana (Fig. 2). Furthermore, the branching pattern of the phylogeny did not follow any geographic pattern: haplotypes found at the same locality may or may not be closely related to each other, while closely related haplotypes may or may not originate from the same area. As a result, specimens of the same morphospecies from the same sampling site frequently had highly divergent haplotypes. In fact, often more than one of the 11 main clades (A–K) as delimited in Figure 2 were present at any given sampling site. Morphologically homogeneous populations of snails containing two or more highly divergent haplotypes were found in Kyushu, Shikoku and Honshu (Figs 1, 2). Along the same lines, sequences from Lake Biwa were found dispersed across the entire mtDNA phylogeny, having sister lineages among samples collected on Honshu, Kyushu and Shikoku (Fig. 2). Conversely, all but one of the main clades were represented in Lake Biwa (Fig. 2).

The 28S dataset contained sequences from 157 specimens that represented 37 individual genotypes (GT 1–37; Table 1). Sequences of the basal semisulcospirids Juga acutifilosa and Hua jacqueti were used as outgroups to root the tree (Fig. 3). The oviparous, non-Japanese semisulcospirids ‘Huaprasongi, Koreoleptoxis globus ovalis, Koreoleptoxis amurensis, Koreanomelania nodifila and Koreanomelania nodiperda were basal to a monophyletic Semisulcospira clade. The latter included the divergent Korean Semisulcospira genotypes identified by Lee et al. (2007) (GT20, GT25), a well-differentiated subclade containing sequences of S. tegulata and S. extensa from Korea (GT10) and an unresolved clade containing all remaining genotypes from Korea and Japan. Nodal bootstrap support was generally rather low.

Figure 3.

Best maximum-likelihood phylogram for 28S sequences of Semisulcospira. Hua jacqueti and Juga acutifilosa were used as outgroups to root the tree. Scale indicates inferred 0.9% sequence divergence.

Figure 3.

Best maximum-likelihood phylogram for 28S sequences of Semisulcospira. Hua jacqueti and Juga acutifilosa were used as outgroups to root the tree. Scale indicates inferred 0.9% sequence divergence.

DISCUSSION

Sequence diversity and timing of major phylogenetic splits

Japanese Semisulcospira species are essentially undifferentiated in the nuclear 28S gene, which was only phylogenetically informative above the genus level (Fig. 3). In contrast, deep lineage differentiation has been recovered in the mitochondrial genome. However, this haplotype differentiation is inconsistent with the patterns of morphological variation and spatial distribution. In most cases only two specimens with corresponding shell morphology were sequenced from each sampling site, but in nine out of 21 local populations these specimens represented different haplotype lineages. In the Lake Biwa drainage, sampling was denser and up to three different haplotype lineages were retrieved from specimens of the same morphotype found at the same site (Fig. 1). Harbouring nearly all main haplotype lineages (marked with red dot in Fig. 2), Lake Biwa forms a reservoir of haplotype diversity within Japan. Because of the limited sampling of sequences from drainages outside Lake Biwa, the local haplotype diversity was probably not completely sampled at most collection sites. To achieve such a comprehensive sampling of the entire haplotype diversity throughout Japan, many more specimens per locality from many more localities would need to be sequenced.

The earliest known Japanese fossils attributable to Semisulcospira were reported by Matsuoka & Taguchi (2013) when describing ‘Sulcospira nagiensis’ from the ‘early Middle Miocene’ of the Katsuta Group at Kaki, southwestern Japan. This fossil species is hery transferred from Sulcospira Troschel, 1858 to Semisulcospira, for two reasons. First, the shell bears strong resemblance to Recent members of the genus, exhibiting typical features such as an elongate-conical shape, flattened whorls, narrow suture and spiral cords. Second, Sulcospira is a pachychilid genus endemic to Southeast Asia (Java to southwestern China) and is not known from the Japanese fauna, either Recent or fossil (Köhler & Dames, 2009). The record of S. nagiensis provides a minimum age for the radiation of Semisulcospira on Japan. However, the evolutionary history of Semisulcospira might extend even further back in time as suggested by Strong & Köhler (2009). Thus, one can conclude that the main haplotype lineages in Japan probably diverged many millions of years ago. Based on this premise, it is fair to postulate that the deepest splits within the mitochondrial tree occurred much earlier than 4 Ma when the earliest precursor of Lake Biwa, Palaeolake Biwa, was formed (Takahashi, 2012).

Incongruence between mtDNA phylogeny and current taxonomy

Notably, the viviparous genus Semisulcospira as currently circumscribed appears to be polyphyletic in the mtDNA trees with respect to the oviparous Koreoleptoxis (Fig. 2) and Koreanomelania (16S tree only; Supplementary Material Fig. S1). By contrast, Semisulcospira is supported as monophyletic in the 28S tree (Fig. 3). This nonmonophyly in the mtDNA trees is surprising, because these genera are well differentiated from each other by different reproductive modes (viviparous vs oviparous).

With respect to the Japanese members of Semisulcospira, the most striking observation is that divergent morphotypes frequently occupy closely related or even identical branches in the mtDNA tree while, conversely, specimens with identical shell morphology are repeatedly seen to occupy unrelated positions. For example, morphologically very similar specimens from the same locality bear highly distinct haplotypes, such as all S. reiniana specimens from Kyushu (marked with blue square in Fig. 2), while highly morphologically divergent specimens from different locations bear very similar haplotypes (such as the representative shells in Fig. 2). In general, nonmonophyly of morphospecies in mitochondrial phylogenies is not a rare phenomenon and has been attributed to a wide range of causes. The mode of mitochondrial DNA inheritance could be one reason why mtDNA phylogenies may not accurately reflect the relationships of their host organisms, involving phenomena such as retention of ancestral polymorphisms, introgression, paternal leakage, heteroplasmy and even recombination (Edwards & Beerli, 2000; Funk & Omland, 2003; Ballard & Whitlock, 2004; White et al., 2008). Also, the presence of morphologically cryptic species or, conversely, taxonomic over-splitting of lineages, could result in the apparent mismatch of mtDNA trees and morphology-based identifications. Such mismatch has been observed in other freshwater cerithioideans, such as pachychilids (Köhler & Deein, 2010) and pleurocerids (Whelan & Strong, 2015). However, the sheer scale of incongruence between morphology, distribution and mitochondrial cohesion in Semisulcospira from Japan is unparalleled in any of these cases. The magnitude of inconsistency between morphological and mtDNA markers poses a significant challenge to the question of how many species of Semisulcospira there are in Japan and how they can be recognized.

Taxonomic issues: problems in species delimitation and recognition

The incorrect identification or delimitation of species could be the most trivial cause for the observed incongruence between the mtDNA tree and current taxonomy. Since the mid-19th century, almost 40 nominal species-group names have been introduced for Semisulcospira from Japan, many of which have subsequently been relegated to synonymy (Burch & Davis, 1967; Davis, 1969, 1972; Watanabe, 1984; Watanabe & Nishino, 1995). This extent of taxonomic over-splitting is certainly not unusual among gastropods, which traditionally have been identified mainly by their shell. However, the taxonomy and systematics of Semisulcospira in Japan were studied extensively between the late 1960s and mid-1980s by means of morphological, karyological and protein-electrophoretic methods. Thus, the currently accepted taxonomy, even if controversial in some details, stands on rather solid foundations. In the most comprehensive revision to date, Davis (1969) highlighted the number of chromosomes, cords and ribs on the adult shell, as well as number, size and shell sculpture of embryos as defining characters to distinguish species. Other adult shell characters, however, are subject to large phenotypic variation and therefore less reliable.

Notably, the karyotypic variation in Japanese Semisulcospira is comparable with that in only a few extreme cases in the animal kingdom, such as in certain insects (Cook, 2000) and mice (Hauffe & Searle, 1993), and is in marked contrast to the comparatively low karyotypic variation reported in the closely related Pleuroceridae from the eastern USA (Dillon, 1991). For example, among several widely recognized Japanese species, chromosome numbers can vary by several orders of magnitude: S. reiniana (haploid chromosome number: n = 20), S. kurodai and S. libertina (n = 18), S. multigranosa (n = 14–16), S. nakasokae (n = 13), S. decipiens, S. niponica and S. reticulata (n = 12, each with different numbers of metacentric and acrocentric chromosomes), S. habei (n = 7), and S. morii (n = 16) (Burch, 1968; Davis, 1969; Watanabe, 1984). Based on this karyotypic variation, Davis (1969) recognized two species complexes: the S. libertina group including species with greater numbers (n = 18–20), such as S. libertina, S. reiniana and S. kurodai, and the S. niponica group including species with fewer chromosomes (n = 7–14), such as S. niponica, S. decipiens, S. reticulata, S. habei and S. multigranosa. The S. niponica group encompasses species endemic to Lake Biwa and its outflow, the river Seta-gawa. In contrast, members of the S. libertina complex occur primarily outside Lake Biwa, although S. reiniana and S. libertina are found in streams that discharge into the lake and in immediately adjacent areas of the lake. The S. niponica group has been formally elevated to the subgenus Biwamelania (Matsuoka, 1985). However, because such a treatment likely renders the remaining species a nonmonophyletic assemblage, I consider Biwamelania a synonym of Semisulcospira (see also Kamiya et al., 2011).

Nakamura & Ojima (1990) demonstrated that in Semisulcospira changes in chromosome numbers are not accompanied by changes in cellular DNA content, thus ruling out genomic hybridization as cause of the karyotypic differentiation. Rather, they suggested that a reduction in chromosome numbers had occurred in Lake Biwa endemics as a result of Robertsonian translocation (i.e. the breaking and rearrangement of chromosomal arms) leading to the fusion of many smaller chromosomes into fewer, larger ones. In general, minor chromosomal rearrangements occur frequently even within species (e.g. Porter & Sites, 1985; Phillips & Kapuscinksi, 1987) and do not always prevent gene flow between karyotypic races (Andersson et al., 2004). However, such rearrangements can indeed have implications for speciation and radiation by causing genetic incompatibility (Rowell, Rockman & Tait, 2002). In my view, the observed karyotypic differences in Semisulcospira underpin the distinctiveness of at least two widespread species, for which the names S. libertina and S. reiniana are commonly used, and for at least six endemic species in Lake Biwa, namely S. niponica, S. decipiens, S. reticulata, S. habei, S. multigranosa and S. morii.

While these species are likely genetically incompatible owing to their different karyotypes, none of them formed a monophyletic cluster in the mitochondrial tree (when more than one specimen was sequenced). Because karyological methods could not be employed to confirm species identifications (preserved material was examined), the question arises whether there is a correlation between karyotypes and shell morphology. I identified species by means of adult and embryonic shell morphology using the key provided by Davis (1969). However, the rather large intraspecific variability of many species in conjunction with the often rather subtle differences between species rendered species identification a sometimes almost impossible task. This is true in particular for S. libertina and S. reiniana, which are nearly indistinguishable in adult shell morphology (Fig. 4, for example). According to Burch (1968) and Davis (1969), the species differ mainly in size and sculpture of the embryonic shell and in number of juveniles in the brood pouch. Consequently, I identified specimens whose embryos had well-developed axial ribs as S. reiniana, and those without as S. libertina. However, throughout the studied samples, embryos were found to vary continuously from small to large in size, smooth to ribbed in sculpture, and few (<100) to many (>800) in numbers per female. Based on these observations, I consider it questionable if S. libertina and S. reiniana can be distinguished reliably by embryonic shell morphology.

Figure 4.

Shells of sequenced specimens of Semisulcospira included in this study. A.S. niponica.B–E.S. decipiens.F–K.S. multigranosa.L–N.S. reticulata.O, P.S. habei yamaguchi.Q.S. morii.R–W.S. libertina.X–AB.S. reiniana.AC–AF.S. kurodai.AG–AI.S. nakasokae.A. ZMB 114575. B. ZMB 114571a. C. ZMB 114580. D. ZMB 114718a. E. ZMB 114724a. F. ZMB 114574. G. ZMB 114569. H. ZMB 114590. I. ZMB 114728a. J. ZMB 114769a. K. ZMB 114762a. L. ZMB 114581. M. ZMB 114578. N. ZMB 114572. O. ZMB 114724b. P. ZMB 114724c. Q. ZMB 114577. R. ZMB 114703a. S. ZMB 114705a. T. ZMB 114710b. U. ZMB 114710c. V. ZMB 114738a. W. ZMB 114780b. X. ZMB 114588. Y. ZMB 114706a. Z. ZMB 114737b. AA. ZMB 114721a. AB. ZMB 114779a. AC. ZMB 114713a. AD. ZMB 114713b. AE. ZMB 114754a. AF. ZMB 114754b. AG. ZMB 114704a. AH. ZMB 114704b. AI. ZMB 114732a. For localities refer to Table 1.

Figure 4.

Shells of sequenced specimens of Semisulcospira included in this study. A.S. niponica.B–E.S. decipiens.F–K.S. multigranosa.L–N.S. reticulata.O, P.S. habei yamaguchi.Q.S. morii.R–W.S. libertina.X–AB.S. reiniana.AC–AF.S. kurodai.AG–AI.S. nakasokae.A. ZMB 114575. B. ZMB 114571a. C. ZMB 114580. D. ZMB 114718a. E. ZMB 114724a. F. ZMB 114574. G. ZMB 114569. H. ZMB 114590. I. ZMB 114728a. J. ZMB 114769a. K. ZMB 114762a. L. ZMB 114581. M. ZMB 114578. N. ZMB 114572. O. ZMB 114724b. P. ZMB 114724c. Q. ZMB 114577. R. ZMB 114703a. S. ZMB 114705a. T. ZMB 114710b. U. ZMB 114710c. V. ZMB 114738a. W. ZMB 114780b. X. ZMB 114588. Y. ZMB 114706a. Z. ZMB 114737b. AA. ZMB 114721a. AB. ZMB 114779a. AC. ZMB 114713a. AD. ZMB 114713b. AE. ZMB 114754a. AF. ZMB 114754b. AG. ZMB 114704a. AH. ZMB 114704b. AI. ZMB 114732a. For localities refer to Table 1.

Similarly, the ‘typical forms’ of S. niponica, S. decipiens, S. multigranosa, S. reticulata and other Lake Biwa endemics are distinguished by rather subtle differences in shell sculpture, size and shape (Fig. 4). The study of large series revealed rather continuous variation between smooth, nodulose and ribbed forms with shells of varying width (Fig. 4), and it appears doubtful if adult shell features are really sufficient for identification in these species. However, the failure always to distinguish correctly between similar species cannot explain why morphologically divergent specimens repeatedly cluster closely together on the mitochondrial tree.

Apart from misidentifications, the possible existence of morphologically cryptic species also merits consideration and authors have hypothesized the existence of cryptic Semisulcospira species in the past. For example, in S. nakasokae the chromosome number in populations from the river Seta-gawa is n = 13 (Burch, 1968) but in Lake Biwa it is n = 19 (Kobayashi, 1986), implying the existence of an unrecognized species. Similarly, S. reiniana from the Lake Biwa drainage was found to contain two sympatric forms, which can be differentiated only by means of protein electrophoresis and juvenile shell morphology (Urabe, 1993). Postulating cryptic species might explain some of the observed discordance in the mtDNA tree, but in turn aggravates the difficulties with species recognition.

In summary, taxonomic problems may be causing some amount of the apparent incongruence between taxonomy and phylogeny, but they clearly cannot explain it entirely. In fact, I argue that taxonomic difficulties are not at the core of the observed widespread incongruence. This conclusion is based mainly on the lack of any phylogeographic structure in the examined dataset hinting at specifics of mtDNA inheritance as a significant factor.

Gene tree–species tree incongruence

Moore (1995) outlined why the branching topology of any one gene tree may differ from that of the species tree: the evolutionary divergence of new alleles, or haplotypes, is not constrained by the time of speciation—the origin of polymorphism. If polymorphisms are resolved by the extinction and fixation of alternative alleles, then the topology of the species tree will be imposed on the gene tree because homologous gene sequences will coalesce in the most recent common ancestor of any two species (i.e. lineage sorting sensuAvise et al., 1987). If, however, ancient polymorphisms coexisted for periods longer than the time between speciation events, then the order of speciation may not be reflected in the gene tree. Moore (1995) argued that mtDNA trees resolve species relationships more reliably than nuclear (nDNA) genes, because mtDNA has a coalescence time one quarter of that of nDNA. This assertion is based on the fact that the effective population size of mtDNA is one quarter that of nDNA by virtue of being both haploid and transmitted only through females.

However, there is a growing body of evidence that under various circumstances mtDNA markers may be unreliable for resolution of species boundaries (e.g. Funk & Omland, 2003). For example, factors that increase the effective population size of mtDNA, even to the extreme that it becomes larger than that of nDNA, render mtDNA trees less reliable (Hoelzer, 1997). Hoelzer (1997) argued that in populations containing many more females than males, with males having varying reproductive success, the effective population size of nDNA is greatly reduced with the result that the coalescence time of mtDNA becomes longer than that of nDNA. Hoelzer (1997) suggested that in population of 100 breeders, 88 of which are female, the effective population size of mtDNA would exceed that of nDNA. In addition, it has been argued that the effective population size in spatially fragmented populations with small migration rates can be substantially greater than the actual number of individuals (Nei & Takahata, 1993). Both factors might apply to Semisulcospira, potentially resulting in very large effective population sizes and in prolonged coalescence times in mtDNA. Although I was unable to find published data on sex ratios in Semisulcospira species, in the current study I found evidence of pronounced female-biased sex ratios: out of 50 dissected specimens 35 were females bearing eggs or developing young, and only 15 individuals were either males or immature nongravid females. Thus, as documented in some pleurocerids (Aldridge, 1982; Ciparis, Henley & Voshell, 2012), sex ratios in Semisulcospira may be skewed towards females, potentially reaching levels at which the effective population size of mtDNA becomes larger than that of nDNA.

The sample size in the present study is too small for general conclusions, but future studies should carefully examine the sex ratios in Semisulcospira populations as a potential cause for the retention of ancestral haplotype polymorphisms. During my field surveys I found Semisulcospira to be very common and abundant throughout freshwater habitats in Japan. Thus, the effective population size in Semisulcospira species may already be large based on the large size of the actual population. Last, but not least, one could argue that populations are fragmented along the boundaries of rivers and river catchments. That such fragmentation translates into high levels of polymorphisms has been suggested by other population-genetic studies of freshwater snails (e.g. Jarne & Delay, 1991).

Unusually high intraspecific mtDNA divergences in freshwater gastropods like pleurocerids and rissooideans have previously been attributed to the rentention of mtDNA polymorphism (Dillon & Frankis, 2004; Wilke et al., 2006). However, such polymorphisms have not yet been related to taxonomic incongruence at the interspecific level. In fact, ancestral polymorphism has been dismissed as a potential cause for taxonomic incongruence in Thai pachychilids of the genus Brotia, because the coalescence time of neutral alleles was considered to be too short to enable the retention of genetic polymorphisms over very long times (Köhler & Deein, 2010; Köhler, Panha & Glaubrecht, 2010). Indeed, from a theoretical point of view, it would require very unusual, if not extreme, circumstances to maintain ancestral polymorphisms in the mitochondrial genome over the course of millions of years (Takahata & Nei, 1990). Given the possibly Miocene origin of the present haplotype divergence in Semisulcospira, it appears questionable if population size and structure of Semisulcospira is ‘sufficiently exceptional’ to allow such widespread and deeply rooted ancestral polymorphism in the mitochondrial genome to be retained over millions of years.

In summary, population size and structure in Semisulcospira might be favourable for the retention of ancestral polymorphisms in mtDNA markers. If present, such polymorphisms could be another important source of incongruence in the mtDNA-based phylogenies. However, this phenomenon fails to explain incongruence at the deepest phylogenetic splits within Semisulcospira.

When studying the phylogenetic structure among Korean Semisulcospira species, Miura et al. (2013) found that several Korean mtDNA lineages were short-branched members of Japanese mtDNA clades. They concluded that these patterns were indicative of historic introduction of Japanese haplotypes into Korean populations, which must have occurred between 0.3 and 3 Ma. If that is correct, then probably the Japanese mtDNA haplotypes have since been retained within the Korean gene pools. This brings us to the last phenomenon potentially relevant for the present case—introgression of foreign haplotypes into local gene pools. Miura et al. (2013) concluded that in freshwater Cerithioidea in general, among-drainage migration, and potentially also introgression, could prove to be major contributing factors in many cases of heightened within-population mtDNA diversity. Introgression has also been discussed as a cause for incongruence between mtDNA phylogenies and morphology-based taxonomy in pachychilid gastropods such as Brotia (Glaubrecht & Köhler, 2004; Köhler & Deein, 2010; Köhler et al., 2010). For pachychilids it was postulated that speciation was essentially driven by geographical separation and that, if no effective isolating mechanisms had evolved in allopatry, secondary contact then allowed the introgression of neutral markers in zones of contact (Köhler & Deein, 2010). Because most species of Semisulcospira do not occur in allopatry, this scenario may not apply to the present case. However, introgression may have played a role in the persistence of Japanese haplotypes in Korea. To understand the direction and amount of gene flow between the Japanese species, important questions to be answered are which factors drove speciation in Semisulcospira and what determined current distribution patterns? Only then can we hope to disentangle this most perplexing case of mtDNA incongruence.

Outlook

The currently available information on the biology and nuclear differentiation of Semisulcospira species is not sufficiently complete to resolve fully the present case. In order to achieve a better understanding of species boundaries in Semisulcospira, future studies are needed, combining comparative morphology with karyology and multilocus DNA phylogenies. The application of population-genomic methods may be necessary to disentangle reliably the patterns of morphological and genetic differentiation and to estimate important population-genetic parameters, such as effective population size and migration rates (Luikart et al., 2003). Next-generation sequencing methods offer promising new avenues to resolve the phylogenetic patterns within this group, to uncover possible cases of introgressive hybridization and to delimit species (McCormack et al., 2013).

SUPPLEMENTARY MATERIAL

Supplementary Material is available at Journal of Molluscan Studies online.

ACKNOWLEDGEMENTS

I wish to thank M. Grygier, M. Matsuda and N. Katsuki (Lake Biwa Museum) for providing logistic assistance in the field. I thank Adnan Moussalli (Melbourne) for fruitful discussions and also an anonymous reviewer and Associate Editor Ellen Strong for helpful and constructive comments. This work was funded through research grant KO 3381 3/1 by the Deutsche Forschungsgemeinschaft (DFG), Bonn, Germany.

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