ISSN 1346-7565 Acta Phytotax. Geobot. 71 (2): 129–146 (2020) doi: 10.18942/apg.201919
Phylogenetic Position, Divergence Time and Ancestral Distribution of Impatiens hypophylla Makino (Balsaminaceae)
1,* 2 3 Kaori Murayama-Takeshita , Mikio Watanabe And Noriyuki Fujii
1Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Chuo-ku, Kumamoto 860- 8555, Japan. *[email protected] (author for correspondence); 2Department of Biology, Aichi Kyoiku University, Kariya-shi, Aichi 448-8542, Japan; 3Faculty of Advanced Science and Technology, Kumamoto University 2-39-1 Kurokami, Chuo-ku, Kumamoto 860-8555, Japan
Most ‘Sohayaki elements’ have been considered to be relict species that originated from ancestors in eastern to southwestern China during the Tertiary Period. Impatiens hypophylla Makino (Balsamina- ceae), an endemic Japanese species, is considered to be a representative Sohayaki element. To assess the evolutionary history of the Sohayaki elements, we determined the phylogenetic position of I. hypophylla and estimated its divergence time as well as its ancestral distribution. Three species of Impatiens (I. hy- pophylla, I. textorii Miq., and I. ohwadae M. Watan. et Seriz.) in Japan form a clade (hereafter called ‘Japanese Impatiens’), which is sister to I. tienmushanica Y. L. Chen, a species endemic to eastern China. Japanese Impatiens and eastern Chinese species likely diverged approximately 5.68 million years ago (mya) (atpB–rbcL) and 7.37 mya (ITS), followed by further divergence of Japanese Impatiens (I. hypo- phylla, I. textorii, and I. ohwadae) at approximately 3.96 mya (atpB–rbcL) and 5.27 mya (ITS). An esti- mation of ancestral distribution using S-DIVA revealed that the common ancestor of Japanese Impatiens and the eastern Chinese species occurred in eastern China and the Japanese islands, with the most recent common ancestor of Japanese Impatiens being in the Japanese islands. As a consequence, the common ancestor of Japanese Impatiens and the eastern Chinese species would have widely expanded their dis- tribution in eastern China and the Japanese islands through the East China Sea region in the Tertiary Period. The ancestral range was probably divided into Chinese and Japanese populations due to a vicar- iance event between mainland China and the Japanese islands. Thereafter, I. hypophylla and other Japa- nese Impatiens differentiated in the Japanese islands.
Keywords: divergence time, Impatiens hypophylla, Japanese islands, phylogeny, phylogeography, sec- ondary calibration, Sohayaki elements
Japan consists of continental islands located as corridors for biotic interchange (reviewed by at the easternmost region of the Asian continent. Qiu et al. 2011). However, despite past connection The flora of Japan has been asserted to be strong- with the continent, numerous endemic species ly related to the flora of the continent (Hotta 1974, have diversified in Japan. Approximately 40% of Maekawa 1998), thus considered to be a part of the plant species and more than 20 genera (e.g. the Sino-Japanese floristic region (Good 1974, Alecterurus, Perillula, Diaspananthus, Mirica- Takhtajan 1986). Recent phylogeographic studies calia) are endemic to Japan (Hotta 1974, G. Mu- have postulated the origin of the Japanese flora by rata 1977, J. Murata 2000, Kato 2011). Therefore, considering scenarios of past connections be- to understand the evolutionary history of the Jap- tween the Asian continent and the Japanese is- anese flora, it is essential to consider both the in- lands (Li et al. 2008, Qiu et al. 2009a, b). Such fluence of past connections with the continent studies have suggested, for example, that land and the effect of geographic isolation that facili- bridges across the East China Sea (ECS) served tated the evolution of endemism. 130 Acta Phytotax. Geobot. Vol. 71
Many endemic species occur in the Sohayaki studies of P. arguta used genetic variation in nu- region, which is situated on the Pacific side of Ja- clear/chloroplast DNA sequences and nuclear mi- pan and occupying mainly the Kii peninsula, crosatellites to determine that the ancestral popu- Shikoku, and Kyushu (Maekawa 1949, Yoshino lations had been widely distributed across the 1980, Kato 1985, Yahara 1986, 1989, Washitani ECS land bridge in the Early Pleistocene (1.3–2.0 2004). This region is warm and moist due to the mya, Kimura 1996, 2003) and then strictly allo- marine climate and is the only location in Japan patrically during the Middle Pleistocene after that has not been submerged by the sea since the submergence of the land bridge (ca. 0.89 ± 0.38 Tertiary Period 2.6–66 million years ago (mya) mya) (Qi et al. 2014). Although these two studies (Murata 2004). Koidzumi (1931) defined the en- suggested a Quaternary origin for the Sohayaki demic plants in this region as the ‘Sohayaki ele- elements and are inconsistent with the early hy- ments,’ a plant group regarded as among the most pothesis of a Tertiary origin, the two species are ancient in Japan (Murata & Koyama 1976). This widespread and not endemic to Japan. Hence, group is floristically linked to a region extending further studies of Sohayaki elements using en- from eastern to southwestern China and charac- demic Japanese species are necessary to examine terized by several monotypic families and genera the early hypothesis of their origin. (Hara 1959, Murata 1968, 2004, Maekawa 1977, Impatiens hypophylla Makino (Balsaminace- Xie 1997). According to previous studies (Koid- ae) is an annual herb endemic to the Sohayaki re- zumi 1931, Maekawa 1949, 1977, Hara 1959, Hot- gion (Akiyama 1999, Kadota 2017) and is a repre- ta 1967, 1974, Murata 1968, Murata & Koyama sentative Sohayaki element (Koidzumi 1931, 1976), approximately 130 species can be consid- Hara 1959, Murata & Koyama 1976, Kadota ered to be Sohayaki elements, of which 63% are 2017). Although there have been several molecu- endemic to Japan. Therefore, most Sohayaki ele- lar phylogenetic analyses of Impatiens (Fujihashi ments appear to be relict endemic species that et al. 2002, Yuan et al. 2004, Janssens et al. 2006, originated from ancestors from eastern and/or 2007, 2012, Ruchisansakun et al. 2015, Yu et al. southwestern China during the Tertiary Period 2016), the phylogenetic position of I. hypophylla (Hara 1959, Murata 1968, 2004, Maekawa 1977, remains to be explored. Because a previous study Xie 1997). estimated the divergence time of Impatiens using Two phylogeographic studies focused on the the fossil record of related plant groups (Janssens Sohayaki elements Kirengeshoma palmata Ya- et al. 2009), phylogenetic studies of I. hypophylla tabe (Hydrangeaceae) and Platycrater arguta will enable us to estimate the divergence time of Siebold et Zucc. (Hydrangeaceae) (Qiu et al. one of the Sohayaki elements. Therefore, I. hypo- 2009a, Qi et al. 2014). Although these two spe- phylla provides an appropriate study system to cies are considered to be Sohayaki elements address the evolutionary history of the Sohayaki (Koidzumi 1931, Maekawa 1949, 1977, Hara elements. 1959, Hotta 1967, Murata & Koyama 1976), they The aims of the present study are i) to clarify are not Japanese endemic, but are disjunctly dis- the phylogenetic position of the endemic Japa- tributed in Japan (Honshu, Kii, Shikoku and Ky- nese species I. hypophylla, ii) to estimate the di- ushu) and Asian continent (Qiu et al. 2009a, Qi et vergence time from its relatives on the Asian con- al. 2014). Qiu et al. (2009a), using variation in tinent, iii) to infer the ancestral distribution of I. chloroplast DNA (cpDNA) and inter-simple se- hypophylla and related species and iv) to examine quence repeats, revealed that the migration of an- whether the evolutionary history of I. hypophylla cestral populations of K. palmata were from Ja- is consistent with the hypothesis that the Sohaya- pan to mainland China via the ECS region during ki elements diverged from their ancestors in east- the Early to Middle Pleistocene, with Japanese ern to southwestern China during the Tertiary and Chinese populations then separating in the Period. Middle Pleistocene (ca. 0.45 ± 0.197 mya). The June 2020 Murayama & al.―Phylogeny of Japanese ‘Sohayaki elements’ 131
Table 1. Materials and their sources, analyzed for the nrDNA and cpDNA of Japanese Impatiens. Accession Nos. Species / population and locality Collecter and voucher ITS atpB–rbcL Impatiens hypophylla Makino Namino, Aso-shi, Kumamoto, Japan K. Murayama & N. Fujii K04 (KUMA) LC465159 LC465198 Mt. Nokanoike, Miyoshi-shi, Tokushima, Japan K. Murayama & N. Fujii K47 (KUMA) LC465168 LC465206 Mitsue-mura, Uda-gun, Nara, Japan K. Murayama & K. Yamawaki K76 (KUMA) LC465170 LC465208 I. hypophylla var. microhypophylla (Nakai) H. Hara Mt. Chausu-dake, Toyone-mura, Aichi, Japan M. Watanabe s.n., Sep. 15, 2015 (AICH) LC465173 LC465211 Mt. Shaka-dake, Yame-shi, Fukuoka, Japan M. Watanabe s.n., Oct. 07, 2017 (AICH) LC465174 LC465212 I. textorii Miq. Nojiri, Takamori-machi, Kumamoto, Japan K. Murayama & N. Fujii K29 (KUMA) LC465175 LC465213 I. ohwadae M. Watan. et Seriz. Watarase retarding basin, Fujioka-machi, Tochigi, Japan M. Watanabe & S. Serizawa S80359 (AICH) LC465176 LC465214 I. noli-tangere L. Gokanosho, Yatsushiro-shi, Kumamoto, Japan K. Murayama & N. Fujii K08 (KUMA) LC465177 LC465215
Materials and Methods mixer mill (Multi-Beads Shocker MB455U; Yas- ui Kikai Corp., Osaka, Japan), total genomic We collected leaf samples of Impatiens hypo- DNA was extracted from 0.01 g of the dried pow- phylla var. hypophylla from three districts in Ja- der using a slightly modified version of the cetyl- pan (Kii, Kyushu, and Shikoku) and of I. hypo- trimethyl ammonium bromide (CTAB) method phylla var. microhypophylla (Nakai) H. Hara described by Doyle & Doyle (1987). In the pres- from two regions (Aichi and Fukuoka prefec- ent study, we used the internal transcribed spacer tures) (Table 1). Impatiens hypophylla var. micro- (ITS) region of the nuclear ribosomal DNA (nrD- hypophylla has smaller flowers than var. hypo- NA) and the noncoding region of atpB–rbcL of phylla and has been reported from Shizuoka, cpDNA according to the methods used by Yu et Gifu, and Nagano prefectures, including the So- al. (2016). Polymerase chain reaction (PCR) was hayaki region (Akiyama 1998, 1999, Kadota performed for the ITS and atpB–rbcL regions us- 2017). Two accessions of var. microhypophylla ing the following primers: ITS (ITS4 and ITS5; were used in the present analysis. We also col- White et al. 1990) and atpB–rbcL (IMP–atpB lected the three additional species of Impatiens in and IMP–rbcL; Janssens et al. 2006). PCR, se- Japan; I. noli-tangere L., I. ohwadae, and I. texto- quencing, and the assembly procedure were per- rii (Table 1). Impatiens noli-tangere is widely dis- formed as described by Murayama et al. (2019). tributed in Europe, Asia (including Siberia) and The sequence data obtained in the present study North America; I. ohwadae is a Japanese endem- were deposited in the DDBJ/EMBL/GenBank da- ic species of the Kanto district, and I. textorii oc- tabase (Table 1). curs in China, the Korean peninsula, and Japan To determine the phylogenetic position of the (Kadota 2017). A single sample of each species species of Impatiens in Japan, we analyzed their was collected (Table 1). Voucher specimens for phylogeny based on the study of Yu et al. (2016), these collections were deposited in the herbarium in which a comprehensive phylogenetic analysis of the Faculty of Science, Kumamoto University of Impatiens was undertaken. In that study, the (KUMA), and in the herbarium of Aichi Kyoiku phylogenetic analyses using a combined data set University (AICH). of the atpB–rbcL, trnL–F, and ITS regions re- The sample leaves were dried and preserved vealed eight major clades within Impatiens in silica gel. After powdering the leaves using a (Clades 1, 2A–2G). From the data reported in Yu 132 Acta Phytotax. Geobot. Vol. 71
'Japanese Impatiens' 68/62 Impatiens leptocaulon 84/79 I. macrovexilla I. neglecta I. hypophylla 60/51 72/61 I. pterosepala 90/88 91/86 I. noli-tangere(Japan) (Fukuoka) 94/92 var. micro. I. capensis I. noli-tangere 82/77 I. lateristachys 55/51 I. hypophylla 62/- I. oxyanthera 63/56 I. forrestii (Kii) I. faberi I. imbecilla I. hypophylla 42/- 65/71 I. platychlaena 74/74 65/67 I. principis (Shikoku) 57/62 I. lecomtei I. soulieana I. hypophylla 79/73 I. fissicornis I. davidii var. micro.(Aichi) I. tortisepala Subclade 91/91 90/88 I. hypophylla var. microhypophylla (Fukuoka) D I. hypophylla 55/51 55/51 I. hypophylla (Kii) 58/52 74/74 I. hypophylla (Shikoku) (Kyushu) 91/91 I. hypophylla var. microhypophylla (Aichi) 58/52 I. hypophylla (Kyushu) I. textorii 95/91 I. textorii (Japan) 95/91 86/75 I. ohwadae I. tienmushanica (Japan) 43/- 86/75 I. barbata I. poculifer I. ohwadae 49/- I. corchorifolia I. delavayi 62/60 I. nubigena I. tienmu 96/94 I. platysepala I. chekiangensis shanica 91/89 I. fischeri I. teitensis 54/50 Subclade E I. flanaganae I. tuberosa 89/87 I. inaperta 68/56 I. auricoma 49/- I. meruensis I. pseudoviola 59/56 I. usambarensis I. walleriana I. keilii 95/96 I. congolensis 88/84 I. niamniamensis 59/63 I. bequaertii I. sodenii I. cuspidata I. aureliana 54/53 I. leschenaultii Subclade G 48/- I. balsamina 95/95 I. columbaria I. hians 66/69 I. chinensis I. platypetala I. mengtzeana 99/97 93/90 I. cholorosepala Clade 2 98/98 I. monticola I. gongshanensis I. begoniifolia 93/91 I. napoensis 36/- 66/64 I. trichosepala 97/95 I. xanthina I. campanulata I. duclouxii 65/64 I. chiulungensis 96/97 I. conchibracteata 100/100 I. rubrostriata Subclade F I. hunanensis I. yaoshanensis I. arguta Subclade C 64/64 I. cyanantha I. uliginosa I. aquatilis I. blinii I. drepanophora 64/60 I. nyimana 100/98 I. scutisepala I. taronensis 81/84 I. bicornuta 70/51 I. cyathiflora I. rectangula I. margaritifera I. hypophylla var. microhypophylla (Aichi) 87/84 I. purpurea 76/74 I. siculifer Subclade B I. cymbifera 34/- I. tuberculata 86/86 I. laxiflora I. desmantha 87/84 I. sunkoshiensis 98/95 95/95 I. racemosa I. harae I. radiata 97/96 I. fragicolor 63/62 I. sulcata 96/94 I. parviflora 77/73 I. scullyi 94/94 I. scabrida 74/80 I. falcifer 71/61 I. angulata 65/- I. morsei I. lobulifera 100/100 I. pingxiangensis 94/- I. obesa Subclade A 46/- I. kerriae I. wenshanensis 39/94 I. stenosepala I. wilsonii I. clavigera I. balansae I. tubulosa 44/- I. chishuiensis I. pritzelii Clade 1 82/84 I. spanthulata I. malipoensis I. apalophylla I. hongkongensis Hydrocera triflora 0.009
Fig. 1. Maximum likelihood (ML) tree of Impatiens including Japanese species using the atpB–rbcL region of cpDNA se- quence data. The ML tree and most parsimonious (MP) tree showed almost the same topology. Numbers along branches indicate bootstrap probabilities (ML and MP). Values of MP analysis less than 50% were omitted. The clade names (Clades 1, 2 and Subclades A–G) were adopted FIG.from Yu 1 et al. (2016). Japanese samples newly added in this study are masked with gray. An enlargement is in the upper left; we suggest that the clade of Impatiens hypophylla, I. textorii, and I. ohwadae as ‘Japanese Impatiens’ and ‘I. hypophylla var. micro.’ represents the accessions of I. hypophylla var. micro- hypophylla. In the MP analysis, 561 MP trees were obtained; consistency index including uninformative characters (CI) = 0.6684; retention index (RI) = 0.8771. June 2020 Murayama & al.―Phylogeny of Japanese ‘Sohayaki elements’ 133
72/71 Impatiens inaperta I. hypophylla 'Japanese Impatiens' 77/76 I. furcata 100/100 I. andohahelae var. micro.(Fukuoka) 93/86 I. andringitrensis 94/97 99/96 I. gibbosa I. hypophylla 46/- I. subabortiva (Kii) 30/- I. amoena 40/- I. miniata 100/100 I. hypophylla I. baroni 79/75 I. manaharensis (Shikoku) 33/- 98/99 I. sambiranensis 72/73 I. firmula I. hypophylla 79/- I. anovensis var. micro.(Aichi) 93/92 I. fuchsioides 83/77 I. percrenata 99/99 100/100 I. vilersi 91/92 I. hypophylla 3/- I. sodenii 100/100 I. auricoma (Kyushu) I. tuberosa I. aureliana 93/71 I. textorii I. kilimanjari (Japan) 14/- 99/99 I. hians 100/100 I. usambarensis 100/100 I. textorii I. walleriana 6/- I. keilii 71/61 I. congolensis 72/65 100/100 I. bombycina I. ohwadae I. parasitica Subclade 85/67 19/- 46/52 I. niamniamensis 90/64 31/- 100/100 I. burtonii G I. stuhlmannii I. tienmushanica I. bequaertii 38/- 20/- I. hoehnelii I. balsamina 87/78 16/- I. leschenaultii I. chekiangensis I. pseudoviola 19/- I. cuspidata 87/81 I. meruensis 96/65 I. platypetala I. chinensis 78/92 I. claeri 100/100 I. zenkeri I. columbaria 52/- I. mengtzeana 99/100 I. chlorosepala 99/100 I. monticola 72/53 I. gongshanensis 20/- 86/75 I. xanthina I. begoniifolia 100/99 I. acehensis 100/100 I. napoensis 95/97 I. trichosepala I. levingei 40/- I. campanulata 75/70 65/- I. cordata I. henslowiana 54/64 I. hunanensis 35/62 100/100 I. duclouxii 24/- I. rubrostriata Subclade F I. conchibracteata 100/100 56/64 I. teitensis 42/- I. fischeri 66/- I. rothii Subclade E I. flanaganae I. arguta 57/61 I. capensis Subclade C 92/100 I. noli-tangere (Japan) 90/75 I. noli-tangere 83/61 I. tortisepala 98/87 I. pterosepala 97/94 I. neglecta I. leptocaulon 70/53 I. macrovexilla 77/55 73/86 I. lateristachys 99/97 55/- I. imbecilla I. faberi Clade 2 93/93 I. piufanensis 96/72 I. oxyanthera 53/- 83/84 31/- I. forrestii I. microstachys I. fissicornis 74/55 I. tayemonii 95/93 I. pritzelii 99/100 I. davidii 80/62 90/86 I. lecomtei Subclade I. soulieana 99/99 I. nubigena D 78/68 I. delavayi 68/55 I. poculifer 100/99 I. chiulungensis I. corchorifolia I. barbata 90/92 I. hypophylla var. microhypophylla (Fukuoka) 77/71 94/97 I. hypophylla (Kii) 100/100 I. hypophylla (Shikoku) I. hypophylla var. microhypophylla (Aichi) 91/92 99/99 I. hypophylla (Kyushu) 93/71 72/65 100/100 I. textorii(Japan) 40/100 I. textorii 85/67 I. ohwadae I. tienmushanica I. chekiangensis 74/- I. morsei 93/96 I. angulata 99/100 I. lobulifera Subclade 100/100 I. obesa A 96/100 I. pingxiangensis 43/- I. kerriae 100/100 I. aquatilis 79/74 I. cyanantha 100/100 I. uliginosa 30/- I. yaoshanensis 72/77 I. drepanophora 67/- I. rectangula 49/- I. taronensis 100/100 I. scutisepala I. holocentra 13/- I. nyimana 0/- I. margaritifera 99/100 I. eubotrya 19/- 46/- I. siculifer I. principis 99/100 I. harae 80/77 I. radiata 40/- 40/- I. racemosa 68/65 I. urticifolia Subclade B 99/100 I. laxiflora 55/56 58/62 I. desmantha 99/99 I. sunkoshiensis I. tuberculata 88/70 I. blinii 83/91 I. cymbifera 94/98 I. parviflora 56/- I. brachycentra 100/100 I. scullyi 55/- I. amphorata 100/100 I. sulcata 55/55 I. fragicolor 76/70 97/98 I. falcifer I. scabrida I. bicornuta 80/87 I. wenshanensis I. stenosepala Subclade A 62/59 I. tubulosa 50/- I. wilsonii I. chishuiensis 68/64 I. hongkongensis 76/61 I. malipoensis 0/- I. apalophylla Clade 1 0.08 100/100 I. spathulata I. balansae I. clavigera Hydrocera triflora
Fig. 2. Maximum likelihood (ML) tree of ImpatiensFIG. including 2 Japanese species using the ITS region of nrDNA sequence data. The ML tree and most parsimonious (MP) tree showed almost the same topology. Numbers along branches indicate boot- strap probabilities (ML and MP). Values of MP analysis less than 50% were omitted. The clade names (Clades 1, 2 and Subclades A–G) were adopted from Yu et al. (2016). Japanese samples newly added in the present study are masked with gray. An enlargement is shown on the upper left side; we suggest that the clade of Impatiens hypophylla, I. textorii, and I. ohwadae as ‘Japanese Impatiens’ and ‘I. hypophylla var. micro.’ represents the accessions of I. hypophylla var. microhy- pophylla. In the MP analysis, 3932 MP trees were obtained; consistency index including uninformative characters (CI) = 0.2298; retention index (RI) = 0.7223. 0.90134 1 I. chekiangensisActa Phytotax. Geobot. Vol. 71
I. hypophylla 1 var. micro. (Fu.) 0.98 Impatiens neglecta 1 I. pterosepala (2.78 mya) 0.95 I. hypophylla 0.99 I. macrovexilla III 0.95 I. leptocaulon (Kii) 1 I. noli-tangere(Japan) 1 I. noli-tangere (3.96 mya) 0.98 I. hypophylla 1 I. capensis 0.83 I. oxyanthera 1 (Shikoku) 0.99 I. forrestii I. hypophylla 0.33 I. faberi II 0.96 0.42 I. lateristachys 0.85 (5.68 mya) 1 (Kyushu) 0.83 I. imbecilla 1 I. platychlaena I. hypophylla 0.99 I. principis I var. micro. (Ai.) 0.87 I. lecomtei 1 I. soulieana 1 1 I. ohwadae 0.39 0.64 I. davidii 0.29 I. fissicornis I. tortisepala I. textorii 0.88 I. nubigena 0.36 I. delavayi I. tienmu 0.97 0.51 I. corchorifolia I. barbata shanica 0.48 I. poculifer 0.90 1 I. chekiangensis I. platysepala I. tienmu 1 I. hypophylla var.(Fu.) 1 I. teitensis 0.95 I. hypophylla(Kii) 0.99 0.98 I. hypophylla(Shi.) 0.85 1 0.96 I. hypophylla (Kyu.) I. fischeri I. hypophylla var.(Ai.) 1 1 I. ohwadae I. textorii I. flanaganae I. tienmushanica 1 I. teitensis 0.99 I. fischeri 0.34 I. flanaganae I. congolensis 0.34 I. congolensis 1 1 I. niamniamensis 1 I. keilii I. niamniamensis 0.18 I. bequaertii 1 0.98 I. usambarensis 0.26 I. walleriana I. keilii 0.14 I. sodenii 0.34 I. leschenaultii 0.22 I. cuspidata I. bequaertii 0.23 1 I. balsamina 0.18 0.99 I. aureliana 0.34 I. tuberosa 1 I. inaperta 0.98 I. usambarensis 0.20 0.93 I. auricoma 0.89 I. meruensis 0.26 I. pseudoviola I. walleriana 0.80 0.99 I. chinensis I. platypetala 0.32 1 I. columbaria 0.14 I. hians I. sodenii I. campanulata 1 1 0.32 I. monticola 0.56 I. mengtzeana 0.34 I. leschenaultii 1 I. gongshanensis 0.3 I. chlorosepala 0.22 1 1 I. napoensis I. cuspidata 0.86 I. begoniifolia 1 I. trichosepala 1 I. xanthina 0.23 I. balsamina 0.99 0.61 I. conchibracteata 1 I. duclouxii 0.99 I. chiulungensis 1 I. rubrostriata I. aureliana I. hunanensis I. arguta 0.19 I. aquatilis 0.34 I. tuberosa 0.93 0.39 I. uliginosa I. yaoshanensis 1 0.07 I. cyanantha I. inaperta 1 I. bicornuta I. cyathiflora 0.20 0.93 0.09 0.21 I. margaritifera 1 I. auricoma 0.09 I. drepanophora 0.03 1 I. scutisepala 0.89 1 0.03 I. taronensis I. rectangula I. meruensis I. blinii I. nyimana 0.57 0.33 I. desmantha I. pseudoviola 1 I. laxiflora 0.80 0.10 I. tuberculata 1 I. racemosa 0.99 I. chinensis 0.69 1 I. harae 0.14 I. radiata I. sunkoshiensis I. platypetala 1 1 I. siculifer 1 1 I. purpurea 0.32 I. cymbifera [13.9ｰ26.45 mya] 1 I. columbaria 1 I. fragicolor 0.63 I. sulcata 1 ☆ 1 1 I. scullyi I. parviflora I. hians0.98 1 I. falcifer I. scabrida 1 I. angulata I. campanulata 0.85 I. morsei 1 I. lobulifera 1 0.29 I. obesa I. monticola I. pingxiangensis 1 0.32 0.71 I. kerriae 0.66 I. wenshanensis [16.9ｰ27.21 mya] 0.56 I. stenosepala I. mengtzeana 0.16 I. spathulata 0.03 I. pritzelii 1 0.04 I. wilsonii I. gongshanensis 0.11 0.16 I. tubulosa I. chishuiensis 0.69 0.06 I. clavigera 1 I. balansae I. chlorosepala I. malipoensis 0.67 I. hongkongensis I. apalophylla 0.30 1 I. napoensis Hydrocera triflora 0.86 I. begoniifoliaMiocene Pliocene Pleistocene 1 I. trichosepala Tertiary Quaternary
20 I. xanthina15 10 5 0 (mya) 0.99 0.61 I. conchibracteata Fig. 3. BEAST-derived1 chronograms of ImpatiensI. duclouxii including Japanese species using the atpB–rbcL region of cpDNA sequence data. Numbers on branches indicate posteriorI. chiulungensis probabilities.FIG. 3 Values less than 0.5 are omitted. Blue bars indicate the 95% highest posterior density (HPD)1 credibility intervals for node ages. Arrowheads indicate divergence points between Japa- nese Impatiens and Chinese endemic speciesI. rubrostriata (I), divergence time of I. hypophylla from I. textorii and I. ohwadae (II), and diversification within I. hypophylla (III). White star represents the calibration point of the divergence time of Impatiens: 22.5 million years ago (mya) (22.5 ± 5.6 mya) given by Janssens et al. (2009). Japanese samples newly added in the present study are masked with gray. ‘I. hypophylla var. micro.’ represents the accessions of I. hypophylla var. microhypophylla. Number on the lower part of the figure indicates geological age (mya). June 2020 Murayama & al.―Phylogeny of Japanese ‘Sohayaki elements’ 135
I. corchorifolia 1 0.92 Impatiens furcata 1 I. inaperta 1 I. andohahelae I. hypophylla 1 I. andringitrensis I. gibbosa 0.33 (Kii) 0.41 I. subabortiva (1.19 mya) I. hypophylla 0.49 I. amoena (5.27 mya) III 1 0.88 I. miniata (Shi.) I. baroni 0.96 I. manaharensis 1 I. hypophylla 0.91 1 I. sambiranensis var. micro.(Fu.) 0.97 I. firmula (7.37 mya) II 1 1 I. fuchsioides 1 I. hypophylla 0.89 I. anovensis var. micro.(Ai.) 1 I. vilersi 1 0.35 I. percrenata I. hypophylla I. sodenii (8.91 mya) I (Kyu.) 0.32 I. kilimanjari 1 I. textorii I. aureliana I. tuberosa 0.92 1 0.82 I. auricoma I’ I. textorii(Japan) 0.59 I. parasitica 0.32 0.72 I. bombycina I. ohwadae 0.86 I. congolensis 1 1 I. keilii I. niamniamensis I. tienmushanica 0.47 1 I. stuhlmannii 0.18 0.6 I. burtonii I. chekiangensis I. bequaerti 0.98 I. hoehnelii 0.09 1 I. usambarensis 1 1 I. hians I. walleriana I. meruensis 0.78 I. pseudoviola 1 0.73 I. leschenaultii 0.49 I. cuspidata I. balsamina I. lobulifera 1 1 I. platypetala I. chinensis 1 1 1 I. zenkerii I. pingxiangensis 1 I. claerii I. columbaria I. obesa 0.53 0.36 I. chlorosepala 1 I. mengtzeana 1 I. monticola I. kerriae 0.78 I. gongshanensis I. xanthina 1 1 1 I. begoniifolia I. yaoshanensis 0.96 I. napoensis I. acehensis 0.37 I. trichosepala I. uliginosa 0.55 I. henslowiana 1 0.99 I. campanulata 0.96 I. cordata I. aquatilis I. levingei 0.83 I. hunanensis I. cyanantha 0.58 I. duclouxii 0.75 1 I. rubrostriata I. conchibracteata I. drepanophora 1 I. fischeri 0.75 I. teitensis 0.97 I. rothii I. rectangula 0.2 I. arguta I. flanaganae I. taronensis 0.58 I. capensis 1 1 I. noli-tangere(Japan) I. noli-tangere I. holocentra 1 I. tortisepala 1 I. pterosepala I. scutisepala 1 I. neglecta 0.81 I. leptocaulon I. macrovexilla I. margaritifera 1 1 I. oxyanthera 0.84 I. forrestii 1 I. piufanensis I. nyimana 0.97 I. imbecilla 1 1 I. lateristachys 0.89 I. radiata 0.75 I. faberi I. microstachys 0.24 I. tayemonii I. harae 0.96 I. pritzelii 1 I. fissicornis 1 I. davidii I. racemosa0.98 1 I. lecomtei I. soulieana I. urticifolia 1 I. delavayi 1 I. nubigena 0.95 I. poculifer I. eubotrya 1 I. chiulungensis 0.75 I. corchorifolia 1 I. barbata I. principis 0.33 I. hypophylla(Kii) 1 I. hypophylla(Shi.) 1 I. hypophylla var.(Fu.) I. siculifer 1 I. hypophylla var.(Ai.) 1 I. hypophylla(Kyu.) I. laxiflora 1 I. textorii 0.6 0.92 1 I. textorii(Japan) 1 I. ohwadae I. desmantha I. tienmushanica I. chekiangensis I. sunkoshiensis 0.54 I. morsei 1 I. angulata 1 I. lobulifera I. tuberculata1 1 I. pingxiangensis I. obesa I. kerriae 0.74 I. cymbifera 1 I. yaoshanensis 1 I. uliginosa 0.94 1 I. aquatilis I. blinii I. cyanantha 1 I. drepanophora I. flacifer 0.91 I. rectangula 0.78 I. taronensis 1 I. holocentra I. scabrida I. scutisepala 0.84 I. margaritifera I. sulcata 0.88 I. nyimana 0.52 1 I. radiata 0.99 I. harae I. fragicolor 1 I. racemosa 0.99 I. urticifolia 1 I. eubotrya I. brachycentra 0.39 1 I. principis I. siculifer [13.44ｰ26.34 mya] 1 I. laxiflora I. parviflora 1 0.77 I. desmantha 1 1 I. sunkoshiensis ☆ I. amphorata I. tuberculata 1 I. cymbifera 1 I. blinii 1 I. flacifer I. scullyi 1 I. scabrida 1 1 I. sulcata I. bicornuta 0.76 I. fragicolor 1 I. brachycentra 0.23 I. parviflora 0.32 I. stenosepala 1 I. amphorata I. scullyi I. wenshanensis I. bicornuta 1 I. stenosepala [16.9ｰ27.22 mya] I. wenshanensis I. chishuiensis 1 I. chishuiensis 0.92 I. hongkongensis 0.99 I. tubulosa I. hongkongensis 0.2 I. wilsonii 0.15 I. clavigera I. tubulosa 0.47 I. balansae 1 I. apalophylla 0.94 I. malipoensis I. wilsonii I. spathulata I. clavigera Hydrocera triflora Miocene I. balansae Pliocene Pleistocene Tertiary I. apalophylla Quaternary I. malipoensis 20 15 I. spathulata10 5 0 (mya) Hydrocera triflora Fig. 4. BEAST-derived chronograms of Impatiens includingFIG. Japanese 4 species using the ITS region of nrDNA sequence data. Numbers on branches indicate posterior probabilities. Values less than 0.5 are omitted. Blue bars indicate the 95% highest posterior density (HPD) credibility intervals for node ages. Arrowheads indicate divergence points between the Japanese Impatiens and Chinese endemic species (I. tienmushanica) (I), divergence time of I. hypophylla from I. textorii and I. ohwadae (II), and diversification withinI. hypophylla (III). In addition, the node I’ shows divergence between I. chekian- gensis and the clade of Japanese Impatiens and I. tienmushanica. White star represents the calibration point of the diver- gence time of Impatiens: 22.5 million years ago (mya) (22.5 ± 5.6 mya) given by Janssens et al. (2009). Japanese samples newly added in the present study are masked with gray. ‘I. hypophylla var. micro.’ represents the accessions of I. hypo- phylla var. microhypophylla. Number on lower part of the figure indicates geological age (mya). 136 Acta Phytotax. Geobot. Vol. 71 et al. (2016), we excluded several samples con- the same sequence data sets from phylogenetic taining great amounts of missing sequence data analyses as mentioned above. Hydrocera triflora and used 112 and 145 species of Impatiens as well was used as an outgroup for this analysis. Bayes- as the outgroup species, Hydrocera triflora (L.) ian relaxed dating was conducted using the Wight & Arn. for the atpB–rbcL and ITS regions, Bayesian Evolutionary Analysis Sampling Trees respectively (Appendix 1). We excluded the se- (BEAST) v.1.8.4 software package (Drummond quence of the trnL–F region from the present & Rambaut 2007). BEAST is known as the most analysis because the samples were fewer than for common and useful software for estimating di- the other two regions (67 species) in Yu et al. vergence time (e.g., Yang et al. 2016, Ebersbach (2016). The eight accessions of the Japanese spe- et al. 2017, Gutiérrez-Ortega et al. 2018). Jans- cies of Impatiens were also included in the data sens et al. (2009) estimated the divergence time set (Table 1). In the present study, we used aligned of Impatiens using 11 fossil records from the As- sequence data for 916 bp of the atpB–rbcL region terids. According to Janssens et al. (2009), we set for the cpDNA and 755 bp of the ITS region for the prior parameters of crown age of Impatiens as the nrDNA. In the atpB–rbcL region, the portion 22.5 mya (22.5 ± 5.6 mya) (white stars in Figs. 3 of unknown homology (349−381 bp) was re- & 4). We assumed the birth-death speciation moved from the first alignment data (949 bp). We model (Gernhard 2008) and the uncorrelated re- constructed the phylogenetic trees of the atpB– laxed clock model with log normal nodes (Drum- rbcL and ITS regions separately, because the mond et al. 2006) for each atpB–rbcL and ITS combined data of the two regions showed a sig- region, where the best model was selected from nificant value in the partition homogeneity test (p all plausible combinations of assumptions (Yule < 0.01) (Mickevich & Farris 1981) implemented and birth-death speciation models; strict clock, in PAUP* v.4.0b10 (Swofford 2003). relaxed clock with log normal nodes, and relaxed The phylogenies were determined using max- clock with exponential nodes). In addition, we imum parsimony (MP) in PAUP (Swofford 2003) used the substitution model for the GTR+G mod- and maximum likelihood (ML) using PhyML el for the atpB–rbcL region and the GTR+I+G v.3.0 (Guindon et al. 2010). In the MP analysis, model for the ITS region, as mentioned in the trees were constructed on the basis of substitu- phylogenetic analysis using ML method de- tion data and using the heuristic search option scribed above. MCMC search was run for a 1.0 × with MulTrees off. The addition sequence option 107 chain length. We checked the convergence was set at random. Clade support was estimated and sufficient effective sample size (ESS) of each using bootstrap analysis (Felsenstein 1985) based parameter using Tracer v.1.6 (ESS > 200) (Ram- on 1,000 replicates. In the analysis, we used the baut et al. 2014). A phylogenetic tree, as well as heuristic option for the type of bootstrap search. divergence time, was estimated using TreeAnno- In the ML analysis, the best-fitting evolutionary tator v.1.8.4 (Drummond et al. 2012), discarding model was estimated on the basis of the Smart 10% of the trees as burn-in. Model Selection in PhyML (Lefort et al. 2017) To infer the ancestral distribution of I. hypo- (atpB–rbcL, GTR+G model; ITS, GTR+I+G phylla and related species, we used RASP v.4.2 model). We conducted tree searching of the ML (Yu et al. 2015) with statistical dispersal-vicari- tree using the nearest neighbor interchange (NNI) ance analysis (S-DIVA, Yu et al. 2010). We used option. The branch supports were calculated us- the data set of Subclade D (Figs. 1 & 2) (including ing the bootstrap method with 1,000 replicates. outgroup species: atpB–rbcL, I. flanaganae To estimate the divergence time of the species Hemsl.; ITS, I. angulata S. X. Yu, Y. L. Chen & of Impatiens in Japan, we used the secondary cal- H.N. Qin) and ran the analysis on the tree ob- ibration method (Shaul & Graur 2002) with the tained from BEAST. The distribution area of atpB–rbcL region of cpDNA and the ITS region each species was divided into fourteen main re- of nrDNA separately. For the analyses, we used gions: A, Southern China; B, Western China; C, June 2020 Murayama & al.―Phylogeny of Japanese ‘Sohayaki elements’ 137
Eastern China; D, Northern China; E, Central hypophylla (atpB–rbcL, 74/74%; ITS, 100/100%), China; F, Tibet and Mongolia; G, Japanese is- and the other was the clade of I. ohwadae and I. lands; H, Korean peninsula; I, Russia; J, Central textorii (atpB–rbcL, 86/75%; ITS, 100/100%). In Asia; K, North America; L, Europe; M, South- the tree of the ITS region, the two Chinese en- eastern Asia; N, Africa (atpB–rbcL) and Taiwan demic species, I. tienmushanica Y. L. Chen and I. (ITS). The original distribution of the species was chekiangensis Y. L. Chen, both of which are only assigned according to the distribution area de- in Zhejiang Province in eastern China were basal scribed by Chen et al. (2007), Yu (2012), The to Japanese Impatiens (Fig. 2). In particular, Jap- Plant List (2013) and Kadota (2017). anese Impatiens formed a clade that was sister to the accession of I. tienmushanica (72/65%), while I. chekiangensis was sister to the clade of Japa- Results nese Impatiens and I. tienmushanica with rela- tively high probabilities (85/67%). The sister rela- Both phylogenetic trees using the atpB–rbcL tionship among Japanese Impatiens and I. tien- and ITS regions revealed two major clades mushanica was also supported by the tree of the (Clades 1 & 2) (Figs. 1 & 2). Clade 1 was a small atpB–rbcL region with high probabilities clade composed of 9–10 species. Clade 2 includ- (95/91%) (Fig. 1). ed many species of Impatiens and was divided In the divergence time estimations (Figs. 3 & into seven subclades (Subclades A−G), as in the 4), the Japanese Impatiens (I. hypophylla, I. tree of Yu et al. (2016). However, several species ohwadae, and I. textorii) and the eastern Chinese (I. chiulungensis Y. L. Chen, I. principis Hook. f., endemic (I. tienmushanica) diverged in the Late I. wenshanensis S. H. Huang in the atpB–rbcL Miocene (node I). The age of the node I was esti- tree, and I. pritzelii Hook. f. in the ITS tree) were mated as 5.68 (95% highest posterior density, positioned in different clades in comparison with 3.07–9.16) mya in the atpB–rbcL region, and 7.37 the findings of Yu et al. (2016). Furthermore, in (4.3–11.55) mya in the ITS region. Additionally, the ITS region, the species of Subclade A (Yu et in the ITS region, the divergence time of I. cheki- al. 2016) were divided into two clades (Fig. 2). angensis and the clade of Japanese Impatiens and These differences may be the result of differenc- I. tienmushanica (node I’, Fig. 4) was estimated es in the method of analysis; we separated se- as 8.91 (5.24–13.17) mya. The mean divergence quence data for the atpB–rbcL and ITS regions, time of I. hypophylla from I. ohwadae and I. tex- while Yu et al. (2016) combined sequence data for torii (node II) was estimated to be 3.96 (1.97–6.6) the atpB–rbcL, ITS, and trnL–F regions. mya in the atpB–rbcL tree, and 5.27 (2.72–8.61) The accessions of the species of Impatiens in mya in the ITS region, which corresponds to the Japan (I. hypophylla, I. noli-tangere, I. ohwadae, Pliocene Period. Diversification among the acces- and I. textorii) were included in Subclade D of sions of I. hypophylla (node III) started from Clade 2 with the following bootstrap probabili- around the Late Pliocene to Middle Pleistocene: ties: atpB–rbcL, ML/MP = 55/51%; ITS, ML/MP 1.19 (0.43–2.4) mya in the ITS tree, 2.78 (1.23– = 90/92% (Figs. 1 & 2). In Subclade D, three spe- 4.84) mya in the atpB–rbcL tree. cies of Impatiens in Japan (I. hypophylla, I. ohwa- Ancestral area reconstructions with RASP dae, and I. textorii) formed a monophyletic group using S-DIVA are shown in Figs. 5 & 6. Focusing (hereafter called ‘Japanese Impatiens’) with high on the clade of Japanese Impatiens and the east- probabilities (atpB–rbcL, 91/91%; ITS, 91/92%). ern Chinese species, both analyses using the The accession of I. noli-tangere in Japan formed atpB–rbcL and ITS regions inferred that the com- a clade with those in China with high probabili- mon ancestor of Japanese Impatiens and I. tien- ties (atpB–rbcL, 91/86%; ITS, 92/100%). In Japa- mushanica was in eastern China and the Japanese nese Impatiens, two clades were clearly identified islands (node 32, CG in Fig. 5 & node 33, CG in with high probabilities: one was the clade of I. Fig. 6). In the analysis using the ITS region, it 138 Acta Phytotax. Geobot. Vol. 71
Region 0.99 (BE) Impatiens leptocaulon =unknown BEK 1 *
1.0 (A) I. macrovexilla A=Southern China BEL 3 (C) I. neglecta 2 AB BF 0.98 0.97 (ABCE) I. pterosepala 6 ABC BK (ABCDEFGHIJKLM) I. noli-tangere 4 ABCDEFGHIJKLM BKL 1.0 1.0 5 (ABCDEFGHIJKLM) I. noli-tangere 0.25 8 (KL) I. capensis ABCE BL (B) I. imbecilla ABCK BM 7 1.0 11 0.44 (B) I. lateristachys ABCL C=Eastern China
0.88 (BE) I. oxyanthera 9 ABE CDGH
0.99 10 (BM) I. forrestii ABEK CG (B) I. faberi 0.42 17 ABEL (B) I. platychlaena D=Northern China 1.0 12 ABK DE 0.99 13 (B) I. principis
0.95 14 (B) I. lecomtei ABL E=Central China 0.83 18 1.0 ACE F=Tibet, Mongolia 16 (B) I. soulieana (ACE) I. davidii B=Western China G=Japan 0.78 15 (DE) I. fissicornis BC H=Korean (B) I. tortisepala BCE I=Russia 0.33 25 0.72 (B) I. corchorifolia 19 BCEK J=Central Asia 0.63 (B) I. barbata 21 BCEL (BF) I. nubigena K=North America 0.93 0.96 20 22 (BF) I. delavayi BCG KL
0.57 24 (B) I. poculifer vicariance BCGN L=Europe
1.0 (C) I. platysepala BCK M=Southeastern Asia 23 dispersal 1.0 33 (C) I. chekiangensis BCL N=Africa (G) I. hypophylla (Kii) 1.0 26 BE 0.96 27 (G) I. hypophylla var. micro.(Fukuoka)
1.0 (G) I. hypophylla (Shikoku) 29 (G) I. hypophylla var. micro.(Aichi) 'Japanese Impatiens' 28 34 1.0 0.97 1.0 31 (G) I. hypophylla (Kyushu)
1.0 (CDGH) I. textorii 32 1.0 30 (G) I. ohwadae (C) I. tienmushanica (N) I. flanaganae (outgroup: Clade E)
Fig. 5. RASP analysis consensus tree showing S-DIVA results for ancestral distributional reconstruction of Subclade D (see, Fig. 1) of Impatiens using the atpB–rbcL region of cpDNA. Numbers along branches indicate posterior probabilities from BEAST analysis. The settings of the distribution areas of each species are written to the left of the species name, as re- quired in RASP. Numbers in circles indicate node numbers and correspond to those in Appendix 2. Two or more upper- case letters (e.g. AB, ABC) indicate combined regions, simulated by the software to be merged ancestral regions in the past. The proportion of ancestral regions for eachFIG. node can be found5 in Appendix 2. Estimated events of vicariance (green circle) or dispersal (blue circle) are indicated at nodes and shown outside the main circle corresponding to the ancestral region. Color of the main circle at nodes corresponds to the color bar key on right side of the figure. Asterisk represents an unknown distribution. The clade for Japanese Impatiens is shown with a gray background. ‘I. hypophylla var. micro.’ represents the accessions of I. hypophylla var. microhypophylla. June 2020 Murayama & al.―Phylogeny of Japanese ‘Sohayaki elements’ 139
(BE) Impatiens leptocaulon Region 0.61 1 =unknown BM (A) I. macrovexilla * 1.0 3 A=Southern China (ABCE) I. pterosepala C=Eastern China 2 1.0 (C) I. neglecta AB CDGH 0.98 7 (KL) I. capensis 0.41 4 ABC CG 1.0 5 (ABCDEFGHIJKLM) I. noli-tangere ABCDEFGHIJKLM D=Northern China 1.0 6 (ABCDEFGHIJKLM) I. noli-tangere (B) I. tortisepala ABCE DE (BM) I. forrestii 0.99 14 ABCM DEN 1.0 8
9 (BE) I. oxyanthera 1.0 ABE E=Central China (AE) I. piufanensis 1.0 12 (B) I. imbecilla ACE EN 10 1.0 (B) I. lateristachys AE F=Tibet, Mongolia 1.0 13 11 0.7 19 1.0 (B) I. faberi AM G=Japan (B) I. microstachys (DE) I. fissicornis B=Western China H=Korean 0.99 15 1.0 16 (N) I. tayemonii BC I=Russia
1.0 17 (ACE) I. davidii BCE J=Central Asia 1.0 18 (B) I. lecomtei 25 BDGL K=North America 0.47 (B) I. soulieana (BF) I. delavayi BE KL 1.0 20 1.0 (BF) I. nubigena 21 BF L=Europe
0.88 22 (B) I. poculifer BJLM M=Southeastern Asia 1.0 23 (B) I. chiulungensis vicariance
1.0 24 (B) I. corchorifolia BK N=Taiwan dispersal (B) I. barbata BL 1.0 35 (G) I. hypophylla (Kii) 0.34 26 (G) I. hypophylla var. micro.(Fukuoka) 27 1.0 (G) I. hypophylla (Shikoku) 1.0 29 (G) I. hypophylla var. micro.(Aichi) 28 1.0 'Japanese Impatiens' 1.0 (G) I. hypophylla (Kyushu) 32
36 1.0 (CDGH) I. textorii 30 1.0 1.0 (CDGH) I. textorii 33 31 0.89 (G) I. ohwadae 34 1.0 (C) I. tienmushanica (C) I. chekiangensis (AM) I. angulata (outgroup: Clade A)
Fig. 6. RASP analysis consensus tree showing S-DIVA results for ancestral distributional reconstruction of Subclade D (see, Fig. 2) of Impatiens using the ITS region of nrDNA. Numbers along branches indicate posterior probabilities from BEAST analysis. Settings of the distribution areas of each species are written to the left of the species name, as required in RASP. Numbers in circles indicate node numbers and correspond to those in Appendix 2. Two or more uppercase letters (e.g. AB, ABC) indicate combined regions, simulated by the software to be merged ancestral regions in the past. Proportion of an- cestral regions for each node can be found in AppendixFIG. 2. Estimated6 events of vicariance (green circle) or dispersal (blue circle) are indicated at nodes and shown outside the main circle corresponding to the ancestral region. Color of main circle at nodes corresponds to the color bar key on right side of figure. Asterisk represents an unknown distribution. The clade for Japanese Impatiens is shown with a gray background. ‘I. hypophylla var. micro.’ represents the accessions of I. hypo- phylla var. microhypophylla. 140 Acta Phytotax. Geobot. Vol. 71 was inferred that the common ancestor of Japa- tions split into Chinese mainland species and Jap- nese Impatiens, I. tienmushanica, and I. chekian- anese species due to vicariance. Thereafter, the gensis was in eastern China (node 34, C in Fig. 6). ancestral Japanese Impatiens persisted in the Jap- On the other hand, the distribution of the most anese islands during the Pliocene, from which the recent common ancestor of Japanese Impatiens Japanese endemic I. hypophylla diverged and was estimated to be in Japan (node 31, G in Fig. 5 survived in the Sohayaki region. & node 32, G in Fig. 6). In addition, the vicari- As mentioned above, I. hypophylla is a repre- ance event was estimated to be at the divergence sentative Sohayaki element (Koidzumi 1931, between Japanese Impatiens and the eastern Chi- Hara 1959, Murata & Koyama 1976, Kadota nese species, I. tienmushanica (node 32 in Fig. 5 2017), which are hypothesized to be relict endem- & node 33 in Fig. 6). The dispersal event was es- ic species that originated from ancestors distrib- timated to be at the divergence of I. textorii and/ uted in eastern to southwestern China during the or I. ohwadae (node 30 in Figs. 5 & 6) in the re- Tertiary Period (Hara 1959, Murata 1968, 2004, sults from both the atpB–rbcL and ITS regions. Maekawa 1977, Xie 1997). The present study re- vealed that Japanese Impatiens including I. hypo- phylla, and their Chinese relatives diverged Discussion around the Late Tertiary Period (Figs. 3 & 4). Furthermore, this study inferred that the com- Based on geological evidence, there have mon ancestor of the Japanese Impatiens and the been three main stages of land bridge formation eastern Chinese I. tienmushanica was widely dis- between mainland China and the Japanese is- tributed in eastern China and the Japanese is- lands since the Late Miocene, with most of the lands (Figs. 5 & 6). Additionally, the RASP anal- ECS floor being exposed approximately 5.0–7.0 ysis using the ITS region estimated that the com- mya, 1.3–2.0 mya, and 0.015–0.2 mya (Kimura mon ancestor of Japanese Impatiens and the east- 1996, 2003, Qi et al. 2014). The present study re- ern Chinese I. tienmushanica and I. chekiangen- vealed that Japanese Impatiens, including I. hy- sis was previously distributed in eastern China pophylla and the eastern Chinese species, likely (Fig. 6). Therefore, the results obtained from I. diverged 5.68 (atpB–rbcL)–7.37 mya (ITS), dur- hypophylla and its relatives in the present study ing the Late Miocene of the Tertiary Period, and generally support the early hypothesis of the ori- I. hypophylla likely diverged from other Japanese gin of the Sohayaki elements, although dispersal species (I. ohwadae and I. textorii) from the Late from China was insufficiently demonstrated be- Miocene to the Pliocene of the Tertiary Period: cause of lack of evidence. 3.96 (atpB–rbcL) and 5.27 mya (ITS) (Figs. 3 & In previous phylogeographic studies of So- 4). Additionally, this study inferred that the com- hayaki elements, the ancestral populations of Ki- mon ancestor of Japanese Impatiens and the east- rengeshoma palmata migrated to mainland Chi- ern Chinese species was widely distributed na from Japan in the Middle Pleistocene (Qiu et throughout mainland China and the Japanese is- al. 2009a), while Platycrater arguta was widely lands, and that the vicariance event was estimat- distributed across the ECS land bridge and ed at the time of divergence of Japanese Impa- evolved strictly allopatrically during the Middle tiens and the eastern Chinese I. tienmushanica Pleistocene (Qi et al. 2014). In contrast, the pres- (Figs. 5 & 6). Therefore, the common ancestor of ent study inferred that the common ancestor of Japanese Impatiens and the eastern Chinese spe- Japanese Impatiens (including I. hypophylla) and cies was plausibly distributed across the ECS re- eastern Chinese species was in eastern China and gion through the first stage (5.0–7.0 mya) of the the Japanese islands during the Late Miocene of land bridge mentioned above in the Late Miocene the Tertiary Period, and that I. hypophylla and to Early Pliocene. After the land bridge disap- other Japanese Impatiens differentiated in the peared, it was inferred that the ancestral popula- Japanese islands from the Late Miocene to the June 2020 Murayama & al.―Phylogeny of Japanese ‘Sohayaki elements’ 141
Pliocene (Figs. 3–6). Accordingly, the Sohayaki cess. J. Theor. Biol. 253: 769–778. elements are plausibly species with various his- Good, R. 1974. The Geography of Flowering Plants. fourth edition. Longman, London. torical backgrounds (including ancestral origins, Guindon, S., J. F. Dufayard, V. Lefort, M. Anisimova, W. divergence times, and migration routes). Since Hordijk & O. Gascuel. 2010. New algorithms and there have been only three case studies on the methods to estimate maximum-likelihood phyloge- topic, further studies are required to assess the nies: assessing the performance of PhyML 3.0. Syst. evolutionary history of the Sohayaki elements. Biol. 59: 307–321. Gutiérrez-Ortega, J. S., M. M. Salinas-Rodríguez, J. F. Martínez, F. Molina-Freaner, M. A. Pérez-Farrera, A. We thank Sumio Sei, Tadashi Kariyazaki, Masashi Tashi- P. Vovides, Y. Matsuki, Y. Suyama, T. A. Ohsawa, Y. ro, Ayako Maeda, Kazumi Fujikawa, Yasushi Ibaragi, Watano & T. Kajita. 2018. 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Received May 10, 2019 ; accepted October 30, 2019.
Appendix 1. DNA sequences obtained from DDBJ/EMBL/GenBabk for estimating the phylogenetic position of Japanese Impatiens. Taxon Sources or origin atpB–rbcL ITS Impatiens acehensis Grey-Wilson Indonisia: Gunung Sinabung AY348739 I. amoena H. Perrier Madagascar: Antsatrotro AY348795 I. amphorata Edgew. Western Himalayas AY348740 I. andohahelae Eb. Fisch. & Rahelivololona Madagascar: Andohahela AY348741 I. andringitrensis H. Perrier Madagascar: Ambatofitorahana AY348742 I. angulata S. X. Yu, Y. L. Chen & H. N. Qin China: Guangxi KP776010 KP776060 I. anovensis H. Perrier Madagascar: Ambanizana AY348743 I. apalophylla Hook. f. China: Guangxi KP776011 KP776061 I. aquatilis Hook. f. China: Yunnan DQ147811 AY348745 I. arguta Hook. f. & Thomson China: Yunnan DQ147812 AY348746 I. aureliana Hook. f. China: Yunnan DQ147814 AY348747 I. auricoma Baill. Bot. Gard. Marburg (Cult.) DQ147815 AY348748 I. balansae Hook. f. China: Yunnan KP776012 KP776062 I. balsamina L. Kruidtuin Leuven (Cult.) DQ147816 AY348749 I. barbata H. F. Comber China: Yunnan DQ147818 AY348750 I. baronii Baker Madagascar: Ranomafana AY348751 I. begoniifolia S. Akiyama & H. Ohba China: Yunnan DQ147819 AY348752 I. bequaertii De Wild. Bot. Gard. Koblenz Univ. (Cult.) DQ147820 AY348753 I. bicornuta Wall. China: Yunnan DQ147821 AY348754 I. blinii Lévl. China: Guangxi KP776013 KP776063 I. bombycina W. Lobin & E. Fischer African origin: Bot. Gard. Koblenz Univ. (Cult.) AY348755 I. brachycentra Kar. & Kir. Central Asia AY348756 I. burtonii Hook. f. African origin: Bot. Gard. Koblenz Univ. (Cult.) AY348757 I. campanulata Wight South Indian origin: Ray Morgan: U. K. (Cult.) DQ147822 AY348758 I. capensis Meerb. North American origin: Holden Arboretum (Cult.) DQ147823 AY348759 I. chekiangensis Y. L. Chen China: Zhejiang KP776014 KP776064 I. chinensis L. China: Yunnan DQ147825 AY348761 I. chishuiensis Y. X. Xiong China: Guizhou KP776015 KP776065 I. chiulungensis Y. L. Chen China: Sichuan KP776016 KP776066 I. chlorosepala Hand.-Mazz. China: Guangxi KP776017 KP776067 I. claeri N. Halle African origin: Bot. Gard. Koblenz Univ. (Cult.) AY348763 I. clavigera Hook. f. China: Guangxi KP776018 KP776068 I. columbaria J. J. Bos African origin: Nat. Bot. Gard. Meise (Cult.) DQ147828 AY348764 I. conchibracteata Y. L. Chen China: Yunnan DQ147829 AY348765 I. congolensis G. M. Schulze & R. Wilczek African origin: Bot. Gard. Koblenz Univ. (Cult.) DQ147830 AY348766 I. corchorifolia Franch. China: Yunnan DQ147831 AY348767 I. cordata Wight Indian origin: Ray Morgan: UK (Cult.) AY348768 144 Acta Phytotax. Geobot. Vol. 71
Appendix 1. Continued. Taxon Sources or origin atpB–rbcL ITS I. cuspidata Wight & Arn. South Indian origin: Ray Morgan: U. K. (Cult.) DQ147832 AY348769 I. cyanantha Hook. f. China: Yunnan DQ147833 AY348770 I. cyathiflora Hook. f. China: Yunnan DQ147834 I. cymbifera Hook. f. China: Xizang KP776019 KP776069 I. davidii Franch. China: Fujian KP776020 KP776070 I. delavayi Franch. China: Yunnan DQ147836 AY348773 I. desmantha Hook. f. China: Yunnan DQ147837 AY348774 I. drepanophora Hook. f. China: Yunnan DQ147838 AY348776 I. duclouxii Hook. f. China: Guangxi KP776021 KP776071 I. eubotrya Miq. Indonisia: Gunung Sinabung AY348777 I. faberi Hook. f. China: Sichuan DQ147841 AY348778 I. falcifer Hook. f. China: Xizang KP776022 KP776072 I. firmula Baker Madagascar: Ranomafana AY348780 I. fischeri Warb. Africa DQ147843 AY348781 I. fissicornis Maxim. China: Shaanxi DQ147844 AY348782 I. flanaganae Hemsl. African origin: Roy. Bot. Gard. Edinburgh (Cult.) DQ147846 AY348783 I. forrestii Hook. f. ex W. W. Smith China: Yunnan DQ147847 AY348784 I. fragicolor C. Marquand & Airy Shaw China: Xizang KP776023 KP776073 I. fuchsioides H. Perrier Madagascar: Tsaratanana AY348785 I. furcata H. Perrier African origin: Bot. Gard. Koblenz Univ. (Cult.) AY348786 I. gibbosa H. Perrier Madagascar: Tsaratanana AY348787 I. gongshanensis Y. L. Chen Myanmar: Putao KP776024 KP776074 I. harae H. Ohba & S. Akiyama China: Xizang KP776025 KP776075 I. henslowiana Arn. Sri Lanka origin: Bot. Gard. Koblenz Univ. (Cult.) AY348790 I. hians Hook. f. African origin: Bot. Gard. Berlin (Cult.) DQ147849 AY348791 I. hoehnelii T. C. E. Fr. African origin: Bot. Gard. Koblenz Univ. (Cult.) AY348792 I. holocentra Hand.-Mazz. China: Yunnan AY348793 I. hongkongensis Grey-Wilson China: Hongkong KP776027 KP776076 I. hunanensis Y. L. Chen China: Guangxi KP776028 KP776077 I. imbecilla Hook. f. China: Sichuan DQ147851 AY348796 I. inaperta H. Perrier Madagascar DQ147852 AY348797 I. keilii Gilg. atpB–rbcL: South China Bot. Gard. Guangzhou KP776029 AY348798 (Cult.), ITS: Tanzania (Africa) origin: Bot. Gard. Koblenz Univ (Cult.) I. kerriae Craib Thailand: Qingmai DQ147853 AY348799 I. kilimanjari Oliver Africa AY348800 I. lateristachys Y. L. Chen & Y. Q. Lu China: Sichuan KP776030 KP776078 I. laxiflora Edgew China: Guangxi KP776031 KP776079 I. lecomtei Hook. f. China: Yunnan DQ147855 AY348802 I. leptocaulon Hook. f. China: Guangxi KP776032 KP776080 I. leschenaultii Wall. South Indian origin: Ray Morgan: U. K. (Cult.) DQ147856 AY348803 I. levingei Gamble ex Hook. f. Indian origin: UK (Cult.) AY348804 I. lobulifera S. X. Yu, Y. L. Chen & H. N. Qin China: Guangxi KP776033 KP776081 I. macrovexilla Y. L. Chen China: Guangxi KP776034 KP776082 I. malipoensis S. H. Huang China: Guangxi KP776035 KP776083 I. manaharensis Baill. Madagascar AY348805 I. margaritifera Hook. f. China: Xizang KP776036 KP776084 I. mengtzeana Hook. f. China: Yunnan DQ147858 AY348806 I. meruensis Gilg. Tanzanian origin: Ray Morgan: U. K. (Cult.) DQ147859 AY348807 I. microstachys Hook. f. China: Sichuan KP776085 I. miniata Grey-Wilson Madagascar: Tsaratanana AY348809 I. monticola Hook. f. China: Sichuan DQ147860 AY348810 I. morsei Hook. f. China: Guangxi KP776037 KP776086 I. napoensis Y. L. Chen China: Yunnan DQ147861 AY348811 I. neglecta Y. L. Xu & Y. L. Chen China: Anhui KP776038 KP776087 I. niamniamensis Gilg. African origin: Nat. Bot. Gard. Meise (Cult.) DQ147862 AY348812 I. noli-tangere L. China: Guangxi KP776039 KP776088 I. nubigena W. W. Smith China: Sichuan KP776040 KP776089 I. nyimana C. Marquand & Airy-Shaw China: Xizang KP776041 KP776090 June 2020 Murayama & al.―Phylogeny of Japanese ‘Sohayaki elements’ 145
Appendix 1. Continued. Taxon Sources or origin atpB–rbcL ITS I. obesa Hook. f. China: Guangxi KP776042 KP776091 I. oxyanthera Hook. f. China: Sichuan DQ147865 AY348814 I. parasitica Bedd. Indian origin: Ray Morgen: UK (Cult.) AY348815 I. parviflora DC. Belgium: Leuven DQ147866 AY348816 I. percrenata H. Perrier Madagascar: Andohahela AY348817 I. piufanensis Hook. f. China: Guangxi KP776094 I. pingxiangensis H. Y. Bi & S. X. Yu China: Guangxi KP776043 KP776093 I. platychlaena Hook. f. China: Sichuan DQ147867 I. platypetala Lindl. Bali: Indonesian origin: Ray Morgan: U. K. (Cult.) DQ147868 AY348819 I. platysepala Y. L. Chen China: Anhui KP776044 I. poculifer Hook. f. China: Yunnan DQ147870 AY348820 I. principis Hook. f. China: Guangxi KP776026 KP776096 I. pritzelii Hook. f. China: Hubei KP776045 AY348821 I. pseudoviola Gilg. African origin: Roy. Bot. Gard. Edinburgh (Cult.) DQ147871 AY348822 I. pterosepala Hook. f. China: Hubei KP776046 KP776097 I. purpurea Hand.-Mazz. China: Yunnan DQ147872 I. racemosa DC. atpB–rbcL: China: Xizang, ITS: China: Yunnan DQ147873 KP776098 I. radiata Hook. f. atpB–rbcL: China: Sichuan, ITS: China: Xizang KP776047 AY348824 I. rectangula Hand.-Mazz. China: Yunnan DQ147874 AY348825 I. rothii Hook. f. Ethiopia AY348827 I. rubrostriata Hook. f. China: Yunnan DQ147876 AY348828 I. sambiranensis H. Perrier Madagascar: Tsaratanana AY348829 I. scabrida DC. atpB–rbcL: Himalayan origin: Holden arboretum DQ147877 KP776099 (Cult.), ITS: China: Xizang I. scullyi Hook. F. China: Xizang KP776048 KP776100 I. scutisepala Hook. f. China: Yunnan DQ147878 AY348830 I. siculifer Hook. f. China: Guangxi KP776049 KP776101 I. sodenii Engl. and Warb. ex Engl. African origin: Roy. Bot. Gard. Edinburgh (Cult.) DQ147879 AY348832 I. soulieana Hook. f. China: Sichuan DQ147880 AY348833 I. spathulata Y. X. Xiong China: Guangxi KP776050 KP776102 I. stenosepala Pritz. ex Diels China: Shaanxi DQ147881 AY348835 I. stuhlmannii Warb. African origin: Bot. Gard. Koblenz Univ. (Cult.) AY348836 I. subabortiva H. Perrier Madagascar: Tsaratanana AY348837 I. sulcata Wall. China: Xizang KP776051 KP776103 I. sunkoshiensis S. Akiyama, H. Ohba & Wakab. China: Xizang KP776052 KP776104 I. taronensis Hand.-Mazz. China: Yunnan DQ147882 AY348838 I. tayemonii Hayata Taiwan AY348839 I. teitensis Grey-Wilson African origin: Bot. Gard. Koblenz Univ. (Cult.) DQ147883 AY348840 I. textorii Miq. Japan AY348841 I. tienmushanica Y. L. Chen China: Zhejiang KP776053 KP776105 I. tortisepala Hook. f. China: Sichuan KP776054 KP776106 I. trichosepala Y. L. Chen China: Yunnan DQ147885 AY348843 I. tuberculata Hook. f. & Thomson China: Xizang KP776055 KP776107 I. tuberosa H. Perrier Madagascan origin: Bot. Gard. Univ. Kopenhagen DQ147886 AY348844 (Cult.) I. tubulosa Hemsl. ex F. B. Forbes & Hemsl. China: Guangxi KP776056 KP776108 I. uliginosa Franch. China: Yunnan DQ147887 AY348845 I. urticifolia Wall. China: Xizang KP776109 I. usambarensis Grey-Wilson atpB–rbcL: African origin:Roy. Bot. Gard. Edinburgh DQ147890 AY348847 (Cult.), ITS: African origin: Bot. Gard. Koblenz Univ. (Cult.) I. vilersi Costantin & Poisson Madagascar: Andohahela AY348848 I. walleriana Hook. f. atpB–rbcL: African origin: Nat. Bot. Gard. Meise DQ147892 AY348849 (Cult.), ITS: Kenya I. wenshanensis S. H. Huang China: Guangxi KP776057 KP776110 I. wilsonii Hook. f. China: Sichuan KP776058 KP776111 I. xanthina H. F. Comber China: Yunnan DQ147893 AY348850 I. yaoshanensis K. M. Liu & Y. Y. Cong China: Sichuan KP776059 KP776112 I. zenkeri (Warb.) Grey-Wilson African origin: Bot. Gard. Koblenz Univ. (Cult.) AY348852 Hydrocera triflora (L.) Wight & Arn. Sri Lanka DQ147895 AY348853 146 Acta Phytotax. Geobot. Vol. 71
Appendix 2. Proportion of ancestral regions of each node in RASP analysis.
1. atpB–rbcL (Fig. 5) 2. ITS (Fig. 6) node number proportion node number proportion 1 AB 50%, ABE 50% 1 AB 50%, ABE 50% 2 ABC 20%, ABCE 20%, BC 20%, BCE 20%, 2 ABC 20%, ABCE 20%, BC 20%, BCE 20%, C 20% C 20% 3 ABC 20%, ABCE 20%, B 20% BC 20%, 3 ABC 20%, ABCE 20%, B 20%, BC 20%, BCE 20% BCE 20% 4 B 33.33%, K 33.33%, L 33.33%, * 0.01% 4 * 99.20%, BJLM 0.8% 5 BK 20%, BKL 20%, BL 20%, K 20%, L 20% 5 * 99.21%, BDGL 0.79% 6 ABCK 6.67%, ABCL 6.67%, ABEK 6.67%, 6 B 33.33%, BK 33.33%, BL 33.33% ABEL 6.67%, ABK 6.67%, ABL 6.67%, B 7 B 100% 6.67%, BCL 6.67%, BCEK 6.67%, BCEL 6.67%, BCK 6.67%, BEK 6.67%, BEL 6.67%, 8 B 100% BK 6.67%, BL 6.67% 9 AB 33.33%, ABE 33.33%, BE 33.33%, * 0.01% 7 B 100% 10 B 100% 8 B 100% 11 B 100% 9 B 100% 12 B 100% 10 B 100% 13 B 100% 11 B 100% 14 B 100% 12 B 100% 15 DEN 50%, EN 50% 13 B 100% 16 E 100% 14 B 100% 17 BE 100% 15 E 100% 18 B 100% 16 BE 100% 19 B 100% 17 B 100% 20 B 100% 18 B 100% 21 B 100% 19 B 100% 22 B 100% 20 B 100% 23 B 100% 21 B 100% 24 B 100% 22 B 100% 25 B 100% 23 C 100% 26 G 100% 24 BC 100% 27 G 100% 25 B 100% 28 G 100% 26 G 100% 29 G 100% 27 G 100% 30 G 100% 28 G 100% 31 G 100% 29 G 100% 32 G 100% 30 G 100% 33 CG 100% 31 G 100% 34 C 100% 32 CG 100% 35 BC 100% 33 BCG 100% 36 ABCM 100% 34 BCGN 100% The distribution area was divided into main fourteen regions: A, Southern China; B, Western China; C, Eastern China; D, Northern China; E, Central China; F, Tibet and Mongolia; G, Japanese islands; H, Korean peninsula; I, Russia; J, Central Asia; K, North America; L, Europe; M, Southeastern Asia; N, Africa (atpB–rbcL) and Taiwan (ITS). Asterisk (*) represents an unknown distribution.