bioRxiv preprint doi: https://doi.org/10.1101/2020.04.25.060632; this version posted April 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Original article 2 Geographic and subsequent biotic isolations led to a diversity anomaly of Heterotropa 3 (Aristolochiaceae) in insular versus continental regions of the Sino-Japanese Floristic 4 Region 5 6 7 Author affiliations 8 Daiki Takahashi1*, Yu Feng2, Shota Sakaguchi1, Yuji Isagi3, Ying-Xiong Qiu2, Pan Li2, Rui-Sen 9 Lu2, Chang-Tse Lu4, Shih-Wen Chung5, Yang-Shan Lin6, Yun-Chao Chen6, Atsushi J. Nagano7, 10 Lina Kawaguchi7, Hiroaki Setoguchi1 11 1Graduate school of Human and Environmental studies, Kyoto University, Japan; 2Systematic & 12 Evolutionary Botany and Biodiversity Group, MOE Laboratory of Biosystem Homeostasis and 13 Protection, College of Life Sciences, Zhejiang University, China; 3Graduate school of 14 Agriculture, Kyoto University, Japan; 4Department of Biological Resources, National Chiayi 15 University, Taiwan; 5Herbarium of Taiwan Forestry Research Institute, Taiwan; 6Miaoli Distinct 16 Agricultural Research and Extension station, Taiwan; 7Faculty of Agriculture, Ryukoku 17 University, Japan 18 *Corresponding author: [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.25.060632; this version posted April 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 19 20 Abstract 21 The Sino-Japanese Floristic Region is highly diverse with respect to temperate plants. 22 However, the reasons for this diversity are poorly understood because most studies have 23 only considered geographic isolation caused by climatic oscillations. Heterotropa 24 (genus Asarum; Aristolochiaceae) diverges here and shows high species diversity in 25 insular systems (63 species) compared to continental areas (25 species). Heterotropa 26 shows low dispersal ability with small distribution ranges, implying diversification by 27 geographic events, and high floral diversity, implying pollinator-mediated 28 diversification. To reveal how abiotic and biotic factors have shaped the diversity 29 anomaly of Heterotropa, we conducted phylogenetic analysis using ddRAD-seq and 30 chloroplast genome datasets including 79 species, estimation of floral trait evolution, 31 and comparison of isolation factors within clades based on distribution range and floral 32 trait analysis. Phylogenetic analysis indicates that Heterotropa originated in mainland 33 China and expanded to the Japanese Archipelago in the Miocene, and the major clades 34 almost correspond to geographic distributions. Floral traits evolved repeatedly in the tip 35 nodes within the clades. Although the major clades include a high proportion of species 36 pairs showing isolation by floral traits, there are no conditional relationships between 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.25.060632; this version posted April 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 37 two isolation factors, indicating that most species pairs with floral trait isolation are 38 distributed allopatrically. The repeated exposure and submergence of land-bridges 39 caused by climatic oscillations would have led to significant population fragmentation 40 in insular systems. Thus, the diversity anomaly of Heterotropa would have resulted 41 from geographic and climatic events during the Miocene, while subsequent repeated 42 floral trait evolution would have followed geographic isolation during the Pleistocene. 43 44 Keywords 45 Sino-Japanese Floristic Region, biotic and abiotic factors, ddRAD-seq, Asarum, 46 Heterotropa 47 48 49 50 51 52 53 54 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.25.060632; this version posted April 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 55 56 Introduction 57 The Sino-Japanese Floristic Region (SJFR) extends from the eastern Himalayas to the 58 Japanese Archipelago through south and central China (Takhtajan, 1969; Wu & Wu, 59 1996). This region can boast one of the most diverse temperate floras anywhere in the 60 world and has high endemism (Wu & Wu, 1996). This diversity has been thought to be 61 linked to climatic and physiographical complexity and historical environmental changes 62 associated with the Pleistocene (< 2.6 Mya) climatic oscillations (Qian & Ricklefs, 63 2000). During glacial periods, when the climate of this region was cooler by ca. 4-6 °C 64 and sea level was approximately 130m lower than its present level, temperate plants in 65 this region retreated to refugia at lower altitudes or southern parts (Harrison, Yu, 66 Takahara, & Prentice, 2001; Tsukada, 1984). During the interglacial periods, expansion 67 to higher altitudes or northern parts would have occurred. In the eastern island systems, 68 sea level changes due to climatic oscillations have caused repeated formation and 69 division of land-bridges in the East China Sea (Ujiie, 1990), and these events have 70 provided opportunities for population expansion and fragmentation (Qiu, Fu, & Comes, 71 2011). Many phylogeographic studies have revealed that the present interspecific and 72 intraspecific genetic structures of temperate plants in this region reflected the range 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.25.060632; this version posted April 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 73 shifts caused by climatic oscillations (Li, Yan, & Ge, 2012; Sakaguchi et al., 2012; 74 Setoguchi et al., 2006; Wang et al., 2015; Yang et al., 2017). It has been considered that 75 these climatic and associated environmental changes during the Pleistocene triggered 76 range fragmentation, vicariance, and population isolation (Qiu et al., 2011). It has also 77 been hypothesised that allopatric speciation would be a major mode of speciation in the 78 temperate plants of this region (Qian & Ricklefs, 2000). 79 Although the importance of geographic isolation as a major isolation 80 mechanism in plants has been addressed (Boucher, Zimmermann, & Conti, 2016; 81 Esselstyn, Timm, & Brown, 2009; Govindarajulu, Hughes, & Bailey, 2011; Verboom, 82 Bergh, Haiden, Hoffmann, & Britton, 2015), recent studies in other regions have 83 implied that biotic factors also promote species diversification (Lagomarsino, 84 Condamine, Antonelli, Mulch, & Davis, 2016; L. Lu et al., 2019). In particular, floral 85 trait evolution has been thought to promote speciation through segregation of gene flow 86 by pollinator shifts (Armbruster, 2014; Van der Niet, Peakall, & Johnson, 2014). 87 Previous studies have shown that the tempo and pattern of floral trait evolution varies 88 distinctively among lineages, and floral trait evolution has been implicated in shaping 89 patterns of species diversification (Givnish et al., 2015; Jaramillo & Manos, 2001). It 90 has been considered that biotic drivers can play complementary roles to abiotic factors 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.25.060632; this version posted April 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 91 in reproductive isolation; that is, biotic factors can facilitate reproductive isolation even 92 without geographical isolation (Rundle & Nosil, 2005). To fully understand the 93 diversification process of species groups, it is essential to reveal the relative 94 contributions of biotic and abiotic drivers. However, many studies conducted in the 95 SJFR have only discussed the role of allopatric fragmentation due to geographic and 96 climatic events, and few studies have considered other factors as drivers of the 97 diversification of the temperate plants. In addition, although the high endemism in the 98 SJFR provides an outstanding opportunity for investigating the evolutionary history of 99 plant diversification (Yang et al., 2017), to date, most phylogeographic studies in the 100 region have focused on individual species or only small groups, including fewer than 10 101 taxa (But Mitsui et al., 2008; Yoichi, Jin, Peng, Tamaki, & Tomaru, 2017). Thus, our 102 knowledge of the diversification process of temperate plants in the SJFR remains 103 fragmentary, due to a lack of integrative multidimensional studies of morphology, 104 phylogeny, biogeography, and ecology with adequate sampling of diversified groups. 105 In this study, we focused on the Heterotropa (genus Asarum; Aristolochiaceae) 106 as a model group endemic to the SJFR, and one of the most speciose warm-temperate 107 plant groups (comprising approximately 90 species) in this region (Sugawara, 2006). 108 Taxa of Heterotropa are low-growing, rhizomatous herbs that grow in shaded 6 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.25.060632; this version posted April 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 109 understories, and are