Genetic and reproductive characterization of distylous Primula reinii in the Hakone volcano, Japan: implications for conservation of the rare and endangered plant

Main Article Content

Hiroaki Setoguchi
Kaoruko Kurata
Kazuhiro Fukushima
Masaya Yamamoto
Honami Sugawara


Genetic and ecological evaluation are crucial in effective management of rare and endangered species, including those exhibiting complex breeding systems such as distyly.  We studied a threatened distylous herb Primula reinii in the Hakone volcano, central Japan, to obtain baseline information of reproductive and genetic status towards conservation.  In two representative populations inhabiting a central cone and somma of the volcano, population size, floral morph ratio, stigmatic pollen deposition, and fruit-set were measured.  Using microsatellite markers, we evaluated genetic diversity, structure and differentiation of populations.  Population bottlenecks and historical changes in population size were also estimated from genotype data.  We found significant deviation from equal morph ratios in the central cone population, which also exhibited skewed mating success together with a high frequency of pollination within the same morph.  These trends were not detected in the somma population.  From genetic insights, the central cone population showed slightly lower genetic diversity, whereas no significant deviation from Hardy-Weinberg equilibrium was found in either population.  The estimated moderate genetic differentiation and admixed genetic structure suggest recent lineage divergence and/or gene flow between populations.  While robust evidence for a recent bottleneck was not obtained in our analyses, a clear signature of historical population contraction was detected in the central cone population. Our findings suggest that the skewed morph ratio strongly influenced the reproduction of small and isolated populations in the short-term, highlighting the vulnerability of distylous plant populations under ongoing anthropogenic pressure.

Article Details



Barret, S.C. (1989). The evolutionary breakdown of heterostyly, pp. 151−169. In: Bock J.H & Y.B. Linhart (eds.). The Evolutionary Ecology of Plants. Westvier Press, USA, 614pp.

Barrett, S.C. (2002). Evolution of sex: the evolution of plant sexual diversity. Nature Reviews Genetics 3: 274.

Barrett, S.C. & J.S. Shore (2008). New insights on heterostyly: comparative biology, ecology and genetics, pp. 3−32. In: Franklin‐Tong V. (ed.). Self-incompatibility in Flowering Plants. Springer, Germany, XLI+313pp.

Brys, R., H. Jacquemyn & T. Beeckman (2008). Morph‐ratio variation, population size and female reproductive success in distylous Pulmonaria officinalis (Boraginaceae). Journal of Evolutionary Biology 21: 1281−1289.

Byers, D.L. & T.R. Meagher (1992). Mate availability in small populations of plant species with homomorphic sporophytic self-incompatibility. Heredity 68: 353−359.

Cornuet, J.M. & G. Luikart (1996). Description and power analysis of two tests for detecting recent population bottlenecks from allele frequency data. Genetics 144: 2001–2014.

Dempster, A.P., N.M. Laird & D.B. Rubin (1977). Maximum likelihood from incomplete data via the EM algorithm. Journal of the Royal Statistical Society. Series B (methodological) 39: 1–22.

Earl, D.A. & B.M. vonHoldt (2012). STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the Evanno method. Conservation Genetics Resources 4: 359–361.

Estoup, A., P. Jarne & J.M. Cornuet (2002). Homoplasy and mutation model at microsatellite loci and their consequences for population genetics analysis. Molecular Ecology 11: 1591–1604.

Evanno, G., S. Regnaut & J. Goudet (2005). Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Molecular Ecology 14: 2611–2620.

Excoffier, L., G. Laval & S. Schneider (2005). Arlequin (version 3.0): An integrated software package for population genetics data analysis. Evolutionary Bioinformatics 1: 47–50.

Frankham, R., D.A. Briscoe & J.D. Ballou (2002). Introduction to conservation genetics. Cambridge university press, UK.

García‐Robledo, C. & F. Mora (2007). Pollination biology and the impact of floral display, pollen donors, and distyly on seed production in Arcytophyllum lavarum (Rubiaceae). Plant Biology 9: 453–461.

Garza, J.C. & E.G. Williamson (2001). Detection of reduction in population size using data from microsatellite loci. Molecular Ecology 10: 305–318.

Hedrick, P.W. (2005). A standardized genetic differentiation measure. Evolution 59: 1633–1638.

Heuch, I. (1979). Equilibrium populations of heterostylous plants. Theoretical Population Biology 15: 43–57.

Hodgins, K.A. & S.C. Barrett (2006). Female reproductive success and the evolution of mating‐type frequencies in tristylous populations. New Phytologist 171: 569–580.

Ishihama, F., S. Ueno, Y. Tsumura & I. Washitani (2006). Effects of density and floral morph on pollen flow and seed reproduction of an endangered heterostylous herb, Primula sieboldii. Journal of Ecology 94: 846-855.

Jackson, P.W. & K. Kennedy (2009). The global strategy for plant conservation: a challenge and opportunity for the international community. Trends in Plant Science 14: 578–580.

Jombart, T. (2008). adegenet: a R package for the multivariate analysis of genetic markers. Bioinformatics 24: 1403–1405.

Kéry, M., D. Matthies & B. Schmid (2003). Demographic stochasticity in population fragments of the declining distylous perennial Primula veris (Primulaceae). Basic and Applied Ecology 4: 197–206.

Kitamoto, N., M. Ueno, Y. Tsumura, I. Washitani & R. Ohsawa (2005). Development of microsatellite markers in Primula sieboldii E. Morren, a threatened herb. Japanese Journal of Conservation Ecology 10: 47–51.

Kobayashi, M. (1997). 14C ages of pyroclastic-flow deposits from central cones on the western slope of old somma of Hakone volcano, central Japan. Bulletin of Volcanological Society of Japan 42: 355–358.

Kobayashi, M. (1999). Tephrochronology and eruptive activity on Hakone volcano in the past 50 ka. The Quaternary Research 38: 327–343.

Kobayashi, M., K. Mannen, M. Okuno, T. Nakamura & K. Hakamata (2006). The Owakidani Tephra group: A newly discovered post-magmatic eruption product of Hakone Volcano, Japan. Bulletin of Volcanological Society of Japan 51: 245–256.

Kopelman, N.M., J. Mayzel, M. Jakobsson, N.A. Rosenberg & I. Mayrose (2015). Clumpak: a program for identifying clustering modes and packaging population structure inferences across K. Molecular Ecology Resources 15: 1179–1191.

Marshall, J., B. Kingsbury & D. Minchella (2009). Microsatellite variation, population structure, and bottlenecks in the threatened copperbelly water snake. Conservation Genetics 10: 465–476.

Matsumura, C. & I. Washitani (2000). Effects of population size and pollinator limitation on seed-set of Primula sieboldii populations in a fragmented landscape. Ecological Research 15: 307–322.

Meirmans, P.G. & P.H. van Tienderen (2004). GENOTYPE and GENODIVE: two programs for the analysis of genetic diversity of asexual organisms. Molecular Ecology Notes 4: 792–794.

Meeus, S., O. Honnay, R. Brys & H. Jacquemyn (2012). Biased morph ratios and skewed mating success contribute to loss of genetic diversity in the distylous Pulmonaria officinalis. Annals of Botany 109: 227–235.

Ministry of the Environment (2019). The Japanese red lists 2019. Retrieved from

Moritz, C. (1994). Defining ‘evolutionarily significant units’ for conservation. Trends in Ecology & Evolution, 9, 373–375.

Nagai, M. & M. Takahashi (2008). Geology and eruptive history of Hakone volcano, central Japan. Research Report of the Kanagawa Prefectural Museum Natural history, 13: 25–42.

Nagasawa, K., H. Setoguchi, M. Maki, H. Goto, K. Fukushima, Y. Isagi, Y. Suyama, A. Matsuo, Y. Tsunamoto, K. Sawa & S. Sakaguchi (2020). Genetic consequences of plant edaphic specialization to solfatara fields: Phylogenetic and population genetic analysis of Carex angustisquama (Cyperaceae). Molecular Ecology 29(17): 3234–3247.

Nikolic, N. & C. Chevalet (2014). Detecting past changes of effective population size. Evolutionary Applications 7: 663–681.

Osawa, T. & S. Inohara (2008). Understanding current status and factors of degradation of threatened plants in the Hakone region of the Fuji-Hakone-Izu National Park −Investigation using park volunteer research data. Japanese Journal of Conservation Ecology 13: 179–186.

Padilla, D.P., L.G. Spurgin, F.A. Fairfield, J.C. Illera & D.S. Richardson (2015). Population history, gene flow, and bottlenecks in island populations of a secondary seed disperser, the southern grey shrike (Lanius meridionalis koenigi). Ecology and Evolution 5: 36–45.

Pedersen, J.L., S.E. Macdonald & S.E. Nielsen (2016). Reproductive ecology of the distylous species Houstonia longifolia: implications for conservation of a rare species. Botany 94: 983–992.

Peery, M.Z., R. Kirb, B.N. Reid, R. Stoelting, E.L.E.N.A. Doucet‐Bëer, S. Robinson, C. Vásquez-Carrillo, J.N Pauli & P.J. Palsbøll (2012). Reliability of genetic bottleneck tests for detecting recent population declines. Molecular Ecology 21: 3403–3418.

Piry, S., G. Luilart & J.M. Cornuet (1999). BOTTLENECK: a computer program for detecting recent reductions in the effective population size using allele frequency data. Journal of Heredity 90: 502–503.

Pritchard, J.K., M. Stephens & P. Donnelly (2000). Inference of population structure using multilocus genotype data. Genetics 155: 945–959.

R Core Team (2018). R: A Language and Environment for Statistical Computing. Retrieved from

Rao, S. & J.P. Strange (2012). Bumble bee (Hymenoptera: Apidae) foraging distance and colony density associated with a late-season mass flowering crop. Environmental entomology 41: 905–915.

Raymond, M. & F. Rousset (1995). Genepop (Version-1.2) –population-genetics software for exact tests and Ecumenicism. Journal of Heredity 86: 248–249.

Richards, J. (2003). Primula. Timber Press, USA.

Takakura, K. (2011). Improved method of plant DNA extraction using glass-fiber filter. Bunrui, 11: 139–149.

Tanaka, N. (2008). Plants in Hakone [translated from Japanese]. Newsletter of the Kanagawa Prefectural Museum of Natural History, 14: 21.

Thompson, J.D., S.C. Barrett & A.M. Baker (2003). Frequency–dependent variation in reproductive success in Narcissus: implications for the maintenance of stigma–height dimorphism. Proceedings of the Royal Society of London. Series B: Biological Sciences 270: 949–953.

Tóth, E.G., F. Tremblay, J.M. Housset, Y. Bergeron & C. Carcaillet (2019). Geographic isolation and climatic variability contribute to genetic differentiation in fragmented populations of the long-lived subalpine conifer Pinus cembra L. in the western Alps. BMC Evolutionary Biology 19: 190.

Ueno, S., Y. Tsumura & I. Washitani (2003). Development of microsatellite markers in Primula sieboldii E. Morren, a threatened Japanese perennial herb. Conservation Genetics 4: 809–811.

Ueno, S., N. Kitamoto, R. Ohsawa, Y. Tsumura & I. Washitani (2006). Nine additional microsatellite markers for Primula sieboldii E. Morren. Conservation Genetics 6: 1063–1064.

Ueno, S., Y. Yoshida, Y. Taguchi, M. Honjo, N. Kitamoto, I. Washitani, R. Ohsawa & Y. Tsumura (2009). Development of 120 microsatellite markers for Primula sieboldii E. Morren for linkage mapping. Conservation Genetics 10: 1945–1952.

van Rossum, F. & L. Triest (2006). Within-population genetic variation in the distylous Primula veris: Does floral morph anisoplethy matter in fragmented habitats?. Perspectives in Plant Ecology, Evolution and Systematics 7: 263–273.

van Rossum, F., S.C. De Sousa & L. Triest (2006). Morph-specific differences in reproductive success in the distylous Primula veris in a context of habitat fragmentation. Acta Oecologica 30: 426–433.

Wang, Y., Q.F. Wang, Y.H. Guo & S.C. Barrett (2005). Reproductive consequences of interactions between clonal growth and sexual reproduction in Nymphoides peltata: a distylous aquatic plant. New Phytologist 165: 329–336.

Washitani, I. (1996). Predicted genetic consequences of strong fertility selection due to pollinator loss in an isolated population of Primula sieboldii. Conservation Biology 10: 59–64.

Washitani, I., M. Kato, J. Nishihiro & K. Suzuki (1994). Importance of queen bumble bees as pollinators facilitating inter‐morph crossing in Primula sieboldii. Plant Species Biology 9: 169–176.

Washitani, I., F. Ishihama, C. Matsumura, M. Nagai, J. Nishihiro & M. Nishihiro (2005). Conservation ecology of Primula sieboldii: synthesis of information toward the prediction of the genetic/demographic fate of a population. Plant Species Biology 20: 3–15.

Wilcock, C. & R. Neiland (2002). Pollination failure in plants: why it happens and when it matters. Trends in plant science 7: 270–277.

Williamson-Natesan, E.G. (2005). Comparison of methods for detecting bottlenecks from microsatellite loci. Conservation Genetics 6: 551–562.

Wyatt, R. & R.L. Hellwig (1979). Factors determining fruit set in heterostylous bluets, Houstonia caerulea (Rubiaceae). Systematic Botany 4: 103–114.

Yamamoto, M., K. Horita, D. Takahashi, Y. Murai & H. Setoguchi (2018). Floral morphology and pollinator fauna of sister species Primula takedana and P. hidakana in Hokkaido Island, Japan. Bulletin of the National Museum of Nature and Science Series B 44: 97–103.

Yamamoto, M., K. Kurata & H. Setoguchi (2017). Conservation genetics of an ex situ population of Primula reinii var. rhodotricha, an endangered primrose endemic to Japan on a limestone mountain. Conservation Genetics 18: 1141–1150.

Yamamoto, M., M. Yasui, H. Setoguchi & K. Kurata (2013). Conservation of an endangered perennial herb, Primula reinii var. rhodotricha. Journal of Nature Restoration and Conservation 6: 23–29.

Yoichi, W., X.F. Jin, C.I. Peng, I. Tamaki & N. Tomaru (2017). Contrasting diversification history between insular and continental species of three‐leaved azaleas (Rhododendron sect. Brachycalyx) in East Asia. Journal of Biogeography 44: 1065–1076.