Evolutionary origin of a periodical mass‐flowering plant

Abstract The evolutionary origin of periodical mass‐flowering plants (shortly periodical plants), exhibiting periodical mass flowering and death immediately after flowering, has not been demonstrated. Within the genus Strobilanthes (Acanthaceae), which includes more than 50 periodical species, Strobilanthes flexicaulis on Okinawa Island, Japan, flowers gregariously every 6 years. We investigated the life history of S. flexicaulis in other regions and that of closely related species together with their molecular phylogeny to reveal the evolutionary origin of periodical mass flowering. S. flexicaulis on Taiwan Island was found to be a polycarpic perennial with no mass flowering and, in the Yaeyama Islands, Japan, a monocarpic perennial with no mass flowering. Molecular phylogenetic analyses indicated that a polycarpic perennial was the ancestral state in this whole group including S. flexicaulis and the closely related species. No distinctive genetic differentiation was found in S. flexicaulis among all three life histories (polycarpic perennial, monocarpic perennial, and periodical plant). These results suggest that among S. flexicaulis, the periodical mass flowering on Okinawa Island had evolved from the polycarpic perennial on Taiwan Island via the monocarpic perennial in the Yaeyama Islands. Thus, the evolution of life histories could have taken at the level of local populations within a species.


| INTRODUC TI ON
Periodical mass-flowering plants (called shortly periodical plants), such as bamboos (subfamily Bambusoideae, Poaceae) and Strobilanthes (Acanthaceae), flower and die gregariously and periodically at certain intervals of longer than 2 years (120 years at most) (Janzen, 1976;McClure, 1966). These periodical mass-flowering plants are referred to as periodical plants because their life cycles are similar to those of periodical cicadas, which exhibit periodical mass emergence at 13-or 17-year intervals in North America (Alexander & Moore, 1962;Simon, 1988). These periodical organisms including periodical plants and cicadas are characterized by the following life history traits: periodicity (reproduction according to a cycle of longer than 2 years), mass emergence and mass flowering (almost all individuals in a population synchronously reproduce), and semelparity and monocarpy (one reproduction event per lifetime).
In plants, the intervals between the two mass-flowering events are not regular in non-periodical plants (Sakai et al., 1999;Vander Wall, 2002). Monocarpic perennials are also found in a wide range of families; many of them lack mass flowering (Young & Augspurger, 1991). Other than bamboos and Strobilanthes, monocarpic perennials with synchronized flowering have been reported in several groups such as Cerberiopsis in Apocynaceae (Burd, Read, Sanson, & Jaffré, 2006;Read, Sanson, Burd, & Jaffré, 2008), and Tachigali in Fabaceae (Forget, Kitajima, & Foster, 1999;Kitajima & Augspurger, 1989). If mass flowering occurs periodically (the fixed intervals), these plants should be categorized as periodical plants. However, whether their intervals are periodical or not is unknown.
The periodicity of synchronized flowering is also known in annuals and strict biennials (almost all individuals flower and die in the second year from germination) (Kelly, 1985). Annuals flower synchronously in the same season every year. Most individuals of several strict biennial species flower synchronously every 2 years (Kelly, 1985). Flowering of these annuals and biennials is induced by seasonal changes or individual size (Harper, 1977;Rees, Sheppard, Briese, & Mangel, 1999;Rose, Rees, & Grubb, 2002). In contrast, flowering of periodical plants is induced by time from germination, but not by seasonal changes or individual size (Janzen, 1976). It is indicated by transplantation or propagation of cutting that does not affect timing of flowering in periodical plants (Kakishima, Yoshimura, Murata, & Murata, 2011;Tanimoto & Kobayashi, 1998;Watanabe, Ueda, Manabe, & Akai, 1982). Therefore, periodicity of more than 2 years is unique and found only in periodical plants. Thus, the evolution of periodicity of more than 2 years is a key event in the evolution of periodical plants.
The definition of periodical plants used in this report is as follows: periodicity (more than 2 years fixed year intervals of flowering after germination), mass flowering (almost all individuals in a population synchronously flower) and monocarpy (one flowering event per lifetime). In this definition, monocarpic perennials with either mass flowering or fixed three-or-more year periodicity are not considered a periodical plant. We here exclude annuals and strict biennials from periodical plants, because of the lack of yearly counting mechanisms, as explained above.
The evolutionary origins of periodical organisms are still unknown, although several hypotheses have been proposed. In periodical cicadas, the acquisition of periodicity is suggested to have preceded the selection of 13-or 17-year cycles (Ito et al., 2015;Sota et al., 2013;Tanaka, Yoshimura, Simon, Cooley, & Tainaka, 2009;Yoshimura, 1997). However, the evolutionary history of periodical cicadas has not been verified, because non-periodical, closely related species have not been found. In bamboo, a theoretical study based on phylogenetic inference suggests that the length of massflowering cycles (the interval between two consecutive mass-flowering events) has been multiplied in several groups, for example, from 15-year cycles to 30-, 60-, 120-year ones (Veller, Nowak, & Davis, 2015). Although the molecular phylogeny suggests cycle elongation in bamboos, the assumed annual ancestral species is unknown. Not that the information of closely related non-periodical species has not been enough to verify the ancestral state of these periodical organisms.
To identify the evolutionary pathway from the ancestral state to the periodical mass flowering (emergence), the genus Strobilanthes is a prospective group with more than 50 periodical mass-flowering species (Daniel, 2006;Janzen, 1976;Wood, 1994). Strobilanthes is distributed in East, Southeast and South Asia and includes approximately 400 species (Hu, Deng, & Wood, 2011). A molecular phylogenetic study indicated that periodical mass flowering has evolved several times independently in Strobilanthes (Moylan, Bennett, Carine, Olmstead, & Scotland, 2004). Here, we focus on Strobilanthes flexicaulis, a subshrub distributed in Japan (Okinawa Island and the Yaeyama Islands) and Taiwan (Taiwan Island) (Seok, Hsieh, & Murata, 2004;Wood & Scotland, 2003;Figures 1a,b and 2a). On Okinawa Island, this species exhibits periodicity by flowering gregariously every 6 years (Kakishima et al., 2011;Figure 1a (Herrera, Jordano, Guitián, & Traveset, 1998;Kelly, 1994;Kelly & Sork, 2002;Silvertown, 1980;Webb & Kelly, 1993 Table S3). In the mass-flowering years, we arbitrarily chose and labeled representative flowering or fruiting individuals in the observation area of each population on Okinawa Island because too many individuals were flowering (Supporting Information Table   S3). The data of OK1-3 from 2009 to 2011 was already published in the previous paper (Kakishima et al., 2011). We checked whether the labeled individuals survived with flowers, survived without flowers or died in the flowering season of the following year (1 year after labeling) to examine whether they are mono-

| DNA extraction and sequencing
Total genomic DNA was extracted from silica gel-dried leaf tissue using the method using CTAB (Doyle & Doyle, 1987) with slight modifications after pretreatment with HEPES buffer (pH 8.0) (Setoguchi & Ohba, 1995). The sequences determined in this study were registered in the DNA Data Bank of Japan (DDBJ), which is linked to GenBank, and their accession numbers are provided with the sample information in Supporting Information Table S3.
The PHOTOTROPIN2 (PHOT2) gene was selected as a nuclear DNA (nDNA) marker because PHOT2 has been successfully used to reconstruct the phylogeny of Verbenaceae in Lamiales, including Acanthaceae (Yuan & Olmstead, 2008). Phototropins are blue-light receptors that control a range of responses that optimize the photosynthetic efficiency of plants (Christie, 2007). The fragments between exon 10 and exon 14 of the PHOT2 gene (five exons and four introns) were initially amplified by polymerase chain reaction (PCR) using the primers 10F and 14R (Yuan & Olmstead, 2008 When overlapping double peaks were found in the obtained electropherograms, we used the TA-cloning system (Invitrogen, Carlsbad, CA, USA) for sequencing. At least 16 clones per sample were chosen and sequenced using the same procedure as in the first PCR, followed by direct sequencing. If the nucleotides in the cloned sequences were not detected by direct sequencing, they were considered PCR errors.
The PCR cycling conditions included an initial step for 2 min at 95°C, followed by 40 cycles of 45 s at 95°C, 45 s at 52°C, and 1 min at 60°C, with a final extension for 10 min at 60°C. Because this region was GC rich, the extension temperature was set lower than general conditions. The PCR products were purified with ExoSAP-IT (USB, OH, USA) and directly sequenced with the same primers as used for PCR, and primer trnS1 (AATGTAAGGAGTCTGTCTTC) was designed for this study and used as necessary. The sequencing was performed in the same manner as nuclear DNA sequencing. Two other regions (trnG-trnR and matK) were amplified by PCR using primers trnG_1F and trnR_22R for trnG-trnR (Tripp, 2007) and matK-AF and matK-8R (Ooi, Endo, Yokoyama, & Murakami, 1995). The PCR cycling conditions were as follows: an initial step for 2 min at 95°C, followed by 40 cycles of 45 s at 95°C, 45 s at 52°C, and 1 min at 72°C, with a final extension for 10 min at 72°C.

| Molecular phylogenetic analyses
Monophyly of the Parachampionella group, including S. flexicaulis, was supported in a previous study (Seok et al., 2004). Four Strobilanthes species from Japan and Taiwan were selected based on the previous study, and S. formosana and S. dimorphotricha were used as outgroup species (24 plant materials in total; Supporting Information Table S3). The sequences were aligned using ClustalW 1.8 (Thompson, Higgins, & Gibson, 1994) and then adjusted manually. The aligned lengths of the combined cpDNA sequences and the PHOT2 sequences were 2,772 and 2,270 bp, respectively. The phylogenetic trees based on cpDNA and nDNA were constructed separately using a Bayesian approach with MrBayes 3.1.2 (Ronquist & Huelsenbeck, 2003) and maximum-likelihood (ML) phylogenetic analysis using RAxML (Stamatakis, 2014). In the Bayesian phylogenetic analysis, the F81 model for nucleotide substitutions was selected for the tree based on the Bayesian information criterion (BIC) using the program Kakusan4 (Tanabe, 2011). Based on the selected model, we performed two separate runs of the Metropolis-coupled Markov chain Monte Carlo (MCMCMC) analysis, each with a random starting tree and four chains (one cold and three hot). The MCMCMC was 10 million generations long, and the chain was sampled every 1,000th generation from the cold chain. The first 2,500 sample trees (25% of the total 10,000 sample trees) were discarded as burn-in after checking that the average standard deviation of the split frequencies (ASDSF) reached a stationary state at <0.01. A 50% majority consensus tree of the output tree file from MrBayes was generated using FigTree ver. 1.3.1 (Rambaut, 2009). The ML phylogenetic analyses were implemented in RAxML 8 (Stamatakis, 2014) with a GTR+G likelihood model for nucleotide substitutions. The ML bootstrap proportions (BPs) and trees were obtained by simultaneously running rapid bootstrapping with 10,000 iterations followed by a search for the most likely tree. In the Bayesian analysis, the 50% majority-rule consensus tree of all of the post-burn-in trees was depicted with Bayesian posterior probabilities (PPs). All the clades in the ML tree were recognized in the Bayesian tree; therefore, the BPs were plotted on the Bayesian trees. The statistically parsimonious networks were constructed using TCS version 1.21 (Clement, Posada, & Crandall, 2000). The incongruence length difference (ILD) test (Farris, Källersjö, Kluge, & Bult, 1994 was performed to check a significant incongruence between the molecular phylogenetic trees based on cpDNA and nDNA sequences.

| RE SULTS
Three The survival rate of procumbent S. rankanensis cannot be measured because of vegetative propagation, although we confirmed that S. rankanensis was alive at the same place where flowering S. rankanensis had observed in the previous year.
We reconstructed molecular phylogenetic trees within the monophyletic Parachampionella group (Seok et al., 2004) with two outgroup species based on three regions (trnSG, matK, trnGR) of cpDNA and one nDNA gene, PHOT2. The sequences of cpDNA and nDNA were combined because the ILD test (Farris, Källersjö, Kluge, & Bult, 1994 showed no significant incongruence between the molecular phylogenetic trees based on cpDNA and nDNA sequences (p = 1.000) (Supporting Information Figure S1a  Evolution of monocarpy Acquisition of periodicity and synchronicity phenotypes, gene expression, and genomes among these closely related populations for the identification of the responsible genes (Ellegren et al., 2012).

| D ISCUSS I ON
Our findings, a periodical plant evolved from a polycarpic perennial via a monocarpic perennial, differ from the hypothesis in which the ancestral monocarpic annuals evolved into periodical plants via life cycle multiplication from one year to many years (Veller et al., 2015). The difference between our findings and the life cycle multiplication hypothesis proposed for Bamboos questions whether life cycle multiplication is responsible for the origin of periodicity or only for the elongation of life cycles once short cycle periodicity is established. Life cycle elongation by multiplication of intervals may have occurred after the evolution of periodical plants in Strobilanthes (Janzen, 1976). Different evolutionary mechanisms between Bamboos and Strobilanthes might have happened in the evolution of periodicity and life cycle elongation.
The several factors that might have derived the evolution of periodicity in S. flexicaulis can be considered. The cost of reproduction may play an important role in the evolution between polycarpy and monocarpy (Young & Augspurger, 1991). To verify the cost of reproduction, we need to examine the size of flowering individuals, the number of flowers and fruits per individual and the survival rates over individual lifetime. Two population-level factors have been proposed to explain mass flowering (masting) in forests: pollination efficiency and predator satiation (Janzen, 1976;Kelly, 1994). These factors might have contributed to the evolution of mass flowering in S. flexicaulis on Okinawa Island (Kakishima et al., 2011). We need to examine further whether these factors are indeed working as selection on Okinawa Island, but not in the Yaeyama Islands and Taiwan Island. Future detailed study may elucidate the causal mechanisms underlying the evolution of periodical plants from polycarpic perennials via monocarpic perennials.

ACK N OWLED G M ENTS
We are grateful to K. Nakajima, S. Matsumura, M. Yamada, M.
Muramatsu, S. Tsai, G. Song, M. Okazaki and C.F. Hsieh for supporting the field observations. This study was supported by the Asahi Glass Foundation (to SK); the collaborative research program of the National Institute for Basic Biology (NIBB) (to SK and JY); the Japan Advanced Plant Science Network (to SK); the Collaborative Research of Tropical Biosphere Research Center, University of the Ryukyus (to SK); and JSPS KAKENHI Grant Numbers JP20370032 (to JM), JP22255004, JP22370010, JP26257405, and JP15H04420 (to JY), and JP26840126, JP13J03600 and JP17K15182 (to SK). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

CO N FLI C T O F I NTE R E S T
None declared.

AUTH O R CO NTR I B UTI O N S
SK, JM, and JY conceived the study. SK, YL, TYAY, and PL conducted the field works. SK conducted the laboratory works. SK, TI, and YO conducted the analyses. SK, YO, MH, JM, and JY wrote the manuscript. All authors read, revised, and approved the manuscript.