ISSN 1346-7565 Acta Phytotax. Geobot. 70 (2): 87–102 (2019) doi: 10.18942/apg.201823
Geographic Distribution of Ploidy Levels and Chloroplast Haplotypes in Japanese Clerodendrum trichotomum s. lat. (Lamiaceae)
1,* 2 3 4 5 Leiko Mizusawa , Naoko Ishikawa , Okihito Yano , Shinji Fujii and Yuji Isagi
1Faculty of Human Development and Culture, Fukushima University, 1 Kanayagawa, Fukushima 960-1296, Japan. * [email protected] (author for correspondence); 2Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Tokyo 153-8902, Japan; 3Faculty of Biosphere-Geosphere Science, Okayama University of Science, 1-1 Ridai-cho, Kita-ku, Okayama 700-0005, Japan; 4Faculty of Human Environments, University of Human Environments, 6-2 Kamisanbonmatsu, Motojuku-cho, Okazaki, Aichi 444-3505, Japan; 5Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan
Clerodendrum trichotomum s. lat., under which many infraspecific taxa have been recognized, includes both tetraploid and diploid individuals, although chromosome numbers and geographic variation in ploi- dy levels have not been investigated in the Japanese archipelago. The geographic distribution of ploidy levels and chloroplast haplotypes of four Japanese taxa of C. trichotomum s. lat., based on chromosome counts, flow cytometry, and genotyping of five microsatellite loci is reported. It was determined that Japanese C. trichotomum var. trichotomum and var. yakusimense are tetraploid (2n = 104), while var. es- culentum and C. izuinsulare are diploid (2n = 52). The diploid taxa are distributed only on the southern edge of the Japanese archipelago, while tetraploid C. trichotomum is distributed widely. Such distribu- tion patterns may be formed by temperate forest shrinkage during, and tetraploid expansion after, glacial periods. Thirteen haplotypes were detected, and were divided into the following three clades: (1) Japa- nese C. trichotomum var. trichotomum and C. izuinsulare, (2) C. trichotomum var. yakusimense and var. esculentum, and (3) Chinese C. trichotomum. Two haplotypes were shared between diploid and tetra- ploid lineages, suggesting multiple polyploidization events in C. trichotomum s. lat.. Inconsistency be- tween nuclear and chloroplast phylogenetic trees suggests a past inter-lineage hybridization event in C. trichotomum s. lat.
Key words: chloroplast phylogeny, Clerodendrum izuinsulare, Clerodendrum trichotomum, Cleroden- drum trichotomum var. esculentum, Clerodendrum trichotomum var. fargesii, Clerodendrum trichoto- mum var. yakusimense, diploid, polyploid, Sino-Japanese region, tetraploid
Clerodendrum trichotomum Thunb. s. lat. is a Ohwi with glabrous and thick leaves; var. escul- deciduous pioneer shrub that grows to 5 m tall. It entum Makino with acute apex and cordate base is common in disturbed secondary forests in both of the leaves, and var. izuinsulare (K. Inoue, M. cool temperate and warm temperate climates and Haseg. et S. Kobay.) H. Ohba et S. Akiyama with is widely distributed in the Sino-Japanese region: short style and glabrous leaves (Ohwi 1953, Walk- southern China, Korean Peninsula, Taiwan, and er 1976, Yamazaki 1993, Ohba & Akiyama 2002). the Japanese archipelago (Yamazaki 1993). It is Although Ohba & Akiyama (2002) proposed highly variable in morphology and many infra- treating populations on the Izu islands as C. tri- specific taxa have been recognized (Ohwi 1953, chotomum var. izuinsulare, here we treat them as Yamazaki 1993, Zhuang 1999). In the Japanese C. izuinsulare Inoue, Haseg. et Kobay. following archipelago, at least four infraspecific taxa were Inoue et al. (1997). The diagnostic characteristics recognized: var. trichotomum with pubescent of the infraspecific taxa are variable and appear leaves and long style; var. yakusimense (Nakai) to be continuous between the taxa (Mizusawa & 88 Acta Phytotax. Geobot. Vol. 70
Miyajima unpublished), and have caused confu- the findings with the chloroplast genetic structure sion in their identity in local floras (Hatushima in the wider Japanese archipelago to better under- 1975, Yamazaki 1993, Shimabuku 1997, Shiroka- stand the morphological polymorphism and di- wa 2001, Miyata 2003, Fujii 2006). versification process in C. trichotomum s. lat., a Polyploidization is known to be a key factor highly variable species that includes many infra- that contributes to plant speciation and differen- specific taxa. tiation (Ramsey & Schemske 1998, Soltis et al. 2004). Previous studies of the chromosomes of Clerodendrum trichotomum s. lat. have reported Materials and Methods that C. trichotomum s. lat. includes diploid and tetraploid individuals, although those reports Target taxa and sample identification have been restricted to studies in China and from In this study we included Clerodendrum tri- some locations in Japan. Diploids (2n = 52) are chotomum Thunb. var. trichotomum, C. trichoto- distributed widely in China (Zeng et al. 2011), mum var. yakusimense (Nakai) Ohwi, C. trichot- while Bowden (1940, 1945) reported 2n = 92 for omum var. esculentum Makino, and C. izuinsu- two individuals of C. trichotomum from Japan lare Inoue, Haseg. et Kobay. The geographic dis- and cultivated in Brooklyn and Chapel Hill, USA. tribution of each variety is summarized in Fig. 1 During microsatellite marker isolation, we (Makino 1917, Nakai 1924, Hatushima 1975, generated data that suggested C. izuinsulare is Ohwi 1953, Yamazaki 1993, Inoue et al. 1997, diploid whereas var. trichotomum is tetraploid, at Shimabuku 1997, Shirokawa 2001). In southern least in the Izu islands (Mizusawa et al. 2011). We Kyushu and the Nansei islands, varieties trichot- genotyped 36 and 42 individuals of var. trichoto- omum, esculentum, and yakusimense may be mum and C. izuinsulare, respectively, for 19 mic- sympatric. Although Walker (1976) treated var. rosatellite loci and detected three or four alleles yakusimense as a synonym of C. trichotomum in some loci in each individual of var. trichoto- var. fargesii (Dode) Rehder, var. fargesii is re- mum, but only one or two in each individual of C. ported to occur locally in the mountains of west- izuinsulare (Mizusawa et al. 2011). The microsat- ern China (Sargent 1917). Here, we focus on the ellite genotype of var. yakusimense, a naturalized populations in southern Kyushu to the Nansei is- population in the Izu islands, also suggested that lands, which we refer to as var. yakusimense, fol- it was a tetraploid (Mizusawa 2018). These previ- lowing Ohwi (1953). The distribution of C. tri- ous studies suggest that geographic variation in chotomum s. lat. has not been sufficiently investi- the ploidy levels of C. trichotomum s. lat. corre- gated in the southern portion of the region and on late with the infraspecific taxa recognized. How- Taiwan. ever, ploidy levels have not been considered in re- Identification of the samples was based on the gard to the infraspecific taxa of C. trichotomum following diagnostic features. Variety trichoto- s. lat. mum has pubescent leaves and style protruding Ploidy and DNA variation is sometimes effec- ca. 40 mm beyond the pink corolla tube with tive at elucidating the taxonomy of infraspecific white petal. Clerodendrum var. yakusimense has taxa. For example, a combined approach of ploidy flowers similar to those of var. trichotomum, but and DNA variation improved the understanding can be identified by the thick, glabrous leaves. of infraspecific taxa in Aucuba, Damnacanthus, Clerodendrum var. esculentum has pubescent and Cayratia in the Sino-Japanese region, as well leaves with acute apex and cordate base, some- as in Clerodendrum trichotomum s. lat. (Ohi et what shorter inflorescences, and slender and acu- al. 2003, Naiki & Nagamasu 2004, Ishikawa et minate sepals (Makino 1917), but its floral mor- al. 2014). phology has not been described in detail. In Shi- Here, we report the ploidy levels of the Japa- koku, Kyushu, and the Nansei islands, we identi- nese taxa of C. trichotomum s. lat. and correlate fied potentially sympatric populations of var. tri- June 2019 Mizusawa & al.– Distribution of Ploidy and Haplotypes in Clerodendrum trichotomum s.l. 89 chotomum and var. esculentum based on the larg- of staining, the fluorescence intensity of each cell est leaf on a shoot. Clerodendrum izuinsulare can was measured using a BD Accuri C6 Flow Cy- be easily distinguished from the other taxa by its tometer (BD Biosciences, CA, USA). In the histo- glabrous leaves, short style and white corolla tube gram of the fluorescence intensity, only the peak (Inoue et al. 1997). region data was used to calculate the mean fluo- Voucher specimens have been deposited in rescence intensity. one of the following herbaria: Kyoto University (KYO), Osaka Museum of Natural History Estimation of geographic distribution of ploidy (OSA), Tokyo Metropolitan University (MAK), levels in C. trichotomum s. lat. and Faculty of Symbiotic Systems Science, Fuku- The geographic distribution of ploidy levels in shima University (FKSE) (Tables 1, 2). Japanese C. trichotomum s. lat. was estimated us- ing five microsatellite markers (ct028, ct041, Determination of ploidy level of each taxon ci111, ci141, and ci144; Mizusawa et al. 2011) The ploidy level of each taxon was estimated based on the methods of Yagi et al. (2009) and by counting the chromosomes, and/or flow cyto- Vergilino et al. (2009). The microsatellite mark- metric analysis (FCA). The number of individu- ers are highly polymorphic and co-dominant als tested in each assay and the results are sum- DNA markers. The number of alleles at each lo- marized in Table 1. cus is one or two in diploid individuals and up to Chromosome counts were performed accord- four in tetraploid individuals. The five loci were ing to the following procedure: first, the root tips chosen from a primer list (Mizusawa et al. 2011) were cut from potted plants between 9:00 and with the condition that they had at least four al- 11:00 am and incubated with a 2 mM solution of leles in a population of C. izuinsulare that ap- 8-oxyquinoline for 3 h at 18 °C. Subsequently, the peared to be diploid. root tips were fixed with 45% acetic acid for 10 s In a preliminary assay, ploidy level was esti- at room temperature (ca. 15–25 °C), then incu- mated using the five microsatellite loci on 15 in- bated in 1 N HCl for 10 min at 60 °C. After stain- dividuals whose ploidy levels were known based ing with Schiff's Reagent (Wako Pure Chemical on chromosome counts or FCA. Based on the pre- Industries, Osaka, Japan) for 1 h at room temper- liminary assay, the maximum number of alleles ature (ca. 15–25 °C), the slides were stained with at each locus was one or two in diploid individu- 1 % acetic orcein and observed after using the als and three or four in tetraploid individuals (Ta- squash technique. ble 1). In the main assay, individuals with one or Flow cytometric analysis (FCA) was conduct- two alleles at each locus were considered diploid, ed using the following procedure: fresh leaves and individuals with three or four alleles at one or from potted plants were rapidly frozen in liquid more loci were considered tetraploid. The main nitrogen. Subsequently, leaf segments (ca. 1 cm × assay was conducted on 86 individuals of Clero- 0.5 cm) were crushed in liquid nitrogen using a dendrum trichotomum s. lat. collected from a mortar and pestle, after which 500 μL of buffer wide area of the Japanese archipelago, two indi-
(10 mM Tris-HCl, pH 8.0; 2 mM MgCl2; 0.1% viduals from China, and two individuals from Triton X-100; 10 µL/mL RNase; 40 mg/mL poly- Korea (Table 2). vinylpyrrolidone; 10 µL/mL β-mercaptoethanol) The genotype determination based on the mi- was added to the contents of the mortar to isolate crosatellite markers was carried out according to the nuclei. The suspension was filtered using 30- the following procedure. The genomic DNA was μm Partec CellTrics disposable filters (Sysmex, extracted from the dried leaves using the Gentra Kobe, Japan) to remove the unnecessary pieces of Puregene Tissue Kit (QIAGEN, Venlo, Nether- tissue and incubated at 37 °C for 30 min. There- lands). The polymerase chain reaction (PCR) was after, 50 μL of propidium iodide solution was performed according to the standard protocol of added to the filtrate to stain the nuclei. After 2 h the QIAGEN Multiplex PCR Kit (QIAGEN, Ven- 90 Acta Phytotax. Geobot. Vol. 70
N 0 1,000 km 45N
3. Sado 1. Hokkaido 9. Korea 5. Western Honshu
2. Eastern Honshu 35N 4. Izu Islands
10. China 7. Kyushu
6. Shikoku
8. Nansei Islands C. trichotomum C. trichotomum var. yakusimense C. trichotmum var. esculentum 25N C. izuinsulare 125E 135E 145E
Fig. 1. Geographic distribution of Clerodendrum trichotomum s. lat. in the Japanese archipelago and the names of regions (Makino 1917, Nakai 1924, Ohwi 1953, Hatushima 1975, Yamazaki 1993, Inoue et al. 1997, Shimabuku 1997). White- dashed line indicates the Itoigawa-Shizuoka tectonic line. Distributions in Taiwan and areas south of the Nansei islands are not fully understood. lo, Netherlands) in a final volume of 10 μL, which Tate & Simpson 2003), and rpS16 intron (Small et contained 5 ng of the extracted DNA, 5 μL of 2X al. 2005). The three regions were amplified by Multiplex PCR Master Mix, and 0.2 μM of each PCR using a QIAGEN Multiplex PCR Kit (QIA- multiplexed primer. The PCR amplification was GEN, Germantown, MA, USA). The reaction so- performed using the following reaction condi- lutions for PCR contained 2–5 ng of extracted tions: initial denaturation at 95 °C for 15 min, fol- DNA, 5 µL of 2X Multiplex PCR Master Mix, lowed by 25 cycles of denaturation at 94 °C for 30 and 0.25 µM of the forward and reverse primers. s, annealing at 57 °C for 1 min 30 s, extension at PCR was conducted using the following reaction 72 °C for 1 min, and a final extension at 60 °C for program: initial denaturation at 95 °C for 15 min, 30 min. The size of the PCR products was mea- followed by 35 cycles of denaturation at 94 °C for sured using the ABI PRISM 3130 Genetic Ana- 30 s, annealing at 57 °C for 1 min, extension at 72 lyzer and Gene Mapper software (Applied Bio- °C for 1 min, followed by a final extension at 72 systems, Foster City, CA, USA). °C for 7 min. The PCR products were cleaned us- ing Exonuclease I (TaKaRa, Otsu, Shiga, Japan) Sequencing of chloroplast DNA for phylogenetic and a TSAP (Promega Corporation, Madison, analysis WI, USA). The purified templates were dye-la- To create a phylogenetic tree of C. trichoto- beled using BigDye (Applied Biosystems, Foster mum s. lat., we sequenced three regions of the City, CA, USA) and sequenced with an ABI 3130 chloroplast DNA: the psbJ–petA intron (Shaw et automated DNA sequencer (Applied Biosystems, al. 2007), psbA–trnHGUG spacer (Sang et al. 1997, Foster City, CA, USA). For sequencing, we newly Fig. 1 June 2019 Mizusawa & al.– Distribution of Ploidy and Haplotypes in Clerodendrum trichotomum s.l. 91
Table 1. Ploidy levels and maximum number of alleles per locus in five microsatellite loci. The numbers of samples on each assay are shown in parentheses. "Flow" means the result of the flow cytometric analysis. "Tet" means tetraploid and "Dip" means diploid. The numbers in the "SSR" column mean the maximum numbers of alleles per locus in each individual. Taxa Location information Ploidy information (N) Voucher ID Location Latitude Longitude Chromosome Flow SSR var. trichotomum Aizuwakamatu-shi, 37.49 139.93 2n = 104 (1) Tet (1) 4 (1) L. Mizusawa & K. Shuto 151108000_4 Fukushima (FKSE) Wakasa-cho, Fukui 35.47 135.86 - Tet (4) 4, 4 (2) L. Mizusawa 160501_000_1 (FKSE) L. Mizusawa 160501_000_2 (FKSE) Kitashirakawa, Kyoto 35.03 135.79 2n = 104 (1) Tet (3) 3, 3, 4 (3) L. Mizusawa140719_215 (FKSE) Yasuura-cho, Hiro- 34.27 132.72 2n = 104 (1) Tet (1) - - shima Kamae-cho, Ohita 32.81 131.94 2n = 104 (1) Tet (4) 4 (1) Sakaguchi s.n. (FKSE 98452) Busan, Korea 35.02 128.84 - Tet (2) - L. Mizusawa & H. J. Choi 160810_299_1 (FKSE) L. Mizusawa & H. J. Choi 160810_299_2 (FKSE) Namyangju-si, Korea 37.74 127.17 - Tet (1) - - Mt. Emei, China 29.51 103.33 - Dip (1) - - var. yakusimense Amami-Ohshima 28.27 129.37 2n = 104 (2) Tet (5) 4, 4, 4 (3) L. Mizusawa 14082621_212 (FKSE) Island L. Mizusawa 14082620_4 (FKSE) var. esculentum Kuro-shima Island 30.83 129.96 2n =52 (2) Dip (6) 2, 2 (2) L. Mizusawa & M. Miyajima 171025410_6 (FKSE) L. Mizusawa & M. Miyajima 171025409 (FKSE) C. izuinsulare Miyake-jima Island 34.09 139.52 2n = 52 (2) Dip (7) 2, 2, 2 (3) L. Mizusawa 14091239 (FKSE) L. Mizusawa 14091239_209 (FKSE) designed two forward primers, petA_227 (TTC model, and separate model, were compared based GGT TTT CCA GAT GTA GT) in the psbJ–petA on BIC4 using Kakusan4/Aminosan (Tanabe intron and rpS16-F-378 (ACG AGG CAC CGA 2011). A phylogenetic tree was estimated using AGT AAT GT) in the rpS16 intron. MrBayes v.3.2.6 x64, (Ronquist et al. 2012). Trac- er v.1.6 (Rambaut et al. 2014) was used to check Phylogenetic analysis based on chloroplast hap- the convergence of likelihood, and to decide the lotypes and nuclear genotypes number of steps for burn-in. The first 1,000,000 A Bayesian phylogenetic tree was constructed steps were burn-in. The reproducibility of each based on the chloroplast haplotype sequences. clade was checked by Phylogears 2 v.2.0.2016.09.06 The chloroplast DNA (cpDNA) sequences were (Tanabe 2008). aligned using MUSCLE (Edgar 2004) in MEGA Additionally, a parsimonious haplotype net- 5 (Tamura et al. 2011). Indels, simple sequence work was drawn by TCS v.1.21 (Clement et al. repeats (SSRs), and an inversion were removed 2000) for the cpDNA data set. The TCS algo- from the sequences before constructing the phy- rithm does not limit mutation types to a single logenetic tree, because the algorithm adopted in nucleotide mutation; therefore, the indel informa- the following software assumed a single nucleo- tion was used to draw the haplotype network, tide mutation. Finally, 1,325 positions were used while SSRs and an inversion were not used. The to construct the Bayesian phylogenetic tree. Four mutation type of SSRs, bidirectional stepwise mutation models, a none-partitioned model, par- mutation (Valdes et al. 1993), is not appropriate titioned equal mean rate model, proportional for estimating the parsimonious network. An in- 92 Acta Phytotax. Geobot. Vol. 70
Table 2. Abbreviations and geographic information on sampling location, and number of individuals collected from each lo- cation. "SSR" indicate numbero of samples used for microsatellite genotyping and “cp” indicate number for chloroplast DNA sequencing. Clerodendrum trichotomum var. trichotomum, var. yakusimense, var. esculentum, and C. izuinsulare are shown as "tri", "yak", "esc", and "izu", respectively. Region Geographic information Location Abbreviation Lat Lon Taxon SSR cp Voucher ID Hokkaido Hakodate, Hokkaido HOK 41.75 140.70 tri 1 1 Eastern Honshu Tazawako lake, Akita AK1 39.71 140.63 tri 1 1 K. Shutoh et al. s.n. (FKSE 90346) Akita Univ., Akita AK2 39.39 140.08 tri 3 3 - Yamagata YMA 38.35 140.42 tri 1 1 K. Sawa s.n. (FKSE 90672) Fukushima FKS 37.68 140.39 tri 2 2 L. Mizusawa s.n. (FKSE 88200) Tsukawa, Niigata NG1 37.69 139.42 tri 3 2 L. Mizusawa & K. Shutoh s.n. (FKSE 90669) L. Mizusawa & K. Shutoh s.n. (FKSE 90670) Uonuma, Niigata NG2 37.34 139.10 tri 1 1 - Joetsu, Niigata NG3 37.28 138.47 tri 1 1 S. Fujii 12856 (OSA) Kanazawa-city, Ishikawa ISI 36.56 136.66 tri 1 1 - Saitama SAI 35.89 139.63 tri 1 1 L. Mizusawa 080801 (FKSE) Mimomi-Sakura, Chiba CHB 35.70 140.15 tri 1 1 - Miura peninsula ZUS 35.30 139.58 tri 2 2 - Izu peninsula (Inland side) IP1 35.09 138.88 tri 2 2 - Izu peninsula (Pacific side) IP2 34.90 139.05 tri 3 4 - Sadogashima-island Sado island SAD 38.09 138.44 tri 2 0 S. Fujii 12817 (KYO) S. Fujii 12838 (KYO) Izu Islands Oh-shima island OH 34.73 139.40 tri 3 3 - Nii-jima island NI 34.37 139.27 tri 1 2 L. Mizusawa 08072723 (FKSE) Nii-jima island NI 34.37 139.27 izu 3 3 L. Mizusawa 08072727 (FKSE) L. Mizusawa 0807279 (FKSE) L. Mizusawa 08072732 (FKSE) Kozu-shima island KO 34.21 139.15 tri 2 3 - Kozu-shima island KO 34.21 139.15 izu 2 2 L. Mizusawa0806051 (FKSE) Mikura-jima island MK 33.87 139.60 izu 3 3 - Hachijo-jima island HA 33.11 139.80 izu 4 4 L. Mizusawa 0806141 (FKSE) L. Mizusawa 0806144 (FKSE) L. Mizusawa 08061412 (FKSE) Western Honshu Kyoto KYO 35.31 135.72 tri 3 3 - Izumo-city, Shimane SM1 35.47 132.76 tri 2 1 S. Fujii 11066 (OSA) Ota-city, Shimane SM2 35.17 132.50 tri 1 1 S. Fujii 11038 (KYO) Okazaki-city, Aichi AI1 34.89 137.26 tri 1 1 S. Fujii 14765 (OSA) Minamichita-cho, Aichi AI2 34.66 136.98 tri 3 1 S. Fujii 11125 (KYO) Minamiise-cho, Mie ME1 34.28 136.57 tri 4 3 S. Fujii 11166 (OSA), S. Fujii 12939 (KYO) S. Fujii 10999 (KYO), S. Fujii 11341 (KYO) Owase, Mie ME2 34.15 136.29 tri 3 3 S. Fujii 14731 (OSA), S. Fujii 14733 (MAK) Kushimoto, Wakayama WAK 33.46 135.80 tri 4 2 S. Fujii 12959 (MAK) S. Fujii 12960 (KYO, OSA) S. Fujii 12975 (KYO, MAK) S. Fujii 12976 (OSA) Shikoku Muroto, Kochi SK1 33.25 134.18 tri 1 1 S. Fujii 13604 (OSA) Tosa, Kochi SK2 33.49 133.38 tri 2 1 S. Fujii 13597 (OSA), S. Fujii 13591 (OSA) Southern tip SK3 32.75 132.69 tri 1 0 S. Fujii 12105 (KYO, MAK) Southern tip SK3 32.75 132.69 esc 4 1 S. Fujii 12083 (MAK, OSA) S. Fujii 12117 (KYO, MAK) S. Fujii 12100 (KYO, MAK) S. Fujii 12133 (KYO, MAK, OSA) Kyushu Kumamoto KYU 32.82 130.73 tri 1 1 - Nansei Islands Yaku-shima island YAK 30.34 130.52 yak 2 3 L. Mizusawa 08071410 (FKSE) L. Mizusawa 08071443 (FKSE) Kuro-shima island KRO 30.83 129.96 esc 2 2 S. Fujii 9631 (KYO,OSA) S. Fujii 9987 (KYO, MAK, OSA) Akuseki-jima island AKU 29.45 129.60 esc 1 1 S. Fujii 11500 (KYO, MAK, TSN) Takara-jima island TAK 29.15 129.20 esc 1 1 S. Fujii 11568 (KYO, OSA) Amami-ohshima island AMA 28.27 129.37 yak 3 3 - Korea Mt. Jiri, Korea KRA 35.28 127.56 tri 1 2 - China Maijishan, China MAJ 34.37 106.01 tri 1 2 - Hubei, China HUB 31.50 110.35 tri 1 3 - Cultivated in Kyoto Botani- cal Gardens - - esc 1 0 - Total 86 79 June 2019 Mizusawa & al.– Distribution of Ploidy and Haplotypes in Clerodendrum trichotomum s.l. 93 version in the psbA–trnHGUG spacer was also re- 4.69% to 11.94%, with an average of 7.31% (SE = moved to draw the haplotype network because 0.24). Therefore, fluorescence intensity ratios this inversion includes SSRs. We judged that this among the Japanese taxa were as follows: 1.78 in inversion region is too complex to use in estimat- Japanese var. trichotomum/C. izuinsulare, 1.64 in ing the most parsimonious network. Japanese var. trichotomum/var. esculentum, 1.85 A nuclear neighbor-joining tree was con- in var. yakusimense/C. izuinsulare, and 1.71 in structed based on the genotypes of the five micro- var. yakusimense/var. esculentum. satellite loci. All calculations to construct the nu- From the microsatellite assays of 86 samples, clear phylogenetic tree were performed on R v. 10–34 alleles were detected in the five loci (Table x64 3.1.2 (R Core Team 2014) using the add-on 3). Within each individual, the maximum number packages. The input file in the loci format was of alleles per locus was one or two in C. izuinsu- converted to the genind format using packages lare, Chinese var. trichotomum, and var. esculen- pegas v.0.10 (Paradis 2010) and adegenet v.2.0.1 tum, indicating diploids, whereas three or four in (Jombart 2008, Jombart & Ahmed 2011). The var. yakusimense and Japanese and Korean var. package poppr v.2.5.0 (Kamvar et al. 2014, Kam- trichotomum indicated tetraploids. The diploid var et al. 2015) constructed neighbor-joining tree individuals were distributed throughout the Izu with a bootstrap value based on the genind format islands, southern Shikoku, the Nansei islands, input file, using Bruvo’s distance as the genetic and China. They showed a disjunct distribution distance among individuals. Bruvo’s distance within Japan. The tetraploid individuals were dis- was devised to calculate the genetic distance tributed throughout the Korean peninsula and in based on microsatellite genotype data, and can be a wide area in the Japanese archipelago. The dip- apply to samples that include multiple ploidy lev- loid and tetraploid individuals were sympatric in els (Bruvo et al. 2004). By considering visibility, southern Shikoku and on two of the Izu islands a dendrogram was drawn by the ape package v.3.3 (Fig. 4). (Paradis et al. 2004) based on the Bruvo’s dis- tances calculated by poppr. The output trees Phylogenetic relationship and geographic distri- showed the same topology between ape and pop- bution of chloroplast haplotypes pr. Thirteen haplotypes were detected in all 79 samples as follows: A1, A2, A3, A4, A5, A6, D7, B8, B9, B10, B11, C12, and C13. The haplotypes Results were divided into four clades, reflecting their geographical distribution (Figs. 5–7). Clade-A in- Ploidy level and geographic distribution of each cluded the haplotypes of C. izuinsulare and Japa- taxon nese and Korean var. trichotomum, excluding a Chromosome counts indicated that Cleroden- sample from Kyushu. Clade-B included the hap- drum izuinsulare and var. esculentum were dip- lotypes of var. yakusimense and var. esculentum. loid with 2n = 52, whereas the Japanese var. tri- Clade-C included the haplotypes of the Chinese chotomum and var. yakusimense were tetraploid samples. Clade-D included a haplotype of var. with 2n = 104 (Fig. 2). Flow cytometric analysis trichotomum from Kyushu. A1 was shared be- identified the ploidy level of each individual (Fig. tween C. izuinsulare and the Japanese and Kore- 3, Table 1). The means of fluorescence intensity an var. trichotomum. B9 was shared between var. in FCA were 76.80 × 103 (SE = 3.31 × 103) in C. yakusimense and var. esculentum. izuinsulare, 83.27 × 103 (SE = 3.50 × 103) in var. esculentum, 136.70 × 103 (SE = 5.58 × 103) in Jap- Neighbor-joining tree based on microsatellite anese var. trichotomum, and 142.28 × 103 (SE = genotypes 7.24 × 103) in var. yakusimense, with coefficient The neighbor-joining tree based on microsat- of variation values (CV-value) ranging from ellite genotypes indicated that diploid individuals 94 Acta Phytotax. Geobot. Vol. 70
2n = 104
10μm
C. trichotomum var. trichotomum
2n = 104
10μm C. trichotomum var. yakusimense
2n = 52
10μm C. trichotomum var. esculentum
2n = 52
10μm C. izuinsulare
Fig. 2. Chromosome counts of Japanese Clerodendrum trichotomum var. trichotomum, var. yakusimense, var. esculentum, and C. izuinsulare from Fukui Prefecture, central Honshu, Amami-Oshima in the Nansei islands, Kuroshima in the Nan- sei islands, and Miyakejima in the Izu islands, respectively. June 2019 Mizusawa & al.– Distribution of Ploidy and Haplotypes in Clerodendrum trichotomum s.l. 95
Table 3. Number of detected alleles of each loci on diploid
800 C. trichotomum var. trichotomum (N = 23) and tetraploid (N = 63). Number of shared al- leles between the two ploidy levels were shown as
600 “Shared”. Detail information of each loci have been written in Mizusawa et al. (2011). Number of alleles 400 Diploid Tetraploid Locus Shared (N = 23) (N = 63)
200 ci111 6 10 6 ct028 12 14 9 0 ci144 9 13 9
800 var. yakusimense ct041 12 33 11 ci141 12 20 10 600
400 formed clades of each taxon: Clerodendrum izuinsulare, var. esculentum and Chinese var. tri-
200 chotomum. Clerodendrum izuinsulare and Chi- nese var. trichotomum formed a sister clade. Most 0 individuals of var. yakusimense were included in
700 var. esculentum a clade, but one individual was nested in the clade Cell count
600 of Japanese var. trichotomum (Fig. 8). Korean var. trichotomum was also nested in the clade of Japanese var. trichotomum. The Japanese var. tri- 400 chotomum was in multiple clades. The bootstrap values were as low as 50% or less, except for the
200 Chinese var. trichotomum and C. izuinsulare (Fig. 8). 0
800 C. izuinsulare Discussion 600 Ploidy levels of each taxon of Japanese C. tricho-
400 tomum s. lat. Chromosome counts, flow cytometric analy- ses and microsatellite genotyping revealed that 200 both diploid and tetraploid taxa of C. trichoto- mum s. lat. occur in Japanese. Our counts of the 0 diploid chromosome number of 2n = 52 con- firmed the previous report by Zeng et al. (2011). DNA amount (FLA-2) In contrast, our tetraploid chromosome counts of 2n = 104 were larger than those of Bowden (1940, Fig. 3. Comparison of amount of DNA between diploid Clerodendrum trichotomum var. esculentum and C. 1945), perhaps due to different methods used at izuinsulareFig3 and tetraploid C. trichotomum var. trichoto- that time (1940s), which did not include the mum and var. yakusimense based on flow cytometric squash technique (Bowden 1940). Clerodendrum analysis. Clerodendrum trichotomum var. trichotomum, var. yakusimense, var. esculentum, and C. izuinsulare var. trichotomum was heterogeneous in ploidy were sampled from Fukui Prefecture in western Hons- level, with ploidy levels showing an allopatric hu, Amami-Oshima in the Nansei islands, Kuroshima in distribution. Diploid populations were in China the Nansei islands, and Miyakejima in the Izu islands, respectively. 96 Acta Phytotax. Geobot. Vol. 70
Number of samples Muximum number of 500 km microsatellite alleles in a locus
1 1 4 2 2 3 AK1 3 HOK AK2 FKS 4 NG1 CHB YMA SAI OH 5 SAD IP2 NG2 NG3 ZUS Izu ISI KYO Populations of Izu Islands SM1 IP1 NI 40N AI1 Izu KRA SM2 KO SK1 KYU AI2
HUB SK2 ME1 Izu MK Esc SK3 KRO Yak MAJ YAK Izu ME2 HA Esc AKU Esc Esc TAK WAK 30N Yak 140E AMA
110E 120E 130E Esc Cultivated in Kyoto Botanical Gardens
Fig. 4. Geographic distribution of ploidy levels based on maximum number of microsatellite alleles on each locus. Individuals Fig4with one or two (white or light gray) alleles on each locus were considered diploid; individuals with three or four (dark gray or black) alleles on one or more loci were considered tetraploid. Size of the pie chart indicates number of samples. Abbreviation of locations is shown beside each pie chart in regular font; taxa are in italic font. ‘Yak’ indicates C. trichoto- mum var. yakusimense, ‘Esc’ indicates C. trichotomum var. esculentum, and ‘Izu’ indicates Clerodendrum izuinsulare. Pie chart without indication of taxon represents C. trichotomum var. trichotomum. whereas tetraploid populations were on the Ko- gesii from western China is therefore necessary. rean Peninsula and widespread in the Japanese We found both var. esculentum and C. izuin- archipelago. Further morphological investigation sulare to be diploid. Clerodendrum var. esculen- might be necessary to confirm the validity of tum is characterized by leaves with and acute treating the two groups, diploid Chinese plants apex and cordate base, usually elongated triangu- and the tetraploid Japanese var. trichotomum, as lar in shape and often pubescent branches (Maki- the same taxon. no 1917, Ohwi 1953, Shimabuku 1997, Fujii Clerodendrum var. yakusimense is also tetra- 2006); C. izuinsulare is characterized by gla- ploid, with 2n = 104. Walker (1976) treated var. brous leaves and short style and petals. Although yakusimense as a synonym of var. fargesii, al- the short style and petals were considered to be though the chromosome number of var. fargesii diagnostic for C. izuinsulare, Fujii (2006) report- was reported to be 2n = 24 (Patermann 1935). ed that the floral morphology of a var.esculentum Completely different chromosome numbers for in the northern Nansei islands was similar to C. the same infraspecific taxon are implausible. izuinsulare. To clarify the relationship between Confirming the chromosome number for var.far - the two diploid taxa, further study of the mor- June 2019 Mizusawa & al.– Distribution of Ploidy and Haplotypes in Clerodendrum trichotomum s.l. 97
B10 Var. yakusimense (Nansei Isls.), 4x B11 1.000 B B9 Var. esculentum (Nansei Isls.), 2x B8
D7 D Var. trichotomum (Japan), 4x A5 Var. trichotomum (Korea), 4x A1 0.997 C. izuinsulare (Izu Isls.), 2x A A6 0.998 A2 A3 A4
0.998 C13 Var. trichotomum (China), 2x C C12
C. bungey Fig5
6.0 * 10-4
Fig. 5. Bayesian phylogenetic tree based on sequences of 13 chloroplast haplotypes. Numbers given at internal nodes are boot- strap values of each branch. Capital letters (A, B, C, & D) under internal nodes are clade names. phology of var. esculentum is necessary. ploidy levels Inoue et al. (1997) treated Clerodendrum Our microsatellite data suggest that tetraploid izuinsulare as a distinct species, while Ohba & individuals are widespread, whereas Japanese Akiyama (2002) treated it as a variety of C. tri- diploid individuals are restricted to the southern chotomum. Our results based on ploidy level and region of Japan. The two diploid taxa, C. izuinsu- nuclear neighbor-joining tree support the treat- lare and var. esculentum, belong to different hap- ment of Inoue et al. (1997). Despite the overlap- lotype groups, A and B. They also differ from the ping flowering season and shared pollinators Chinese diploid var. trichotomum in group C within sympatric populations (Mizusawa et al. (Fig. 6). That the two Japanese haplotype groups 2014), the clear clustering, even in sympatric pop- formed different clades from each other and from ulations of C. izuinsulare and var. trichotomum, the Chinese haplotype group implies that groups indicate low gene flow between the two taxa (Fig. A and B differentiated before colonization of the 8). The difference in ploidy level may contribute Japanese archipelago (Fig. 5). to a clear genetic differentiation between C. The restricted and disjunct distribution on the izuinsulare and Japanese var. trichotomum. To southern edge of the two diploid taxa, var. escul- elucidate the taxonomic status of C. izuinsulare, entum and C. izuinsulare, suggests that those comparative studies with other diploid and tetra- taxa are remnants of the past. The present north- ploid taxa are necessary. ern limit of the Japanese tetraploid var. trichoto- mum is in the temperate mixed forests of south- Geographic distribution and establishment of ern Hokkaido. Climate change during the last 98 Acta Phytotax. Geobot. Vol. 70
A2 glacial maximum (LGM) caused temperate mixed forests to decline on the southern coastal A3 A1 t t area of Japan up to the Tohoku region (Tsukada 1984), while warm temperate forests remained A4 A6 t only on the southern edges, such as on the Izu t t, d Peninsula, Kii Peninsula, southern Shikoku, southern Kyushu, and the Nansei islands (Kamei
A5 1981). The present distribution of diploid var. es- t culentum and Clerodendrum izuinsulare matches well with the past refugia of warm temperate ele- C13 C12 ments during the LGM. D7 t d d The relict-like distribution of diploid lineages
B8 B9 B11 Fig. 6. Haplotype network based on chloroplast sequences. Colored d t, d t circles indicate haplotypes; circle size indicates the number of individuals. Closed circles indicate undetected haplotypes from present or past. Solid line between haplotypes indicates one mu- tation; the dashed line indicates one indel. Lower case ‘d’ indi- cates haplotypes detected only in diploid individuals; ‘t’ indi- cates haplotypes detected only in tetraploid individuals; ‘dt’ in- B10 dicate haplotypes shared between diploid and tetraploid individ- t uals.
Number of samples Haplotypes 500 km
1 A1 B8
2 A2 B9
50N 3 A3 B10 AK1
HOK A4 B11 AK2 4 FKS A5 C12
YMA CHB A6 C13 SAI NG1 OH Fig6 IP2 D7 NG2 NG3 Izu
ZUS Populations of Izu Islands KYO ISI NI
SM1 IP1 40N Izu SM2 KRA AI1 KYU KO AI2 HUB SK2 SK1 Izu Esc ME1 MK KRO Esc Yak SK3 MAJ YAK Esc ME2 AKU Izu HA 140E Esc WAK TAK 30N Yak AMA
110E 120E 130E
Fig. 7. Geographic distribution of chloroplast haplotypes. Location name is shown beside each pie chart in regular font; taxon is shown in italic font. ‘Yak’ indicates Clerodendrum trichotomum var. yakusimense, ‘Esc’ indicates C. trichotomum var. esculentum, and ‘Izu’ indicates C. izuinsulare. Pie charts without indication of taxon represent C. trichotomum var. tri- chotomum. Fig 7 June 2019 Mizusawa & al.– Distribution of Ploidy and Haplotypes in Clerodendrum trichotomum s.l. 99
var. esc 6 66 8 * 8 6 var. yak 8 8 var. tri (China) 8 8 10 8 5 5 5 5 3 8 2 5 5 4 4 4 4 4 2 10 4 2 5 4 5 92 2 5 4 4 9 4 2 4 56 4 43 73 4 4 5 6 2 2 7 4 C. izui 2 2 2 56 4 6 5 5 2 2 66 68 2 2 2 53 2 5 5 2 2 5 2 2 55 4 5 6 5 2 5 5 2 8 1 6 var. yak
Fig. 8. Neighbor-joining tree based on five microsatellite loci genotypes. Clerodendrum trichotomum var. yakusimense, var. esculentum, Chinese var. trichotomum, and C. izuinsulare are shown as ‘var. yak,’ ‘var. esc,’ ‘var. tri (China),’ and ‘C. izui’, respectively. Branches without indication of taxon are tetraploid var. trichotomum. Numbers on internal nodes are bootstrapFig8 values of each branch. Bootstrap values of 50 or more are shown in this figure. Numbers on tips of nodes indi- cate sampling regions defined in Figure 1 (1: Hokkaido, 2: Eastern Honshu, 3: Sado, 4: Izu islands, 5: Western Honshu, 6: Shikoku, 7: Kyushu, 8: Nansei islands, 9: Korea, and 10: China). Node color indicates haplotype group: red for group A, blue for group B, green for group C, and brown for group D. Asterisk indicates cultivated individual of var. esculentum in Kyoto Botanical Garden, Kyoto, Japan, its origin unknown. with a wider distribution of polyploid lineage is pines. Ploidy information may help to reveal the common in plants (Fearn 1972, Thompson & Lu- detailed distribution of var. yakusimense and var. maret 1992, Kataoka et al. 2010, and Yano et al. esculentum in the southern islands. 2014). The distribution of Japanese diploid and tetraploid populations of Clerodendrum trichoto- Multiple polyploidization and formation of tetra- mum s. lat. follows the same pattern. After the ploid lineages LGM, tetraploid populations may have expanded The Japanese diploid lineages share their hap- to northern Japan while diploids remained in lotypes with their respective geographically close southern refugia. te Beest et al. (2012) have point- tetraploid lineages. Haplotype A is shared be- ed out the possibility that polyploid lineages are tween C. izuinsulare and Japanese var. trichoto- adaptive and invasive in a wider range of envi- mum, and haplotype B between var. esculentum ronments compared to their ancestral diploid lin- and var. yakushimense (Fig. 7). Although it is un- eages. The apparently smaller range of distribu- clear which haplotype groups were ancestral be- tion of the tetraploid var. yakusimense than the cause the haplotype phylogenetic tree showed diploid var. esculentum is probably due to of a non-hierarchical topology, at least three tetra- lack of information on its distribution in the is- ploidization events were suggested in the lineag- lands to the south, such as Taiwan and the Philip- es of the A1, A5, and B9 haplotypes (Figs. 5 & 6). 100 Acta Phytotax. Geobot. Vol. 70
Haplotypes in tetraploid A2, A3, A4, A6, B10, such phylogenetic discordance. In other words, it and B11 could have been generated from one of is possible that ancestral alleles lost in C. izuinsu- the three haplotypes, A1, A5, and B9. lare may be conserved in Japanese var. trichoto- The haplotype data suggest that the Japanese mum. The low value of the fluorescence intensity diploid lineages were involved in the establish- ratios described above can also be interpreted as ment of the tetraploid lineages. However, the Jap- a result of genome reduction after polyploidiza- anese diploid lineages were suspected to be not tion. Gene reduction after polyploidization has the only ancestors of the tetraploid lineages, be- been suggested in a wide range of angiosperm cause the fluorescence intensity ratio on the FCA lineages (Raina et al. 1994, Eilam et al. 2009, was smaller than the expected value of the two Buggs et al. 2012). It is not easy to distinguish the when using either Clerodendrum izuinsulare or effects of incomplete lineage sorting and hybrid- var. esculentum as the denominator. We carried ization, but some studies have been conducted in out additional experiments on one sample of each an attempt to solve this problem (Joly et al. 2009). taxon by pseudo-internal standardization using Additional information is necessary to elucidate either Arabidopsis thaliana or Triticum aestivum the cause of the phylogenetic discordance be- as size standards with three replicates to confirm tween the chloroplast and nuclear genomes in C. that the C-value ratios were also smaller than the trichotomum s. lat.. expected values of the two in all combinations between diploids and tetraploids (1.71–1.81). The diploid C. trichotomum from a wide area of Chi- This work was supported by the Competitive Research na showed a slight difference in chromosome size Funds for Fukushima University (17RI025, 17RK015), SICORP Program of the Japan Science and Technology among regions (Zeng et al. 2011). Our results Agency (4-1403), and Environment Research and Tech- from fluorescence intensity was slightly different nology Development Fund, Ministry of the Environment between two diploids, C. izuinsulare and var. es- (4-1605). Dr. Shingo Kaneko (Fukushima University) culentum. The diploids of Clerodendrum tricho- helped us to conduct DNA sequencing and microsatellite tomum s. lat. are probably heterogeneous Karyo- genotyping. The flow cytometric analysis was conducted with the help of Drs. Rina Hoshino, Hirokazu Tsukaya, type analysis of both diploid and tetraploid taxa Takashi Shiga, and Keiichi Okazaki. Dr. Goro Kokubu- may reveal their relationships with the Chinese gata gave us helpful suggestions for counting the chromo- populations. Comparing genome size by FCA is somes. Dr. Kazuhiko Hoshizaki, Dr. Kohtaroh Shutoh, more practical, because the high number and Dr. Mizuki Inoue, Dr. Shota Sakaguchi, Dr. Tetsuro Yo- small size of the chromosomes makes karyotype shikawa, Kazuyoshi Sawa, Masako Shutoh, Masashi Yokogawa, and Yoshihiro Matsuura, helped us to collect analysis difficult. leaf samples. Keiju Kishikawa cared for the plant material Although the bootstrap values on the nuclear used for chromosome counts and the flow cytometric phylogenetic tree were low, the mismatch be- analysis. tween the nuclear and chloroplast phylogenetic trees may also reflect the involvement of unsam- pled diploid lineages in the establishment of the Japanese tetraploid lineages. The Japanese var. References trichotomum and C. izuinsulare formed a clade in a chloroplast phylogenetic tree but not in a nucle- Bowden, W. M. 1940. Diploidy polyploidy and winter ar phylogenetic tree. Hybridization among lin- hardiness relationships in the flowering plants. Amer. J. Bot. 27: 357–371. eages in target taxa can cause phylogenetic dis- Bowden, W. M. 1945. A list of chromosome numbers in cordance between chloroplast and nuclear phylo- higher plants II. Menispermaceae to Verbenaceae. genetic trees because of the different modes of Amer. J. Bot. 32: 191–201. inheritance of the chloroplast and nuclear ge- Bruvo, R., N. K. Michiels, T. G. D’souza & H. Schulen- nomes (Seehausen 2004). burg. 2004. A simple method for the calculation of microsatellite genotype distances irrespective of Incomplete lineage sorting can also cause ploidy level. Molec. Ecol. 13: 2101–2106. June 2019 Mizusawa & al.– Distribution of Ploidy and Haplotypes in Clerodendrum trichotomum s.l. 101
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Received May 26, 2018; accepted November 14, 2018