Origin of Cultivated Citrus (Rutaceae) Documented by the Contents of Internal Transcribed Spacer Sequences (ITS) in Nuclear Ribosomal DNA

Home , Aurantioideae, Citreae, Citrus lucida, Citrus macroptera, Papeda (citrus)

J. Jpn. Bot. 88: 222–238 (2013)

a, a b c Hiroki Yamaji *, Kenji Kondo , Takeshi Kuniga , Hirohisa Nesumi , b a a Toshio Yoshida , Kazunori Hashimoto and Osami Takeda

aBotanical Raw Materials Research Department, Botanical Raw Materials Division, Tsumura & Co., 3586, Yoshiwara, Ami-machi, Ibaraki, 300-1192 JAPAN; bCitrus Research Station, National Institute of Fruit Tree Science, National Agriculture and Food Research Organization, 485-6, Nakamachi, Okitsu, Shimizu-ku, Shizuoka, 424-0292 JAPAN; cShikoku Research Center, Nation Agricultural Research Center for Western Region, Senyû, Zentsûji, Kagawa, 765-8508 JAPAN *Corresponding author: [email protected]

(Accepted on May 23, 2013)

In Citrus cultivars, mandarins, pummelos, citrons, and kumquat/papeda groups were estimated to contribute as parental species for almost Citrus cultivars in precedent studies. In order to verify the hypothesis and to estimate the parental wild species of each Citrus cultivar, this study documented variation of the internal transcribed spacer (ITS) ribotypes of nuclear ribosomal DNA in 61 accessions of 33 species two varieties belonging to major Citrus cultivars, wild Citrus, and related genera. Six species and one variety, e.g., C. deliciosa, C. medica, C. maxima, had non-additive or almost non-additive sequences, probably consisting of a single ribotype. They were thought to have developed not through hybridization but by selection, and were probably putative parental wild species of Citrus cultivars or their direct descendants. In contrast, most Citrus cultivars, e.g., C. limon, C. sinensis, C. aurantium, showed additive states in a number of sites, and consisted of 2–4 major phylogenetically distinct ribotypes. They were estimated as hybrid origin. In the Citrus species examined in this study, 18 ribotypes were recognized, and were classified into nine phylogenetically distinct groups. Among them, four groups were regarded as correspondent to mandarins, pummelos, citrons, and kumquat/papeda groups as well as precedent hypotheses. To the contrary, the rest five groups were probably not included in any putative parental species estimated in precedent studies. Therefore, in addition to foregoing four parental species, at most five putative parental species are presumably existent or were probably existent in the past, and participated in the development of cultivars of Citrus. Origins of major Citrus cultivars were discussed based on ribotype combinations.

Key words: Citrus cultivar, hybrid origin, internal transcribed spacer (ITS) of nuclear ribosomal DNA.

Species belonging to Citrus L. (Rutaceae) and Poncirus Raf.) are the most popular fruits and its allied genera (e.g., Fortunella Swingle of the world (Iwamasa 1976, 1999), and many

—222— August 2013 The Journal of Japanese Botany Vol. 88 No. 4 223 kinds are cultivated for various purposes. et al. 1993) suggested the basic, true species The taxonomy of these species has been a hypothesis proposed by Scora (1975) and challenge for botanists because the number of Barrett and Rhodes (1976). Moreover, Torres et species recognized in Citrus s. str. differs among al. (1978) have estimated parental combinations systems. Swingle (1946) and Swingle and Reece using isozyme patterns; C. aurantifolia (1967) recognized 16 species that are wild or (Christm.) Swing. and C. limon have been naturally hybridized. They did not recognize suggested to be a hybrid of species belonging to almost all artificially created cultivars as taxa. subgenus Papeda and C. medica, C. sinensis and In contrast, Tanaka (1954, 1977) has treated C. aurantifolia, respectively. Also, C. aurantium artificially created cultivars as species equally has been estimated to be a hybrid between C. with the wild ones, and recognized as many as maxima and C. reticulata (Scora 1975, Torres 162 species. Such discrepancies between the et al. 1978). These findings are also supported previous taxonomic systems are due to whether by chemical variations in polymethoxyflavones they recognize cultivars as taxa. Furthermore (Mizuno et al. 1991). as causative role for confusion, Swingle (1946) Federici et al. (1998) have estimated the and Swingle and Reece (1967) assigned species phylogenetic relationships of Citrus and its names to representative cultivated species from related genera using RFLP and RAPD analyses, ancient days such as Citrus aurantium L., C. and showed that most species described as sinensis (L.) Osb., and C. limon (L.) Burm. f. hybrids in the preceding reports had no unique as in wild species while the other cultivars were fragments but high heterozygosity indices. described without taxonomic position. Such However, this study is difficult to interpret imbalanced treatments are due to lack of clarity because they postulated hybridism of each over of hybridism. If they are hybrid origin, species in advance. Araújo et al. (2003) showed which wild species participated as parent of each a phylogenetic relationship of subfamily cultivar? based on noncoding sequences of chloroplast Thus, the number and identities of parental DNA. In their results, Citrus did not compose wild species that have contributed to the a monophyletic clade but instead a clade development of Citrus cultivars is the center of with Poncirus, Fortunella, and Microcitrus. controversy. Chemotaxonomic (Scora 1975) and Bayer et al. (2009) also reported Citrus was numerical taxonomic (Barrett and Rhodes 1976) paraphyletic with seven related genera based examinations have indicated that three species, on three noncoding region of chloroplast DNA. citron (C. medica L.), pummelo (C. maxima However, this study did not document reticulate (Burm.) Merr.), and mandarin (C. reticulata evolution of Citrus including a putative artificial Blanco), are the parental species, which are hybridization event because chloroplast genomes defined by Swingle and Reece (1967). Other are commonly inherited only maternally. cultivated Citrus species in subgenus Citrus Among the precedent studies, Barkley et are believed to be hybrids derived from these al. (2006) is the most comprehensive both parental species , belonging to subgenus Papeda, in sample size and in number of molecular or the other related genera (Barkley et al. 2006). markers. They used 24 simple sequence repeat Recent molecular analyses can reveal (SSR) markers on as many as 370 Citrus the processes of crossbreeding and reticulate accessions. In this study, a Model-based evolution of plants more explicitly (Arnold clustering approach identified five populations; 1997). For Citrus cultivars, the results of mandarins, pummelos, citrons, trifoliates, fraction I protein analysis (Handa et al. 1986) and kumquat/papeda group. Among the five and mitochondrial DNA analysis (Yamamoto populations, trifoliates (Poncirus) were supposed 224 植物研究雑誌第 88 巻第 4 号 2013 年 8 月 not to contribute to many Citrus cultivars. These estimated through classification of the ribotypes. basic species coincide with those of Scora Li et al. (2010) conducted cloning analysis (1975) and Barrett and Rhodes (1976). Both for ITS region for 30 accessions in addition to the phylogenetic and model-based clustering AFLP, three intergenic region sequence analysis analyses supported the hypothesis that there are of cpDNA. This study showed that five old only a few naturally occurring species of Citrus, cultivars; C. aurantifolia, C. limon, C. paradisi, and most of these Citrus cultivars arose through C. sinensis and C. aurantium were hybrid origin, various hybridization events between the and as with Barkley et al. (2006), four parental naturally occurring species. However, Barkley et species; mandarin, citron, papeda and pummelo alʼs (2006) study seems predetermined because participated to generate these cultivars. However, it selected putative non-hybrid strains to estimate Li et al. (2010) did not consider phylogenetically phylogenetic relationships. distinct ribotypes from the putative four species. Therefore, we searched for hidden, preserved Moreover, as only Poncirus was accepted as molecular traits of original species from putative outgroup; it probably misled phylogenetic hybrids, cultivated Citrus species. Among relationships of ribotypes because six other the many molecular markers, the internal genera formed a monophyletic clade with a part transcribed spacer (ITS) regions of nuclear of Citrus based on cpDNA sequences (Araújo et ribosomal DNA (nrDNA) can in many cases al. 2003, Bayer et al. 2009). provide direct evidence of hybridization, Our goals in the present study are (1) to reticulate evolution, and putative parental document variation of ribotypes in major Citrus species (Baldwin et al. 1995, Sang et al. 1995, cultivars in the world and in Japan in comparison Wendel et al. 1995, Soltis et al. 1998, Yamaji et with an evident hybrid cultivar, wild species and al. 2005, 2007). This information is available related genera, (2) to estimate nature and number because the ITS sequences exist in nrDNA as of parental species that have participated in a tandem multicopy array, maintaining more the development of Citrus cultivars based on than two different ribotypes and traces of past phylogenetic relationship of their ribotypes, (3) hybridization as heterozygosity of different to verify effectivity of ITS analysis for parental ribotypes. Therefore, even in diploid hybrids like estimation of diploid hybrids between more than Citrus, more than two ribotypes from more than two species analysis, (4) to estimate the parental two parental species are likely to be maintained. wild species of each Citrus cultivar. When ITS polymorphisms within an individual are observed, cloning of PCR products and Materials and Methods sequencing of multiple clones are effective in Plant materials dissecting the polymorphism (Hershkovitz et Sixty-one individuals were sampled in this al. 1999, Kita and Ito 2000, Yamaji et al. 2007). study. Their sources and voucher information In Citrus species, Matsuyama et al. (1993) are listed in Table 1. For taxonomic names, reported polymorphisms in nrDNA using PCR- we adopted the system by Tanaka (1977). In RFLP, and Xu et al. (2006) applied this marker this study, 15 species and two varieties were to estimate parent species of C. changshanensis represented for cultivars in the world and in using ITS additivities recognized by direct Japan, one species for evident hybrid cultivars, sequence analysis. eight species for wild Citrus species, and nine Therefore, ITS markers are expected to species for related genera. Discrimination be useful in determining the ITS ribotypes of between cultivated / wild species in Citrus is original species of Citrus cultivars, and the referred from Tanaka (1954). For outgroups, number of the original species would then be Glycosmis pentaphylla (Retz.) DC. and Murraya August 2013 The Journal of Japanese Botany Vol. 88 No. 4 225

Table 1. The origin and voucher specimens of each sample examined in this study. All voucher specimens are deposited in THS. “Okitsu” indicates “Okitsu branch, National Institute of Fruit Tree Science, Shizuoka, Japan”. “Ibaraki” indicates “Ibaraki, Japan”. Infrageneric taxa within Citrus is followed by Tanaka (1954). * followed by Tanaka (1954).

Taxon wild / cultivated * Origin Voucher Accession No. Glycosmis pentaphylla (Retz.) DC. wild Cultivated in Okitsu (Species No. 6219001, strain No. W0001) THS 78604 AB456043 Murraya paniculata (L.) Jack. wild Cultivated in Okitsu (Species No. 6211001, strain No. A0001) THS 78610 AB456044 Swinglea glutinosa Merr. wild Cultivated in Okitsu (Species No. 6227001, strain No. B0001) THS 78607 AB456045 Feroniella lucida Swingle wild Cultivated in Okitsu (Species No. 6221001, strain No. B0002) THS 78605 AB456046 Atalantia monophylla DC. wild Cultivated in Okitsu (Species No. 6223001, strain No. W0001) THS 78606 AB456047 Severinia buxifolia (Poir.) Tenore wild Cultivated in Okitsu (Species No. 6213001, strain No. A0001) THS 78611 AB456048 Eremocitrus glauca (Lindl.) Swingle wild Cultivated in Okitsu (Species No. 6208001, strain No. W0004) THS 78609 AB456049 Poncirus trifoliata (L.) Raf. wild Cultivated in Okitsu (Species No. 6205001, strain No. A0005) THS 77968 AB456050 Fortunella japonica Swing. wild Cultivated in Okitsu (Species No. 6203001, strain No. A0001) THS 77967 AB456051 Citrus Subg. Archicitrus Sect. Papeda C. macroptera Montr. wild Cultivated in Okitsu (Species No. 6201043, strain No. A0001) THS 77959 AB456052 C. hystrix DC. wild Cultivated in Okitsu (Species No. 6201039, strain No. A0001) THS 77958 AB456053 Sect. Limonellus C. aurantifolia (Christm.) Swing. possibly wild Cultivated in Okitsu (Species No. 6201003, strain No. A0002) THS 77969 AB456054 C. limettioides Tan. cultivated Cultivated in Okitsu (Species No. 6201051, strain No. A0001) THS 77962 AB456055 C. montana Tan. cultivated Cultivated in Okitsu (Species No. 6201053, strain No. A0001) THS 77963 AB456057 Sect. Citrophorum C. medica L. wild Cultivated in Okitsu (Species No. 6201057, strain No. B0001) THS 77964 AB456058 C. medica L. var. sarcodactylis cultivated Cultivated in Okitsu (Species No. 6201061, strain No. A0001) THS 78614 AB456059 C. limon (L.) Burm. f. 1 wild Cultivated in Okitsu (Species No. 6201013, strain No. A0026) THS 77973 AB456060 C. limon (L.) Burm. f. 2 wild Cultivated in Okitsu (Species No. 6201013, strain No. ID-1123) THS 77974 AB456061 Sect. Cephalocitrus C. maxima (Burm.) Merr. 1 wild Cultivated in Okitsu (Species No. 6201007, strain No. A0009) THS 77970 AB456062 C. maxima (Burm.) Merr. 2 wild Cultivated in Okitsu (Species No. 6201007, strain No. A0005) THS 77971 AB456063 C. paradisi Macf. 1 cultivated On-market product in Ibaraki THS 76547 AB456064 C. paradisi Macf. 2 cultivated Cultivated in Okitsu (Species No. 6201019, strain No. A0005) THS 78617 AB456065 C. hassaku Hort. ex Y. Tanaka 1 cultivated On-market product in Ibaraki, produce of Wakayama, Japan THS 74872 AB456066 C. hassaku Hort. ex Y. Tanaka 2 cultivated On-market product in Ibaraki THS 76551 AB456067 C. hassaku Hort. ex Y. Tanaka 3 cultivated Cultivated in Okitsu (Species No. 6201009, strain No. A0001) THS 77972 AB456068 Sect. Aurantium C. natsudaidai Hayata 1 cultivated On-market product in Ibaraki, produce of Kumamoto, Japan THS 74865 AB456069 C. natsudaidai Hayata 2 cultivated Cultivated in Okitsu THS 35626 AB456070 C. natsudaidai Hayata 3 cultivated On-market product in Ibaraki, produce of Yamaguchi, Japan THS 76498 AB456071 C. natsudaidai Hayata 4 cultivated On-market product in Ibaraki THS 76549 AB456072 C. natsudaidai Hayata 5 cultivated Cultivated in Okitsu (Species No. 6201017, strain No. B0029) THS 77975 AB456073 C. aurantium L. 1 cultivated Cultivated in Ibaraki THS 74873 AB456074 C. aurantium L. 2 cultivated Cultivated in Ibaraki THS 00823 AB456075 C. aurantium L. 3 cultivated On-market product in Ibaraki, produce of Yamaguchi, Japan THS 76497 AB456076 C. aurantium L. 4 cultivated Cultivated in Okitsu (Species No. 6201121, strain No. A0005) THS 77979 AB456077 C. neo-aurantium Hort. ex Tan. cultivated Cultivated in Shizuoka THS 38174 AB456078 C. sinensis (L.) Osb. cultivated Cultivated in Okitsu (Species No. 6201023, strain No. A0102) THS 77977 AB456079 C. sinensis (L.) Osb. var. brasiliensis Tan. 1 cultivated On-market product in Ibaraki THS 76548 AB456080 C. sinensis (L.) Osb. var. brasiliensis Tan. 2 cultivated On-market product in Ibaraki, produce of California, USA THS 74868 AB456081 C. iyo Hort. ex Tan. 1 cultivated On-market product in Ibaraki THS 74864 AB456082 C. iyo Hort. ex Tan. 2 cultivated On-market product in Ibaraki THS 76545 AB456083 C. iyo Hort. ex Tan. 3 cultivated Cultivated in Okitsu (Species No. 6201133, strain No. A0001) THS 78618 AB456084 Subg. Metacitrus Sect. Osmocitrus C. junos Siebold ex Miq. cultivated Cultivated in Okitsu (Species No. 6201151, strain No. A0004) THS 77981 AB456085 Sect. Acrumen C. unshiu Marc. 1 cultivated On-market product in Ibaraki, produce of Shizuoka, Japan THS 74869 AB456088 C. unshiu Marc. 2 cultivated Cultivated in Okitsu (Species No. 6201035, strain No. B0076) THS 77978 AB456089 C. reticulata Blanco 1 cultivated On-market product in Ibaraki THS 74863 AB456090 C. reticulata Blanco 2 cultivated Cultivated in Okitsu THS 35631 AB456091 C. reticulata Blanco 3 cultivated Cultivated in Okitsu (Species No. 6201021, strain No. 0026 ID481) THS 77976 AB456092 C. deliciosa Ten. 1 cultivated Cultivated in Okitsu THS 35632 AB456093 C. deliciosa Ten. 2 cultivated Cultivated in Okitsu (Species No. 6201187, strain No. A0001) THS 77982 AB456094 C. tachibana (Makino) Tan. 1 wild Japan, Shizuoka, Matsuzaki THS 74888 AB456095 C. tachibana (Makino) Tan. 2 wild Cultivated in Okitsu (Species No. 6201217, strain No. A0001) THS 77965 AB456096 C. tachibana (Makino) Tan. 3 wild Cultivated in Okitsu (Species No. 6201217, strain No. B0010-ID497) THS 77966 AB456097 C. kinokuni Hort. ex Tan. 1 cultivated Cultivated in Okitsu THS 35635 AB456098 C. kinokuni Hort. ex Tan. 2 cultivated On-market product in Ibaraki THS 39252 AB456099 C. kinokuni Hort. ex Tan. 3 cultivated Cultivated in Okitsu (Species No. 6201205, strain No. W0009) THS 77984 AB456100 C. kinokuni Hort. ex Tan. 4 cultivated Cultivated in Okitsu (Species No. 6201205, strain No. W0011-ID529) THS 77983 AB456101 C. sunki Sakurai wild Cultivated in Okitsu THS 35636 AB456102 C. amblycarpa Ochse cultivated Cultivated in Okitsu (Species No. 6201227, strain No. A0001) THS 78616 AB456105 Hybrid cultivars C. unshiu × reticulata ‘Siranuhi’ 1 cultivated On-market product in Ibaraki, produce of Kumamoto, Japan THS 74861 AB456106 C. unshiu × reticulata ‘Siranuhi’ 2 cultivated On-market product in Ibaraki THS 76546 AB456107 226 植物研究雑誌第 88 巻第 4 号 2013 年 8 月 paniculata (L.) Jack were used following Araújo have been defined by S1 nuclease mapping. All et al. (2003). unambiguous sequences were aligned manually. When ambiguous (additive) sites were found, DNA isolation and amplification they were coded according to the IUPAC (IUB) Total genomic DNA was isolated from codes. 200 to 300 µg of fresh or dried leaf tissues or fresh peel by DNeasy® Plant Mini Kit (Qiagen) Cloning following its protocol. The isolated DNA was We cloned the PCR products of the ITS resuspended in 100 µL TE. PCR amplifications regions of putative hybrid samples. These were achieved using universal primers, ITS5 (5’- putative hybrids had been inferred from GGA AGT AAA AGT CGT AAC AAG G-3’; nucleotide additivities in more than one site White et al. 1990) and ITS4 (5’-TCC TCC GCT found in the direct sequencing. PCR products TAT TGA TAT GC-3’; White et al. 1990). We were ligated into the pT7Blue cloning vector used the following thermocycle protocol: (94˚C, in the Perfectly Blunt Cloning Kit according 2 min) × 1 cycle, (94˚C, 1.5 min; 48˚C, 2 min; the manufacturer’s instructions (Novagen Inc.). 72˚C, 3 min) × 30 cycles, and (72˚C, 15 min) Resulting recombinant plasmids were used to × 1 cycle. The PCR reaction mixture consisted transform the competent cells included in the kit. of 10 × gene-Taq Buffer (Nippon Gene) 5 µL, The transformation mix was incubated in 250 dNTP mix (Nippon Gene) 4 µL, forward primer µL SOC medium for 1 hr at 37˚C on a rotary (ITS5: 10 pmol/µl) 1µL, reverse primer (ITS4: shaker, then plated on LB agar with 50 µg / 10 pmol/µl) 1 µL, gene-Taq (Nippon Gene) 0.25 mL ampicillin. 18–48 colonies were randomly µL, DMSO 5 µL, D.D.W. 32.5 µL, and template selected and were put into PCR reaction DNA 1.25 µL. PCR products were separated buffer. PCR was conducted using the same from other by-products using 2% TAE agarose protocol for the direct sequencing with a slight gel electrophoreses. The desired bands were cut modification; D.D.W. was increased to 33.75 out and purified using GFXTM PCR DNA and µL, and template DNA was omitted for the Gel Band Purification Kit (Amersham biotech). picked up colonies. The amplified ITS regions were purified directly using GFXTM PCR DNA Sequencing the PCR products and a Gel Band Purification Kit (Amersham We sequenced the purified PCR products biotech). Sequencing and alignments of clones using the BigDye Terminator ver. 3.1 Cycle were performed in the same way, utilizing direct Sequencing Kit and the Model 3100 automated sequencing. sequencer (Applied Biosystems) following the manufacturerʼs instructions. For sequencing, Phylogenetic analysis we used two internal primers, ITS2 (5’-GCT Phylogenetic relationships were analyzed GCG TTC TTC ATC GAT GC-3’; White et al. using the maximum-parsimony (MP) method 1990) and ITS3 (5’-GCA TCG ATG AAG AAC using PAUP 4.0b10 (Swofford 2004). Additive GCA GC-3’; White et al. 1990), in addition state characters (e.g., R = A + G) and indels to the primers used for amplification. The were used in addition to the four nucleotides sequences were aligned manually. To determine of DNA, and were weighted equally. The MP the boundaries separating the rRNA genes from analyses were conducted through a heuristic the ITS1 and ITS2 regions, we compared the search with a TBR (tree-bisection-reconnection) sequences with those of Daucus carota and branch swapping option. In each analysis, 100 Vicia faba (Yokota et al. 1989), in which the rounds of random sequence additions were limits of mature 18S, 5.8S, and 26S rRNAs performed to identify multiple islands of equally August 2013 The Journal of Japanese Botany Vol. 88 No. 4 227 most parsimonious trees (Maddison 1991). less than 10 additive sites. Among them, C. Bootstrap analysis (Felsenstein 1985) with 1000 medica, C. sunki, C. deliciosa, C. kinokuni and C. replications and 100 rounds of random sequence amblycarpa had no additive sites. In contrast to additions for each replication was performed the species mentioned above, the other species, with the same program. which were major part of Citrus cultivars had more than nine additive sites, and were supposed Results to be of hybrid origins. ITS region sequence alignment and nucleotide Only two species showed intraspecific site variation variation: C. tachibana and C. sinensis. In C. All sequences obtained by direct sequencing tachibana, which is a wild species distributed were deposited in the DDBJ/EMBL/GenBank in Japan (Yamazaki 1989), incomplete database under the accession numbers listed in substitutional variations were recognized in Table 1. seven sites (Fig. 1). In C. sinensis, C. sinensis In many of the cultivar species, their var. brasiliensis 2 had three unique incomplete sequence signals were not clear from the middle substitutional sites compared to the other two part in direct sequence analyses. This lack of samples. clarity is probably due to the polymorphism of indels within nrDNA repeats or between nrDNA Composition of the ITS ribotypes in putative loci in different chromosomes (Booy et al. 2000). hybrids and phylogenetic reconstruction In such cases, we joined the forward and reverse A total of 18–48 ITS clones were sequenced sequences with “N.” As in Citrus aurantifolia, C. for each of 27 samples including Fortunella limettioides, and C. limon, indel polymorphism japonica. The sequence variations of clones in by more than one nucleotide probably caused the three example samples are shown in Fig. 2. In obscurity; we therefore aligned their sequences Citrus aurantium, the sequences of 13, 3, and 10 with the other species and filled up the gap with clones were identical and were named “ribotype “N”. E”, “ribotype O”, and “ribotype P”, respectively. The number of nucleotide substitutions and We consider these to be “major” that involved indels within Citrus totalled 69: 30 in ITS1, more than one identical clone. They were 5 in 5.8S, 33 in ITS2, and 1 in partial 26S, therefore named based on their phylogenetic respectively (Fig. 1). In all but four of the sites, positions in the following analysis (Fig. 4). In incomplete substitutions such as additive states contrast, the other three clones were unique (e.g., ‘G’ and ‘R’ (=‘G’ + ‘A’)) were observed. sequences, but had no unique substitutions. We All the additive states were composed of call these “minor” ribotypes that had only one only two nucleotides. Such additive sites are unique clone. Almost all of the minor ribotypes supposed to indicate that more than one ribotype are chimeric, i.e., consisting of an admixture of differing at these sites is involved within a single sequences of the major ribotypes. Also, in C. individual (Baldwin et al. 1995, Sang et al. 1995, natsudaidai and C. limon, three major ribotypes, Campbell et al. 1997, Yamaji et al. 2007). “F”, “I,”, “N”, and two major ribotypes, “E” and The numbers and positions of the additive “R”, were detected, respectively. sites varied depending on the species. Among Consequently, all of the samples examined the Citrus species, all the putative wild species were composed of 2–4 “major” ribotypes and (C. macroptera, C. hystrix, C. montana, C. sunki, 1–11 “minor” ribotypes. In the Citrus and and C. tachibana), and six cultivars (C. medica, Fortunella species examined in this study, 18 C. medica var. sarcodactylis, C. maxima, C. (ribotype A to R) and 3 (Fortunella japonica deliciosa, C. kinokuni and C. amblycarpa) had A, B, and C) major ribotypes were recognized, 228 植物研究雑誌第 88 巻第 4 号 2013 年 8 月 238 植物研究雑誌第 88 巻第 4 号 2013 年 8 月

ITS1 5.8S

position 000000000000000111111111122222 23444 023566666777899001223345900235 99012 sample 283045689179745476795671739573 86502

Poncirus trifoliata CCCGTGCCGTGGCCCCCACCCGCGCCCCCTCCGCG Fortunella japonica * 25 CCCGTGCCGTAGCCCCCACCCGCGCCCCCTCCGCG Citrus Subg. Archicitrus Sect. Papeda C. macroptera CCCGGATCGTGGCATCCACCCGCGC Y CCCCCCGCG C. hystrix C Y CGGATCGTGG Y CTCCACCCGCGCCCCCCCCG YR Sect. Limonellus C. aurantifolia * 26 YY CGGATCGTGG Y C Y CCA YY CGC R CC NNNNN CGCG C. limettioides * 22 YY CG KRY CGT R GCCC Y CA YY CGC R CC NNNNN CGCG C. montana CCTGGATCGTGGTCTCCACCCCCGCCCCCCCCGCG Sect. Citrophorum C. medica TTCGGATCGTGGCCCCCATTCGC ACC--CTCCGCG C. medica var. sarcodactylis TTCGGATCGTGGCCCCCATTCGCACC--CTCCGCG C. limon 1 YY CG KRY CGT R GCCC Y CA YY CGC R CC NNNNN CGCG C. limon 2 * 27 YY CG KRY CGT R GCCC Y CA YY CGC R CC NNNNN CGCG Sect. Cephalocitrus C. maxima1 * 31 CCCGGACCGTGGCCCCCACCTGCGCCCTCTCCGCG C. maxima2 CCCGGACCGTGGCCCCCACCTGCGCCCTCTCCGCG C. paradisi 1 * 44 CCCG KR CCG YR GCCC Y CACC Y GCGCCC Y CTCCGCG C. paradisi 2 CCCG KR CCG YR GCCC Y CACC Y GCGCCC Y CTCCGCG C. hassaku 1 * 20 CCCGGACCGTGGCCCCCACC Y GCG YY C Y CT Y CGCG C. hassaku 2 * 18 CCCGGACCGTGGCCCCCACC Y GCG YY C Y CT Y CGCG C. hassaku 3 * 24 CCCGGACCGTGGCCCCCACC Y GCG YY C Y CT Y CGCG Sect. Aurantium C. natsudaidai 1 CCCG KR CCGT R GCCC Y C M CC Y GCGCCC Y CTCCGCG C. natsudaidai 2 * 26 CCCG KR CCGT R GCCC Y C M CC Y GCGCCC Y CTCCGCG C. natsudaidai 3 * 20 CCCG KR CCGT R GCCC Y C M CC Y GCGCCC Y CTCCGCG C. natsudaidai 4 * 21 CCCG KR CCGT R GCCC Y C M CC Y GCGCCC Y CTCCGCG C. natsudaidai 5 * 27 CCCG KR CCGT R GCCC Y C M CC Y GCGCCC Y CTCCGCG C. aurantium 1 * 19 CCCG KR CCGT R GCCC Y CACC Y GCGCC MYY TCCGCG C. aurantium 2 * 31 CCCG KR CCGT R GCCC Y CACC Y GCGCC MYY TCCGCG C. aurantium 3 * 22 CCCG KR CCGT R GCCC Y CACC Y GCGCC MYY TCCGCG C. aurantium 4 * 28 CCCG KR CCGT R GCCC Y CACC Y GCGCC MYY TCCGCG C. neo-aurantium * 29 CCCG KR CCGT R GCCC Y CACC Y GCGCC MYY TCCGCG C. sinensis * 24 CCCG KRY CG YR GCCC Y CACC Y GCG YY C Y CTC Y GCG C. sinensis var. brasiliensis 1 CCCG KRY CG YR GCCC Y CACC Y GCG YY C Y CTC Y GCG C. sinensis var. brasiliensis 2 * 47 CCC RKRY CG YR GCCC Y CACC Y G M G YY C Y CTC Y GCG C. iyo 1 CCCG KR CCG YR GCCC Y CACC Y GCGCCC Y CTCCGCG C. iyo 2 * 18 CCCG KR CCG YR GCCC Y CACC Y GCGCCC Y CTCCGCG C. iyo 3 CCCG KR CCG YR GCCC Y CACC Y GCGCCC Y CTCCGCG Subg. Metacitrus Sect. Osmocitrus C. junos * 31 CCCGTGC Y GTAGCCC YY ACCCGCGC Y CCCTCCGCG Sect. Acrumen C. unshiu 1 * 48 CCCG KR CCGT R GCCC Y CACCCGCG YY CCCT Y CGCG C. unshiu 2 * 22 CCCG KR CCGT R GCCC Y CACCCGCG YY CCCT Y CGCG C. reticulata 1 CCCG KR CC RYR GCCC Y CACC Y GCGCCC Y CTCCGCG C. reticulata 2 * 29 CCCG KR CC RYR GCCC Y CACC Y GCGCCC Y CTCCGCG C. reticulata 3 * 28 CCCG KR CC RYR GCCC Y CACC Y GCGCCC Y CTCCGCG C. deliciosa 1 CCCGTGCCGTAGCCCTCACCCGCGCCCCCTCCGCG C. deliciosa 2 CCCGTGCCGTAGCCCTCACCCGCGCCCCCTCCGCG C. tachibana 1 CCCGTGCCGTAGCCCTCACCCGCGCCCCCTCCGCG C. tachibana 2 CCCGTGCCGTAGCCC Y CACC Y GCGCCCCCTCCGCG C. tachibana 3 CCCG K GCCGTA K CCC Y CACCCGCGCCCCCTCCGCG C. kinokuni 1 CCCGTGCCGTAGCCCTCACCCGCGCCCCCTCCGCG C. kinokuni 2 CCCGTGCCGTAGCCCTCACCCGCGCCCCCTCCGCG C. kinokuni 3 CCCGTGCCGTAGCCCTCACCCGCGCCCCCTCCGCG C. kinokuni 4 CCCGTGCCGTAGCCCTCACCCGCGCCCCCTCCGCG C. sunki CCCGTGCCATAG CCCTCACCCGCGCCCCCTCCGCG C. amblycarpa CCCGTGCCGTAGCCCTCACCCGCGCCCCCTCCGCG Hybrid cultivars C. unshiu × reticulata ‘Siranuhi’ 1 CCCG KR CC R T R GCCC Y CACCCGCG YY CCCTCCGCG C. unshiu × reticulata ‘Siranuhi’ 2 * 39 CCCG KR CC R T R GCCC Y CACCCGCG YY CCCTCCGCG

Fig.Fig. 1. 1. Character Character states states of of each each sample sample inin variablevariable sites within Citrus.. CharacterCharacter statesstates in in an an additive additive condition condition of of more more than than oneone base base are are reversed reversed in in black black and and white, white, andand thosethose inin derivativederivative states are shaded. Asterisks (*)(*) andand theirtheir leftneighbouring numbers numbersindicate indicatesamples samplesthat were that examined were examined in cloning in analysis, cloning andanalysis, the number and the of number clones sequenced.of clones sequenced. August 2013 The Journal of Japanese Botany Vol. 88 No. 4 229 238 植物研究雑誌第 88 巻第 4 号 2013 年 8 月

ITS1 5.8S

position 000000000000000111111111122222 23444 023566666777899001223345900235 99012 sample 283045689179745476795671739573 86502

Fig. 1. Character states of each sample in variable sitesFig. within1. Continued. Citrus. Character states in an additive condition of more than one base are reversed in black and white, and those in derivative states are shaded. Asterisks (*) and their left numbers indicate samples that were examined in cloning analysis, and the number of clones sequenced. 230 植物研究雑誌第 88 巻第 4 号 2013 年 8 月

ITS1 5.8S ITS2 26S

position 000000000000000111111111122222 2 3 4 4 4 4 44444455555555555555666666666666 6 023566666777899001223345900235 9 9 0 1 2 2 35567700123455556789001122234444 6 sample 283045689179745476795671739573 8 6 5 0 2 6 11523949390412676247092756852357 0 ribotype

Citrus limon 2

direct Y Y CG KR Y CGT R GCCC Y CA Y Y CGC R CC NN CTCCGCGCC--CCCCCC M TCCGCGT N ACCC RK GC R A Y KMG Y 13 CCCGTGCCGTAGCCCTCACCCGCGCCCCCT CCGCG CC- -CCCCCCCTCCGCGTAACCCGGGCGACGAG T E 1 CCCGTGCCGTAGCCCTCACCCGCGCCCCCT CCGCG CC--CCCCCCCTCCGCGT-ACCCATGCAATTCA C 1 TTCGGATCGTGGCCCCCACCCGCGCCCCCT CCGCG CC- -CCCCCCCTCCGCGTAACCCGGGCGACGAG T 1 TTCGGATCGTGGCCCCCATTCGCACCCCCT CCGCG CC--CCCCCCATCCGCGT-ACCCATGCAATTCA C 8 TTCGGATCGTGGCCCCCATTCGCACC- -CT CCGCG CC- -CCCCCCATCCGCGT-ACCCATGCAATTCA C R

Citrus natsudaidai 4

direct C C C G KR CCGT R GCCC Y C M CC Y GCGCCC Y CTCCGCGCC--C Y CC Y C M TCCGCGT N A Y CC RKR C R ACGAGT 8 CCCGTGCCGTAGCCCTCACCCGCGCCCCCT CCGCG CC- -CCCCCCCTCCGCGTAATCCGGGCGACGAG T F 1 CCCGGACCGTGGCCCCCCCCCGCGCCCCCT CCGCG CC- -CCCCTCATCCGCGTAATCCGGGCGACGAG T 5 CCCGGACCGTGGCCCCCCCCCGCGCCCCCT CCGCG CC- -CCCCTCATCCGCGT-ACCCATACAACGAG T I 1 CCCGGACCGTGGCCCCCACCTGCGCCCTCT CCGCG CC--CTCCCCATCCGCGT-ACCCATACAACGAG T 1 CCCGGACCGTGGCCCCCACCTGCGCCCC-T CCGCG CC--CTCCCCATCCGCGT-ACCCATGCAACGAG T 6 CCCGGACCGTGGCCCCCACCTGCGCCCTCT CCGCG CC- -CTCCCCATCCGCGT-ACCCATGCAACGAG T N

Citrus aurantium 2

direct C C C G KR CCGT R GCCC Y CACC Y G CGCC M Y Y TCCGCGCC--C Y CCCC M TCCGCGT N ACCC RK GC R ACGAGT 13 CCCGTGCCGTAGCCCTCACCCGCGCCCCCT CCGCG CC- -CCCCCCCTCCGCGTAACCCGGGCGACGAG T E 1 CCCGGACCGTGGCCCCCACCTGCGCCCTCT CCGCG CC--CTCCCCATCCGCGT-ACCCATGCAACGAG T 1 CCCGTGCCGTAGCCCTCACCCGCGCCCCCT CCGCG CC--CCCCCCCTCCGCGT-ACCCATGCAACGAG T 3 CCCGGACCGTGGCCCCCACCTGCGCCATCT CCGCG CC- -CTCCCCATCCGCGT-ACCCATGCAACGAG T O 1 CCCGGACCGTGGCCCCCACCTGCGCCATCT CCGCG CC--CTCCCCATCCGCGT-ACCCATGCGACGAG T 10 CCCGGACCGTGGCCCCCACCTGCGCCCTTT CCGCG CC- -CTCCCCATCCGCGT-ACCCATGCAACGAG T P

Fig. 2. Example of ribotype components in heterogeneous Citrus species recognized by cloning analysis. Clones in the same states in the variable sites were bundledFig. 2. and areExample indicated ofin largeribotype letters (majorcomponents ribotype), andin heterogeneousclones with unique combinationsCitrus species in the variablerecognized sites are by indicated cloning in small analysis. letters (minor Clones in ribotype).the Minor same ribotypes states were in theplaced variable between thesites major were ribotypes. bundled Sites that and are are different indicated between inthe largemajor ribotypesletters are(major framed ribotype), in dashed lines. and An clones asteriskwith indicates unique that the combinations major ribotype “F+R” in theis probably variable a chimera sites of are ribotypes indicated F and R. in small letters (minor ribotype). Minor ribotypes were placed between the major ribotypes. Sites that are different between the major ribotypes are framed in dashed lines. An asterisk indicates that the major ribotype “F+R” is probably a chimera of ribotypes F and R. respectively. Their sequences were deposited in sequences of ribotypes D, E, F, and R were the DDBJ/EMBL/GenBank database under the completely congruent with those of C. sunki, C. accession numbers listed in Fig. 3 for ribotype A deliciosa, C. amblycarpa, C. kinokuni, and C. to R, and AB456108, AB456109, and AB456110 medica, respectively. for Fortunella japonica A, B and C. In the MP trees, Citrus was scattered into Nucleotide variation between the ribotypes two clades (clade A and B). Clade A included in Citrus and the phylogenetic relationships of species belonging to Eremocitrus, Microcitrus, Citrus and its allied species as estimated by the Poncirus, Fortunella, and ribotypes A–G with maximum-parsimony (MP) method are shown comparatively high bootstrap value (71%). in Figures 3 and 4, respectively. The ribotypes Ribotype C–F formed a monophyletic clade “C– were named in alphabetical order, following the F.” In Clade B, ribotype R was sister to the other order in the MP strict consensus tree. ribotypes, and three clades were recognized; In all the ribotypes of Citrus species, the subgenus Papeda clade (C. hystrix, C. montana, lengths of the ITS1, 5.8S, and ITS2 were 221 C. macroptera, and ribotype H), clade “I–L” to 250 base pairs (bp), 164 bp, and 215 to 217 (ribotype I–L), and clade “M–Q” (ribotype M– bp, respectively. Ribotype R, which had 29 Q). Clade “I–L” was further divided into two bp deletions in ITS1, was recognized in C. clades: ribotype I and clade “J–L” because of its aurantifolia, C. limettioides, and C. limon, with comparatively low bootstrap support (59%). these deletions presumably inhibiting their Figure 5 shows the major ribotype content of detection of the sequences in direct sequencing each sample. In all of the samples under cloning analyses. Among the ribotypes of Citrus, the analysis, 2–4 major ribotypes were recognized. August 2013 The Journal of Japanese Botany Vol. 88 No. 4 231

ITS1 5.8S ITS2 26S

position 000000000000000111111111122222 23444 444444455555555555555666666666666 6 023566666777899001223345900235 99012 235567700123455556789001122234444 6 Taxa Ribotype 283045689179745476795671739573 86502 611523949390412676247092756852357 0 Accession No.

A CCCGTGCTGTAGCCCCTACCCGCGCCCCCTCCGCGCC--CCCCCCCGCCGCGCAACCCGGGCGTCGAGC AB456112 B CCCATGCCGTAGCCCCCACCCGAGCCCCCTCTGCGCC--CCCCCGCTCCGCGTAACCCGGGCGACGAGC AB456111 C CCCGTGCCGTAGCCCTCACCCGCGCTCCCTCCGCGCC--CCCCCCCTCCGCGTAACCCGGGTGACGAGT AB456114 C. sunki D CCCGTGCCATAGCCCTCACCCGCGCCCCCTCCGCGCC--CCCCCCCTGCGCGTAACCCGGGCGACGAGT AB456115 C.deliciosa E CCCGTGCCGTAGCCCTCACCCGCGCCCCCTCCGCGCC--CCCCCCCTCCGCGTAACCCGGGCGACGAGT AB456116 C.kinokuni F CCCGTGCCGTAGCCCTCACCCGCGCCCCCTCCGCGCC--CCCCCCCTCCGCGTAATCCGGGCGACGAGT AB456117 G CCCGTGCCGTAGCCCCCACCCGCGCCCCCTCCGCGCC--TCCCCCCTCCGCGCAACCCGGGCAACGAGC AB456113 H CCCGGATCGTGGTCTCCACCCGCGCCCCCCCCGCGCCCACCTCCCATCCGCGT-ACCCATGCAACGAGT AB456118 I CCCGGACCGTGGCCCCCCCCCGCGCCCCCTCCGCGCC--CCCCTCATCCGCGT-ACCCATACAACGAGT AB456119 J CCCGGATCGTGGCCCCCACCCGCGTTCCCTCCGCGCG--CCCCTCATCCGCGT-ACCCATGAAACGAGT AB456120 K CCCGGACCGTGGCCCCCACCCGCGTTCCCTTCGCGCG--GCCCTCATCCGCGT-ACCCATGTAACGAGT AB456121 L CCCGGACCGTGGCCCCCACCCGCGTTCCCTCCGCGCG--GCCCTCATCCGCGT-ACCCATGTAACGAGT AB456122 M CCCGGACCGTGGCCCCCACCTGCGCCCTCTCCGCGCC--CTCCCCATCCGTGT-ACCCATGCAACGAGT AB456123 N CCCGGACCGTGGCCCCCACCTGCGCCCTCTCCGCGCC--CTCCCCATCCGCGT-ACCCATGCAACGAGT AB456124 O CCCGGACCGTGGCCCCCACCTGCGCCATCTCCGCGCC--CTCCCCATCCGCGT-ACCCATGCAACGAGT AB456125 P CCCGGACCGTGGCCCCCACCTGCGCCCTTTCCGCGCC--CTCCCCATCCGCGT-ACCCATGCAACGAGT AB456126 Q CCCGGACCGCGGCCCCCACCTGCGCCCTCTCCGCGCC--CTCCCCATCCGCGT-TCCCATGCAACGAGT AB456127 C.medica R TTCGGATCGTGGCCCCCATTCGCACC--CTCCGCGCC--CCCCCCATCCGCGT-ACCCATGCAATTCAC AB456128

Fig. 3. Ribotypes in Citrus species recognized by cloning analysis. The sites shown are the same as those in Figs. 1 and 2. A ribotype with the taxa name on the right side indicates that the same, nonadditive sequences were recognized in these taxa in direct sequence analyses.

All of the samples belonging to the same species hystrix, C. maxima, C. reticulata, C. indica and were composed of the same ribotypes except C. tachibana had one ribotype, respectively. We for C. sinensis. Citrus sinensis var. brasiliensis could not compare our result with that of Li et 2 had ribotype A in addition to the content of al. (2010) because no sequences are deposited in C. sinensis. Among the 18 ribotypes, ribotypes DDBJ/EMBL/GenBank database. A, C, and G were recognized only in C. junos, In plants, concerted evolution is thought to and ribotypes H and I were detected only in C. homogenize polymorphisms in the ITS region aurantifolia and C. natsudaidai, respectively. within a lineage (Hillis and Dixon 1991, Baldwin The other ribotypes were observed in more et al. 1995, Alvarez and Wendel 2003). Though than one taxon; ribotype E was recognized in as almost all species are diploids in Citrus (2n = many as 12 taxa. 18; Iwamasa 1976), their clonal propagation by apomictic development of nucellar embryos is Discussion observed in a major part of Citrus (Spiegel-Roy Variation and heterogeneity of ITS ribotypes in and Goldschmidt 1996), which may stabilize Citrus and perpetuate hybrid taxa (Scora 1975), are Fifteen of 24 species and two varieties of presumed to have preserved their additive ITS Citrus examined in this study had more than sequences. one phylogenetically distinct ribotype. Ribotype polymorphism within individuals was estimated Putative parental species of Citrus cultivars to be generated as additivities between divergent In Citrus, species with non-additive lineages through gene flow and hybridization sequences probably consist of a single ribotype, (Malik et al. 1973, Scora 1975, Torres et al. and are thought to have developed not through 1978, Mizuno et al. 1991, Federici et al. 1998). hybridization but by selection. In this study, Li et al. (2010) also showed that C. limon, C. Citrus montana, C. sunki, C. deliciosa, C. jambhiri, C. bergamia, C. limonia, C. sinensis, amblycarpa, C. kinokuni, C. medica and C. C. paradisi and C. aurantium had multiple, medica var. sarcodactylis were examples of phylogenetically distinct ribotypes, and that these species. The sequence of C. sunki showed C. medica, C. macroptera, C. micrantha, C. a non-additive ITS sequence that was the 232 植物研究雑誌第 88 巻第 4 号 2013 年 8 月

>53 Glycosmis pentaphylla

>22 Murraya paniculata

>19 S winglea glutinosa

>2 >49 Feroniella lucida

>21 Atalantia monophylla 75 >10 S everinia buxifolia >9 4 E remocitrus glauca

>10 Microcitrus australasica 65

12 P oncirus trifoliata >3 1 4 ribotype A PPWS 1

71 6 Fortunella japonica A 4 65 1 3 3 Fortunella japonica B

5 ribotype B PPWS 2 >4 1

2 ribotype C Clade A Citrus sunki, ribotype D 85 2 2 2 MANDARIN Citrus deliciosa, ribotype E

1 Citrus kinokuni, ribotype F

5 Fortunella japonica C

1 ribotype G PPWS 3 97 >10 2 C itrus hystrix 90 C itrus montana 2 55 3 PAPEDA 1 ribotype H 55 3 1

2 C itrus macroptera

2 ribotype I PPWS 4 1 59 1 >1 ribotype J 89 >3 1 ribotype K PPWS 5 78 1 ribotype L Clade B 1 1 ribotype M 87 >3 ribotype N

94 3 1 ribotype O PUMMELO

1 ribotype P ribotype Q 2 5 change Citrus medica, ribotype R CITRON 38

Fig. 4. Phylogenetic relationships in Citrus and its allies based on MP analysis of ITS1, 5.8S, ITS2, and partial 26S of nuclear ribosomal DNA. The tree is one of 34 equally parsimonious trees of 406 steps (CI = 0.73; RI = 0.70). Bootstrap values are given above branches of the clades. Length and numbers below branches indicate character support for clades. Putative parental wild species (PPWS) estimated in this study are indicated on the right side. Two large clades (A, B) are indicated on the right brackets. August 2013 The Journal of Japanese Botany Vol. 88 No. 4 233

Phylogenetic position

Ribotype A BCDEFGHIJKLMNOP Q R

Putative Parental Intrageneric PPWS5 PPWS1 PPWS2 PPWS3 PPWS4

Wild species CITRON PAPEDA PUMMELO classification Sample MANDARIN Sect. Limonellus C. aurantifolia ○ ○ C. limotteoides ○ ○ ○ Sect. Citrophorum C. medica, C. medica var. sarcodactylis ○ C. limon 2 ○ ○ Sect. Cephalocitrus C. maxima 1 ○ ○ C. paradisi 1 ○ ○ ○ C. hassaku 1, 2, 3 ○ ○ ○ ○ Sect. Aurantium C. natsudaidai 2, 3, 4, 5 ○ ○ ○ C. aurantium 1, 2, 3, 4 ○ ○ ○ C. neo-aurantium ○ ○ ○ C. sinensis ○ ○ ○ C. sinensis var. brasiliensis 2 ○ ○ ○ ○ C. iyo 2 ○ ○ ○ Sect. Osmocitrus C. junos ○ ○ ○ ○ Sect. Acrumen C. unshiu 1, 2 ○ ○ ○ C. reticulata 1, 3 ○ ○ ○ C. deliciosa 1, 2, C. amblycarpa ○ C. kinokuni 1, 2, 3, 4 ○ C. sunki ○ Hybrid cultivars C. unshiu × reticulata ‘Siranuhi’ 2 ○ ○ ○ ○

Fig. 5. Ribotypes recognized in each sample. The phylogenetic position of each ribotype and corresponding putative wild species on the upper side refer to Fig. 4. *Tanaka (1954). same as that of ribotype D. Citrus deliciosa and considered to be of hybrid origin, and their C. amblycarpa had the same sequence as that ribotypes were thought to be derived from other of ribotype E, and the sequence of C. kinokuni non-additive PPWS. For example, C. sinensis was the same as that of ribotype F. In the same and C. limon had ribotype E as one of the way, C. medica and C. medica var. sarcodactylis PPWS, C. deliciosa, and were estimated to have had the same sequence as that of ribotype R. developed through hybridization as C. deliciosa Citrus maxima is a naturally distributed species or its closely related species as a parental species. and is thought to be a parental species of Citrus However, 14 of 18 ribotypes in Citrus were cultivars (Swingle and Reece 1967, Iwamasa recognized only in cloning analyses, and these 1999). This species contained two closely related ribotypes probably were also originally derived ribotypes, M and N, which were distinguished from other PPWS not examined in this study. only in one site. Therefore, the above six species In order to estimate the number of PPWS are probably putative parental wild species or involved in the formation of Citrus cultivars, their direct descendants. We refer to such species it is probably appropriate to count each clade hereafter as “PPWS” . rather than to count each ribotype. Because these In contrast, species with more than PPWS probably contain intraspecific variation one ribotype except C. maxima were as one of the wild species, C. tachibana (see 234 植物研究雑誌第 88 巻第 4 号 2013 年 8 月

Fig. 3). As this study examined 3 of 6 species in Papeda species. We call this PPWS “PAPEDA” subgenus Papeda, 9 of 10 species in subgenus hereafter. Citrus recognized by Swingle and Reece (1967) Ribotypes O, P, and Q formed a clade with in addition to almost all major Citrus cultivars, ribotypes M and N in C. maxima, and these five major ribotypes originated from PPWS were ribotypes were distinguished from each other expected to be recognized. by one or two substitutions, which suggested As mentioned above, mandarin (C. that they had derived from a single species. reticulata), citron (C. medica) and pummelo As it includes pummelo (C. maxima) we call (C. maxima) were regarded as main foundation this PPWS “PUMMELO” hereafter. Because species in previous studies (Scora 1975, Barrett C. maxima does not develop nucellar embryos and Rhodes 1976, Handa et al. 1986, Yamamoto (Iwamasa 1976), this species is presumed to et al. 1993). Papeda species is also estimated as a contain a comparatively large amount of genetic PPWS (Barkley et al. 2006, Li et al. 2010). variation. Citrus sunki, C. deliciosa, C. amblycarpa, The remaining seven ribotypes did not C. kinokuni, and ribotype C were supposed to coincide with PPWS estimated in precedent have originated from a single species because hypothesis. Ribotypes A, B, and G were thought they were different in a few sites and constituted to have originated from distinct wild species, a single clade (Fig. 5). These species were respectively (PPWS 1, 2 and 3), because they probably developed not through hybridization were discriminated from each other at several but by selection, and in each species nrDNA sites and did not form a clade. Ribotype B was variation was thought to be fixed into a single recognized only in C. sinensis var. brasiliensis ribotype. Therefore, they are probably as and its evident hybrid cultivar, ‘Shiranuhi’. intraspecific variation like one of the wild Ribotypes A and G were found only in C. junos. species, C. tachibana. This hypothesis also Ribotypes J, K, and L constituted a clade, and coincides with Swingle and Reece’s (1967) were discriminated at only three sites. Ribotype finding that all of the species were deposited J was recognized in C. limettioides, C. sinensis, into mandarin cultivars. Therefore, we call this C. sinensis var. brasiliensis, ribotypes K and L PPWS “MANDARIN” hereafter. Clade G were found in C. hassaku, C. unshiu, etc., and indicated in ITS phylogram of Li et al. (2010) these three ribotypes were supposed to have coincided with MANDARIN. Citrus tachibana derived from a single wild species (PPWS 5). was estimated to form a clade with these species, In contrast, ribotype I was recognized only in and to have possibly originated from the same C. natsudaidai, and was discriminated from PPWS: MANDARIN. ribotypes J, K, and L in six sites. As the bootstrap In contrast, ribotype R, which was value of clade ‘I–J’ was comparatively low (59%; recognized in C. medica, C. limon, etc., was Fig. 3), ribotype I was supposed to have derived phylogenetically distinct, which supported the from a distinct wild species (PPWS 4). hypothesis that citron (C. medica) originated If one wild species retained single or from one of the distinct wild species. Therefore, phylogenetically similar ribotypes, PPWS 1–5 we call this PPWS “CITRON” hereafter. are extant or existed in the past, and participated Ribotype H was included in a clade with in the development of Citrus cultivars though three species belonging to subgenus Papeda, they could not be identified from extant wild or though it was not identical to any species, and cultivated species examined in this study. was thought to have been derived from the other species in subgenus Papeda, or from other Effectiveness of ITS analysis for parental intraspecific variation of the three subgenus estimation of diploid hybrids between more than August 2013 The Journal of Japanese Botany Vol. 88 No. 4 235 two species analysis of the direct parental species, and C. unshiu Citrus unshiu × reticulata ‘Siranuhi’ is a probably shares a common parental species. recently made hybrid variety of C. unshiu × C. Citrus natsudaidai was estimated to be of hybrid sinensis ‘Kiyomi’ and C. reticulata (Matsumoto origin between MANDARIN (especially C. 2001), and contains four ribotypes, B, D, K kinokuni), PUMMELO, and unique putative and L. Among them, ribotype B and D were parental species, PPWS 4. shared with C. sinensis var. brasiliensis and Citrus sinensis is regarded as being of hybrid C. reticulata, respectively, and ribotypes origin between C. maxima and mandarin species K and L were both shared with C. unshiu, (Tanaka 1954, Iwamasa 1999). This study which supports the effectiveness of using showed that C. sinensis was probably derived ITS sequences to examine hybrid content for from a hybrid between MANDARIN (especially Citrus. Moreover, this result confirmed that C. deliciosa and C. amblycarpa), PPWS 5, and more than two ribotypes from discrete parental PUMMELO. For C. sinensis var. brasiliensis, species are maintained in diploid hybrid plants PPWS 2 also probably played a role. This result because the ITS sequences exist in nrDNA as conflicts with that of Barkley et al. (2006), which a tandem multicopy array at a chromosomal report that oranges (C. sinensis) had one of the locus of multiple loci (Baldwin et al. 1995). The lowest heterozygosity levels among the hybrid remaining three ribotypes; E, J, Q, which were groups, and no intraspecific polymorphism. recognized above the three parental species were Citrus iyo is expected as being of hybrid origin not detected possibly because of the probable between C. sinensis and mandarin species. In this limit of cloning analysis or of split of pairing study, ribotype E belonging to MANDARIN, L chromosome, or concerted evolution. to PPWS 5, and Q to PUMMELO were detected in C. iyo. Though ribotype E and Q were shared Origin of hybrid cultivars of Citrus with C. sinensis, ribotype belonging to PPWS 5 Citrus aurantifolia is referred to as being were different from C. sinensis, but same with of hybrid origin between subgenus Papeda C. unshiu. It is possible C. unshiu or common species or its allies and C. medica (Scora 1975, parental species participated for generating C. Torres et al. 1978, Nicolosi et al. 2000, Berkley iyo. et al. 2006). Our result clearly supported these Citrus junos was found to be composed of results, as C. aurantifolia was found to be of four ribotypes (A, C, E, G), and was estimated hybrid origin between PAPEDA (similar species to be a hybrid between PPWS 1, 3, and to subgenus Papeda ones) and CITRON (C. MANDARIN. Citrus unshiu was estimated medica). Citrus limon has been reported to be to be of hybrid origin between MANDARIN of hybrid origin between C. medica and C. (especially C. kinokuni) and PPWS 5. aurantifolia by Swingle and Reece (1967) and Citrus paradisi has been estimated to be Malik et al. (1973). In contrast, Torres et al. of hybrid origin between C. maxima and C. (1978) estimated this species as being of hybrid sinensis (Swingle and Reece 1967, Scora et al. origin between C. sinensis and C. aurantifolia. 1982). We showed that C. paradisi is composed We showed that C. limon is a hybrid between of MANDARIN (especially C. deliciosa and MANDARIN (C. deliciosa or its allies) and C. amblycarpa), and PUMMELO (ribotype CITRON. M in C. maxima and Q in C. sinensis, etc.). Citrus hassaku was estimated to be of hybrid Therefore, C. paradisi is possibly a hybrid origin between PPWS 5 and PUMMELO between C. sinensis and C. maxima, which (ribotype M, N in C. maxima). Therefore, for supports the results of precedent studies (Scora C. hassaku, C. maxima was estimated to be one et al. 1982), or is possibly a simple hybrid 236 植物研究雑誌第 88 巻第 4 号 2013 年 8 月 between MANDARIN and PUMMELO. Citrus C. T. 2006. Assessing genetic diversity and population aurantium and C. neo-aurantium had the same structure in a citrus germplasm collection utilizing simple sequence repeat markers (SSRs). Theor. Appl. ribotype contents, and were predicted to be Genet. 112: 1519–1531. hybrids between MANDARIN (especially C. Barrett H. C. and Rhodes A. M. 1976. A numerical deliciosa and C. amblycarpa) and PUMMELO. taxonomic study of affinity relationships in cultivated Citrus reticulata was estimated to be a hybrid Citrus and its close relatives. Syst. Bot. 1: 105–136. between MANDARIN and PUMMELO. Bayer R. J., Mabberley D. J., Morton C., Miller C. H., Sharma I. K., Pfeil B. E., Rich S., Hitchcock R. and Ribotypes D and E were shared with C. sunki Sykes S. 2009. A molecular phylogeny of the orange and C. deliciosa, respectively, and ribotype Q subfamily (Rutaceae: Aurantioideae) using nine was shared with C. paradisi, C. sinensis and C. cpDNA sequences. Amer. J. Bot. 96: 668–685. iyo, suggesting its complicated origin. Booy G., Schoot J. V. and Vosman B. 2000. Heterogeneity of the internal transcribed spacer 1 (ITS1) in Tulipa Citrus paradisi, C. aurantium, C. neo- (Liliaceae). Pl. Syst. Evol. 225: 29–41. aurantium, C. reticulata were all estimated to be Campbell C., Wojciechowski M. F., Baldwin B. G., Alice hybrid between MANDARIN and PUMMELO, L. A. and Donoghue M. J. 1997. Persistent nuclear however, they were different in ribotype ribosomal DNA sequence polymorphism in the components. It probably indicated that different Amelanchier agamic complex (Rosaceae). Mol. Biol. Evol. 14: 81–90. strains wish a single species played a role for Federici C. T., Fang D. Q., Scora R. W. and Roose M. L. generating these Citrus cultivars. 1998. 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山路弘樹 a，近藤健児 a，國賀武 b，根角博久 c，吉田俊雄 b，橋本和則 a，武田修己 a：核リボゾーム DNA 中の ITS 領域の組成により明らかとなったカンキツ類栽培種の起源カンキツ類 Citrus は世界で最も生産されている果物て，もしも 1 つのグループが 1 つの母種由来だとすると，の一つであり多数の品種を擁している．いままで多くの従来考えられてきた母種マンダリン，シトロン，パペダ，研究において，マンダリン，シトロン，ポメロ（ザボン），ポメロの 4 種に加えて，最大で 5 種の母種（推定母種 1 パペダの４種が栽培種の形成に関与した母種だと推定～ 5）が栽培種の起源に関わった可能性がある．されてきた．本研究では同仮説の検証，栽培カンキツ各交配育種の経緯が明らかな不知火（デコポン）は 4 種の起源解明を目的に，世界および日本で栽培されてい種のリボタイプをもち，交配親であるウンシュウミカる代表的な栽培種 15 種 2 変種，交配経緯が明らかな交ン，ポンカン，スイートオレンジそれぞれのリボタイプ配品種 1 種，野生種 8 種，Citrus 近縁属 9 種，合計 61 の一部を持っていた．ほとんどが 2 倍体種よりなるカサンプルについて，交配種の母種推定に有効な核リボゾンキツ類において，ITS リボタイプの組成を調査するこーム DNA 中の ITS 領域を調査した．このうち 27 サンとにより，3 種以上の交配親が関与した場合でも，そのプルについてはクローニング解析により，リボタイプの複雑な交雑経緯を推定するのに有効だった．これは ITS 構成を調査した．領域がタンデムリピートするマルチコピーよりなるた ITS 領域においてゲノム内多型がほとんど見られなかめと考えられる．ったのは栽培種の中ではチチュウカイマンダリン，キ各栽培種の ITS リボタイプ構成より，ライムはシトシュウミカン，スンキ，Citrus amblycarpa，シトロン，ロンとパペダの雑種由来であることが示され，同様にレザボンのみであり，これらは単一の野生種由来と推定さモンはシトロン＋マンダリン（特にチチュウカイマンダれた．これに対してレモン，スイートオレンジ，サワーリン），ハッサクは推定母種 5 ＋ポメロ，ナツミカンはオレンジ，ポンカンなど多くの栽培種では，多くの変異マンダリン（特にキシュウミカン）＋ポメロ＋推定母種サイトで多型を示し，クローニング解析で 2 ～ 4 種の 4 と示された．スイートオレンジはマンダリン＋推定母系統的に異なるリボタイプを併せ持つことが確認され種 5 ＋ポメロであり，ネーブルオレンジではさらに推た．合計 18 種のリボタイプが調査した種から見出され，定母種２が関与していた．イヨカンはマンダリン＋推定それらは系統的に 9 グループに分けられた．母種 5 ＋ポメロ，ユズは推定母種 1 ＋ 3 ＋マンダリンこのうち，チチュウカイマンダリン，キシュウミカンであり，ウンシュウミカンはマンダリン（特にキシュなどと同一ないし近縁な 4 リボタイプは従来の推定でウミカン）＋推定母種 5 由来と推定された．グレープの母種の一種「マンダリン」，シトロンと同一の 1 リボフルーツ，サワーオレンジ（ダイダイ類），ポンカンは，タイプは同じく「シトロン」，パペダ類に近縁な 1 リボいずれもマンダリン＋ポメロ由来と推定されたが，構成タイプは「パペダ」，ポメロ類と同一ないし近縁な 5 リリボタイプが異なることから，マンダリン，ポメロ中のボタイプは「ポメロ」に該当すると考えられた．残り 7 異なる系統を母種としていることが示唆された．リボタイプは系統的に 5 グループに分かれ，ITS のゲノ（a ツムラ生薬研究部，ム内多型を示さない種の中で，これらと同一ないし高い b 農研機構果樹研究所カンキツ研究興津拠点，類似度を示すものは本研究では見出されなかった．従っ c 農研機構近畿中国四国農業研究センター）