HYBRID ORIGINS of IXERIS NAKAZONEI (ASTERACEAE) Botanical Journal of the Linnean Society, 2003, 141, 379–387

Blackwell Science, LtdOxford, UKBOJBotanical Journal of the Linnean Society0024-4074The Lin- nean Society of London, 2003 141

Original Article

HYBRID ORIGINS OF IXERIS NAKAZONEI (ASTERACEAE) Botanical Journal of the Linnean Society, 2003, 141, 379–387. With 5 figures T. DENDA and M. YOKOTA

Hybrid origins of Ixeris nakazonei (Asteraceae, Lactuceae) in the Ryukyu Archipelago, Japan: evidence from molecular data

TETSUO DENDA* and MASATSUGU YOKOTA

Laboratory of Ecology and Systematics, Faculty of Science, University of the Ryukyus, Senbaru 1, Downloaded from https://academic.oup.com/botlinnean/article/141/3/379/2433721 by guest on 01 October 2021 Nishihara, Okinawa 903–0213, Japan

Received August 2002; accepted for publication November 2002

The proposed hybrid origin of Ixeris nakazonei collected in the Ryukyu Archipelago (Okinawajima) and the Atsumi Peninsula (Koijigahama) of central Japan was examined using nuclear DNA (nDNA) and chloroplast DNA (cpDNA) markers. The results indicated that most tetraploids of I. nakazonei (2n = 32) had nDNA of both putative parents, I. debilis (2n = 48) and I. repens (2n = 16). The ﬁrst tetraploid of I. nakazonei must have arisen directly from inter- speciﬁc hybridization between I. debilis and I. repens, considering the intermediacy of the chromosome number. All tetraploids on Okinawajima had cpDNA of I. repens, while those in the Atsumi Peninsula had cpDNA of I. debilis, indicating that the tetraploid has at least two independent origins. All hexaploids of I. nakazonei (2n = 48), on the other hand, had cpDNA of I. debilis, but some of the hexaploids had nDNA of both putative parents. Ixeris debilis and I. repens may also be involved in the origin of the hexaploid, although the establishment process of this cytotype is still obscure. Since the hexaploid of I. nakazonei is cytologically indistinguishable from I. debilis, it can backcross with I. debilis. Introgression from diploid I. repens to hexaploid I. debilis mediated by I. nakazonei possibly occurs on Okinawajima. © 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 141, 379– 387.

ADDITIONAL KEYWORDS: chromosome number – introgression – multiple origins – molecular marker – natural hybridization – polyploidy – secondary contact.

INTRODUCTION islands lying between the Japan Archipelago and Taiwan, this species locally occurs in the Atsumi Pen- Natural hybridization appears to have occurred in insula of central Japan (Denda & Yokota, 2000). numerous plant groups and is widely recognized as a Kitamura (1942) inferred that I. nakazonei arose significant evolutionary force leading to the formation from interspecific hybridization between I. debilis A. of new taxa (e.g. Arnold, 1992). In the genus Ixeris Gray and I. repens A. Gray based on their morpholog- (Asteraceae, Lactuceae), which comprises approxi- ical characteristics. Ixeris repens is a typical maritime mately 20 species distributed in the far east to south- plant widely distributed from Kamchatka through eastern Asia, several natural hybrid taxa have been Japan to Indochina. This species is clearly distin- reported (Kitamura, 1942). Ixeris nakazonei guished from other Ixeris by its palmate radical (Kitamura) Kitamura is one of the examples that have leaves, long vegetative underground stems, short beak a putative hybrid origin. Although I. nakazonei was of achene, and characteristic large involucral size. On believed to be endemic to the Ryukyu Archipelago, the other hand, I. debilis, distributed from Japan to which comprises approximately 200 subtropical Korea and southern China, is distinguished from I. repens by its long petiolate radical leaves, relatively long achene beak, and small involucral size. This species is usually found as a common weed of inland hab- *Corresponding author. E-mail: [email protected] itat, such as along footpaths between paddy fields, and

380 T. DENDA and M. YOKOTA thus it is ecologically isolated from I. repens to some due to gene conversion and/or backcrossing to one of extent. Ixeris debilis, however, often grows on the the parents, we investigated the pollen stainability as coastal dunes in the Ryukyu Archipelago and often an useful index to evaluate the fertility of I. nakazonei encounters I. repens in that habitat. Ixeris nakazonei, as well as that of I. debilis and I. repens. On the other usually found growing among both of the putative par- hand, chloroplast restriction site variation between ents on the coastal dunes, is characterized by its irreg- I. debilis and I. repens had been previously character- ularly pinnatifid leaves, long vegetative underground ized in the intergenic spacer between atpB and rbcL of stems, long achene beak, and relatively large imbri- cpDNA digested with the restriction endonuclease cate outer involucral bracts. The gross morphology of MboI (Pak & Ito, pers. comm.). Chloroplast DNA I. nakazonei is intermediate between that of I. debilis markers are useful in determining the parentage of and I. repens. hybridization because they are generally maternally Cytological study partly supports the putative inherited. The aim of this study is to verify the pro- hybrid origin of I. nakazonei. Ixeris debilis is hexap- posed hybrid origin of I. nakazonei using both nuclear Downloaded from https://academic.oup.com/botlinnean/article/141/3/379/2433721 by guest on 01 October 2021 loid with 2n = 48, and I. repens is diploid with 2n = 16 and chloroplast DNA markers. (Pak & Kawano, 1990). Ixeris nakazonei has the same basic chromosome number of x = 8, but intraspecific chromosome number variation, 2n = 32 (tetraploid), MATERIAL AND METHODS 40 (pentaploid) and 48 (hexaploid), has been found in some populations (Denda & Yokota, 1999). All these PLANT MATERIAL cytotypes occur in Okinawajima, the largest island in Since we were unable to obtain the pentaploid because the Ryukyu Archipelago, but only the tetraploid was of rareness, only the tetraploid and hexaploid of found in the Atsumi Peninsula (Denda & Yokota, 1999, I. nakazonei as well as the putative parents were used 2000). The tetraploid of I. nakazonei always occurs in the present study. In total, 187 plants composed of intermingling with I. debilis and I. repens, and the 24 tetraploids and 20 hexaploids of I. nakazonei from chromosome number of 2n = 32 is numerically inter- four localities (Hentona, Higashiemae, Ikei and mediate between those of the putative parents. The Komesu) on Okinawajima in the Ryukyu Archipelago, tetraploid of I. nakazonei is therefore believed to have eight tetraploids of I. nakazonei from one locality arisen from hybridization between I. debilis and (Koijigahama) in the Atsumi Peninsula of central I. repens, or I. repens and a hexaploid of I. nakazonei Japan, 79 plants of I. debilis from 27 localities, and 56 (Denda & Yokota, 1999, 2000). It is difficult, however, plants of I. repens from 21 localities were collected and to clarify the relationship between I. nakazonei and its analysed (Fig. 1, Appendix). In four localities on Oki- putative parents based only on cytological features nawajima as well as in one locality of the Atsumi because of the relatively small chromosome size and Peninsula, I. nakazonei occurred sympatrically with similar karyotypes. Consequently, many questions I. debilis and I. repens, and it was distinguished from remain regarding the origin and establishment of both of the putative parents based on its irregularly I. nakazonei. pinnatifid leaves (Fig. 2). The elucidation of the hybrid origin hypothesis has been aided by the ability to make comparisons between the putative parents and their derivatives at NUCLEAR DNA ANALYSIS the molecular level. Both nuclear DNA (nDNA) and Total DNAs were isolated from fresh leaves by CTAB chloroplast DNA (cpDNA) markers have yielded method (Doyle & Doyle, 1987). The IGS region was important insights concerning hybridization in plants. amplified first by long-distance PCR using primers Various kinds of nDNA markers have been used to 26S-IGS and 18S-IGS, whose sequences were homolo- analyse plant hybrid systems. The external tran- gous to portions of 26S and 18S nrDNA (Baldwin & scribed spacer (ETS), which is located at the 3¢ portion Markos, 1998). PCR mixture contained the following of intergenic spacer (IGS) between 26S and 18S reagents: 72 mL of distilled H2O, 10 mL of 10X PCR nuclear ribosomal DNA (nrDNA), appears to be more buffer, 10 mL of 2 mM dNTPs, 2 mL of 4 pM solution of variable and phylogenetically informative than the each primer, 1 mL of KOD Dash (Toyobo), and 2 mL of internal transcribed spacer (ITS) region at lower tax- total DNA template. Thermal cycle consisted of 25 onomic levels in Asteraceae (Linder et al., 2000). In cycles of 30 s at 94∞C and 8 min at 72∞C. To construct the present study, nDNA markers diagnostic for the PCR primers of ETS region, PCR products of the I. debilis or I. repens were screened using polymerase IGS region were partially sequenced using primer ETS- chain reaction–restriction fragment length polymor- Hel-1 designed by Baldwin & Markos (1998). Based phism (PCR–RFLP) analysis of the ETS region. Since on these primary sequence data, two PCR primers, the parental nrDNAs in fertile hybrid taxa are occa- ETS-F2 (5¢-ATTCGTAACACGTTGCTTGCGACG-3¢) sionally homogenized among successive generations and 18S-R1 (5¢-GCAGGATCAACCAGGTAGCA-3¢),

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Figure 1. Collection localities of Ixeris nakazonei (solid triangles), I. debilis (solid circles) and I. repens (open circles). Detailed localities are given in the Appendix. were synthesized, and the 3¢ portion of the ETS region ium bromide, and restriction site variation was was amplified (Fig. 3). PCR mixture contained the fol- detected under ultraviolet light. lowing reagents: 73.5 mL of distilled H2O; 10 mL of 10X PCR buffer; 10 mL of 2 mM dNTPs; 2 mL of 4 pM solution of each primer; 1 mL of rTaq polymerase (Toyobo); CHLOROPLAST DNA ANALYSIS and 0.5 mL of PCR product from amplification of the For PCR amplification of the intergenic spacer IGS region. Thermal cycle consisted of 20 cycles of 1 s between atpB and rbcL of cpDNA, we designed prim- at 94∞C, 1 s at 60∞C, and 1 s at 72∞C. Hereafter, the 3¢ ers that have homologous sequences to portions of the portion of the ETS region amplified by PCR is simply atpB and rbcL genes. The sequence of atpB primer is designated as the ETS region for convenience. 5¢-CCCTCTGTAGCACTCATAGC-3¢ and that of rbcL PCR products of the ETS region were cleaned using primer is 5¢-GAGTTACTCGGAATGCTGCC-3¢. PCR QIAquick PCR Purification Kit (Qiagen) following rec- mixture contained the following reagents: 62 mL of dis- ommendations of the manufacturer. Purity of the PCR tilled H2O; 12 mL of 25 mM MgCl2; 10 mL of 10X PCR products were checked by electrophoresing 5 mL on 4% buffer; 10 mL of 2 mM dNTPs; 2 mL of 4 pM solution of polyacrylamide gel and staining with DNA silver each primer; 1 mL of rTaq polymerase (Toyobo); and staining kit (Atto). The remaining PCR products were 1 mL of total DNA template. Thermal cycle consisted of digested with the restriction enzyme EcoRV. Digested 25 cycles of 1 min at 94∞C, 1min at 55∞C, and 2 min at PCR products were electrophoresed on 4% NuSieve 72∞C. PCR products were concentrated to 10 mL by GTG agarose gel (FMC). Gels were stained with ethid- EtOH precipitation and digested using the restriction

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Figure 2. Ixeris nakazonei, I. debilis and I. repens in Komesu on Okinawajima. (A) I. nakazonei (tetraploid) with characteristic irregularly pinnatiﬁd leaves. (B) Sympatric occurrence of I. debilis (above) and I. repens (below). Scale bars = 2 cm.

Figure 3. PCR amplified region of the ETS region and relative position of PCR primers used in this study. enzyme MboI. MboI digests of the intergenic spacer each plant, and more than 1000 pollen grains were between atpB and rbcL were separated on a 2% aga- counted for calculating the percentage of normal pol- rose gel. The gels were stained with ethidium bromide len grain. Fully stained pollen grains are not neces- and viewed under UV light. sarily fertile, and the pollen stainability does not mean the actual fertility in the strict sense. However, the percentage of fully stained pollen grains should be POLLEN STAINABILITY proportional to that of the actual pollen fertility. Thus, Tetraploid and hexaploid plants of I. nakazonei as well we did not attempt germination tests in the present as I. debilis and I. repens were collected from nine study. localities on Okinawajima for an observation of pollen stainability. Mature pollen grains were harvested from flower-heads in full bloom and fixed in 70% eth- RESULTS anol. Pollen grains were stained following the procedure of Alexander (1980) and classified into two NUCLEAR DNA ANALYSIS categories: (1) normal, nuclei and cytoplasm were fully PCR products of the ETS region were approximately stained; or (2) abnormal, nuclei and cytoplasm were 270 base pairs (bp) in length, and neither inter- nor poorly stained or the pollen grain content was low or intraspecific length variations were detected by 4% non-existent. At least two slides were prepared for polyacrylamide gel electrophoresis (data not shown).

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Figure 4. EcoRV digests of the ETS region for Ixeris repens (lane 1: Sesoko, lane 12: Komesu), I. debilis (lane 2: Sannai, lane 11: Kobata), and tetraploid (lane 3: Hentona, lanes 4 and 5: Komesu, lane 6: Koijigahama) and hexaploid (lanes 7 and 8: Hentona, lanes 9 and 10: Komesu) of I. nakazonei electrophoresed on 4% NuSieve agarose gel. ‘M’ indicates the 100 bp DNA ladder as a molecular size marker.

The ETS region was digested into two fragments of c. apparently derived from the 860 bp fragment (Fig. 5, 160 bp and 110 bp in all plants of I. repens (Fig. 4, lanes 3 and 12). Since no intraspecific restriction site lanes 1 and 12), while for all plants of I. debilis inves- variation was found in each of I. debilis and I. repens, tigated the ETS region was not visibly digestable with the 720 bp and 140 bp fragments of I. debilis and the EcoRV (Fig. 4, lanes 2 and 11). The 270 bp fragment of 860 bp fragment of I. repens were used as cpDNA I. debilis and the 160 bp and 110 bp fragments of markers that were diagnostic for each species. I. repens were consistent in all individuals of each spe- All tetraploids of I. nakazonei collected on Okinawa- cies, and therefore used as nDNA markers that were jima possessed cpDNA marker of I. repens (Fig. 5, diagnostic for each species. lanes 4–6). In contrast, those tetraploids collected in For I. nakazonei, 28 of the 32 tetraploids and 11 of the Atsumi Peninsula clearly had cpDNA markers of the 20 hexaploids collected on Okinawajima, and all I. debilis (Fig. 5, lane 7). Hexaploids of I. nakazonei tetraploids collected in the Atsumi Peninsula had investigated in this study all had cpDNA markers of nDNA markers of both I. debilis and I. repens (Fig. 4, I. debilis (Fig. 5, lanes 8–11). lanes 3, 5, 6, 8 and 10). They clearly possessed the 160 bp and 110 bp fragments from I. repens together with the 270 bp fragment from I. debilis. The remain- POLLEN STAINABILITY ing tetraploids and hexaploids of I. nakazonei pos- The average pollen stainability in I. debilis, I. repens sessed only the nDNA marker of I. debilis or possessed and hexaploids of I. nakazonei was more than 90% the nDNA marker of both putative parents but that of (Table 1). While average stainability in tetraploids of I. repens was only weakly recognized (Fig. 4, lanes 4, 7 I. nakazonei (c. 65%) was lower than the other three and 9). We did not find tetraploids and hexaploids that taxa, a high stainability (more than 80%) was had only the nDNA marker of I. repens. observed in some tetraploids (Table 1). A tetraploid plant collected from Hentona on Okinawajima showed a particularly high pollen stainability of 93.2%. CHLOROPLAST DNA ANALYSIS The PCR product of the intergenic spacer region between atpB and rbcL was approximately 1180 bp in DISCUSSION length (Fig. 5, lanes 1 and 14), and neither inter- nor intraspecific length variations were found by agarose HYBRID ORIGINS OF THE TETRAPLOID gel electrophoresis. The PCR product of I. repens was PCR–RFLP analysis of the ETS region indicates that digested into two fragments, 860 bp and 320 bp the majority of tetraploids of I. nakazonei have nDNA (Fig. 5, lanes 2 and 13). Three digested fragments of both I. debilis and I. repens. Considering the chromo- were present following restriction analysis of some number intermediacy (Denda & Yokota, 1999) in I. debilis, however, including the 320 bp fragment addition to the results of the molecular analysis found in I. repens plus 720 bp and 140 bp fragments presented here, the tetraploid of I. nakazonei must

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Figure 5. MboI digests of the intergenic spacer region between atpB and rbcL for Ixeris repens (lane 2: Matsumoto, lane 13: Komesu), I. debilis (lane 3: Sannai, lane 12: Hioki), and tetraploid (lane 4: Hentona, lanes 5 and 6: Komesu, lane 7: Koijigahama) and hexaploid (lanes 8 and 9: Hentona, lanes 10 and 11: Komesu) of I. nakazonei electrophoresed on 2% agarose gel. Lanes 1 and 14 indicate the undigested PCR product of the intergenic spacer region between atpB and rbcL, and ‘M’ indicates the 100 bp DNA ladder as a molecular size marker.

Table 1. Pollen stainability of I. nakazonei and its puta- and I. repens, some tetraploids of I. nakazonei have tive parents high pollen stainability without amphidiploidization. Indeed, we occasionally observed mature fruit of the Normal pollen (%) No. of tetraploid in the field. Recombination among succes- individuals sive generations of the tetraploid may rearrange Taxa Average (SD)* Max. Min. investigated chromosomal segments, resulting in at least partial fertility of this cytotype. This is also consistent with Ixeris nakazonei the result that some tetraploids only have nDNA of tetraploid 65.0 (18.3) 93.2 34.9 28 I. debilis, although they are morphologically indistin- hexaploid 92.4 (11.6) 99.1 75.1 20 guishable from the other tetraploids that have nDNA I. debilis 95.9 (3.4) 99.8 90.8 20 of both parents. In some hybrid taxa, concerted evolution, via gene conversion or unequal crossing-over, can I. repens 94.5 (6.9) 99.4 72.0 34 homogenize different parental genomes, and the result may be that only one parental genome may be *SD: standard deviation seen in the hybrid (Hillis et al., 1991; Wendel, Schnabel & Seelanan, 1995). The nDNA of I. repens, which was initially involved in the tetraploid of have originated from interspecific hybridization I. nakazonei, is likely to be homogenized and nearly between I. debilis and I. repens. This interpretation is replaced by that of I. debilis through recombination consistent with the previous observation that the tet- among successive generations of the tetraploid raploid occurs only in the sympatric localities of cytotype. I. debilis and I. repens (Denda & Yokota, 1999, 2000). PCR–RFLP analysis of cpDNA provides further Polyploidy is in general induced by somatic chromo- information about parentage of the hybridization. The some doubling or union of unreduced gametes (e.g. tetraploid on Okinawajima has cpDNA of I. repens, Ramsey & Schemske, 1998). In the case of while that in the Atsumi Peninsula has cpDNA of I. nakazonei, however, the tetraploid has directly I. debilis. Since cpDNA is in general maternally inher- arisen from the union of reduced gametes of hexaploid ited, the tetraploid on Okinawajima was derived I. debilis and diploid I. repens. As a result, the first through hybridization with I. repens functioning as produced tetraploid of I. nakazonei would be expected the maternal parent. In contrast, in the Atsumi Pen- to have the chromosomes of I. debilis and I. repens in insula, I. debilis must have been pollinated by the proportion of three to one, respectively, since the I. repens at the hybridization event. The tetraploid of former is hexaploid and the latter is diploid. Despite I. nakazonei has at least two independent origins. this disproportion of chromosomal composition, in Ixeris debilis often dominates and intermingles with addition to the genome difference between I. debilis I. repens in the coastal dunes on Okinawajima, while

© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 141, 379–387 HYBRID ORIGINS OF IXERIS NAKAZONEI (ASTERACEAE) 385 in Koijigahama of the Atsumi Peninsula, only a few tion is refuted by the fact that hexaploids of plants of I. debilis are found on the margin of a mari- I. nakazonei investigated in the present study all have time forest adjacent to a large population of I. repens cpDNA of I. debilis, despite the fact that all tetra- spreading on the coastal dunes (Denda & Yokota, ploids on Okinawajima have cpDNA of I. repens. Con- 2000). It is possible that since there is a greater supply sequently, the establishment process of hexaploid of of pollen from the dominant species, it will preferen- I. nakazonei is still obscure, and further study is tially act as the paternal parent at the hybridization needed to clarify the origin of this cytotype. event from which the tetraploid was derived. In the present study, the hexaploid of I. nakazonei Multiple origins of polyploid taxa have been was distinguished from I. debilis based on its irregu- reported in many plant groups such as Elatostema larly pinnatifid leaves, following Kitamura’s descrip- (Tamaki, Denda & Yokota, 2001), Heuchera (Segraves tion (Kitamura, 1942). However, morphological et al., 1999), Senecio (Ashton & Abbott, 1992) and intermediacy often exists between these two taxa. Tragopogon (Soltis & Soltis, 1989; Cook et al., 1998). Since the hexaploid of I. nakazonei has normal pollen Downloaded from https://academic.oup.com/botlinnean/article/141/3/379/2433721 by guest on 01 October 2021 Of these examples, the allotetraploid Tragopogon spe- stainability and is cytologically similar to I. debilis, cies represent remarkable examples of recurrent for- having the same chromosome number of 2n = 48 and mation on a local scale (Soltis & Soltis, 1989, 1991). the same karyotypes (Denda & Yokota, 1999), the Considering the fact that the tetraploid of I. nakazonei hexaploid of I. nakazonei can freely backcross with always grows with its parental taxa in distant locali- sympatric I. debilis. As a result, the species boundary ties on Okinawajima (Denda & Yokota, 1999), recur- between I. nakazonei and I. debilis becomes somewhat rent formation on a small geographical scale may also obscure at the hexaploid level. The hexaploid of be applicable to the tetraploid on this island. Although I. nakazonei partly takes in the nDNA of I. repens, and cpDNA analysis is inconclusive in this regard because thus the backcross between the hexaploid of all tetraploids on Okinawajima have cpDNA of I. nakazonei and I. debilis may cause the introgression I. repens, further molecular study may show recurrent from the diploid I. repens to the hexaploid I. debilis formation of this cytotype on such a small island. over the cytological barrier. Introgression between taxa of different ploidy levels has been reported from several plant groups. In Vaccinium, genome exchange HYBRIDITY OF THE HEXAPLOID AND POSSIBLE easily occurs between the tetraploid taxa and sympa- INTROGRESSION tric diploid taxa via unreduced gametes (Qu, Hancock The hexaploid of I. nakazonei is cytologically indistin- & Whallon, 1998). The process of possible introgres- guishable from I. debilis (Denda & Yokota, 1999), but sion from I. repens to I. debilis must be rather com- some hexaploids also have nDNA of both I. debilis and plex, including tetraploid formation via interspecific I. repens. Ixeris debilis and I. repens are both involved hybridization between I. debilis and I. repens, and fol- in the origin of the hexaploid. It is not likely that, con- lowing hexaploid formation possibly in the tetraploid sidering their chromosome number, the hexaploid of population. In a previous survey (Denda & Yokota, I. nakazonei (2n = 48) has directly arisen from inter- 1999), I. debilis was found in nine of the ten localities specific hybridization between I. debilis (2n = 48) and investigated on Okinawajima. Ixeris debilis is often I. repens (2n = 16). It has been suggested that, within more dominant in the coastal dunes of the Ryukyu a polyploid population, the union of reduced and unre- Archipelago than is the typical maritime plant duced gametes sometimes generates a new cytotype of I. repens. Despite that, I. debilis prefers an inland higher ploidy level (Ramsey & Schemske, 1998). Auto- habitat in other areas of Japan. Introgression from hexaploid formation in the tetraploid populations has I. repens to I. debilis may have an effect on the alter- been reported from several plant taxa. For example, nation of habitat preference of I. debilis from inland to 2% of the progeny of autotetraploid Beta vulgaris coastal dunes in the Ryukyu Archipelago. Linn. was found to be of the hexaploid cytotype (Hornsey, 1973), and 1% hexaploid plants were recovered from the progeny of tetraploid Medicago SECONDARY CONTACT IN THE RYUKYU ARCHIPELAGO sativa Linn. (Bingham, 1968). Further, hexaploid It is noteworthy that the hybrid between I. debilis and progeny were recovered from crosses between the tet- I. repens is found only in the Ryukyu Archipelago raploid species Hieracium praealtum Gochn. and except for a rare example in the Atsumi Peninsula of H. caespitosum Dahlst. (Chapman & Bicknell, 2000). central Japan. Is there any reason why I. debilis Autohexaploid formation in tetraploid populations is comes into contact and crosses with I. repens in this not necessarily a rare event in flowering plants, and it area? Interspecific hybridizations between taxa hav- is possible that the hexaploid of I. nakazonei has ing different habitat preferences have been reported arisen from the fusion of reduced (2x) and unreduced from several plant taxa distributed in the Ryukyu (4x) gametes of the tetraploid. However, this presump- Archipelago; e.g. Pteris (Denda, Tomiyama & Yokota,

2001), Eurya and Vaccinium (Tateishi et al., 2001a,b). Denda T, Yokota M. 2000. A new locality of Ixeris nakazonei In the case of Pteris, P. ryukyuensis Tagawa usually (Asteraceae) from the Atsumi Peninsula, Aichi Pref., Japan. grows in alkaline soils in open locations, whereas Journal of Phytogeography and Taxonomy 48: 91–92 (in P. semipinnata Linn. prefers to grow in acidic soils in Japanese). more or less shaded habitats. The former species, how- Doyle JJ, Doyle JL. 1987. A rapid DNA isolation procedure ever, comes into contact with the latter in the central for small quantities of fresh tissue. Phytochemical Bulletin part of Okinawajima, where alkaline and acidic soils 19: 11–15. occur side by side. It seems likely that, in a small Hillis DM, Moritz C, Porter CA, Baker RJ. 1991. Evidence island such as Okinawajima, various habitats are for biased gene conversion in concerted evolution of ribosomal DNA. Science 251: 308–310. often contiguous like a miniature garden, giving rise Hornsey KG. 1973. The occurrence of hexaploid plants among to opportunities for secondary contact between taxa autotetraploid populations of sugar beet (Beta vulgaris L.), having different habitat preferences.

and the production of tetraploid progeny using a diploid pol- Downloaded from https://academic.oup.com/botlinnean/article/141/3/379/2433721 by guest on 01 October 2021 linator. Caryologia 26: 225–228. ACKNOWLEDGEMENTS Kitamura S. 1942. Expositiones Plantarum Novarum Orien- tali – Asiaticarum VII. Acta Phytotaxonomica et Geobotanica We wish to express our sincere gratitude to Dr Jae- 11: 120–133. Hong Pak of the Kyung-Pook National University, Linder CR, Goertzen LR, Heuvel BV, Francisco-Ortega Korea and Professor Motomi Ito of the University of J, Jansen RK. 2000. The complete external transcribed Tokyo, Japan for their kindness in providing us with spacer of 18S-26S rDNA: Amplification and phylogenetic the significant information about the cpDNA marker utility at low taxonomic levels in Asteraceae and closely of the genus Ixeris. This study was partly supported by allied families. Molecular Phylogenetics and Evolution 14: a Grant-in-Aid for Encouragement of Young Scientists 285–303. (no. 11740473) and for Scientific Research (no. Pak JH, Kawano S. 1990. Biosystematic studies on the genus 14540648) from the Japan Society for the Promotion of Ixeris (Compositae-Lactuceae) II. Karyological analyses. Science. Cytologia 55: 553–570. Qu L, Hancock JF, Whallon JH. 1998. Evolution in an auto- REFERENCES polyploid group displaying predominantly bivalent pairing at meiosis: genomic similarity of diploid Vaccinium darrowi Alexander MP. 1980. A versatile stain for pollen, fungi, yeast and autotetraploid V. corymbosum (Ericaceae). American and bacteria. Stain Technology 55: 13–18. Journal of Botany 85: 698–703. Arnold ML. 1992. Natural hybridization as an evolutionary Ramsey J, Schemske DW. 1998. Pathway, mechanisms, and process. Annual Review of Ecology and Systematics 23: 237– rates of polyploid formation in flowering plants. Annual 261. Review of Ecology and Systematics 29: 467–501. Ashton PA, Abbott RJ. 1992. Multiple origins and genetic Segraves KA, Thompson JN, Soltis PS, Soltis DE. 1999. diversity in the newly arisen allopolyploid species, Senecio Multiple origins of polyploidy and the geographic structure cambresis Rsser (Compositae). Heredity 68: 25–32. of Heuchera grassulariifolia. Molecular Ecology 8: 253–262. Baldwin BG, Markos S. 1998. Phylogenetic utility of the Soltis DE, Soltis PS. 1989. Allopolyploid speciation in Trago- external transcribed spacer (ETS) of 18S-26S rDNA: congru- pogon: Insights from chloroplast DNA. American Journal of ence of ETS and ITS trees of Calycadenia (Compositae). Botany 76: 1119–1124. Molecular Phylogenetics and Evolution 10: 449–463. Soltis PS, Soltis DE. 1991. Multiple origins of the allotetra- Bingham ET. 1968. Aneuploids in seedling populations of tet- ploid Tragopogon mirus (Compositae): rDNA evidence. Sys- raploid alfalfa, Medicago sativa. Crop Science 8: 571–574. tematic Botany 16: 407–413. Chapman H, Bicknell R. 2000. Recovery of a sexual and an Tamaki K, Denda T, Yokota M. 2001. Origin of triploids of apomictic hybrid from crosses between the facultative Elatostema suzukii (Urticaceae) on Okinawa Island, the apomicts Hieracium caespitosum and H. praealtum. New Ryukyus. Journal of Plant Research 114: 377–380. Zealand Journal of Ecology 24: 81–85. Tateishi Y, Yokota M, Shinjo K, Hiraiwa A, Niiro Y. 2001a. Cook LM, Soltis PS, Brunsfeld SJ, Soltis DE. 1998. Mul- Flora of Iwotorishima Island, the central Ryukyus and dis- tiple independent fromations of Tragopogon tetraploids persal modes of its components. Biological Magazine of Oki- (Asteraceae): evidence from RAPD markers. Molecular Ecol- nawa 39: 49–76 (in Japanese). ogy 7: 1293–1302. Tateishi Y, Yokota M, Shinjo K, Hiraiwa A, Niiro Y. Denda T, Tomiyama H, Yokota M. 2001. An intersectional 2001b. Taxonomically or phytogeographically noteworthy hybrid in the genus Pteris (Pteridaceae) on Okinawa Island: vascular plants from Iwotorishima Island, the central evidence from nuclear and chloroplast DNA. Acta Phytotax- Ryukyus. Biological Magazine of Okinawa 39: 77–92 (in onomica et Geobotanica 52: 159–165. Japanese). Denda T, Yokota M. 1999. A cytological study of Ixeris naka- Wendel JF, Schnabel A, Seelanan T. 1995. Bidirectional zonei (Asteraceae; Lactuceae) in Okinawa Island, the interlocus concerted evolution following allopolyploid speci- Ryukyus. Acta Phytotaxonomica et Geobotanica 50: 35–42. ation in cotton (Gossypium). Proceedings of the National

Academy of Sciences of the United States of America 92: 280– City, Hiroshima Pref., TD980144 (2); (16) Sendo, Mito 284. Town, Shimane Pref., TD980145 (3); (17) Himi, Saijo City, Ehime Pref., TD980115 (2); (18) Oono, Touyo City, Ehime Pref., TD980116 (2); (19) Sunouchi, APPENDIX Kawauchi Town, Ehime Pref., TD980114 (2); (20) Collection localities and voucher specimens of Hiranonohama, Shimoda, Nakamura City, Kochi I. nakazonei, I. debilis and I. repens are listed. Abbre- Pref., TD980100 (2); (21) Kamisaka, Toyotsu Town, viated names of each locality used throughout this Fukuoka Pref., TD000115(5); (22) Hioki, Shintomi paper are indicated by italics. Numbers in parenthe- Town, Miyazaki Pref., TD 980149(3); (23) Takae, Sen- ses indicate the number of individuals examined. At dai City, Kagoshima Pref., TD980152 (3); (24) Beppu, least one specimen for each locality is deposited in the Ei Town, Kagoshima Pref., TD980157 (2); (25) Yaka, herbarium of the University of the Ryukyus (RYU). Kin Town, Okinawa Pref., TD980143 (5); (26) Senbaru, Nishihara Town, Okinawa Pref., TD000120(2); (27) Downloaded from https://academic.oup.com/botlinnean/article/141/3/379/2433721 by guest on 01 October 2021 I. nakazonei: (1) Hentona, Kunigami Village, Oki- Nakama, Urasoe City, Okinawa Pref., TD000119(2). nawa Prefecture, tetraploid TD000117(2), hexaploid TD000118 (4); (2) Higashiemae, Ie Vil., Okinawa Pref., I. repens: (1) Hamadahama, Hamada, Akita City, tetraploid TD000128(3), hexaploid TD000129(1); (3) Akita Pref., TD990196(2); (2) Katsurahama, Hamada, Ikei, Katsuren Town, Okinawa Pref., tetraploid Akita City, Akita Pref., TD990197(1); (3) Iritahama, TD000124 (7), hexaploid TD000125 (10); (4) Komesu, Yosami, Shimoda City, Shizuoka Pref., TD990105 (2); Itoman City, Okinawa Pref., tetraploid TD000122(12), (4) Aikawa, Matsutou City, Ishikawa Pref., hexaploid TD000123 (5); (5) Koijigahama, the Atsumi TD980095(4); (5) Hamaji, Mikuni Town, Fukui Pref., Peninsula, Aichi Pref., TD000107 (8) TD980097(3); (6) Kumihara, Tahara Town, Aichi Pref., TD000105(3); (7) Koijigahama, Irago, Atsumi Town, I. debilis: (1) Sannai, Akita City, Akita Pref., Aichi Pref., TD000108 (2); (8) Shotenkyo, Kumihama TD990192 (2); (2) Katsurahama, Hamada, Akita City, Town, Kyoto Pref., TD980141(2); (9) Hashi, Goutsu Akita Pref., TD990193 (2); (3) Shirahama, Shimoda City, Shimane Pref., TD980147(2); (10) Sanrigahama, City, Shizuoka Pref., TD990101(2); (4) Tsukumohama, Kiami, Masuda City, Shimane Pref., TD980146(1); (11) Suzaki, Shimoda City, Shizuoka Pref., TD990102(2); Komuronohama, Kubokawa Town, Kochi Pref., (5) Ueda, Numazu City, Shizuoka Pref., TD990112 (2); TD980102(2); (12) Yokohama, Oogata Town, Kochi (6) Matsumoto, Matsuto City, Ishikawa Pref., Pref., TD980101(2); (13) Tomitahama, Shintomi Town, TD980096 (3); (7) Hamaji, Mikuni Town, Fukui Pref., Miyazaki Pref., TD980150(2); (14) Karahama, Ouzu, TD980098 (3); (8) Furuyaishizuka, Kanazu Town, Sendai City, Kagoshima Pref., TD980153(3); (15) Fuki- Fukui Pref., TD980099 (5); (9) Okubo, Tahara Town agehama, Imada, Fukiage Town, Kagoshima Pref., Aichi Pref., TD000112(5); (10) Hiinosekimon, Irago, TD980154(1); (16) Hentona, Kunigami Vil., Okinawa Atsumi Town, Aichi Pref., Yamashiro3859(1); (11) Pref., TD000116 (5); (17) Yonamine, Nakijin Vil., Oki- Koijigahama, Irago, Atsumi Town, Aichi Pref., nawa Pref., TD970047(4); (18) Higashiemae, Ie Vil., TD000109(4); (12) Kobata, Kobe City, Hyogo Pref., Okinawa Pref., TD970042 (2); (19) Sesoko, Motobu TD000127 (5); (13) Kande, Kobe City, Hyogo Pref., Town, Okinawa Pref., TD970046(5); (20) Ikei, Kat- TD000113(6); (14) Jyodo, Okayama City, Okayama suren Town, Okinawa Pref., TD000126 (2); (21) Pref., TD980148 (2); (15) Taguchi, Higashihiroshima Komesu, Itoman City, Okinawa Pref., TD000121(6).