ISSN 1346-7565 Acta Phytotax. Geobot. 67 (3): 133–146 (2016) Acta Phytotaxonomica et Geobotanica doi: 10.18942/apg.201609 Editor-in-Chief TAMURA, Minoru N. (Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan) Hybrid Origin of the Apogamous Fern Dryopteris hondoensis Editors (Dryopteridaceae) EBIHARA, Atsushi (Tsukuba, Japan) NAIKI, Akiyo (Taketomi, Japan) 1,* 2 1 FUKUHARA, Tatsundo (Munakata, Japan) TAKAMIYA, Masayuki (Kumamoto, Japan) KIYOTAKA HORI , YASUYUKI WATANO AND NORIAKI MURAKAMI FUSE, Shizuka (Kyoto, Japan) TSUBOTA, Hiromi (Hiroshima, Japan) IKEDA, Hiroshi (Tokyo, Japan) WATANO, Yasuyuki (Chiba, Japan) KUROSAWA, Takahide (Fukushima, Japan) YONEKURA, Koji (Sendai, Japan) 1 Makino Herbarium, Tokyo Metropolitan University,1-1 Minamiosawa, Hachioji, Tokyo 192-0397, Japan. *[email protected] (author for correspondence); 2Department of Biology, Graduate School of Science, Editorial Board Chiba University, 1-33 Yayoi-cho, Inage, Chiba 263-8522, Japan AZUMA, Hiroshi (Kyoto, Japan) NISHIDA, Harufumi (Tokyo, Japan) BOUFFORD, David E. (Boston, U.S.A.) NISHIDA, Sachiko (Nagoya, Japan) We report that the triploid apogamous species Dryopteris hondoensis Koidz. shares its sequences with FUJII, Shinji (Okazaki, Japan) OHMURA, Yoshihito (Tsukuba, Japan) the D. erythrosora complex (D. erythrosora, D. caudipinna and D. koidzumiana) and D. chinensis at two KAWAKUBO, Nobumitsu (Gifu, Japan) PAK, Jae-hong (Taegu, Korea) nuclear markers (PgiC and GapCp). This is the first report of reticulate evolution between subg. Erythr- MURAKAMI, Noriaki (Hachioji, Japan) PENG, Ching-I (Taipei, Republic of China) ovariae sect. Erythrovariae, to which D. erythrosora, D. caudipinna and D. koidzumiana and D. hon- NAGAMASU, Hidetoshi (Kyoto, Japan) TAKAHASHI, Hideki (Sapporo, Japan) doensis belong, and sect. Aemulae of subg. Dryopteris, to which D. chinensis belongs. Apogamous spe- cies of Dryopteris may hybridize with distantly related species congeners. NAKADA, Takashi (Tsuruoka, Japan) TAN, Benito C. (Berkeley, U.S.A.) Key words: Dryopteridaceae, Dryopteris chinensis, Dryopteris caudipinna, GapCp, PgiC, rbcL, reticu- Acta Phytotaxonomica et Geobotanica (APG) is published in one volume, comprising three issues per late evolution year, by the Japanese Society for Plant Systematics. It is sent to all members of the society. APG is the continuation journal of the Societas Phytogeographica Kyoto, Japan. The journal is open to the fields of systematic botany, phytogeography and closely related disciplines. The Instructions to Authors are Apogamy, or agamospory in ferns, is a type of it extensive morphological and genetic variation available at the cover page 3. All manuscripts should be sent to Editor-in-Chief. asexual reproduction in which unreduced spores and often form species complexes in which mor- Application of admission for membership of the society should be addressed to the Treasurer, Dr. Ha- are formed. The resultant gametophytes produce phological species are difficult to distinguish be- jime Ikeda (Institute of Plant Science and Resources, Okayama University, Chuo 2-20-1, Kurashiki, sporophytes of the next generation without fertil- cause of their continuous variation (Watano & Okayama 710-0046, Japan) and other correspondences the Secretary, Dr. Takashi Shiga (Mathematical ization (Manton 1950). Apogamous reproduction Iwatsuki 1988, Suzuki & Iwatsuki 1990, Lin et al. and Natural Sciences, Faculty of Education, Niigata University, Ikarashi 2no-cho 8050, Niigata 950- is common in ferns. Approximately 10% of all 1995, Grusz et al. 2009, Hori et al. 2014). This is 2181, Japan). The annual fee is 5,000 Yen for Japanese members, 3,000 Yen for student members and species of ferns (Lovis 1977) and 13% of the taxa because apogamous species of ferns can hybrid- 3,000 Yen for foreign members resided in abroad. APG is available by subscription for 8,000 Yen per of Japanese pteridophytes with known mode of ize with other sexual species to produce apoga- year by the Treasurer. reproduction are reported to exhibit apogamous mously reproducible offspring (Lin et al. 1995, reproduction (Takamiya 1996). In apogamous Ebihara et al. 2012, Lee & Park 2013, Hori et al. Reprographic Reproduction outside Japan species, all offspring from a parent are expected 2014). Making a copy of this publication to be clonal with a small amount of genetic varia- Lee and Park (2013) recently attempted to as- Please obtain permission from the following Reproduction Rights Organizations (RROs) to which the copyright tion. Interestingly, Darnaedi et al. (1990) report- sess reticulate evolution in subg. Erythrovariae holder has consigned the management of the copyright regarding reprographic reproduction. ed that Dryopteris yakusilvicola Sa. Kurata, a sect. Variae sensu Fraser-Jenkins (1986; the Obtaining permission to quote, reproduce; translate, etc. triploid apogamous species of recent hybrid ori- Dryopteris varia complex) in Korea. They re- Please contact the Secretary of the society. gin, showed no allozyme variation within the 56 ported that D. sacrosancta (subg. Erythrovariae, Users in countries and regions where there is a local RRO under bilateral contact with Japan Academic Associa- individuals they examined. They considered D. sect. Variae) and D. chinensis (subg. Dryopteris, tion for Copyright Clearance (JAACC). yakusilvicola to have originated from a single hy- sect. Aemulae Fraser-Jenk.) shared the same chlo- Users in countries and regions of which RROs are listed on the following website are requested to contact the re- brid event between the tetraploid sexual species roplast DNA (cpDNA) haplotype. Subsequently, spective RROs directly to obtain permission. D. sparsa and the diploid sexual species D. sa- Hori et al. (2014) reported triploid apogamous in- baei. dividuals of the D. varia complex to have three Japan Academic Association for Copyright Clearance (JAACC) Address 9-6-41 Akasaka, Minato-ku, Tokyo 107-0052 Japan This phenomenon, however, is not common. PgiC sequences, which can be considered homeo- Website http://www.jaacc.jp/ Despite the clonal nature of apogamous repro- logs, on homeologous chromosomes, but not on E-mail [email protected] Fax: +81-3-3475-5619 duction, many apogamous species of ferns exhib- homologous chromosomes. They reported that D. 134 Acta Phytotax. Geobot. Vol. 67 chinensis and Dryopteris sacrosancta and D. ko- mode of several individuals in the samples was bayashii, which Serizawa (2009) separated from estimated by counting the number of spores per D. sacrosancta s.l., shared the same nuclear PgiC sporangium (32, apogamous; 64, sexual) accord- sequences. The data suggest that apogamous D. ing to Manton (1950). All voucher specimens are sacrosancta s.s. and D. kobayashii are hybrids deposited in MAK and/or TNS. between the D. varia complex and D. chinensis, which are distantly related based on the cpDNA Ploidy analyses tree (Ebihara 2011, Hori et al. 2014). Ploidy analyses followed the methods of Hori Furthermore, Hori et al. (2014) reported that et al. (2014). To determine ploidy level, the DNA Dryopteris pacifica (subg. Erythrovariae, sect. content (2C value) of each nucleus extracted from Variae) and the D. erythrosora complex (subg. fresh pinnae was measured once per sample by Erythrovariae, sect. Erythrovariae) also shared flow cytometry using a Cyflow Ploidy Analyzer the same PgiC sequences, suggesting that hybrid- PA-II (Partec, Munster, Germany) and a Cystain ization with members of the D. erythrosora com- UV Precise P kit (Partec). Approximately 100 plex contributed to the diversification of the mm2 of each pinna was torn into several pieces apogamous species of the D. varia complex. and finely chopped with a razor blade in 0.25 mL Thus, these results indicate that phylogenetically of nucleus extraction buffer from the kit. Then, wider taxon sampling is necessary to understand 0.8 mL of staining solution from the kit was add- reticulate evolution in apogamous ferns. ed to the chopped tissues. The crushed tissue and Dryopteris hondoensis Koidz is a triploid buffers were filtered through a 30-μm nylon mesh apogamous species of sect. Erythrovariae, dis- (Partec) before analysis. Approximately 25 mm2 tributed in China, Korea and Japan (Honshu, Shi- of fresh leaf tissues of Nicotiana tabacum L. (2C koku, Kyushu). It is distinguished from other value = 11.71 pg., Narayan 1987) was used as an members section Erythrovariae by its yellowish internal standard. green, dull lamina and mostly plane scales on the rachis of the pinnae (Iwatsuki 1995). In this Molecular analyses study, we determined D. hondoensis to be com- For molecular analyses, a small amount of posed of nuclear DNA sequences from the D. leaf tissue was dried in plastic bags, 20 cm × 10 erythrosora complex (D. erythrosora, D. caudip- cm, using silica gel. Subsequently, total DNA was inna and D. koidzumiana) of sect. Erythrovariae extracted from the dried leaves using cetyltri- and those from D. chinensis of sect. Aemulae. methylammonium bromide solution according to This is the first report of reticulate evolution be- the method of Doyle & Doyle (1987). tween subg. Erythrovariae sect. Erythrovariae The plastid gene rbcL was used in this study and subg. Dryopteris sect. Aemulae. as the cpDNA marker. Polymerase chain reaction (PCR) amplification ofrbcL was performed using the primers aF and cR of Hasebe et al. (1994). Materials and Methods PCR entailed an initial denaturation step at 95°C for 10 min; followed by 35 cycles of denaturation, annealing,