T REPRO N DU LA C The International Journal of Plant Reproductive Biology 6(1) pp. 1-14, 2014 P T I F V O E
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S H T Hybrid Origin of Nephrolepis ×hippocrepicis Miyam. (Nephrolepidaceae)
Tzu-Tong Kao1, Wen-Liang Chiou1, Sheng-Yuan Hsu1, Chun-Ming Chen2, Yi-Shan Chao1 & Yao-Moan Huang 1*
1 Taiwan Forestry Research Institute, 53 Nan-Hai Rd., Taipei 10066, Taiwan
2Dr. Cecilia Koo Botanic Conservation Center, 31 Tongsing Rd., Gaoshu Township, Ping-Tung 90646, Taiwan
*e-mail: [email protected]
Received: 10. 05. 2013; Revised & Accepted: 23.06.2013; Published online: 01.10.2013
ABSTRACT
Plants of Nephrolepis ×hippocrepicis Miyam. found in the Taipei Botanical Garden, northern Taiwan, were characterized by mostly aborted and a few abnormally large-sized spores, closely- adjoining lanceolate triangulate pinnae, reniform indusia, and bi-colored basal scales with caudate apexes. These characters and C-value of N. ×hippocrepicis are intermediate between those of N. biserrata and N. cordifolia. Its bidirectional interspecific hybrid origins between N. biserrata and N. cordifolia have been identified based on the DNA sequences of a chloroplast region atpB-rbcL spacer and two nuclear regions CAS1 (cycloartenol synthase 1) and gapCp (glyceraldehydes-3-phosphate dehydrogenase) genes. Its spore germination rate was less than 1% and none of the gametophyte produced young sporophyte. Instead of sexual reproduction, the species is likely to propagate through runners and new hybridization events.
Keywords: Hybrid, Nephrolepis biserrata, Nephrolepis cordifolia, Nephrolepis ×hippocrepicis
INTRODUCTION brownii (Desv.) Hovenkamp & Miyam. (Knapp 2011). All of these are widely distributed in Taiwan. Some plants of Nephrolepis with abnormal spores were Nephrolepis Schott, a monophyletic genus of discovered on two adjacent palms Phoenix hanceana Nephrolepidaceae (Kramer 1990, Hennequin et al. Naudin and Butia capitata (Mart.) Becc. in 2004 at the 2010) is widely distributed in tropical regions. Species Taipei Botanical Garden (TPBG) (Fig. 1B). They are of this genus are characterized by articulate pinnae, both with morphology intermediate between N. biserrata and creeping and erect rhizomes, and reniform to lunulate N. cordifolia and identified as N. ×hippocrepicis indusia. The monophyly of this genus has been Miyam., a hybrid firstly reported at Ryukyu Island of determined by Hennequin et al. (2010). Japan (Hovenkamp & Miyamato 2005). In Taiwan, three Nephrolepis species recorded are N. This study aims to determine the hybrid origin of N. biserrata (Sw.) Schott; N. cordifolia (L.) C. Presl; and N. ×hippocrepicis at the TPBG by comparing their 2 The International Journal of Plant Reproductive Biology 6(1) pp.1-14, 2014 January, 6 (1) 2014 Hybrid origin of Nephrolepis ×hippocrepicis 3
Table 2 — Primers used to amplify and sequence DNA from members of the Nephrolepis in this study. morphology, C-value, and molecular data. Their atpB-1, rbcL-1, CAS1_2620F, CAS1_3523R, reproductive strategy was also studied. ESGAPCP8F1 and ESGAPCP11R1 (Table 2). The DNA region Primer Primer sequence Primer source primers for the region of CAS1 gene were newly atpB-rbcL atpB-1 5'-ACATCKARTACKGGACCAATAA-3' Chiang et al., 1998 MATERIAL & METHODS designed in this study. These three regions were amplified separately with standard polymerase chain atpB-rbcL rbcL-1 5'-AACACCAGCTTTRAATCCAA-3' Chiang et al., 1998 Plant materials were collected at the TPBG around reaction (PCR) using a T3 Thermocycler (Biometra, CAS1 CAS1_2620F 5'-AGGGCGAATGTGGTGTCAT-3' This study the two palms, No. 4420 (Phoenix hanceana Naudin) Göttingen) under the following conditions: (1) CAS1 CAS1_3523R 5'-ATCTTCATGCCATCTTCTGC-3' This study and No. 4428 (Butia capitata Becc.). Samples were denaturing 5 min at 95 °C; (2) denaturing, annealing, and gapCp ESGAPCP8F1 5'-ATYCCAAGYTCAACTGGTGCTGC-3' Schuettpelz et al., 2008 collected from different trees or different sides and extension 35 cycles; denaturing 45 s at 94 °C; annealing different heights of the same palm to avoid sampling the 45 s at 50 °C (atpB-rbcL), 54 °C (gapCp gene), or 56 °C gapCp ESGAPCP11R1 5'-GTATCCCCAYTCRTTGTCRTACC-3' Schuettpelz et al., 2008 same clone. The voucher specimens were deposited in (CAS1 gene); extension 75 s at 72 °C; (3) final extension the herbarium TAIF, Taiwan Forestry Research Institute 8 min at 72 °C. Some PCR products were sequenced hand. The cladistic analyses of sequencing data were Each of the three species, N. biserrata, N. (Table 1). Morphologies of N. ×hippocrepicis, N. directly, while others were cloned into pGEM-T Easy performed by the maximum parsimony (MP) and the ×hippocrepicis, and N. cordifolia, had ca. 64 spores per biserrata and N. cordifolia were examined and vectors (Promega, Madison). Ligation, transformation, neighbor-joining (NJ) method using PAUP* v. 4.0b10 sporangium (the abortive spores in N. ×hippocrepicis compared. Spore number per sporangium, spore plating and selection of clones followed the instructions (Swofford 2002). All characters were weighted equally were also counted). Germination percentages of their morphology, spore germination percentage, and C-value included with the kit. 30 clones in each sample were and gaps were treated as fifth base (MP) or pair-wise fresh mature spores were 96%, < 1%, and 97%, were determined following Huang et al. (2009) and screened using T7 and SP6 vector primers (Lunt et al. deletion (NJ). The most parsimonious trees were respectively. Only N. biserrata and N. cordifolia Chao et al. (2012). 1999). The plasmid DNAs from the 10 positive clones obtained with heuristic searches of 1000 replicates with Total genomic DNA was extracted using Plant produced sporophytes through their spore cultures. C- were purified using the High-Speed Plasmid Mini Kit random stepwise sequence addition, tree Genomic DNA Mini Kit (Geneaid Biotech Ltd.). A value of N. ×hippocrepicis fell between N. biserrata and (Geneaid, Taipei, Taiwan), and sequenced in an Applied bisection–reconnection (TBR) branch swapping, and chloroplast region atpB-rbcL spacer and two nuclear N. cordifolia (Fig. 6). Biosystems Model 3730 automated sequencer (Applied saving the best tree from each random sequence regions, cycloartenol synthase 1 (CAS1) gene and The two N. ×hippocrepicis populations in the Taipei Biosystems, Carlsbad, CA, USA). All sequences were addition. Bootstrap support values (BS) were calculated glyceraldehydes-3-phosphate dehydrogenase (gapCp) Botanical Garden had different chloroplast atpB-rbcL submitted to GenBank (Table 1) and aligned by BioEdit with 500 replicates. Neighbor-joining analyses were gene, were amplified and sequenced, using primers spacer sequences. The sequences of N. ×hippocrepicis v.7.1.3.0 (Hall, 1999), and the alignment was checked by conducted by calculating Kimura's (1980) 2-parameter plants on P. hanceana were identical with those of N. distance and BS were calculated with 1000 replicates. Table 1 — Information of vouchers in this study. biserrata while the sequences of those on B. capitata Nephrolepis brownii was used as the out group for were identical with N. cordifolia (Fig. 7A). The alleles of Voucher Host tree Host tree GenBank Acession Number phylogenetic analysis. nuclear CAS1 and gapCp genes belong to two different Name Specimen in TAIF* of TPBG** Number atpB.rbcL CAS1 gapCp groups in all sampling N. ×hippocrepicis while the other N. biserrata Y M Huang 1166 Elaeis guineensis Jacq. No. 5167 KF277238 KF277212 RESULTS three species, N. biserrata, N. cordifolia, and N. N. biserrata Y M Huang 1167 Elaeis guineensis Jacq. No. 4455 KF277239 KF277213 brownie, only had one. One group of the N. N. biserrata Y M Huang 1168 Elaeis guineensis Jacq. No. 4454 KF277240 KF277214 KF277227 Morphological comparisons among N. biserrata, N. ×hippocrepicis alleles was clustered with N. biserrata N. biserrata Y M Huang 1174 Phoenix hanceana Naudin No. 4420 KF277241 ×hippocrepicis, and N. cordifolia are summarized in and the other with N. cordifolia (Fig. 7B & C). N. brownii Y M Huang 1172 Phoenix dactylifera L. No. 5201 KF277250 KF277226 KF277237 Table 3 and illustrated in Figs. 2-5. Most characters of N. N. cordifolia Y M Huang 1169 Arenga pinnata (Wurmb) Merr. No. 5117 KF277247 KF277223 KF277236 ×hippocrepicis were intermediate between N. biserrata N. cordifolia Y M Huang 1170 Phoenix dactylifera L. No. 4425 KF277248 KF277224 DISCUSSION N. cordifolia Y M Huang 1171 Phoenix dactylifera L. No. 4425 KF277249 KF277225 and N. cordifolia, except its spores were the largest (if N. ×hippocrepicis Y M Huang 1173 Butia capitata (Mart.)Becc. No. 4428 KF277245 KF277215 KF277228 not aborted). Nephrolepis ×hippocrepicis could be Hybrid Origins of Nephrolepis ×hippocrepicis— KF277219 KF277232 distinguished from N. biserrata and N. cordifolia by According to the NJ trees of chloroplast atpB-rbcL N. ×hippocrepicis Y M Huang 1175 Phoenix hanceana Naudin No. 4420 KF277242 KF277216 KF277229 lanceolate triangulate pinnae (Fig. 2B & 3), reniform spacer sequences and nuclear genes of CAS1 and gapCp KF277220 KF277233 indusia (Fig. 1H), aborted spores (Fig. 4B), and bi- (Fig. 7B & C), Nephrolepis ×hippocrepicis was N. ×hippocrepicis Y M Huang 1176 Phoenix hanceana Naudin No. 4420 KF277243 KF277217 KF277230 colored basal scales with caudate apex (Fig. 5L & P). In originated from the bidirectional hybridization between KF277221 KF277234 addition, the pinnae of N. ×hippocrepicis are closely N. biserrata and N. cordifolia. The chloroplast atpB- N. ×hippocrepicis Y M Huang 1177 Phoenix hanceana Naudin No. 4420 KF277244 KF277218 KF277231 adjoining; the interspaces between adjacent pinnae are rbcL spacer sequences (Fig. 7A) indicate the different KF277222 KF277235 less than half pinna width (Fig. 1E). maternal lineages of N. ×hippocrepicis plants on the N. ×hippocrepicis Y M Huang 1178 Butia capitata (Mart.) Becc. No. 4428 KF277246 * TAIF: The herbarium of Taiwan Forestry Research Institute ** TPBG: Taipei Botanical Garden 2 The International Journal of Plant Reproductive Biology 6(1) pp.1-14, 2014 January, 6 (1) 2014 Hybrid origin of Nephrolepis ×hippocrepicis 3
Table 2 — Primers used to amplify and sequence DNA from members of the Nephrolepis in this study. morphology, C-value, and molecular data. Their atpB-1, rbcL-1, CAS1_2620F, CAS1_3523R, reproductive strategy was also studied. ESGAPCP8F1 and ESGAPCP11R1 (Table 2). The DNA region Primer Primer sequence Primer source primers for the region of CAS1 gene were newly atpB-rbcL atpB-1 5'-ACATCKARTACKGGACCAATAA-3' Chiang et al., 1998 MATERIAL & METHODS designed in this study. These three regions were amplified separately with standard polymerase chain atpB-rbcL rbcL-1 5'-AACACCAGCTTTRAATCCAA-3' Chiang et al., 1998 Plant materials were collected at the TPBG around reaction (PCR) using a T3 Thermocycler (Biometra, CAS1 CAS1_2620F 5'-AGGGCGAATGTGGTGTCAT-3' This study the two palms, No. 4420 (Phoenix hanceana Naudin) Göttingen) under the following conditions: (1) CAS1 CAS1_3523R 5'-ATCTTCATGCCATCTTCTGC-3' This study and No. 4428 (Butia capitata Becc.). Samples were denaturing 5 min at 95 °C; (2) denaturing, annealing, and gapCp ESGAPCP8F1 5'-ATYCCAAGYTCAACTGGTGCTGC-3' Schuettpelz et al., 2008 collected from different trees or different sides and extension 35 cycles; denaturing 45 s at 94 °C; annealing different heights of the same palm to avoid sampling the 45 s at 50 °C (atpB-rbcL), 54 °C (gapCp gene), or 56 °C gapCp ESGAPCP11R1 5'-GTATCCCCAYTCRTTGTCRTACC-3' Schuettpelz et al., 2008 same clone. The voucher specimens were deposited in (CAS1 gene); extension 75 s at 72 °C; (3) final extension the herbarium TAIF, Taiwan Forestry Research Institute 8 min at 72 °C. Some PCR products were sequenced hand. The cladistic analyses of sequencing data were Each of the three species, N. biserrata, N. (Table 1). Morphologies of N. ×hippocrepicis, N. directly, while others were cloned into pGEM-T Easy performed by the maximum parsimony (MP) and the ×hippocrepicis, and N. cordifolia, had ca. 64 spores per biserrata and N. cordifolia were examined and vectors (Promega, Madison). Ligation, transformation, neighbor-joining (NJ) method using PAUP* v. 4.0b10 sporangium (the abortive spores in N. ×hippocrepicis compared. Spore number per sporangium, spore plating and selection of clones followed the instructions (Swofford 2002). All characters were weighted equally were also counted). Germination percentages of their morphology, spore germination percentage, and C-value included with the kit. 30 clones in each sample were and gaps were treated as fifth base (MP) or pair-wise fresh mature spores were 96%, < 1%, and 97%, were determined following Huang et al. (2009) and screened using T7 and SP6 vector primers (Lunt et al. deletion (NJ). The most parsimonious trees were respectively. Only N. biserrata and N. cordifolia Chao et al. (2012). 1999). The plasmid DNAs from the 10 positive clones obtained with heuristic searches of 1000 replicates with Total genomic DNA was extracted using Plant produced sporophytes through their spore cultures. C- were purified using the High-Speed Plasmid Mini Kit random stepwise sequence addition, tree Genomic DNA Mini Kit (Geneaid Biotech Ltd.). A value of N. ×hippocrepicis fell between N. biserrata and (Geneaid, Taipei, Taiwan), and sequenced in an Applied bisection–reconnection (TBR) branch swapping, and chloroplast region atpB-rbcL spacer and two nuclear N. cordifolia (Fig. 6). Biosystems Model 3730 automated sequencer (Applied saving the best tree from each random sequence regions, cycloartenol synthase 1 (CAS1) gene and The two N. ×hippocrepicis populations in the Taipei Biosystems, Carlsbad, CA, USA). All sequences were addition. Bootstrap support values (BS) were calculated glyceraldehydes-3-phosphate dehydrogenase (gapCp) Botanical Garden had different chloroplast atpB-rbcL submitted to GenBank (Table 1) and aligned by BioEdit with 500 replicates. Neighbor-joining analyses were gene, were amplified and sequenced, using primers spacer sequences. The sequences of N. ×hippocrepicis v.7.1.3.0 (Hall, 1999), and the alignment was checked by conducted by calculating Kimura's (1980) 2-parameter plants on P. hanceana were identical with those of N. distance and BS were calculated with 1000 replicates. Table 1 — Information of vouchers in this study. biserrata while the sequences of those on B. capitata Nephrolepis brownii was used as the out group for were identical with N. cordifolia (Fig. 7A). The alleles of Voucher Host tree Host tree GenBank Acession Number phylogenetic analysis. nuclear CAS1 and gapCp genes belong to two different Name Specimen in TAIF* of TPBG** Number atpB.rbcL CAS1 gapCp groups in all sampling N. ×hippocrepicis while the other N. biserrata Y M Huang 1166 Elaeis guineensis Jacq. No. 5167 KF277238 KF277212 RESULTS three species, N. biserrata, N. cordifolia, and N. N. biserrata Y M Huang 1167 Elaeis guineensis Jacq. No. 4455 KF277239 KF277213 brownie, only had one. One group of the N. N. biserrata Y M Huang 1168 Elaeis guineensis Jacq. No. 4454 KF277240 KF277214 KF277227 Morphological comparisons among N. biserrata, N. ×hippocrepicis alleles was clustered with N. biserrata N. biserrata Y M Huang 1174 Phoenix hanceana Naudin No. 4420 KF277241 ×hippocrepicis, and N. cordifolia are summarized in and the other with N. cordifolia (Fig. 7B & C). N. brownii Y M Huang 1172 Phoenix dactylifera L. No. 5201 KF277250 KF277226 KF277237 Table 3 and illustrated in Figs. 2-5. Most characters of N. N. cordifolia Y M Huang 1169 Arenga pinnata (Wurmb) Merr. No. 5117 KF277247 KF277223 KF277236 ×hippocrepicis were intermediate between N. biserrata N. cordifolia Y M Huang 1170 Phoenix dactylifera L. No. 4425 KF277248 KF277224 DISCUSSION N. cordifolia Y M Huang 1171 Phoenix dactylifera L. No. 4425 KF277249 KF277225 and N. cordifolia, except its spores were the largest (if N. ×hippocrepicis Y M Huang 1173 Butia capitata (Mart.)Becc. No. 4428 KF277245 KF277215 KF277228 not aborted). Nephrolepis ×hippocrepicis could be Hybrid Origins of Nephrolepis ×hippocrepicis— KF277219 KF277232 distinguished from N. biserrata and N. cordifolia by According to the NJ trees of chloroplast atpB-rbcL N. ×hippocrepicis Y M Huang 1175 Phoenix hanceana Naudin No. 4420 KF277242 KF277216 KF277229 lanceolate triangulate pinnae (Fig. 2B & 3), reniform spacer sequences and nuclear genes of CAS1 and gapCp KF277220 KF277233 indusia (Fig. 1H), aborted spores (Fig. 4B), and bi- (Fig. 7B & C), Nephrolepis ×hippocrepicis was N. ×hippocrepicis Y M Huang 1176 Phoenix hanceana Naudin No. 4420 KF277243 KF277217 KF277230 colored basal scales with caudate apex (Fig. 5L & P). In originated from the bidirectional hybridization between KF277221 KF277234 addition, the pinnae of N. ×hippocrepicis are closely N. biserrata and N. cordifolia. The chloroplast atpB- N. ×hippocrepicis Y M Huang 1177 Phoenix hanceana Naudin No. 4420 KF277244 KF277218 KF277231 adjoining; the interspaces between adjacent pinnae are rbcL spacer sequences (Fig. 7A) indicate the different KF277222 KF277235 less than half pinna width (Fig. 1E). maternal lineages of N. ×hippocrepicis plants on the N. ×hippocrepicis Y M Huang 1178 Butia capitata (Mart.) Becc. No. 4428 KF277246 * TAIF: The herbarium of Taiwan Forestry Research Institute ** TPBG: Taipei Botanical Garden 4 The International Journal of Plant Reproductive Biology 6(1) pp.1-14, 2014 January, 6 (1) 2014 Hybrid origin of Nephrolepis ×hippocrepicis 5
Fig. 1— Habit and Morphology. (A, D, G) Nephrolepis biserrata. (B, E, H) N. ×hippocrepicis. (C, F, J) N. cordifolia. (A-C) Populations on palms. (D-F) Leaf segments. (G-J) Indusia.
Fig. 2— Nephrolepis ×hippocrepicis Miyam. (A) Habit. (B) Pinna. (C) Indusium. (D-F) Pinna base (from base to apex). 4 The International Journal of Plant Reproductive Biology 6(1) pp.1-14, 2014 January, 6 (1) 2014 Hybrid origin of Nephrolepis ×hippocrepicis 5
Fig. 1— Habit and Morphology. (A, D, G) Nephrolepis biserrata. (B, E, H) N. ×hippocrepicis. (C, F, J) N. cordifolia. (A-C) Populations on palms. (D-F) Leaf segments. (G-J) Indusia.
Fig. 2— Nephrolepis ×hippocrepicis Miyam. (A) Habit. (B) Pinna. (C) Indusium. (D-F) Pinna base (from base to apex). 6 The International Journal of Plant Reproductive Biology 6(1) pp.1-14, 2014 January, 6 (1) 2014 Hybrid origin of Nephrolepis ×hippocrepicis 7 palms P. hanceana and B. capitata. The combining have resulted from the gardeners' weeding, excluding analyses of chloroplast and nuclear DNA data reveal that the young N. ×hippocrepicis. It will be worth to explore those N. ×hippocrepicis on the P. hanceana were the presence of terrestrial N. ×hippocrepicis in the originated from N. biserrata (♀) × N. cordifolia (♂) and natural habitat. those on the B. capitata were originated from N. According to the recent study, N. cordifolia belongs to clade 2 and N. biserrata belongs to clade 3 of the cordifolia (♀) × N. biserrata (♂). Nephrolepis's phylogeny tree (Hennequin et al. 2010). Previous studies show that all known Nephrolepis Most clade 2 species have lunulate indusia and medial species are basically diploid and have basic sorus positions while clade 3 species have reniform or chromosome number 41 (Kramer 1999, Hovenkamp & rounded indusia and submedial to marginal sorus Miyamato 2005) so as the three species in Taiwan (Tsai positions. Nephrolepis ×hippocrepicis has reniform & Shieh 1983). Tetraploids were only reported in N. indusia as clade 3 species, but its overall shape is clearly hirsutula (G. Frost.) Presl and N. pectinata (Willd.) intermediate between those of N. biserrata and N. Schott (Löve et al. 1977). The C-value of N. cordifolia (Fig. 1 G-J). Its sorus position is highly ×hippocrepicis is intermediate between N. biserrata and variable from medial to submarginal. N. cordifolia (Fig. 6), that indicates N. ×hippocrepicis is most likely to be diploid as its parents. Reproduction and distribution — Nephorlepis might In summary, according to the molecular evidences reproduce through spores, tubers, or runners (Steil and the C-value, N. ×hippocrepicis is a diploid species 1952). In this observation, N. ×hippocrepicis failed to originated from bidirectional hybridizations between N. reproduce through spores in the lab. The tubers, which sometimes found on N. cordifolia and its related species biserrata and N. cordifolia. The N. ×hippocrepicis (Hovenkamp & Miyamato 2005), have never been plants in the TPBG have multiple origins and the plants founded on N. ×hippocrepicis. Therefore, N. on P. hanceana and B. capitata have different parental ×hippocrepicis might only reproduce through runners. combination. The dispersal ability of runner reproduction is limited. In the TPBG, N. ×hippocrepicis is epiphytic and its Morphology —The hybrid origin of N. ×hippocrepicis distribution is restricted on the upper trunks of palms. As is also indicated by the evidence of its intermediate a result, N. ×hippocrepicis should rely on new morphology between N. biserrata and N. cordifolia hybridization events to establish new populations. (Table 3). It is much similar with N. biserrata at the first Nephrolepis biserrata is a pantropic species and N. glance by its epiphytic habit, long stipes, long pendulous cordifolia is an Asia-Australasia or Palaeotropic species leaves, fewer leaves per tuft, lamina scales, and lacking which is now spanning the pantropical zone (Hennequin tubers. However, its auricled and closely adjoining et al. 2010). Both species are very common in those pinnae are clearly different from N. biserrata. tropic regions. Nevertheless, N. ×hippocrepicis was only The morphology of the two N. ×hippocrepicis recorded in Ryukyu, Taiwan, and possibly in Thailand (Hovenkamp & Miyamato 2005; Knapp 2011). populations in the TPBG is somewhat different. The N. According to this study, hybridization between N. biserrata × N. cordifolia plants on P. hanceana have biserrata and N. cordifolia happened multiple times in longer leaves and less-auricled middle and basal pinnae the Taipei Botanical Garden. This indicates the than the N. cordifolia × N. biserrata plants on B. capitata hybridization between the two species might not be a (Fig. 3). Because the two populations on different hosts rare event. However, the dispersal ability of N. possess different parental combinations, the cause of the ×hippocrepicis is limited and its habitat is highly morphological differentiation remains further studies. overlapped with its parents, especially with N. biserrata. The epiphytic habit of N. ×hippocrepicis is Nephrolepis biserrata has highly variable morphology Fig. 3— Pinna morphology of selected plants. (A) Nephrolepis cordifolia. (B, C) N. ×hippocrepicis. (D) N. interesting because both epiphytic and terrestrial plants and has similar life form with N. ×hippocrepicis. It is biserrata. The series number indicates the position of pinnae: number 1 is the basal pinna, pinnae with of its both parents, N. biserrata and N. cordifolia are suggested that N. ×hippocrepicis is highly possibly larger number are closer to leaf apex. found. The lacking of terrestrial individual of N. distributed in other tropical regions and just not been ×hippocrepicis in the Taipei Botanical Garden might identified yet. 6 The International Journal of Plant Reproductive Biology 6(1) pp.1-14, 2014 January, 6 (1) 2014 Hybrid origin of Nephrolepis ×hippocrepicis 7 palms P. hanceana and B. capitata. The combining have resulted from the gardeners' weeding, excluding analyses of chloroplast and nuclear DNA data reveal that the young N. ×hippocrepicis. It will be worth to explore those N. ×hippocrepicis on the P. hanceana were the presence of terrestrial N. ×hippocrepicis in the originated from N. biserrata (♀) × N. cordifolia (♂) and natural habitat. those on the B. capitata were originated from N. According to the recent study, N. cordifolia belongs to clade 2 and N. biserrata belongs to clade 3 of the cordifolia (♀) × N. biserrata (♂). Nephrolepis's phylogeny tree (Hennequin et al. 2010). Previous studies show that all known Nephrolepis Most clade 2 species have lunulate indusia and medial species are basically diploid and have basic sorus positions while clade 3 species have reniform or chromosome number 41 (Kramer 1999, Hovenkamp & rounded indusia and submedial to marginal sorus Miyamato 2005) so as the three species in Taiwan (Tsai positions. Nephrolepis ×hippocrepicis has reniform & Shieh 1983). Tetraploids were only reported in N. indusia as clade 3 species, but its overall shape is clearly hirsutula (G. Frost.) Presl and N. pectinata (Willd.) intermediate between those of N. biserrata and N. Schott (Löve et al. 1977). The C-value of N. cordifolia (Fig. 1 G-J). Its sorus position is highly ×hippocrepicis is intermediate between N. biserrata and variable from medial to submarginal. N. cordifolia (Fig. 6), that indicates N. ×hippocrepicis is most likely to be diploid as its parents. Reproduction and distribution — Nephorlepis might In summary, according to the molecular evidences reproduce through spores, tubers, or runners (Steil and the C-value, N. ×hippocrepicis is a diploid species 1952). In this observation, N. ×hippocrepicis failed to originated from bidirectional hybridizations between N. reproduce through spores in the lab. The tubers, which sometimes found on N. cordifolia and its related species biserrata and N. cordifolia. The N. ×hippocrepicis (Hovenkamp & Miyamato 2005), have never been plants in the TPBG have multiple origins and the plants founded on N. ×hippocrepicis. Therefore, N. on P. hanceana and B. capitata have different parental ×hippocrepicis might only reproduce through runners. combination. The dispersal ability of runner reproduction is limited. In the TPBG, N. ×hippocrepicis is epiphytic and its Morphology —The hybrid origin of N. ×hippocrepicis distribution is restricted on the upper trunks of palms. As is also indicated by the evidence of its intermediate a result, N. ×hippocrepicis should rely on new morphology between N. biserrata and N. cordifolia hybridization events to establish new populations. (Table 3). It is much similar with N. biserrata at the first Nephrolepis biserrata is a pantropic species and N. glance by its epiphytic habit, long stipes, long pendulous cordifolia is an Asia-Australasia or Palaeotropic species leaves, fewer leaves per tuft, lamina scales, and lacking which is now spanning the pantropical zone (Hennequin tubers. However, its auricled and closely adjoining et al. 2010). Both species are very common in those pinnae are clearly different from N. biserrata. tropic regions. Nevertheless, N. ×hippocrepicis was only The morphology of the two N. ×hippocrepicis recorded in Ryukyu, Taiwan, and possibly in Thailand (Hovenkamp & Miyamato 2005; Knapp 2011). populations in the TPBG is somewhat different. The N. According to this study, hybridization between N. biserrata × N. cordifolia plants on P. hanceana have biserrata and N. cordifolia happened multiple times in longer leaves and less-auricled middle and basal pinnae the Taipei Botanical Garden. This indicates the than the N. cordifolia × N. biserrata plants on B. capitata hybridization between the two species might not be a (Fig. 3). Because the two populations on different hosts rare event. However, the dispersal ability of N. possess different parental combinations, the cause of the ×hippocrepicis is limited and its habitat is highly morphological differentiation remains further studies. overlapped with its parents, especially with N. biserrata. The epiphytic habit of N. ×hippocrepicis is Nephrolepis biserrata has highly variable morphology Fig. 3— Pinna morphology of selected plants. (A) Nephrolepis cordifolia. (B, C) N. ×hippocrepicis. (D) N. interesting because both epiphytic and terrestrial plants and has similar life form with N. ×hippocrepicis. It is biserrata. The series number indicates the position of pinnae: number 1 is the basal pinna, pinnae with of its both parents, N. biserrata and N. cordifolia are suggested that N. ×hippocrepicis is highly possibly larger number are closer to leaf apex. found. The lacking of terrestrial individual of N. distributed in other tropical regions and just not been ×hippocrepicis in the Taipei Botanical Garden might identified yet. 8 The International Journal of Plant Reproductive Biology 6(1) pp.1-14, 2014 January, 6 (1) 2014 Hybrid origin of Nephrolepis ×hippocrepicis 9
Fig. 4— Spore morphology. (A, D, G, K, N) Nephrolepis biserrata. (B, E, H, L, P) N. ×hippocrepicis. (C, F, J, M, Q) N. cordifolia. (A-C) Spore morphology, most N. ×hippocrepicis spores (B) are aborted. (D-F) Proximal view. (G-J) Proximal surface. (K-M) Distal view. (N-P) Distal surface. (A-C) Bars = 50 μm; (D-Q) Fig. 5— Indumentum. (A, D, G, K, N) Nephrolepis biserrata. (B, E, H, L, P) N. ×hippocrepicis. (C, F, J, M, bars = 5 μm. Q) N. cordifolia. (A-C) Leaf indumentums; (D-F) rachis scales; (G-J) stipe scales; (K-M) basal scales; (N- Q) basal scales (b) and stipe scales (s). (A-C) Bars = 0.5 mm; (D-Q) bars = 1 mm. 8 The International Journal of Plant Reproductive Biology 6(1) pp.1-14, 2014 January, 6 (1) 2014 Hybrid origin of Nephrolepis ×hippocrepicis 9
Fig. 4— Spore morphology. (A, D, G, K, N) Nephrolepis biserrata. (B, E, H, L, P) N. ×hippocrepicis. (C, F, J, M, Q) N. cordifolia. (A-C) Spore morphology, most N. ×hippocrepicis spores (B) are aborted. (D-F) Proximal view. (G-J) Proximal surface. (K-M) Distal view. (N-P) Distal surface. (A-C) Bars = 50 μm; (D-Q) Fig. 5— Indumentum. (A, D, G, K, N) Nephrolepis biserrata. (B, E, H, L, P) N. ×hippocrepicis. (C, F, J, M, bars = 5 μm. Q) N. cordifolia. (A-C) Leaf indumentums; (D-F) rachis scales; (G-J) stipe scales; (K-M) basal scales; (N- Q) basal scales (b) and stipe scales (s). (A-C) Bars = 0.5 mm; (D-Q) bars = 1 mm. 10 The International Journal of Plant Reproductive Biology 6(1) pp.1-14, 2014 January, 6 (1) 2014 Hybrid origin of Nephrolepis ×hippocrepicis 11
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1 T 2 3 12 The International Journal of Plant Reproductive Biology 6(1) pp.1-14, 2014 January, 6 (1) 2014 Hybrid origin of Nephrolepis ×hippocrepicis 13
C-value of N. ×hippocrepicis is intermediate Hall TA 1999 BioEdit a user-friendly biological between N. cordifolia and N. biserrata (Fig. 6). sequence alignment editor and analysis program for Nevertheless, C-value of N. exaltata (2C = 19.1 ± 0.35 Windows 95/98/NT. Nucleic Acids Symp. 41 95–98. pg) is almost the same with that of N. biserrata (2C = 19.0 ± 0.20 pg) and significantly larger than N. cordifolia Harris JL & Ingram R 1991 Chloroplast DNA and “Duffii” (2C = 13.6 ± 0.08 pg, Obermayer et al. 2002, biosystematics: the effects of intraspecific diversity and “Duffi” in the original paper might be a typing error), a plasmid transmission. Taxon 40 393–421. cultivar with uncertain origin (Hovenkamp & Miyamato 2005, Hennequin et al. 2010). Nephrolepis exaltata, N. Hennequin S, Hovenkamp P, Christenhusz MJM & biserrata, N. cordifolia, and N. cordifolia “Duffii” are all Schneider H 2010 Phylogenetics and biogeography of diploid, n=41. What makes the C-value so different at a Nephrolepis – A tale of old settlers and young tramps. same ploidy needs to be explored further. Bot. J. Linn. Soc. 164 113–127. Although N. exaltata and N. ×hippocrepicis have similar morphology, the different distribution and the Hovenkamp PH & Miyamoto F 2005 A conspectus of the fertile spores and higher C-value of the former indicate native and naturalized species of Nephrolepis Fig. 6— Relative nuclear DNA contents of their different lineage. Giving more molecular analyses, (Nephrolepidaceae) in the world. Blumea 50 279–322. Nephrolepis cordifolia, N. ×hippocrepicis, and including both chloroplast and nuclear sequences, N. biserrata. should facilitate to decipher the relationships of these Huang YM, Hsu SY, Huang MH & Chiou WL 2009 two species. Reproductive biology of three cheilanthoid ferns in Nephrolepis exaltata and Nephrolepis ×hippocrepicis Taiwan. Int. J. Plant Reprod. Biol. 1 109-116. — Nephrolepis exaltata (L.) Schott is a neotropical Acknowledgements — The authors wish to species with very similar morphology to N. acknowledge Fong-Chin Kuo who first discovered and Javalgekar SR & Mahabale TS 1959 Germination of ×hippocrepicis but rarely naturalized outside its native provided the information of N. ×hippocrepicis in the spores and prothalli in two species of Nephrolepis, N. range (Hovenkamp & Miyamato 2005). This species TPBG. We also thank Teng-Yi Huang and Jia-Wen Ko exaltata Schott. and N. acuta Presl. Proceed. Nat. Inst. was proposed to be originated from N. cordifolia for assisting the spore cultures, Li-Yaung Kuo for flow Sci. India 25 333–338. crossing with N. biserrata because it shares chloroplast cytometer operation, Te-Yen Tang for assisting the DNA sequences (rbcL, rps4 + rps4-trnS, trnG intron + molecular experiments, Che-Wei Lin for drawing Fig. 2, Kimura M 1980 A simple method for estimating trnG-trnR) with N. cordifolia and its morphology and Ralf Knapp and Pi-Fong Lu for sharing their evolutionary rates of base substitutions through intermediate between N. biserrata and N. cordifolia opinions of the morphologies on those species in this comparative studies of nucleotide sequence. J. Mol. (Hennequin et al. 2010). However, N. cordifolia only study. Evol. 16 111–120. naturalized to neotropical regions recently (Hennequin et al. 2010) and seems unlikely to be a parent of N. LITERATURE CITED Knapp R 2011 Ferns and Fern Allies of Taiwan. KBCC exaltata, which have been in the neotropical for long & Yuan-Liou, Taipei. time already. Different from N. ×hippocrepicis, N. Benedict RC 1916 The origin of new varieties of exaltata spores are fertile (Javalgekar & Mahabale Nephrolepis by orthogenetic saltation I. Progressive Ko WH 2003 Germination of fern spores in natural soils. 1959, Ko 2003). Nephrolepis exaltata was suspected to variations. Bull. Torrey Bot. Club 43 207–234. Amer. Fern J. 93 70–75. be hybrid with many species including N. biserrata and Fig. 7— The NJ trees based on (A) atpB-rbcL intergenic Chao YS, Dong SY, Chiang YC, Liu HY & Chiou WL Kramer KU 1990 Nephrolepidaceae. In Kramer KU & might be a source of the famous cultivar N. spacer, (B) CAS1 gene, and (C) gapCp gene sequences 'bostoniensis' (Benedict 1916, Morton 1958, Nauman 2012 Extreme multiple reticulate origins of the Pteris Green PS (eds.) The Families and Genera of Vascular of three species of Nephrolepis. MP topologies of these cadieri complex (Pteridaceae). Int. J. Mol. Sci. 13 4523- 1979, Wagner Jr. et al. 1999). Its spores are 32-50 μm in Plants. Vol. 1 Pteridophytes and Gymnosperms. DNA regions were the same as their NJ topologies. 4544. Springer, New York Pp 188–190. length (41 μm in average) with variable sculpturing Bootstrap values of MP/NJ are shown on branches. (Nauman 1981), which is similar with the rare Plants' name followed by the host number listed in Chiang TY, Schaal BA, Peng CI 1998 Universal primers Liew FS 1977 Scanning electron microscopical studies “morphologically normal” spores of N. ×hippocrepicis. Table1. for amplification and sequencing a noncoding spacer on spores of Pteridophytes. 11. The family Oleandraceae between the atpB and rbcL genes of chloroplast DNA. (Oleandra, Nephrolepis and Arthropteris). Gard. Bull. Bot. Bull. Acad. Sin. 39 245–250. Sing. 30 101–110. 12 The International Journal of Plant Reproductive Biology 6(1) pp.1-14, 2014 January, 6 (1) 2014 Hybrid origin of Nephrolepis ×hippocrepicis 13
C-value of N. ×hippocrepicis is intermediate Hall TA 1999 BioEdit a user-friendly biological between N. cordifolia and N. biserrata (Fig. 6). sequence alignment editor and analysis program for Nevertheless, C-value of N. exaltata (2C = 19.1 ± 0.35 Windows 95/98/NT. Nucleic Acids Symp. 41 95–98. pg) is almost the same with that of N. biserrata (2C = 19.0 ± 0.20 pg) and significantly larger than N. cordifolia Harris JL & Ingram R 1991 Chloroplast DNA and “Duffii” (2C = 13.6 ± 0.08 pg, Obermayer et al. 2002, biosystematics: the effects of intraspecific diversity and “Duffi” in the original paper might be a typing error), a plasmid transmission. Taxon 40 393–421. cultivar with uncertain origin (Hovenkamp & Miyamato 2005, Hennequin et al. 2010). Nephrolepis exaltata, N. Hennequin S, Hovenkamp P, Christenhusz MJM & biserrata, N. cordifolia, and N. cordifolia “Duffii” are all Schneider H 2010 Phylogenetics and biogeography of diploid, n=41. What makes the C-value so different at a Nephrolepis – A tale of old settlers and young tramps. same ploidy needs to be explored further. Bot. J. Linn. Soc. 164 113–127. Although N. exaltata and N. ×hippocrepicis have similar morphology, the different distribution and the Hovenkamp PH & Miyamoto F 2005 A conspectus of the fertile spores and higher C-value of the former indicate native and naturalized species of Nephrolepis Fig. 6— Relative nuclear DNA contents of their different lineage. Giving more molecular analyses, (Nephrolepidaceae) in the world. Blumea 50 279–322. Nephrolepis cordifolia, N. ×hippocrepicis, and including both chloroplast and nuclear sequences, N. biserrata. should facilitate to decipher the relationships of these Huang YM, Hsu SY, Huang MH & Chiou WL 2009 two species. Reproductive biology of three cheilanthoid ferns in Nephrolepis exaltata and Nephrolepis ×hippocrepicis Taiwan. Int. J. Plant Reprod. Biol. 1 109-116. — Nephrolepis exaltata (L.) Schott is a neotropical Acknowledgements — The authors wish to species with very similar morphology to N. acknowledge Fong-Chin Kuo who first discovered and Javalgekar SR & Mahabale TS 1959 Germination of ×hippocrepicis but rarely naturalized outside its native provided the information of N. ×hippocrepicis in the spores and prothalli in two species of Nephrolepis, N. range (Hovenkamp & Miyamato 2005). This species TPBG. We also thank Teng-Yi Huang and Jia-Wen Ko exaltata Schott. and N. acuta Presl. Proceed. Nat. Inst. was proposed to be originated from N. cordifolia for assisting the spore cultures, Li-Yaung Kuo for flow Sci. India 25 333–338. crossing with N. biserrata because it shares chloroplast cytometer operation, Te-Yen Tang for assisting the DNA sequences (rbcL, rps4 + rps4-trnS, trnG intron + molecular experiments, Che-Wei Lin for drawing Fig. 2, Kimura M 1980 A simple method for estimating trnG-trnR) with N. cordifolia and its morphology and Ralf Knapp and Pi-Fong Lu for sharing their evolutionary rates of base substitutions through intermediate between N. biserrata and N. cordifolia opinions of the morphologies on those species in this comparative studies of nucleotide sequence. J. Mol. (Hennequin et al. 2010). However, N. cordifolia only study. Evol. 16 111–120. naturalized to neotropical regions recently (Hennequin et al. 2010) and seems unlikely to be a parent of N. LITERATURE CITED Knapp R 2011 Ferns and Fern Allies of Taiwan. KBCC exaltata, which have been in the neotropical for long & Yuan-Liou, Taipei. time already. Different from N. ×hippocrepicis, N. Benedict RC 1916 The origin of new varieties of exaltata spores are fertile (Javalgekar & Mahabale Nephrolepis by orthogenetic saltation I. Progressive Ko WH 2003 Germination of fern spores in natural soils. 1959, Ko 2003). Nephrolepis exaltata was suspected to variations. Bull. Torrey Bot. Club 43 207–234. Amer. Fern J. 93 70–75. be hybrid with many species including N. biserrata and Fig. 7— The NJ trees based on (A) atpB-rbcL intergenic Chao YS, Dong SY, Chiang YC, Liu HY & Chiou WL Kramer KU 1990 Nephrolepidaceae. In Kramer KU & might be a source of the famous cultivar N. spacer, (B) CAS1 gene, and (C) gapCp gene sequences 'bostoniensis' (Benedict 1916, Morton 1958, Nauman 2012 Extreme multiple reticulate origins of the Pteris Green PS (eds.) The Families and Genera of Vascular of three species of Nephrolepis. MP topologies of these cadieri complex (Pteridaceae). Int. J. Mol. Sci. 13 4523- 1979, Wagner Jr. et al. 1999). Its spores are 32-50 μm in Plants. Vol. 1 Pteridophytes and Gymnosperms. DNA regions were the same as their NJ topologies. 4544. Springer, New York Pp 188–190. length (41 μm in average) with variable sculpturing Bootstrap values of MP/NJ are shown on branches. (Nauman 1981), which is similar with the rare Plants' name followed by the host number listed in Chiang TY, Schaal BA, Peng CI 1998 Universal primers Liew FS 1977 Scanning electron microscopical studies “morphologically normal” spores of N. ×hippocrepicis. Table1. for amplification and sequencing a noncoding spacer on spores of Pteridophytes. 11. The family Oleandraceae between the atpB and rbcL genes of chloroplast DNA. (Oleandra, Nephrolepis and Arthropteris). Gard. Bull. Bot. Bull. Acad. Sin. 39 245–250. Sing. 30 101–110. 14 The International Journal of Plant Reproductive Biology 6(1) pp.1-14, 2014 January, 6 (1)
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Nauman CE 1979 A new Nephrolepis hybrid from Tryon AF & Lugardon B 1991 Spores of the Florida. Amer. Fern J. 69 65–70. Pteridophyta. Springer, New York.
Nauman CE 1981 Spore morphology of the genus Tsai JL & Shieh WC 1983 A Cytotaxonomic survey of Nephrolepis in Florida. Pollen et Spores 23 219–224. the Pteridophytes in Taiwan (1) material collection and chromosome observation. J. Sci. Eng. 20 137–159. Obermayer R, Leitch IJ, Hanson L & Bennett MD 2002 Nuclear DNA C-values in 30 species double the familial Wagner Jr. WH, Wagner FS, Palmer DD & Hobdy RW representation in pteridophytes. Ann. Bot. 90 209–217. 1999 Taxonomic notes on the Pteridophytes of Hawaii- II. Contr. Univ. Mich. Herb. 22 135–187. Schuettpelz E, Grusz AL, Windham MD, Pryer KM 2008 The utility of nuclear gapCp in resolving polyploid fern origins. Syst. Bot. 33 621–629.