The Hybrid Origin of Adiantum meishanianum (Pteridaceae): A Rare and Endemic Species in Taiwan

Adiantum meishanianum is an endemic species distributed only in Meishan village, Kaohsiung, Taiwan. Because its sporangia contain only abortive spores, A. meishanianum has been regarded as having a hybrid origin, presumably with A. caudatum, A. malesianum, and A. philippense as the putative parents. The aim of this study is to confirm the hybrid origin and determine the parental lineages of A. meishanianum by examining cytology, reproductive modes, and using molecular phylogenetics. We found that the sequences of two chloroplast regions, rps16-matK intergenic spacer and the matK gene, are identical between A. meishanianum and A. malesianum, suggesting A. malesianum is the maternal parent. The nuclear phylogeny reconstructed based on the low-copy marker, CRY2 first intron, reveals that A. meishanianum has three types of sequences: one type groups with sequences of sexual diploid individuals of A. philippense and the other two group with sequences of the sexual tetraploid A. malesianum, indicating that A. philippense is the paternal species. Our data further imply that the extant A. meishanianum probably originated from a single hybridization event, and its rarity is likely due to the limited distribution of the paternal parent. Keywords—Adiantum malesianum, Adiantum philippense, CRY2 first intron, cryptic species, hybridization. Adiantum meishanianum F. S. Hsu ex Y. C. Liu & W. L. Chiou (Pteridaceae), a rare species endemic to Taiwan (Liu et al. 2009), has only been found in Meishan village, Kaohsiung City, Taiwan, with a single population, and is considered as critically endangered (Wang et al. 2012). Hsu (1993) first reported that A. meishanianum is a triploid species based on a somatic chromosome count (2n = 90). Lee et al. (2007) later found that A. meishanianum produces only aborted spores, which either fail to germinate or develop into abnormal gametophytes, suggesting that this species is a sterile triploid hybrid. The persistence and expansion of the population of A. meishanianum seems to rely solely on vegetative propagation via adventitious buds borne on the elongated rachis tips. Among the once-pinnate species of Adiantum in Taiwan, A. caudatum L., A. malesianum Ghatak. and A. philippense L. have been hypothesized as the parental candidates due to their morphological similarities, including the long pinna stalk (3–4 mm), the semi-orbiculate basal pinnae, and the adventitious bud on the elongated rachis (Hsu 1993; Lee et al. 2007; Liu et al. 2009). This study aims to resolve the hybrid origin ofA.meishanianum by reconstructing the phylogenies based on both chloroplast and nuclear DNA sequences of all once-pinnate Adiantum species in Taiwan. The ploidy and reproductive modes of the candidate parental species, as determined by the spore number per sporangium and cytological evidence, were further examined to confirm the hybrid relationships. Materials and Methods Chloroplast dataset—We sampled 17 Adiantum species, representing all major clades in the genus (Lu et al. 2012) and including all of the once-pinnate species in Taiwan (Knapp 2011). DNA was extracted using a modified CTAB procedure (Wang et al. 2004). We adopted the strategy of Kuo et al. (2011) to design specific primers for the chloroplast DNA (cpDNA) regions, matK and rps16-matK intergenic spacer (IGS). First, we applied universal primers (“FERN chlB fYAA”, “FERN rps16 fQCGR”, “FERN rps16 fSRQE”, “FERmatK fEDR”, and “FERmatK rAGK”) to generate sequences of matK, rps16 second exon and rps16-matK IGS regions of selected Adiantum species. Based on the sequences obtained, we designed three specific primers: “Adn rps16 fEET”, “Adn matK rRLF”, and “Adn matK fHIS”. The PCR reactions were performed in 15 mL reaction volumes, including 20 ng genomic DNA, 1 + PCR buffer, 200 mM dNTP, 15 pmol of each primer, and 0.5 U polymerase (GENET BIO ExPrime Taq DNA Polymerase, Korea). The detailed information of these primers and PCR conditions can be found in Table 1. Nuclear dataset—Phylogenies based on biparentally inherited DNA markers, particularly low-copy nuclear genes, provide an efficient way to disentangle the reticulate relationships caused by hybridization and/or polyploidization (Ebihara et al. 2005; Adjie et al. 2007; Li et al. 2012; Sessa et al. 2012). Only recently have such molecular markers become readily available to fern biologists (Rothfels et al. 2013). However, the universality of these regions and developed primers for these nuclear genes usually vary among fern lineages (Chen at al. 2012; Rothfels et al. 2013). As a case study, we developed a novel low-copy nuclear marker: the first intron of the cryptochrome gene (CRY), which encodes a blue light photoreceptor. It is an ideal nuclear marker for Adiantum species because the copy number and exon/intron positions are known from A. capillus-veneris L. (Kanegae and Wada 1998; Imaizumi et al. 2000). Among the non-coding regions of the five A. capillus-veneris CRY genes, the first intron is most stable in its exon/intron boundary. Thus, based on the published A. capillus-veneris CRY1–5 coding sequences, we first designed one degenerate primer set to target the first intron (“Adn CRY fPEE” and “Adn CRY rDLL”). By electrophoresis in 1 + TBE 0.8% agarose gel, we verified, isolated, and sequenced the PCR products of 400 bp, which contains only the amplicons from CRY2. After we obtained partial CRY2 first exon and second exon sequences from A. caudatum, A. meishanianum, and A. philippense, a specific primer set to amplify once-pinnate Adiantum CRY2 first intron was designed: “Adn CRY2 fHLN” and “Adn CRY2 rVKQ”. All PCR reactions were carried out as described above. Other details of primer information and PCR conditions can be found in Table 1. To distinguish different sequence types after PCR (i.e. alleles or homeologs), single-strand conformation polymorphism (SSCP) was used when the direct sequencing of amplicons of “Adn CRY2 fHLN + Adn CRY2 rVKQ” failed. The protocol of SSCP electrophoresis followed Ebihara et al. (2005). Before SSCP electrophoresis, the PCR products were purified using a Gel/PCR purification kit (Genomics, Taipei, Taiwan) and eluted with ddH2O. After SSCP electrophoresis, all gel slices containing the separated single-strand DNA products (i.e. bands) were purified by the same purification kit, and were re-amplified and sequenced. Voucher information of the materials used in chloroplast or nuclear DNA analyses of this study is summarized in the Appendices. Phylogenetic analyses—DNA sequences were aligned using ClustalW implemented in BioEdit (Hall 1999) and the alignments were edited