Corresponding author: Kiyotaka Hori (
Academic editor: Thais Almeida
We analyzed the phylogeny of the
Hori K, Murakami N (2019) Origin of the
In sexually reproductive fern species, meiosis produces 64 haploid spores per sporangium following mitotic divisions of spore mother cells four times, and each mother cell contains half of the parental chromosome number (
Apogamous reproduction is not an unusual feature in ferns. Approximately 3% of all fern species (
Hybridization patterns with germination from which apogamous species are derived. (
The ploidy level and reproductive mode of the
Species | Reproducive mode | ploidy level | Refereces |
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apogamous | 3× |
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apogamous | 3× |
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apogamous | 3× |
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apogamous | 4× |
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apogamous | 3× |
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apogamous | 3× |
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apogamous | 3× |
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apogamous | 3× |
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sexual | 2× |
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sexual | 4× |
|
In this study, all 10 species of the
We counted mitotic chromosomes from
For molecular analyses, total DNA was extracted from silica-dried leaves using cetyltrimethylammonium bromide solution, according to
PCR amplification was performed using PrimeSTAR Max DNA Polymerase (Takara, Kyoto, Japan). PCR entailed an initial denaturation step at 95 °C for 10 min, followed by 35 cycles of denaturation, annealing, and elongation steps at 98 °C for 10 s, 55 °C for 5 s, and 72 °C for 5 s, respectively, using a Model 9700 thermal cycler (Applied Biosystems, Foster City, CA, USA).
Gel electrophoresis of
PCR products were purified using ExoSAP-IT (USB, Ohio, USA) or Illustra ExoStar 1-Step (GE Healthcare, Wisconsin, USA) and used as templates for direct sequencing. Reaction mixtures for sequencing were prepared using the BigDye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems). The reaction mixtures were analyzed using an ABI 3130 Genetic Analyzer (Applied Biosystems). All plant samples were classified based on their PCR-SSCP banding patterns, and each band was DNA sequenced.
For phylogenetic analyses, the sequences were typified and made non-redundant by removing duplicate sequences. Only one sequence representing each allele for
The ploidy level and reproductive mode of
We sequenced 719–748 bp of the
To define allelic types of the
To divide each allele number with the alphabet, we used clades supported by
In total, five types of plastid
Allelic constitution of
A 50% majority consensus tree resulting from Bayesian Markov chain Monte Carlo Bayesian (
Full-data set of a 50% majority consensus tree resulting from Bayesian Markov chain Monte Carlo Baysean (
50% majority consensus tree resulting from Bayesian Markov chain Monte Carlo Bayesian (
Full-data set of a 50% majority consensus tree resulting from Bayesian Markov chain Monte Carlo Baysean (
Reproductive mode, ploidy level, plastid haplpotype (
Voucher | Locality | Species | rep. | ploi. |
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Okinawa pref.: Kunigami village, Mt. Yonahadake |
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apo. | α1 |
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Okinawa pref.: Nago city, Genka |
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α1 |
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Kagoshima pref.: Amami city, Sumiyou village, Santaro-touge, 350m alt. |
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sex. | 2× | α2 |
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Kagoshima pref.: Amami city, Naze |
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sex. | 2× | α1 |
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Kagoshima pref.: Yakushima Is, Koseda, 70m alt. |
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α1 |
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Kagoshima pref.: Yakushima Is, Koseda, 70m alt. |
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α1 |
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Kagoshima pref.: Yakushima Is, Hara, 80m alt. |
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α1 |
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Mie pref.: Minamimuro county, Kiho-cho |
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apo. | 3× | β1 |
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Fukuoka pref.: Kasuya county, Hisayama-machi, 140m alt. |
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β1 |
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Fukuoka pref.: Kasuya county, Hisayama-machi, 140m alt. |
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β1 |
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Mie pref.: Minamimuro county, Kiho-cho, 70m alt. |
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β1 |
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Kagoshima pref.: Yakushima Is, Isso-river, 390m alt. |
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γ2 |
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Kasgoshima pref.: Amami city, Naze, Honchya-touge, 250m alt. |
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sex. | 2× | δ1 | D1 |
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Kasgoshima pref.: Amami city, Sumiyou village, Santaro-touge, 350m alt. |
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sex. | 2× | δ1 |
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Kasgoshima pref.: Amami city, Naze, Ooaza-asato |
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sex. | 2× | δ4 |
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Kagoshima pref.: Yakushima Is, Koseda, 70m alt. |
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β2 |
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Kagoshima pref.: Yakushima Is, Koseda, 71m alt. |
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apo. | α1 |
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Kagoshima pref.: Yakushima Is, Isso-river, 200m alt. |
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apo. | δ2 |
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Okinawa pref.: Nago city, Genka | apo. | α1 |
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Kagoshima pref: Yakushima Is, Tabugawa, 200m alt. |
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β1 |
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Mie pref.: Minamimuro county, Kiho-cho |
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β1 |
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Mie pref.: Minamimuro county, Kiho-cho |
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β1 |
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Kagoshima pref.: Yakushima Is, Miyanoura river, 20m alt. |
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β1 |
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Kagoshima pref.: Tokunoshima Is, Mt. Inokawadake, 200m alt. |
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apo. | 3× | δ1 |
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Kagoshima pref.: Tokunoshima Is, Mt. Inokawadake, 200m alt. |
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apo. | 3× | δ2 | B2D1 |
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Chiba pref.: Katori county, Tako-machi, Hayashi |
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δ3 |
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Okinawa pref.: Nago city, Genka |
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apo. | δ1 | B2D2 | |
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Fukuoka pref.: Kasuya county, Hisayama-machi, 140m alt. |
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δ3 | B2D2 | ||
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Yamaguchi pref.: Nagato city, Ichinoo |
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δ3 | B2D2 | ||
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Mie pref.: Minamimuro county, Kiho-cho |
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apo. | 3× | δ5 |
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Shizuoka pref.: Shimoda city, Renndaiji-onsenn, 100m alt. |
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apo. | 3× | δ5 | B2D3 |
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Kagoshima pref.: Tokunoshima Is, Mt. Inokawadake, 200m alt. |
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apo. | 3× | δ1 |
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Mie pref.: Minamimuro county, Kiho-cho |
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apo. | 3× | δ3 |
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Mie pref.: Minamimuro county, Kiho-cho |
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apo. | 3× | δ3 |
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Kagoshima pref.: Kagoshima city, Chuzann-cho, Takinoshita-river | apo. | 3× | δ1 |
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Okinawa pref.: Kunigami village, Mt. Yonahadake | γ1 |
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|||
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Okinawa pref.: Kunigami village, Mt. Yonahadake | γ1 | CK | |||
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Mie pref.: Minamimuro county, Kiho-cho | δ3 |
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|||
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Mie pref.: Minamimuro county, Kiho-cho | apo. | 3× | δ1 | D2H2 | |
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Kumamoto pref.: Amakusa city | apo. | 3× | δ1 |
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Mie pref.: Minamimuro county, Kiho-cho |
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ε |
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Kanagawa pref.: Minamiashigara city, Kano |
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sex. | 4× | ε |
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Figure
The reticulogram of the
The allelic constitution in
The allelic constitution of
Allelic constitution in other species suggested that there were six undetected parental species which have only one allele E, F, G, J, K, or H. This study could not resolve ploidy and reproductive mode of these species. Tentatively, in the reticulogram (Figure
According to the relationships of diploid sexual species and triploid apogamous species, allelic inheritance patterns in the
Continuous morphological variation in the
We are grateful to the following individuals for their assistance in collecting plant materials: Mr. S. Saito of Kyushu University, Prof. M. Takamiya, and Mr. K. Hatake of Kumamoto University; Prof. Shunsuke Serizawa of Faculty of Education, Aichi University of Education; and Mr. K. Ohora and Mr. N. Kanemitsu of the Nippon Fernist Club. We are also grateful to Prof. M. Takamiya of Kumamoto University for providing his cytological data and advice during this study. In addition, this study was partly supported by a Grant-in-Aid for JSPS Fellows No. 26-1720 and 18K14785 to K. H. and Scientific Research No. 25291089 and by Grant-in-Aid for Scientific Research No. 16H04835 to N. M.
A1, LC468160; A2, LC468161; A3, LC468162; A4, LC468163; A5, LC468164; A6, LC468165; B1, LC468167; B2, LC468168; B3, LC468169; C, LC468166; D1, LC468172; D2, LC468173; D3, LC468174; E, LC468170; F, LC468171; G, LC468175; H1, LC468180; H2, LC468181; J, LC468176; K, LC468177;
α1, LC468195; α2, LC468196; β1, LC468199; β2, LC468200; γ1, LC468197; γ2, LC468198; δ1, LC468201; δ2, LC468202; δ3, LC468203; δ4, LC468204; δ5, LC468205; ε, LC468208;