Deparia × nanakuraensis K.Hori (Athyriaceae), a new hybrid pteridophyte from Japan

Abstract I describe Deparia × nanakuraensishyb. nov. and discuss differences in morphological characteristics between parental species D. pterorachis and D. viridifrons with chromosome counting, plastid, and nuclear DNA markers. The new hybrid is endemic to the eastern and northern parts of Japan. Based on the criteria of the International Union for Conservation of Nature and Natural Resources, this new species is here considered Data Deficient. The ploidy level is diploid sterile.


Introduction
The genus Deparia Hook. & Grev. is one of the largest groups in the Athyriaceae family. It contains 60-90 species mostly in East Asia, with some species distributed in Africa, western Indian Ocean, northeastern North America, the Hawaiian Islands, Australia, New Zealand, and South Pacific Islands (Kato 1984;Rothfels et al. 2012;He et al. 2013;Kuo et al. 2016Kuo et al. , 2018PPG I 2016;Moran et al. 2019).
For the conservation assessment, the area of occupancy (AOO) and extent of occurrence (EOO) were estimated using GeoCAT (Bachman et al. 2011), default settings for grid size were applied. In addition, mitotic chromosomes from D. ×nanakuraensis were counted.
To observe mitotic chromosomes, root tips were collected in the field, and pretreated with 0.004 M 8-hydroxyquinoline for 6 h at approximately 17-20 °C. After fixation in ethanol and acetic acid (3:1) for 15-30 min, the root tips were hydrolyzed in 1 N HCl at 60 °C for 1-3 min and then squashed in 2% aceto-orcein solution. The chromosomes were observed under a microscope (Leica DM2500) and then photographed by using a digital camera (Leica MC170 HD).
For the molecular analyses, total DNA was extracted from silica-dried leaves using cetyltrimethylammonium bromide solution, according to Doyle and Doyle (1990).
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 AK1 PCR products was performed using gels of 50% MDE gel solution (Lonza) containing 2% glycerol at 15 °C for 16 h at 300 V, followed by silver staining. For sequencing of the bands separated on the gels, the polyacrylamide gel was dried after silver staining by sandwiching the gel between Kent paper and a cellophane sheet on an acrylic backplate at 55 °C for 4 h. To extract the DNA, a piece of the DNA band was peeled from the dried gel using a cutter knife and incubated in 50 μL of Tris-EDTA buffer (10-mM Tris-HCl and 1-mM EDTA, pH 8.0) at 4 °C overnight. The supernatant solution was used as a template for further PCR amplification with the same primer set employed for initial PCR amplification.
PCR products were purified using 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).

Molecular analysis
The accession numbers of DNA sequences in the datasets were shown in Appendix I. The sequences were aligned using MUSCLE (Edgar 2004) and assessed with Bayesian inference (BI) analysis using MrBayes 3.2.6 (Ronquist et al. 2012), maximum parsimony (MP), and maximum likelihood (ML) analysis using the MEGA X software (Kumar et al. 2018). Indels were treated as missing characters in all analyses. In the BI analysis, the best-fit model (trnL-F: HKY+I model; AK1: HKY model) of sequence evolution for each DNA region was selected using jModelTest 2.1.10 (Darriba et al. 2012). Four Markov chain Monte Carlo chains were run simultaneously and sampled every 100 generations for 1 million generations in total. Tracer 1.7.1 (Rambaut et al. 2018) was used to examine the posterior distribution of all parameters and their associated statistics, including estimated sample sizes. The first 2,500 sample trees from each run were discarded as burn-in periods. The MP tree was obtained using the Tree-Bisection-Regrafting (TBR) algorithm (Nei and Kumar 2000) at search level 3, at which the initial trees were obtained by the random addition of sequences (100 replicates). The confidence level of the monophyletic groups was estimated with 1,000 MP bootstrap pseudo-replicates. In ML analysis, the best-fitting model of sequence evolution for each marker was selected using MEGA; Tamura 3-parameter + I model was used for trnL-F and HKY model for AK1. Initial trees for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood approach and then selecting the topology with superior log likelihood value. The bootstrap method with 1,000 replications was employed to estimate the confidence levels of monophyletic groups in MP and ML analysis.

Chromosome count
Mitotic metaphase chromosome number observed in an individual of D. ×nanakuraensis (Hori 3391) was 2n = 80 ( Figure 1). This individual had shrunken sporangium with no spores. The basic chromosome numbers of the genus Deparia is x= 40 (Sano et al. 2000;Rothfels et al. 2012), and suitably, this sample was found to be a sterile diploid.

Plastid and nuclear DNA phylogenetic trees
We sequenced 653-746 bp of the trnL-F intergenic spacer from different specimens. The aligned trnL-F matrix was 765 bp, of which 121 characters (15%) were parsimony-informative. For the AK1 intron, we sequenced 338-590 bp of the intron for each specimen, yielding a 604 bp aligned matrix, of which 74 characters (12%) were parsimony-informative. The
Distribution and ecology. Deparia ×nanakuraensis is known from the eastern and northern part of Honshu in Japan ( Figure 5). It was observed to grow on soil under deciduous forest (Figure 6) or planted coniferous forest containing Cryptomeria japonica. This hybrid is endemic to Japan. In the type locality, this hybrid comprised a population of over 30 individuals with juveniles ( Figure 7) although parents of D. viridifrons and D. pterorachis were both absent, and sporangium had no spores. However, it is expected that Deparia ×nanakuraensis can reproduce young individuals from buds on its rhizome (Figure 8).
Conservation status. IUCN Red List Category. Based on estimates from Geo-CAT, the EOO of D. ×nanakuraensis was 46,321 km 2 . The known AOO of D. ×nanakuraensis was 44 km 2 . The localities correspond to less than 20 points, but I could not check the population size on each locality. Therefore, available information is inadequate to support the assessment of its extinction risk. According to the IUCN (2012) criteria, the category of Data Deficient (DD) is appropriate.
The ploidy level of this hybrid is the same as its parents because D. viridifrons and D. pterorachis are both sexual diploid (Kurita 1963;Mitui 1966Mitui , 1968Mitui , 1970Hirabayashi 1970). In addition, this can be the first report of a diploid sterile hybrid of the genus Deparia from Japan although several hybrids have been described (Ebihara 2017).
In conclusion, this study described Deparia ×nanakuraensis based on morphology, cytology, and molecular DNA analysis. The morphological characteristics were intermediate between its parents D. viridifrons and D. pterorachis. This hybrid can produce young individuals from buds on its rhizome. Based on the criteria of the International Union for Conservation of Nature and Natural Resources, this new species is here considered Data Deficient. This hybrid can be the first report of diploid sterile hybrid of the genus Deparia from Japan. In future studies, it is expected that more hybrids of the genus Deparia will be discovered and described from Japan.