Under the cooperative project with Jiulianshan National Nature Reserve (#202102220001), several botanical explorations were made to the Jiulianshan National Nature Reserve and extensive field investigations of the new species were conducted from 2020 to 2021 by us. The gross morphology of the new species was photographed and the quantitative characters were measured when conducting field investigations. Herbarium specimens were collected and deposited in the herbaria NF and SYS. Herbarium abbreviations follow Thiers (2020). A systematic examination of online digital images of the A. normale complex available on CVH (https://www.cvh.ac.cn/) and GBIF (https://www.gbif.org/) was carried out. Scanning Electron Microscope (SEM) was used to take spore images of the new species. Mature spores obtained from herbarium specimen were mounted on specimen tabs and then coated with platinum in a sputter coater. Observations were conducted using an ESEM-Quanta 200 (FEI, Hillsboro, Oregon, US) with 15 Kv at Nanjing Forestry University, Nanjing, China. ImageJ software (Rasband 1997–2017) was used to measure the morphological data and the SEM micrographs.

The young root tips of the new species were pretreated with a mixture of 2mM 8-hydroxyquinoline solution and 0.2% colchicine solution (volume ratio = 1: 1) for 3 h and then fixed in Carnoy’s solution for 5 h. The tips were then macerated in 1 N HCl at 60 °C for 10 minutes and then squashed in 2% aceto-orcein. The chromosomes of the two samples of the new species were counted and photographed using a light microscope (Olympus, Japan).

Phylogenetic analyses of the A. normale complex were performed to study the interspecific relationships among the new species and other members in the complex based on sequences of seven plastid markers (atpB, rbcL, rps4, rps4-trnS, trnL, trnL-F, and trnG; with trnL and trnL-F combined and rps4 and rps4-trnS combined) and one low-copy nuclear gene, pgiC. Total genomic DNA was extracted from silica gel-dried leaves using the modified 2 ×CTAB procedure of Doyle and Doyle (1987). Primers and PCR protocols followed Chang et al. (2018) and Xu et al. (2020). For the nuclear gene pgiC, the purified nuclear DNA products were ligated into a pUCm-T Vector (Sangon, Shanghai, China). Ten positive clones for each individual were randomly selected for sequencing. Except for the new species, all other DNA sequences of the A. normale complex used in this study were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/) following Chang et al. (2018) and Xu et al. (2020). Voucher information and GenBank accession numbers are provided in Suppl. material 1.

The newly generated sequences were assembled and edited using Sequencher V.4.14 (GeneCodes Corporation, Ann Arbor, Michigan). All sequences of each gene were initially aligned with MAFFT v.7 (Katoh and Standley 2013) and manually adjusted in BioEdit (Hall 1999). The aligned sequences of seven plastid genes were then concatenated using PhyloSuite (Zhang et al. 2020). Independent phylogenetic analyses of the plastid and nuclear datasets were conducted using RAxML (Stamatakis 2006) and MrBayes v. 3.2.7a (Ronquist and Huelsenbeck 2003) on the Cipres web server (Miller et al. 2010), respectively. The maximum likelihood (ML) tree searches were performed using RAxML-HPC2 on XSEDE with 1000 bootstrap replicates. The models of nucleotide substitution for the combined plastid DNA dataset (TIM2+I+G) and the nuclear pgiC (F81+I+G) were selected independently under the Akaike Information Criterion (AIC) using jModelTest v. 3.7 (Darriba et al. 2012). The temperature parameter of Bayesian inference (BI) set to 0.2, and other priors set to their default values. Two independent runs, each with four chains (one cold, three heated), were conducted, each beginning with a random tree and sampling one tree every 1000 generations of 10 000 000 generations. Convergence among runs and stationarity was assessed using TRACER v.1.4 (Rambaut and Drummond 2007), and the first 25% was discarded as burnin. The remaining trees were used to calculate a 50% majority-rule consensus topology and posterior probabilities (PP).

The spores of the new species Asplenium jiulianshanense were well-developed (Fig. 5A). We counted the spore number per sporangium under a light microscope and found that each sporangium contains 64 spores. Therefore, we assumed that it is sexually reproducing. Spore size is considered a good indicator of ploidy level when compared with close relatives in the A. normale complex (Chang et al. 2013, 2018). We measured the spore size of the new species (37‒43 µm, Fig. 5) and calculated the mean spore size (39.8 µm) based on 20 mature spores using ImageJ. It has a larger spore size than most members of the complex, indicating polyploidy (Fujiwara et al. 2017; Chang et al. 2018). In addition, our cytological study confirmed that the chromosome number of the new species is 2n = 144 (Fig. 6), which indicates that it is a tetraploid species.

The 98 aligned plastid gene sequences are 5,583 bp in length, with 382 parsimony informative sites in total. The tree topologies by the ML and BI analyses were generally concordant when using the concatenated plastid dataset. The major clades of the phylogeny reconstructed in this study were also congruent with those of previous studies (Chang et al. 2018, 2020; Xu et al. 2020). Owing to only one gene region of rbcL with 525 bp being available from NCBI for A. minutifolium Kanem. & Tagane, four collections of the new species and one collection of A. minutifolium together formed a well-supported clade nested within clade III, and this well-supported clade was resolved as sister to A. kiangsuense (Fig. 3).

The nuclear dataset included 54 aligned sequences in total. The total alignment was 851 bp in length, with 98 parsimony informative sites. All the three pgiC alleles of the new species were nested within clade A. Within clade A, one allele of the new species was well resolved as sister to A. kiangsuense, while the other two alleles were closely related to some alleles of A. boreale (Ohwi ex Sa. Kurata) Nakaike (Fig. 4).

Figure 4. 

The phylogenetic position of Asplenium jiulianshanense K.W.Xu & G.L.Xu based on the low-copy nuclear gene pgiC. The numbers associated with branches are maximum likelihood bootstrap (MLBS) values followed by Bayesian inference posterior probabilities (PP). “*” indicates MLBS = 100% or PP = 1. Black vertical bars indicate those major clades identified by Chang et al. (2018).

Figure 5. 

Asplenium jiulianshanense K.W.Xu & G.L.Xu A–C spore morphology of the new species D rhizome scale. Scale bars: 300 μm (A); 10 μm (B, C); 1 mm (D).

Figure 6. 

Chromosomes of Asplenium jiulianshanense K.W.Xu & G.L.Xu in mitotic root-tip cells, 2n = 144. Scale bars: 20 μm.

The A. normale complex is widely distributed in south and southeast Asia, tropical east Africa, and tropical Pacific islands (Chang et al. 2013; Lin and Viane 2013). However, the species delimitation and taxonomic treatment within the complex have been controversial until an integrative taxonomic approach was employed (Wu 1999; Chang et al. 2013, 2018; Lin and Viane 2013). The treatment of one species A. normale with some varieties was generally accepted by former studies (Kurata 1963; Ito 1972; Wu 1999). Recent integrative taxonomic studies using cytological, morphological, and DNA sequence data revealed that recurrent reticulation events occurred among members of this species complex (Chang et al. 2013, 2018). The status of an increasing number of taxa within the species complex has been confirmed using integrative taxonomy in recent studies (Chang et al. 2013, 2018, 2020; Fujiwara et al. 2017, 2020).

Chang et al. (2018) recognized seven species (A. boreale, A. guangdongense, A. kiangsuense, A. normaloides, A. normale, A. pifongiae, A. pseudonormale) of the A. normale complex in China. Morphologically, the new species A. jiulianshanense collected from the Jiulianshan National Nature Reserve is most similar to A. kiangsuense in having plants less than 15 cm tall, rachises adaxially without a deep furrow, pinnae elliptic to trapeziform-oblong, and 3–4 sori per pinna in the middle part of frond (Fig. 1). However, the new species can be easily distinguished from A. kiangsuense by fronds with only one gemma at the distal end of rachis, 8‒20(‒22) pinnae pairs, pinna margins entire to sinuate, exospore length 37‒43 µm (Fig. 1). We also checked the synonyms (e.g. A. gulingense Ching & S. H. Wu, A. hangzhouense Ching & C. F. Zhang, A. parviusculum Ching) related to A. kiangsuense. All of them lack gemmae at the distal end of rachis and those pinnae in the lower part of the frond are spreading and divaricate, while A. jiulianshanense usually has only one gemma at the distal end of rachis and pinnae in the lower part of the frond are spreading horizontally or reflexed. Within taxa bearing gemmae on the rachis, the gemmae of the new species only occur at the distal end of rachis, which is also different from A. pseudonormale. The new species can also be distinguished from A. normaloides and A. normale by having plants less than 15 cm tall (vs. more than 20 cm tall in A. normaloides and more than 15 cm tall in A. normale) and 3–4 sori per pinna in the middle part of the frond (vs. 10–14 sori per pinna in A. normaloides and usually more than 4 sori per pinna in A. normale). In addition, Kanemitsu et al. (2017) described a new species A. minutifolium of the A. normale complex from Thailand, which is also similar to A. jiulianshanense in morphology. However, A. jiulianshanense is distinct from A. minutifolium by usually having one gemma at the distal end of each rachis (vs. with no buds on the rachis in A. minutifolium), pinna margins entire to sparsely crenate (vs. pinna margins entire or slightly undulate in A. minutifolium), and sori arranged oppositely (vs. usually arranged in a row in A. minutifolium).

The reticulate relationships of the A. normale complex have been well investigated using evidence from molecular, cytological, and morphological data (Chang et al. 2013, 2018, 2020; Fujiwara et al. 2017). Both diploids and tetraploids were confirmed to exist in the complex and multiple tetraploid species were revealed to originate via autopolyploidy and allopolyploidy (Chang et al. 2013; Fujiwara et al. 2017). For example, the allotetraploid species A. serratipinnae T.Fujiw. & Watano originated from their diploid parents of A. normale and A. oligophlebium (Fujiwara et al. 2017, 2020), and autotetraploid species A. kiangsuense and A. boreale Nakaike were revealed by the evidence that their two pgiC copies nested within the same group (Chang et al. 2013).

For the case of the new species A. jiulianshanense, both the spore size and the chromosome number indicate that it was a tetraploid species. The count of 64 spores per sporangium suggests that the new species has sexual reproduction. Phylogenetically, though only one gene region rbcL with 525 bp was available for A. minutifolium and all the 525 sites were identical to those of A. jiulianshanense and A. kiangsuense, A. jiulianshanense and A. minutifolium together were well resolved as a distinct clade sister to A. kiangsuense based on the phylogeny reconstructed using seven maternally inherited plastid genes (Fig. 3). As for the phylogeny reconstructed using the low-copy nuclear pgiC gene, one allele was resolved as sister to the autotetraploid species A. kiangsuense and the other two alleles were nested together with the autotetraploid A. boreale (Fig. 4). The plastid and nuclear phylogenies together suggest that the present new species is derived from hybridization between autotetraploids A. kiangsuense and A. boreale. In any case, the tetraploid taxa in the A. normale complex still need more study. More collections of additional species, including molecular data and chromosome counts, are still needed to analyze their phylogenetic position and better understand their pathways of reticulate evolution.