﻿Angiopterisnodosipetiolata (Marattiaceae), a new fern species from Yunnan, China

﻿Abstract Angiopterisnodosipetiolata Ting Wang tris, H.F.Chen & Y.H.Yan, a new fern of Marattiaceae, is described and illustrated. Morphologically, A.nodosipetiolata is similar to A.chingii with more than one naked pulvinus on the stipe and numerous jointed hairs on the undersides of the mature pinnae. However, the pinnae of A.nodosipetiolata are lanceolate and can reach up to 4–6 pairs, whereas they are elliptic and occur in 2–3 pairs in A.chingii. Phylogenetic and genetic distance analysis, based on the plastid genomes, also indicates that A.nodosipetiolata is not closely related to A.chingii. Currently, there are ca. 500 mature individuals in Gulinqing Nature Reserve and we suggest A.nodosipetiolata should be categorised as an Endangered (EN) species according to the criteria of IUCN.


Morphological analysis
Field observations were conducted in China starting in 2022.Observation of herbarium specimens were carried out at KUN, PE and PYU and the voucher specimens of Angiopteris nodosipetiolata were deposited at IBSC, CSH and SWFU.Petiole scales and the hairs on the pinnae surface were observed using an OLYMPUS-SZ61 stereoscopic microscope and an OLYMPUS-BX43 biological microscope.The ornamentation of spores was examined with a JSM-6360LV Scanning Electron Microscope.

Taxon sampling, DNA extraction and sequencing
In this study, five new Angiopteris plastomes were sequenced and completely assembled.Detailed information on the samples is provided in Table 1.All leaf samples were frozen in liquid nitrogen and stored at −80 °C.Total DNA was extracted from the leaves using a modified CTAB method and sequenced using short reads produced by the NovaSeq 6000 platform (2 × 150 bp) by Novogene (Beijing, China).

Phylogenetic analyses
In this study, we analysed the phylogenetic relationship of 15 Angiopteris species by combining newly-obtained data with publicly available complete plastome data from the National Center for Biotechnology Information (NCBI; https://www.ncbi.nlm.nih.gov/).To reduce the effect of different plastid regions on phylogenetic inference, we constructed the phylogeny of Angiopteris, based on the complete plastid genome sequences and coding sequences (CDS).Firstly, we used Homblocks (Bi et al. 2018) to automatically recognise locally collinear blocks and excavate core conserved fragments (protein coding genes, conserved non-coding regions and rRNA genes) amongst complete plastid genomes; these were used as "complete plastomes" for phylogenetic analysis (note that these therefore were shorter than the complete sequenced plastomes in Table 1).Next, we used the "get_annotated_regions_from_gb.py"script (https://github.com/Kinggerm/PersonalUtilities/) to extract 84 CDSs from all plastid genomes and used MAFFT v.7.475 (Katoh and Standley 2013) and trimAl (Capella-Gutiérrez et al. 2009) for alignment and trimming.We used Maximum Likelihood (ML) and Bayesian Inference (BI) methods for phylogenetic construction.The best-fit model of evolution of ML and BI methods were selected by ModelTest-NG (Darriba et al. 2020) under the Bayesian Information Criterion (BIC) with " -T raxml" and "-T mrbayes" parameters, respectively.ML analyses were conducted with RAxML v.8.2.10 (Stamatakis 2014) and node support was assessed using rapid bootstrap (RBS) analysis with 1000 pseudo-replicates.BI analyses were constructed with MrBayes v.3.2 (Ronquist et al. 2012), using ten million generations and sampling trees every 1000 generations.Two runs each with three heated and one cold chain were performed in parallel.Each chain started with a random tree and the first 25% of sampled generations were discarded as burn-in to construct a majority-rule consensus tree and estimate the posterior probabilities (PP).The convergence of runs was assumed when the average standard deviation of split frequencies dropped below 0.01 according the MrBayes manual.

Genetic distance estimation
The intraspecific genetic distances for both complete plastomes and 84 CDSs of Angiopteris nodosipetiolata were calculated using the two-parameter (K2P) model in MEGA 11.0 (Tamura et al. 2021).We also computed interspecific genetic distances between A. nodosipetiolata and three morphologically similar species, A. latipinna, A. subrotundata and A. chingii.Subsequently, the "ggplot2" (Wickham 2011) and "ggpubr" (https://rpkgs.datanovia.com/ggpubr/)package in R was employed to analyse and visualise potential significant differences between the intraspecific and interspecific genetic distances using the Wilcoxon test.

Phylogenetic relationships within Angiopteris
Complete plastid genomes and 84 CDSs were each used to construct phylogenetic relationships.These had total lengths of 110,566/71,779 bp, amongst which, there were 358/199 bp, 1,012/512 bp and 109,196/71,068 bp of parsimony-informative sites, singleton sites and constant sites, respectively (Table 2).Phylogenetic relationships derived from different regions displayed identical topologies in both Bayesian Inference (BI) and Maximum Likelihood (ML) trees (Fig. 1).The results indicated that specimens of the newly-discovered species, Angiopteris nodosipetiolata, form a monophyletic group with high support (RBS = 100/99, PP = 1/1) and this was recovered as sister to A. danaeodes, rather than a close relative of A. latipinna, A. subrotundata or A. chingii.

Genetic Distance within Angiopteris
The genetic distances between A. nodosipetiolata and three Angiopteris species ranged from 0.0005881 to 0.0010409 for complete plastid genome and from 0.0005296 to 0.0009341 for 84 CDSs.In contrast, the intraspecific genetic distances amongst A. nodosipetiolata sequences were notably smaller, measuring 0.0000452 for complete plastid genome and 0.0000697 for 84 CDSs.These intraspecific distances within A. nodosipetiolata were significantly smaller (P < 0.01) than the distances observed between A. nodosipetiolata and A. latipinna, A. subrotundata or A. chingii (Fig. 2).Diagnosis.Angiopteris nodosipetiolata is morphologically quite similar to A. chingii J.M. Camus in terms of having more than one naked pulvinus on the stipe and numerous joint-like hairs on the undersides of the mature pinnae.However, the former's pinnae are lanceolate, occurring in 4-6 pairs, in contrast with the elliptical pinnae of the latter, which consist of only 2-3 pairs.Judging from the shape of laminae, A. nodosipetiolata also closely resembles  Nonetheless, these two species exhibit only one naked pulvinus on the stipe and the surfaces of their mature pinnae, apart from the mid-rib, are smooth and hairless (Table 3).The phylogenetic and genetic distance analysis also showed that A. nodosipetiolata is not the closest relative of A. latipinna, A. subrotundata or A. chingii (Figs 1, 2).Description.Plants terrestrial, 70-120 cm tall.Rhizomes long creeping.Stipes 40-70 cm long, 0.7-1.5 cm in diam., with 2-3 naked pulvini (occasionally with 1); stipe scales peltate, brown, lanceolate, margins with hair-like outgrowths, apex acuminate, cells clathrate and elongate.Laminae 35-50 × 25-30 cm; once pinnate, pinnae 4-6 pairs, lanceolate, ca.15-20 × 3.5-4.5 cm, bases round-cuneate, margins coarsely dentate, apices caudate, densely covered with jointed hairs on the undersides of the mature pinnae.Veins free, simple or bifurcate, false veins absent.Sori medial, yet closer to the main vein, ranging in length from ca. 0.5 cm on the basal pinnae to ca. 1.8 cm in the middle pinnae.Exospores with forked rod-like ornamentation.
Geographical distribution.Currently, Angiopteris nodosipetiolata is only found in Gulinqing Nature Reserve of Yunnan Province based on our current knowledge and may represent a species endemic to Yunnan, China.
Ecology.Angiopteris nodosipetiolata is observed at the edge of montane forests, growing at an elevation of approximately 1400 m.

Vernacular name. 瘤柄观音座莲 (liu bing guan yin zuo lian).
Conservation status.There are ca.500 mature individuals of Angiopteris nodosipetiolata has been found at the type locality and it thrives in forest edge areas that are susceptible to human disturbance.The status of the new species should be classified as Endangered (EN), based on current information and following the International Union for Conservation of Nature and Natural Resources guidelines.In addition, it should be listed and protected as second grade Wild Plants Under State Protection like all other taxa in Angiopteris.

Discussion
Cryptic species are phenotypically highly similar species (Struck and Cerca 2019), but which represent distinct evolutionary lineages (Mayo 2022).In recent years, an increasing number of cryptic species that were hidden in plain sight have been unveiled, driven in part by the rise of DNA barcoding (Ni et al. 2012;Tyagi et al. 2019;Labe et al. 2022;Takenaka et al. 2023).Notable groups that include cryptic species encompass organisms like Isoetes Linnaeus (1753: 1100; Gu et al. (2022)), Ceratopteris Brongn. (1822: 186;Yu et al. (2021)) and jellyfish (Moura et al. 2023).Angiopteris nodosipetiolata shares a remarkable morphological resemblance with A. chingii, like the presence of more than one naked pulvinus on the stipe and numerous jointed hairs on the undersides of the mature pinnae (Ching 1958;He and Christenhusz 2013).This likeness renders A. nodosipetiolata susceptible to potential misidentification as A. chingii.Upon re-examining the morphological characteristics of A. nodosipetiolata and A. chingii, the pinnae morphology (lanceolate vs. elliptic) and the arrangement of pinna pairs (4-6 vs. 2-3) can serve as distinguishing characteristics (Table 3).Besides, a deeper insight obtained from phylogenetic analysis in plastid genomes also provides a new perspective (Figs 1, 2).Contrary to expectations of a close relationship with A. latipinna, A. subrotundata and A. chingii, A. nodosipetiolata is considered the sister clade to A. danaeodes.The distinguishing feature is that A. danaeodes has only one naked pulvinus on the stipe, pinnae 2-3 pairs and the surfaces of their mature pinnae, apart from the mid-rib, are smooth and hairless.In contrast, A. nodosipetiolata has more than one naked pulvinus on the stipe and numerous joint-like hairs on the undersides of the mature pinnae.
Relying on macroscopic morphological classification in the past has frequently resulted in treating groups with subtle morphological differences as a single species due to subjective interpretations of morphology (Gu et al. 2022).For instance, Didymochlaena Desvaux (1811: 303) was historically considered to contain only one (PPG I 2016) or six species (Shang et al. 2020), based on morphological characters, while a recent study utilising morphological and molecular variation has identified 22 species within the genus (Shang et al. 2023), marking a threefold increase in the previously known number of species.Similarly, Ptisana soluta (Compton) Murdock & Perrie (2023:53), a member of the Marattiaceae, was long regarded as the widespread South Pacific species P. salicina (Sm.)Murdock (2008b: 746) in New Caledonia.However, a recent study, based on sequence data and morphology, suggested it to be an endemic species with a vulnerable conservation ranking (Shepherd et al. 2023).Thus, integrating research results from morphological classification, molecular systematics, ecology and other fields, holds significant guiding significance for the rapid and accurate identification of cryptic species (Kabus et al. 2023;Meghana et al. 2023).

Conclusion
Utilising interdisciplinary approaches to investigate species boundaries forms the foundation for the proper understanding, conservation and utilisation of biodiversity resources.Whether in natural reserves or in urban and rural areas with frequent human activity, it is believed that there are additional undiscovered groups, akin to the Angiopteris nodosipetiolata.We hope that these groups are not lost to extinction before humanity can gain a comprehensive understanding of them.

Figure 1 .
Figure 1.Maximum Likelihood and Bayesian Inference tree of Angiopteris species, based on (A) complete plastid genome sequences and (B) 84 CDSs.Maximum Likelihood bootstrap support (RBS) and Bayesian Inference posterior probability (PP) are given above the branches.NA: written abbreviation for not available, used to show the sampling site cannot be provided.

Figure 3 .
Figure 3. Angiopteris nodosipetiolata Ting Wang tris, H.F.Chen & Y.H.Yan A, B habitat C, D, E lamina F portion of stipes showing pulvini G rhizome H, I sporangia J pinnae backside, showing a dense covering of hairs K exospores L petiole scales.

Table 1 .
List of vouchers used in this study.

Table 2 .
The detailed parameters for the phylogenetic tree construction.

Table 3 .
Morphological comparison of Angiopteris nodosipetiolata and its similar taxa.