﻿Lepisorusmedioximus (Polypodiales, Polypodiaceae), a new species from Shan State of Myanmar

﻿Abstract A new species of the species-rich fern genus Lepisorus (Polypodiales, Polypodiaceae) has been found to occur in Shan state, Myanmar. Lepisorusmedioximus is described based on morphological characters and phylogenetic evidence. Phylogenetic analyses showed that the specimens of L.medioximus formed a distinct clade nested in the Pseudovittaria clade. The morphological comparison demonstrated that the species is distinct from phylogenetically related species, namely L.elegans, L.contortus, and L.tosaensis, in the morphology of the rhizome scales, size, and shape of the lamina, position of sori, and paraphyses.


Introduction
The genus Lepisorus (J. Sm.) Ching (Polypodiaceae) occurs throughout Eastern and Southern Asia with range extensions towards the Pacific islands including Hawai'i and towards tropical Afromadagascar (Ching 1933;Zink 1993;Wang et al. 2012). Taking into account the various taxonomic studies and the recent proposal to expand the generic circumscription by including all genera of the tribe Lepisoreae such as Lemmaphyllum C. Presl, Lepidomicrosorium Ching & K.H. Shing, Neocheiropteris Christ, Neolepisorus Ching, Paragramma (Blume) T. Moore, and Tricholepidium Ching (Zhao et al. 2020) Lepisorus can be currently recognized as one of the most species-rich genera among genera in Polypodiaceae, comprising ca. 90 species in 18 sections (PPGI 2016;Fujiwara et al. 2020;Zhao et al. 2020). Whereas the core of the genus Lepisorus (Lepisorus s.s.) is easily recognized by its unique suite of morphological characters including creeping rhizomes covered by clathrate scales, simple leaves, and sori covered with scale-like paraphyses (Ching 1933;Qi et al. 2013;Zhao et al. 2020), some controversy still exists concerning the broader circumscription to avoid the need to recognize the genus Ellipinema Li Bing Zhang & Liang Zhang . Despite significant progress (Wang et al. 2010a;Wei et al. 2017;Zhang et al. 2020;Zhao et al. 2020), taxonomic uncertainty is arguably not restricted to the generic classification but affects the estimation of the total species diversity that is expected to be still underestimated due to the difficulty in the taxonomic classification of this genus. Several characters utilized as key information on species delimitation show high variation within some species (Wang et al. 2010b). In particular, the identification of species relies on a few diagnostic characters such as the shape of the lamina, position of sori, and structure of the rhizome scales and paraphyses. Unfortunately, these characters are hardly diagnosable in the field. As a consequence, some species have been frequently misidentified or overlooked, as exemplified by the recent reclassification of Japanese L. thunbergianus (Kaulf.) Ching and relatives (Fujiwara et al. 2018), and the rediscovery of Lepisorus cespitosus Y.X. Lin previously known only as type specimens (Fujiwara et al. 2020).
Yunnan has been increasingly recognized as the diversity hotspot of Lepisorus s.s (Wang et al. 2012;Fujiwara et al. 2020). While sufficient explorations of the Lepisorus diversity have been made available for China and India, this cannot be claimed for the regions south and southwest of Yunnan, namely Laos and Myanmar. These two countries as well as Thailand show extremely low species diversity of the genus despite being expected to harbor a notable diversity of this genus (Suppl. material 1: Table S1). This can be attributed to not only the difficulty in the taxonomic classification of this genus but also the underestimation of whole fern species diversity due to fewer flora surveys previously conducted. Thus, overlooked species are expected to occur in these regions.
The Shan state of Myanmar is the focus of this study. The Shan state covers 155,800 km 2 which is almost a quarter of the whole area of Myanmar and is mainly comprised of a hilly plateau bordering Yunnan, China in the north, Laos in the east, and Thailand in the south. Shan state has been in historical times much less surveyed than Yunnan Province of China although the latter is known for its rich diversity of ferns including Lepisorus. Thus, we expect to retrieve not only new records but also some new fern species that are putative endemics to Shan State. To make this expectation tested, floristic inventories were carried out across the Shan state in September 2019. Two unusual specimens of Lepisorus were collected in Pin Laung Township, Ka Thaung (upper) located in the southern part of the state, which were recognized as a putative new species. This proposal was studied by consulting checklists of Myanmar and adjacent areas (Dickason 1946;Lindsay and Middleton 2012;Qi et al. 2013;Khine et al. 2017;Khine and Schneider 2020;Hori 2021;Vongthavone et al. 2021), and careful comparison of morphological characters with previously described species by consulting specimens and species protologues (e.g., Ching 1933;Bir and Trikha 1969;Lin 1996, 1997;Lin 2000;Qi et al. 2013). Besides morphological diagnostics, we employed DNA sequences to identify genotypic evidence supporting these two specimens as distinct species from any other species that are previously described.

Morphology
The morphology of the two specimens of Lepisorus sp. (Hori et al. 108225 and 108229) was compared to descriptions and specimens of species sharing similarities in the main diagnostic features, namely rhizome scales, the size and shape of the lamina, the position of sori, and paraphyses. The morphological observation was conducted using a stereomicroscope. Voucher specimens were deposited in MBK, HITBC, and RAF.

DNA extraction and chloroplast DNA region sequencing
Total DNA for each of the two specimens was extracted from silica dried leaves using the CTAB method according to Doyle and Doyle (1987). Four plastid regions, rbcL gene, rbcL-atpB intergenic spacer, rps4-trnS intergenic spacer, and trnL-trnF region including the trnL intron and the trnL-trnF intergenic spacer were amplified according to the protocol provided (Wang et al. 2010b) using ExTaq (TaKaRa Bio Inc., Shiga, Japan). The PCR products were purified using Illustra ExoStar 1-Step (GE Healthcare, Wisconsin, USA) and used as templates for Sanger sequencing. Reaction mixtures for sequencing were prepared using the SuperDye v3.1 Cycle Sequencing Kit (ADS). The reaction mixtures were analyzed using an ABI 3130 Genetic Analyzer (Applied Biosystems).
ML analyses were performed using IQ-TREE v.1.6 (Nguyen et al. 2015) with default settings. 1000 ultrafast bootstrap replicates were conducted to infer node robustness (Hoang et al. 2018). For the BI method, MrBayes 3.2.6 (Ronquist et al. 2012) was employed by applying two runs of four MCMC chains for 100,000,000 generations with samples taken every 1000 generations. Tracer 1.6 (Rambaut and Drummond 2013) was used to evaluate the samples trees with a focus on convergence. The first 25% were discarded as burn-in. The MP analysis was performed using a heuristic approach with TBR branch-swapping, as implemented in MEGAX (Kumar et al. 2018). Ten initial trees were generated by the addition of randomly selected sequences. The robustness of each branch was assessed by bootstrap analysis calculating 1000 replicates.

Results and discussion
The combined dataset of rbcL, rbcL-atpB, rps4-trnS, and trnL-F contained 4,617 bp of which 744 sides were parsimoniously informative. The optimal log-likelihood for the reconstructed phylogeny inferred by the ML method was ln = -21430.138. The topologies were congruent among the phylogenetic hypothesis obtained using the three distinct phylogenetic inference approaches. The result showed that two specimens of Lepisorus from the Shan state of Myanmar formed a clade with a bootstrap value of 100% (ML ultrafast bootstrap value = 100%, MP bootstrap value = 100%) and BI posterior probability of p = 1.0. This clade was nested in the subclade of the sect. While the latter two species were highly distinct from the new species in their linear to linear-lanceolate lamina, the accumulated substitution event causing a rather long branch separated the two specimens from L. elegans-the morphologically most similar species of this clade (Fig. 1).

Lepisorus medioximus
Etymology. The epithet 'medioximus' refers to the sori attached to the middle location on lamina.
Distribution. This species is only known from the type locality in Myanmar, Shan state. Note. Until now, we have not discovered additional specimens from other localities despite our exhaustive search focusing on herbarium specimens collected in all parts of Myanmar and the Yunnan province of China. We specifically checked not only the Myanmar Lepisorus specimens deposited to the Makino Botanical Garden (MBK), the Institute of Botany, Chinese Academy of Sciences at Beijing (PE) and the Royal Botanic Gardens (K) but also the Lepisorus specimens of Dickason collection deposited in the United States National Herbarium (US), the Natural History Museum (BM), and Naturalis Biodiversity Center (L). Given the observation of more than 50 individuals of the species at the type locality, we expected this species to be abundant in this poorly collected area. Further inventories in Shan state and the adjacent areas should be necessary to find new localities of the species and evaluate the conservation status of the species. Reflecting our limited knowledge, the IUCN red list status of this species is given as "Data Deficient" instead of "Critical Endangered". The latter status would assume a restriction of this species range to the two localities recorded.