﻿Two new species in the fern genus Lomariopsis (Lomariopsidaceae) from East Asia

﻿Abstract Two East Asian Lomariopsis (Lomariopsidaceae, Polypodiales) species, Lomariopsismoorei and Lomariopsislongini, which were previously misidentified as L.spectabilis, are here described as new species based on evidence from morphological characters and a molecular phylogeny. The two species differ from the three other described species in East Asia by their venation, pinna shapes, and perine morphology. A phylogeny based on a combined dataset of three chloroplast regions (rbcL+ rps4-trnS + trnL-L-F) showed that L.moorei and L.longini each formed a well-supported monophyletic group which was distantly related to both L.spectabilis and the other morphologically similar East Asian species, L.boninensis.


Introduction
Lomariopsis Fée is the most species rich genus in the fern family Lomariopsidaceae and contains approximately 60 spp., accounting for 85% of the family (PPG I 2016). This genus has a wide distribution in tropical and subtropical regions; there are 15 species in the Neotropics (Moran 2000), nine species in Africa (Roux 2009), 11 species in the islands of the Indian Ocean (Holttum 1939b;Roux 2009;Rakotondrainibe and Jouy 2017), and 12 species in Asia and the Oceanian region (Holttum 1932(Holttum , 1939a(Holttum , 1966(Holttum , 1978. The latest phylogeny of Lomariopsis included 24 species (ca. 40% of the species diversity in Lomariopsis), but only two species from Asia and the Oceanian region have been sampled (Chen et al. 2017) while the vast majority (ca. 10 species) from these areas have not yet been surveyed. In addition, because gametophytes of Lomariopsis species are able to establish as long-lived, asexual colonies in the wild (Watkins and Moran 2019), several species are found as gametophyte-only populations, which is called independent gametophytes (Pinson et al. 2017). In Japan and Taiwan, gametophytes of unknown species have been also reported (Ebihara et al. 2013;Wu et al. in press), a finding which further points out that the efforts of systematics research for Asian Lomariopsis remains inadequate, and there might have been undocumented and cryptic species.
To investigate phylogenetically Lomariopsis from these poorly sampled areas, we sampled most Asian and Oceanian species, including all species in East Asia where two previously unidentified species were discovered. They both had been misidentified as L. spectabilis Mett. One was from Chiayi County in Taiwan and Hainan Island in China, and the other one was from northern Vietnam and west southern China. They are superficially similar to two Asian species, L. boninensis Nakai and L. spectabilis in morphology. In this study, we presented a new Lomariopsis phylogeny supplied with comprehensive East Asian sampling, and reevaluated diagnostic characters leading to the description of these species.

Materials and methods
Perine morphology and spore number in sporangia Spores were taken from mature sporangia and fixed on double-sided tape, and then gold coated with a sputter-coater for 1-3 min. Spores were subsequently examined with a tabletop SEM (TM 3000; Hitachi, Ibaraki, Japan).
To examine the spore number per sporangium, at least five mature, unopened sporangia per specimen were collected. These sporangia were broken individually, and we counted the number of spores inside under a stereomicroscope.

DNA extraction and chloroplast DNA region sequencing
Twenty-nine samples were included in our molecular phylogenetic study. Voucher information is provided in Appendix 1 (i.e., those samples noted with *). Total DNA extraction was done following the modified CTAB protocol of Kuo (2015). Three chloroplast (cp) regions were amplified and sequenced: trnL-L-F (trnL gene + trnL-trnF intergenic spacer), the gene rbcL, and rps4-trnS (rps4 gene + intergenic spacer), which were also used in previous phylogenies of Lomariopsis and Lomariopsidaceae (Rouhan et al. 2007;Li et al. 2009;Chen et al. 2017). The primers used for PCR amplification and sequencing were: FernL 1Ir1 (Li et al. 2010) and f (Taberlet et al. 1991) for trnL-L-F; rps5 (Nadot et al. 1994) and trnS (Souza-Chies et al. 1997) for rps4-trnS; af (Hasebe et al. 1994) and 1379R (Pryer et al. 2001) for rbcL. PCR amplifications were prepared in 15 μL reactions each containing 20 ng of genomic DNA, 1× SuperRed PCR Master Mix RED (TOOLS, Newtaipei City, Taiwan) and 0.5 μM of each primer. A typical amplification program began with one initial denaturation step for 5 min at 94 °C then 35 cycles of 1 min at 94 °C, 30 s at 55 °C, and 1 min at 72 °C followed by a final extension of 10 min at 72 °C and was performed on a SimpliAmp Thermal Cycler. PCR products were cleaned using ExoSAP-IT (Thermo Fisher Scientific, Waltham, Massachusetts, USA), and then sequenced with the same PCR primers with an ABI 3730XL (Thermo Fisher Scientific, Waltham, Massachusetts, USA) by the Genomics BioSci. & Tech. company in Taiwan. GenBank accession numbers of the sequences are listed in Appendix 1.

Phylogenetic analyses
In total, we sampled 35 Lomariopsis species, including African/Malagasy and Neotropical members sequenced in previous studies (Rouhan et al. 2007;Lehtonen and Cárdenas 2019), and representatives from the three remaining Lomariopsidaceae genera (Chen et al. 2017) as outgroups. Importantly, our Lomariopsis sampling covered almost all Asian and Oceanian species (Holttum 1932, 1939a, 1939b, Moran 2000, including four of which were phylogenetically investigated for the first time. Before this study, three species were known to be distributed in East Asia: L. lineata (C.Presl) Holttum (syn. L. cochinchinensis Fée,), L. chinensis Ching, and L. boninensis. The materials of East Asian "L. spectabilis" belonged to either L. boninensis or one of the two new species described here. Except for L. chinensis, all East Asian species were included in our sampling. Voucher information for all samples is provided in Appendix 1. The sequences were aligned using MUSCLE (Edgar 2004) as implemented in AliView (Larsson 2014). The alignment of every coding gene was further divided into three partitions based on the codon positions. The portions of rps4-trnS IGS (intergenic spacer), trnL gene, and trnL-F IGS were each treated as an independent partition as well. In the phylogenetic analyses, each partition was assigned the appropriate substitution model, which was inferred by ModelFinder (Kalyaanamoorthy et al. 2017) and using the Bayesian information criterion (BIC, Schwarz 1978).
We used IQtree 1.6.8 (Nguyen et al. 2015) to infer maximum likelihood (ML) phylogenies with 1,000 standard bootstrap replicates. The Bayesian phylogenetic analysis was performed using Mr Bayes 3.2.7 (Ronquist et al. 2012). Two simultaneous runs were carried out with four chains (5 × 10 6 generations each). Each chain was sampled every 1,000 generations. Log likelihoods of MCMC runs were inspected in Tracer 1.6 (Rambaut and Drummond 2013) to confirm their convergence. The first 25% of the generations were conservatively discarded as burn-in.
Etymology. The lanceolate shape of the terminal pinnae of sterile leaves is similar to the holy lance, which is also called Lance of Longinus. Diagnosis. Lomariopsis moorei is most similar to L. boninensis, but scales on stipes are narrower (usually < 2 mm) in L. moorei (Fig. 5B) and broader (usually > 2 mm) in L. boninensis. The swollen ring at the region of articulation on the abaxial side (especially upper pinnae) is more obvious in L. moorei (Table 1; Fig. 6A).
Paratypes. Taiwan Distribution. Taiwan (Chiayi County) and China (Hainan Is.). Ecology. In shaded places, understory of evergreen broadleaf forests, below 1,000m in elevation. Etymology. The name moorei is dedicated to Dr./Mr. Shann-Jye Moore , an enthusiastic fern taxonomist and knowledgeable pteridologist from Taiwan. The Mr. Shann-Jye Moore Memorial Scholarship has been established by the Taiwan Society of Plant Systematics to commemorate his passions, and to support Taiwanese students studying the systematics of ferns and lycophytes.
Note. We have not yet found entire sporophyll from the type locality, but fallen fertile pinnae on14 Aug 2020 (Fig. 3), which contained intact sporangia with green spores. Although mature sporophytes were found to have a restricted distribution in Taiwan, independent gametophytes of this species were found throughout Taiwan Main Is using a DNA-identification approach to survey gametophyte populations (Wu et al. in press).

Discussion
In previous molecular phylogenies of Lomariopsis, none of Oceanian species were included, and L. lineata and L. spectabilis (including the misidentified L. boninensis and L. longini) were the only Asian species (Rouhan et al. 2007;Li et al. 2009;Chen et al. 2017). Here, with a comprehensive sampling in these areas, the present phylogeny ( Fig. 1) provides new insights into the evolutionary relationships and systematics for Lomariopsis species from these areas. In the present tree, the nine Asian/Oceanian species are retrieved into two well-supported clades. The first clade consists of L. boninensis only, while the second clade accommodates the remaining eight species. These Asian and Oceanian clades are either nested within, or closely related to other paleotropical species (Africa and Madagascar), but their inter-clade relationships remain unclear (Fig. 1). Data from additional genetic regions will be necessary to better resolve the uncertainties of these nodes, and hence to confirm biogeographical origin(s) of Asian/Oceanian taxa. Among all six described East Asian species, L. chinensis is the only one missing from the current phylogeny. To the best of our knowledge, this species has only been collected once, as the type collections. Despite the lack of phylogenetic information, L. chinensis is morphologically unique in the genus and easily distinguished from other Lomariopsis species because of its reticulate leaf venation. Lomariopsis species diversity in Asia and Oceania could still be underestimated, and more undocumented species could be eventually revealed by phylogenetic analyses using multiple specimens in each morphologically-defined species, similar to the case of discovering the two new species here described. Indeed, L. moorei and L. longini, together with L. boninensis, are genetically distant taxa in East Asia even if all three have been long misidentified and confused under a single name of the South East Asian species, L. spectabilis, due to their overall similar morphology (DeVol and Kuo 1975;Tsai and Shieh 1994;Iwatsuki et al. 1995;Yang and Liu 2002;Li et al. 2009;Phan 2010;Xing et al. 2013;Knapp 2014;Chen et al. 2017;Ebihara 2017;TPG 2019). However, clear molecular phylogenetic results spurred us to seek other characters supporting the distinction between these lineages, and these actual species now can be identified based on microscopic characters (Table 1). These characters include perine ornamentation, which has been revealed to have highly diversified forms in Lomariopsis (Rouhan et al. 2007). Additionally, we found that the spore number per sporangium varies among these species, which can also help in distinguishing species. However, unlike most cases in ferns of the Polypodiales, such a reduction in the number of spores in sporangia (e.g., 64 to 32) may not represent a reproductive switch to apomixis for Lomariopsidaceae (Chen et al. 2017). Further cytological investigations, e.g., through flow cytometry to infer both spore vs. leaf genome sizes , are necessary to clarify whether changes in the two phenomena (i.e., spore number per sporangium and reproductive mode) are linked in these Lomariopsis species.
Key to Lomariopsis longini, L. moorei, L. spectabilis, and other morphologically close species in East Asia