Pteris latipinna sp. nov. (Pteridaceae), a new species segregated from Pteris fauriei

Abstract Pteris fauriei is widely distributed in Eastern Asia and has high morphological variation. Some morphologically similar plants related to this species are difficult to distinguish. We showed that the new Pteris species from Taiwan, previously identified as P. fauriei, can be morphologically distinguished by its wide pinnae, larger terminal pinnae than the lateral pinnae in sterile fronds, and triangular basal segments of the lateral pinnae. It was confirmed that this species is phylogenetically separated from the other East Asian Pteris species, except for a morphologically distinct species P. arisanensis, by means of chloroplast genes, rbcL and matK. The new species is named as Pteris latipinna sp. nov., referring to its wide pinnae. Here, we provide a key to facilitate the identification of the morphologically similar Pteris species in Asia. The morphological descriptions, images, ecology, and distribution are also presented.


Introduction
Pteris fauriei Hieron. is widely distributed in Eastern Asia. Two varieties of P. fauriei have been confirmed, and both varieties have different cryptic characteristics and prefer different niches. Pteris fauriei Hieron. var. fauriei, apomictic and triploid (2n = 87), usually has herbaceous laminae and prefers cooler sites; P. fauriei var. minor Hieron., sexual and diploid (2n = 58), usually has coriaceous laminae and is found in warmer sites (Huang et al. 2006;Huang et al. 2007). In Taiwan, some undescribed Pteris plants (Fig. 1), usually regarded as P. fauriei var. fauriei, with herbaceous laminae were found in understory of evergreen forests. However, those plants have wider laminae and pinnae than other bipinnatifid Pteris species recorded in Taiwan. Outside Taiwan, P. natiensis Tagawa, a Japanese endemic fern (Iwatsuki 1995), apomictic and diploid (Nakato and Ebihara 2016), is the most morphologically similar species in East Asia.
Pteris fauriei and morphologically similar Pteris species are phylogenetically close. Chao et al. (2014) revealed that the clade (A1, including those species with bipinnatified laminae mostly) arose more recently than most of other clades in Pteris. Because of similar morphology, the characteristics to delimitate species need to be examined and compared in detail, such as venation, scale color, shapes of pinnae and segments, and pinnae stalked or sessile (Chao et al. 2013).
In this study, we clarified the morphological and phylogenetic characteristics of the undescribed Pteris plants, in comparison with P. fauriei, P. natiensis, and related bipinnatifid Pteris species from East Asia, including P. wulaiensis C.M. Kuo endemic to Taiwan; P. arisanensis Tagawa, P. biaurita L., P. kawabatae Sa. Kurata, P. kiuschiuensis Hieron., and P. oshimensis Hieron. distributed in China and Japan; and P. boninensis H. Ohba, P. laurisilvicola Sa. Kurata, P. satsumana Sa. Kurata, and P. yakuinsularis Sa. Kurata endemic to Japan (Iwatsuki 1995;Liao et al. 2013). On the basis of morphological and molecular data, the taxonomic treatments were applied.

Phylogenetic analyses
To clarify the phylogenetic relationships of the undescribed plants, 34 other Pteris taxa with bipinnatifid laminae were sampled. Three Pteris species, P. grevilleana, P. longipinna, and P. venusta, were used as outgroups. These bipinnatifid and outgroup species belong to clades A1 and A2, respectively, according to the phylogenetic tree of Pteris . Vouchers and GenBank accession numbers are listed in Appendix 1. Total genomic DNA was extracted from young fronds, following a modified cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle 1990). Two chloroplast genes, rbcL and matK, were amplified using the PCR primers for rbcL and matK as per Chao et al. (2014). Alignment was performed with ClustalW (Thompson et al. 1994) and manually edited using BioEdit 7.1.3 (Hall 1999). Gaps were treated as missing data.
Maximum likelihood (ML) analyses were performed using GARLI v.2.0.1019 (Zwickl 2006). Ten independent runs were conducted using automatic termination following 10,000 generations without a significant (lnL increase of 0.01) change in topology. To calculate ML bootstrap support for each node, 1,000 bootstrap replicates were performed with automatic termination at 10,000 generations, under one run.

Morphology
The distinct morphologies that distinguished the undescribed species from other bipinnatifid Pteris species are its wide pinnae, up to 7 cm wide, and fewer pairs of lateral Ovate to lanceolate, more and less narrowed at base Width of lateral pinna 3-7 cm 2-3.5 cm 1-3 cm 3-5 cm pinnae, only 2-5 pairs ( Fig. 1). Furthermore, its terminal pinnae of sterile fronds are larger than the lateral pinnae (Table 1). In Taiwan, these characteristics can separate the undescribed species from P. fauriei var. fauriei and P. fauriei var. minor (these two taxa were illustrated by one of their type materials, Figs. S1 and S2, respectively). An endemic species in Japan, Pteris natiensis (illustrated by holotype, KYO, Fig. S3), also has sterile fronds with slightly larger terminal pinnae than the lateral pinnae. Its pinnae are slightly narrower than those of the undescribed species (3-5 cm vs. 3-7 cm), and the basal pinna-segments are adnate to the rachis whereas they are not adnate to the rachis in the undescribed species (Table 1). Another specific trait of the undescribed species is the triangular (vs. falcate) basal segments of the lateral pinnae, which could be used to identify the new species from other similar species, including P. fauriei and P. natiensis (Table 1). The triangular and falcate basal segments are resulted by the longer costa adnate with the segments of the undescribed species and shorter costa adnate with the segments of the other species, respectively.

Phylogeny and chloroplast DNA differences
Genetic data and the accession numbers of the sequences are listed in Appendix 1. The chloroplast DNA (cpDNA) alignment matrix of rbcL (1,278 bp) and matK (900 bp) contained a total of 2,178 characters with 121 parsimony-informative sites. The loglikelihood score for the most likely ML tree was -5304.42470.
The phylogenetic tree ( Fig. 2) infers that the Pteris species with bipinnatifid laminae formed one monophyletic group (the clade of ingroup taxa), as revealed in the previous Pteris phylogeny ). The undescribed taxon and P. fauriei were divided into two different clades, Clade I and II. In Clade I, the undescribed taxon shared identical cpDNA sequences with P. arisanensis, although they can be separated by their morphologies, such as venation and lamina shape (Fig. S4). The undescribed taxon cpDNA differed from P. natiensis, P. wulaiensis (Fig. S5), and P. yakuinsularis cpDNA by one nucleotide substitution, and from P. laurisilvicola cpDNA by two nucleotide substitutions. In Clade II, P. fauriei var. fauriei, P. fauriei var. minor, and P. oshimensis shared identical cpDNA sequences.
Other Distribution. Taiwan (Fig. 4). Ecology. In shaded places, understory of evergreen broad leaf forests, below 1,000 m in elevation.
Etymology. The specific epithet 'latipinna' refers to its wide pinnae. Preliminary conservation assessment. We investigated the distribution of P. latipinna Y.S.Chao & W.L.Chiou, sp. nov. in Taiwan. To date, only a few small populations are recorded. However, the 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.

Discussion
A new species, P. latipinna Y.S.Chao & W.L.Chiou, sp. nov., growing understory of forests in Taiwan was found and identified in this study. Pteris latipinna is the largest species among the bipinnatifid Pteris species with single-axis in Taiwan. There were 29 Pteris species recorded in the Flora of Taiwan (Shieh 1994), and several new species and new records have been recently found (Chao et al. 2013;Chao et al. 2015;Ebihara et al. 2014;Knapp 2011;Knapp and Hsu 2017). In this study, we describe one more new species, and thus in total, 36 Pteris species, including infraspecies, have been documented in Taiwan. Although the ploidy of P. latipinna is not known, with the similar morphology and apomitic reproductive mode, it is inferred that those species possibly evolved through a complex reticulate hybridization-polyploidization speciation. Those apomicitic Pteris species have also been suggested with possible hybrid origins (Chao et al. 2012a;Chao et al. 2012b;Walker 1979). Pteris latipinna has 32 spores per sporangium, which is thought as apomictic (Chao et al. 2010;Huang et al., 2006;Nakato 1975;Walker 1979). For those species in the same clade (Clade I) of P. latipinna, it is reported that P. laurisilvicola is diploid and triploid and apomictic (Nakato 1996;Nakato and Ebihara 2016); P. natiensis and P. wulaiensis are diploid (Huang et al. 2011;Kurita 1962;Nakato and Ebihara 2016); P. yakuinsularis are triploid (Nakato and Ebihara 2016); P. arisanensis is tetrapolyploid (Tsai and Shieh 1984). Remarkably, P. latipinna and P. arisanensis have the same cpDNA characteristics although their morphologies are clearly different. They have different lamina shapes (wide ovate for P. latipinna vs. ovate for P. arisanensis) and venation (free veins in P. latipinna vs. costal areolae in P. arisanensis) (Fig. S4). Similarly, in Clade II, P. oshimensis does not morphologically resemble P. fauriei but share identical cpDNA sequences. More cpDNA and nuclear DNA markers are needed to clarify the relationships among these species in P. fauriei complex.
In this study, taxa in Clade I and Clade II compose Pteris fauriei complex because they are morphologically similar and phylogenetically close with Pteris fauriei. All of them are distributed in Asia, mostly in Japan and Taiwan. Interestingly, distributions of most of those species are limited: Pteris latipinna and P. wulaiensis are endemic in Taiwan; P. boniensis, P. natiensis, and P. yakuinsularis are endemic in Japan (Iwatsuki 1995;Shieh 1994). This pattern of distribution implies those species arose in a small area within a short time recently .
The traits useful for separating P. latipinna from the similar species are used in a key for identification of this species as shown below.