ISSN 1346-7565 Acta Phytotax. Geobot. 71 (1): 13–21 (2020) doi: 10.18942/apg.201910 Asplenium serratipinnae (Aspleniaceae: Polypodiales), a New Allotetraploid Species in the A. normale Complex 1,†,* 1 2 1 Tao Fujiwara , junki ogiso , sadamu maTsumoTo and YasuYuki waTano 1Department of Biology, Graduate School of Science, Chiba University, Yayoi-cho, Inage, Chiba 263-8522, Japan; 2Department of Botany, National Museum of Nature and Science, 4-1-1 Amakubo, Tsukuba 305-0005, Japan. †Present Address: Center for Integrative Conservation, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Mengla 666303, Yunnan, China. *[email protected] (author for correspondence) Asplenium serratipinnae (Aspleniaceae: Polypodiales), an allotetraploid species between the diploid race of A. normale and A. oligophlebium is described as new. It is endemic to Japan and morphologically most similar to A. normale, but differs in having narrower pinnae with an auriculate to hastate acroscopic base and deeply serrated margins. Key words: Aspleniaceae, Asplenium normale, polyploidy, taxonomy, tetraploid Asplenium L. (Aspleniaceae), one of the most Chang et al. (2013) collected samples of these species-rich genera of ferns, comprises approxi- species from regions throughout Japan, China, mately 700 species (PPG1 2016, Smith et al. Southeast Asia, Hawaii and Africa and conducted 2006). It occurs in all tropical and temperate re- phylogenetic analyses using chloroplast DNA gions. Asplenium is characterized by having the (cpDNA) and nuclear gene markers. Chang et al. highest proportion of polyploids among derived (2013) showed that recurrent reticulation events ferns (Schneider et al. 2017). In ferns, many spe- occurred among diploid members of this species cies complexes were formed through frequent re- complex and that the tetraploid race of A. nor- ticulate evolutionary events, e.g. the Appalachian male in Japan was an allotetraploid between a Asplenium complex (Werth et al. 1985), the New diploid race of A. normale in China and Southeast Zealand Asplenium complex (Shepherd et al. Asia and an unknown diploid race of A. boreale. 2008), and the A. monanthes complex (Dyer et al. In addition, Chang et al. (2013) suggested that A. 2012). The Asplenium normale complex belongs boreale was an autotetraploid and A. shimurae to the ‘black-stemmed’ spleenwort group (Sch- was an allotetraploid between an unknown dip- neider et al. 2005). In Japan, three tetraploid spe- loid race corresponding to a clade of A. shimurae cies, A. normale D. Don, A. boreale (Ohwi ex Sa. in their cpDNA phylogeny and the diploid race of Kurata) Nakaike and A. shimurae (H. Itô) Na- A. normale in China and Southeast Asia. Recent- kaike, and a diploid species, A. oligophlebium ly, Chang et al. (2018) recognized six diploid spe- Baker, have been recognized in the complex. cies within this complex, including two novel Their recognition is supported by sufficient dif- species, A. normaloides Y. Fen Chang & H. Sch- ferences in rbcL gene sequences (Murakami et neid. corresponding to the diploid race in the al. 1999), sterility of their hybrids (Matsumoto et clade of A. shimurae in the cpDNA phylogeny, al. 2003) and distinct flavonoid patterns (Iwashi- and A. guangdongense Y. Fen Chang & H. Sch- na & Matsumoto 1994, Matsumoto et al. 2003). neid. corresponding to the diploid race of A. boreale, 14 Acta Phytotax. Geobot. Vol. 71 by employing their ‘diploid first’ approach with 12 samples of cryptic species II, four samples of an integrated taxonomic analysis including cyto- cryptic species III, one sample of the tetraploid logical, morphological and phylogenetic analy- Asplenium normale from Taiwan (an allotetra- ses. Although diploid species or races have been ploid between the diploid race of A. normale and discovered in almost all major clades in the cpD- A. guangdongense) and four samples of the dip- NA phylogeny of this complex through their ef- loid A. normale from Taiwan (Appendix 1). All forts, the classification of the tetraploid lineages samples used in the present study were collected remains a major problem. by Fujiwara et al. (2017). We obtained a digital The Asplenium normale complex in Japan ex- image of a frond from a dried specimen of each hibits high variation in flavonoid content (Iwash- sample by using a scanner (GT-X980; Epson, Na- ina et al. 1990, 1993, Iwashina & Matsumoto gano, Japan). A middle pinna representative of 1994, Matsumoto et al. 2003, Fujiwara et al. each sample was selected and its morphological 2014). Tetraploid plants of A. normale can be di- characteristics were analyzed using ImageJ soft- vided into eight chemotypes, one of which has ware (Schneider et al. 2012). the same flavonoid composition as A. oligophle- To measure the shape and size of the pinnae, bium (Iwashina & Matsumoto 1994, Fujiwara et we marked the following points on each pinna al. 2014). By integrating our results from popula- (Fig. 1): the apex (A), the base (B), the intersect- tion genetics, chemotaxonomy and phylogenet- ing points of a perpendicular line at the midpoint ics, three cryptic species (I–III) with distinct fla- of line segment AB with the acroscopic and ba- vonoid compositions within Japanese tetraploid siscopic margins of the pinna (C and D), and the A. normale were noted (Fujiwara et al. 2017) The apex of the acroscopic basal lobe (E). Line seg- study revealed that cryptic species I and III origi- ments AB and CD were defined as pinna length nated from hybridization between the diploid and pinna width, respectively. Angles CAD and race of A. normale and A. guangdongense (treat- EBD were defined as the angle of the apex and the ed as an unknown diploid race of A. boreale in angle of the base, respectively. With respect to Fujiwara et al. 2017), whereas cryptic species II the extent of tooth development on the pinna mar- originated from hybridization between the dip- gin, we measured the relative area (S) and relative loid race of A. normale and A. oligophlebium. Ad- perimeter (L) of the pinna and defined the teeth ditionally, it was noted that cryptic species II index as L2S-1, which was expected to increase could be distinguished by morphological charac- with teeth development. Principal component teristics of the pinnae (Fujiwara et al. 2017). analysis (PCA) was conducted with R v.3.3 (R In this study, we measured morphological fea- Development Core Team 2013), using six vari- tures to discriminate cryptic species II (an allo- ables: 1) pinna length, 2) pinna width, 3) pinna tetraploid with A. oligophlebium as one of its dip- length to pinna width ratio (pinna length/pinna loid parents) from the diploid race of A. normale width), 4) apex angle, 5) base angle, and 6) the and the other allotetraploids with a different pa- tooth index. Differences in morphological char- rental species pair (the diploid race of A. normale acteristics between groups were tested for statis- and A. guangdongense) in the A. normale com- tical significance using Welch’s t test (Welch plex From our findings, we determined that cryp- 1947). tic species II represents a distinct species, which For the scanning electron microscope (SEM) we describe here as new. images, the spores were transferred from dried specimens to double-sided carbon tape on an alu- minum specimen holder. The samples were then coated with gold using a sputter coater (JEOL Materials and Methods JFC-1100; JOEL. Tokyo, Japan) and imaged us- ing a SEM (JEOL JSM-6510A; JOEL) at an accel- We analyzed 48 samples of cryptic species I, erating voltage of 15 kV. February 2020 Fujiwara & al. — A New Allotetraploid Species in Asplenium normale Complex 15 ently similar to A. normale × A. oligophlebium, which is a putative triploid hybrid (Ebihara 2016), but can be distinguished by the normally shaped spores 33–41.5 μm in diameter (Fujiwara et al. 2017, Fig. 1 and Supplemental Data). The present study revealed morphological differences between cryptic species II and the other samples of Asplenium normale. Cryptic species II is also unique among tetraploid taxa in Fig. 1. Diagram of pinna showing points used for morpho- having A. oligophlebium as one of its diploid par- logical measurements. ents. We therefore describe the allotetraploid spe- cies between the diploid race of A. normale and A. oligophlebium as a new species, A. serratipin- Results and Discussion nae T. Fujiw. & Watano. It should be noted that A. serratipinnae appears to be endemic to Japan, The six morphological characteristics were based on the specimens we examined, as is one of summarized using PCA; the first two axes, PC1 its diploid parents, A. oligophlebium (Nakaike and PC2, explained 62.3% and 26.1% of the vari- 1992). Genetically, A. serratipinnae is character- ation, respectively (Fig. 2). PC1 correlated posi- ized by sharing identical sequence alleles with A. tively with the pinna length to width ratio and the oligophlebium at all three nuclear loci examined tooth index, and negatively with the apex and (LFY, PgiC and NIA) (Fujiwara et al. 2017). Al- base angles. PC2 was negatively correlated with though Chang et al. (2013, 2018) examined the the two size variables, pinna length and width. PgiC gene in samples containing tetraploid A. The PCA result indicated that the samples could normale that were widely collected from China not be separated into the tetraploid cryptic spe- and Hawaii, samples that shared alleles with A. cies nor the diploid Asplenium normale, but rath- oligophlebium were not reported. This may be er into two groups: group 1, which contained all another indication that A. serratipinnae occurs samples of cryptic species I, cryptic species III only in Japan. and the Taiwanese A. normale samples including Chang et al. (2018) recognized ten species in diploids, and group 2, which contained only cryp- the A. normale complex; nine of them being ei- tic species II.