Taxonomic revision of the Asplenium wrightii complex (Aspleniaceae) with reinstatement of A. alatulum and A. subcrenatum

Abstract The Asplenium wrightii complex is morphologically variable and difficult in species delimitation. Owing to lack of comprehensive sampling in phylogenetic studies, the taxonomy of this complex remains unresolved. Based on extensive field observations, specimen examination and our recent molecular data, the present study aims to clarify the identities of three species of Asplenium in this complex from Asia. Our study revealed that A. alatulum and A. subcrenatum, previously treated as synonyms of A. wrightii, should be reinstated. A taxonomic revision of the three species, including their type information, detailed descriptions, voucher specimens, distribution, ecology, as well as taxonomic notes, is carried out.


Introduction
Asplenium L. (Aspleniaceae) comprises more than 700 species of epilithic, epiphytic or terrestrial herbs, distributed throughout temperate and tropical regions of the world (Kramer and Viane 1990). A number of systematists worked on the taxonomy of the
Rhizome scales were soaked in distilled water for 24 hours and then mounted on glass slides. The morphology of rhizome scales was observed and photographed using a stereo light microscope (LEICA S8APO).
Scanning Electron Microscope (SEM) images were taken of the spores and stomata of Asplenium alatulum, A. subcrenatum and A. wrightii. Spore and pinna samples, obtained from herbarium specimens, were mounted on specimen tabs and then coated with platinum in a sputter coater. Observations were conducted using a JSM-633OF SEM (JEOL Ltd., Akishima, Tokyo, Japan) scanning electron microscope with 10 kV at Sun Yat-Sen University, Guangzhou, China (Figs 1, 2).

Results
Though the density and shape of stomata are similar amongst Asplenium alatulum, A. subcrenatum and A. wrightii, the size of stomata is distinguishable amongst the three species. Asplenium alatulum has the largest stomata (60-70 μm in length and Table 1. List of the 14 species included in Asplenium ser. Wrightiana by Wu (1999) in alphabetical order, references being given in the right-hand column. Species indicated by asterisk were treated as synonyms of A. wrightii by Lin and Viane (2013). Four species, recognised by Lin and Viane (2013), are indicated with boldface. 40-45 μm in width, Fig. 1B, C, E, F), while the other two species A. subcrenatum and A. wrightii have similar size of stomata (45-65 μm in length and 25-35 μm in width, Fig. 1H, I, K, L). In addition, the rhizomes, scales, rachis wings, pinna pairs, pinna margins and perispores of the three species are very diverse, but relatively stable within species in morphology (Figs 1,2,4,5,6). Our results confirmed that A. alatulum and A. subcrenatum, previously treated as synonyms of A. wrightii, should be reinstated as distinct species. Asplenium subcrenatum is not restricted to their type locality, but it has an extensive geographical distribution in south-western China and northern Vietnam.
Asplenium alatulum is distinct from A. wrightii in having erect to decumbent rhizome (vs. erect rhizome), oblong cells of scales (Fig. 2C, F, I) (vs. quadrangle cells of scales, Fig. 2A, D, G), winged rachis (vs. winged towards apex) and fewer pinna pairs.  Phylogenetically, our earlier molecular work resolved A. alatulum in a distinct clade, sister to the clade containing A. wrightii and A. × shikokianum (Xu et al. 2020). One accession from the Taiwan Island was nested within A. alatulum, indicating that this species should also be distributed in Taiwan.  Fig. 3B.
Distribution and habitat. Asplenium subcrenatum is distributed in China and Vietnam. It grows as an epiphyte on tree trunks or occurs on rocks by stream-sides in the evergreen broad-leaved forests of limestone areas (Fig. 5A).  Note. Just like Asplenium alatulum, a comprehensive taxonomic study of A. subcrenatum was scarce after this species was described. Lin and Viane (2013) treated it as a synonym of A. wrightii, based only on macromorphological data. Both our molecular data (Xu et al. 2020) and micromorphological data in this study (Figs 1, 2) support the distinction of A. subcrenatum.
Asplenium subcrenatum is different from A. wrightii in having its stipe and rachis covered with reddish-brown scales (Fig. 2B, E, H) (vs. brown to dark brown scales, Fig. 2A, D, G), scale margins nearly entire (vs. glandular margins or long-fibrillose) and pinna margins almost entire to crenate-sinuate (vs. serrate to coarsely dentate). Meanwhile, the perispores of A. subcrenatum are different from those of A. wrightii. The folds of A. subcrenatum are cristato-alate and areolofenestrate and the margins of the folds are echinulate, while the folds of A. wrightii are cristato-alate and imperforate and the margins of the folds are echinulate or approximately laevigate.