Rediscovery of Angiopteris tonkinensis (Marattiaceae) after 100 years, and its revision

Abstract The border area between south-eastern Yunnan, China and northern Vietnam is one of the regions with richest biological diversity including that of the fern genus Angiopteris (Marattiaceae). Based on the analysis of morphology and DNA sequences of multiple chloroplast regions (atpB, rbcL, rps4-trnS spacer and trnL-F spacer), we revised Angiopteris tonkinensis (Hayata) J.M.Camus and proposed a new combination Angiopteris tamdaoensis (Hayata) J.Y.Xiang & T.Wang, comb. nov., which was previously regarded as a synonym of A. tonkinensis. We found support for a monophyletic Angiopteris including Protomarattia. This discovery adds two new distribution sites of A. tonkinensis, one in China (Malipo, Yunnan) and one in Vietnam (Quan Ba, Ha Giang). We suggest A. tonkinensis should be categorised as Critically Endangered (CR) species according to the criteria of IUCN.


Introduction
The fern genus Angiopteris Hoffmann (1796) comprises about 30-40 species in the world and 28 species (17 endemic) in China (He and Christenhusz 2013). The border area between south-eastern Yunnan, China and northern Vietnam is one of the regions with the richest biological diversity including that of Angiopteris. According to He and Christenhusz (2013), there are 16 species (five endemic) in this area. The endemic species in this area include Angiopteris bipinnata (Ching) J.M.Camus (Camus 1989), A. dianyuecola Z.R.He & W.M.Chu (He and Chu 2006), A. latipinna (Ching) Z.R.He, W.M.Chu & Christenh. (He and Christenhusz 2013), A. sparsisora Ching (Ching 1982) and A. subrotundata (Ching) Z.R. He & Christenhusz (He and Christenhusz 2013).
During our fieldwork in Malipo (south-western China) on 18 May 2018 and Quan Ba (northern Vietnam) on 10 Oct 2019, two small populations of ferns caught our attention (Fig. 1). We identified them as Protomarattia tonkinensis Hayata (1919), a very rare species known only from the type specimen collected in Monte Tamdao (Tonkin) of Vietnam and that had never been recorded again since 1919.
In this study, we aimed to explore the identity of the materials collected from Malipo, Yunnan, China and Quan Ba, Ha Giang, Vietnam by means of morphological and phylogenetic studies. We inferred the phylogeny of Protomarattia tonkinensis and Archangiopteris tamdaoensis based on four chloroplast regions (atpB, rbcL, rps4-trnS and trnL-F), then we revised their taxonomic status.

Morphological analysis
The Voucher specimens were deposited at Southwest Forestry University (SWFU), the herbarium of Institute of Ecology and Biological Resources, Hanoi, Vietnam (HN) and Shanghai Chenshan Botanical Garden Herbarium (CSH). For morphological comparisons, primary literature (Hayata 1919) and the specimens were critically checked. Petiole scales were observed with a Nikon ECLIPES E100 biological microscope. Sporangia and venation of Protomarattia tonkinensis were observed with a LEICA M165 FC stereoscopic fluorescence microscope. The ornamentation of spores was observed with a ZEISS electron scanning microscope.

Phylogenetic analysis
We analysed 19 samples of Marattiaceae with DNA sequences of four chloroplast regions (atpB, rbcL, rps4-trnS spacer and trnL-F spacer). Fourteen sequences of seven species were newly generated for this study and their voucher information and GenBank accession numbers are presented in Table 1. Additional sequences of five species were downloaded from GenBank and their GenBank accession numbers are presented in Table 2.
Total genomic DNA was extracted from silica-gel dried leaves using the TSINGKE plant DNA extraction kit (generic). The sequences were amplified using the primers designed by previous studies: primers e and f for trnL-F gene (Li and Lu 2006), F1 and R4 for rbcL gene (Murdock 2008a), rps5' (Nadot et al. 1994) and trnS R (Smith and Cranfill 2002) for rps4-trnS, atpB-F1 (Murdock 2008a) and atpE 384R (Wolf 1997) for atpB. Sequencing was performed using the ABI 3730XL DNA analyser (Applied Biological Systems, Foster City, CA, USA).
Sequences were assembled and edited with SeqMan (Burland 1999) and then aligned and manually adjusted on Mega7.0 (Kumar et al. 2016). To estimate phylogenetic relationships, we applied Maximum Likelihood (ML) analysis with concatenated DNA datasets. PartitionFinder2 (Lanfear et al. 2016) was used to select a subset scheme and substitution models as assessed by the Bayesian Information Criterion (BIC). The best-fit scheme proposed two subsets: (rps4-trnS, trnL-F) and (atpB, rbcL). The best-fit model for subset1 was GTR+G and for subset2 was GTR+I+G. Maximum Likelihood analyses were performed using IQ-TREE v.1.6.8 (Nguyen et al. 2015) with 1000 thorough bootstrap replicates. Bootstrap values were labelled on the tree branches.

Endangered categories analysis
Following the Red List Categories and Criteria (IUCN 2001), we used the GEO-CAT tool (http://geocat.kew.org/; Bachman et al. 2011) to assess the current status of Poromarattia tonkinensis.

Results
The spores of Protomarattia tonkinensis were roundish-oblong. The ornamentation of the external perispore was coarsely echinate with spines occasionally forked at their apices and fused at their bases. Scales peltate, reddish-brown lanceolate, margin entire to sparsely denticulate, apex acuminate and scale cells elongate (Fig. 2). Protomarattia tonkinensis is morphologically quite similar to Archangiopteris tamdaoensis Hayata (Table 3), especially the horizontal dorsiventral rhizomes and simply pinnate fronds, but the former has submarginal synangia, ca. 4-6 mm, sporangia fully fused into synangia which lack pedicels, whereas the latter has medial sori, ca. 7-10 mm, sporangia respectively fused at base into receptacles (Hayata 1919; He and Christenhusz 2013). The combined data matrix included up to 2788 nucleotides for each of the 19 sequences of 12 species. The phylogenetic analyses (ML) resolved eight accessions of Protomarattia tonkinensis in a clade clearly separated from Archangiopteris tamdaoensis (Fig.  3). Angiopteris, Archangiopteris and Protomarattia form a monophyletic group with high support value, which proved that they were more closely related than with Ptisana.

Discussion
In the field, the populations of Protomarattia tonkinensis have eight mature individuals in Malipo and seven mature individuals (clones of the same individual excluded) in Quan Ba. Before our study, the species had not been recorded since its publication about 100 years ago. The assessment result shows that P. tonkinensis should be assessed as Critically Endangered (CR). More fieldwork is needed in similar forest regions in south-western China, Myanmar and Vietnam to confirm its distribution and conservation status.
There have been a number of controversies surrounding Protomarattia tonkinensis since its publication. Some suggested that it should be treated as a synonym of Archangiopteris tamdaoensis (Christensen and Tardieu-Blot 1935;Copeland 1947;Ching 1958;Ching 1959;Chen 1964;Camus 1989;He and Christenhusz 2013), while others regarded P. tonkinensis as a "good" species and argued that Protomarattia should be treated as a different genus (Pichi-Sermolli 1968,   Numbers on branches are support values of ML, * means 100%.