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Research Article
The identity of Hypolepis robusta, as a new synonym of Hypolepis alpina (Dennstaedtiaceae), based on morphology and DNA barcoding and the new distribution
expand article info Morigengaowa‡§, Jun-Jie Luo§|, Ralf Knapp, Hong-Jin Wei§, Bao-Dong Liu‡, Yue-Hong Yan§, Hui Shang§
‡ Harbin Normal University, Harbin, China
§ Shanghai Chenshan Plant Science Research Centre, Chinese Academy of Sciences, Shanghai, China
| Shanghai Normal University, Shanghai, China
¶ Muséum National d’Histoire Naturelle, Eberbach, Germany

Corresponding author: Bao-Dong Liu ( 99bd@163.com )

Corresponding author: Hui Shang ( shanghui@csnbgsh.cn )

Academic editor: Blanca León
© 2018 Morigengaowa, Jun-Jie Luo, Ralf Knapp, Hong-Jin Wei, Bao-Dong Liu, Yue-Hong Yan, Hui Shang.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Morigengaowa, Luo J-J, Knapp R, Wei H-J, Liu B-D, Yan Y-H, Shang H (2018) The identity of Hypolepis robusta, as a new synonym of Hypolepis alpina (Dennstaedtiaceae), based on morphology and DNA barcoding and the new distribution. PhytoKeys 96: 35-45. https://doi.org/10.3897/phytokeys.96.23470
Open Access

Abstract

Based on field observations and examinations of herbarium specimens (including type material), consulting the original literature and molecular phylogenetic analysis of the rbcL and trnL-F sequences, it is concluded that Hypolepis robusta is conspecific with Hypolepis alpina and is here formally treated as a synonym of it. Additionally H. alpina is reported with new distribution records in Guangdong, Guangxi and the Hainan Island of China, respectively.

Keywords

Hypolepis alpina , molecular phylogenetic, synonym, taxonomy, Type material

Introduction

Hypolepis Bernh. (1805) is one of the largest genera in the family Dennstaedtiaceae, with approximately 80 species (PPG I 2016) widespread in tropical and southern temperate parts the world, mainly in tropical Asia and tropical America, but the exact number of species in China is still unclear (Brownsey 1987, Ching 1959, Xing et al. 2013). Amongst them, Hypolepis alpina (Blume) Hook. was initially described as Cheilanthes alpina Blume from Java in the first publication relating to the ferns of Malaya (Blume 1828). It was later transferred to Hypolepis by Hooker (1858) in the last comprehensive treatment of the genus (Brownsey 1987). Afterwards, one endemic species in the Taiwan province of China, Hypolepis alte-gracillima Hayata (1915), was reduced to a synonymy of H. alpina, according to the Flora of Taiwan (Shieh 1975). In addition to Taiwan, H. alpina is also distributed in Indonesia, Japan, Malaysia, Papua New Guinea and Philippines (Brownsey 1987, Fig. 1). Subsequently, the species (as H. alte-gracillima) was found in Gongshan County, in the Yunnan Province of China and recorded in Flora Yunnanica (Chu et al. 2006) as having a Yunnan-Taiwan discontinuous distribution. Another endemic species, H. robusta W. M. Chu was described for Yunnan (Chu et al. 2006). This name was treated as a synonymy of H. polypodioides (Blume) Hook. (Fraser-Jenkins 2008). Xing et al. (2013) cited a null name, (“H. robusta Hayata”) as a synonym of H. polypodioides in Flora of China, but Hayata’s name has not nomenclatural bearings nor taxonomic implications for Chu’s name. However, even Chu’s H. robusta is easily distinguishable from H. polypodioides in morphology as an obviously different species. Hypolepis robusta has densely multicellular brown glandular hairs and sori protected by well-developed reflexed adaxial indusium, whereas H. polypodioides has abundantly colourless non-glandular hairs and sori unprotected or occasionally protected by slightly reflexed green lamina segments. In June 2017, as part of the floristic inventory of Yunnan, H. robusta was collected at its type locality, Fugong County and H. alpina was collected at its recorded locality in Gongshan County. In addition, during the field work from 2013 to 2017, several specimens of H. alpina were collected from Taiwan as well as several others that were initially identified as H. robusta in Guangdong, Guangxi, Hainan Island and other locations of Yunnan. After conducting field observations, examinations of the herbarium specimens (including both types studied) and consulting the original literature (Hooker 1858, Chu 1992), it was suspected that H. robusta is conspecific with H. alpina. Therefore, the identity of H. robusta was determined by a more detailed examination of the morphology and molecular phylogenetic analysis.

Figure 1. 

The distributions of Hypolepis alpina noted by Brownsey (1987, blue line) and new record localities since then (red stars), using a map available from http://219.238.166.215/mcp/index.asp.

Materials and methods

Morphological studies

For morphological comparisons, herbarium specimens or high-resolution images of specimens in CSH, K, KUN, L, P, PE, PYU, TAI, TAIF and US were critically checked. Field observations and collections were made in Guangdong, Guangxi, Hainan Island, Taiwan and Yunnan of China (Suppl. material 1: Table S1).

Molecular phylogenetic studies

Nineteen specimens were sampled, including the outgroup taxa Blotiella stipitata (Alston) Faden and Histiopteris incisa (Thunb.) J. Sm., Pteridium aquilinum subsp. wightianum (J. Agardh) W.C. Shieh. Total genomic DNA was extracted from silica gel-dried leaves by using a DNA secure Plant Kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s protocols. The PCR reactions were performed in a Veriti 96-Well Thermal Cycler. Two plastid markers were amplified, the rbcL gene and the trnL-trnF intergenic spacer. Primers used for amplification and sequencing were: rbcL primers 1379R and 1F (Little and Barrington 2003) and trnL-F primers trn-F and trn-r1 (Taberlet et al. 1991, Li et al. 2011). The amplification profiles were: initial denaturation (94 °C, 3 min) followed by 29 cycles of amplification, hybridisation and extension (94 °C, 45 s; 52 °C, 30 s; 72 °C, 1.5 min) and 10 min of final extension at 72 °C for rbcL, initial denaturation (95 °C, 3 min) followed by 35 cycles of amplification, hybridisation and extension (95 °C, 30 s; 52 °C, 30 s; 72 °C, 1 min) and 10 min of final extension at 72 °C for trnL-trnF. Sequencing was conducted using an ABI 3730xl DNA analyser (Applied Biosystems, Invitrogen, Foster City, CA, USA).

Phylogenetic analyses

Sequences were assembled and edited with SeqMan (DNA STAR package; DNA StarInc., Madison, WI, USA), aligned by Bio Edit (Hall 1999) and adjusted manually where necessary. All sequences are available from GenBank (Table 1).

Table 1.
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Plant materials, voucher information, and GenBank accession numbers of the samples used in the phylogenetic analyses.a

Taxon Voucher Locality Geographic coordinates GenBank accession number
rbcL trnL-F
Hypolepis glandulifera Brownsey & Chinnock BLD01 Bali, Indonesia NA MG944782 MG944788
Hypolepis robusta W.M. Chu DRS005 Darong Mountain, Guangxi, China NA MG944773 MG944789
Hypolepis punctata (Thunb.) Mett. ex Kuhn FLX6 Hunan, China NA MG944784 MG944790
Hypolepis tenuifolia (G. Forst.) Bernh. HN31 Wuzhishan Mountain, Hainan, China 18°55'1"N, 109°42'13"E MG944786 MG944791
Hypolepis robusta W.M. Chu HND6 Bawang Mountain, Hainan, China 19°07'26"N, 109°04'46"E MG944774 MG944792
Hypolepis alpina (Blume) Hook. Knapp4486 Yilan County, Taiwan, China 24°49'N, 121°41'E MG944769 MG944794
Hypolepis robusta W.M. Chu SG958 Shengtang Mountain, Guangxi, China NA MG944777 MG944801
Blotiella stipitata (Alston) Faden SG1185 Kenya NA MG944780 MG944795
Pteridium aquilinum subsp. wightianum (J. Agardh) W.C. Shieh SG1760 Yunnan, China NA MG944787 MG944796
Hypolepis robusta W.M. Chu SG1812 Ada Village, Fugong County, Yunnan, China 26°49'5.6964"N, 98°53'36.715"E MG944776 MG944797
Hypolepis alpina (Blume) Hook. SG1838 Dulongjiang Village, Gongshan County, Yunnan, China 27°41'11.004"N, 98°16'54.340"E MG944771 MG944798
Hypolepis alpina (Blume) Hook. SG1871 Dulongjiang Village, Gongshan County, Yunnan, China 27°54'49.306"N, 98°20'37.03"E MG944772 MG944799
Hypolepis resistens (Kunze) Hook. SG2900 Bawangling Mountain, Hainan, China 19°5'28"N, 109°10'59"E MG944785 MG944800
Hypolepis polypodioides (Blume) Hook. SIWS28 Sulawesi, Indonesia NA MG944783 MG944802
Histiopteris incisa (Thunb.) J. Sm. WYD016 Guangdong, China NA MG944781 MG944804
Hypolepis robusta W.M. Chu WYD574 Dawu Mountain, Guangdong, China NA MG944778 MG944805
Hypolepis alpina (Blume) Hook. YYH11628 Xitou Village, Nantou County, Taiwan, China NA MG944770 MG944803
Hypolepis robusta W.M. Chu YYH12064 Mengsong Village, Jinghong City, Yunnan, China NA MG944775 MG944793
Hypolepis robusta W.M. Chu ZXC8465 Gulinqing Village, Maguan County, Yunnan, China 22°51'43.64"N, 104°0'15.59"E MG944779 MG944806

Note: NA = not available.

a: Specimens are deposited at the Shanghai Chenshan Botanical Garden Herbarium (CSH), except for voucher Knapp 4486, which is deposited at the Muséum National d'Histoire Naturelle (P).

For phylogeny reconstructions, two methods were used, maximum likelihood (ML) and Bayesian Inference (BI). The ML analyses were conducted with RAxML-HPC BlackBox8.2.10 (Stamatakis 2014). For the Bayesian analyses, the best-fitting models (HKY+G) were selected using jModeltest2 web server under the Bayesian Information Criterion (BIC) (Darriba et al. 2012). Four chains were used with random initial trees as BI settings. Trees were generated for 1,000,000 generations and sampling was conducted every 100 generations. Before stationarity was conducted, the first 2,500 trees were discarded as burn-in trees and the remaining trees were used to construct the majority-rule consensus trees. The remaining trees were used to construct a consensus tree. ML bootstrap values and BI posterior probabilities were labelled on the tree branches.

DNA barcoding analyses

For species delimitation between H. alpina and the other species of Hypolepis, the DNA barcoding gap method, based on the Kimura two parameter (K2P) distance, was used. Intra- and inter-taxa genetic distances were evaluated using MEGA 5.0 (Tamura et al. 2011).

Results

A total of 19 new sequences amongst the total of 19 specimens were generated in the cpDNA matrix of rbcL and trnL-F containing 2,166 bp characters with 374 variable sites and 149 parsimony-informative sites. The optimal ML tree showed a negative log-likelihood score (-lnL) of 5577.824547 and the Bayesian tree was consistent with the ML tree. The statistical support is shown along the branches (ML/BI). Individuals of H. alpina and H. robusta formed a highly supported monophyletic group with an MLBS of 100 as sister clades of H. tenuifolia. Moreover, all rbcL and trnL-F sequences of the H. robusta, from type locality, were identical to those of H. alpina from Taiwan. The sequences of H. robusta from Guangdong, Guangxi and from Hainan Island were also clustered in the H. alpina clade, which had an MLBS of 100 (Fig. 2).

Figure 2. 

Phylogeny of 16 Hypolepis samples and Blotiella stipitata, Histiopteris incisa, and Pteridium aquilinum subsp. wightianum based on rbcL and trnL-F. Bootstrap values and Bayesian posterior probabilities are shown along branches (ML/BI).

No differences were observed in the rbcL and trnL-F barcoding sequences of both H. alpina and H. robusta, except that two specimens have two base differences respectively. The genetic distance between H. robusta and H. alpina ranges from zero to 0.002. Their inter-taxon distances were significantly larger than their intra-taxon distances compared with the other species of Hypolepis and the ratio between the minimum inter-taxon distance and the maximum intra-taxon distance is 11 (Fig. 3).

Figure 3. 

Distribution of intra-taxa (black) and inter-taxa (grey) Kimura two parameter (K2P) distances based on rbcL and trnL-F sequences as barcode. Hypolepis alpina and Hypolepis robusta versus the other species of Hypolepis.

Discussion

Hypolepis robusta was first reported by Chu (1992), being endemic to the Yunnan Province (Chu et al. 2006). After carefully comparing the type (including holotype and lectotype) of H. robusta and H. alpina, it was found that their morphological characteristics, e.g. the adventitious bud at stipe base, frond size, indusium and others (lamina, stipe, hair), are basically the same.

One of the main differences of H. robusta and H. alpina (H. alte-gracillima), mentioned in the key in Flora Yunnanica, is that the former has a few adventitious buds growing on both sides of the stipe base (Chu et al. 2006). However, when several specimens were examined in the herbarium and those from the authors’ own collection, it was found that H. alpina also has this feature (Fig. 4D). Therefore, it is concluded that the character used in the description is not relevant for distinguishing between H. robusta and H. alpina. Moreover, other Asian species of Hypolepis also develop adventitious buds, such as H. pallida (Blume) Hook. and H. tenuifolia (G. Forster) Bernhardi.

Figure 4. 

Hypolepis alpina. A Frond size (photographed by H. Shang in Fugong) B Lamina (photographed by R. Knapp in Nantou) C Hair (photographed by R. Knapp in Nantou) D The adventitious bud at stipe base (photographed by H. Shang in Fugong) E Indusium (photographed by R. Knapp in Nantou).

Another character used to support H. robusta as a new species was its larger size than H. alpina. The latter was reported at higher altitudes in the Malaysian region, from about 1,500–3,500 m and also as low as 1,100 m on Mt Kinabalu in Borneo (Brownsey 1987). However, there is considerable variation between plants from the highest elevations in New Guinea, which have rather smaller fronds and a dense covering of chestnut-brown non-glandular hairs, to those at lower altitudes in the northern part of its range (notably Taiwan), which have large fronds and very few chestnut hairs (Brownsey 1987). According to the description in Flora Yunnanica, H. robusta has a little larger frond than H. alpina (H. alte-gracillima). The field observation showed that H. robusta always occurs at altitudes about 1,000 m or even lower (Fig. 4A) and this is in accordance with the correlation between the altitudes and frond sizes mentioned in previous literature.

The characters of the indusium have been widely used in fern taxonomy. According to the previous literature of H. alpina and H. robusta (Brownsey 1987, Chu et al. 2006), they could be distinguished morphologically as follows: H. robusta has white indusium with marginal laceration, but H. alpina has a reflexed broad green lamina flap. Based on careful observations of all available material, it was found that their indusia are both half membranaceous at the margins and still green at the base (Fig. 4E). However, when the sori mature, the membranaceous margin becomes lacerated or exfoliated and the base can lose its chlorophyll, thus turning white. This difference may therefore be due to the fact that the descriptions have been made at different periods for the same species, a fact which had been previously ignored.

In addition to the morphological identification, a molecular phylogenetic analysis was also undertaken. The phylogenetic analysis of the rbcL and trnL-F sequences strongly supported the monophyly of H. alpina and H. robusta as a phylogenetic species with a wide distribution and distantly related to H. polypodioides (Fig. 2). The DNA barcoding analysis based on the K2P model revealed a significant gap between the inter-taxon and intra-taxon genetic distances, the distance in the H. robusta and H. alpina clade range from zero to 0.002, which is much lower than the inter-taxon distance and, in particular, the genetic distance between the H. alpina from Taiwan and the H. robusta from its type locality in Yunnan is zero (Fig. 3).

To sum up, not only does the morphological comparison identify H. robusta and H. alpina as conspecies, but also the phylogeny analysis identifies these as conspecies. Therefore, H. robusta is here reduced to a synonym of H. alpina. Consequently, H. alpina has three new distribution records in Guangdong, Guangxi and Hainan Island of China (Fig. 1). The new distribution records of H. alpina fill in gaps of the disjunct distribution defined in previous studies.

Taxonomic treatment

Hypolepis alpina (Blume) Hook. (1852: 63)

Hypolepis alpina (Blume) Hook. (1852: 63). Cheilanthes alpina Blume (1828: 138). Cheilanthes dissecta Hook. & Arn. (1841: 75). Hypolepis dissecta (Hook. & Arn.) Brack. (1854: 89–90). Hypolepis alte-gracillimaHayata (1915: 295–297).

Type: Indonesia. Java: Jawa Barat, Gede, Blume C. L. (Lectotype: L-0051753!, L-0051754!).

= Hypolepis robusta W. M. Chu (1992: 36), syn. nov.

Type

China. Yunnan: Fugong County, 1980, W. M. Chu (Holotype: PYU-01017821!, PYU-01017822!, PYU-01017823!, PYU-01017824!).

Fronds up to 1.7 m high. Rhizome long-creeping, 2–10 mm diameter, densely covered in red-brown hairs up to 3 mm long. Stipes reddish-brown, 12–70 cm long, 1.5–13 mm diameter, grooved adaxially, covered in red-brown non-glandular hairs up to 2 mm long and shorter glandular hairs, few adventitious buds at both sides of the stipe base; lamina ovate in outline, 3– or 4–pinnate, 20–80 (–130) cm × 10–90 cm, rachis red-brown or chestnut-brown at base, becoming chestnut-brown or yellow-brown at apex, densely covered in red-brown or chestnut-brown glandular hairs up to 0.5 mm long with occasional much longer non-glandular hairs; primary pinnae 15–30 pairs, opposite or sub-opposite, the largest at or near base, ovate to narrowly triangular, 10–52 cm × 3–28 cm; secondary pinnules narrowly ovate to ovate, 2–14 cm × 0.8–5 cm; ultimate pinnules to 10 mm × 5 mm. Sori circular or ovate, originating away from margins, without hairs between sporangia, protected by reflexed adaxial indusium, green at base and half membranaceous at margin, when the sori turn mature, the membranaceous margin becomes lacerated or exfoliated and the base part may turn white. Spores very pale under light microscope, perispores with interconnecting flattened projections, (32–) 34–37 (–40) µm × (20–) 21–25 (–28) μm.

Distribution

China (Guangdong, Guangxi, Hainan, Taiwan, Yunnan), Indonesia, Japan, Malaysia, Papua New Guinea, Philippines.

Acknowledgements

We thank Yu-Feng Gu, Ke-Rui Huang, Xi-Le Zhou and Zu-Xia He for their help in field investigations; special thanks go to Yea-Chen Liu, Kuan-Yu Shen, Da-Jun Lin and Yi-Hsuan Hsu for their help in field investigations and for taking photographs of specimens in Taiwan. We are grateful to the editor Dr. Blanca León and two anonymous reviewers for their very valuable suggestion in improve our manuscript. This study was supported by a grant from the National Natural Science Foundation of China (NSFC) to Hui Shang (# 31700170) and Science and Technology Basic Work (2013FY112100).

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Supplementary material

Supplementary material 1 

Table S1

Morigengaowa, Jun-Jie Luo, Ralf Knapp, Hong-Jin Wei, Bao-Dong Liu, Yue-Hong Yan, Hui Shang

Data type: (measurement/occurence/multimedia/etc.)

Explanation note: Herbarium specimens information of Hypolepis alpina and Hypolepis robusta samples checked in this study.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
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