CC BY
9
Turczaninowia 25, 4: 155-163 (2022) DOI: 10.14258/turczaninowia.25.4.17 http://turczaninowia.asu.ru
ШШ ISSN 1560-7259 (print edition)
M TURCZANINOWIA
¡¡j| ISSN 1560-7267 (online edition)
УДК 582.394.74:581.96(515.7)
Asplenium delinghaense, a new species from western part of Qilian Mountains in Qinghai-Xizang Plateau
S.-Q. Liang1' 2 3 5, S.-B. Zhang4, X.-C. Zhang1' 2> 6*
1 Institute of Botany, Chinese Academy of Sciences, Nanxincun, 20, Xiangshan, Haidian District, Beijing, 100093, China
2 China National Botanical Garden, Wofosi Road, Haidian District, Beijing, 100093, China
3 University of Chinese Academy of Sciences, Yuquan Road, 19(A), Shijingshan District, Beijing, 100049, China
4 Qinghai Provincial Association for Wildlife and Plants Conservation, Xichuan South Road, 25, Chengxi District, Xining, 810008, China
5ORCID iD: https://orcid.org/0000-0002-3917-5479
6E-mail: zhangxc@ibcas.ac.cn; ORCID iD: https://orcid.org/0000-0003-3425-1011
* Corresponding author
Keywords: alpine plants, Aspleniaceae, China, East Asia, polyploidy.
Summary. A new species of the genus Asplenium L. from northern Qinghai, A. delinghaense S. Q. Liang et X. C. Zhang, is described and illustrated here. This new species resembles A. iskardense Viane et Reichst., A. daghestanicum Christ, and A. neovarians Ching but differs in frond and perispore morphology. Molecular phylogenetic evidence supported the close relationship of A. delinghaense with members of the A. varians complex and the A. pekinense complex and indicated an allotetraploid origin of it.
Asplenium delinghaense - новый вид из западной части хребта Циляньшань (Цинхай-Тибетское плато)
С. Ц. Лян1, 2, 3, Ш. Б. Чжан4, С. Ч. Чжан1, 2
1 Институт ботаники Академии наук Китая, ул. Нансинцун, д. 20, Сяншань, район Хайдиан, г. Пекин, 100093, Китай
2Китайский национальный ботанический сад, ул. Вофосы, район Хайдиан, г. Пекин, 100093, Китай
3 Университет Академии наук Китая, ул. Ючан, д. 19(А), район Шицзиншань, г. Пекин, 100049, Китай
4 Ассоциация охраны дикой природы и растений провинции Цинхай, ул. Юж. Сичуан, д. 25, район Ченси, г. Синин, 810008, Китай
Ключевые слова: высокогорные растения, высокогорья, Восточная Азия, Китай, полиплоидия, Aspleniaceae.
Аннотация. Описан и проиллюстрирован новый вид рода Asplenium L. из северного Цинхая - A. delinghaense S. Q. Liang et X. C. Zhang. Новый вид напоминает A. iskardense Viane et Reichstein, A. daghestanicum Christ и A. neovarians Ching, но отличается формой вай и морфологией периспория. Молекулярно-филогенетические данные подтверждают тесную связь A. delinghaense с видами комплексов A. varians и A. pekinense и указывают на его аллотетраплоидное происхождение.
Поступило в редакцию 01.07.2022 Принято к публикации 16.11.2022
Submitted 01.07.2022 Accepted 16.11.2022
Introduction
Asplenium L. is one of the richest cosmopolitan genera among extant ferns, represented by a total of ca. 700 species (Kramer, Viane, 1990; Schneider et al., 2004; Rothfels et al., 2012; Lin, Viane, 2013). The Qinghai-Xizang Plateau is a diversification center for high altitudes species of Asplenium, especially the Himalayas which surround the western and southern edges of it (Kramer, Viane, 1990; Wu, 1999). In China, the field investigation of the Qinghai-Xizang Plateau has been mainly focused on Xizang and Sichuan, whereas Qinghai, a vast area showing a rugged landscape in the northeast of the Qinghai-Xizang Plateau, still lacks a more fully exploration. According to Flora Qinghaiica (Mei, 1997), only three Asplenium species were recorded in this province, which can hardly match the observed morphological diversity and variance. During a comprehensive field exploration throughout Qinghai organized recently by Mr. Sheng-Bang Zhang, a small Asplenium plant growing in dolomite-marble crevices was collected from the Cypress Mountain in Delingha. Cypress Mountain is located in the western section of Qilian Mountain and adjacent to Qaidam Basin in the south. Qilian Mountain lies on the northeastern edge of the Qinghai-Xizang Plateau, and abundant glaciers make it an important "solid water reservoir" nourishing the vegetation in surrounding regions. The climate in the Qaidam Basin is quite xeric and windy, therefore, ferns hardly survive there, except for very few drought-resisting species such as Equisetum avense. The present Asplenium specimen from Delingha resembles species of ser. Variantia Ching et S. H. Wu, a group including a complex assemblage of small ferns mainly distributed in the high-altitude area of the Qinghai-Xizang Plateau and adjacent temperate regions (Ching and Wu, 1985; Wu, 1999); it was different from known species by morphology, cytology, and molecular phylogeny. Therefore, we describe it as a new species named Asplenium delinghaense S. Q. Liang et X. C. Zhang and present the result here.
Material and methods
Morphological study
Specimens for morphological studies were obtained from our collections and herbarium PE. High resolution scanned images from websites of herbaria BR, K, and P were also used in morphological comparison. Morphological characteristics of frond were observed and photographed with a Leica
S9D stereo microscope. Spore size and surface ornamentation were observed by light microscopy (LM) and scanning electron microscopy (SEM) using a Leica DM4000 microscope and a Hitachi S-4800 field emission SEM, respectively. For LM, untreated spores were collected and embedded in neutral balsam. We randomly selected more than 50 spores to measure the length of the exospore under LM; measurements are given in the following format: (minimum) mean minus standard deviation (s)-mean plus s (maximum). For SEM, unopened mature sporangia were broken on a specimen stub to release spores, then coated with platinum.
Cytological study
In Asplenium, monoploid nuclear DNA content (1Cx-value) is relatively stable among related species, therefore, we inferred the ploidy level of our samples through comparing the 2C-value estimated by flow cytometry (FCM) with the data of well-studied species. FCM investigations were performed with a BD LSRFortessa flow cytometer. We selected Zea mays ssp. mays "B73" (5.64 pg/2C, Diez et al., 2013) or Capsicum annuum var. annuum (6.76 pg/2C, Moscone et al., 2003) as the internal standard and propidium iodide as the nucleic acid dye. Suspension of cell nuclei was prepared by chopping tissues of silica gel-dried sample with fresh internal standard in a petri dish containing modified Galbraith's buffer (provided by the Plant Science Facility of IBCAS). For each sample, the measurement was repeated for three times. Mean and s were both calculated to represent the nuclear DNA content of the sample.
Phylogenetic analyses
We included a total of16 individuals, representing 13 species of Asplenium ser. Variantia Ching et S. H. Wu from China and adjacent regions, including A. tenuicaule, A. neovarians, A. varians, A. kuk-konenii, A. altajense, A. kansuense, A. anogrammoi-des, A. pekinense, A. sarelii, A. fugax, A. capillipes, A. pulcherrimum, and A. tenuifolium. A. incisum was chosen as an outgroup. Detailed voucher information and GenBank accession numbers were listed in Appendix.
Total genomic DNA was isolated from silica gel-dried material using the Plant Genomic DNA Kit (Tiangen Biotech, Beijing, China) following the manufacturer's protocol. Two plastid DNA fragments (rbcL and rpl32-trnP) and one nuclear gene fragment (pgiC) were amplified by PCR and sequenced for phylogenetic analyses. Primers, experiment conditions and alignment processes
were all followed Liang et al. (2019). IQ-TREE v.1.6.8 (Nguyen et al., 2015) was used to reconstruct maximum likelihood (ML) phylogeny based on concatenated plastid DNA sequences and nuclear gene sequences, respectively.
Results and discussion
Based on gross morphology (Fig. 1), Asplenium delinghaense is very similar to A. iskardense Viane et Reichst. (holotype: K001092507, image online!)
Fig. 1. Asplenium delinghaense S. Q. Liang et X. C. Zhang, sp. nov.: A-B - Adaxial and abaxial views of individual 1 of the holotype, Sheng-Bang Zhang et al. 7617 (PE); C-D - Individual 2 and 3 of the holotype; E - Habit of a living plant in the original habitat; F - Variation in frond morphology of the holotype; G - Habitat of the species in Delingha, Qinghai.
and A. daghestanicum Christ (holotype: P00622782, image online!; isotype: BR0000024934701, image online!), two species in need of further field investigation to clarify the distribution range. Up to now, A. iskardense is only known from the type locality in Pakistan (Viane, Reichstein, 2003); A. daghestanicum has been found in Daghestan and Chad (Viane, 1987), thus, it is expected to have a wider distribution. A. delinghaense can be distinguished from them by the stipe a little bit
FCM results showed that the nuclear DNA content of A. delinghaense is 19.4 ± 1.3 pg/2C, which is similar to the level of related tetraploid species in Liang et al. (2021). As cytotypes can often be distinguished by spore sizes (Sleep, Reichstein, 1984), the length of exospore, (29)35-41(47) ^m,
shorter than lamina, larger spore size and different perispore morphology (Fig. 2H). A. delinghaense also resembles A. neovarians Ching (holotype: PE01895940!), a species known only from few collections in NW China (Ching, Wu, 1985; Lin, Viane, 2013). However, our phylogenetic results indicated that they are diverged on genetic level (Figs. 3-4). In addition, they are also different in exospore size, perispore morphology and number of pinnae pairs.
is also an indicator of high ploidy of this species comparing with related taxa (Lin, Viane, 2013). In the plastid DNA phylogeny (Fig. 3), A. delinghaense showed close maternal relationship with A. fugax, A. capillipes, and members of the A. pekinense complex (Lin, Viane, 2013; Liang et al., 2021). However, in the
Fig. 2. Micromorphology of Asplenium delinghaense S. Q. Liang et X. C. Zhang, sp. nov. (Sheng-Bang Zhang et al. 7617 (PE), individual 1): A - Rhizome scales; B - Fibrillar scales on the lower stipe; C-E - Differences in the degree of division among the third, second and basal pinnae; F - Optical section of spore showing exospore contour and the thickness of perispore folds; G-H - LM and SEM photos of spore showing perispore morphology.
nuclear DNA phylogeny, two different copies were we concluded that A. delinghaense is probably an detected. One of them showed a position similar to allotetraploid that originated from hybridization of that of the plastid result, whereas the other clustered A. tenuicaule and an unknown taxon closely related with A. tenuicaule var. subvarians and one of its to A. fugax, A. capillipes, or to members of the putative tetraploid offspring, A. kansuense (Liang et A. pekinense complex. al., 2021) (Fig. 4). According to the above evidence,
| A. kansuense LSQ17, Zhouqu, Gansu A. tenuicaule var. subvarians M3A1376, Altai, Russia A. neovarians 12264, Banma, Qinghai A. neovarians 6106, Baiyu, Sichuan A. kukkonenii WR0494-1, Kunming, Yunnan A. tenuicaule var. tenuicaule 8978, Kangding, Sichuan A. tenuicaule var. tenuicaule LSQ8, Baoji, Shaanxi A. varians A55, Kunming, Yunnan
A. delinghaense sp. nov. 7617, Delingha, Qinghai — A. fugax s.n.-7, Yanbian, Sichuan -A. capillipes 8670, Wenxian, Gansu
43
100
100
A. anogrammoides 8042, Beijing A. pekinense 8044, Beijing A. sarelii WR0485-5, Wanzhou, Sichuan A. altajense LUEP796, Buryatia, Russia A. tenuifolium 8438, Maguan, Yunnan
A. pulcherrimum 8059B, Duyun, Guizhou
1 A. incisum A0007, Jinzhai, Anhui 0.02
Fig. 3. Maximum likelihood (ML) phylogeny of plastid DNA dataset showing the position of Asplenium delinghaense. Bootstrap support values are shown above branches.
Taxonomic treatment
Asplenium delinghaense S. Q. Liang et X. C. Zhang, sp. nov. (Figs. 1-2).
Diagnosis. Asplenium delinghaense resembles A. iskardense and A. daghestanicum but can be distinguished by its lophate perispore with costate-cristate ridges (with narrow dentate crests and swollen fold base) and some perforations near the base. In comparison, ridges of perispore are costate and costate-cristate with narrower fold base in the latter two species. The ratio between
the length of lamina and stipe is around 1.0 in A. delinghaense, whereas the ratio is ca. 0.5 and ca. 1.4 in A. iskardense and A. daghestanicum, respectively. A. delinghaense is also similar to A. neovarians, and their distribution overlapped in NW China. Comparing with A. neovarians, A. delinghaense has longer stipes, more pinnae pairs and perispore with narrower and crests broader folds. In addition, the spore size of A. delinghaense is the largest among all four species, though they were all estimated to be tetraploid.
89
93 68
I j
100 I
I /
S8|
' /
95 93
91 j
A. kansuense LSQ17, Zhouqu, Gansu A. tenuicaule var. subvarians M3A1376, Altai, Russia A. delinghaense sp. nov. 7617, Delingha, Qinghai A. neovarians 12264, Banma, Qinghai A. neovarians 6106, Baiyu, Sichuan A. neovarians 12264, Banma, Qinghai A. neovarians 6106, Baiyu, Sichuan A. altajense L1JEP796, Buryatia, Russia
A. kukkonenii WR0494-1, Kunming, Yunnan A. varians A55, Kunming, Yunnan A. tenuicaule var. tenuicaule 8978, Kangding, Sichuan A. kansuense LSQ17, Zhouqu, Gansu A. anogrammoides 8042, Beijing A. tenuicaule var. tenuicaule LSQ8, Baoji, Shaanxi — A. kukkonenii WR0494-1, Kunming, Yunnan A. varians A55, Kunming, Yunnan A. delinghaense sp. nov. 7617, Delingha, Qinghai A. fugax s.n.-7, Yanbian, Sichuan A. capillipes 8670, Wenxian, Gansu
A. anogrammoides 8042, Beijing A. pekinense 8044, Beijing A. sarelii WR0485-5, Wanzhou, Sichuan A. sarelii WR0485-5, Wanzhou, Sichuan A. pekinense 8044, Beijing A. altajense LLJEP796, Buryatia, Russia A. tenuifolium 8438, Maguan, Yunnan
I A.
99 I
■ A. pulcherrimum 8049B, Duyun, Guizhou
A. pulcherrimum 8049B, Duyun, Guizhou
■ A. incisum A0007, Jinzhai, Anhui 0.02
Fig. 4. ML phylogeny of nuclear DNA dataset showing the position of two different copies of Asplenium delinghaense. Bootstrap support values are shown above branches.
Holotype: "China, Qinghai, Delingha, Baishushan (Cypress Mountain) 37°29'24"N, 97°22'19"E, alt. 3610 m. V 2021. Sheng-Bang Zhang et al. 7617" (PE!).
Description. Plants 5-8 cm tall. Rhizome erect, short, apex scaly; rhizome scales (1.5)2.1-4.0(4.4) x (0.3)0.4-0.6(0.7) mm wide at the base, brown, narrowly triangular to linear-subulate, base
cordate, margin nearly entire, apex acuminate, terminating in a long apical tail. Fronds firmly herbaceous, green, brown green when dry, more than ten usually, tufted; stipes 3-5 cm long, generally a little bit shorter than laminae, slender, adaxially sulcate, abaxially dark brown at the base, upward green, base with few narrowly triangular scales, toward the rachis covered with more sparsely fibrillar scales; rachis structure similar to that of distal part of stipe. Laminae 32-52 x 9-14 mm, narrowly triangular, base bipinnate and nearly symmetrical, becoming pinnate toward the apex, apex acute-acuminate with triangular apical segment ca. 6-10 x 2-5 mm; pinnae 6 to 8 pairs, opposite to subopposite, deltate-ovate to rhombic, shortly (up to 0.5 mm) stalked, basal pinnae pinnate, 0.8-1.1 cm remote from the next pair and usually slightly larger, the second, third, fourth, and fifth pinnae slightly smaller and pinnatisect to pinnatilobate; only basal pinnae with a pair of independent pinnules, the basal acroscopic pinnules largest, sessile or slightly stipitate, base broadly cuneate, apex serrate with long and sharp teeth, apex obtuse. Veins slightly raised adaxially, biforked or simple, not reaching margin. Sori 1-3 per segment, linear, 1-3 mm, submedial on veins, confluent at maturity but not covering the upper half of the segment; indusia white-gray, semi-elliptic, membranous, entire, opening toward costa or costule, persistent. Spores dark brown, perispore lophate with costate-cristate folds, average exospore length (29)35-41(47) |im.
Etymology. Delinghaense is derived from the type locality, Delingha, a transliteration of a Mongolian word means "golden world".
Distribution and habitat. Asplenium delinghaense is known only from the Cypress Mountain (Baishushan) geopark north of Delingha, Qinghai, NW China, growing on steep dolomite-marble, at an elevation of 3610 m.
Conservation status. Asplenium delinghaense is known only from its type locality, Cypress Mountain (Baishushan) geopark north of Delingha, located at the northeastern edge of the Qaidam Basin, where is quite xeric. Exact population size of this new species is unknown, because it is adaptive to the barren rocky south face slope of the alpine karst mountain, which is massive and difficult for people to climb.
Acknowledgements
This work was supported by the National Plant Specimen Resource Center Project (NPSRC) (E0117G1001) and "Investigation of Wild Ferns Diversity of Qinghai Province" supported by Qinghai Shanshui Natural Resources Survey and Planning Institute. We appreciate Mr. Ji-Yuan Zhang, the chairman of the Delingha Municipal CPPCC and the Vice Chairman of the Qinghai Photography Association, for his guidance on ecology. We also thank Jin-Dan Zhang and Xiu-Ping Xu from the Plant Science Facility of the Institute of Botany, Chinese Academy of Sciences for their technical assistance on flow cytometry and scanning electron microscopy.
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Appendix
Specimen information and GenBank accession number. Asterisks (*) and en-dashes (-) indicate newly
generated and missing sequences, respectively.
Taxon Voucher Country rbcL rpl32-trnP pgiC
Asplenium altajense (Komar.) Grubov A. I. Shmakov et al. MEP796 (PE) Tunkinsky, Buryatia Republic, Russia MK828788 MK828931, MK828932
Asplenium anogrammoides Christ X.-C. Zhang 8042 (PE) Beijing, China - MK828796 MK828859, MK828860
Asplenium capillipes Makino X.-C. Zhang et S.-Q. Liang 8670 (PE) Wenxian, Gansu, China MN688475 MN688506 MN688448
Asplenium delinghaense S. Q. Liang et X. C. Zhang S.-B. Zhang et al. 7617 (PE) Delingha, Qinghai, China OP795813* OP795810* OP795818*, OP795819*
Asplenium fugax Christ X.-H. Jin s. n.-7 (PE) Yanbian, Sichuan, China MN688476 MN688507 MN688449
Asplenium incisum Thunb. C.-F. Zhao A0007 (PE) Jinzhai, Anhui, China - MK828792 MK828854
Asplenium kansuense Ching X.-C. Zhang et S.-Q. Liang LSQ17 (PE) Zhouqu, Gansu, China MK828812 MK828878, MK828879
Asplenium kukkonenii Viane et Reichst. R. Wei & Q.-P. Xiang WR0494-1 (PE) Kunming, Yunnan, China MK828829 MK828900, MK828901
Asplenium neovarians Ching S.-B. Zhang et al. 12264 (PE) Banma, Qinghai, China OP795811* OP795808* OP795814*, OP795815*
Asplenium neovarians Ching X.-C. Zhang 6106 (PE) Baiyu, Sichuan, China OP795812* OP795809* OP795816*, OP795817*
Asplenium pekinense Hance X.-C. Zhang 8044 (PE) Beijing, China - MK828798 MK828861, MK828862
Asplenium pulcherrimum (Baker) Ching ex Tardieu Z.-Y. Guo 8059B (PE) Duyun, Guizhou, China MN688469 MN688498 MN688429, MN688430
Asplenium sarelii Hook. R. Wei WR0485-5 (PE) Wanzhou, Chongqing, China MK828807 MK828871, MK828872
Appendix (continued)
Taxon Voucher Country rbcL rpl32-trnP pgiC
Asplenium tenuicaule var. subvarians (Ching) Viane A. I. Shmakov et al. МЭА1376 (PE) Chemalsky, Altai Republic, Russia MK828846 MK828925
Asplenium tenuicaule var. tenuicaule Hayata X.-C. Zhang et al. 8978 (PE) Kangding, Sichuan, China - MK828821 MK828893
Asplenium tenuicaule var. tenuicaule Hayata S.-Q. Liang LSQ8 (PE) Baoji, Shaanxi, China - MK828818 MK828889
Asplenium tenuifolium D. Don X.-C. Zhang et al. 8438 (PE) Maguan, Yunnan, China MN688478 MK828791 MK828922
Asplenium varians Wall. ex Hook. et Grev. C.-F. Zhao A55 (PE) Kunming, Yunnan, China - MK828837 MK828909, MK828910
