A new species, Ptilagrostis contracta, endemic to Qinghai-Tibet Plateau is described and illustrated. It is distinguished from other species in Ptilagrostis by having contracted panicles, 1-geniculate awns with hairy columns and scabrous bristles and evenly pubescent lemmas. Evidence from lemma epidermal pattern, cytology and molecular phylogenetic analyses based on the nuclear ITS sequence data confirm its systematic position in Ptilagrostis.
Citation: Zhang Z-S, Li L-L, Chen W-L (2017) Ptilagrostis contracta (Stipeae, Poaceae), a New Species Endemic to Qinghai-Tibet Plateau. PLoS ONE 12(1): e0166603. https://doi.org/10.1371/journal.pone.0166603
Editor: Fan Chen, Institute of Genetics and Developmental Biology Chinese Academy of Sciences, CHINA
Received: July 14, 2016; Accepted: October 15, 2016; Published: January 6, 2017
Copyright: © 2017 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All DNA sequences used in this article are available from the National Center for Biotechnology Information Search database (accession number KX539299). Other relevant data are within the paper and its Supporting Information files.
Funding: This work was funded by the National Natural Science Foundation of China (grant nos. 31070168, 31470300, 31110103911, and 30990241), http://www.nsfc.gov.cn/publish/portal1/; and Science and Technology Basic Work (grant no. 2013FY112100), http://most.gov.cn/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of the authors are articulated in the 'author contributions' section.
Competing interests: The authors have declared that no competing interests exist.
Ptilagrostis Griseb. is a small genus of approximately 11 high altitude species, widely accepted on the basis of its distinctive morphological characters, cytological evidence [1–6] and molecular data [7, 8]. Ptilagrostis is disjunctive in Asia and North America [5, 9–12] with the diversity center in the Qinghai-Tibet Plateau, where approximately eight taxa occur. They grow in various alpine habitats, including meadows, grasslands, shrubby and even Abies forests.
During several botanical expeditions to Sichuan Province in 2013–2015, some unusual populations of Ptilagrostis were discovered in Litang and Kangding counties, located in the southeastern Qinghai-Tibet Plateau. The plants can be distinguished from all the other known Ptilagrostis species by having contracted panicles, 1-geniculate awns with pubescent columns and scabrid bristles and evenly pubescent lemmas. We determined that these populations represent a hitherto undescribed species and named as P. contracta. It is strongly supported to be a member within Ptilagrostis by the evidence from lemma epidermal pattern, cytology and molecular phylogenetic analyses based on the nuclear ITS sequence data. The detailed description of the new species is given below.
Material and Methods
All the collecting locations of the new species reported in this study are not in any natural conservation area and no specific permissions were required for these locations. And our field studies did not involve any endangered or protected species.
A total of five populations of new species were collected and examined during the field work conducted in the Qinghai-Tibet Plateau from 2013 to 2015. The specimens from PE, HNWP, KUN, CDBI were carefully checked, as well as the specimens borrowed from HITBC, B, CAS, GOET and K.
Matured caryopses and voucher specimen were collected by the authors during the field trip in Litang. Voucher specimen: CHINA. Sichuan, Litang, Long. 100.41842 E, Lat. 29.72025 N, 3701 m, 26 September 2014, Z. S. Zhang & L. L. Li 341, deposited in the Herbarium, Institute of Botany, CAS (PE).
Vigorous root tips obtained from germinated caryopses were pre-treated in an ice water mixture at 0° for 24 hours, then fixed in Carnoy’s fluid (3: 1 ethanol: glacial acetic acid) at 4° for at least 30 min. They were then digested at 37° in a combination (1:1) of 2.5% cellulase and 1.25% pectinase for 1 hour before being stained with improved carbol-fuchsin solution  and squashed for cytological observation. Permanent slides were made using the standard liquid nitrogen method. The photographs were taken using a Zeiss Axio Imager A1 camera. The karyotype was analyzed following Li and Chen .
Lemma epidermal pattern
Lemma ultra structure was studied by using dry mature caryopses sampled from the type specimen. To remove epicuticular wax the lemmas were cleaned in xylene for an hour. Samples were mounted and then covered with gold from a vacuum spray gun (type S-800, HITACHI). All the observation and photographs were made at varying magnifications by using scanning electron microscope (Hitachi S-4800 FESEM, Hitachi, Tokyo, Japan).
In order to determine the systematic position of the new species, one new ITS sequence, for Ptilagrostis contracta, was added to a data alignment of 48 selected accessions from GenBank of Ptilagrostis species and selected species of 23 other Stipeae genera, with Phaenosperma globosum Munro ex Benth. as an outgroup [7, 8]. GenBank accession numbers and voucher information for the materials used in this study are provided in S1 Table.
DNA extraction, amplification and sequencing.
Total genomic DNA of Ptilagrostis contracta was extracted from silica gel-dried leaves collected in the field using a Plant Genomic DNA Kit according to the instructions of the manufacturer (Tiangen Biotech, Beijing, China). ITS sequence data was employed to infer phylogeny in this study. They were amplified and sequenced using primer pairs ITS1 (AAGTCGTAACAAGGTTTCCGTAGGTG)  / ITS4 (TCCTCCGCTTATTGATATGC) . The amplification parameters were: initial denaturation phase of 4 min at 94°C; followed by 30 cycles of denaturation at 94°C for 30 s, annealing phase at 55°C–57°C for 30 s, extension phase at 72°C for 1 min 10 s, and final extension at 72°C for 6 min. Automated sequencing was performed by Sino Geno Max Inc. (Beijing, China). Sequences of P. contracta were deposited at GenBank (S1 Table).
Sequence data were edited and assembled in the ContigExpress program of the Vector NTI Suite v.6.0 (Informax, North Bethesda, Maryland, U.S.A.). Sequences alignment was done manually using BioEdit v.184.108.40.206 . All gaps were treated as missing data.
Maximum likelihood (ML) analysis was performed using RAxML 7.2.7  from CIPRES cluster web servers  with a K80+G model suggested by jModelTest  according to the Akaike information criteria [21, 22]. Bootstrap probability (BP) values were calculated for 1000 replicates.
In the Bayesian analysis, the K80+G model was selected by the program jModelTest according to the Bayesian information criteria . Markov chain Monte Carlo (MCMC) iterations with four chains were conducted for 3 000 000 generations, sampling a tree every 1000 generations, with the program MrBayes 3.2.3 . The first 10% trees were discarded as burn-in and the remaining trees were used to determine the posterior probabilities (PP) for branches.
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The chromosome number of Ptilagrostis contracta is 2n = 22 (Fig 1G), same as the previous observation on other species of Ptilagrostis [2, 8]. Ptilagrostis contracta has medium and small chromosomes and the length of chromosomes varies from 1.4 μm to 3.6 μm. The karyotype is 2n = 14m+6sm+2st (Fig 1H). Acronyms: m, metacentric chromosome; sm, submetacentric chromosome; st, acrocentric chromosome.
A-B. habitat; C. contracted panicle; D. base of panicle branch (pulvinae absent); E. spikelets; F. floret (lemma evenly pubescent); G. chromosomes; H. karyotype; I. Lemma epidermal pattern. Scale bar in D represents 2 mm; in E, F represent 5 mm; in G, H represent 5 μm.
Lemma epidermal pattern
Ptilagrostis contracta has typical Ptilagrostis saw-like lemma epidermal pattern (Ptilagrostis SLP) . The fundamental cells (FC) are 3–9 times longer than the short cells (SC); side wall of FC sinuous, slightly thickened; SC padded by silica bodies (SB), alternated with cork cell (CC) or not; SB rectangular or oblong with 2–4 shallow contractions; prickles (P) and macro hairs (MH) present (Fig 1I).
BI and ML analyses (Fig 2) produced highly congruent topologies. As shown in Fig 2, several monophyletic clades can be recognized although the internal relationship among the clades are not well-resolved in Stipeae. Monophyletic core Ptilagrostis clade including the type of Ptilagrostis namely P. mongholica (Turcz. ex Trin.) Griseb. plus other eight species of Ptilagrostis were well supported (74/0.99). However P. kingii (Bol.) Barkworth was resolved closely related to Piptatheropsis Romasch., P.M. Peterson & Soreng. Significantly, the accession of Ptilagrostis contracta deeply nested within the core Ptilagrostis clade.
A. habit; B. glume; C. floret; D. palea; E. callus F. outer surface of ligule; G. inner surface of ligule. Scale bar in B, C, F, G represent 5 mm; in D represent 2 mm; in E represent 0.5 mm (Drawn by Z. S. Zhang).
Type:—CHINA. Sichuan: Litang, 3701 m, 26 September 2014, Z. S. Zhang & L. L. Li 341 (holotype PE!).
Ptilagrostis contracta differs from the other species of Ptilagrostis in having contracted panicles, 1-geniculate awns with hairy columns and scabrous bristles and evenly pubescent lemmas. In gross morphology, the new species resembles to P. yadongensis Keng f. & J. S. Tang and Stipa subsessiliflora (Rupr.) Roshev. (≡Ptilagrostis subsessiliflora (Rupr.) Roshev.) [6, 25, 26] in having contracted panicles. For distinguishing these 3 narrow panicled species, a key is provided here.
Key to Ptilagrostis contracta, P. yadongensis and Stipa subsessiliflora
Perennial, densely tufted, basal branching intravaginal; culms erect, 60–105 cm tall, ca. 2–3 mm in diameter, 3-noded. Leaf sheaths nearly smooth; leaf blades slender, fold, up to 43 cm, 0.6–1 mm wide, nearly smooth; ligule lanceolate, 1–2.5 mm. Panicles contracted, 13–31 cm long; branches often paired, appressed to the rachis, pulvinae at the base of the branches absent; lower part bare, upper part with few spikelets. Spikelets 10–14 mm long, yellowish-brown or purple at base; glumes subequal, lanceolate-oblong, membranous, hyaline, midrib obvious, lateral veins inconspicuous, lower 10–14 mm long, upper 10–14 mm long; calluses obtuse, ca. 0.5 mm long; lemmas 6–9 mm long, evenly pubescent with hairs ca. 0.5 mm long, apex 2-toothed, teeth 0.3–1 mm long; awns persistent, 1.3–2.0 cm long, 1-geniculate, columns twisted, hairs ca. 0.5–0.7 mm long, bristles scabrous, hairs ca. 0.1 mm long; paleas subequal to lemmas; anthers ca. 2.5 mm long, bearded at apex.
Distribution and habitat.
Ptilagrostis contracta grows under the birch forests or in the Rhododendron thickets at about alt. 3500–4300 m. It is endemic to Qinghai-Tibet Plateau and currently found only in Sichuan province, China (Fig 4).
Ptilagrostis contracta is currently known from 9 locations in Sichuan province within the regions of Dawu, Kangding, Litang and Zamtang. The number of mature individuals is estimated to be ca. 18,000 in the populations. The populations in Kangding and Litang are all close to roads or villages in habitats seriously disturbed by human activities. One population in Kangding (Z. S. Zhang & L. L. Li 375) possesses no more than 20 mature individuals. The population size of P. contracta will likely drop to 10,000 in the near future and is close to qualifying for a Vulnerable (VU) species according to the criteria C (IUCN red list categories and criteria, Version 3.1, Second edition) . So P. contracta was categorized as a Near Threatened (NT) species (A taxon is Near Threatened when it has been evaluated against the criteria but does not qualify for Critically Endangered, Endangered or Vulnerable now, but is close to qualifying for or is likely to qualify for a threatened category in the near future) .
Additional specimens examined.
CHINA. Sichuan: Daocheng Xian & Litang Xian, Hei-tze-shan, 3700 m, 29 August 1934, C. S. Liu 1425 (PE); Dawu Xian, 4300 m, 15 September 1974, Anonymous 5974 (PE); Kangding Xian, 3574 m, 28 September 2014, Z. S. Zhang & L. L. Li 366 (PE); Kangding Xian, 3545 m, 28 September 2014, Z. S. Zhang & L. L. Li 371 (PE); Kangding Xian, 3924 m, 29 September 2014, Z. S. Zhang & L. L. Li 375 (PE); Litang Xian, 3728 m, 24 September 2014, Z. S. Zhang & L. L. Li 336 (PE); Litang Xian, 4100 m, 24 September 1973, Sichuan Vegetation Survey Team 3987 (PE, N, KUN); Zamtang Xian, 4100 m, 17 July 1975, Anonymous 9320 (PE).
Ptilagrostis contracta is strongly supported within Ptilagrostis by our lemma epidermal pattern (LEP) and cytology (2n = 22) data. Molecular evidence from ITS sequence data also support the generic affiliation of P. contracta. Romaschenko et al  reported the incongruence between nuclear and plastid tree in tribe Stipeae, Ptilagrostis species formed one clade in ITS tree but separated into two clades in plastid tree. To resolve the phylogentic problem of Ptilagrostis, further genetic analyses are needed.
The new species, Ptilagrostis contracta is similar to the other species of Ptilagrostis by haing intravaginal basal branches, thin leaves, lanceolate ligules, blunt calluses and hyaline glumes but differs by 1-geniculate awns with hairy columns and scabrous bristles and evenly pubescent lemmas which expended the morphological variation of Ptilagrostis.
This study was supported in part by National Natural Science Foundation of China granted to DYH: (grant nos.: 31110103911), YFW: (grant nos.: 30990241) http://www.nsfc.gov.cn/publish/portal1/; Science and Technology Basic Work to DYH: (grant no.: 2013FY112100) http://most.gov.cn/. We acknowledge the curators of B, CAS, CDBI, GOET, HITBC, HNWP, K, KUN, PE for making the collections available for this study and the source (papers) of all the GenBank data we used in this article. Also we acknowledge two anonymous reviewers for comments that led to improvements of the manuscript.
- Conceptualization: WLC.
- Data curation: ZSZ.
- Formal analysis: ZSZ.
- Funding acquisition: WLC.
- Investigation: ZSZ LLL.
- Methodology: WLC.
- Project administration: WLC.
- Resources: ZSZ LLL.
- Supervision: WLC.
- Validation: WLC.
- Visualization: ZSZ WLC.
- Writing – original draft: ZSZ WLC.
- Writing – review & editing: ZSZ WLC.
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