Ptilagrostis contracta (Stipeae, Poaceae), a New Species Endemic to Qinghai-Tibet Plateau

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.

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.

Ethics statements
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. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111

Material collection
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. 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 4f or 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 [13] 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 [14].

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).

Molecular systematics
Taxon sampling. 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) [15] / ITS4 (TCCTCCGCTTATTGATATGC) [16]. 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).
In the Bayesian analysis, the K80+G model was selected by the program jModelTest according to the Bayesian information criteria [23]. 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 [24]. 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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In addition, the new name contained in this work has been submitted to IPNI, from where it will be made available to the Global Names Index. The IPNI LSIDs can be resolved and the associated information viewed through any standard web browser by appending the LSID contained in this publication to the prefix http://ipni.org/. The online version of this work is archived and available from the following digital repositories: PubMed Central and LOCKSS.

Lemma epidermal pattern
Ptilagrostis contracta has typical Ptilagrostis saw-like lemma epidermal pattern (Ptilagrostis SLP) [8]. 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).  (Fig 1 and Fig 3).  [6,25,26] in having contracted panicles. For distinguishing these 3 narrow panicled species, a key is provided here. 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) [27]. 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) [27].

Discussion
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 [28] 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.
Supporting Information S1 Table. GenBank accession numbers and voucher information for the materials used in this study. (XLSX)