Gentianella macrosperma, a new species of Gentianella (Gentianaceae) from Xinjiang, China

Abstract Gentianella macrosperma Ma ex H.F. Cao, J.D. Ya & Q.R. Zhang, a new species of Gentianaceae from Xinjiang, Northwest China is described and illustrated. This new species is unique in having equal length of corolla lobe and corolla tube, nectaries located at the throat of the corolla tube and large seeds up to 1.6 mm in diameter. In addition, an updated identification key to the Chinese species of Gentianella is provided.


Introduction
Gentianella Moench (Gentianaceae) consists of approximately 300 species distributed from the temperate, arctic and alpine regions of the Northern Hemisphere, to South America, Australia and New Zealand (Pringle 2017). About 70% of species (ca. 200 spe-cies) occur in South America, where new species continue to be discovered (Pfanzelt et al. 2015;Pringle 2015Pringle , 2017Pringle and Grant 2017). Molecular phylogenetic studies indicated that Gentianella was polyphyletic, and the new circumscription of Gentianella s. str. contains species with one nectary per petal lobe Kadereit 2001, 2002). However, the taxonomic placement of the Asiatic species with two nectaries per corolla lobe has yet to be determined. Before the phylogenetically-based concept of Asiatic gentianellas proposed, the description of this genus published in Flora of China (Ho and Pringle 1995) remains applicable in the present context. There are 10 species of Gentianella reported from China and mainly distributed in northern China and alpine areas of southwest China mountains (Ho andPringle 1995, Chen et al. 2011).
During the field expedition to west of Xinjiang, China, an unusual species of Gentianaceae was collected. Its corolla campanulate without plicae and fringed scale, lobed to middle of corolla, two nectaries per corolla lobe located at the corolla tube fit the main characters of Gentianella. Subsequent morphological investigation and molecular study supported this species as new to science and described here.

Materials and methods
Specimen collections of Gentianella were carefully examined, especially the relevant species, including G. holosteoides Schott & Kotschy ex N.M. Pritch., G. longicarpa (Gilli) Holub, G. sibirica (Kusn.) Holub, G. stoliczkae (Kurz ex C.B. Clarke) Holub and G. umbellata (M. Bieb.) Holub. Collections at the following herbaria (BM, FR, GH, GLM, HIMC, HNWP, JE, K, E, KFTA, KUN, MA, MPU, MW, P, PE, PEY, W, WAG) were checked on-site and via Chinese Virtual Herbarium (CVH, http://www. cvh.ac.cn/), Global Biodiversity Information Facility (GBIF, https://www.gbif.org/) and Global Plants on JSTOR (https://plants.jstor.org/). The high-resolution images of type specimen of G. sibirica (LE01043410, LE01043411, LE00050650) were obtained from curators of LE. Relevant literatures were investigated (Gillett 1957;Shishkin and Bobrov 1967;Omer et al. 1988;Ho and Pringle 1995;Omer 1995;Struwe et al. 2002;Aitken 2007;Chen et al. 2011;Mohd et al. 2018). Line drawings, description and most of photographs were based on the latest collections (J.D. Ya et al. 17CS16327), except that the images of seeds were from the type specimen (Shun-Li Chen Tianyi281, PE00029466). The conservation status of the new species was evaluated according to the guidelines of the IUCN Red List Categories and Criteria (IUCN 2017) Fresh leaves of this new species were dried immediately by using silica gel for DNA extraction. Genomic DNA extraction, amplification and DNA sequencing of ITS and the plastid matK followed the protocol described by Xi et al. (2014) and sequences of relevant species were downloaded from GenBank (Appendix 1).
The molecular phylogenetic tree of 88 species representing 13 genera of Gentianaceae was reconstructed using Bayesian Inference (BI) and Maximum Likelihood (ML). Chelonanthus alatus (Aubl.) Pulle (Gentianaceae: Helieae) was chosen as outgroup (Figure 1). ITS and matK datasets were combined for analysis. BI analysis was performed using MrBayes 3.26 (Ronquist and Huelsenbeck 2003). Markov Chain Monte Carlo (MCMC) analysis was performed using MrBayes for 10,000,000 generations for the combined dataset, with two simultaneous runs, with each run comprising four incrementally heated chains. BI analysis was started with a random tree and sampled every 1000 generations. The combined dataset was partitioned and the best-fit DNA substitution model for two DNA regions using Bayesian Information Criterion (BIC) was estimated using jModeltest 2 (Darriba et al. 2012). ML analysis was conducted with RAxML 8.2.10 (Stamatakis et al. 2008) using the GTR substitution model with gamma-distributed rate heterogeneity amongst sites and the proportion of invariable sites estimated from the data. Support values for nodes/clades were estimated from 1000 bootstrap replicates.

Results
The ITS matrix was 689 bp in length including 376 variable sites and 266 parsimonyinformative sites and the matK matrix was 821 bp in length including 286 variable sites and 198 parsimony-informative sites. The best-fit BIC model of ITS and matK datasets was SYM+G and TVM+G, respectively. The major-rule consensus tree of both BI and ML analyses with support values is shown in Figure 1.
Phenology. Flowering and fruiting from June to September.

Discussion
It was Prof. Yu-Quan Ma (also as Yu Chuan Ma), a specialist of Gentianaceae, who first recognised this plant as a distinct new species and inscribed the name "Gentianella macrosperma Ma" on the specimen kept at PE. Later the same year, he proposed another name "Gentianella procumbens Ma" to the same collections, corresponding to its procumbent stems. However, both names were never published. Based on field observation and specimen examination, procumbent stems occurred occasionally in some individuals, the character of larger seeds being easily distinguished from other Gentianella species.
In all the known Chinese species of Gentianella, the length of corolla lobes is shorter than that of the corolla tube and nectaries which are located at the base or middle of the corolla tube. The same length of corolla lobes and corolla tube and nectaries positioned at the throat of the corolla tube make G. macrosperma a distinctive species amongst them. Its large seeds up to 1.6 mm in diameter are perhaps unique amongst the Asiatic species of Gentianella.
G. macrosperma is similar in size and shape of the corolla lobe to G. sibirica and G. longicarpa, but further differs from them both in the lack of rosette basal leaves, predominant 4-merous flowers and smaller corolla, no more than 5 mm long, except the corolla lobed to the middle, nectaries position and seeds size. Gentianella longicarpa, which is endemic to Afghanistan, is also distinct from G. macrosperma in its light-pink, pale blue or lilac-violet flower and larger corolla up to 8 mm long and all calyx lobes are shorter than the corolla tube. G. macrosperma is similar in habit and inflorescences to G. umbellata and G. stoliczkae. The flower of G. umbellate is larger than those of G. macrosperma and, although the size of the corolla lobe in the two species overlaps, the corolla lobe is much shorter than the corolla tube in G. umbellate. In G. stoliczkae, flowers are in densely clustered cymes, the corolla are generally much larger up to 20 mm long with various colours from purple, pink, pale blue to yellow and the capsule has a short gynophore ca. 1-2 mm long.
The molecular evidence shows that G. macrosperma has the closest relationship with G. holosteoides which is native to Turkey and Pakistan and they also share similar floral whorls and basal leaves shape, but plants of G. holosteoides are smaller in stature, no more than 5 (7) cm height; it further differs from G. macrosperma in its smaller basal leaves, larger flowers with corolla lobes shorter than corolla tube, nectaries position at corolla base and smaller, numerous seeds. A detailed morphological comparison is given in Table 1.
Von Hagen and Kadereit (2001) proposed Gentianella s. str. to only include species with one nectary per petal lobe, however, G. umbellata and G. stoliczkae represented in their study are both binectariate species. Current molecular analyses also shows the binectariate G. macrosperma clustered into von Hagen and Kadereit's Gentianella s. str. A careful selection of species across wider geographic regions of this genus and data from more nuclear and chloroplast sequences may clarify the generic circumscription in Gentianella.

Key to species of Gentianella in China
The following key is based on Flora of China (Ho and Pringle 1995), Flora of the U.S.S.R. (Shishkin and Bobrov 1967) and other literature (Omer et al. 1988;Aitken 2007;Chen et al. 2011). It includes 11 species of Gentianella in China. Plant 12-40 cm tall, nectaries close to the throat of corolla tube, seeds 1.  Corolla lobes 2.0 mm long, the same length as corolla tube 1.5-3.0 mm long, much shorter than corolla tube 2-3 mm long, shorter than corolla tube ca. 2 mm long, much shorter than corolla tube 3-7 mm long, much shorter than corolla tube 2-3(4) mm long, much shorter than corolla tube