Tongoloa arguta (Apiaceae), a new species from southwest China

Abstract A new species Tongoloa arguta (Apiaceae) is described and illustrated in this article. The new species grows in alpine bushes and meadows in south-western China. It resembles T. silaifolia, but differs from the latter by the length of the stem, ultimate segments of leaf and rays of the umbel. Phylogenetic analysis, based on nuclear ribosomal DNA internal transcribed spacer (ITS) sequences, is provided, as well as comparative morphology between related species.


Introduction
Tongoloa H.Wolff (Apiaceae) is a genus comprising about 15-20 species distributed mainly in southwest China, with a few species extending westwards to central Nepal (Watson 1999;Pan and Watson 2005;Zhou et al. 2009). Tongoloa species are characterised by having conic taproots, inflated and membranous leaf sheaths, cordate fruit base and filiform fruit ribs (Wolff 1925;Mukherjee and Constance 1991;Pimenov and Kljuykov 2000;Pan and Watson 2005). Some species have been described in Pimpinella L. due to the morphological similarity (Boissieu 1902(Boissieu , 1906. The genus Tongoloa was formally established by Wolff (1925) and accepted as an inde-pendent genus (Pimenov and Kljuykov 1995;Pimenov 2017). Molecular phylogenetic analyses, based on limited materials of nuclear ribosomal DNA internal transcribed spacer (ITS) and chloroplast markers, indicated that Tongoloa is part of the East Asia clade of Apioideae (Apiaceae) (Zhou et al. 2009;Downie et al. 2010).
So far, 15 species of Tongoloa have been identified from different regions of China (Pimenov 2017), most of which being known from the Hengduan Mountains. While studying specimens in herbariums (CDBI, PE), we noticed several interesting specimens of Tongoloa collected from Sichuan and Yunnan, which have short stems and fewer rays of the umbel (3-8). Through field investigation and anatomical study, we confirmed that this species does not match any previously-published description of Tongoloa found from southwest China to central Himalaya. Further molecular analysis revealed significant differences between this species and its relatives. The results allow us to infer that these newly-collected specimens from Sichuan and Yunnan belong to a new species.

Materials and methods
We collected an unknown Tongoloa species from several populations in Yunnan and Sichuan Provinces. In addition to the samples collected in the field, the type specimens of Tongoloa and high-resolution type specimen photos were examined, including the specimens deposited in K, P, E, B, A, GB, LD, MW, NY, GH, W, US, PE, KUN, CDBI, WUK and HNWP. Considering the similarity between the new species and T. silaifolia, as well as other related species, we compared their morphological characteristics. The fresh fruits were preserved with formaldehyde-acetic acid-alcohol (FAA) for anatomical study. The mericarp transverse sections were examined using a stereomicroscope (Nikon SMZ25, Japan) after safranin O-fast green staining.
A plant genomic DNA kit (CWBIO, China) was used to extract total DNA from silica-dried leaves. Referring to the previous studies (White et al. 1990;Zhou et al. 2009), we used nuclear ribosomal DNA internal transcribed spacer (ITS) sequences for phylogenetic inference. Amplification was undertaken using a volume of 30 µl with 15 µl 2 × Taq MasterMix (CWBIO, China), 10 µl ddH 2 O, 1.5 µl forward primer, 1.5 µl reverse primer and 2 µl total DNA. The PCR reaction was performed in Geneamp PCR System 9700 (USA) with initial denaturation at 95 °C for 2 min, 35 cycles of 94 °C for 60 s, 52.5 °C for 45 s and 72 °C for 60 s and a final extension of 72 °C for 7 min. PCR products were sent to BGI (China) for sequencing. The GenBank accession numbers and sample information of the ITS sequences used in this study are shown in Table 1.
To determine the systematic position of the new species, 37 ITS sequences with accession numbers were obtained from GenBank, including 9 species of Tongoloa ( Table 1). Taxa of Chamaesium clade were selected as the outgroup (Downie et al. 2010). Maximum Likelihood (ML) analyses with GTR + G + I model and 1000 bootstrap (BS) replicates was performed using MEGA7 (Kumar et al. 2016). Bayesian Inference (BI) analysis was conducted with MrBayes version 3.2 (Ronquist et al. 2012) and the Markov Chain Monte Carlo (MCMC) search was performed for 1 × 10 8 generations.
Etymology. The species epithet "arguta" was given to describe the acute tips of the ultimate segments of leaves.
Phenology. The species was observed flowering from August to September and fruiting from September to October.  Conservation status. Tongoloa arguta is common in some alpine bushes and meadows at an altitude of about 4300 m in Yunnan and Sichuan, where human activities and especially yak grazing pose a potential threat to its survival. We categorise T. arguta as Near Threatened (NT), according to IUCN (2019).