Cyclorhizapuana (Apiaceae), a new species from Sichuan, China

Abstract A new species, Cyclorhizapuana J. Zhou & Z.W. Liu (Apiaceae) from Sichuan Province of China, is described and illustrated here. Morphological comparisons with congeneric species revealed that it is distinguished by its slender habit, sparse annular scars, 4-pinnatisect leaf blade with ultimate segments linear (2–4×0.5–1 mm), subequal rays, oblong fruits with slightly thickened ribs, obconic stylopodium and slightly concave seed face. A molecular analysis based on nuclear ribosomal DNA internal transcribed spacer (ITS) sequences indicated that C.puana is genetically distinct from the other two species of the genus. A distribution map, as well as an updated key, are provided for the species of Cyclorhiza.


Introduction
Cyclorhiza M.L. Sheh & R.H. Shan is a small genus of Apiaceae subfamily Apioideae, with two species currently recognised (Sheh and Shan 1980;Sheh and Watson 2005;Pimenov 2017). It is distributed in southwest China and characterised by carrot-like roots with prominent annular scars, bracts and bracteoles usually absent, yellow petals and fruits subpentagonal in cross section (Sheh and Shan 1980;Sheh and Watson 2005). Previous phylogenetic studies indicated that the two species of Cyclorhiza con-stituted a highly-supported monophyletic clade in the tribe Komarovieae and showed a sister group relationship to Calyptrosciadium Rech. f. & Kuber from SW Asia (Zhou et al. 2009;Downie et al. 2010;Zhou et al. 2020).
During a botanical survey to examine Apiaceae in Sichuan Province of China, we discovered a small population of Cyclorhiza, whose morphology was clearly distinct from the other species of the genus. We checked all the collections of Cyclorhiza at PE and KUN, and digital resources from CVH and GBIF. We noticed a specimen identified as C. waltonii in CSH, whose morphology is exactly the same as in our collection. Further examination of morphological characters, coupled with molecular evidence, convinced us that this plant is a distinct new species, which is described and illustrated here.

Morphological studies
The morphological characters were examined based on collected specimens. Fruits were taken from dry specimens and studied using a stereo microscope. Herbarium specimens were deposited at KUN. Morphological comparisons with the related C. waltonii (H. Wolff) M.L. Sheh & R.H. Shan and C. peucedanifolia (Franch.) Constance are provided in Table 1 and Fig. 1.

Phylogenetic analysis
For the new species, we used our own collection and the specimen deposited as CSH.
Cyclorhiza waltonii and C. peucedanifolia each included two new accessions to examine the possible infraspecific molecular variation (Table 2). In addition, thirty-two ITS sequences were obtained from GenBank to determine the phylogentic position of the new species. The taxa chosen represented a broad cross-section of sampling from the tribe Komarovieae and other clades or tribes of Apioideae, identified in previous phylogenetic studies Zhou et al. 2020). Two species of Bupleureae were used to root the tree. The final data matrix comprised a total of 38 accessions. Detailed information about herbarium vouchers, GenBank accession numbers, and literature citations of previously published sequences for those taxa considered in this study are listed in Table 2.
The Plant Genomic DNA Kit (Tiangen Biotech) was used to isolate DNA from materials of silica-gel-dried and herbarium specimens, and nuclear ribosomal DNA internal transcribed spacer (ITS) sequences were used for phylogenetic inference. Detailed information on PCR amplification and sequencing strategies was obtained from Zhou et al. (2008). Phylogenetic analyses were conducted using both Maximum Likelihood (ML) and Maximum Parsimony (MP). ML analyses were performed using MEGA7 (Kumar et al. 2016), with the GTR + G + I model and 1000 bootstrap (BS) replicates. Parsimony analysis was performed using PAUP* v. 4.0b10 (Swofford 2003). For the heuristic search, 100 random addition sequence replicates, tree bisection-reconnection (TBR), saving multiple trees and ACCTRAN optimisa-tion were chosen. Gaps were treated as missing data. Bootstrap values were calculated from 1000 replicate analyses using TBR branch swapping and simple stepwise-addition of taxa.

Molecular phylogenetic analysis
The final aligned data matrix contained 629 positions, in which 256 were parsimony informative. The new species yielded high sequence divergence values with the other two species of the genus, i.e. C. waltonii (5.21%) and C. peucedanifolia (4.39-4.60%). The phylogeny showed that two accessions of the new species formed a strongly-supported monophyletic group and constituted a sister branch of Cyclorhiza species within the tribe Komarovieae (Fig. 2). Each of the three species was resolved as monophyletic groups, with the infraspecific divergence values ranging between 0.00-0.58%.  Diagnosis. Cyclorhiza puana resembles C. waltonii but differs from the latter in its long-cylindric roots with sparse annular scars (vs. stout, branched near stem into a cluster of several long, woody, carrot-like roots with prominent annular scars), smaller ultimate segments 2-4 × 0.5-1 mm (vs. 4-20 × 2-6 mm), rays subequal (vs. unequal), stylopodium obconic (vs. low-conic) and seed face slightly concave (vs. deeply sulcate).
Etymology. The species epithet "puana" is given in honour of Prof. Pu Fading (1936-) for his outstanding contributions to the Chinese Apiaceae. Vernacular name. The Chinese name is given as "炉霍环根芹" (lú huò huán gēn qín), referring to the locality where the type specimen was collected.  Phenology. Flowering from June to July, and fruiting from July to September. Distribution and habitat. The new species is distributed in Sichuan Province, China. It grows in the alpine open mixed forests at elevations of 3000-3200 m (Fig. 3).
Conservation status. So far, only two populations with no more than ten individuals have been found. Through further investigations, more populations may be discovered to assess its conservation status. Based on the available data, the new species can be assessed as Data Deficient (DD) on the basis of recommendations of the International Union for Conservation of Nature (IUCN 2019).

Discussion
Due to its topographical and climatic heterogeneity, the Hengduan Mountains of the Sino-Himalayas is one of the richest regions across China in terms of biodiversity (Ying and Zhang 1984;Pu 1993 (Sheh and Watson 2005). The new species seems to be relatively rare and known only from two localities in Sichuan Province, where it grows in alpine open mixed forests at elevations of 3000-3200 m. Generally, the three species share a similar ecology and habitat. Cyclorhiza has been regarded as a well-defined genus since it was established by Sheh and Shan in 1980. The new species possesses typical characteristics of the genus, such as taproots with prominent annular scars, bracts and bracteoles usually absent, and fruits subpentagonal in cross section, with variation in some characters (e.g. the seed face slightly concave, and the stylopodium obconic).
The specimen from CSH identified as Cyclorhiza waltonii actually corresponds to C. puana. The confusion between these two species is likely driven by the similar morphology of the leaf blades, i.e., ultimate segments. However, the ultimate leaf segments in C. puana are smaller, 2-4 × 0.5-1 mm, whereas those in C. waltonii are 4-20 × 2-6 mm (Sheh and Shan 1980). Additionally, C. puana can be distinguished by obconic stylopodium (vs. low-conic), ribs filiform, prominent, slightly thickened (vs. filiform, prominent, narrowly winged) and seed face slightly concave (vs. deeply sulcate). The genetic difference between these two species is relatively large (> 5%), further supporting their separate status.