Taxonomic Identity of Carpinus dayongina Franchet (Betulaceae)

Abstract Carpinus polyneura and C. dayongina are recognised as separate species in Flora of China. In this study, the results of an examination of literature, morphological comparison and phenetic clustering of nuclear ITS sequences suggest that C. dayongina is conspecific with C. polyneura. Thus, we propose reducing C. dayongina to a synonym of C. polyneura.


Introduction
Carpinus (Linnaeus 1753) is a large genus in the family Betulaceae. It contains about 50 species distributed across the Northern Hemisphere (Asia, Europe, North America) (Li and Skvortsov 1999;Holstein and Weigend 2017), 36 of which occur in China (including 30 endemic species) (Li and Skvortsov 1999;Tong et al. 2014;Lu et al. 2017;Lu et al. 2018;Lu 2020). Although some Carpinus species have been studied (Hu 1948;Hu 1964;Li 1979;Ill and Chang 1997;Li and Skvortsov 1999), this genus is still taxonomically problematic because of the lack of comprehensive field investigations and analyses of morphological characters for some species.
Carpinus polyneura Franchet was described, based on collections (Farges, s.n.) (Fig. 1A) from Chengkou County, Sichuan Province (now Chengkou County, Chongqing City) (Franchet 1899). Although there are some morphological variations amongst populations of this species due to its wide distribution, the higher density of leaf veins and setiform serrate leaf margin make it clearly distinguishable from the other species of Carpinus (Hu 1964).
In the protologue, Carpinus polyneura is described as having lanceolate or ovatelanceolate with a long-acuminate apex and simply serrate margin and nutlets that are mainly villous at the apex. Carpinus dayongina K. W. Liu & Q. Z. Lin was described, based on several collections ( Fig. 1B-D) from Tianmenshan, Dayong County, Hunan Province (now Tianmenshan, Zhangjiajie City, Hunan Province) (Liu and Lin 1986). Liu and Lin (1986) stated that C. dayongina was similar to C. polyneura, but could be distinguished by its narrower leaves, shorter infructescence, smaller bracts and narrower leaves. They cited two collections, i.e. K. W. Liu 33359 and Y. T. Xiao 40700, with the former designated as holotype. The collection, Y. T. Xiao 40700, contains a total of six specimens (here considered as duplicates). The specimen with the barcode CSFI017465 ( Fig. 1C) bears leaves that are clearly wider in shape (ovate-lanceolate) than those of the other five specimens (narrow-lanceolate) and it was identified as C. polyneura previously (by Qi Cheng Jing in June 1984). Li and Skvortsov (1999) pointed out that C. polyneura has leaves with doubly setiform serrate margin and nutlets that are pubescent, while C. dayongina has narrower leaf blades with simply setiform serrate margin and nutlets that are only villous at the apex (Table 1).
When revising the species of Carpinus in China, we noticed that these two species are very similar to each other. This made us speculate that the two are possibly conspecific, although Li and Skvortsov (1999) followed Liu and Lin (1986) and treated them as separate species in Flora of China.

Morphological analysis
Specimens of C. polyneura and C. dayongina deposited in the herbaria CSFI, HHBG, HIB, HNWP, IBK, IBSC, IFP, KUN, LBG, NAS, P, PE, SHM, SZ and WUK were studied and field investigations in Guizhou, Hubei, Hunan and Zhejiang to study C. polyneura and C. dayongina had been conducted in recent years. The morphological characteristics of the two species were also documented via photography and some of the physical features were measured (Table 2). Abbreviations for the names of herbaria in this study refer to the Herbarium Index Database (http://sweetgum. nybg.org/science/ih/).  Li and Skvortsov (1999

Molecular analysis based on nuclear ribosomal ITS sequences
Twelve individuals from five populations of the two species (Table 3), including nine individuals of C. polyneura (P1-P9) and three individuals from the type locality of C. dayongina (D1-D3), respectively, were sampled. Fresh leaves were collected from each individual. Coordinates and altitude information were recorded by using a hand-held GPS. All voucher specimens were stored in Nanjing Forestry University (NF). DNA was extracted using the modified CTAB method (Doyle and Doyle 1990). PCR amplifying and sequencing of the ITS fragment refer to Lu et al. (2016). We made an alignment of 12 newly-sequenced ITS fragments and 10 ITS sequences of C. polyneura, C. mollicoma (Hu 1949), C. rupestris (Camus 1929) and C. tschonoskii (Maximowicz 1881b) that were downloaded from NCBI (Table 4). We used Mega X (Kumar et al. 2018) to construct a neighbor-joining (NJ) tree (Saitou and Nei 1987) using pairwise deletion and the P-distance model. Bootstrap values were set to 1000 to calculate the support values.

Results and discussion
Re-collections of material from the type localities and further field investigation showed that the leaf shapes are quite variable in the same area, from ovate-lanceolate to lanceolate, then narrow-lanceolate (Fig. 2). After a thorough examination of more specimens, we found that, not only leaf blade shape, but also infructescence length and bract size of C. dayongina, are all within the limits of C. polyneura (Table 5). After review of the type specimens, we found the leaf margins of C. polyneura and C. dayongina have both simply and doubly setiform serration (Table 5). In addition, by carefully searching for comprehensive and extensive groups of specimens, performing field investigations and measuring morphological characteristics, we found that the indumentum of their nutlets is also variable, from being villous at the apex and glabrous, sparsely villous or pubescent in the remaining part (Fig. 3, Table 5). Therefore, this character can also not be regarded as a useful character to differentiate these two taxa.  Phenetic comparison of ITS sequences showed that the samples of C. dayongina from Tianmenshan population and those of C. polyneura from other regions are mixed with each other (Fig. 4).
We therefore conclude that C. dayongina and C. polyneura are conspecific. According to ICN (McNeill et al. 2018), the earlier published C. polyneura has priority over C. dayongina and thus, C. dayongina is reduced to a synonym of C. polyneura herein.