Carpinus tibetana (Betulaceae), a new species from southeast Tibet, China

Abstract A new species Carpinus tibetana Z. Qiang Lu & J. Quan Liu from southeast Tibet is described and illustrated. The specimens of this new species were previously identified and placed under C. monbeigiana Hand.-Mazz. or C. mollicoma Hu. However, the specimens from southeast Tibet differ from those of C. monbeigiana from other regions with more lateral veins (19–24 vs 14–18) on each side of the midvein and dense pubescence on the abaxial leaf surface, while from those of C. mollicoma from other regions differ by nutlet with dense resinous glands and glabrous or sparsely villous at apex. Principal Component Analyses based on morphometric characters recognise the Tibetan populations as a separate group. Nuclear ribosomal ITS sequence variations show stable and distinct genetic divergences between the Tibetan populations and C. monbeigiana or C. mollicoma by two or three fixed nucleotide mutations. Phylogenetic analysis also identified three respective genetic clusters and the C. mollicoma cluster diverged early. In addition, the Tibetan populations show a disjunct geographic isolation from the other two species. Therefore, C. tibetana, based on the Tibetan populations, is here erected as a new species, distinctly different from C. monbeigiana and C. mollicoma.


Introduction
The birch family (Betulaceae) comprises six genera and approximately 167 species (Christenhusz and Byng 2016). In this family, the hornbeams in the genus Carpinus (Linnaeus, 1753) are small to medium-size trees (Li and Skvortsov 1999;Holstein and Weigend 2017). In Flora of China, 33 hornbeam species are described and 28 of which are endemic (Li and Skvortsov 1999). The endemic species C. monbeigiana Hand.-Mazz. is mainly distributed in southeast (SE) Tibet and northwest (NW) Yunnan. This species is recognised due to the leaves doubly or simply setiform serrate along the margin, nutlets with dense resinous glands, peduncles and rachises with densely yellow hirsute and densely hispidulous bracts with an inflexed auricle at the base of the inner margin. However, a small number of specimens from SE Tibet were also identified as C. mollicoma Hu because of the numerous lateral veins and dense pubescence on the abaxial leaf surface (Li and Skvortsov 1999). Another species, C. viminea Wall. ex Lindl. is also distributed to SE Tibet and NW Yunnan (Wu 1991, Li andSkvortsov 1999). However, C. viminea is distinctly different from both C. monbeigiana and C. mollicoma with the long leaf petiole and a lobe at the base of the inner margin of bract. After examining all specimens of C. monbeigiana and C. mollicoma preserved in the Chinese Virtual Herbarium (http://www.cvh.org.cn) and Lanzhou University (LZU) in 2015, we found that the specimens from Tibet under C. monbeigiana or C. mollicoma might stand as a new species because they are clearly different from specimens of the two species collected from Yunnan ( Figure 1). In order to further test this hypothesis, we conducted field surveys and an examination of morphological variation and genetic divergence. All lines of evidence support the establishment of a new species to accommodate the Tibetan populations as distinct from both C. monbeigiana and C. mollicoma.

Field surveys
After examining Carpinus specimens preserved in KUN and PE (Table 1), we found that the nutlet sizes of C. monbeigiana become stable after July. This was further confirmed by the measurements of the nutlet sizes of C. monbeigiana collected between July and September in 2015 from the same locality (Xishan, Kunming, Yunnan Province). Hence, collections from before July were excluded in our measurements of the morphological variation of specimens. We conducted the field surveys in Tibet and Yunnan from July to September in 2015 and 2016 in order to collect enough samples from different individual trees for morphological analyses and later genetic analyses. For the latter purpose, fresh leaves of each tree were immediately dried by silica gel in a plastic bag. All sampled populations of C. monbeigiana and C. mollicoma in the field are listed in Table 2. Voucher specimens were deposited in Lanzhou University Herbarium (LZU).

Morphological analysis
A total of 90 specimens (19 from southeast Tibet, 17 for C. mollicoma and 54 for C. monbeigiana) from individual trees were used for morphological comparisons. We examined morphological variations within and between the Tibetan populations and C. monbeigiana and C. mollicoma from other regions (Table 1) and measured 22 characters for morphological Principal Component Analyses (PCA) ( Table 3).

Genetic analysis
For genetic analyses of the nuclear ITS region, 33 individuals from 7 populations of three groups were used. Amongst them, 8 individuals from two populations were collected from southeast Tibet while 9 individuals for C. mollicoma and 16 individuals for C. monbeigiana. Carpinus viminea was also included because this species also occurs in SE Tibet and NW Yunnan (Wu 1991, Li andSkvortsov 1999). Total DNA was extracted from 15-25 mg silica gel dried leaves using the modified CTAB method (Doyle and Doyle 1990). Nuclear ribosomal ITS sequence was used to confirm the species status of the Tibetan populations because the sequence variation of this fragment is stable within and between species with high species discrimination power (Lu et al. 2016). PCR amplifying and sequencing of the ITS fragment followed Lu et al. (2016). All newly available ITS sequences were uploaded to GenBank under the accession numbers KY436145-KY436155 and KY683787-KY683789. We used RAxML-8.1.17 (Stamatakis 2014) to conduct the Maximum likelihood (ML) analyses under the GTR + G model. Bootstrap replicates (1000) were set to calculate the support values.

Results
Morphologically, the Tibetan populations (Table 1; Figures 2-3) differ distinctly from those of C. monbeigiana from Yunnan with more lateral veins (19-24 vs 14-18) on each side of the midvein and more densely pubescent on the abaxial leaf surface and the difference was also found in the narrower distance between lateral veins (4-5 mm vs 5-8 mm) and smaller nutlet (Table 3). Meanwhile, plants of the Tibetan populations also differ from C. mollicoma by the nutlet having dense resinous glands and being glabrous or sparsely villous at apex. The difference was also found in the size of infructescence (2.5-4.5 cm × 1-1.5 cm vs 4-7 cm × 1.5-2.5) and bract (0.9-1.9 cm × 0.4-0.6 cm vs 1.5-1.9 cm × 0.6-0.9 cm). A Principal Component Analyses (PCA) distinguished samples from the two species and the Tibetan populations into three dif- Table 2. Locations of the sampled populations from which individuals were used for genetic analyses of the nuclear ribosomal ITS sequence variations.

Species (individual number)
Location  Figure 4). The first principal component axis (PC1; accounting for 43.16% of the variation) significantly separated C. mollicoma from C. monbeigiana and Tibetan populations, where there was a slight overlap between them. However, the second principal component axis (PC2; 14.51%) significantly separated the Tibetan populations from the other two species. Genetically, the aligned 33 ITS sequences were 611 base pairs in length. In addition, three ITS sequences from C. monbeigiana were also downloaded from NCBI (AF432043, AF432044 and AF432048). In total, 16 types were identified from these   sequences and the individual number of shared types is presented in Table 5. Phylogenetic analysis of these sequences suggested that the sampled individuals of C. monbeigiana, C. mollicoma and the Tibetan populations separated into three genetic clades with C. mollicoma diverging first and C. monbeigiana and plants from the Tibetan populations forming a sister relationship ( Figure 5). The sequence variations of the Tibetan individuals showed a combination of the mutations found for C. mollicoma or C. monbeigiana (Table 2). Geographically, all specimen records in the present study and those from Chinese Virtual Herbarium (http://www.cvh.org.cn/) suggested that the Tibetan populations are disjunct in geographical distributions from both C. monbeigiana and C. mollicoma ( Figure 6). Table 5. Nuclear ribosomal ITS sequence variations between three closely related species. The fixed nucleotide mutations were presented in bold type. Three ITS sequences (Type 5 and Type 6) of Carpinus monbeigiana (from Yunnan) were downloaded from NCBI (AF432043, AF432044 and AF432048).

Discussion
Different species concepts emphasise the different criteria used to define and publish a new species (Wheeler and Meier 2000). An integrative practice using multiple criteria to circumscribe species boundaries will produce relatively objective and operational taxonomy (Su et al. 2015, Hu et al. 2015, Liu 2016Lu et al. 2017). In this study, we demonstrated that the Tibetan populations previously placed under C. monbeigiana or C. mollicoma should be erected as a distinct new species based on the following lines of evidence. Firstly, these populations were obviously distinct from those of C. monbeigiana by the more lateral veins and dense pubescence on the abaxial leaf and from C. mollicoma by the nutlet with dense resinous glands and glabrous or sparsely villous at apex. All statistical analyses of the morphological traits clustered them into three separated groups. These populations seem to be characterised by a morphological combination of the other two species. Secondly, genetic divergences amongst these three groups are distinct; all of the sampled individuals from the Tibetan populations have a combination of unique genetic mutations that are found in the other two species but in a combination that is distinct from them. Phylogenetic analysis of nuclear ribosomal ITS sequence variations suggested that all sampled individuals from the Tibetan populations comprised a genetic cluster which seems to be more closely related to C. monbeigiana than to C. mollicoma. Finally, the Tibetan populations occupy a distinct distribution disjunct from others of C. monbeigiana and C. mollicoma. All lines of evidence suggest that the divergence amongst these populations is consistent with warranting three distinct species. Given this, we here recognise the Tibetan populations as a new species. In addition, this new species probably originated through the geographic isolation from hybrid (homoploid or allopolyploid) speciation between C. monbeigiana and C. mollicoma because of its morphological and genetic combination of the other two species. However, this hypothesis needs further tests from multiple lines of evidence, including both chromosomal and population genetic observations. Description. Trees to 10 m tall, deciduous; bark grey, smooth. Branchlets dark grey, densely yellow or white pubescent when young, glabrescent the next year. Stipules deciduous. Petiole 5-8 mm, densely white or yellow puescent; leaves alternate, leaf blade ovate-elliptic or elliptic, usually 5-8 cm × 2-3 cm, abaxially sericeous-villous along veins, pubescent elsewhere, base rounded or rounded-cuneate, margin irregularly and doubly setiform mucronate serrate, apex attenuate-acuminate or caudateacuminate; lateral veins (17) 19-23 on each side of midvein. Male inflorescence pendulous, spicate-cymose, cylindric, enclosed by buds during winter, with many overlapping bracts, 1.5-5.0 cm × 5.0-8.0 mm when mature; flowers without bracteoles, inserted at base of bracts. Female inflorescence terminal or axillary on dwarf shoots, racemose; flowers paired; bracts leaflike, complanate, overlapping. Mature infructescence 5-10 cm × 2.0-3.5 cm; peduncle ca. 1.2 cm, densely yellow hirsute; bracts of female flowers loosely overlapping, 1.5-1.9 cm × 6-8 mm, abaxially densely yellow hirsute along reticulate veins, outer margin coarsely dentate, without basal lobe, inner margin entire, with inflexed basal auricle, apex acuminate or caudate-acuminate; veins 5-6. Nutlet ovoid-ellipsoid, 3.2-3.6 mm × 2.2-2.5 mm, glabrous or sparsely villous at apex, densely brown or orange resinous glandular, prominently 8-or 9-ribbed. Fl. Apr-May, fr. Jul-Sep.

Taxonomic treatment
Etymology. Due to its narrow distribution in Tibet, we give the specific epithet (Carpinus tibetana) referring to the name of the Xizang Autonomous Region (Tibet) of China where it is distributed.

1
Bracts with lobes at bases of inner and outer margins; petioles slender, (1.