Dichocarpum hagiangense—a new species and an updated checklist of Ranunculaceae in Vietnam

Dichocarpum hagiangense from Ha Giang province, northern Vietnam is described and illustrated. Diagnostic features of the new species are a short rhizomatous stem, (2–)3-foliolate or simple leaves, and pink-purple flowers. The described species is distinct from closely allied D. trifoliolatum in having longer sepals, shape and obcordate apex of petal limbs, shorter flower stem, number and tooth shape of basal leaves; it differs from D. basilare and D. carinatum in having stem leaf, retuse apex and longer of central leaflet, number and (2–)3-foliated (or simple) of leaf. With the support of molecular data, the new species was clearly distinguished from other species in the Dichocarpum group by eight autapomorphic characters in nrITS sequence. A key to all species of Dichocarpum is provided. We suggest the IUCN conservation status of D. hagiangense to be “Critically Endangered”. A newest checklist of the family Ranunculaceae in Vietnam is updated.


INTRODUCTION
The flowering plant family Ranunculaceae comprises about 60 genera and 2,500 species worldwide distribution but mainly in East Asia (Tamura, 1993;Wang et al., 2001). In Vietnam, Ranunculaceae has the presence of 11 genera and about 40 species .
In Vietnam, some specimens of Ranunculaceae with the same label (No. 3725) have been collected by P.A. Pételot since 1930 from Sa Pa town, Lào Cai province and deposited in Muséum national d'Histoire naturelle [MNHN-P-P00194832, MNHN-P-P00194833]. The specimens were first identified as Isopyrum adiantifolium Hook.f. & Thomson (1855: 42)  , but were later determined as I. sutchuenense Franch (1894: 284). In 1973, Lauener defined these specimens as Dichocarpum sutchuenense (Franch.) W.T. Wang & P.K. Hsiao (1964: 328). In ''Cay co Viet Nam: an illustrated of flora of Vietnam'',  only recorded this species (Fig. 1). After a botanical exploration in Ha Giang province in 2001, Phan, Averyanov & Nguyen (2001) Hsiao in a cloud forest at an elevation of about 1,500 m a.s.l. (Fig. 1). In addition, in 2002, Averyanov, Loc, and Doan found an unknow Dichocarpum species in Van Ban district, Lao Cai province (Fig. 1). The plants had light blue-violet flowers growing on open wet granite rocks of a high waterfall at elevation 1300 m a.s.l. The specimens depositing at HN, LE, and MO should be examined (HAL 2212). To date, there are only two species of Dichocarpum recorded in Vietnam Phan, Averyanov & Nguyen, 2001). The genus is still scarcely known in the country.

DNA extraction and sequencing
Total DNA was extracted from dried leaves using the DNeasy Plant Minikit. The ITS region was amplified using the forward primer dichFb 5 -CCT GCT CAA GCA GAA CGA C-3 and dichRb 5 -TTG ACA TGC TTA AAT TCA GC-3 designed based on the ITS sequence of Dichocarpum spp. obtained from GenBank. The PCR protocol comprised an initial denaturation at 95 • C for 3min, 35 cycles of 50s at 95 • C, 40 s annealing temperature for the primer at 51 • C, 50s extension at 72 • C, and 10min final extension at 72 • C, then 4 • C until used. After purification, DNA fragments were sequenced with a BigDye Terminator Cycle Sequencing Ready Reaction kit and run on an ABI PRISM 3100 Genetic Analyzer.
The sequence was deposited in Genbank under accession number MT739412. The ITS sequence of D. hangiangensis was aligned using Clustal X 1.64 (Thompson et al., 1997) with ITS sequences of other species of Dichocarpum and Isopyrum manshuricum (EF437119) used as outgroup taxa (Xiang et al., 2017). The distance and equally weighted maximum parsimony (MP) and maximum likelihood (ML) analyses were performed using PAUP* (4.0 beta ver.) (Swofford, 1998). A heuristic search procedure was used with the following settings: ten replicates of random taxon addition, tree-bisection reconnection branch swapping, multiple trees retained, no steepest descent, and accelerated transformation. Gaps were treated as missing data, and there were no indels within the alignment for the Dichocarpum spp. sampled. Bootstrap analysis was carried out with 100 replicates. For ML analysis, the substitution model that best fitted the data set was determined by the Akaike information criterion (AIC) with MODEL Test 3.7 (Posada & Crandall, 1998). Bootstrap analysis with 100 replicates was conducted to assess the degree of support for ML tree clades.

Checklist preparation
The updated checklist is prepared by reviewing all scientific names of Ranunculaceae which had recorded in Vietnam from mainly four monographs -''Flore générale de l'Indo-Chine 1'' , ''Supplément a la flore générale de l'Indo-Chine 1'' , ''Cây có Viê . t Nam: an Illustrated Flora of Vietnam 1'' , and ''Checklist of Plant Species of Vietnam 2'' . The most widely accepted classification system, APG4 (Chase et al., 2016) is applied for the checklist. All the scientific names were nomenclature checked according to Shenzen code of International Association for Plant Taxonomy (Turland et al., 2018) together with online consulted from World Flora Online, The Plant List, and International Plant Names Index websites. The invalid names and cultivation species are not recorded in the checklist.
The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants (ICN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. In addition, new names contained in this work which have been issued with identifiers by IPNI will eventually be made available to the Global Names Index. The IPNI LSIDs can be resolved and the associated information viewed through any standard web browser by appending the LSID contained in this publication to the prefix ''http://ipni.org/''. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central, and CLOCKSS.