Chayamaritia vietnamensis (Gesneriaceae), a new species from Son La Province, northern Vietnam

Abstract Chayamaritia vietnamensis, a new species from Son La Province, northern Vietnam, is described and illustrated. The phylogenetic study revealed that the new species is most closely related to C. banksiae and C. smitinandii. The morphological comparison suggests it as the third new species of Chayamaritia and distinguished from C. banksiae and C. smitinandii by a combination of morphological characters of leaf blades, bracts, calyx and corolla, especially its peltate leaf blades. This species is provisionally assessed as endangered (EN B1ab(iii), B2ab(iii)) using IUCN Categories and Criteria. Information on ecology, phenology and an identification key for the known Chayamaritia species are also provided.


Introduction
The genus Chayamaritia D.J. Middleton & Mich.Möller (Gesneriaceae) was originally established and described, based on molecular and morphological data (Middleton et al. 2015). The genus Chayamaritia comprises two species and is hitherto known only in Laos and Thailand. A thickened rhizomatous prostrate stem, along with alternately arranged leaves and the imbricate sepals characterise the genus (Middleton et al. 2015). The type species, Chayamaritia smitinandii (B.L.Burtt) D.J.Middleton, was initially placed in the genus Chirita Buch.-Ham. (Burtt, 2001), then transferred to the genus Henckelia Spreng. (Weber et al. 2011) and eventually separated as a new genus in 2015, together with the newly-described species, Chayamaritia banksiae D.J.Middleton (Middleton et al. 2015). Chayamaritia banksiae and C. smitinandii are endemic to Laos and Thailand, respectively (Middleton et al. 2015).
During a joint Sino-Vietnamese botanical survey for plant biodiversity in northern Vietnam in late October 2019, we collected an unknown Gesneriaceae plant. In the Xuan Nha Nature Reserve, Moc Chau District, Son La Province, northern Vietnam, we discovered this plant that looked like a Chayamaritia species. Detailed comparison of these specimens with the type specimens and protologues of known Chayamaritia species showed that these specimens neither fitted the existing protologues nor conformed to the type specimens of these species. Although, the leaf indumentum and inflorescence of the unknown plant was most similar to those of C. banksiae and C. smitinandii, it could be easily distinguished from the latter two by the combination of several morphological characters of the leaf blades, bracts, calyx and corolla, especially its peltate leaf blade. Thus, we confirmed that it represented a new species of Chayamaritia, which is the first Chayamaritia species from Vietnam. The description, illustration, information on ecology, phenology and provisional conservation assessment using IUCN (2019) of the proposed new species are provided. Furthermore, an identification key to the known Chayamaritia species is given.

Plant material
Herbarium materials were studied from the following herbaria: E, IBK, US and VNMN (herbarium acronyms according to Index Herbariorum; Thiers 2019). The macromorphological features were observed, based on the specimen sheets and notes in both the field and the conservation nursery at the Gesneriad Conservation Center of China and the National Gesneriaceae Germplasm Bank at GXIB. Micromorphological features were analysed and photographed using an optical microscope (Stemi DV4, LEICA S8 AP0, Jena, Germany).
These morphological characters of newly-proposed species were compared with those of the two known Chayamaritia species from protologues, type specimens and living plants. The description of the new species followed the terminology used by Harris and Harris (2001) and Wang et al. (1998). Assessment of the conservation status of the new species was according to Categories and Criteria of the IUCN (2019).

DNA extraction, PCR amplification and sequencing
Leaves were dried using silica gel for DNA extraction (Chase and Hills 1991). Genomic DNA was extracted using the CTAB protocol (Doyle and Doyle 1987). To confirm the placement of this new species, we performed phylogenetic studies of DNA sequence data obtained from the nuclear ribosomal internal transcribed spacer (ITS) region and the plastid trnL-F intron spacer (trnL-F). Given the phylogenetic studies of Middleton et al. (2015), we sampled two species (three accessions) from Chayamaritia and the new species as ingroup and ten species from all closely related and morphologically similar genera, including Allostigma (one species), Deinostigma (two species), Loxostigma (two species), Petrocosmea (two species), Pseudochirita (two varieties) and Primulina (two species) as outgroup (Middleton et al. 2015). DNA extraction, PCR amplification and sequencing were performed following Wei et al. (2013). The species name, voucher specimens and GenBank accession numbers of DNA sequences used in this study are listed in Table 1.

Phylogenetic analysis
The sequence data were edited and assembled using Lasergene Navigator 7.1 (DNAstar, Madison, Wisconsin, USA). Two datasets (ITS and trnL-F) were aligned independently using MAFFT version 7.0 (Katoh and Standley 2013) with default settings. The two best-supported tree topologies from Maximum Likelihood (ML) analyses of ITS and trnL-F were compared visually for topological incongruences. As there were no hard incongruences , phylogenies were reconstructed, based on the combined dataset using ML and Bayesian Inference (BI). BI was performed using MRBAYES v.3.2.7 (Ronquist et al. 2012). Best-fitting models for the BI analysis were obtained independently for each gene region using MODEL-TEST v.3.7 (Posada and Buckley 2004). GTR+G and GTR+I were the best-fitting models for ITS and trnL-F, respectively. One cold and three incrementally heated Markov Chain Monte Carlo (MCMC) chains were run for five million generations and trees were sampled every 1,000 generations (5,000 trees sampled in total). The first 1250 trees (25%) were discarded as burn-in prior to calculating the BI consensus trees and posterior probabilities (PP) (See Suppl. material 1: log file). The ML analyses were performed in RAxML using raxmlGUI (Silvestro and Michalak 2012), with GTRGAMMA setting and 1,000 bootstrap replicates.
Phenology. Flowering occurs from October to December and fruiting from November to January.
Etymology. The specific epithet "vietnamensis" is derived from Vietnam, which holds the first discovered and only known location for the species.
Distribution and habitat. Chayamaritia vietnamensis is hitherto only known from the type locality, Xuan Nha Nature Reserve, Moc Chau District, Son La Province, northern Vietnam. It grows on rock surfaces surrounded by limestone areas in a subtropical evergreen seasonal rain forest.
Conservation status. Chayamaritia vietnamensis is known from two small-sized populations in the Xuan Nha Nature Reserve's buffer zone. The EOO and AOO of the new species are about 6.15 km 2 and 2.2 km 2 , respectively. Furthermore, the natural habitat is mostly disturbed by local farmers who impose intense pressure on the remaining primary forest patches, by converting the natural habitat of the species into cornfields. Thus, following the IUCN Red List Categories and Criteria (IUCN 2019), it is provisionally assessed as endangered (EN B1ab(iii), B2ab(iii)) Identification key for the three known Chayamaritia species (Fig. 3

Discussion
From the viewpoint of morphology, the newly-described species should be treated as a species of Chayamaritia, because it exhibits a suite of morphological characters that are diagnostic to the genus and should not be established as a distinct genus. It has a thickened rhizomatous prostrate stem, alternately arranged leaves and imbricate sepals (Middleton et al. 2015). The current molecular work also confirmed that the new species is sister to a clade comprising the two known Chayamaritia species (C. banksiae and C. smitinandii). A detailed morphological comparison of the new species with its two relatives is summarised in Table 2. This new species is the first Chayamaritia species reported from Vietnam. In order to facilitate identification, we also provide an identification key to all known Chayamaritia species.

Conclusion
In the present study, we discovered and described a new species of Chayamaritia from Vietnam, based on both morphological and molecular evidence. This newly-described species further suggests floristic similarities amongst countries of the Indochinese Peninsula. Our new finding provides an essential addition to the ongoing project of 'Flora of Vietnam' and 'Flora of Cambodia, Laos and Vietnam'.