﻿Hedyotiskonhanungensis (Rubiaceae): A new species from the central highlands of Vietnam

﻿Abstract A new species of Hedyotis L. (Rubiaceae), Hedyotiskonhanungensis B.H. Quang, T.A. Le, K.S. Nguyen & Neupane, is described and illustrated from the central highlands of Vietnam based on morphological and phylogenetic evidence. The new species belongs to the morphologically diverse tribe Spermacoceae (ca. 1000 species) of the family Rubiaceae, which is represented by 70–80 species in Vietnam. The phylogenetic analysis, based on four DNA regions (ITS, ETS, petD, rps 16), confirms the new species’ placement within the genus Hedyotis – one of the largest genera in the tribe, comprising ca. 180 species across Asia and the Pacific. Hedyotiskonhanungensis is morphologically distinct from all southeastern Asian Hedyotis L. in its set of traits such as leaf type (shape and thickness), growth habit, and floral parts (color of inflorescence axis and the shape of calyx lobes). The new species shows similarities with Hedyotisshenzhenensis, H.shiuyingiae, and H.yangchunensis from China in its herbaceous habit, fleshy ovate leaf blades, and dark purple floral parts, but it is phylogenetically distinct and can be distinguished from them by the following combination of morphological traits: habit with slightly smaller stature (<25 cm), broadly ovate or deltoid stipules with cuspidate apex and entire margin, and ovate or nearly ovate calyx lobes.


Introduction
The Asian-Pacific genus Hedyotis L. (ca. 180 species) lies within a polymorphic, mainly herbaceous tribe Spermacoceae (ca. 1000 species) of the family Rubiaceae. In Vietnam, this tribe is represented by 70-80 species belonging to the genera Dimetia  (Govaerts et al. 2022). The genus Hedyotis and its related genera that form the Hedyotis-Oldenlandia complex have a long history of taxonomic confusion and disagreement due to the use of inconsistent and overlapping characters in the generic delimitations. The genus Hedyotis is currently treated in a narrow sense based on the molecular phylogenies (Guo et al. 2013;Wikström et al. 2013;Neupane et al. 2015) where members of it are united by their predominantly shrubby to tree-like habit, septicidal capsules, flattened seeds, and tropical upland distributions in Asia and the Pacific. The species of this genus are mostly found in the mid or high-elevation slopes (to 4000 m a.s.l.) of Asia (Sri Lanka, southern India, SE China, Indochina, Malesia), Papuasia, and NW Pacific (Micronesian islands). In Vietnam there are about 20 species reported in the flora of this country (Pitard 1922;Pham 2003;Tran 2005;Do et al. 2013;Govaerts et al. 2022) but the actual number could be as high as 40 species since many species listed under Oldenlandia share the diplophragmous capsule characteristic of Hedyotis (pers. obs.).
During a botanical field survey in Kon Ha Nung Biosphere Reserve (UNESCO 2021) of the highlands of Central Vietnam, we came across a population of Spermacoceae (collection number LTA 531) with the following morphological features: perennial herbs, opposite obovate to nearly oval leaf blades, interpetiolar entire stipules with cuspidate apex, 4-merous flowers, bilobed stigmas, inferior ovary, and many-seeded, diplophragmous capsules with persistent ovate or nearly oval calyx lobes. The morphological features, specifically the presence of diplophragmous capsules, observed in the new population of Spermacoceae align with the diagnostic characteristics of the genus Hedyotis as outlined in Wikström et al. (2013) and Neupane et al. (2015). Following a thorough review of the taxonomic literature on the genus Hedyotis in Vietnam and surrounding regions, including Pitard (1922), Fukuoka (1970), Pham (2003), Tran (2005), Chen and Taylor (2011), Wikström et al. (2013), andNeupane et al. (2015), as well as a comprehensive morphological comparison with closely related species such as Hedyotis shenzhenensis T.Chen, H. shiuyingiae T.Chen, and H. yangchunensis Ko & Zhang, and utilizing phylogenetic analysis to determine its placement within the Spermacoceae, we have determined that our specimens represent a previously undescribed species within the genus Hedyotis. This species is hereby named Hedyotis konhanungensis.

Morphological study
Our collected specimens were compared with all described species from southeast Asia and southern China by studying relevant literature and examining digital herbarium images. Morphological characters were recorded using Nikon SMZ745/SMZ745T stereoscopic microscope and photographs of vegetative and floral parts were taken both in the field and from the samples preserved in 70% ethanol using Canon EOS 7D. The type specimens have been stored in the following three herbaria (acronyms follow Thiers 2022, continuously updated): Vietnam National Museum of Nature (VNMN), Institute of Ecology and Biological Resources, Vietnam Academy of Sciences and Technology (HN), and Kon Ka Kinh National Park.

Phylogenetic analysis
To establish its phylogenetic position within the tribe Spermacoceae, total genomic DNA was extracted from silica-dried material with the DNeasy Plant Kit (Qiagen, Valencia, California, U.S.A.). Four DNA regions (nuclear genome: ITS, ETS; plastid genome: petD, rps16) that were used in our earlier studies (Neupane et al. 2015, Neupane et al. 2017, were selected for amplification from this sample. Amplifications were performed in a 25 μl reaction mixture composed of 1 μl of each primer (10 μM), 1 μl of DNA template, 12.5 μl of GoTaq Green Master Mix (Promega, Madison, Wisconsin, U.S.A.), 9.5 μl of water. The amplification protocol for nuclear and chloroplast regions follows Kårehed et al. (2008) and Groeninckx et al. (2009) respectively. Amplified PCR products were purified using ExoSAP-IT PCR Product Cleanup (Thermo Fisher Scientific) following the manufacturer's protocols and sequenced at Apical Scientific Company for sequencing (Selangor, Malaysia).
We added the DNA sequences of our sample to our existing DNA data matrix (Suppl. material 1). The GenBank accession numbers for our sample were OQ401065, OQ401066, OQ458733, and OQ401067, which correspond to the rps16, petD, ITS, and ETS regions, respectively. We also included additional sequences representing the ITS, petD, and rps16 regions for Hedyotis yangchunensis, Hedyotis shenzhenensis, and Hedyotis shiuyingiae, obtained from Guo et al. (2013) and available in GenBank. Each of these DNA regions was aligned using MAFFT v.7 (Katoh and Standley 2013) and concatenated into a single matrix Suppl. material 2) to infer the phylogeny. Phylogenetic analysis was conducted on the combined matrix under the maximum likelihood (ML) framework with four partition schemes (ITS, ETS, petD, rps16) for their own substitution model (GTR+GAMMA) parameters using RAxML v.8.2.12 (Stamatakis 2014). The RAxML tree search and bootstrap analysis (1000 rapid bootstrap replicates for clade support) were conducted on CIP-RES (Miller et al. 2010).

Hedyotis konhanungensis
Diagnosis. Hedyotis konhanungensis is similar to H. shenzhenensis, H. shiuyingiae and H. yangchunensis from southeastern China (Guangdong and Hongkong) in the morphology of the leaf blades, floral bracts, dichasial cymes, and fruits, but differs from them by its broadly ovate or deltoid (vs. triangular or broadly triangular) stipules with entire (vs. hairy or lacerated) margins and cuspidate (vs. acute) apex, suborbicular or broadly oval (vs. subovate or ovate to lanceolate) lowest floral bracts, ovate or nearly oval (vs. triangular or subulate to lanceolate) persistent calyx lobes on fruits, and stamens in long-styled flowers inserted in lower ¼ or near the base (vs. at the middle or near the mouth) of the corolla tube (Table 1).

Distribution and ecology.
Hedyotis konhanungensis is recorded only from the type locality in the Kon Pne Commune of the Central Highlands of Vietnam, which is part of the Annamite Range. This range is known for its rich biodiversity and high number of endemic species (Averyanov et al. 2003). The species grows in the understorey of the evergreen forests in the valleys or on flat areas to slopes of sandstone mountains. Within its occupancy areas, the new species was associated with some shrubs or herbs such as Pavetta bauchei Bremek., Lasianthus biflorus (Blume) M.Gangop. & Chakrab., Staurogyne sp., Popowia sp., Huperzia sp.

Discussion
The genus Hedyotis, as currently circumscribed, corresponds to a primarily woody genus characterized by "diplophragmous" capsules (septicidally dehiscent capsules that separate into two distinct valves, as described by Wight and Arnott in 1834) and "fruiticosa type" seeds (dorsiventrally flattened seeds with a ventral hilar ridge topped by a punctiform apical hilum, as described by Terrell and Robinson in 2003). The members of this hyperdiverse genus (approximately 180 species) primarily occupy the mountains of Asia and the Micronesian Islands (in the northwestern Pacific). The molecular dating analysis suggests that Hedyotis split from its sister lineages of approximately 10 species (the African/Malagasy Agathisanthemum group and the African-tropical Asian-North American Edrastima group) nearly 27 million years ago (Neupane et al. 2017). The phylogenetic comparative analysis of evolutionary correlations shows that the evolution and diversification of the hyperdiverse Hedyotis along tropical mountain orogeny are strongly linked to the formation of a woody habit and many narrow endemic species (Neupane et al. 2017). The new species Hedyotis konhanungensis, found in the mountains of central Vietnam, is another example of a tropical montane species in this genus. This species is resolved within the clade containing other southeast Asian species and is unique among the Indochinese Hedyotis in its thick and fleshy oval leaves and completely dark purple floral parts (calyx and corolla) including the inflorescence stalk. Despite the geographic distance, its morphological similarity with Chinese members and non-monophyletic group of H. shenzhenensis and H. shiuyingiae with H. yangchunensis (Fig. 1) suggests the independent evolution of fleshy leaves and somewhat stunted growth habit among these species. Here we provide both morphological and molecular evidence to confirm the novelty of this taxon and propose it as species new to science.