Curcuma tuanii (Zingiberaceae) a new species of subgenus Ecomata from northern Vietnam based on morphological and molecular evidence

ABSTRACT A new species, Curcuma tuanii (Zingiberaceae), is described and illustrated from Hoa Binh Province northern Vietnam based on both the morphological and molecular data. The species is morphologically compared with its closest allies from subgenus Ecomata. Additionally, the anatomical structures of the species were provided.


Introduction
The genus Curcuma L. (Zingiberaceae) contains many taxa of economic, medicinal, ornamental, and cultural importance (Záveská et al. 2012).This genus comprises approximately 120 currently accepted species (Leong-Škorničková et al. 2007;2015a).However, the exact number of species is still controversial.Curcuma is one of the largest genera in the Zingiberaceae family.Its species are widely distributed in South East Asia, with a few species occurring in northern Australia and the islands of the South Pacific (Záveská et al. 2012).The highest genus diversity is observed in India and Thailand, with at least 40 species in each country.Then the number of native species follows Myanmar, Bangladesh, Indonesia, and Vietnam (Leong-Škorničková et al. 2008).
In the course of the progressing revision of the genus Curcuma for the Flora of Cambodia, Laos, and Vietnam, 12 new Curcuma species from subgenus Ecomata (Záveská et al. 2012) et al. (2022).Additionally, Thailand is also recognized as the region with the highest species richness for subgenus Ecomata with 29 species (Saensouk et al. 2022).
Acta Botanica Brasilica, 2023, 37: e20230028 During floristic conservation in Hoa Binh Province, Vietnam in 2019, the first and the third authors collected a taxon of Curcuma subgen.Ecomata extremely similar to C. sahuynhensis in appearance.After a critical examination of morphology enforced by molecular analysis, we conclude it is a new species for science.Thus, the present work aims to (1) reconstruct the phylogeny of genus Curcuma to clarify the phylogenetic position of the new species and its closely related taxa, (2) provide the taxonomic treatment with descriptions, anatomical features of leaves and rhizome, photographs, distribution, ecology, conservation assessment, and morphological comparison of new species with its allies.

Molecular phylogenetic analyses
We extracted total genomic DNAs from silica gel-dried leaves of the collection and generated 11 new sequences for five Curcuma species belonging 'Pierreana' group subgenus Ecomata.The 4 primer sequences and protocols of genes ITS, matK, psbA-trnH, and trnL-F used for PCR and sequencing in this study followed the protocols described in Záveská et al. (2012).In total, 36 species of Curcuma including 2 individuals of the new species C. tuanii were sampled for phylogenetic analyses.The Alpinia conchigera Griff., A. galanga (L.) Willd., Camptandra parvula (King ex Baker) Ridl., Larsenianthus careyanus (Benth.& Hook.f.) W.J.Kress & Mood were used as outgroups.The sequences used in this study showed in Table S1.Sequences were aligned in Geneious v.8.0.5 using MUSCLE (Kearse et al. 2012).The combined dataset consists of 5181 base pairs.Two methods, maximum likelihood (ML) and Bayesian inference (BI), were used to construct the phylogenetic relationships of Curcuma on the CIPRES Science Gateway Portal (Miller et al. 2010).Individual analyses of chloroplast and ITS detected no well-supported topological conflicts (i.e., incongruences among individual DNA regions with BS < 70%; Hillis & Bull 1993).We thus conducted further phylogenetic analyses for the combined data set of five DNA regions using both ML and Bayesian inference (BI) methods.The ML analyses were conducted in RAxML 8.2.12 (Stamatakis 2006) using the nucleotide substitution models estimated separately for each gene region under the Akaike Information Criterion (AIC) in jModeltest 2.1.6(Darriba et al. 2012) and applying 1,000 bootstrap replicates.The BI analysis was performed on MrBayes 3.2.6 (Ronquist et al. 2012) using the nucleotide substitution models that estimated separately each gene region by jModeltest 2.1.6(Darriba et al. 2012).Two independent runs, each comprising four Markov chain Monte Carlo (MCMC) chains, were conducted.We ran the MCMC for 20 million generations, and trees were sampled every 2000 generations.Tracer v.1.6was used to check the effective sample sizes (ESSs) of all relevant parameters (>200) (Rambaut et al. 2014).We removed the first 25% of sampled generations as burn-in to obtain the majority-rule consensus tree and Bayesian posterior probabilities (PP).The final result was visualized in FigTree v.1.4.0 (Rambaut 2009).

Morphological analyses
All measurements of morphological characteristics were carried out in the field based on living flowering plants used for the type preparation.Some rhizomes were collected and grown in the garden of the Faculty of Pharmacognosy and Traditional Medicine, Hanoi University of Pharmacy to examine the stability of characteristics.The plant terminology follows Beentje (2016) and the previous work of Nguyen et al. (2022).Voucher herbarium specimens are stored at VNMN and HNU herbarium.All the photos were taken with a Nikon D300S fitted with an AF-S 60mm f/2.8 Macro lens.
Conservation status study: The preliminary conservation assessment follows the guidelines of IUCN (2022).

Anatomical method
Anatomical studies were carried out using fresh as well as preserved specimens.The specimens (leave and rhizome) were cut by hand into thin slices (about 10-20 μm).Next, these transverse sections were used for staining.These samples were cleared in 5.0% (w/v) chloramine-T for 10 minutes.Then they are subsequently acidulated in 5.0% (w/v) acetic acid for 15 minutes.Those slices were dyed in safranin and mounted on a clear glass slide using glycerin.The sections were subsequently observed under Leica EZ4 stereoscope (Leica Microsystem, Heerbrugg, Switzerland) with 5X to 100X magnifications.

Phylogenetic relationships
The topology from ML and BI analyses of the combined matrix were highly congruent, we thus present the BI tree with BS and PP values in Fig. S1.The molecular results indicate four well supported major clades within Curcuma representing: 'Curcuma', 'Hitcheniopsis', 'Ecomata' and 'Pierreana' groups (Fig. 1).These results are in congruence with Záveská et al. (2012).
The new species of Curcuma was placed together with some native Vietnamese species classified in 'Pierreana' group belonging to subgen.Ecomata (Fig. 1).In this clade, C. xanthella and C. newmanii were weakly supported as sisters to the remaining species.While, four species including C. vitellina, C. sahuynhensis, C. pambrosima and the new species C. tuanii formed a subclade with supported by the BI analysis (Fig. 1).Curcuma tuanii is recovered with strong support, the two individuals of the new species formed a separate clade.
Acta Botanica Brasilica, 2023, 37: e20230028 On the other hand, C. sahuynhensis and C. pambrosima were recovered as closely related species by molecular data (Fig. 1).By comparing specimens in the herbaria, the description in literature (Leong-Škorničková et al. 2015b), and our observation in the field, the two species C. tuanii and C. sahuynhensis share some morphological characters such as color and shape of bracts, rich yellow staminodes as long as labellum, and L-shaped anther.However, the molecular data indicated that C. sahuynhensis is most closely to C. pambrosima than C. tuanii and C. vitellina.In addition, C. tuanii and C. cotuana share several morphological characters, but the molecular data reveals that these two species are not so closely related (Fig. 1).Thus, the morphological differences between C. tuanii and closest relatives (C.sahuynhensis, C. vitellina and C. pambrosima) are discussed below and presented in Table 1.

Taxonomic treatment
Distribution and IUCN preliminary assessment:-Curcuma tuanii has only been recorded in north Vietnam.The distribution range of C. tuanii is located in northern Vietnam and well isolated from populations of relative species in Central and South Vietnam (Fig. 5).The distribution ranges of these four relative taxa are nearby but do not overlap.Curcuma sahuynhensis and C. pambrosima distributions are limited to the eastern coast of South Central.Whereas, C. cotuana and C. vitellina occur in the western montane forest of Central and South Vietnam, respectively.
According to the data obtained from local people, at the type locality in Hoa Binh Province where we have counted about 500 adult individuals, this new species forms several subpopulations within the area of Hien Luong Commune (Hoa Binh Province, Da Bac District) with no imminent threat.The forest, where this species grows, is protected by local community.Two paratype specimens of this species were collected from plants cultivated in Nghe An and Ha Tinh Provinces by local people from the wild population in Can Loc District, Ha Tinh Province, near border to Nghe An Province.We propose conservation status for the new species as Data Deficient (DD) following the IUCN criteria (2022) due to the lack of verified data on its current distribution and population size.

Anatomical feature of Curcuma tuanii
Anatomical characters of leaf:-Transverse sections of C. tuanii leaf are symmetrical, passing through a midrib and lamina are shown in Fig. 6.
Midrib and vascular bundles: The epidermis is a single-layer tissue consisting of polygon-shaped cells, whose walls vary in size and are totally soaked with cellulose.Short unicellular trichomes are observed sparsely.The upper collenchyma consists of polygonshaped and diverse-magnitude cells, which are arranged tightly into 5-15 layers.In the leaf midrib, ground tissue is made up of parenchyma cells which are thinwalled and polygonal.Some cells contain oil globules or oleoresin.The upper parenchyma arranged tightly into 5-15 layers.Whereas the lower one was 3-5.Vascular bundles are the closed type with 1-3 metaxylem vessels and phloem forming a cap-like structure.3-5 layers of fibrous sclerenchymatous cells are observed below large veins.In this each vascular bundle, the xylem is located in the superior position while the phloem is located in the inferior site.It is covered by a ring of the irregularly-sized sclerenchymatous sheath, which included 3-5 layers of lignified polygonal cells.Xylem included 1-3 primary veins and 2-4 secondary veins, which are nearly roundshaped, lignified, and randomly distributed.Phloem is larger than xylems in every bundle and consists of cellulose-covered polygonal cells, which are made up of 7-10 layers.There are two types of vascular bundles that differ in size and position: 10-13 larger vascular bundles formed a single conspicuous abaxial arc toward the lower epidermis, alternating with air chambers and embedded in collenchyma observed.Smaller vascular bundles are scattered in ground tissue.Where as, the central midrib has 4-6 smaller phloem-xylem bundles scattered in the upper parenchyma.The air chamber is a kind of aerenchyma formed through a tight arrangement of about 30-40 circular-shaped and unequal-size cells.Leaf blade: A transverse section through the leaf blade is composed of 5-7 layers of collenchymatous cells.The adaxial and abaxial epidermis consisted of a single layer, polygonal-slightly elongated cells.It is covered with a moderately thin cuticle.Below both of the epidermal layers, there are thin-walled, large, irregular polygonal cells of single layer hypodermis.The layers of palisade cells are observed on the upper surface continuously in leaf lamina but observed on the ventral side where the main vascular bundle, towards ventral side.Beneath the palisade mesophyll is the spongy mesophyll cells consisting of 3-5 layers, with irregular shapes.Oleoresin cells are scattered in layers of mesophyll cells.
Anatomical characters of the rhizome:-Transverse section of the rhizome is circular in outline.The outermost layer is the periderm which consists of more layers of rectangular and tangentially elongated cells.The outer layer is a slightly cutinized epidermis, which is built by a single rectangular or polyhedral cell layer; the exodermis (suberized tissue) is located under the epidermis, consisting of 1-3 layers of polygonal cells, scramble; the third layer is a multilayered hypodermis consisting of 4-6 rectangular or polygonal layers.Followed to these is a wide cortex with irregularly scattered vascular bundles.Each vascular bundle is nearly circular and composed of groups of xylem entirely surrounded by phloem.Xylem consists of parenchyma, vessels, tracheids, and fibers.Metaxylem consists of 1-2(-4) lignified polygonal vessels and their magnitudes are 1-2 times larger than protoxylem.Where as, the protoxylem consists of 2- Curcuma tuanii (Zingiberaceae) a new species of subgenus Ecomata from northern Vietnam based on morphological and molecular evidence around the vascular bundles is absent.There is a singlelayered endodermis called Casparian strips composed of thin-walled rectangular cells separating the cortical region and stellar region.In the stellar region, inner vascular bundles are similar to those in the cortex and also present scattered inside.Parenchymatous cells in both regions are thin-walled, polygonal to circular, and also filled with starch grains.Some of these reproductive cells contain deposition of orangered substance oleoresin.Numerous oleoresin cells are scattered throughout the rhizome.Some other cells contain pale yellow oil globules (Fig. 7).

Figure 1 .
Figure 1.Majority rule consensus tree from the Bayesian inference based on the combined datasets of four DNA regions (ITS, matK, psbA-trnH, and trnL-F).ML bootstrap values and posterior probabilities (PP) of the BI analysis are presented above the branches.Support values less than 50% are indicated with "-".

Figure 6 .Figure 7 .
Figure 6.Transverse section of midrib: A. overall view, B. upper epidermis layer, C. a unit of the single conspicuous abaxial arc including vascular bundle, lignified pericyclic fiber, and air chamber, D. detail of a vascular bundle scattered in ground tissue, E. detail of a main vascular bundle in the single conspicuous abaxial arc.Transverse section of leaf passing through lamina: F. with lower epidermis & G. with upper epidermis.Note: e: epidermis, t: trichomes, x: xylem, p: phloem, vb: vascular bundle, lpf: Lignified pericyclic fiber (along with vascular bundle/ sclerenchymatous cells), pc: parenchymatous cells, h: hypodermis, pm: palisade mesophyll, o: oleoresin/oil globules, sm: spongy mesophyll, ac: air chamber.Photos by Nguyen Ngoc Anh, correction and design by Hoang Tuan Nguyen & Danh Duc Nguyen.

Table 1 .
Comparison of the morphologically vegetative and reproductive characteristics of Curcuma tuanii with its closest relatives: C. sahuynhensis, C. vitellina, C. pambrosima and C. cotuana.