A new seed-setting species from the polyploid genus Curcuma of the ginger family (Zingiberaceae) based on morphological and molecular data

A new seed-setting species from the polyploid genus Curcuma of the ginger family (Zingiberaceae) based on morphological and molecular data | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A new seed-setting species from the polyploid genus Curcuma of the ginger family (Zingiberaceae) based on morphological and molecular data Juan Chen, Jia-Wei Yan, Hui-Hong Wang, Si-Jin Zeng, Lin-Ya Zeng, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5287647/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The polyploid genus Curcuma L. is an economically important, yet taxonomically rather difficult genus, mainly distributed in South and Southeast Asia. Several Chinese important traditional medicinal herbs are from Curcuma , such as “jianghuang姜黄”, “yujin郁金” and “ezhu莪术”. During field investigation of plant resources in Yunnan, the distribution center of the genus in China, an unknown flowering and seed-setting species of Curcuma was discovered. Its morphological characters were assessed for further taxonomic treatment and molecular analysis was conducted to ascertain its phylogenetic position within the genus as well. Its genome size, chromosome number and ploidy level were evaluated by k -mer distribution analysis and cytological method. Results This species resembles Curcuma longa but can be distinctly differed in its yellow rhizomes, sometimes with pubescent abaxial surfaces, green or sometimes with vary faint and narrower purple stripes at the midrib, white tinged with pale purple to pale purple coma bracts. Its chromosome number is 2 n = 42. The haploid genome size estimation of Curcuma flavescens based on k -mer distribution is 874.19 Mb. Smudgeplot analysis suggested it is a diploid heterozygous genome (AB). Plastid phylogenomic analyses indicated that this new species is embedded within subg. Curcuma . The comprehensive phylogenetic studies conducted on Curcuma species using nrITS regions showed it is nested with C. montana , a species from India and Bangladesh. Moreover, morphological analysis further reinforced the distinctiveness of this species from C. montana . It revealed several key differences across various anatomical features such as the color of rhizomes, the indumentum of leaves and the morphology of inflorescence and flowers. Our findings make a strong case for using next-generation sequencing to explore phylogenetic relationships and identify new species. Conclusion The morphological and molecular evidences support the recognition of Curcuma flavescens as a new species. This provides a good diploidy material for the further breeding work in the genus Curcuma , and might also contribute to the study of the polyploid origin in this genus. Curcuma Genome size Ploidy level New species Plastid genome nrITS region Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Polyploidy occurs in many taxa, is particularly wide-spread in flowering plants, and is a prominent feature of the chromosome evolution of higher plants. At least half of the known angiosperm species have experienced polyploidy in their evolutionary history (Hieter & Griffiths 1999 ; Shaked et al. 2001 ; Xing et al. 2010 ). Polyploidy has also played a significant role in evolution and diversification of various members of Zingiberaceae (see Mukherjee 1970 ; Lim 1972 ; Poulsen 1993 ; Chen and Chen 1984 ; Takano 2001 ; Takano & Okada 2002 ), including Curcuma L. (Joseph et al. 1999 ; Ardiyani 2002 ; Sirisawad et al. 2003 ; Leong-Škorničková et al. 2007 ; Chen et al. 2013 ). Curcuma , one challenging genus in the family Zingiberaceae, is mainly distributed in South and Southeast Asia with many species extending to China, Australia, and the South Pacific. Recent estimates of the total species number vary from 50 (Larsen et al. 1998 ; Wu & Larsen 2000 ), to 100 (Sirirugsa 1996 ) or 120 (Leong-Škorničková et al. 2007 ). Most Curcuma species are of polyploid origin, often sterile and reproducing mainly vegetatively, with turmeric ( C. longa ), one of the highly polyploid species, being the best known (Leong-Škorničková et al. 2007 ; Záveská et al. 2016 ). Few species seem to be diploid, reproducing sexually (Leong-Škorničková et al. 2007 ). The widely accepted basic chromosome number is x = 21 (Islam 2004 ; Puangpairote et al. 2016 ) but Leong-Škorničková et al. ( 2007 ) suggested that x = 7 should be considered the primary basic chromosome number. The most recent phylogeny of the genus (Záveská et al. 2012 ; Leong- Škorničková et al. 2015) retained two previously accepted subgenera and proposed one subgenus mainly based on spur types: (1) subg. Curcuma L., characterized by conspicuous coma bracts, epigynous glands and two forward-facing anther spurs, 2 n = 42, 63, 84, 105; (2) subg. Hitcheniopsis (Baker) K. Schum., characterized by the lack of epigynous glands and the presence of anther spurs, 2 n = 20, 22, 24, 28, etc.; (3) subg. Ecomatae Škorničk. & Šída f., characterized by epigynous glands, two L-shaped spurs and the lack of conspicuous coma bracts, 2 n = 42. Although several studies have attempted to reveal the evolutionary process in the genus and detect possible hybrids and their parental species (Záveská et al. 2011 , 2012 , 2016 ; Skopalíková et al. 2023 ), most of the relationships at the species level are unresolved, especially for those triploid plants. Such ability to reliably identify hybrids and their parental species may have direct significance in subsequent research towards better utilization of Curcuma species. China is one of the important distribution areas of Curcuma species. Nowadays, 24 names have been associated with Chinese Curcuma species, 16 of which are based on types from China (Tong 1997 ; Ye et al. 2008 ; Chen & Xia 2013 ; Chen et al. 2019 , 2021 ; Zhang et al. 2019 ). Several species have been used as Chinese herbal medicine for more than a thousand years. For example, “jianghuang姜黄” from the rhizome of Curcuma longa L., “片姜黄” from the sliced rhizome of C. wenyujin Y.H.Chen & C.Ling, “莪术” from the rhizomes of C. phaeocaulis Valeton, C. wenyujin or C. kwangsiensis S.G.Lee & C.F.Liang, and “郁金” from the tubers of C. wenyujin , C. longa , C. kwangsiensis or C. phaeocaulis are officially recorded in Chinese Pharmcopoeia (China Pharmacopoeia Committee 2020 ). The previous cytological analysis of many Curcuma species from China showed that Curcuma flaviflora S.Q.Tong was diploid with 2 n = 2 x = 42, C. nankunshanensis N.Liu, X.B.Ye & Juan Chen, C. ruiliensis N.H.Xia & Juan Chen and C. kwangsiensis S.G.Lee & C.F.Liang was tetraploid with 2 n = 4 x = 84. The rests were all triploid (2 n = 3 x = 63) (Ye et al. 2008 ; Chen et al. 2013 , 2019 , 2021 ). During the revision of Chinese Curcuma species begun from 2007, a seed-setting Curcuma species with much branched rhizomes and terminal inflorescences belonging to subg. Curcuma from Puwen of Xishuangbanna in Yunnan attracted our attention. Since only four members of C . subg. Curcuma from China can produce viable seeds, namely C . exigua , C. kwangsiensis , C. nankunshanensis and C. ruiliensis , this species as well as its ploidy level was worthy of study. Among them, only C. exigua has many branched rhizomes (like the rhizomes of C. longa ) and terminal inflorescences, while the other species all have ovoid rhizomes which are unbranched or few branched and bear both lateral and terminal inflorescences. After further studies including morphological and phylogenetic analyses, it is concluded that the material from Puwen represented a new species which is described and illustrated below. Its genome size, chromosome number and ploidy level were also reported to facilitate further breeding and polyploid study in this genus. Methods Morphological and taxonomic analyses During the revision of Curcuma species, names of taxa currently recognized as members of Curcuma have been investigated. Types and original material including herbarium sheets, drawings/icons and manuscripts were searched and checked. All specimens of Curcuma species in the following herbaria in China were examined: AU, EMA, FJIDC, GXMG, GXMI, HITBC, IBK, IBSC, IMC, IMDY, KUN, NAS, PE, SYS and TAI. Additional digitized material from China and neighboring countries (Thailand, Laos, Myanmar, India and Vietnam) in the following herbaria has also been studied: AAU, BM, C, E, G, K, L, P, US, and WU. The description of this new species was based on the specimens collected from the type locality. The style of description followed Chen et al. ( 2021 ) and the terminology in general followed Beentje ( 2016 ). Chromosome cytology Chromosome counts were conducted on somatic metaphases using the root tip squash technique outlined by Chen et al. ( 2013 ). Actively growing root tips of cultivated plants introduced from the field were used to count the chromosome numbers. The number of chromosomes was determined from five complete well-spread mitotic plates. The voucher specimen is deposited at IBSC ( J.Y. Jin JYJ 054 − 7 ). The k -mer analysis Availability of Next Generation Sequencing data provides a means for genome size estimation by k -mer analysis. Silica-gel dried leaves were sent to Novogene (Tianjin, China) to extract total genomic DNA for library preparation for genome skimming sequencing. Paired-end (150 bp) sequencing was conducted by Novogene Corporation Inc. on the Illumina HiSeq2000 platform (Illumina, Inc., San Diego, CA, USA), generating approximately 100 Gb raw data for one sample of the new species. The raw reads were further curated to remove low-quality reads and 3′ adapter contamination using fastp ( https://github.com/OpenGene/fastp ). Clean reads were used for k -mer counting using Jellyfish 2.2.7 (Marçais & Kingsford 2011 ). The k -mer size of 17 and the ‘jellyfish count -m 17 and -C’ command was used to create the histogram data. The Jellyfish count output was then exported to k -mer histogram using the ‘jellyfish histo’ command. The output file was used as an input file for the software developed by Novogene Corporation Inc. to calculated the depth distribution map of k -mer. The heterozygosity rate was calculated according to Marcais & Kingsford (2011). The ploidy level of this new species was also evaluated by Smudgeplot v0.2.5 (Ranallo-Benavidez et al. 2020 ) ( https://github.com/KamilSJaron/smudgeplot ). The k -mer frequencies were first generated using KMC v3.1.1( https://github.com/tbenavi1/KMC ) with parameters ‘-k21 -t10 -m64 -ci1 -cs10000 @FILES kmcdb tmp’ and then converted to k -mer frequency histogram using parameters ‘kmc_tools transform kmcdb histogram species_k21.hist -cx10000’. The ‘smudgeplot.py cutoff species_k21.hist L/U’ command was then used to estimate k- mer coverage thresholds from the histogram file. The k -mers in the coverage ranging from L to U were extracted with the command ‘kmc_tools transform’ and ‘KMC_dump’ command was used to reduce the file to compute set of k -mer pairs. The Smudgeplot showing proposed ploidy level was then generated with coverages of identified k -mer pairs (i.e., species_coverages.tsv file) using ‘smudgeplot.pyplot’ command. Taxon sampling To reveal the approximate phylogenetic relationship within Curcuma , two datasets including plastid genomes and nrITS sequences respectively were utilized. Firstly, 10 newly sequenced samples and 13 released plastid genomes of 23 species representing three subgenera of Curcuma were sampled based on previous phylogenetic relationships reported in Záveská et al. ( 2012 ). Four species ( Hedychium villosum Wall., Kaempferia elegans Wall., Pyrgophyllum yunnanesnsis (Gagnep.) T.L.Wu & Z.Y.Chen and Zingiber mioga Roscoe) from Zingiberaceae were also downloaded from GenBank as outgroups according to phylogenetic relationships reported in Kress et al. ( 2002 ). Then, the dataset of ITS sequences of 65 samples representing 51 seed-setting species from three subgenera of Curcuma were used for molecular analyses. Two species ( Hedychium villosum and Zingiber mioga ) were selected as outgroups. Since high intra-individual ITS polymorphism has been documented in Záveská et al. ( 2016 ), 1‒3 individuals per species have been sampled for ITS analysis. Sequences of C. flavescence , C. exigua and C. woodii were extracted from the assembled nrDNA sequences. Other available sequences were downloaded from GenBank. Detailed information of all species sampled and sequences used are available in Additional file 1: Table S1 & S2. DNA extraction, sequencing, assembly and annotation Silica-gel dried leaves were sent to Novogene for genome skimming sequencing. Paired-end (150 bp) sequencing was conducted on the Illumina HiSeq2000 platform as mentioned above, generating approximately 20 Gb raw data for each sample. After quality control of the raw data, all paired reads were extracted for plastid and nrDNA assembly using GetOrganelle 1.7 (Jin et al. 2020 ). Plastomes were annotated with the Plastid Genome Annotator and GeSeq with default settings (PGA; Tillich et al. 2017 ; Qu et al. 2019 ) and manually corrected using Geneious Prime 2019 ( https://www.geneious.com ). The plastid genome of C. longa (MK965541) and nrDNA sequences of C. montana Roxb. (JQ409939.1) were downloaded from GenBank as the references. Phylogenetic analysis Complete plastomes were aligned with MAFFT v. 7 using the FFT-NS-i ×1000 strategy (Katoh & Standley 2013 ). The ITS regions were extracted from the nrDNA sequences and aligned with MAFFT v. 7 with default parameters and manually adjusted in Geneious. Phylogenetic analyses were performed under maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI). The MP analysis was performed using PAUP* 4.0a169 (Swofford 2003 ). The test settings included 1000 replications of randomly added sequences and a heuristic search with tree bisection-reconnection branch swapping. The ML analysis was conducted using the IQ-TREE with the TVM + F + R2 model (CP), TNe + I + G4 model (ITS) and 1000 bootstrap iterations. Nodes with UFboot values ≥ 95 were regarded as well-supported. The BI analysis was performed using MrBayes 3.2.7 (Ronquist et al. 2012 ). The following settings were applied: sampling frequency = 100, temp = 0.1, and number of Markov chain Monte Carlo generations = 6000000. The first 1500000 trees were discarded as burn-in to ensure that the chains reached stationarity. A majority-rule consensus tree was constructed based on the trees sampled after generation 4500000. Nodes with posterior probability (PP) values of 0.95 and greater were regarded as significant. Results and discussion Taxonomic treatment Curcuma flavescens Juan Chen & N.H.Xia sp. nov. ( Figs. 1 , 2 ) Type CHINA. Yunnan, Xishuangbanna, Jinghong, Puwen, 958 m a.s.l., 2 Aug 2021, J. Chen & J.W. Yan 21080205A (holotype: IBSC!, isotypes: KUN, PE). Diagnosis It is similar to C. longa in its somewhat white coma bracts but differs in yellow (vs. orange to orange-red) rhizomes, green upper side of lamina sometimes with purple bands (vs. absence of purple bands), glabrous to puberulent (vs. glabrous) lower sides of leaf blades, white tinged with pale purple to pale purple (vs. white, whitish green or green) coma bracts. Description Rhizomatous herbs, 60‒90 cm tall. Main rhizome 3.2‒5.2 × 2.2‒3 cm, ovoid, yellow internally; lateral rhizomes 1.0‒1.5 cm in diam., fleshy, dark brown externally, yellow internally, becoming paler or white at tip; roots many, 2‒3 mm in diam. at top, tuberous and fleshy, brown to white externally, white internally; root tubers many, 2.1‒3 × 0.9‒1.1 cm, obovate to fusiform, brown to white externally, white internally. Leafy shoots usually with 3‒5 leaves at anthesis; pseudostems composed of 2‒3 leafless sheaths and 3‒5 leafy sheaths, green or brown, puberulent; ligules 1‒2 mm in height, bilobed, pubescent on margin; petioles up to 26 cm long, green, glabrous; lamina 24‒62 × 7‒11 cm (measured at anthesis), elliptic, base acuminate, slightly oblique, apex attenuate with uppermost tip glabrous, margin entire and glabrous, adaxial surface green, sometimes the leaf blades at the lower parts with faint and narrow (2‒5 mm wide) purple stripes at the midrib but the new leaves green, glabrous, abaxial surface normally glabrous rarely puberulent. Inflorescence terminal; spike 11‒16 × 4‒6.5 cm, consisting of 20‒30 bracts; peduncle hidden within pseudostems, up to 27 cm long, green, glabrous; coma bracts 3.5‒5 × 0.9‒1.9 cm, elliptic, obliquely spreading, base involuted, apex obtuse or acute and mucronate, white or white with pale purple tip to pale purple, glabrous to sparsely puberulent on both sides; fertile bracts 3.5‒4.2 × 1.4‒2.1 cm, ovate, mostly erect, pale green, connate to 1/3 above base, base involuted, apex obtuse, glabrous to sparsely puberulent on both sides; cincinni with 3–5 flowers at base of inflorescence, 1–2 flowers at top; bracteoles one per flower, the first and largest bracteole 2.5‒2.8 × 1.2‒1.6 cm, the remaining bracts gradually decrease in size, elliptic, apex acute, white, glabrous on both sides. Flowers 4.6‒5.2 cm long, much exserted from bracts. Calyx 8‒10 mm long, tubular, inconspicuously tridentate, apex obtuse, white, glabrous externally. Floral tube 2.7‒3.3 cm long, narrowly cylindrical at base, funnel-shaped at apex, yellowish white, glabrous externally, villous at throat; lateral corolla lobes 1.2‒1.5 × 0.6‒0.9 cm, elliptic, apex obtuse and slightly concave, white, glabrous on both sides; dorsal lobe 1.3‒1.8 × 0.9‒1.2 cm, elliptic and concave, white, glabrous on both sides, apex mucronate, mucro 2‒3 mm long, white, sparsely pubescent. Lateral staminodes 1‒1.6 × 0.5‒0.7 cm, lanceolate, white, glabrous on both sides; labellum 1.2‒1.8 ×1.5‒1.7 cm, square-shaped, median lobes with obtuse apices and exceeding the laterals, with a 1‒2 mm long incision, white, two yellow bands at center, glabrous on both sides. Stamen 1.1‒1.3 cm long; filament 7‒9 mm long, broad, flat, white; anther 4‒5 mm long, yellowish white, spur short, 2‒3 mm long, yellowish white, anther crest absent. Epigynous glands 2, 4–5 mm long, linear. Style white, glabrous; stigma ca. 1 mm wide, capitate, white, ostiole ciliate. Fruit a globular trilocular capsule, white, pubescent externally; seeds irregularly obovoid, ca. 5 mm long, cream to light brown, shiny, enclosed in translucent white, laciniate aril. Phenology Flowering in July to August; fruiting in August to September. Etymology The specific epithet is derived from the yellow color of rhizome from this new species. Chinese vernacular name and uses 柠檬黄姜黄 [níng méng huáng jiāng huáng]. No use has been reported. Distribution and conservation status This species is endemic to China. A recent field survey conducted at the type locality revealed the existence of several populations consisting of about 100 adult individuals, thriving within rubber plantations or along roadsides, at an elevation of ca. 958 m a.s.l. Remarkably, a single specimen collected from Lancang fits the characteristics of this species. However, given the scarcity of information regarding the distribution (extent of occurrence (EOO) or areas of occupancy (AOO)) and population sizes of the species, the conservation status of C. flavescens is temporarily assessed here as Data Deficient (DD), following the IUCN Red List Categories and Criteria (IUCN 2012 ). Additional specimens examined China. Yunnan: Lancang, 26 Aug 1957, G.S. Sin 486 (KUN); Xishuangbanna, Jinghong, Puwen, 958 m a.s.l., 2 Aug 2021, J. Chen & J.W. Yan 21080205B (IBSC) ; ibid., 6 Sep 2020, S.J. Zeng & L.Y. Zeng 20090604 (IBSC); ibid., 6 Aug 2022, J.Y. Jin JYJ053-1, JYJ053-5 & JYJ 054 − 7 (IBSC). Notes As in the other seed-setting species of Curcuma , such as C. kwangsiensis and C. ruiliensis , characters of this new species such as the purple stripes (presence or absence) on the upper surface of leaf blades, the leaf indumentum (glabrous on both sides or glabrous adaxially but pubescent abaxially) and coma color (white, pink to dark violet) are variable, while the stature, the rhizome shape and color, the lamina shape, the fertile bract shape and color, the color and shape of labellum and lateral staminodes are stable within the population observed. Curcuma flavescens shares some similarities with C. exigua , yet it exhibits distinct characters by its much thicker and stronger pseudostems, broader leaf blades, sometimes with pubescent abaxial surfaces, green or sometimes with vary faint and narrower purple stripes (vs. green with obvious purple stripes) at the midrib, larger inflorescence, and ovate (vs. lanceolate) fertile bract with obtuse (vs. acute) apex and much involuted base. It can be readily distinguished from C. ruiliensis by much branched and yellow rhizomes, slender stature, shorter ligule, ovate fertile bracts with obtuse apex, much broader coma bracts, only bearing terminal inflorescence and being diploid not tetraploid. A detailed comparison is provided in Table 1 . Table 1 Morphological comparison of the diagnostic characters of Curcuma flavescens and closely related species. Characters C. flavescens C. longa C. exigua C. ruiliensis Plant height (cm) 60‒90 80‒120 40‒80 85‒110 Rhizome branch Much branched Much branched Much branched Rarely branched Rhizome color Yellow Orange to orange -red Yellow Pale yellow or white Ligule length (mm) 1‒2 ca. 2 1‒2 5‒6 Leaf blade Size 24‒62 × 7‒11 33–60 × 10–20 19‒22 × 5‒7 47–62 × 9–11 Shape Elliptic Elliptic Elliptic to lanceolate Elliptic Color Green or with 2‒5 mm purple stripes Green Green with 5‒6 mm purple stripes Green with ca. 2 cm purple stripes Indumentum Glabrous or rarely pubescent abaxially Glabrous Glabrous Glabrous adaxially, pubescent abaxially Inflorescence Position Terminal Terminal Terminal Terminal and lateral Size (cm) 11–16.5 × 5–8.5 12.5–16 × ca. 9 9.5–10 × 3.5–5.5 12.5–17 × 5–7 Bract number 20–30 25–40 (–80) ca. 11 38–45 Coma bract shape Elliptic, base involuted, apex obtuse Elliptic, base involuted, apex acute to obtuse Oblong to lanceolate Lanceolate, obliquely spreading, apex acute Size (cm) 3.5‒5 × 0.9‒1.9 5–6 × 1.7–2.5 3.8–4.2 × 0.8–1.0 4.8–5.9 × 0.8–1.1 Color White or tinged with pale purple to pale purple White, whitish green or green White tinged with purple Purple Fertile bract Size (cm) 3.5‒4.2 × 1.4‒2.1 3.8–4 × 2.4–3 2.5–3 × 1.2–1.6 4.1–4.5 × 1.6–2.2 Apex shape Obtuse Acute to obtuse Acute Narrowly acute Bracteole size 2.5‒2.8 × 1.2‒1.6 ca. 2.3 × 1.0 ca. 2.1 × 0.7 1.3–1.5 × 0.5–0.6 Flower color White White White Yellow Diploid level 2 x 3 x Unknown, seed-setting 4 x It is also closely related to C. montana Roxb. in the appearance of inflorescences. However, this species has prominently exserted flowers and usually glabrous leaf blades on both sides instead of being pubescent on the abaxial side. It has yellow labellum and lateral staminodes differing from white color in C. montana . Other distinguishing features are the presence of yellow rhizomes and green fertile bracts, setting it from C. montana with light-orange-yellow rhizomes and white fertile bracts. Chromosome cytology The chromosome number of Curcuma flavescens was determined to be 2 n = 42 (Fig. 1 J). It is either diploid or hexaploid according to the different understanding of the basic chromosome number ( x = 21 or 7) (Leong-Škorničková et al. 2007 ; Chen et al. 2013 ; Puangpairote et al. 2016 ). The k -mer analysis The k -mer ( k = 17) depth and frequency results showed that the genome size of C. flavescens is 874.19 Mb with a heterozygosity rate of 1.61% (Fig. 3 A). The k -mer depth distribution histograms (Fig. 3 A) also revealed a unique bimodal profile with a high peak around 16 × coverage and a shorter peak around 31 ×. This could be evidence of a highly heterogeneous genome. The ploidy level was further analyzed using the reported method for reference-free profiling of polyploid genomes, suggesting that the possibility of AB is 0.48 (Fig. 3 B), which proposed that it is diploid. Considering the basic chromosome number in Curcuma species was 21; therefore, it is a diploid plant (2 n = 2 x = 42). The plastid genome features of Curcuma flavescens The complete plastid genome of Curcuma flavescens is 162,133 bp in length and has a circular and quadripartite structure with a large single-copy (LSC) region (87,013 bp) and a small single-copy (SSC) region (15,622 bp), which are separated by a pair of inverted repeats (IRs) regions (29,749 bp) (Fig. 4 ). The size range of this sequenced cp genome is almost agreed with those of other Zingiberaceae species (Cui et al. 2019 ; Gui et al. 2020 ; Liang & Chen 2021 ). The GC content is 36.2% in the complete plastid genome, while that in the LSC, SSC, and IR regions are 34.0%, 29.8%, and 41.2%, respectively, which is also in accordance with previous studies (Cui et al. 2019 ; Gui et al. 2020 ; Liang & Chen 2021 ). The IR region has higher GC content than other regions, possibly because of the higher GC content of tRNA and rRNA that located in that region (Table 2 ). The cp genome contains 133 functional genes including 87 protein-coding genes (PCGs), 38 tRNA genes and eight rRNA genes. Among these genes, 93 are unique genes and 20 are duplicated genes. The 93 unique genes contain 71 CDS and 22 tRNA genes. Twenty repeat genes including all rRNA genes located in IR region, and the ycf1 gene spans the SSC and IR region (Table 2 and Fig. 4 ). Similar results had also been reported in the relevant studies of Gui et al. (202) and Lin et al. ( 2024 ). Table 2 List of the annotated genes in the chloroplast genome of Curcuma flavescens . Category Groups of genes Name of genes Self‑replication Ribosomal RNA rrn4.5, rrn5, rrn16, rrn23 Transfer RNA trnA-UGC , trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnG-GCC, trnG-UCC , trnH-GUG , trnI-CAU , trnI-GAU , trnK-UUU, trnL-UAG , trnL-CAA , trnL-UAA, trnM-CAU , trnN-GUU , trnP-UGG, trnQ-UUG, trnR-UCU , trnR-ACG , trnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU , trnT-UGU, trnfM-CAU, trnV-UAC , trnV-GAC , trnW-CCA, trnY-GUA Small subunit of ribosome rps2, rps3, rps4 , rps7 , rps8, rps11 , rps12 , rps14, rps15, rps16, rps18 , rps19 Large subunit of ribosome rpl2 , rpl14, rpl16, rpl20, rpl22 , rpl23 , rpl32, rpl33, rpl36 RNA polymerase subunits rpoA, rpoB, rpoC1, rpoC2 Photosynthesis Photosystem I psaA, psaB, psaC, psaI, psaJ Photosystem II psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ Subunits of cytochrome petA, petB, petD, petG, petL, petN ATP synthase atpA, atpB, atpE, atpF, atpH, atpI NADH‑dehydrogenase NdhA , ndhB , ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK Other genes Rubisco large subunit rbcL Translational initiation factor infA Maturase K matK Envelope membrane protein cemA Acetyl‑CoA carboxylase accD Proteolysis clpP Cytochrome c biogenesis ccsA Unknown Conserved open reading frames ycf1 , ycf2 , ycf3, ycf4 Note: the bold genes indicate they have two copies. The phylogenetic position of Curcuma flavescens As indicated by prior researches (Kress et al. 2002 ; Záveská et al. 2012 ; Liang & Chen 2021 ), the phylogenomic trees recovered three main clades with high support: Curcuma subg. Curcuma , C . subg. Hitcheniopsis and C. subg. Ecomatae . However, analyses of the two data sets focused on the Curcuma yielded little different topologies because C. subg. Hitcheniopsis is sister to C. subg. Ecomatae in the plastid genomes dataset while C. subg. Hitcheniopsis is cluster with C . subg. Curcuma and C. subg. Ecomatae is the basal group in the ITS dataset. The phylogenetic trees based on plastid genomes and ITS regions both indicated that C. flavescens is clustered within C . subg. Curcuma (BS = 100%, Figs. 5 & 6 ). This species is distant from those related species, such as C. longa , C. exigua and C. ruiliensis (Fig. 5 ) in the phylogenetic tree recovering by plastid genomes. The results from ITS analyses of 51 seed-setting species shows that C. flavescens is nested with C. montana , a species currently only known from India and Bangladesh, and far from those seed-setting species from China, e.g., C. exigua , C. ruiliensis and C. kwangsiensis (Fig. 6 ). Therefore, based on results of morphological, cytological and molecular analyses, a new seed-setting species with 2 n = 42 is proposed here. Abbreviations IBSC South China Botanical Garden, Chinese Academy of Sciences, Herbarium KUN Kunming Institute of Botany, Chinese Academy of Sciences, Herbarium PE Institute of Botany, the Chinese Academy of Sciences, Herbarium BI Bayesian inference ML Maximum likelihood MP Maximum parsimony LSC Large single-copy SSC Small single-copy IR Inverted repeat PCG Protein-coding gene Declarations Supplementary information Additional file 1: Table S1. Ten Curcuma samples newly sequenced for plastid genomes and five of them for nrITS regions in this study, their collection numbers and GenBank accession numbers. Table S2. 17 plastid genomes and 62 nrITS sequences downloaded from NCBI database. Acknowledgements We are grateful to the curators of the herbaria mentioned in the “Materials and methods” part for allowing us to examine the specimens or providing the high-resolution images of specimens. Author contributions SJZ and LYZ first discovered and collected the new species in the field survey. JC and JWY conducted the molecular experiment. HHW conducted the cytological study. EWT and HHW analyzed the data. JC drafted the manuscript. NHX and JC designed and supervised the study. JC provided the funding. NHW, EWT and JC reviewed the manuscript. All authors have read and agreed with the submission of this manuscript. All authors read and approved the final manuscript. Funding This study was supported by the National Natural Science Foundation of China (Grant nos. 32070223, 31200161), Guangdong Flagship Project of Basic and Applied Basic Research (2023B0303050001) and Biological Resources Program, Chinese Academy of Sciences (Grant no. KFJ-BRP-017-19). Availability of data and materials Specimens are deposited at the herbarium IBSC. DNA sequences are deposited at GenBank. Author information Authors and Affiliations State Key Laboratory of Plant Diversity and Specialty Crops, South China Botanical Garden, Chinese Academy of Sciences, 510650, Guangzhou, People’s Republic of China Juan Chen, Jia-Wei Yan, Si-Jin Zeng, Nian-He Xia South China National Botanical Garden, 510650, Guangzhou, China Juan Chen, Jia-Wei Yan, Si-Jin Zeng, Nian-He Xia School of Traditional Chinese Medicine, Southern Medical University, 510515, Guangzhou, People’s Republic of China Hui-Hong Wang, En-Wei Tian Huizhou Forestry Science Research Institute (Huizhou Botanical Garden Management Service Center), Huizhou, 516001 Lin-Ya Zeng Corresponding author Correspondence to Nian-He Xia ( [email protected] ) Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests All authors declare that there is no competing interest. 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Supplementary Files Additionalfile1.docx Additional file 1: Table S1 Ten Curcuma samples newly sequenced for plastid genomes and five of them for nrITS regions in this study, their collection numbers and GenBank accession numbers. Table S2. 17 plastid genomes and 62 nrITS sequences downloaded from NCBI database. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5287647","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":376372644,"identity":"461666cc-8e1c-4ff5-a114-094ec42cfa04","order_by":0,"name":"Juan Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYDACCShmY2ZsfABkJhCvhY+9udmAeC0gIMdzvA3EJqyFf3bzsQeWbXZ5bBKJbZU/aizyGNgPP2D4uQOPJXeOpRtItiUXg7Tc5jkmUczAk2bA2HsGtxYDiRwzCck25sQ2kBbGBonEBoYcBmbGNnxa8r8BtdSDtRT+BGnhf0NISw4bUMvhxDaeg20MvCAtEgRskbiRZiYhce54Yht7Y7M0yC9sEs8MDvbi0cI/I/mZtERZdeL8ZvaHH3/U1OXx8yc/fPATjxYQYJZA5rEB8QH8GhgYGD8QUjEKRsEoGAUjGwAAtwBJbB6IFwMAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-1766-1329","institution":"South China Botanical Garden","correspondingAuthor":true,"prefix":"","firstName":"Juan","middleName":"","lastName":"Chen","suffix":""},{"id":376372645,"identity":"8dfa4d93-f414-40e9-b56f-3fac981da7af","order_by":1,"name":"Jia-Wei Yan","email":"","orcid":"","institution":"South China Botanical Garden","correspondingAuthor":false,"prefix":"","firstName":"Jia-Wei","middleName":"","lastName":"Yan","suffix":""},{"id":376372646,"identity":"e20f3b0e-51b4-41ed-a113-5e40ba018278","order_by":2,"name":"Hui-Hong Wang","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hui-Hong","middleName":"","lastName":"Wang","suffix":""},{"id":376372647,"identity":"c87edcfc-b457-446a-ab10-5190df70fed0","order_by":3,"name":"Si-Jin Zeng","email":"","orcid":"","institution":"South China Botanical Garden","correspondingAuthor":false,"prefix":"","firstName":"Si-Jin","middleName":"","lastName":"Zeng","suffix":""},{"id":376372648,"identity":"2e93394f-75eb-48b2-82a0-289fccd30fdc","order_by":4,"name":"Lin-Ya Zeng","email":"","orcid":"","institution":"Huizhou Forestry Science Research Institution","correspondingAuthor":false,"prefix":"","firstName":"Lin-Ya","middleName":"","lastName":"Zeng","suffix":""},{"id":376372649,"identity":"a0c176e1-aea2-436a-bda9-1ee17603d54f","order_by":5,"name":"En-Wei Tian","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"En-Wei","middleName":"","lastName":"Tian","suffix":""},{"id":376372650,"identity":"a78ceb63-208e-4aa7-8fa8-8c7d593002eb","order_by":6,"name":"Nian-He Xia","email":"","orcid":"https://orcid.org/0000-0001-9852-7393","institution":"South China Botanical Garden","correspondingAuthor":false,"prefix":"","firstName":"Nian-He","middleName":"","lastName":"Xia","suffix":""}],"badges":[],"createdAt":"2024-10-18 08:10:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5287647/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5287647/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":71509471,"identity":"e366c964-6d2c-4372-9d2d-ecb3d4a53e2c","added_by":"auto","created_at":"2024-12-16 10:12:14","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6698162,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eCurcuma flavescens\u003c/em\u003efrom type locality. \u003cstrong\u003eA\u003c/strong\u003e Habit; B Inflorescence (side view); \u003cstrong\u003eC\u003c/strong\u003e Inflorescence with leaves (top view); \u003cstrong\u003eD\u003c/strong\u003e Fertile bract (left) and coma bract (right);\u003cstrong\u003eE\u003c/strong\u003e Expanded labellum; \u003cstrong\u003eF\u003c/strong\u003e Bracteoles and flowers; \u003cstrong\u003eG\u003c/strong\u003e Flower dissection; \u003cstrong\u003eH\u003c/strong\u003e Cross section of rhizome and tubers; \u003cstrong\u003eI\u003c/strong\u003e Seeds (left) and fruits (right);\u003cstrong\u003e J\u003c/strong\u003e Chromosome number 2\u003cem\u003en\u003c/em\u003e = 42.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5287647/v1/65b5db0d23fdc6234795ee33.jpg"},{"id":71509921,"identity":"83da8ea5-6009-4bc3-bc10-8c48524ea8e7","added_by":"auto","created_at":"2024-12-16 10:20:14","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":683984,"visible":true,"origin":"","legend":"\u003cp\u003eIllustration of \u003cem\u003eCurcuma flavescens\u003c/em\u003e. \u003cstrong\u003eA\u003c/strong\u003e Whole plant; B Rhizomes (side view); \u003cstrong\u003eC\u003c/strong\u003e Fertile bract (left) and coma bract (right); \u003cstrong\u003eD\u003c/strong\u003e Bracteoles (left) and flowers (right);\u003cstrong\u003e F\u003c/strong\u003e Expanded labellum; \u003cstrong\u003eG\u003c/strong\u003e Ovary with two epigynous glands, style and stigma (left) and calyx (right); \u003cstrong\u003eH\u003c/strong\u003e Seeds and fruits. Drawn by D.H. Cui.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5287647/v1/ea5857499dfe277acbf46220.jpg"},{"id":71509920,"identity":"c7438ca6-aaf0-410a-857d-e39407eb2175","added_by":"auto","created_at":"2024-12-16 10:20:14","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":633163,"visible":true,"origin":"","legend":"\u003cp\u003eGenome size and ploidy analysis. \u003cstrong\u003eA\u003c/strong\u003e The \u003cem\u003ek\u003c/em\u003e-mer (k = 17) frequency distribution. Two primary \u003cem\u003ek\u003c/em\u003e-mer peaks are present, indicating that the genome is diploid; \u003cstrong\u003eB\u003c/strong\u003e The ploidy analysis for reference-free profiling of polyploid genomes based on Smudgeplot. The Smudgeplot shows the frequency of \u003cem\u003ek\u003c/em\u003e-mer pairs within the genome, with darker colors indicating the group is less frequent and bright yellow indicating the group is more frequent. When visualized, the plot shows distinct “smudges” representing each \u003cem\u003ek\u003c/em\u003e-mer pair with the greatest of density of \u003cem\u003ek\u003c/em\u003e-mers relating to the ploidy level of the genome.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5287647/v1/cf8e51e002126032ffccdf03.jpg"},{"id":71509470,"identity":"3b3e8325-575a-49ee-b2d5-4fc7025eaf16","added_by":"auto","created_at":"2024-12-16 10:12:14","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1629799,"visible":true,"origin":"","legend":"\u003cp\u003eCircular plastid genome map of \u003cem\u003eCurcuma flavescens\u003c/em\u003e. Genes drawn within the circle are transcribed clockwise, while those drawn outside are transcribed counterclockwise. Genes of different functional groups are colored by different colors. The darker gray in the inner circle corresponds to the DNA GC content, while the lighter gray corresponds to the DNA AT content.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5287647/v1/3cd59e280820f10631507cfb.jpg"},{"id":71509473,"identity":"2c9d1a77-0064-4f7e-91dc-ba49d6f8c7c0","added_by":"auto","created_at":"2024-12-16 10:12:14","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":340183,"visible":true,"origin":"","legend":"\u003cp\u003eThe phylogenetic position of \u003cem\u003eCurcuma flavescens\u003c/em\u003e in the genus \u003cem\u003eCurcuma\u003c/em\u003e based on the maximum likelihood (ML), maximum parsimony (MP) and Bayesian inference (BI) method using plastid genomes. The bootstrap values are indicated at nodes. Red, yellow and blue represent the bootstrap values of ML, BI and MP, respectively. The triangle represents that the bootstrap values of nodes was less than 75%.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5287647/v1/92781184fb996c14111dab00.jpg"},{"id":71509474,"identity":"a2e220e9-b195-431d-bd97-a45a39268d0e","added_by":"auto","created_at":"2024-12-16 10:12:14","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3920155,"visible":true,"origin":"","legend":"\u003cp\u003eThe phylogenetic position of \u003cem\u003eCurcuma flavescens\u003c/em\u003e in the genus \u003cem\u003eCurcuma\u003c/em\u003e based on the maximum likelihood (ML), maximum parsimony (MP) and Bayesian inference (BI) method using ITS regions of 51 seed-setting species. The bootstrap values are indicated at nodes. Red, yellow and blue represent the bootstrap values of ML, BI and MP, respectively. The triangle represents that the bootstrap values of nodes was less than 75%.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5287647/v1/5f234fe123d1d36255c12b25.jpg"},{"id":74440139,"identity":"993530c3-362f-4fba-8b38-d5cc49cdc030","added_by":"auto","created_at":"2025-01-22 09:58:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":15030158,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5287647/v1/ba19dba1-fe1a-49f6-afbf-8ff1f5e8db4d.pdf"},{"id":71509467,"identity":"41a259e5-981b-42cb-9aa0-d551cf5b7fb6","added_by":"auto","created_at":"2024-12-16 10:12:14","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":31233,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 1: Table S1 Ten\u003cem\u003e Curcuma \u003c/em\u003esamples newly sequenced for plastid genomes and five of them for nrITS regions in this study, their collection numbers and GenBank accession numbers. Table S2. 17 plastid genomes and 62 nrITS sequences downloaded from NCBI database.\u003c/p\u003e","description":"","filename":"Additionalfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5287647/v1/dbb4f255b3ae535e2354f76c.docx"}],"financialInterests":"","formattedTitle":"A new seed-setting species from the polyploid genus Curcuma of the ginger family (Zingiberaceae) based on morphological and molecular data","fulltext":[{"header":"Background","content":"\u003cp\u003ePolyploidy occurs in many taxa, is particularly wide-spread in flowering plants, and is a prominent feature of the chromosome evolution of higher plants. At least half of the known angiosperm species have experienced polyploidy in their evolutionary history (Hieter \u0026amp; Griffiths \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Shaked et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Xing et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Polyploidy has also played a significant role in evolution and diversification of various members of Zingiberaceae (see Mukherjee \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1970\u003c/span\u003e; Lim \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1972\u003c/span\u003e; Poulsen \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Chen and Chen \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Takano \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Takano \u0026amp; Okada \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), including \u003cem\u003eCurcuma\u003c/em\u003e L. (Joseph et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Ardiyani \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Sirisawad et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Leong-Škorničkov\u0026aacute; et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eCurcuma\u003c/em\u003e, one challenging genus in the family Zingiberaceae, is mainly distributed in South and Southeast Asia with many species extending to China, Australia, and the South Pacific. Recent estimates of the total species number vary from 50 (Larsen et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Wu \u0026amp; Larsen \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), to 100 (Sirirugsa \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1996\u003c/span\u003e) or 120 (Leong-Škorničkov\u0026aacute; et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Most \u003cem\u003eCurcuma\u003c/em\u003e species are of polyploid origin, often sterile and reproducing mainly vegetatively, with turmeric (\u003cem\u003eC. longa\u003c/em\u003e), one of the highly polyploid species, being the best known (Leong-Škorničkov\u0026aacute; et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Z\u0026aacute;vesk\u0026aacute; et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Few species seem to be diploid, reproducing sexually (Leong-Škorničkov\u0026aacute; et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The widely accepted basic chromosome number is \u003cem\u003ex\u003c/em\u003e\u0026thinsp;=\u0026thinsp;21 (Islam \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Puangpairote et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) but Leong-Škorničkov\u0026aacute; et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) suggested that \u003cem\u003ex\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7 should be considered the primary basic chromosome number.\u003c/p\u003e \u003cp\u003eThe most recent phylogeny of the genus (Z\u0026aacute;vesk\u0026aacute; et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Leong- Škorničkov\u0026aacute; et al. 2015) retained two previously accepted subgenera and proposed one subgenus mainly based on spur types: (1) subg. \u003cem\u003eCurcuma\u003c/em\u003e L., characterized by conspicuous coma bracts, epigynous glands and two forward-facing anther spurs, 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;42, 63, 84, 105; (2) subg. \u003cem\u003eHitcheniopsis\u003c/em\u003e (Baker) K. Schum., characterized by the lack of epigynous glands and the presence of anther spurs, 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;20, 22, 24, 28, etc.; (3) subg. \u003cem\u003eEcomatae\u003c/em\u003e Škorničk. \u0026amp; Š\u0026iacute;da f., characterized by epigynous glands, two L-shaped spurs and the lack of conspicuous coma bracts, 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;42. Although several studies have attempted to reveal the evolutionary process in the genus and detect possible hybrids and their parental species (Z\u0026aacute;vesk\u0026aacute; et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Skopal\u0026iacute;kov\u0026aacute; et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), most of the relationships at the species level are unresolved, especially for those triploid plants. Such ability to reliably identify hybrids and their parental species may have direct significance in subsequent research towards better utilization of \u003cem\u003eCurcuma\u003c/em\u003e species.\u003c/p\u003e \u003cp\u003eChina is one of the important distribution areas of \u003cem\u003eCurcuma\u003c/em\u003e species. Nowadays, 24 names have been associated with Chinese \u003cem\u003eCurcuma\u003c/em\u003e species, 16 of which are based on types from China (Tong \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Ye et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Chen \u0026amp; Xia \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Several species have been used as Chinese herbal medicine for more than a thousand years. For example, \u0026ldquo;jianghuang姜黄\u0026rdquo; from the rhizome of \u003cem\u003eCurcuma longa\u003c/em\u003e L., \u0026ldquo;片姜黄\u0026rdquo; from the sliced rhizome of \u003cem\u003eC. wenyujin\u003c/em\u003e Y.H.Chen \u0026amp; C.Ling, \u0026ldquo;莪术\u0026rdquo; from the rhizomes of \u003cem\u003eC. phaeocaulis\u003c/em\u003e Valeton, \u003cem\u003eC. wenyujin\u003c/em\u003e or \u003cem\u003eC. kwangsiensis\u003c/em\u003e S.G.Lee \u0026amp; C.F.Liang, and \u0026ldquo;郁金\u0026rdquo; from the tubers of \u003cem\u003eC. wenyujin\u003c/em\u003e, \u003cem\u003eC. longa\u003c/em\u003e, \u003cem\u003eC. kwangsiensis\u003c/em\u003e or \u003cem\u003eC. phaeocaulis\u003c/em\u003e are officially recorded in Chinese Pharmcopoeia (China Pharmacopoeia Committee \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The previous cytological analysis of many \u003cem\u003eCurcuma\u003c/em\u003e species from China showed that \u003cem\u003eCurcuma flaviflora\u003c/em\u003e S.Q.Tong was diploid with 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2\u003cem\u003ex\u003c/em\u003e\u0026thinsp;=\u0026thinsp;42, \u003cem\u003eC. nankunshanensis\u003c/em\u003e N.Liu, X.B.Ye \u0026amp; Juan Chen, \u003cem\u003eC. ruiliensis\u003c/em\u003e N.H.Xia \u0026amp; Juan Chen and \u003cem\u003eC. kwangsiensis\u003c/em\u003e S.G.Lee \u0026amp; C.F.Liang was tetraploid with 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4\u003cem\u003ex\u003c/em\u003e\u0026thinsp;=\u0026thinsp;84. The rests were all triploid (2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3\u003cem\u003ex\u003c/em\u003e\u0026thinsp;=\u0026thinsp;63) (Ye et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDuring the revision of Chinese \u003cem\u003eCurcuma\u003c/em\u003e species begun from 2007, a seed-setting \u003cem\u003eCurcuma\u003c/em\u003e species with much branched rhizomes and terminal inflorescences belonging to subg. \u003cem\u003eCurcuma\u003c/em\u003e from Puwen of Xishuangbanna in Yunnan attracted our attention. Since only four members of \u003cem\u003eC\u003c/em\u003e. subg. \u003cem\u003eCurcuma\u003c/em\u003e from China can produce viable seeds, namely \u003cem\u003eC\u003c/em\u003e. \u003cem\u003eexigua\u003c/em\u003e, \u003cem\u003eC. kwangsiensis\u003c/em\u003e, \u003cem\u003eC. nankunshanensis\u003c/em\u003e and \u003cem\u003eC. ruiliensis\u003c/em\u003e, this species as well as its ploidy level was worthy of study. Among them, only \u003cem\u003eC. exigua\u003c/em\u003e has many branched rhizomes (like the rhizomes of \u003cem\u003eC. longa\u003c/em\u003e) and terminal inflorescences, while the other species all have ovoid rhizomes which are unbranched or few branched and bear both lateral and terminal inflorescences. After further studies including morphological and phylogenetic analyses, it is concluded that the material from Puwen represented a new species which is described and illustrated below. Its genome size, chromosome number and ploidy level were also reported to facilitate further breeding and polyploid study in this genus.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMorphological and taxonomic analyses\u003c/h2\u003e \u003cp\u003eDuring the revision of \u003cem\u003eCurcuma\u003c/em\u003e species, names of taxa currently recognized as members of \u003cem\u003eCurcuma\u003c/em\u003e have been investigated. Types and original material including herbarium sheets, drawings/icons and manuscripts were searched and checked. All specimens of \u003cem\u003eCurcuma\u003c/em\u003e species in the following herbaria in China were examined: AU, EMA, FJIDC, GXMG, GXMI, HITBC, IBK, IBSC, IMC, IMDY, KUN, NAS, PE, SYS and TAI. Additional digitized material from China and neighboring countries (Thailand, Laos, Myanmar, India and Vietnam) in the following herbaria has also been studied: AAU, BM, C, E, G, K, L, P, US, and WU.\u003c/p\u003e \u003cp\u003eThe description of this new species was based on the specimens collected from the type locality. The style of description followed Chen et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and the terminology in general followed Beentje (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eChromosome cytology\u003c/h3\u003e\n\u003cp\u003eChromosome counts were conducted on somatic metaphases using the root tip squash technique outlined by Chen et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Actively growing root tips of cultivated plants introduced from the field were used to count the chromosome numbers. The number of chromosomes was determined from five complete well-spread mitotic plates. The voucher specimen is deposited at IBSC (\u003cem\u003eJ.Y. Jin JYJ 054\u0026thinsp;\u0026minus;\u0026thinsp;7\u003c/em\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe\u003c/b\u003e \u003cb\u003ek\u003c/b\u003e\u003cb\u003e-mer analysis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAvailability of Next Generation Sequencing data provides a means for genome size estimation by \u003cem\u003ek\u003c/em\u003e-mer analysis. Silica-gel dried leaves were sent to Novogene (Tianjin, China) to extract total genomic DNA for library preparation for genome skimming sequencing. Paired-end (150 bp) sequencing was conducted by Novogene Corporation Inc. on the Illumina HiSeq2000 platform (Illumina, Inc., San Diego, CA, USA), generating approximately 100 Gb raw data for one sample of the new species. The raw reads were further curated to remove low-quality reads and 3\u0026prime; adapter contamination using fastp (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/OpenGene/fastp\u003c/span\u003e\u003cspan address=\"https://github.com/OpenGene/fastp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Clean reads were used for \u003cem\u003ek\u003c/em\u003e-mer counting using Jellyfish 2.2.7 (Mar\u0026ccedil;ais \u0026amp; Kingsford \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The \u003cem\u003ek\u003c/em\u003e-mer size of 17 and the \u0026lsquo;jellyfish count -m 17 and -C\u0026rsquo; command was used to create the histogram data. The Jellyfish count output was then exported to \u003cem\u003ek\u003c/em\u003e-mer histogram using the \u0026lsquo;jellyfish histo\u0026rsquo; command. The output file was used as an input file for the software developed by Novogene Corporation Inc. to calculated the depth distribution map of \u003cem\u003ek\u003c/em\u003e-mer. The heterozygosity rate was calculated according to Marcais \u0026amp; Kingsford (2011).\u003c/p\u003e \u003cp\u003eThe ploidy level of this new species was also evaluated by Smudgeplot v0.2.5 (Ranallo-Benavidez et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/KamilSJaron/smudgeplot\u003c/span\u003e\u003cspan address=\"https://github.com/KamilSJaron/smudgeplot\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The \u003cem\u003ek\u003c/em\u003e-mer frequencies were first generated using KMC v3.1.1(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/tbenavi1/KMC\u003c/span\u003e\u003cspan address=\"https://github.com/tbenavi1/KMC\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with parameters \u0026lsquo;-k21 -t10 -m64 -ci1 -cs10000 @FILES kmcdb tmp\u0026rsquo; and then converted to \u003cem\u003ek\u003c/em\u003e-mer frequency histogram using parameters \u0026lsquo;kmc_tools transform kmcdb histogram species_k21.hist -cx10000\u0026rsquo;. The \u0026lsquo;smudgeplot.py cutoff species_k21.hist L/U\u0026rsquo; command was then used to estimate \u003cem\u003ek-\u003c/em\u003emer coverage thresholds from the histogram file. The \u003cem\u003ek\u003c/em\u003e-mers in the coverage ranging from L to U were extracted with the command \u0026lsquo;kmc_tools transform\u0026rsquo; and \u0026lsquo;KMC_dump\u0026rsquo; command was used to reduce the file to compute set of \u003cem\u003ek\u003c/em\u003e-mer pairs. The Smudgeplot showing proposed ploidy level was then generated with coverages of identified \u003cem\u003ek\u003c/em\u003e-mer pairs (i.e., species_coverages.tsv file) using \u0026lsquo;smudgeplot.pyplot\u0026rsquo; command.\u003c/p\u003e\n\u003ch3\u003eTaxon sampling\u003c/h3\u003e\n\u003cp\u003eTo reveal the approximate phylogenetic relationship within \u003cem\u003eCurcuma\u003c/em\u003e, two datasets including plastid genomes and nrITS sequences respectively were utilized. Firstly, 10 newly sequenced samples and 13 released plastid genomes of 23 species representing three subgenera of \u003cem\u003eCurcuma\u003c/em\u003e were sampled based on previous phylogenetic relationships reported in Z\u0026aacute;vesk\u0026aacute; et al. (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Four species (\u003cem\u003eHedychium villosum\u003c/em\u003e Wall., \u003cem\u003eKaempferia elegans\u003c/em\u003e Wall., \u003cem\u003ePyrgophyllum yunnanesnsis\u003c/em\u003e (Gagnep.) T.L.Wu \u0026amp; Z.Y.Chen and \u003cem\u003eZingiber mioga\u003c/em\u003e Roscoe) from Zingiberaceae were also downloaded from GenBank as outgroups according to phylogenetic relationships reported in Kress et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Then, the dataset of ITS sequences of 65 samples representing 51 seed-setting species from three subgenera of \u003cem\u003eCurcuma\u003c/em\u003e were used for molecular analyses. Two species (\u003cem\u003eHedychium villosum\u003c/em\u003e and \u003cem\u003eZingiber mioga\u003c/em\u003e) were selected as outgroups. Since high intra-individual ITS polymorphism has been documented in Z\u0026aacute;vesk\u0026aacute; et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), 1‒3 individuals per species have been sampled for ITS analysis. Sequences of \u003cem\u003eC. flavescence\u003c/em\u003e, \u003cem\u003eC. exigua\u003c/em\u003e and \u003cem\u003eC. woodii\u003c/em\u003e were extracted from the assembled nrDNA sequences. Other available sequences were downloaded from GenBank. Detailed information of all species sampled and sequences used are available in Additional file 1: Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e \u0026amp; S2.\u003c/p\u003e\n\u003ch3\u003eDNA extraction, sequencing, assembly and annotation\u003c/h3\u003e\n\u003cp\u003eSilica-gel dried leaves were sent to Novogene for genome skimming sequencing. Paired-end (150 bp) sequencing was conducted on the Illumina HiSeq2000 platform as mentioned above, generating approximately 20 Gb raw data for each sample. After quality control of the raw data, all paired reads were extracted for plastid and nrDNA assembly using GetOrganelle 1.7 (Jin et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Plastomes were annotated with the Plastid Genome Annotator and GeSeq with default settings (PGA; Tillich et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Qu et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and manually corrected using Geneious Prime 2019 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.geneious.com\u003c/span\u003e\u003cspan address=\"https://www.geneious.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The plastid genome of \u003cem\u003eC. longa\u003c/em\u003e (MK965541) and nrDNA sequences of \u003cem\u003eC. montana\u003c/em\u003e Roxb. (JQ409939.1) were downloaded from GenBank as the references.\u003c/p\u003e\n\u003ch3\u003ePhylogenetic analysis\u003c/h3\u003e\n\u003cp\u003eComplete plastomes were aligned with MAFFT v. 7 using the FFT-NS-i \u0026times;1000 strategy (Katoh \u0026amp; Standley \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The ITS regions were extracted from the nrDNA sequences and aligned with MAFFT v. 7 with default parameters and manually adjusted in Geneious. Phylogenetic analyses were performed under maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI). The MP analysis was performed using PAUP* 4.0a169 (Swofford \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The test settings included 1000 replications of randomly added sequences and a heuristic search with tree bisection-reconnection branch swapping. The ML analysis was conducted using the IQ-TREE with the TVM\u0026thinsp;+\u0026thinsp;F\u0026thinsp;+\u0026thinsp;R2 model (CP), TNe\u0026thinsp;+\u0026thinsp;I\u0026thinsp;+\u0026thinsp;G4 model (ITS) and 1000 bootstrap iterations. Nodes with UFboot values\u0026thinsp;\u0026ge;\u0026thinsp;95 were regarded as well-supported. The BI analysis was performed using MrBayes 3.2.7 (Ronquist et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The following settings were applied: sampling frequency\u0026thinsp;=\u0026thinsp;100, temp\u0026thinsp;=\u0026thinsp;0.1, and number of Markov chain Monte Carlo generations\u0026thinsp;=\u0026thinsp;6000000. The first 1500000 trees were discarded as burn-in to ensure that the chains reached stationarity. A majority-rule consensus tree was constructed based on the trees sampled after generation 4500000. Nodes with posterior probability (PP) values of 0.95 and greater were regarded as significant.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eTaxonomic treatment\u003c/h2\u003e \u003cp\u003e \u003cb\u003eCurcuma flavescens\u003c/b\u003e \u003cb\u003eJuan Chen \u0026amp; N.H.Xia\u003c/b\u003e \u003cb\u003esp. nov.\u003c/b\u003e \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eType\u003c/em\u003e CHINA. Yunnan, Xishuangbanna, Jinghong, Puwen, 958 m a.s.l., 2 Aug 2021, \u003cem\u003eJ. Chen \u0026amp; J.W. Yan 21080205A\u003c/em\u003e (holotype: IBSC!, isotypes: KUN, PE).\u003c/p\u003e \u003cp\u003e \u003cem\u003eDiagnosis\u003c/em\u003e It is similar to \u003cem\u003eC. longa\u003c/em\u003e in its somewhat white coma bracts but differs in yellow (vs. orange to orange-red) rhizomes, green upper side of lamina sometimes with purple bands (vs. absence of purple bands), glabrous to puberulent (vs. glabrous) lower sides of leaf blades, white tinged with pale purple to pale purple (vs. white, whitish green or green) coma bracts.\u003c/p\u003e \u003cp\u003e \u003cem\u003eDescription\u003c/em\u003e Rhizomatous herbs, 60‒90 cm tall. Main rhizome 3.2‒5.2 \u0026times; 2.2‒3 cm, ovoid, yellow internally; lateral rhizomes 1.0‒1.5 cm in diam., fleshy, dark brown externally, yellow internally, becoming paler or white at tip; roots many, 2‒3 mm in diam. at top, tuberous and fleshy, brown to white externally, white internally; root tubers many, 2.1‒3 \u0026times; 0.9‒1.1 cm, obovate to fusiform, brown to white externally, white internally. Leafy shoots usually with 3‒5 leaves at anthesis; pseudostems composed of 2‒3 leafless sheaths and 3‒5 leafy sheaths, green or brown, puberulent; ligules 1‒2 mm in height, bilobed, pubescent on margin; petioles up to 26 cm long, green, glabrous; lamina 24‒62 \u0026times; 7‒11 cm (measured at anthesis), elliptic, base acuminate, slightly oblique, apex attenuate with uppermost tip glabrous, margin entire and glabrous, adaxial surface green, sometimes the leaf blades at the lower parts with faint and narrow (2‒5 mm wide) purple stripes at the midrib but the new leaves green, glabrous, abaxial surface normally glabrous rarely puberulent. Inflorescence terminal; spike 11‒16 \u0026times; 4‒6.5 cm, consisting of 20‒30 bracts; peduncle hidden within pseudostems, up to 27 cm long, green, glabrous; coma bracts 3.5‒5 \u0026times; 0.9‒1.9 cm, elliptic, obliquely spreading, base involuted, apex obtuse or acute and mucronate, white or white with pale purple tip to pale purple, glabrous to sparsely puberulent on both sides; fertile bracts 3.5‒4.2 \u0026times; 1.4‒2.1 cm, ovate, mostly erect, pale green, connate to 1/3 above base, base involuted, apex obtuse, glabrous to sparsely puberulent on both sides; cincinni with 3\u0026ndash;5 flowers at base of inflorescence, 1\u0026ndash;2 flowers at top; bracteoles one per flower, the first and largest bracteole 2.5‒2.8 \u0026times; 1.2‒1.6 cm, the remaining bracts gradually decrease in size, elliptic, apex acute, white, glabrous on both sides. Flowers 4.6‒5.2 cm long, much exserted from bracts. Calyx 8‒10 mm long, tubular, inconspicuously tridentate, apex obtuse, white, glabrous externally. Floral tube 2.7‒3.3 cm long, narrowly cylindrical at base, funnel-shaped at apex, yellowish white, glabrous externally, villous at throat; lateral corolla lobes 1.2‒1.5 \u0026times; 0.6‒0.9 cm, elliptic, apex obtuse and slightly concave, white, glabrous on both sides; dorsal lobe 1.3‒1.8 \u0026times; 0.9‒1.2 cm, elliptic and concave, white, glabrous on both sides, apex mucronate, mucro 2‒3 mm long, white, sparsely pubescent. Lateral staminodes 1‒1.6 \u0026times; 0.5‒0.7 cm, lanceolate, white, glabrous on both sides; labellum 1.2‒1.8 \u0026times;1.5‒1.7 cm, square-shaped, median lobes with obtuse apices and exceeding the laterals, with a 1‒2 mm long incision, white, two yellow bands at center, glabrous on both sides. Stamen 1.1‒1.3 cm long; filament 7‒9 mm long, broad, flat, white; anther 4‒5 mm long, yellowish white, spur short, 2‒3 mm long, yellowish white, anther crest absent. Epigynous glands 2, 4\u0026ndash;5 mm long, linear. Style white, glabrous; stigma ca. 1 mm wide, capitate, white, ostiole ciliate. Fruit a globular trilocular capsule, white, pubescent externally; seeds irregularly obovoid, ca. 5 mm long, cream to light brown, shiny, enclosed in translucent white, laciniate aril.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePhenology\u003c/em\u003e Flowering in July to August; fruiting in August to September.\u003c/p\u003e \u003cp\u003e \u003cem\u003eEtymology\u003c/em\u003e The specific epithet is derived from the yellow color of rhizome from this new species.\u003c/p\u003e \u003cp\u003e \u003cem\u003eChinese vernacular name and uses\u003c/em\u003e 柠檬黄姜黄 [n\u0026iacute;ng m\u0026eacute;ng hu\u0026aacute;ng jiāng hu\u0026aacute;ng]. No use has been reported.\u003c/p\u003e \u003cp\u003e \u003cem\u003eDistribution and conservation status\u003c/em\u003e This species is endemic to China. A recent field survey conducted at the type locality revealed the existence of several populations consisting of about 100 adult individuals, thriving within rubber plantations or along roadsides, at an elevation of ca. 958 m a.s.l. Remarkably, a single specimen collected from Lancang fits the characteristics of this species. However, given the scarcity of information regarding the distribution (extent of occurrence (EOO) or areas of occupancy (AOO)) and population sizes of the species, the conservation status of \u003cem\u003eC. flavescens\u003c/em\u003e is temporarily assessed here as Data Deficient (DD), following the IUCN Red List Categories and Criteria (IUCN \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eAdditional specimens examined\u003c/em\u003e China. Yunnan: Lancang, 26 Aug 1957, \u003cem\u003eG.S. Sin 486\u003c/em\u003e (KUN); Xishuangbanna, Jinghong, Puwen, 958 m a.s.l., 2 Aug 2021, \u003cem\u003eJ. Chen \u0026amp; J.W. Yan 21080205B (IBSC)\u003c/em\u003e; ibid., 6 Sep 2020, \u003cem\u003eS.J. Zeng \u0026amp; L.Y. Zeng 20090604\u003c/em\u003e (IBSC); ibid., 6 Aug 2022, \u003cem\u003eJ.Y. Jin JYJ053-1, JYJ053-5 \u0026amp; JYJ 054\u0026thinsp;\u0026minus;\u0026thinsp;7\u003c/em\u003e (IBSC).\u003c/p\u003e \u003cp\u003e \u003cem\u003eNotes\u003c/em\u003e As in the other seed-setting species of \u003cem\u003eCurcuma\u003c/em\u003e, such as \u003cem\u003eC. kwangsiensis\u003c/em\u003e and \u003cem\u003eC. ruiliensis\u003c/em\u003e, characters of this new species such as the purple stripes (presence or absence) on the upper surface of leaf blades, the leaf indumentum (glabrous on both sides or glabrous adaxially but pubescent abaxially) and coma color (white, pink to dark violet) are variable, while the stature, the rhizome shape and color, the lamina shape, the fertile bract shape and color, the color and shape of labellum and lateral staminodes are stable within the population observed.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCurcuma flavescens\u003c/em\u003e shares some similarities with \u003cem\u003eC. exigua\u003c/em\u003e, yet it exhibits distinct characters by its much thicker and stronger pseudostems, broader leaf blades, sometimes with pubescent abaxial surfaces, green or sometimes with vary faint and narrower purple stripes (vs. green with obvious purple stripes) at the midrib, larger inflorescence, and ovate (vs. lanceolate) fertile bract with obtuse (vs. acute) apex and much involuted base.\u003c/p\u003e \u003cp\u003eIt can be readily distinguished from \u003cem\u003eC. ruiliensis\u003c/em\u003e by much branched and yellow rhizomes, slender stature, shorter ligule, ovate fertile bracts with obtuse apex, much broader coma bracts, only bearing terminal inflorescence and being diploid not tetraploid. A detailed comparison is provided in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMorphological comparison of the diagnostic characters of \u003cem\u003eCurcuma flavescens\u003c/em\u003e and closely related species.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eC. flavescens\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eC. longa\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eC. exigua\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eC. ruiliensis\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlant height (cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60‒90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e80‒120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40‒80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e85‒110\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRhizome branch\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMuch branched\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMuch branched\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMuch branched\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRarely branched\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRhizome color\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYellow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOrange to orange -red\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYellow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePale yellow or white\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLigule length (mm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1‒2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eca. 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1‒2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5‒6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLeaf blade\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSize\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24‒62 \u0026times; 7‒11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33\u0026ndash;60 \u0026times; 10\u0026ndash;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19‒22 \u0026times; 5‒7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e47\u0026ndash;62 \u0026times; 9\u0026ndash;11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShape\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eElliptic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eElliptic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eElliptic to lanceolate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eElliptic\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eColor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGreen or with 2‒5 mm purple stripes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGreen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGreen with 5‒6 mm purple stripes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGreen with ca. 2 cm purple stripes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIndumentum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGlabrous or rarely pubescent abaxially\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGlabrous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGlabrous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGlabrous adaxially, pubescent abaxially\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInflorescence\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePosition\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTerminal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTerminal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTerminal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTerminal and lateral\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSize (cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11\u0026ndash;16.5 \u0026times; 5\u0026ndash;8.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.5\u0026ndash;16 \u0026times; ca. 9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.5\u0026ndash;10 \u0026times; 3.5\u0026ndash;5.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.5\u0026ndash;17 \u0026times; 5\u0026ndash;7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBract number\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20\u0026ndash;30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25\u0026ndash;40 (\u0026ndash;80)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eca. 11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e38\u0026ndash;45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eComa bract shape\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eElliptic, base involuted, apex obtuse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eElliptic, base involuted, apex acute to obtuse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOblong to lanceolate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLanceolate, obliquely spreading, apex acute\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSize (cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.5‒5 \u0026times; 0.9‒1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026ndash;6 \u0026times; 1.7\u0026ndash;2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.8\u0026ndash;4.2 \u0026times; 0.8\u0026ndash;1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.8\u0026ndash;5.9 \u0026times; 0.8\u0026ndash;1.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eColor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWhite or tinged with pale purple to pale purple\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWhite, whitish green or green\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWhite tinged with purple\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePurple\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFertile bract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSize (cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.5‒4.2 \u0026times; 1.4‒2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.8\u0026ndash;4 \u0026times; 2.4\u0026ndash;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.5\u0026ndash;3 \u0026times; 1.2\u0026ndash;1.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.1\u0026ndash;4.5 \u0026times; 1.6\u0026ndash;2.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eApex shape\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eObtuse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAcute to obtuse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAcute\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNarrowly acute\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBracteole size\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.5‒2.8 \u0026times; 1.2‒1.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eca. 2.3 \u0026times; 1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eca. 2.1 \u0026times; 0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.3\u0026ndash;1.5 \u0026times; 0.5\u0026ndash;0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlower color\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWhite\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWhite\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWhite\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYellow\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiploid level\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003cem\u003ex\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003cem\u003ex\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUnknown, seed-setting\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003cem\u003ex\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIt is also closely related to \u003cem\u003eC. montana\u003c/em\u003e Roxb. in the appearance of inflorescences. However, this species has prominently exserted flowers and usually glabrous leaf blades on both sides instead of being pubescent on the abaxial side. It has yellow labellum and lateral staminodes differing from white color in \u003cem\u003eC. montana\u003c/em\u003e. Other distinguishing features are the presence of yellow rhizomes and green fertile bracts, setting it from \u003cem\u003eC. montana\u003c/em\u003e with light-orange-yellow rhizomes and white fertile bracts.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eChromosome cytology\u003c/h3\u003e\n\u003cp\u003eThe chromosome number of \u003cem\u003eCurcuma flavescens\u003c/em\u003e was determined to be 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;42 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ). It is either diploid or hexaploid according to the different understanding of the basic chromosome number (\u003cem\u003ex\u003c/em\u003e\u0026thinsp;=\u0026thinsp;21 or 7) (Leong-Škorničkov\u0026aacute; et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Puangpairote et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe\u003c/b\u003e \u003cb\u003ek\u003c/b\u003e\u003cb\u003e-mer analysis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ek\u003c/em\u003e-mer (\u003cem\u003ek\u003c/em\u003e\u0026thinsp;=\u0026thinsp;17) depth and frequency results showed that the genome size of \u003cem\u003eC. flavescens\u003c/em\u003e is 874.19 Mb with a heterozygosity rate of 1.61% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The \u003cem\u003ek\u003c/em\u003e-mer depth distribution histograms (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) also revealed a unique bimodal profile with a high peak around 16 \u0026times; coverage and a shorter peak around 31 \u0026times;. This could be evidence of a highly heterogeneous genome. The ploidy level was further analyzed using the reported method for reference-free profiling of polyploid genomes, suggesting that the possibility of AB is 0.48 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), which proposed that it is diploid. Considering the basic chromosome number in \u003cem\u003eCurcuma\u003c/em\u003e species was 21; therefore, it is a diploid plant (2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2\u003cem\u003ex\u003c/em\u003e\u0026thinsp;=\u0026thinsp;42).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe plastid genome features of\u003c/b\u003e \u003cb\u003eCurcuma flavescens\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe complete plastid genome of \u003cem\u003eCurcuma flavescens\u003c/em\u003e is 162,133 bp in length and has a circular and quadripartite structure with a large single-copy (LSC) region (87,013 bp) and a small single-copy (SSC) region (15,622 bp), which are separated by a pair of inverted repeats (IRs) regions (29,749 bp) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The size range of this sequenced cp genome is almost agreed with those of other Zingiberaceae species (Cui et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Gui et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Liang \u0026amp; Chen \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The GC content is 36.2% in the complete plastid genome, while that in the LSC, SSC, and IR regions are 34.0%, 29.8%, and 41.2%, respectively, which is also in accordance with previous studies (Cui et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Gui et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Liang \u0026amp; Chen \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The IR region has higher GC content than other regions, possibly because of the higher GC content of tRNA and rRNA that located in that region (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The cp genome contains 133 functional genes including 87 protein-coding genes (PCGs), 38 tRNA genes and eight rRNA genes. Among these genes, 93 are unique genes and 20 are duplicated genes. The 93 unique genes contain 71 CDS and 22 tRNA genes. Twenty repeat genes including all rRNA genes located in IR region, and the \u003cem\u003eycf1\u003c/em\u003e gene spans the SSC and IR region (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Similar results had also been reported in the relevant studies of Gui et al. (202) and Lin et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eList of the annotated genes in the chloroplast genome of \u003cem\u003eCurcuma flavescens\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCategory\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGroups of genes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eName of genes\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSelf‑replication\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRibosomal RNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003errn4.5, rrn5, rrn16, rrn23\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTransfer RNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003etrnA-UGC\u003c/b\u003e, \u003cem\u003etrnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnG-GCC, trnG-UCC\u003c/em\u003e, \u003cb\u003etrnH-GUG\u003c/b\u003e,\u003c/p\u003e \u003cp\u003e\u003cb\u003etrnI-CAU\u003c/b\u003e, \u003cb\u003etrnI-GAU\u003c/b\u003e, \u003cem\u003etrnK-UUU, trnL-UAG\u003c/em\u003e,\u003c/p\u003e \u003cp\u003e\u003cb\u003etrnL-CAA\u003c/b\u003e, \u003cem\u003etrnL-UAA, trnM-CAU\u003c/em\u003e, \u003cb\u003etrnN-GUU\u003c/b\u003e,\u003c/p\u003e \u003cp\u003e\u003cem\u003etrnP-UGG, trnQ-UUG, trnR-UCU\u003c/em\u003e, \u003cb\u003etrnR-ACG\u003c/b\u003e,\u003c/p\u003e \u003cp\u003e\u003cem\u003etrnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU\u003c/em\u003e,\u003c/p\u003e \u003cp\u003e\u003cem\u003etrnT-UGU, trnfM-CAU, trnV-UAC\u003c/em\u003e, \u003cb\u003etrnV-GAC\u003c/b\u003e,\u003c/p\u003e \u003cp\u003e\u003cem\u003etrnW-CCA, trnY-GUA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSmall subunit of ribosome\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003erps2, rps3, rps4\u003c/em\u003e, \u003cb\u003erps7\u003c/b\u003e, \u003cem\u003erps8, rps11\u003c/em\u003e, \u003cb\u003erps12\u003c/b\u003e, \u003cem\u003erps14, rps15, rps16, rps18\u003c/em\u003e, \u003cb\u003erps19\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLarge subunit of ribosome\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003erpl2\u003c/b\u003e, \u003cem\u003erpl14, rpl16, rpl20, rpl22\u003c/em\u003e, \u003cb\u003erpl23\u003c/b\u003e, \u003cem\u003erpl32, rpl33, rpl36\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRNA polymerase subunits\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003erpoA, rpoB, rpoC1, rpoC2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhotosynthesis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhotosystem I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003epsaA, psaB, psaC, psaI, psaJ\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhotosystem II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003epsbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSubunits of cytochrome\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003epetA, petB, petD, petG, petL, petN\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eATP synthase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eatpA, atpB, atpE, atpF, atpH, atpI\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNADH‑dehydrogenase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eNdhA\u003c/em\u003e, \u003cb\u003endhB\u003c/b\u003e, \u003cem\u003endhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOther genes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRubisco large subunit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003erbcL\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTranslational initiation factor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003einfA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaturase K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ematK\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEnvelope membrane protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ecemA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAcetyl‑CoA carboxylase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eaccD\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProteolysis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eclpP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCytochrome c biogenesis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eccsA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConserved open reading frames\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eycf1\u003c/b\u003e, \u003cb\u003eycf2\u003c/b\u003e, \u003cem\u003eycf3, ycf4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eNote: the bold genes indicate they have two copies.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe phylogenetic position of\u003c/b\u003e \u003cb\u003eCurcuma flavescens\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAs indicated by prior researches (Kress et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Z\u0026aacute;vesk\u0026aacute; et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Liang \u0026amp; Chen \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), the phylogenomic trees recovered three main clades with high support: \u003cem\u003eCurcuma\u003c/em\u003e subg. \u003cem\u003eCurcuma\u003c/em\u003e, \u003cem\u003eC\u003c/em\u003e. subg. \u003cem\u003eHitcheniopsis\u003c/em\u003e and \u003cem\u003eC.\u003c/em\u003e subg. \u003cem\u003eEcomatae\u003c/em\u003e. However, analyses of the two data sets focused on the \u003cem\u003eCurcuma\u003c/em\u003e yielded little different topologies because \u003cem\u003eC.\u003c/em\u003e subg. \u003cem\u003eHitcheniopsis\u003c/em\u003e is sister to \u003cem\u003eC.\u003c/em\u003e subg. \u003cem\u003eEcomatae\u003c/em\u003e in the plastid genomes dataset while \u003cem\u003eC.\u003c/em\u003e subg. \u003cem\u003eHitcheniopsis\u003c/em\u003e is cluster with \u003cem\u003eC\u003c/em\u003e. subg. \u003cem\u003eCurcuma\u003c/em\u003e and \u003cem\u003eC.\u003c/em\u003e subg. \u003cem\u003eEcomatae\u003c/em\u003e is the basal group in the ITS dataset. The phylogenetic trees based on plastid genomes and ITS regions both indicated that \u003cem\u003eC. flavescens\u003c/em\u003e is clustered within \u003cem\u003eC\u003c/em\u003e. subg. \u003cem\u003eCurcuma\u003c/em\u003e (BS\u0026thinsp;=\u0026thinsp;100%, Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This species is distant from those related species, such as \u003cem\u003eC. longa\u003c/em\u003e, \u003cem\u003eC. exigua\u003c/em\u003e and \u003cem\u003eC. ruiliensis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) in the phylogenetic tree recovering by plastid genomes. The results from ITS analyses of 51 seed-setting species shows that \u003cem\u003eC. flavescens\u003c/em\u003e is nested with \u003cem\u003eC. montana\u003c/em\u003e, a species currently only known from India and Bangladesh, and far from those seed-setting species from China, e.g., \u003cem\u003eC. exigua\u003c/em\u003e, \u003cem\u003eC. ruiliensis\u003c/em\u003e and \u003cem\u003eC. kwangsiensis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Therefore, based on results of morphological, cytological and molecular analyses, a new seed-setting species with 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;42 is proposed here.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIBSC\u003c/em\u003e\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;South China Botanical Garden, Chinese Academy of Sciences, Herbarium\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eKUN\u003c/em\u003e\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;Kunming Institute of Botany, Chinese Academy of Sciences, Herbarium\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePE\u003c/em\u003e\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Institute of Botany, the Chinese Academy of Sciences, Herbarium\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBI\u003c/em\u003e\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Bayesian inference\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eML\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Maximum likelihood\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMP\u003c/em\u003e\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Maximum parsimony\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLSC\u003c/em\u003e\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Large single-copy\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSSC \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eSmall single-copy\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/em\u003e\u003c/strong\u003eInverted repeat\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePCG\u003c/em\u003e\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Protein-coding gene\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdditional file 1: Table S1. Ten\u003cem\u003e\u0026nbsp;Curcuma\u0026nbsp;\u003c/em\u003esamples newly sequenced for plastid genomes and five of them for nrITS regions in this study, their collection numbers and GenBank accession numbers. Table S2. 17 plastid genomes and 62 nrITS sequences downloaded from NCBI database.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to the curators of the herbaria mentioned in the \u0026ldquo;Materials and methods\u0026rdquo; part for allowing us to examine the specimens or providing the high-resolution images of specimens.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSJZ and LYZ first discovered and collected the new species in the field survey. JC and JWY conducted the molecular experiment. HHW conducted the cytological study. EWT and HHW analyzed the data. JC drafted the manuscript. NHX and JC designed and supervised the study. JC provided the funding. NHW, EWT and JC reviewed the manuscript. All authors have read and agreed with the submission of this manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China (Grant nos. 32070223, 31200161), Guangdong Flagship Project of Basic and Applied Basic Research (2023B0303050001)\u0026nbsp;and\u0026nbsp;Biological Resources Program, Chinese Academy of Sciences (Grant no. KFJ-BRP-017-19).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpecimens are deposited at the herbarium IBSC. DNA sequences are deposited at GenBank.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eState Key Laboratory of Plant Diversity and Specialty Crops, South China Botanical Garden, Chinese Academy of Sciences, 510650, Guangzhou, People\u0026rsquo;s Republic of China\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJuan Chen, Jia-Wei Yan, Si-Jin Zeng, Nian-He Xia\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;South China National Botanical Garden, 510650, Guangzhou, China\u003c/p\u003e\n\u003cp\u003eJuan Chen, Jia-Wei Yan, Si-Jin Zeng, Nian-He Xia\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eSchool of Traditional Chinese Medicine, Southern Medical University, 510515, Guangzhou, People\u0026rsquo;s Republic of China\u003c/p\u003e\n\u003cp\u003eHui-Hong Wang, En-Wei Tian\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Huizhou Forestry Science Research Institute (Huizhou Botanical Garden Management Service Center), Huizhou, 516001\u003c/p\u003e\n\u003cp\u003eLin-Ya Zeng\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Nian-He Xia ([email protected])\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare that there is no competing interest.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003ePublisher\u0026rsquo;s Note\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpringer Nature remains neutral with regards to jurisdictional claims in\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003epublished maps and institutional affiliations.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eArdiyani M (2002) Systematic study of \u003cem\u003eCurcuma\u003c/em\u003e L.: turmeric and its allies. 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Bot J Linnean Soc 165(4): 388\u0026ndash;401. https://doi.org/10.1111/j.1095-8339.2011.01122.x\u003c/li\u003e\n\u003cli\u003eZ\u0026aacute;vesk\u0026aacute; E, F\u0026eacute;r T, \u0026Scaron;\u0026iacute;da O, Krak K, Marhold K, Leong-\u0026Scaron;korničkov\u0026aacute; J (2012) Phylogeny of \u003cem\u003eCurcuma\u003c/em\u003e (Zingiberaceae) based on plastid and nuclear sequences: proposal of the new subgenus\u003cem\u003e Ecomata\u003c/em\u003e. Taxon 61: 747\u0026ndash;763. http://www.jstor.org/stable/41679307\u003c/li\u003e\n\u003cli\u003eZ\u0026aacute;vesk\u0026aacute; E, F\u0026eacute;r T, \u0026Scaron;\u0026iacute;da O, Marhold K, Leong-\u0026Scaron;korničkov\u0026aacute; J (2016). Hybridization among distantly related species: examples from the polyploid genus \u003cem\u003eCurcuma \u003c/em\u003e(Zingiberaceae). Mol Phylogenet Evol 100: 303\u0026ndash;321. http://www.jstor.org/10.1016/j.ympev.2016.04.017\u003c/li\u003e\n\u003cli\u003eZhang LX, Ding HB, Li HT, Zhang ZL, Tan YH (2019) \u003cem\u003eCurcuma tongii\u003c/em\u003e, a new species of \u003cem\u003eCurcuma\u003c/em\u003e subgen. \u003cem\u003eEcomatae\u003c/em\u003e (Zingiberaceae) from southern Yunnan, China. Phytotaxa 395 (3): 241\u0026ndash;247. https://doi.org/10.11646/phytotaxa.395.3.9.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Curcuma, Genome size, Ploidy level, New species, Plastid genome, nrITS region","lastPublishedDoi":"10.21203/rs.3.rs-5287647/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5287647/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe polyploid genus \u003cem\u003eCurcuma\u003c/em\u003e L. is an economically important, yet taxonomically rather difficult genus, mainly distributed in South and Southeast Asia. Several Chinese important traditional medicinal herbs are from \u003cem\u003eCurcuma\u003c/em\u003e, such as \u0026ldquo;jianghuang姜黄\u0026rdquo;, \u0026ldquo;yujin郁金\u0026rdquo; and \u0026ldquo;ezhu莪术\u0026rdquo;. During field investigation of plant resources in Yunnan, the distribution center of the genus in China, an unknown flowering and seed-setting species of \u003cem\u003eCurcuma\u003c/em\u003e was discovered. Its morphological characters were assessed for further taxonomic treatment and molecular analysis was conducted to ascertain its phylogenetic position within the genus as well. Its genome size, chromosome number and ploidy level were evaluated by \u003cem\u003ek\u003c/em\u003e-mer distribution analysis and cytological method.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThis species resembles \u003cem\u003eCurcuma longa\u003c/em\u003e but can be distinctly differed in its yellow rhizomes, sometimes with pubescent abaxial surfaces, green or sometimes with vary faint and narrower purple stripes at the midrib, white tinged with pale purple to pale purple coma bracts. Its chromosome number is 2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;42. The haploid genome size estimation of \u003cem\u003eCurcuma flavescens\u003c/em\u003e based on \u003cem\u003ek\u003c/em\u003e-mer distribution is 874.19 Mb. Smudgeplot analysis suggested it is a diploid heterozygous genome (AB). Plastid phylogenomic analyses indicated that this new species is embedded within subg. \u003cem\u003eCurcuma\u003c/em\u003e. The comprehensive phylogenetic studies conducted on \u003cem\u003eCurcuma\u003c/em\u003e species using nrITS regions showed it is nested with \u003cem\u003eC. montana\u003c/em\u003e, a species from India and Bangladesh. Moreover, morphological analysis further reinforced the distinctiveness of this species from \u003cem\u003eC. montana\u003c/em\u003e. It revealed several key differences across various anatomical features such as the color of rhizomes, the indumentum of leaves and the morphology of inflorescence and flowers. Our findings make a strong case for using next-generation sequencing to explore phylogenetic relationships and identify new species.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe morphological and molecular evidences support the recognition of \u003cem\u003eCurcuma flavescens\u003c/em\u003e as a new species. This provides a good diploidy material for the further breeding work in the genus \u003cem\u003eCurcuma\u003c/em\u003e, and might also contribute to the study of the polyploid origin in this genus.\u003c/p\u003e","manuscriptTitle":"A new seed-setting species from the polyploid genus Curcuma of the ginger family (Zingiberaceae) based on morphological and molecular data","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-16 10:12:09","doi":"10.21203/rs.3.rs-5287647/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"58377db6-bf4c-460d-9442-d7b1e825150f","owner":[],"postedDate":"December 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-22T09:50:29+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-16 10:12:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5287647","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5287647","identity":"rs-5287647","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}