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Article

Inferring the Phylogeny and Divergence of Chinese Curcuma (Zingiberaceae) in the Hengduan Mountains of the Qinghai–Tibet Plateau by Reduced Representation Sequencing

by
Heng Liang
1,†,
Jiabin Deng
2,†,
Gang Gao
3,
Chunbang Ding
1,
Li Zhang
4,
Ke Xu
5,
Hong Wang
6 and
Ruiwu Yang
1,*
1
College of Life Science, Sichuan Agricultural University, Yaan 625014, China
2
School of Geography and Resources, Guizhou Education University, Guiyang 550018, China
3
College of Life Sciences and Food Engineering, Yibin University, Yibin 644000, China
4
College of Science, Sichuan Agricultural University, Yaan 625014, China
5
Sichuan Horticultural Crop Technical Extension Station, Chengdu 610041, China
6
Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement, Institute of Pomology, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China
*
Author to whom correspondence should be addressed.
†
These authors have contributed equally to this work.
Forests 2021, 12(5), 520; https://0-doi-org.brum.beds.ac.uk/10.3390/f12050520
Submission received: 30 March 2021 / Revised: 21 April 2021 / Accepted: 21 April 2021 / Published: 23 April 2021
(This article belongs to the Special Issue Forest Genomics and Transcriptomics)
Browse Figures
Versions Notes

Abstract

:
Clarifying the genetic relationship and divergence among Curcuma L. (Zingiberaceae) species around the world is intractable, especially among the species located in China. In this study, Reduced Representation Sequencing (RRS), as one of the next generation sequences, has been applied to infer large scale genotyping of major Chinese Curcuma species which present little differentiation of morphological characteristics and genetic traits. The 1295 high-quality SNPs (reduced-filtered SNPs) were chosen from 997,988 SNPs of which were detected from the cleaned 437,061 loci by RRS to investigate the phylogeny and divergence among eight major Curcuma species locate in the Hengduan Mountains of the Qinghai–Tibet Plateau (QTP) in China. The results showed that all the population individuals were clustered together within species, and species were obviously separated; the clustering results were recovered in PCA (Principal Component Analysis); the phylogeny was (((((C. Phaeocaulis, C. yunnanensis), C. kwangsiensis), (C. amarissima, C. sichuanensis)), C. longa), (C. wenyujin, C. aromatica)); Curcuma in China originated around ~7.45 Mya (Million years ago) in the Miocene, and interspecific divergence appeared at ca. 4–2 Mya, which might be sped up rapidly along with the third intense uplift of QTP.

1. Introduction

Curcuma, commonly known as turmeric, has been an important flowering plant genus with medicinal, edible, and horticultural utilizations in the Orient from ancient times. Usually Curcuma are distributed in the subtropical and tropical area of Asia [1,2,3], and there are about 10 species in China (C. longa, C. sichuanensis, C. amarissima, C. yunnanensis, C. phaeocaulis, C. kwangsiensis, C. aromatica, C. wenyujin, C. flaviflora, C. exigua, and C. viridiflora) [4]. Among them, C. viridiflora and C. exigua may be extinct [5], C. yunnanensis and C. flaviflora are scarce and difficult to collect [4]. The worldwide Curcuma represent a paraphyletic group involving Hitchenia, Stahlianthus, and Smithatris in Zingibereae according to matK and ITS [6], and it has been classified into four lineages (Hitcheniopsis, Pierreana, Curcuma, and Ecomata) by using ITS and three chloroplast regions (trnL-trnF, psbA-trnH, matK) [2]. Recently, partial lineages in Curcuma have been clarified, though the phylogeny and divergence among some species are still intractable, especially species in China due to the intense uplift of the Qinghai–Tibet Plateau (QTP) [7,8]. Chinese Curcuma species are distributed to increase ranging from Tibet (only herbarium specimens represent this species now) to South China, most growing in the Hengduan Mountains Areas of QTP, which play an important role on species distribution, evolution, and divergence in eastern Asia [9,10,11,12,13].
The internal micro-anatomy of the rhizomes and leaves of Chinese Curcuma show few differences, and different anatomy traits can support different classification. The Chinese Curcuma species were divided into three groups according to the traits of oil cells, vascular bundles, and the number and diameter of xylem vessels (Group I: C. longa and C. sichuanensis, Group II: C. kwangsiensis and C. exigua, Group III: C. wenyujin, C. aromatica, C. phaeocaulis C. zedoaria, and C. yunnanensis) [14,15]. The hair distribution, stoma density and size, epidermic cell shape and size in leaf, were also suggested to be unique features among Chinese Curcuma species (Group I: C. longa, C. wenyujin, and C. sichuanensis; Group II: C. kwangsiensis and C. exigua; Group III: C. aromatica, C. chuanyujin, C. zedoaria, C. phaeocaulis, and C. yunnanensis). The study on pollen morphology showed that Chinese Curcuma species can be divided into two groups, Group I (pollen < 3 μm) including C. amarrisima, C. flaviflora, C. phaecaulis, C. sichuanensis, and C. wenyujin, and Group II (pollen > 3 μm) including C. aromatica, C. yunnanensis, and C. longa [16]. Therefore, the phylogeny of Chinese Curcuma are always controversial and ambiguous for poor diagnostic characters of morphology (aerial part, underground part, floral morphology, etc.) [17]. Cytological studies supported that some species in this genus were polyploid [18,19,20,21]. Chromosome number in Chinese Curcuma were studied to find out that C. kwangsiensis is 2n = 4x = 84 and C. flaviflora is 2n = 2x = 42, and the left of C. longa, C. aromatica, C. sichuanensis, C. elata, C. wenyujin, and C. phaeocaulis are triploid (2n = 3x = 63) [19]. Polyploidy is commonly distributed in Curcuma, e.g., C. longa 2n = 2x/3x/4x = 32/63/64 [19,22,23,24], C. kwangsiensis 2n = 4x = 64/84 [19,23], and C. aromatica 2n = 2x/3x = 42/86 [25,26], making it hard to distinguish them hardly based on ploidy and chromosome number [18,25,26].
In the past decades, molecular phylogeny of Curcuma around the world has been improved, while the phylogeny and divergence of Chinese Curcuma are still unclear [1,2,7]. Two groups, (Group I: C. chuanhuangjiang, C. aromatica, C. yunnanensis, C. kwangsiensis, and C. phaeocaulis; Group II: C. longa, C. sichuanensis, C. amarissima, and C. wenyujin), were divided by using six mtDNA genes [1]. The matK, rbcL, trnH-psbA, trnL-F, and ITS2 were applied to test barcodes within Curcuma collected from Myanmar and China, and without enough evidence to identify each species (no barcoding gaps found in four chloroplast regions and little gaps in ITS2) [7]. Based on chloroplast genome, a recent study showed that eight Curcuma species in China were divided into two groups (Group I including C. wenyujin, C. phaeocaulis, and C. aromatica, Group II including C. longa, C. yunnanensis, C. amarissima, and C. sichuanensis), and C. flaviflora is far away from the aforementioned seven species, clustered together with Zingiber spectablie (a kind of ginger) and Z. officinale [27]. In all, most DNA regions lack enough information on Chinese Curcuma species identification, particularly among related species, such as C. longa and C. sichuanensis, C. aromatica, and C. wenyujin. Therefore, new technologies need to be introduced to infer the phylogeny and divergence of Chinese Curcuma [3,28,29]. High-throughput and next-generation sequencing (NGS) can provide large amounts of genomic data and make great progress on phylogeny, divergence, and biogeography [30,31,32,33]. Reduced Representation Sequencing (RRS), one of the NGS approaches, can produce numerous sequence tags, and is successfully used to polymorphism detection in multi-species coalescent-based tree and to deal tricky plant’s phylogeny and biogeography [34,35,36,37,38,39,40].
The phylogeny and divergence of Chinese Curcuma are still under debate due to similar morphological characteristics, unique geographical and climate conditions of the QTP [5,28,41,42,43]. The rising and climatic fluctuation of Hengduan Mountain Range (presently affected by monsoons from both the Indian and Pacific oceans) and Yunnan–Guizhou Plateau (affected mainly by Pacific monsoons) were induced with rapid uplift of the Himalayas and the QTP [44,45]. In the Pliocene, the third intense uplift of QTP changed the geographical environment, climate, and species distribution/divergence in China and eastern Asia [9,10,46,47]. Curcuma in China are distributed in the QTP generally, such as Sichuan, Yunnan, Zhejiang, Guangdong, and Guangxi, even in Tibet without living individuals collected in recent years (herbarium presented: 02010668, 02010669, 00074714, 00074715, 01376872, 01376873, 01376874, and 01376875 in Herbarium, Institute of Botany, Chinese Academy of Sciences). The uplift of QTP might play a key role in Curcuma evolution and divergence in China.
In this study, RRS was utilized (1) to evaluate the phylogeny of Curcuma species in China and (2) to explore the impact on the evolution and divergence of Chinese Curcuma because of uplift of QTP.

2. Materials and Methods

2.1. Material and Sequencing

A total 60 specimens of eight Curcuma species were used in this study, and Hedychium coronarium (commonly known as white ginger) was used as outgroup (specimens information in Table 1). All specimens used in this study were identified by Prof. Ruiwu Yang with the Sichuan Agricultural University, and were cultivated at the Sichuan Agricultural University Farmland.
The genomic DNA isolation was carried out on fresh leaves by the CTAB method [48]. The RRS libraries for each sample were prepared using the protocol outline as previously described [49]. We used the restriction enzyme PstI (CTGCAG) to digest the extracted genomic DNA from each individual, and then ligated the resulting fragments to a barcode adaptor and a common adaptor with the correct sticky ends. Then, a Qiagen MinElute 96-well PCR purification kit was used in the clean-up step to clean up the products. After PCR, the PicoGreen and a qPCR machine were used to examine the quality of the PCR products. All individuals were pooled into a single RRS library. Sample sequencing was done on Illumina Hiseq PE150 sequencer in Genepioneer Biotechnologies Co. Ltd., Nanjing, China. Raw Reads of the founder lines were deposited in the National Center for Biotechnology Information (NCBI) BioProject ID: PRJNA557061.

2.2. Clustering

The software of pipeline ipyRAD 0.7.29 [50] was used to process the raw data from the Illumina FASTQ files. The pipeline is focusing on preparing RADseq type data for population level analyses [51]. Following seven sequential steps, the ipyRAD pipeline can obtain species or higher variation across clades in clustering and alignment method based on specific parameters in the ipyRAD documentation (https://github.com/dereneaton/ipyrad (accessed on 21 January 2019)). The ipyRAD standard parameter settings were as follows: Nucleotides with Phred scores of <20 were coded as unknown bases (N), and sequences with >5% N’s were thrown out. Sequences were clustered within individuals by 90% similarity via the uclust function in USEARCH [52]. Clusters of less than 10 sequences were discarded and the minimum number of individuals per cluster was set to 5. Heterozygous loci among more than two individuals were discarded. The remaining clusters were treated as loci and assembled into a phylogenetic matrix.

2.3. Concatenation-Based Species Tree Inference

The reduced-filtered SNPs were acquired from the filtered SNPs (QC) via quality control by using PLINK 1.9 with standard parameter settings. The standard parameter settings were as follows: Missingness per marker was set to 0.05. Minor allele frequency was set to 0.05. The model of substitution for data was run in MrModeltest [53] by the Akaike Information Criterion (AIC) and obtained the best model of GTR. The maximum likelihood (ML) was implemented under the GTR nucleotide substitution model in RAxML8.2.8 [54]. The maximum parsimony (MP) tree branch with booststrap support was done in the software PAUP * 4.0a134. Optimal MP trees were searched by a heuristic strategy with 1000 random sequence additions and TBR branch swapping. Bootstrap values were calculated using 1000 replicates, 10 random additions per replicate, and TBR branch swapping. Bayesian inference (BI) tree was carried out using MrBayes 3.1.2 [55]. Markov chain Monte Carlo (MCMC) searches were started from a random tree and run for 3,000,000 generations, where the topologies were sampled every 100 generations. Furthermore, 25% of our individuals (which the first 2500 trees) were discarded as burnin. The Bayesian posterior probabilities of the nodal supports were inferred and the 50% majority-rule consensus tree was constructed based on the rest of trees.

2.4. Population Structure and Divergence Time Inference

Principal component analysis (PCA) is a purely mathematical method that reflects the clustering between groups and is based on the degree of SNPs in different individuals by EIGENSOFT version 7.2.1 [56]. Population structure of 60 Curcuma individuals was obtained by ADMIXTURE 1.3.0 [57]. We predefined the ancestral proportions (K) from 3 to 12, and ran the cross-validation error (CV) procedure. The default settings and methods were used for other parameters.
On the basis of the high-quality SNPs, the BEAST version 2.5.0 was used to estimate Chinese Curcuma species divergence time, and the Bayesian tree was dated by setting divergence time between Hedychium coronarium and Curcuma as 42.4 Mya (37.4–47.4 Mya) [58]. The GTR model for nucleotide substitution and the “Bayesian skyline” tree prior model was confirmed with a standard normal prior. Substitution model and site heterogeneity model were used the optimal model based on the Bayesian analysis to select the model “relaxed clock” and MCMC runs 200 million generations, and every 1000 steps in the individuals were to ensure effective sample size (ESS) in each parameter greater than 100. The output file assessed convergence in Tracer 1.5. The phylogenetic tree used TreeAnnotator 1.5.3 to discard 25% as burnin. Finally, the divergence time was analyzed and obtained in FigTree 1.1.2 [59].

3. Results

3.1. Sequences Discovery and Characterization

A total of 53.15 Gb raw data were produced by RRS (Raw Reads in NCBI BioProject ID: PRJNA557061). Then, 437,061 unique RRS loci across all the individuals were revealed by using the denove clustering method in ipyRAD. The reduced-filtered SNPs (1295 bp) were obtained under quality control (QC) in filtered SNPs by using Plink 1.9 to analyze the phylogeny, evolution, and divergence.

3.2. Phylogenetic Analyses

The MP, ML, and BI trees were obtained by reduced-filtered SNPs with strong support values (Figure 1), and the same clustering results were recovered in PCA (Figure 2). The phylogeny shown respectively in MP, ML, and BI tree were consistent in this study.
In the three phylogenetic trees, all population specimens from the same species were clustered together firstly and named as a clade in this study, and the eight Curcuma species were divided into four groups (including eight clades) with robust support values: Group I (including C. wenyujin clade and C. aromatica clade) with MP BS = 75, ML BS = 99, and BI PP = 86; Group II (C. longa clade) with MP BS = 100, ML BS = 100, and BI PP = 100; Group III (including C. amarissima clade and C. sichuanensis clade) with MP BS = 90, ML BS = 99, and BI PP = 100; and Group IV (including C. kwangsiensis clade, C. yunnanensis clade, and C. phaeocaulis clade) with MP BS = 92, ML BS = 91, and BI PP = 100. C. longa formed a monophyletic branch with MP BS = 89, ML BS = 100, and BI PP = 99; C. wenyujin clade (MP BS = 100, ML BS = 100, and BI PP = 100) was sister to C. aromatica clade (MP BS = 100, ML BS = 100, and BI PP = 97); C. amarissima clade (MP BS = 100, ML BS = 100, and BI PP = 100) clustered with C. sichuanensis clade (MP BS = 100, ML BS = 100, and BI PP = 100); C. kwangsiensis clade, the first branch in Group IV, was sister to C. yunnanensis clade (MP BS = 97, ML BS = 100, and BI PP = 100) and C. phaeocaulis clade (MP BS = 100, ML BS = 100, and BI PP = 100).
To test the evolution of Chinese Curcuma, a Bayesian clustering algorithm with admixed models was used to estimate the ancestral proportions (K) for each specimen (Figure 3). Based on CV error, the K = 12 represented the best model for these 60 samples. When K = 3, the eight species belong to three gene pools (blue gene pools, green gene pools, and red gene pools). C. longa had independent gene pools (blue). The species of C. amarissima, C. sichuanensis, C. kwangsiensis, C. yunnanensis, and C. phaeocaulis shared green gene pools. The red gene pools were shared by C. wenyujin and C. aromatica; When K = 4 to 12, C. longa could be distinguished by constitution of gene pool, and Group I (C. wenyujin and C. aromatica) is close to Group IV (C. kwangsiensis, C. yunnanensis, and C. phaeocaulis).

3.3. Divergence Time Inference

Curcuma occurred in the Miocene (~7.45 Mya) in China (Figure 4). C. longa appeared around 6.43 Mya, the intraspecific of C. longa diversified at ~2.45 Mya from the late Pliocence to Quaternary. Group I (C. amarissima and C. sichuanensis) and II (C. kwangsiensis, C. yunnanensis and C. phaeocaulis) separated at ~4.83 Mya. The divergence in Group I, occurred at ~3.57 Mya in the Pliocence, and the intraspecific diversification of C. sichuanensis and C. amarissima were at ~1.89 Mya and ~0.67 Mya, respectively. In Group II, C. kwangsiensis occurred at ~3.74 Mya, C. yunnanensis and C. phaeocaulis emerged and separated ca 2.79 Mya, and the diversification within species of C. yunnanensis and C. phaeocaulis were at ~0.81 Mya and ~1.45 Mya. C. wenyujin and C. aromatica originated at ~6.08 Mya during the Miocene and Pliocence. The intraspecific diversification within C. wenyujin and C. aromatica were at ~1.17 Mya and ~0.56 Mya, respectively.

4. Discussion

4.1. Application of “Next-Generation” Sequencing in Estimating the Phylogeny and Biological Implication of Curcuma

The recent rapid speciation of species might lead to a mistake in inference by using a single phylogenetic tree, and this might be presented a poor fit in the multi-species coalescent supermatrix data and model [60,61]. A large amount of sequence data is ideal for establishing and reconstructing phylogenetic trees [5,28,41,62,63,64]. In this study, RRS was firstly used to produce big data to analyze the phylogeny and divergence of Chinese Curcuma. More loci and variable sites might improve the phylogeny reconstruction of Chinese Curcuma [5,41]. Compared to limited individuals and data size introduced in some previous studies, more individuals were involved to produce big data for mining more accurate information to recover a well-resolved phylogeny in this study [31].

4.2. Phylogeny Inference

The phylogeny of Curcuma species is difficult to resolve by depending on traditional approaches for their complicated origins (hybridization, introgression, and common appearance in species) [2,8,17]. The genetic relationship among Chinese Curcuma species is very close in this study, which is consistent with several previous studies [1,3,7].
Studies based on various morphological evidences reveal different results and lead to unreliable classifications of Curcuma: Based on pollen grains, Chen and Xia believed C. aromatica, C. yunnanensis, and C. longa had a close relationship, and the relationships among C. amarrisima, C. elata, C. flaviflora, C. phaecaulis, C. sichuanensis, and C. wenyujin were very close and hard to distinguish [16]. These species in Xiao et al. were delineated as Group I: C. longa, C. xanthorrhiza, and C. sichuanensis, Group II: C. kwangsiensis and C. exigua, Group III: C. wenyujin, C. aromatica, C. phaeocaulis C. zedoaria, and C. yunnanensis by using oil cells and vascular bundles [14]; and the morphology of leaves produced different results (Group I: C. longa, C. xanthorrhiza, C. wenyujin, and C. sichuanensis; Group II: C. kwangsiensis and C. exigua; and Group III: C. aromatica, C. chuanyujin, C. zedoaria, C. phaeocaulis, and C. yunnanensis) [15].
On the basis of molecular marker data (isoenzyme and DNA barcode), Deng et al. (2011a and 2011b) and Li et al. believed the relationship between C. longa and C. sichuanensis was very close, and supported that C. sichuanensis originated from the cultivated mutation of C. longa [1,3,65]. The genetic relationships among C. kwangsiensis, C. yunnanensis, C. aromatica, and C. phaeocaulis were poorly supported, and C. wenyujin clustered with C. longa, C. sichuanensis, and C. amarissima [5]. Chloroplast genomes study showed C. aromatica, C. wenyujin, and C. phaeocaulis have a close relationship and supported C. yunnanensis, C. amarissima, C. sichuanensis, and C. longa as a group [27]. In this study, the phylogeny of Chinese Curcuma is (((((C. Phaeocaulis, C. yunnanensis) C. kwangsiensis), (C. amarissima, C. sichuanensis)), C. longa), (C. wenyujin, C. aromatica)). C. longa is a single clade (MP BS = 100, ML BS = 100, and BI PP = 100), separated from C. sichuanensis clade, was sister to C. amarissima clade with strong support (MP BS = 90, ML BS = 99, and BI PP = 100), was inconsistent with the study of Deng et al. (C. sichuanensis clustered together with C. longa) [3]. The clade of C. wenyujin was close to C. aromatica clade [5] in Group I with MP BS = 75, ML BS = 99, and BI PP = 86. Based on RAMP and ISSR markers, C. phaeocaulis and C. yunnanensis had a close relationship [66,67]. In this study, C. kwangsiensis, C. yunnanensis, and C. phaeocaulis clustered together (Group IV) with robust support values (MP BS = 92, ML BS = 91, and BI PP = 100).

4.3. Divergence Time

The estimates of divergence indicated that the earliest appearance of Curcuma in China was in the Miocene. In the Miocene, large-scale orogenesis and geological events frequently emerged and influenced the speciation of plants living in QTP area during that times [68]. During the time, the drought climate (the Asian Monsoon) was related to intense uplift of QTP [69,70]. Curcuma lies dormant in winter, rhizomes fleshy with tuber-bearing roots, and blooms in the rainy season, which is similar to the drought-resistant plants to satisfy such drought climate [17]. The interspecific divergence of Chinese Curcuma occurred in ca. 4–2 Mya, which was coincided with the drought-resistant and deciduous plant sprang up and expanded rapidly in Hengduan Mountains ca. 4–2 Mya [71]. Chinese Curcuma expanded rapidly during this period, and the third intense uplift of QTP sped up their interspecific divergence.
In addition, hybridization and polyploidy play an important role on speciation and diversification [72,73]. According to previous studies on Curcuma, the distribution range overlapped, and the introgressive hybridization existed extensively [74]. Several Chinese Curcuma species were most likely to be of hybrid origin (C. aromatica and C. kwangsiensis) [8]. The cytomixis and chromosome doubling could promote the production of a large number of new phenotypes in a short time, and polyploids tend to be more adaptive than their parents [75,76,77]. Hybridization and polyploidization in plants are commonly distributed in the QTP regions, where the species overlap generally and the environment vary frequently, to satisfy the harsh environment of the QTP regions [78,79]. Chinese Curcuma species are confirmed to be polyploidy, which might help to adapt the harsh living environment at that time [27,74].

4.4. Future Directions

The phylogeny of major Chinese Curcuma species was improved in this study. Chinese Curcuma species are only a subset of Curcuma around the world. More species with population samples, as well as species from the genera of Hitchenia, Stahlianthus, and Smithatris, and more data should be involved to analyze the phylogeny, evolution, and diversification of Curcuma [6].
In addition, Curcuma has a complex evolutionary history; hence, more new methods such as ddRRS, ddRad, and transcriptome should be used to ensure the reliability of data for the phylogeny, evolution, and diversification reconstruction on such recent radiated genera [80].

5. Conclusions

The RRS was firstly involved to improve the phylogeny and divergence of Chinese Curcuma. The third intense uplift of QTP might speed up the interspecific divergence of Curcuma in China. Overall, this study provides valuable information on the origin of Chinese Curcuma.

Author Contributions

H.L. and R.Y. designed the experiments and analyzed the data. H.L. wrote the original manuscript. G.G. and J.D. collected the plant materials. K.X. and R.Y. identified the materials. L.Z., C.D., and H.W. assisted with manuscript preparation. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National modern agricultural industry technology system Sichuan innovation team (SCCXTD-2020-19), the Science and Technology Foundation of Guizhou Province (Qiankehejichu [2017]1138), and the Education Department of Guizhou Province for youth growth on science and technology (No. Qianjiaohe KY[2016](221)).

Data Availability Statement

These plant materials are required for the collection of plant individuals. The plant materials are maintained in accordance with the institutional guidelines of the College of Life Sciences, Sichuan Agricultural University, China. The Raw Reads are available in NCBI BioProject ID: PRJNA557061.

Acknowledgments

This manuscript has been released as a pre-print at https://www.researchsquare.com/article/rs-5880/v1 (accessed on 27 September 2019) [81]. We would like to thank Khawaja Shafique Ahamad (Department of Botany, University of Poonch Rawalakot, Azad Jammu, and Kashmir Pakistan) for improving the quality of the manuscript. We are grateful to Chuanbei Jiang (Genepioneer Biotechnologies Co. Ltd., Nanjing) for assistance in RRS library preparation. We also thank Zhimemg Wang (College of Life Science, Peking University, Beijing) for helping us run pyRAD and kindly providing additional help in assembly programs. My sincere gratitude also goes to Keyan Zhang (Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Yunnan) for valuable comments for this study.

Conflicts of Interest

The authors declare no conflict of interest.

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Forests 12 00520 g001 550
Figure 1. The ML, MP, and BI trees of Chinese Curcuma. The values (maximum likelihood bootstrap/maximum parsimony bootstrap/Bayesian support value) were on the species clade. The Group I consisted of C. wenyujin and C. aromatica; Group II consisted of C. longa; The Group III consisted of C. amrissima and C. sichuanensis; The Group IV consisted of C. kwangsiensis, C. yunnanensis, and C. phaeocaulis.
Figure 1. The ML, MP, and BI trees of Chinese Curcuma. The values (maximum likelihood bootstrap/maximum parsimony bootstrap/Bayesian support value) were on the species clade. The Group I consisted of C. wenyujin and C. aromatica; Group II consisted of C. longa; The Group III consisted of C. amrissima and C. sichuanensis; The Group IV consisted of C. kwangsiensis, C. yunnanensis, and C. phaeocaulis.
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Figure 2. The principle components analysis of Chinese Curcuma. Principal component analysis (PCA) of the 60 individuals and different species with different colors.
Figure 2. The principle components analysis of Chinese Curcuma. Principal component analysis (PCA) of the 60 individuals and different species with different colors.
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Figure 3. Population genetic structure of Chinese Curcuma. When the kinship (K) = 3–12, each vertical bar represented a Curcuma sample and different color represented different putative ancestral background in putative.
Figure 3. Population genetic structure of Chinese Curcuma. When the kinship (K) = 3–12, each vertical bar represented a Curcuma sample and different color represented different putative ancestral background in putative.
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Figure 4. Phylogeny tree resulting from the Bayesian inference and divergency time of Chinese Curcuma in BEAST (Divergence time/Bayesian support). Group I consisted of C. amarissima, C. sichuanensis, C. kwangsiensis, C. yunnanensis, and C. phaeocaulis. Group II was made up of C. kwangsiensis, C. yunnanensis, and C. phaeocaulis.
Figure 4. Phylogeny tree resulting from the Bayesian inference and divergency time of Chinese Curcuma in BEAST (Divergence time/Bayesian support). Group I consisted of C. amarissima, C. sichuanensis, C. kwangsiensis, C. yunnanensis, and C. phaeocaulis. Group II was made up of C. kwangsiensis, C. yunnanensis, and C. phaeocaulis.
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Table
Table 1. Sample of species, with source details and total loci after processing with ipyRAD, of the 61 individuals used in phylogenetic analyses. Each sample has a sample name, species name, collecting information, and loci information.
Table 1. Sample of species, with source details and total loci after processing with ipyRAD, of the 61 individuals used in phylogenetic analyses. Each sample has a sample name, species name, collecting information, and loci information.
SampleSpeciesSource DetailsTotal of Loci after Ipyrad
SC1Curcuma longaMuchuan, Sichuan26,977
SC3Curcuma longaFulu, Sichuan17,898
SC4Curcuma longaCuiping, Sichuan39,772
SC19Curcuma longaYibin, Sichuan30,974
SC18Curcuma longaQianwe, Sichuan26,311
GZ3Curcuma longaXingyi, Guizhou28,912
SC20Curcuma longaShuangliu, Sichuan32,886
YN11Curcuma longaMenga, Yuannan20,606
SC5Curcuma longaDayi, Sichuan34,053
SC21Curcuma sichuanensisMedicinal, Botanical Garden, Guangxi21,536
SC22Curcuma sichuanensisChongzhou, Sichuan33,411
YN29Curcuma sichuanensisYiwu, Yunnan19,503
SC2Curcuma sichuanensisSanjiang, Sichuan26,973
SC23Curcuma sichuanensisCuiping, Sichuan24,230
SC6Curcuma sichuanensisZiyang, Sichuan27,677
YN10Curcuma sichuanensisMenga, Yuannan38,515
SC24Curcuma sichuanensisChongzhou, Sichuan31,072
SC25Curcuma sichuanensisQianwei, Sichuan20,902
SC26Curcuma sichuanensisMuchuan, Sichuan21,588
SC11Curcuma sichuanensisWeiyuan, Sichuan28,768
SC8Curcuma sichuanensisGAP Base, Sichuan30,753
SC9Curcuma sichuanensisSanjiang, Sichuan28,621
SC10Curcuma sichuanensisChongzhou, Sichuan23,043
SC7Curcuma sichuanensisYibing, Sichuan28,068
YN15Curcuma sichuanensisMenglun, Yunnan13,089
GX9Curcuma sichuanensisMedicinal, Botanical Garden, Guangxi31,039
YN32Curcuma amarissimaMengkang, Yunnan16,865
YN7Curcuma amarissimaMenga, Yunnan17,222
YN8Curcuma amarissimaMenga, Yunnan17,923
GD4Curcuma yunnanensisHuaxian, Guangdong32,976
GD5Curcuma yunnanensisHuaxian, Guangdong24,819
GX10Curcuma phaeocaulisMedicinal, Botanical Garden, Guangxi23,145
YN27Curcuma phaeocaulisYiwu, Yunnan22,636
SC14Curcuma phaeocaulisShuangliu, Sichuan34,212
GX11Curcuma phaeocaulisMedicinal, Botanical Garden, Guangxi29,557
SC13Curcuma phaeocaulisJianwei, Sichuan25,208
GX4Curcuma phaeocaulisMedicinal, Botanical Garden, Guangxi35,825
GZ2Curcuma phaeocaulisAnlong, Guizhou31,112
YN13Curcuma phaeocaulisMenga, Yunnan14,425
YN6Curcuma phaeocaulisMengxing, Yunnan21,440
YN31Curcuma phaeocaulisMengkang, Yunnan21,642
SC15Curcuma phaeocaulisShuangliu, Sichuan13,089
YN21Curcuma phaeocaulisXishuangbanna, Yunnan16,744
SC12Curcuma phaeocaulisChongzhou, Sichuan18,289
YN19Curcuma phaeocaulisMenglun, Yunnan13,489
YN16Curcuma phaeocaulisDaluo, Yunnan26,946
YN17Curcuma phaeocaulisXishuangbanna, Yunnan23,180
YN28Curcuma phaeocaulisYiwu, Yunnan28,065
YN4Curcuma kwangsiensisMedicinal, Botanical Garden, Guangxi17,001
GX14Curcuma aromaticaMedicinal, Botanical Garden, Guangxi26,739
YN5Curcuma aromaticaMengkang, Yunnan24,435
SC16Curcuma aromaticaJianyang, Sichuan26,817
YN12Curcuma aromaticaYiwu, Yunnan18,289
ZJ1Curcuma wenyujinTaoshan, Zhejiang, cultivated23,259
ZJ2Curcuma wenyujinMeiyu, Zhejiang27,430
GX12Curcuma wenyujinMedicinal, Botanical Garden, Guangxi18,273
GD2Curcuma wenyujinSanshui, Guangdong17,080
GX1Curcuma wenyujinMedicinal, Botanical Garden, Guangxi22,593
GX3Curcuma wenyujinMedicinal, Botanical Garden, Guangxi28,736
GD3Curcuma wenyujinHuaxian, Guangdong35,582
JHHedychium coronariumYaan, Sichuan13,383
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Liang, H.; Deng, J.; Gao, G.; Ding, C.; Zhang, L.; Xu, K.; Wang, H.; Yang, R. Inferring the Phylogeny and Divergence of Chinese Curcuma (Zingiberaceae) in the Hengduan Mountains of the Qinghai–Tibet Plateau by Reduced Representation Sequencing. Forests 2021, 12, 520. https://0-doi-org.brum.beds.ac.uk/10.3390/f12050520

AMA Style

Liang H, Deng J, Gao G, Ding C, Zhang L, Xu K, Wang H, Yang R. Inferring the Phylogeny and Divergence of Chinese Curcuma (Zingiberaceae) in the Hengduan Mountains of the Qinghai–Tibet Plateau by Reduced Representation Sequencing. Forests. 2021; 12(5):520. https://0-doi-org.brum.beds.ac.uk/10.3390/f12050520

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Liang, Heng, Jiabin Deng, Gang Gao, Chunbang Ding, Li Zhang, Ke Xu, Hong Wang, and Ruiwu Yang. 2021. "Inferring the Phylogeny and Divergence of Chinese Curcuma (Zingiberaceae) in the Hengduan Mountains of the Qinghai–Tibet Plateau by Reduced Representation Sequencing" Forests 12, no. 5: 520. https://0-doi-org.brum.beds.ac.uk/10.3390/f12050520

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