Alpinia Roxb. is the largest and most widespread genus in Zingiberaceae with ca. 250 species, which is distributed in tropical and subtropical Asia, Australia and Pacific Islands1,2. Several species of Alpinia are important for their ornamental, medicine and economic values. Additionally, Alpinia also plays an important ecological role in the understory vegetation of tropical and subtropical forests1. The genus is characterized by their horizontal or pendulous, broad labellum and filament usually shorter than corolla or labellum.
The phylogenetic relationship among Alpinia species has not been clearly understood yet due to missing data and poor sampling. Rangsiruji et al.3 recognized nine clades of Alpinia based on a dataset from 47 species of Alpinia. They demonstrated significant statistical support for several monophyletic groups of species of Alpinia and suggested that this genus may not be monophyletic.
Kress et al.4 addressed the relationships among genera in the Zingiberaceae by using data from both ITS and matK regions. The study showed well-resolved phylogenetic relationships among the genera and provided a new classification of the Zingiberaceae with four subfamilies and four tribes. Kress et al.4 recognized four major clades of Alpinia, demonstrated that a number of the larger genera in the family such as Amomum, Alpinia, Etlingera, Boesenbergia and Curcuma could be non-monophyletic as well as suggested that more extensive sampling is necessary for these genera.
Kress et al.1 conducted the molecular phylogeny of Alpinia based on 72 taxa of Alpinia and using two molecular makers matK and ITS. The results of this study showed that Alpinia is polyphyletic, six clades within Alpinia were recognized. A new classification of the tribe Alpinieae was also provided by this study. However, the monophyly and phylogenetic position of a number of Alpinia species have not yet been established.
Alpinia coriandriodora D. Fang was described by Fang5 and recognized as an endemic species in Guangxi, China. The A. coriandriodora species is of cultural importance, which was used as food and local medicines for hundreds of years. Vu et al.2 reported A. coriandriodora from Vietnam as a new record for the country. The authors suggested that the morphology of A. coriandriodora is similar to A. bambusifolia and only differs in having an elliptic-lanceolate leaf blade, light yellow labellum with red-brown stripes and triangular anther crest. However, the phylogenetic relationship of A. coriandriodora within Alpinia has not been investigated by using molecular data. Furthermore, the status of A. coriandriodora species is endangered and needs to be preserved under the exploitation of people. The present study aims to conduct molecular analyses to understand the phylogenetic position of A. coriandriodora within Alpinia as well as the relationship among other Alpinia species and interpret evolution of the key morphological characters and provide some recommendations for the conservation of the species.
MATERIALS AND METHODS
Study area: The study was carried out at National Key Laboratory of Gen Technology-Institute of Biotechnology, Herbarium (HN) of Institute of Ecology and Biological Resources under the Vietnam Academy of Science and Technology, Faculty of Natural Sciences-Hung Vuong University, Department of Botany, Ha Noi Pedagogical University No. 2 from May, 2019-July, 2020.
Taxon sampling, DNA extraction, amplification and sequencing: Total genomic DNA was isolated from silica gel-dried leaves of the collections GD01LT, GD02LT, GD03LT, GD01XD, GD02XD, GD03XD. The Vouchers of the collections were deposited in the Herbarium (HN) Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology. Total 12 new sequences were generated for two markers matK and ITS. The primers and protocol for PCR and sequencing in this study followed by Kress et al.1.
It was successfully extracted total DNA and conducted a PCR reaction for 06 samples with the two markers matK and ITS. The total DNA without color and soluted in TE. The PCR products have high quality and clear in an electrophoresis gel, sequencing was performed using the primers designed by Kress et al.1. The sequences were aligned in Geneious v.8.0.56.
Molecular phylogenetic analysis: We added the new sequences into a dataset with a global sampling scheme of Alpinia from previous study1. The combined dataset was partitioned into two subsets corresponding to two gene regions and was analyzed with the Maximum Likelihood (ML)7 and Bayesian Inference (BI) methods8. The ML analysis was conducted in RAxML 8.2.107, applying 1,000 bootstrap replicates with the GTR+I+G substitution model selected in jModeltest 2.1.69. The Bayesian analysis was conducted in MrBayes 3.2.68 as implemented on the CIPRES Science Gateway Portal10 based on the same model as in the ML analysis. The Markov chain Monte Carlo (MCMC) algorithm was run for 5,000,000 generations with a total of four chains, starting from a random tree and trees were sampled every 1000 generations.
List of the 7 morphological characters and character states scored for the morphological character evolution analysis of Alpinia
Morphological matrix of Alpinia and outgroups
“?” represents missing data; 01: Leaf tomentum, 02: Inflorescence type, 03: Inflorescence rachis tomentum, 04: Ovary tomentum, 05: Filament, 06: Bract, 07: Staminodes
The program Tracer v.1.611 was used to check that Effective Sample Sizes (ESSs) were attained for all relevant parameters assessed (>200). With the first 25% of sampled generations discarded as burn-in, the 50% majority-rule consensus tree and Bayesian Posterior Probabilities (PP) were obtained using the remaining trees.
Morphological character evolution: We selected 23 species of Alpinia and outgroups for the reconstruction of ancestral characters, all selected species represented for all the sections and clades from the results of Kress et al.1 and this study. Seven morphological characters concerning leaf tomentum, inflorescence type, inflorescence rachis tomentum, ovary tomentum, filament, bract and staminodes were selected for ancestral character optimization (Table 1).
The morphological description of Alpinia used several morphological characters, however, the seven above characters have usually been highlighted in the description and classification of Zingiberaceae and Alpinia2,4,12, thus we corroborated each character stated by examining specimens and consulting published works2,12. The morphological dataset was presented in Table 2. Additionally, it reduced the taxa sampling in the molecular dataset as in the morphological dataset but including Zingiber officinale and Curcuma longa as out groups and two individuals of Alpinia coriandriodora to construct a molecular simplified tree for morphological character evolution analysis.
The evolutionary history of each of the seven characters was traced over the Bayesian 50% majority-rule tree with 24 taxa sampled, using ML approach available in Mesquite v.2.7513. We used the Markov k-state one-parameter model and the option “Trace Character History” to reconstruct each character.
Phylogenetic relationship: Twelve newly generated sequences in this study were submitted to GenBank and the voucher information and GenBank accession numbers of the sequences were presented in Table 3.
|Fig. S1:||Maximum Likelihood (ML) tree derived from analysis of matK gene
Voucher information and GenBank accession numbers for DNA sequences generated in this study
The single data sets of matK and ITS have 3023 and 729 pb respectively, the combined molecular dataset with 3752 (pb) aligned positions across all taxa.
The molecular results of single makers showed lower resolution of relationships within Alpinia than the combined dataset (Fig. 1, S1 and S2). Thus, we used the combined dataset (matK and ITS) to analyze the phylogenetic relationship within Alpinia and systematic position of A. coriandriodora.
|Fig. S2:||Maximum Likelihood (ML) tree derived from analysis of ITS gene
The results from the combined dataset by ML and BI analyses are highly congruent and few differences have low support. Thus, we combined results in ML tree, the detailed phylogenetic relationships within Alpinia are presented in Fig. 1a. The molecular analysis strongly supported Alpinia as a non-monophyletic group (BS: 100%, PP: 1.0) (Fig. 1). Six clades were recognized within Alpinia. A. coriandriodora was supported as a member of Alpinia and placed into clade IV (Fig. 1b).
Character evolution: The reconstruction of the ancestral character state for Alpinia based on the ML method was shown in Fig. 2-5. For characters 1, 3, 4, “glabrous” was inferred to be the ancestral state in the genus Alpinia (Fig. 2, 3).
Reconstructions of characters 1, 3, 4 indicated that the presence of hairs on leaf, inflorescence and ovary was derived more than once in Alpinia clade (Fig. 2, 3). While, for character 5, the presence of filament is not unique in Alpinia and also occurs in Zingiber, Curcuma and Siliquamomum (Fig. 5). Whereas reconstructions of characters 6 indicated that the presence of bracts was inferred as ancestral character, most members of Alpinia have bracts and only few species evolved without bracts (Fig. 5). Reconstructions of characters 2 and 7 are quite complex (Fig. 4), all three types of inflorescence were evolved multiple times in Alpinia, that are the similarity of absent staminodes in character 7. Additionally, some approaches for ex situ conservation of A. coriandriodora were shown in Fig. 6.
Maximum Likelihood (ML) tree derived from analysis of combined dataset matK and ITS, (a) A phylogram overview and (b) Clade including Alpinia coriandriodora
ML bootstrap values and Posterior Probabilities (PP) of the BI analysis are presented above the branches.“–” indicates the support values less than 50%
Results of the present study indicated that suggested Alpinia coriandriodora is close to southern China Alpinia. Morphological character significance suggested that Alpinia adapt to the pollination by insects and need an approach to protect and increase the population. Our molecular results are congruent with Kress et al.1 and Alpinia coriandriodora was strongly supported as a member of clade IV. Fang5 suggested that A. coriandriodora is similar to A. warburgii K. Schum. and A. stachyodes Hance. A. coriandriodora and A. warburgii are similar by their caudate leaf apex, appendage on connective but A. coriandriodora differs from A. warburgii by the spike inflorescence, ternate flowers, presence of bracts and bracteoles, smaller calyx, short corolla lobes with pubescent hairs. While, A. coriandriodora and A. stachyoides are similar by their spike inflorescence and ternate flowers but A. coriandriodora differs from A. stachyodes by petiolate leaf, caudate leaf apex, non-obvious bracts and bracteoles, larger flowers, sinuate margin of labellum, absence of appendage on connective. The two species A. warburgii and A. stachyodes were recognized as members of clade IV by both Kress et al.1 and the present study (Fig. 1, S1 and S2). Moreover, our molecular results indicated that A. coriandriodora was supported as sister to A. stachyodes (Fig. 1, S1 and S2), while A. warburgii was placed far from the clade including A. coriandriodora and A. stachyodes.
Additionally, Vu et al.2 suggested that Alpinia coriandriodora is similar to A. bambusifolia, the endemic species from Guangxi and Guizhou, China.
Character optimization for leaf tomentum (character 1) inferred on a Bayesian majority-rule tree for 24 taxa, based on the maximum likelihood method
However, A. coriandriodora can be easily distinguished with A. bambusifolia by elliptic-lanceolate leaf blade (vs. narrowly lanceolate in A. bambusifolia), yellow calyx from base to middle and purple-red from the middle to the apex (vs. pale purple-red in A. bambusifolia), yellow labellum with red-brown stripes (vs. white with red stripes in A. bambusifolia), triangular and entire anther crest (vs. absent in A. bambusifolia) and globose fruits (vs. cylindrical in A. bambusifolia).
Character optimization for inflorescence rachis tomentum (character 3) and Ovary tomentum (character 4) inferred on a Bayesian majority-rule tree for 24 taxa, based on the maximum likelihood method
However, the molecular data of A. bambusifolia is not available in both previous studies and this study, thus the future study including data of A. bambusifolia should be conducted to determine the phylogenetic relationship of these related species within Alpinia.
The phylogenetic results reported here are an advancement over previous analyses1,4 in terms of taxon and gene sampling. Alpinia coriandriodora is closely related to southern Chinese Alpinia species (Guangdong, Guangxi, Hainan and Yunnan) such as A. japonica, A. coriacea, A. warburgii, A. stachyodes and A. guangdongensis with strong support (BS: 100%, PP: 1.0) (Fig. 1). Moreover, the molecular results supported A. coriandriodora as sister to a subclade within clade IV (including some species above plus others). Furthermore, Alpinia was distributed in whole Vietnam including subtropical and tropical regions2,12,14. Luu et al.14 and Vu et al.2 recorded 34 species of Alpinia for flora of Vietnam. The molecular analyses indicated that 19 of 34 species of Vietnamese Alpinia were placed in clade IV (Fig. 1, S1 and S2). Thus present result suggested that A. coriandriodora also is closely related to Vietnamese Alpinia.
Character optimization for inflorescence type (character 2) and staminodes (character 7) inferred on a Bayesian majority-rule tree for 24 taxa, based on the maximum likelihood method
In fact, Alpinia coriandriodora shares some similar morphological characters with other Alpinia in southern China and northern Vietnam such as inflorescence a panicle, raceme or spike, bracteoles flat or concave, bracts present. Thus, the molecular result of this study supported the treatments of Fang5 and Vu et al.2. Moreover, the results suggested that A. coriandriodora shares the ancestor with southern Chinese Alpinia. Thus future study on divergence time of Alpinia is necessary to clarify the evolutionary history of this genus.
Additionally, phylogenetic results strongly supported Vu et al.2 to recognized the former Zingiber member “Gùng đá” to Alpinia coriandriodora for Vietnamese flora.
The result of character optimizations suggested that the tomentum of leaf (character 1) evolved twice in Alpinia (Fig. 2) but appears in most species from Southeast Asia including A. coriandriodora. While, tomentum of inflorescence and ovary (characters 3, 4) seems to have appeared multiple times in Alpinia (Fig. 3). Character 1 thus can be used to distinguish the Southeast Asia Alpinia including A. coriandriodora from other members. The inflorescence type has been used in both the intergeneric and infrageneric classification of Alpinia and A. coriandriodora1,2.
To evaluate its taxonomic significance within the genus Alpinia, we optimized the inflorescence type with three character states onto a Bayesian majority-rule tree (Fig. 4) which was not presented in previous studies.
Character optimization for filament (character 5) and bract (character 6) inferred on a Bayesian majority-rule tree for 24 taxa, based on the maximum likelihood method
Our reconstruction result suggested that the state of panicle is ancestral in Alpinia. However, raceme and spike have evolved several times within Alpinia. A. coriandriodora shares different evolutionary history based on our character reconstruction to most Southeast Asian Alpinia (Fig. 4). Additionally, our result indicated that the presence of staminodes is an ancestral character of Alpinia and some groups and species have evolved to reduct this part.
Alpinia often has beautiful and colorful flowers12, however, stamens have short or very short filament or sometimes even absent. Our reconstruction of character 5 indicated that the absence of filament is an ancestral character of Alpinia but the presence of filament evolved multiple times and especially in Southeast Asian members of Alpinia (Fig. 5). This character state appears to be an adaptation to the pollination by insects. In addition, almost all the species of Alpinia have bracts, however, some species have evolved to reduct this part (Fig. 5).
The results from molecular analysis of the endangered species Alpinia coriandriodora provided valuable insight for the conservation and management of this species. Our results also suggest that A. coriandriodora might have great evolutionary potentiality and can adapt to varied environmental conditions2,15.
(a) Habitat with fruits, (b) Root, stem and bud, (c) Young tree from micropropagation of Alpinia coriandriodora and (d) Micropropagation of Alpinia coriandriodora in laboratory
||Scale bars are 5 cm
Based on the results of this study and investigations with local people during our field trips which were inferred that genetic factors could not be a reason for the population decline of A. coriandriodora but due to human-related factors, such as habitat degradation and fragmentation and overexploitation and land reclamation, may have contributed to the endangered status of the species.
A. coriandriodora is perennial herbs with small population size and mostly distributed in the border between Vietnam and China (Bac Kan and Guangxi provinces)2,12. Nybom16 suggested that the species with small population size and narrow geographic ranges might face a growing risk of genetic drift and inbreeding recession. Therefore, to protect and increase viable populations of A. coriandriodora, we propose both in situ and ex situ conservation and restoration strategy. In situ conservation, expanding the existing protected areas and therefore the reclamation and restoration of habitats destroyed by local people for farmland expansion are most important15, which may maintain an appropriate, effective population size of A. coriandriodora. For ex situ conservation, seeds or seedlings should be collected for germplasm storage and to maximize the protection of existing genetic diversity. In addition, artificial breeding using biotechnology (splitting buds, micropropagation) should be encouraged for regression and population reconstruction (Fig. 6). Finally, strengthening public outreach and conservation education for people should be enabled to protect the species and ecological system. Our sampling has confirmed identification and systematic position of A. coriandriodora. The present study has clarified phylogeny of A. coriandriodora, however, future work with comprehensive taxon sampling should focus on the phylogeny of the tribe Alpinieae and evolutionary history of Alpinia.
In this study, our molecular analyses supported the recognization of Alpinia coriandriodora for Vietnamese flora. This species has a close genetic relationship with some other Alipinia members that share the same distribution in southern China and northern Vietnam. Character optimizations suggest that the presence of filament could be an adaptation to the pollination by insects for species of Alpinia. Some reasonable conservation strategies are proposed to protect the species.
The present study shows the molecular phylogenetic relationship of A. coriandriodora within Alpinia. Character optimizations indicate evolutionary of morphology and adaptation to the pollination by insects for species of Alpinia. This study provides conservation strategies to protect the species including maintenance of the plant’s natural habitats, seeds or seedlings collection for germplasm storage and artificial breeding using biotechnology.
We acknowledge the Hung Vuong University by funded this study with the grant number (Project grant No. 16/2020/HĐKH-HV20.16) and Vietnam National Foundation for Science and Technology Development (NAFOSTED) with the grant number 106-NN.03-2015.47.