Biosystematics of Alpinia zerumbet Clade in West Malesia.

Abstract

Alpinia Roxb. is a genus of the family Zingiberaceae and tribe Alpinieae. Alpinia classification based on morphological characteristics is not congruent with molecular classification. Incongruence in this genus (including the Alpinia zerumbet clade) is thought to occur because the rate of evolution at the molecular level is not accompanied by morphological changes or changes that do not occur simultaneously. The evolutionary process also causes many morphological variations in one member of this clade, namely Alpinia malaccensis, a species that has two varieties. The taxonomic status of the two varieties is debated as to whether they should be treated as the same or different species. Another problem is harvesting the essential oil of A. malaccensis rhizomes from the forest directly, without cultivation. It is a concern that the existence of this species will decrease. Therefore, several approach studies need to be carried out to create reference data for A. malaccensis conservation and clarify the status of the two varieties and support the classification of the genus Alpinia for the better. The purpose of this study was to analyze the diversity and distribution of members of Alpinia zerumbet clade in West Malesia, analyze genetic variation, predict habitat suitability and essential oil content of A. malaccensis, and phylogenetic analysis of Alpinia based on the plastid genome. The research method for analyzing the diversity and distribution of members of the Alpinia zerumbet clade consisted of sample collection, observation of morphological characteristics, and recording of environmental data. The description of each species and the key to determining the species are constructed. The method for analyzing genetic diversity consisted of DNA isolation from A. malaccensis leaves, DNA amplification using Inter Simple Sequence Repeat (ISSR) marker, electrophoresed, and visualization. DNA band data was used to determine genetic diversity, population structure, and genetic relationships. In the predictions of habitat suitability, the coordinate points of each A. malaccensis accession were determined based on herbarium data and field observations, the environmental variables were taken from Bioclim2. Both data types were analyzed using the MaxEnt software to determine the most influential environmental variables and model the prediction of habitat suitability. The content of volatile compounds in the two varieties of A. malaccensis was taken from rhizomes and prepared for GC-MS analysis. The GC-MS results data were used for profiling the types of compounds produced and their concentrations as well as for grouping volatile compounds. Alpinia phylogenetic analysis was carried out based on the plastid genome. The phylogeny analysis procedure consists of several steps, DNA isolation and purification, DNA sequencing, plastid genome rearrangement, and construction of an Alpinia phylogenetic tree using the Maximum Parsimony method. The Alpinia zerumbet clade in West Malesia consists of 17 species. They share several characteristics: i.e. absent or caducous secondary bracts, small or reduced lateral staminodes, and often having a petaloid and showy labellum. The highest diversity of this clade is the Borneo regions (8 species), while the lowest diversity is Java (2 species). The members are distributed at an elevation of 2 to 1600 m above sea level (m asl). Most species of Alpinia zerumbet clade are found in primary forest and secondary forest (Pinus or Agathis forest). The genetic diversity of eleven A. malaccensis populations in Java Island was amplified using 12 ISSR primers. The parameters of genetic diversity, Shannon information index, and the percentage of polymorphic loci were low for each population, but moderate in total populations. The coefficient of genetic differentiation and molecular variance among the population are higher than that within a population, which indicates a significant genetic variance among eleven populations of A. malaccensis. UPGMA cluster analysis, STRUCTURE, and Principal Component Analysis divided all accessions into three groups. Group I is distinguished from groups II and III based on differences in varieties, while groups II and III are distinguished based on their distribution location in Java. Group I is composed of populations of var. malaccensis, while groups II and III were composed of var. nobilis. The results of the analysis indicated that ISSR markers can be used to distinguish A. malaccensis varieties. The habitat suitability prediction of A. malaccensis using the Maximum Entropy (MaxEnt) program which is integrated with DIVA-GIS result in a MaxEnt model and environmental factors that most influence habitat suitability prediction of A. malaccensis. The MaxEnt model is excellent and accurate, with an Area Under Curve value of 0.954. The environmental factors that most influences the modeling suitability habitat of A. malaccensis are the precipitation of the warmest quarter, minimum temperature of the coldest month, and mean temperature of the driest quarter. A model of A. malaccensis habitat suitability in West Malesia was obtained with the most appropriate locations at several areas in western Sumatra (Bukit Barisan mountains range) and also western and central Java. Analysis of essential compounds of two varieties of A. malaccensis (var. malaccensis and var. nobilis) based on GC-MS instrument showed that the A. malaccensis rhizomes contained 39 compounds and were dominated by terpenes. The compound found in the entire population of A. malaccensis is methyl cinnamate. This compound that has the greatest concentration is in the population of Ujung Kulon National Park, Banten. This study did not find typical compounds that represent the entire population of each variety. Therefore, the type of essential oil in this species cannot be used to distinguish the two varieties. The plastome of A. malaccensis var. nobilis (162,432 bp) is smaller than A. ligulata (163,073 bp). The number of copies of the rps12 and psbB genes and the presence of the orf138 gene can be distinguishing characters of the two taxa above. The phylogenetic results show that the phylogenetic tree construction is similar to that of the previous studies and Alpinia zerumbet clade has a monophyletic branch, while the genus Alpinia Roxb. has polyphyletic branches. Therefore, it is necessary to review the Alpinia genus to determine a better classification for this genus.