Genome Size, Molecular Phylogeny, and Evolutionary History of the Tribe Aquilarieae (Thymelaeaceae), the Natural Source of Agarwood

Abstract

The tribe Aquilarieae of the family Thymelaeaceae consists of two genera, Aquilaria and Gyrinops, with a total of 30 species, distributed from northeast India, through southeast Asia and the south of China, to Papua New Guinea. They are an important botanical resource for fragrant agarwood, a prized product derived from injured or infected stems of these species. The aim of this study was to estimate the genome size of selected Aquilaria species and comprehend the evolutionary history of Aquilarieae speciation through molecular phylogeny. Five non-coding chloroplast DNA regions and a nuclear region were sequenced from 12 Aquilaria and three Gyrinops species. Phylogenetic trees constructed using combined chloroplast DNA sequences revealed relationships of the studied 15 members in Aquilarieae, while nuclear ribosomal DNA internal transcribed spacer (ITS) sequences showed a paraphyletic relationship between Aquilaria species from Indochina and Malesian. We exposed, for the first time, the estimated divergence time for Aquilarieae speciation, which was speculated to happen during the Miocene Epoch. The ancestral split and biogeographic pattern of studied species were discussed. Results showed no large variation in the 2C-values for the five Aquilaria species (1.35-2.23 pg). Further investigation into the genome size may provide additional information regarding ancestral traits and its evolution history.

Keywords: Aquilaria; Gyrinops; ITS gene; chloroplast genes; flow cytometry.

FIGURE 1

Fluorescence intensity histogram peaks showing the number of nuclei per channel as a function of relative fluorescence obtained after flow cytometric analysis of propidium iodide-stained nuclei. (A) Aquilaria hirta, (B) Aquilaria malaccensis, (C) Aquilaria microcarpa, (D) Aquilaria sinensis, (E) Aquilaria subintegra, and (F) Elaeis guineensis, which acted as an external reference standard.

FIGURE 2

Maximum likelihood tree of 15 species from the Aquilarieae tribe based on (A) a combined data set of five chloroplast loci (matK, rbcL, trnL intron, trnL-trnF, and psbC-trnS), and (B) the nuclear region DNA internal transcribed spacer (ITS) region. Gonystylus bancanus, Phaleria macrocarpa, and Wikstroemia ridleyi served as outgroups. Bootstrap values for a majority of divergences are >50%, as indicated on the corresponding nodes.

FIGURE 3

Divergence time estimation for species in the tribe Aquilarieae. Bayesian consensus tree inferred from the combined sequences of chloroplast non-coding genes matK and rbcL, chloroplast gene intron trnL, and chloroplast intergenic spacer region trnL-trnF and psbC-trnS from 15 species in the Aquilarieae tribe. Gonystylus bancanus, Phaleria macrocarpa, and Wikstroemia ridleyi were used as outgroups. The red dot on the node represent estimated divergence time used to calibrate the analysis. The scales below the phylogenetic tree indicate the estimated divergence time in million years ago (Ma), which happened over several epochs. (A, Aquilaria; Gy, Gyrinops; Go, Gonystylus; P, Phaleria; W, Wikstroemia).