Aquilaria, a plant of significant economic and medicinal value, has been a focal point for researchers worldwide aiming to safeguard its genetic resources and establish a scientific foundation for sustainable development and utilization [25, 35, 36]. This global effort has resulted in extensive investigations into the diversity of Aquilaria germplasm resources and the population genetic structure [37–39]. This study contributes valuable insights into the distribution of Aquilaria species across different countries, as outlined in Table 1. Notably, diverse species of Aquilaria coexist in several countries, including the Philippines, Indonesia, Malaysia, and China. The presence of four Aquilaria species in China, notably A. agallocha and A. grandiflora, holds particular significance, given their relative unfamiliarity to Chinese medical practitioners. The observed trend indicates an escalating diversity of Aquilaria species from mainland China to Southeast Asia. Morphological research emerges as a pivotal tool for swiftly assessing the genetic landscape through morphological traits, offering a precise analysis of variation. This method stands out as the most concise and direct approach for detecting genetic diversity, playing a crucial role in both interspecific and intraspecific classification [40, 41]. In this study, the analysis of specimen features allowed for the identification of 14 characteristic Aquilaria species, including five main cultivated species for agarwood production (Fig. 1 and Figure S1). Our study notes that traditional taxonomy indicates A. beccariana belongs to the Malaysian pedigree rather than the Chinese pedigree. Additionally, A. filaria and A. crassna, originating from different lineages, were found to cluster together within the same group. Notably, A. yunnanensis, A. sinensis, and A. beccariana exhibited remarkable similarities in their leaf characteristics. The research presents the conservative distribution information of the five main cultivated Aquilaria species based on the collected specimens. Among them, A. sinensis stands out with its significant quantitative advantage in mainland China. These findings emphasize the importance of understanding the taxonomy and diversity within Aquilaria species.
With the rapid development of molecular biology and genetics research, the research on the genetic diversity of Aquilaria has gone from the morphological level to the molecular level. In the last decade, the application of DNA barcode technology has emerged as a robust method for identifying Chinese herbal medicines. Numerous laboratories have demonstrated its effectiveness in rapidly and reliably identifying these medicines, transcending their diverse appearances [42–46]. The operational procedure typically involves DNA extraction, sequence amplification using universal primers, sequencing, and subsequent database comparison. It is crucial to highlight that the accuracy of identification results is intricately tied to the quality of the sequence database [47, 48]. However, amidst the success, several scientific oversights have surfaced, with a pivotal concern being the efficient and expeditious acquisition of a comprehensive genetic background of a species. This study focuses on investigating the evolutionary trajectory of Aquilaria species through molecular evolution analysis and DNA barcoding. Four specific segments derived from the chloroplast genome, namely ITS, matK, rbcL, and trnL-trnF, were selected as recommended molecular sequences for Aquilaria classification. These sequences were sourced from various Aquilaria species available in the NCBI database and analyzed to assess species genotyping. The findings reveal that A. sinensis is the species of highest research concern, followed by A. crassna, A. malaccensis, A. hirta, and A. subintegra. These five focal species represent the cultivated Aquilaria species found in diverse countries. The importance of studying these particular species is highlighted due to their significant presence and potential economic value. The ITS sequence emerges as the most widely used target among plant taxonomists, reflecting its extensive utilization in Aquilaria species identification. Additionally, the matK, rbcL, and trnL-trnF sequences have also been employed to varying extents in the analysis of 12 species (Table 2). However, it is crucial to note the generally limited number of sequences representing each species, which can sometimes be as low as one. This limitation raises concerns about the accuracy of species information within experimental samples or discrepancies in published data. The examination of haplotypes and SNP% reveals that certain genotypes pose challenges in species differentiation when using ITS, rbcL, and trnL-trnF sequences. The relationship between these highly polymorphic sequences remains unclear, warranting further investigation into potential errors or inconsistencies in species information. Comparative analysis of different sequences based on genetic distances between species, interspecies, and intraspecific distances, highlights the subpar discrimination results of ITS, rbcL, and trnL-trnF sequences. In contrast, the matK sequence shows greater promise in differentiating species within the Aquilaria genus (Fig. 2). The study introduces an innovative approach for evaluating sequence capabilities in species identification through equivalent comparisons of interspecies and intraspecific distances. This methodology provides insights into the performance of different molecular markers. The comparison between matK and rbcL reveals that while they exhibit similar interspecies distances, the intraspecific distance for matK surpasses that of rbcL (Fig. 3). This indicates that matK molecular markers are more effective in distinguishing between different Aquilaria species. The special molecular identification technologies developed for Aquilaria and agarwood products have provided valuable insights into the genetic dynamics within the genus Aquilaria.
As we delve further into the intricacies of molecular taxonomy, it becomes evident that the reliance on a single sequence, such as matK, may have limitations. Various studies have underscored the importance of adopting a multifaceted strategy for accurate species identification [49–53]. Our results demonstrate that a multifaceted approach, beyond reliance on the matK sequence alone, is necessary for strict species identification. The investigation of the trnL-trnF sequence revealed limited polymorphic sites, highlighting the need for further research in this area. Our analysis identified eight stable polymorphic loci within the matK sequence, with three key loci (+ 249, + 435, and + 684) that can be utilized to identify medicinal agarwood in Chinese medicine. Specifically, the thymine at locus + 684 and the guanine at locus + 435 serve as crucial markers for authentic medicinal agarwood. Locus + 249 can also serve as an auxiliary marker for Chinese agarwood identification, as the presence of cytosine at this locus indicates a Chinese mainland pedigree of Aquilaria. Therefore, specific amplification of the target polymorphic regions within the Aquilaria genus, utilizing the mentioned polymorphic loci on the matK sequence derived from agarwood, holds significant practical value. Our results highlight the straightforward identification of Chinese agarwood (A. sinensis) based on distinctive herbaceous characteristics. Further analysis of 36 commodity sources established a robust correlation between three specific loci (+ 684, + 249, & +435) and Aquilaria species in China. PCR results revealed a distinct molecular band pattern, with imported agarwood presenting a 600 bp length band and Chinese agarwood exhibiting a band around 300 bp (Fig. 4D). This emphasizes the utility of targeted PCR sequencing for accurate identification of legally recognized medicinal materials derived from agarwood. The incorporation of variations in 7 sites within the matK sequences allowed for the classification of all 36 samples into 3 distinct pedigrees, aligning with the primary distribution patterns of these species and their respective standard sequences (Figure S2). The clustering analysis has successfully categorized agarwood samples from both Indonesia and Cambodia, enriching our nuanced understanding of their origins. This meticulous approach not only reaffirms the initial classification but also underscores the efficacy of integrating multiple loci for a thorough assessment of agarwood pedigrees and origins. Our findings also reveal a distinct division into two categories among agarwood samples from China, necessitating a reevaluation of previously obtained specimen distribution information, leading to the recognition of the presence of A. agalloca in the Chinese mainland. The integration of matK + trnL-trnF facilitates a stringent differentiation of agarwood products, exhibiting a meaningful categorization into six branches: Vietnam, Cambodia A, Indonesia, Cambodia B, Chinese A, and Chinese B (Fig. 5). This intricate classification suggests a substantial diversity within agarwood products, potentially indicative of variations in origin or other influencing factors. Our results provide valuable insights into the molecular identification technologies for Aquilaria and agarwood products, highlighting the need for a multifaceted approach, specific amplification of target polymorphic regions, and the incorporation of multiple loci for a thorough assessment of pedigree and origin.
As we navigate the complexities of Aquilaria genetics and taxonomy, it is essential to contextualize our findings within the broader body of research in this field. Several studies have underscored the importance of integrating molecular data with ecological and geographical information to elucidate patterns of species divergence and evolution [54–56]. Additionally, recent advancements in next-generation sequencing technologies have opened new avenues for exploring genetic diversity and population structure within Aquilaria species [12, 17, 57–60]. Our molecular clock analyses yielded significant findings related to the divergence time among different haplotypes within the genus. The phylogenetic trees constructed using 13 haplotypes representing 10 species within Aquilaria revealed distinct placements of three haplotypes of A. sinensis on different branches, while A. yunnanensis formed an independent branch adjacent to A. sinensis (Figure S3). Interestingly, A. agallochum and A. rugosa showed a close relationship to A. sinensis, belonging to the same lineage. Additionally, the Median-Joining network map provided a visual representation of the haplotypes, indicating the existence of four distinct pedigrees: the Chinese pedigree (C1&C2), the Indonesian pedigree (B), and the Indochina pedigree (A), corresponding to A. sinensis, A. malaccensis, and A. cumingiana, respectively. Furthermore, our study explored the evolutionary history and geographic origins of agarwood producers, including Phaleria and Aquilaria (Figure S4). By examining the limited fossil record, we estimated that the divergence time between Phaleria and Aquilaria dates back approximately 60.7 Ma, representing the earliest emergence of agarwood producers. We also identified a divergence event within Aquilaria around 14.65 Ma, during the late Miocene epoch. A. hirta emerged as the oldest member of Aquilaria approximately 6.78 Ma ago, while A. crassna (approximately 3.45 Ma) played a crucial role in species diversification within Asian tropical rainforests. Contrary to previous hypotheses, our analysis disproved the notion of Aquilaria originating in India and instead suggested the independent evolution of a tropical rainforest lineage (C2) in China, distinct from pedigrees in the Indochina peninsula (B) and Indonesia (A). The presence of A. rugosa (1.25 Ma) indicated its exclusive distribution in China and the Indochina peninsula (Fig. 6A). Additionally, our findings challenged previous assertions regarding the representative species in the Malay pedigree, G. walla, dating it back to 5.75 Ma. We found notable morphological similarities between G. walla and A. malaccensis, supporting the proposition to classify Gyrinops within the genus Aquilaria. In summary, our study not only provides insights into the evolutionary timeline and geographic origins of Aquilaria species but also challenges prevailing assumptions regarding species classification and geographic distribution. These findings contribute to a better understanding of the genetic background and evolutionary significance of Aquilaria, which can inform conservation efforts and the sustainable management of agarwood resources.