Dna Barcoding of Dillenia Philippinensis Rolfe. (Katmon) and Dillenia Luzoniensis (S. Vidal) Merr. (Malakatmon) From Selected Barangays in Pola, Oriental Mindoro and Quezon City, Ncr, Philippines

Date of Award

5-2022

Document Type

Thesis

Degree Name

Master of Science in Biology

First Advisor

Vivian A. Panes, PhD

Abstract

The concept of DNA barcoding has achieved widespread acceptance as a new and creative means of species identification in the scientific community. Barcoding is a DNA-based identification approach that uses a short segment of a species' genome. The procedure of species identification gives accurate and trustworthy species identification findings because of the availability of numerous bioinformatics tools. Plants barcoding community have agreed on and suggested plastid markers (rbcL, matK) and nuclear markers (ITS) as standard barcodes. This innovative method, however, should not be considered a replacement for Linnaean taxonomy; rather, it should be considered a supplement to Linnaean taxonomy to expedite the species identification process. The study was conducted to determine the identity of Dillenia philippinensis and Dillenia luzoniensis collected from various barangays in the Municipality of Pola, Oriental Mindoro, and Ateneo de Manila University campus.

Dillenia philippinensis Rolfe, locally known as "Katmon" and Dillenia luzoniensis (S. Vidall) Merr. known as “Malakatmon” are endemic plant in the Philippines from the family Dilleniaceae. However, there is currently a scarcity of scholarly material on D. philippinensis and D. luzoniensis barcode sequences. As such, this scarcity is a limitation in the identification and authentication of these species as well as the conservation efforts as it faces a near-threatened status in the wild. Hence, to address this gap, this study was carried out as a groundbreaking research project for the purposes of providing barcode sequence to these taxa of plants. The result of this study showed that genomic DNA extraction for the rbcL, matK, and ITS was 100%

successful as confirmed by agarose gel electrophoresis result. Extracted DNA was also amplified and sequenced using Sanger sequencing, which resulted in 100% amplification and 100% sequencing success. The matK DNA amplicon recorded the highest DNA size at approximately 800 bp with a sequence length ranging from 753 bp to 764 bp, followed by ITS at approximately 750 bp with a sequence length ranging from 659 bp to 786 bp and, lastly, the rbcL DNA amplicon at approximately 550 bp with a sequence length ranging from 553 bp to 560 bp. These molecular sizes are within the range of the expected genomic DNA size and sequencing lengths of all the plant samples. Moreover, results of the Basic Local Alignment Search Tool (BLASTn) demonstrated a sequence identity of 97.38% to 100% with D. philippinensis reference sequences in the GenBank library, with query coverage ranging from 71% to 100% across all the DNA markers used in the study. On the other hand, result of BLASTn for D. luzoniensis shows sequence multiple species hits to different species in the GenBank specifically species of D. excelsa, D. tetrapetala and D. philippinensis. This is due to the fact that there are no available deposited barcode sequences for D. luzoniensis on the GenBank. Furthermore, character-based analysis (Maximum Likelihood) with the Tamura 3- Parameter Model supported by 1000 bootstrap replications determines the confidence estimate of the trees. The rooted phylogenetic tree of rbcL accessions shows that samples of D. philippinensis and D. luzoniensis from collection sites in Pola, Oriental Mindoro, and sites in Quenzon City were grouped into the same clade, showing their relatedness at the genus level. However, this node bifurcates to form two separate species, supported with 65% bootstrap value for the D. philippinensis and a 66% bootstrap value for the D. luzoniensis. This result confirms that D. luzoniensis and D. philippinensis are not related at the species level but related at the genus level. This

information demonstrates the efficacy of the rbcL marker in providing the molecular data for the identification of D. philippinensis as well as D. luzoniensis or other endemic species under Dilleniaceae family. A high-resolution phylogenetic tree based on the matK sequence, on the other hand, revealed that D. philippinensis accessions from Pola Oriental and collections sites in Quezon City were grouped into the same clade with bootstrap supporting values of 98%, indicating their species-level relatedness, while D. luzoniensis accessions were grouped into another clade with 99% bootstrap support. For the phylogenetic analysis using ITS sequences, the accessions of D. philippinensis from Pola, Oriental Mindoro, and sites in Quezon City are grouped into the same clade with bootstrap support of 100% indicating their high confidence of relatedness at the species level. This study suggests that rbcL are efficient in showing the relatedness between the taxa being examined. On the other hand, matK and ITS marker shows the efficacy in distinguishing closely related species of D. philippinensis and D. luzoniensis. The estimation of evolutionary divergence between all the sequences, results showed less than 1% sequence divergence, rbcL discriminated 100% sequence divergence, matK discriminated 73.63%, and 44.87% for ITS. In between 1% to 5% sequence divergence criterion, rbcL discriminated 26.37%, and ITS discriminated 26.92%. Lastly, for more than 5 % sequence divergence, only ITS has successfully discriminated a value of 28.21 %. This study suggests that matK and ITS are more efficient than rbcL in discriminating sequence divergence to this closely related taxon. Nevertheless, rbcL result shows that species of D. philippinensis and D. luzoniensis are closely related taxa provided by low sequence divergence. DNA barcoding was used to successfully identify D. philippinensis and D. luzoniensis gathered from several locations. This molecular data from barcode analysis, will serve as a means of identification for plants that have a limited number or source from the wild. Specially both D. philippinensis and D. luzoniensis are endemic and under threatened and vulnerable categories. Aside from that DNA barcoding information can also be applied as a tool for species authentication particularly when samples have polymorphic characteristics or when phenology is seasonal. Moreover, barcoding can be the identification source of information that can be used to promote sustainable usage and protection for D. philippinensis and D. luzoniensis it faces near threatened and vulnerable status here in the country.

Link to Full Text

Share

COinS