Confirmation of Tuntun Angin (Elaeocarpus floribundus) Taxonomic Status Using matK and ITS Sequences

Tuntun angin is one of important floodplain plants in and around Kajuik Lake located in Riau Province, Indonesia. Morphological identification shows that the scientific name of this plant is Elaeocarpus floribundus. The study aimed to confirm the taxonomic status of tuntun angin using matK and nuclear intergenic spacer (ITS) sequences. The methods included fresh leaf DNA isolation, polymerase chain reaction, electrophoresis, sequencing, and data analysis using BLASTn program and MEGA software version 6.06 programs. The results showed that the matK sequence (519 bp) of tuntun angin had highest similarity to E. floribundus matK sequence that was available in GenBank. It was supported by the high max score (937), low Evalue (0.0), high identity value (100%), and high query cover (100%). However, the ITS sequence of tuntun angin did not show similarity to E. floribundus ITS sequence because there was no database of the sequence in GenBank. This study was able to confirm the taxonomic status of tuntun angin as E. floribundus using matK sequence and also showed that morphological and molecular identification techniques were complementary to each other. Moreover, this study enriched the DNA sequence database of E. floribundus in GenBank which will be useful for this species’ molecular identification.

Such plants play important roles in maintaining the quality and the quantity of river water, resisting erosion, supplying nutrient for fish and other animals in river, and providing sites of spawning and nursery for fish. Fish migrates from the river body to the floodplain lake when the water in river overflows in rainy season (Zeug & Winemiler, 2008). The fish look for roots or other parts of plant which are submerged to put their eggs and hide from predators (Pease et al., 2006). One of fish that migrated to Kajuik Lake is selais fish which is a Riau endemic fish (Elvyra & Yus, 2012). Not only fish, others animals like birds, crabs, shrimps, and insects also live in floodplain ecosystem (Banner & MacKenzie, 1998;Price & Lovett, 2002).
Tuntun angin grows in and around Kajuik Lake has been identified as Elaeocarpus floribundus based on morphological characters (Elvyra & Yus, 2012). It is a kind of tree that can grow in tropical floodplain forest (Brahma et al., 2013;Rahman & Vacik, 2015) and a member of Elaeocarpaceae. The fruit and the leaf of this plant are rich in vitamin and antioxidant which are useful for treatments of various diseases (Khomdram et al., 2014;Rahman & Vacik, 2015). Sitakunda people in India use this plant as medicine, fodder, and firewood (Rahman & Vacik, 2015). E. floribundus could be found among others in Pangandaran, Jawa Barat, Indonesia (Rosleine & Suzuki, 2012), Manipur, India (Khomdram et al., 2014), and Singapore (Erickson et al., 2014).
Before DNA barcoding technique developed rapidly, plant characterization and identification process was commonly conducted based on morphological characters (Cheong & Ranghoo-Sanmukhiya, 2013;Snow et al., 2016;Roslim et al., 2016b) because they were easier and cheaper. Nevertheless, not all plant species can be characterized and or identified based on morphological characters due to some characters which the ex-
The fresh leaves were used to extract total DNA using DNeasy plant mini kit with the procedure according to manufacturing instruction (Qiagen). The quality and the quantity of total DNA were predicted using electrophoresis on 1.2% agarose gel in 1X TBE buffer (Tris-Borate-EDTA pH 8.0) at 65 volt for 30 minutes.
A primer pairs of matK_Ef_F/matK_ Ef_R was used to amplify matK sequence and ITS_Ef_F/ITS_Ef_R to amplify ITS sequence. Amplification was carried out in a reaction volume 50 μl containing 1X buffer PCR (plus Mg 2+ ) (Thermo Scientific), 0.1 mM dNTPs (Thermo Scientific), 2.4 μM forward primer, 2.4 μM reverse primer, 2 U Dream Taq DNA polymerase (Thermo Scientific), and 1 ng total DNA, and aquabidestilata (Porebski et al., 1997). The PCR conditions were as follows: pre-PCR at 95°C for 5 minutes followed by 35 cycles consisting of denaturation at 95°C for 45 seconds, primer annealing for 45 seconds at 47°C for ITS primer pair and 48°C for matK primer pair, and primer elongation or extension at 72°C for 1 minute 30 seconds. After that, post PCR was performed at 72°C for 10 minutes.
The PCR products were then migrated on 1.2% agarose gel in 1X TBE buffer, at 65 volt for one hour. The gel was then stained with 5 μg/ml of ethidium bromide, visualized on UV lamp (Wi-seUv WUV-M20, Daihan Scientific), and recorded.
The PCR products were then sent to PT Genetika Science Indonesia in Jakarta to be purified and sequenced at 1 st Base Malaysia. The DNA sequencing is a technique to determine the order of nucleotides precisely within a DNA molecule and the sequencing was performed in two direction using PCR primers.
The DNA sequences were then analyzed and aligned using BLASTn (Basic Local Alignment Search Tool) (Altschul et al., 1997) and MEGA software versi 6.06.

Tuntun angin description
Tuntun angin is an annual tree and has brown hard lignosus wood with grey spots. Tree height is about 14-16 meter and can reach 49 meter. The stem is round with rough surface. The green leaves are arranged spirally, 3-5 cm in size, ovalis or ellipticus, acuminate tip, obtusus leaf base, and serrated leaf edges. The leaf vein is pinnate. The branches are monopodial. The white colour flowers form inflorescentia racemus with the bowl-like base and the triangular narrow sepals. The stamens are more than 20. Ovarium is superior. The flower is 5-7.5 cm long. The flower is bisexual, i.e. the stamen and the pistile are in a flower.The fruit is 2.5-3.2 cm in size, drupe in shape, narrowed at the base, bluntly pointed at the apex, light green in young fruit, and yellowish green in mature fruit. The flesh of fruit is acidic (Figure 1 and Figure 2). Local people around Kajuik Lake use the fruit as bait for fishing. The extract of the fruits is good for skin and used as an anti-aging herb (Oyen, 2011). The leaves are used to treat rheumatic, dysentry, and diarrhea diseases, while an infusion of the bark and leaves is used as a mouthwash (Uddin, 2014). Total DNA molecules and DNA fragment of matK and ITS The total DNA molecules obtained in this study were intact, not smeared, and enough for PCR requirement. The PCR products of matK and ITS fragments ( Figure 3) were thick enough for sequencing requirement. The thick and single band of PCR products will produce good sequences (Nugraha et al., 2014;Roslim et al., 2016a;Roslim, 2016).

Analysis of matK and ITS sequences of tuntun angin
The matK sequence obtained in this study is 519 bp in size (GenBank accession number: KX365743) while ITS region 438 bp (GenBank accession number: KX365744) (Figure 4). Alignment analysis of both sequences using BLASTn showed that the matK sequence of tuntun angin was 100% similar to matK sequence of E. floribundus availabled in GenBank database (Table 1). However, the ITS sequence of tuntun angin was not similar to ITS sequence of E. floribundus because there was no database of the sequence in GenBank. This study showed that the determination of a plant species' name using the molecular identification technique must consider some BLAST parameters, for example, high max score, high total score, high query cover, low E-value (0.0), and 100% identity (Madden, 2013;Roslim, 2016;Roslim et al., 2016a). If the identity value does not reach 100%, then the species' name or taxonomic status of sample observed can not be determined. In addition, this study also showed that the molecular identification technique should be supported by the availability and the abundance of DNA sequences database (Will & Rubinoff, 2004;DeSalle, 2006;Hajibabaei et al., 2007;Roslim, 2016), due to the fact that DNA sequence of organism identified must be aligned and compared to the DNA sequence database which was generated from known species (Hajibabaei et al., 2007). Until now (updated in October 1 st , 2016), the DNA sequences database of Elaeocarpus which are available in GenBank have been very abundant, i.e. 12406 data. The number of E. floribundus DNA sequences is 5 data which include rbcL (3 data) and matK (2 data).
The matK gene is a gene with high varia-tion compared to others in a plant chloroplast genome and can be used for identification and verification of plant species. The matK gene encodes maturase-like protein that helps splicing its own and other genes in chloroplast (Lambowitz & Zimmerly, 2004;Zoschke et al., 2010). The matK gene had been used for molecular identification and phylogenetic analysis of legum (Legumenusae) (Wojciechowski et al., 2004), angiosperm (Yu et al., 2011), and some spesies in genus Vicia (Raveendar et al., 2015). In this study, the use of matK as DNA barcode proved to be able to verify the taxonomic status of tuntun angin which has previously been identified morphologically as E. floribundus by Elvyra & Yus (2012) at Universitas Andalas (UNAND), Padang. This verification is helpful to improve public knowledge that the matK sequence could indeed be used for plant molecular identification. The advantages of the plant molecular identifi-cation using DNA barcoding technique are as follows: (1) the plant molecular identification could be performed by anyone, either taxonomist or non taxonomist; (2) unlike identification using morphological characters, the DNA sequence or the DNA barcode is not influenced by the environment; and (3) the molecular identification is easy and fast if the DNA sequence database of target species is provided. Until now, the ITS database of E. floribundus in GenBank has not been available. Therefore, this research has a novelty, i.e. enhanced the DNA sequence database of E. floribundus in public database.
Similar research has been conducted by Kolondam et al. (2012) to confirm the taxonomic status of payus limondok orchid (Phaius tancarvilleae) using DNA barcode of rbcL and matK. The result showed that matK sequence is more capable to be a DNA barcode for payus limondok orchid than rbcL sequence. Bangol et al. (2014) also

Phylogenetic analysis
The DNA sequences of tuntun angin and some species in genus Elaeocarpus were then converted into distance matrix (Table 2) and dendrogram ( Figure 5) and they will describe similarity or relationship between accessions. The farther the genetic distance between accessions showed the differences of a large number of nucleotide and far relationship between the two. The genetic distance between tuntun angin and E. floribundus was 0.0 and it means that they are very similar based on the matK sequence. They were also very similar based on morphological characters. Furthermore, phylogenetic analysis showed that tuntun angin was clustered in one group and had closer relationship with E. floribundus rather than others. This result was in accordance with the BLASTn analysis results and it strengthens the taxonomic status of tuntun angin as E. floribundus.
Since the first time it was developed, the DNA barcode aimed to help species identification using standardized short DNA sequence with minimum length which was deposited in major public database, such as the National Center for Biotechnology Information's GenBank (NCBI, GenBank), the European Molecular Biology Laboratory (EMBL), and the Barcode of Life Data System, and had other corroborating data about the species. Today, the existence of the DNA barcode in public database has made a possibility for some people to acces the Linnaean taxonomic system, like conservationists, agencies deal with control of pests, food safety, and invasive species, geneticists, etc. (Hebert & Gregory, 2005). The universal and standardized DNA barcodes for plant molecular identification are rbcL and matK (CBOL Plant Working Group, 2009), while ITS region is the alternative DNA sequence (Bolson et al., 2015). Inspite of the combinati-on of DNA barcodes, also the plant molecular identification must be complemented or combined to other information about the species identified, for examples, morphological characters, developmental stages, and ecology (Smith et al., 2005). It because, morphological and molecular identification techniques were complementary to each other and the morphological identification technique cannot be subtituted by molecular identification technique (Will & Rubinoff, 2004). Furthermore, domain of taxonomy is the discovery of species and domain of DNA barcoding is identification of species which depends on established taxonomy. Therefore, molecular identification technique still needs other corroborating data (DeSalle, 2006).

CONCLUSION
This study was able to confirm the taxonomic status of tuntun angin as E. floribundus using matK sequence and to enrich database of E. floribundus ITS sequence in GenBank. The success to confirm the taxonomic status of tuntun angin was supported by the availibility of matK sequence of E. floribundus in GenBank. In addition, morphological and molecular identification techniques were complementary to each other. Further experiments to enrich the DNA sequence database of E. floribundus therefore it supports the plant molecular identification is recommended.