Malays. Appl. Biol. (2018) 47(3): 39–48

GENETIC VARIATION OF SAGO PALM (Metroxylon sagu Rottb.) SAMPLES FROM , USING INTERNAL TRANSCRIBED SPACER ANALYSIS

ENGUITO, R.Z.C. and NOVERO, A.U.*

Department of Biological Sciences and Environmental Studies, College of Science and Mathematics, University of the Philippines Mindanao *E-mail: [email protected]

Accepted 6 May 2018, Published online 30 June 2018

ABSTRACT

The sago palm is an emerging important food and energy source in the Philippines. There is limited information on the nature of its distribution in the Philippines. This study presents a phylogenetic tree hinting of the possible influence of geographical distribution to Internal Transcribed Spacer (ITS) polymorphism in sago palm. Internal Transcribed Spacer (ITS) Analysis was employed to assess intra-individual heterogeneity and genetic divergence within the sago morphotypes. The variables under observation were armature type (spiny or non-spiny), maturity, and environment, and combination of these variables yielded eight morphotype samples. Deduced gene sequences of the samples showed a relatively low mean GC% of 50.5%, compared to expected 65.8% from the Metroxylon salomonense (Warb.) Becc. outgroup. This phenomenon was attributed to possible cytosine methylation that led to mutability of GC base pair. Furthermore, branching pattern of ITS sequence divergence for both Neighbour-Joining and Maximum Likelihood methods was observed to be influenced by geographic distribution as non-spiny samples collected from Prosperidad clustered into a monophyletic group with the M. salomonense (Warb.) Becc. Outgroup from Papua New Guinea while the spiny samples from City diverged into a different group. The sago palm spine is a morphological trait that could be used for the differentiation of sago morphotypes within a small population of limited diversity. This trait is of particular interest in breeding due to its possible influence on important agronomic traits such as yield and resistance to pests and diseases.

Key words: Sago palm, plant spine, ITS analysis, phylogenetic analysis, Metroxylon sagu Rottb.

INTRODUCTION various plant species such as cucumber fruits, wherein spine formation is controlled by two genes Sago palm (Metroxylon sagu Rottb.), a member of (Hutchins, 1940). In a study by Park et al. (2004), the Arecaceae Family is only harvested from wild different genes were observed to be associated with stands in the Philippines. Although well-known and spine formation in Daucus carota hydrolase cultivated in Malaysia and Indonesia for its many associated with the cell wall, tail-fiber assembly uses, sago palm is relatively unheard of in the protein, transcriptional regulatory protein, herberine country. Ehara (2018) commented that sago palm’s bridge enzyme, S-adenosyl methionine synthase increasing potential for use as carbohydrate source (SAMS), transketolase and phenylalanyl tRNA may be inevitable as the palm can grow in poor and synthetase beta chain. Among the genes identified, lowly utilized lands. SAMS would be pivotal to the methylation and M. sagu Rottb. is a potential starch-resource expression of genes, since it acts as methyl donor alternative that would address the continuous that controls gene expression. growth of population and exhaustion of known Spiny and spineless sago palms were originally starch sources. However, armature (presence of classified as different species of Metroxylon, namely spines) poses a problem for the optimum utilization Metroxylon rumphii and Metroxylon sagu, of sago starch. Various studies had identified respectively (Beccari, 1918). Random amplified specific genes that contribute to spine formation in DNA polymorphism (RAPD) analysis performed by Ehara (2009) among sago palm collected from * To whom correspondence should be addressed. Indonesia, Malaysia and Mindanao, Philippines, 40 GENETIC VARIATION OF SAGO PALM (Metroxylon sagu Rottb.) USING ITS ANALYSIS showed that spiny and spineless sago belong to one among multiple successions of Vigna radiata, and cluster. Zymogram patterns of sago palm in Thailand further analysis showed that variants are hetero- showed no significant difference, concluding that duplex ITS fragments. Wild and cultivated forms of sago palm cultivars are identical (Boonsermsuk et four Vigna species revealed substantial intra-species al., 1995). Furthermore, Amplified Fragment Length divergence from the ten Vigna species under study. Polymorphism (AFLP) analysis employed by Kjær However, among the four species, intra-individual et al. (2004), suggested that variation in armature ITS variation was observed in only one species. The does not correlate with the underlying genetic heterogeneity was further assessed with reverse variation in sago palm. These suggest that M. sagu transcriptase-PCR (RT-PCR) analysis, which showed Rottb. belongs to only one species, wherein that the regulation of loci is controlled by epigenetic spineless palms can produce spiny seedlings gene silencing since rDNA repeat units in certain (Rauwerdink, 1986; Ehara et al., 1998) or vice versa intra-individual ITS variants were transcriptionally (Jong, 1995). High performance liquid chromato- inactive. Intra-species hybridization and slow graphy was also reported in the analysis of DNA ‘molecular drive’, in relation to incomplete methylation, wherein aside from the type of homogenization, are responsible for the observed armature, the condition of the environment and phenomenon (Saini et al., 2008). maturity of the palm was also considered (Novero ITS analysis can also be used in investigating et al., 2012). The results showed significant intraspecies variability among different variants and differences in DNA methylation of spiny and non- cultivars. Freitas et al. (2013) studied genetic spiny sago palms. However, the results did not diversity of Vigna unguiculata (cowpea) cultivars conform to the generally observed inverse using ITS. Significant variability among cultivars relationship of DNA methylation and gene was determined upon amplification and sequencing expression, wherein transcriptionally silent genes of the ITS1 and ITS2 regions. are highly methylated than active genes (Singal ITS analysis was employed to investigate the & Ginder 1999). genetic structure of Acrocomea aculeata (macaw ITS (internal transcribed spacer) refers to a piece palm; Family Arecaceae) groups by analyzing of non-coding RNA situated between structural individual polymorphism of different ITS regions. ribosomal RNAs (rRNA), which are found as Using five ITS primer sets, DNA from 42 leaf samples repeating units that are arranged in tandem arrays confirmed genetic diversity among and within (Liu et al., 2009) and are not included into mature populations (Vieira et al., 2017). ribosomes (Baldwin et al., 1995). Baker et al. (2000) utilized both cpDNA rps16 intron and nrDNA ITS region on the phylogenetic MATERIALS & METHODS reconstruction of the species under subfamily Calamoideae. M. sagu Rottb. was included among Twenty-four sago palm leaf samples, three from each the species subjected to ITS analysis and the data location for the two sago morphotypes (spiny and can be utilized to serve as theoretical results, non- spiny) at different stages of maturity the exploiting it as basis for experimental comparison. juvenile (~2 months old) and mature (~5 years old) The 22 genera investigated indicated that concerted (Table 1), were collected from del Sur, evolution had not effectively homogenized the ITS Tagum, and Sta Cruz, Davao del repeats since high levels of intra-individual Sur (Figure 1). Prosperidad, , polymorphism was observed. However, no 8.6024°N, 125.8716°E, is a landlocked municipality significant emphasis was given to the polymorphism situated near the Agusan River. It has Type II climate in sago palm armature. Thus, the present study with no dry season. Wet season is characterized by utilized the internal transcribed spacer region to heavy precipitation. Tagum, Davao del Norte, investigate intra-individual heterogeneity of sago 7.4482°N, 125.8094°E, also experiences significant palm morphotypes. rainfall. Its “dry’ months are also beset with rainfall. Fama et al. (2000) reported high level of Sta. Cruz, , 6.8813°N, 125.3686°E, intra-individual ITS1 polymorphism in Caulerpa is characterized by wet (May to October) and dry racemosa complex. ITS analysis was also employed (November to April) seasons. in the study of intraspecies variation in Dioscorea DNA was extracted using a modified protocol spp. (Munirah et al., 2014) and Fructus evodiae (Liu of Angeles et al. (2005). The reagents for DNA et al., 2009). However results showed no distinct extraction included 200 μL 20% SDS, and 0.1 g of variation of species under consideration. PVP. A freshly prepared 0.4% mercapthoethanol was Saini et al. (2008) showed the extent of intra- added to the solution. About 1 g of the leaf sample individual and intra-species variation among the and the freshly prepared extraction solution were Asiatic Vigna species (subgenus Ceratotropis) using added to a pre-chilled mortar and pestle. The nuclear rDNA ITS. ITS polymorphism was observed homogenized mixture was then transferred to 1.5- GENETIC VARIATION OF SAGO PALM (Metroxylon sagu Rottb.) USING ITS ANALYSIS 41

Table 1. DNA samples of different sago palm morphotypes of different maturity stages as obtained from different environments

Designated No. of Armature type Environment Maturity Sampling Location Code samples

Non-Spiny sago Dry environment Juvenile NsDJ Prosperidad, Agusan del Sur 3 palm Mature NsDM Prosperidad, Agusan del Sur 3 Wet environment Juvenile NsWJ Prosperidad, Agusan del Sur 3 Mature NsWM Prosperidad, Agusan del Sur 3

Spiny sago palm Dry environment Juvenile SDJ La Filipina, Tagum City 3 Mature SDM La Filipina, Tagum City 3 Wet environment Juvenile SWJ La Filipina, Tagum City 3 Mature SWM Sta. Cruz, Davao del Sur 3

Fig. 1. Sago palm sampling sites: Prosperidad, Agusan del Sur, 8.6024°N, 125.8716°E; Tagum City, Davao del Norte, 7.4482°N, 125.8094°E; and, Sta. Cruz Davao del Sur, 6.8813°N, 125.3686°E. mL microcentrifuge tubes, vortexed for 5 s, and Amplification of the internal transcribed spacer incubated in a dry bath at 65°C for 1 h. The samples (ITS) region using polymerase chain reaction was were then centrifuged 17,000 x g for 15 min at 4°C. carried out using the protocol of Baker et al. (2000). After centrifugation, 500 μL of the supernatant was ITS1 (forward): 5’-TCCGTAGGTGAACCTGCGG-3’ distributed to 1.5mL micro-centrifuge tubes. In each and ITS4 (reverse): 5’-TCCTCCGCTTATTGA- microcentrifuge tube, 123 μL of 3M sodium acetate TATGC-3’ primer pairs (White et al., 1990) were and 500 μL of cold absolute isopropanol were used to amplify the ITS1, ITS2 and 5.8S regions of added. The samples were then stored at -20°C for the rRNA and parts of the 18S and 26S rRNA. A 24 h. After incubation, samples were centrifuged at 25 μL reaction mixture was prepared containing 10,000 x g at 4°C for 15 min. The supernatant was 1.5 mM MgCl2, 0.3 μM of each primer, 2.5 units of carefully decanted to isolate the precipitate. DNA Taq polymerase, 0.1 mM each dNTP and 25 ng DNA pellets were washed with 70% ethanol and air-dried template. ITS fragments were amplified using the for 20 min. The pellets were re-dissolved with the following parameters: initial denaturation step of addition of 50 μL of sterile ultrapure water. Quality 97°C for 1 min, 1 cycle; denaturation at 97°C for 1 of samples was assessed visually in gel electro- min, 1 cycle; annealing temperature gradient from phoresis and quantified using a spectrophotometer. 50–54°C for 1 min to set optimum annealing 42 GENETIC VARIATION OF SAGO PALM (Metroxylon sagu Rottb.) USING ITS ANALYSIS temperature; and extension at 72°C for 2 min, RESULTS having 27–30 cycles. Final extension step at 72°C for 7 min for 1 cycle. The quality of the PCR The eight amplified ITS fragments varied in terms amplicons was checked using agarose gel electro- size and band intensity, with NsDJ, NsDM, and phoresis in 1.0% agarose in 1X SB buffer at 50V for NsWM samples having the highest band intensity 100 min. Bands were was then viewed under UV (Figure 2). Furthermore, the NsDM sample exhibited transilluminator. PCR products were sequenced by two amplicons, indicating the presence of hetero- the Philippine Genome Center. duplex ITS variant. Multiple sequence alignment was done using ClustalW on the obtained contig sequences and Analysis of Aligned Contig Sequences sequences of Metroxylon sagu Rottb. (AJ242106.1) Manual trimming of the sequences by locating and Metroxylon salomonense (Warb.) Becc. ITS1 and ITS4 primer locations yielded the contig (AJ242107.1) (Baker et al., 2000) to serve as sequences for the eight sago morphotype samples outgroup, using MEGA 7 software (Kumar et al., corresponding with the expected product sizes. 2015). Using the same software, phylogenetic Multiple sequence alignment using ClustalW analysis was employed on the aligned sequences. generated an aligned sequence with a size length of A Kimura 2-parameter model (Kimura, 1980) was 987 bp. Table 2 summarizes the aligned sequence used to generate pairwise evolutionary distance via characteristics with designated locations of 18s transition and transversion substitution. Phylo- rRNA (partial sequence), ITS1, 5.8s rRNA, ITS2, and genetic tree was constructed by Neighbor-joining 26s rRNA (partial sequence). The 18s rRNA region analysis using Maximum Composite Likelihood of the aligned sequences corresponded with the method with 1000 bootstrap replicates, Maximum expected sequence location from base number 1 to Likelihood method using Jukes-cantor analysis with 118 with a size of 118 bp. However, the specific 1000 bootstrap replicates. location of ITS1, 5.8s rRNA, and ITS2 was not

Fig. 2. Gel profile of ITS amplicons of the sago palm morphotypes. R1: NsDJ; R2: NsDM; R3: NsWM; R4: NsWJ; R5: SWM; R6: SWJ; R7: SDM; and R8: SDJ.

Table 2. Comparative analysis of expected and observed site location of ITS1 and ITS2

PARAMETER SEQUENCE LOCATION/SITE LENGTHa Sample 18S rRNA ITS1 5.8S rRNA ITS2 26S rRNA

Expected Metroxylon salomonense 1 to 118 119 to 393 394 to 557 558 to 804 805 to 828 site location (AJ242107.1) (118 bp) (275 bp) (164 bp) (247 bp) (24 bp) and size range (Outgroup) Metroxylon sagu 1 to 119 120 to 394 395 to 558 559 to 806 807 to 828 (AJ242106.1) (119 bp) (275 bp) (164 bp) (248 bp) (22 bp)

Observed Aligned sequences of 1 to 118 193 to 940 (747 bp)b 941 to 987 site location sago morphotype (118 bp) (highly variable site) (47 bp) and size range samples; this study alength range of partial sequences for 18s rRNA and 26s rRNA; bundefined site location for ITS1, 5.8s rRNA, and ITS2. GENETIC VARIATION OF SAGO PALM (Metroxylon sagu Rottb.) USING ITS ANALYSIS 43 determined from the obtained sequence alignment (Figure 3) supported the observed divergence in the but was marked by highly variable sequences from Neighbor-Joining tree (Figure 4), as the same the eight sago morphotype samples. branching pattern was observed. The NsDM sample Further analysis of aligned contig sequences still obtained the highest homology with the generated the nucleotide composition of each outgroup with 95% bootstrap percentage, followed sample (Table 3). High degrees of variation in by SWM (36%), NsWJ (23%), NsDJ and NsWM thymine (T), cytosine (C), adenine (A), and guanine (8%). The only difference between the NJ tree and (G) percentages were observed for the eight samples. the ML tree was the clustering of samples, as the Mean adenosine (17.0%) and guanine (31.7%) NsDJ and NsWM (31%) and SDM and SDJ (35%) percentages of the sago morphotype samples clustered into a single group. The comparison of showed high correlation with A (19.6%) and G the eight derived ITS sequences from this study with (33.7%) percentage of Metroxylon salomonense two sequences found in the database is shown in (Warb.) Becc. However, the obtained mean T Figure 5. (32.5%) percentage was significantly higher than the expected T (14.6%) percentage, while the observed mean C (18.9%) percentage was lower than DISCUSSION the expected C (32.1%) percentage. The obtained GC% percentage for the morphotype samples was ITS analysis can be successfully employed in the significantly lower (mean GC% = 50.5%) than the investigation of intraspecies variability among expected GC% of 65.8. Figure 5 presents the different variants and cultivars. In Vigna sequences of eight ITS fragments isolated from sago unguiculata (cowpea) cultivars, significant palm with their corresponding assigned Genbank variability among cultivars was determined upon Accession Numbers. amplification and sequencing of the ITS1 and ITS2 regions (Freitas et al., 2013). Fama et al. (2000) also Phylogenetic analysis reported high level of intra-individual ITS1 Neighbour-joining (NJ) tree (Figure 3) shows polymorphism in Caulerpa racemosa complex. decreasing bootstrap percentage. The pattern of Saini et al. (2008) showed the extent of intra- ITS sequence divergence was observed to be individual and intra-species variation among the influenced by geographical distribution, as non- Asiatic Vigna species (subgenus Ceratotropis) using spiny samples collected from Prosperidad clustered nuclear rDNA ITS. ITS polymorphism was observed into a monophyletic group with the M. salomonense among multiple successions of Vigna radiata, and outgroup from Papua New Guinea, while spiny further analysis showed that variants are hetero- samples from Tagum City diverged into a different duplex ITS fragments. Wild and cultivated forms of group. However, the SWM sample from Sta. Cruz, four Vigna species revealed substantial intra-species Davao del Sur clustered with the outgroup, while divergence from the ten Vigna species studied. NsWM sample from Prosperidad clustered with the However, among the four species, intra-individual spiny samples. The Maximum Likelihood (ML) tree ITS variation was observed in only one species.

Table 3. Nucleotide composition of the sequenced M. sagu Rottb. ITS fragments.

Sampling Length (nt) Samplea T (U) C A G GC % Location analyzed

Metroxylon salomonense 14.6 32.1 19.6 33.7 65.8 828 (Outgroup); AJ242107.1 Metroxylon sagu; AJ242106.1 16.4 31.0 19.5 33.1 64.1 828 NsDJ Prosperidad 31.8 21.1 17.7 29.4 50.5 541 NsDM Prosperidad 29.8 23.0 15.7 31.5 54.5 527 NsWM Prosperidad 41.7 19.8 11.7 26.8 46.6 597 NsWJ Prosperidad 29.3 15.1 18.5 37.0 52.1 621 SWM Sta. Cruz 32.6 18.1 17.4 31.8 49.9 723 SWJ Tagum City 31.5 18.5 21.2 28.8 47.3 764 SDM Tagum City 34.7 14.8 15.7 34.8 49.6 580 SDJ Tagum City 28.4 20.4 17.8 33.4 53.8 574 aNS=non-spiny or S=spiny; D=dry or W= wet; (J=juvenile or M=mature); bComputed mean excludes Mextroxylon salomonense (Warb.) Becc. (AJ242107.1) and Metroxylon sagu Rottb. (AJ242106.1). 44 GENETIC VARIATION OF SAGO PALM (Metroxylon sagu Rottb.) USING ITS ANALYSIS

Fig. 3. Maximum Likelihood analysis of evolutionary divergence between M. sagu Rottb. morphotype samples using Jukes-Cantor model using 1000 bootstrap replicates.

Fig. 4. Neighbour-Joining analysis using Maximum Composite Likelihood method for computation of evolutionary distance with 1000 bootstrap replicates.

Analysis of folk varieties of M. sagu Rottb from two In our study, the variable regions ITS1 and locations in Malay Archipelago revealed genetic ITS2 were highly variable, concurring with the variation. The folk varieties could be divided into findings of Freitas et al. (2013) and Fama et al. two groups, one group comprised of the Demai (2000). Moreover, the observed size of ITS1, 5.8s variety and the second group comprised 16 rRNA, and ITS2 was 747 bp, which was higher than individuals of other varieties. A 9-base substitution the expected size of 686 bp from Metroxylon was found to exist in the samples’ ITS regions salomonense (AJ242107.1) and 687 bp from (Kumekawa et al., 2013). Metroxylon sagu (AJ242106.1). The obtained GENETIC VARIATION OF SAGO PALM (Metroxylon sagu Rottb.) USING ITS ANALYSIS 45

Fig. 5. Comparison of derived M.sagu ITS sequences (this study) with two Metroxylon sequences found in the database.

(Guanine + Cytosine) GC% percentage for the observed are high mutability of GC base pair due morphotype samples was significantly lower (mean to cytosine methylation (Pfeifer, 2006) and environ- GC% = 50.5%) than the expected GC% of 65.8. GC mental stressors such as cold and dry climates content have significant implication to evolutionary (Šmarda et al., 2014). divergence of species as GC-rich regions are subject These bootstrap percentages of the sago to mutation leading to heterogeneity of genome morphotype samples are significantly lower than within species (Vinogradov, 2003). Possible factors the bootstrap confidence limit (BCL) of 95% (Klein that could have contributed to the low mean GC% & Klein, 1991). According to Nei & Kumar (2000), 46 GENETIC VARIATION OF SAGO PALM (Metroxylon sagu Rottb.) USING ITS ANALYSIS only bootstrap values with 95% or higher percentage samples could be attributed to the decrease in GC% are considered to produce an accurate and robust tree with a value of 46.6% compared to other non-spiny topology. This would indicate that the obtained sample with a GC% value ranging from 50.5% to NJ tree showed low robustness and low support 54.5% (Table 3). The 46.6% GC content of the tree topology. However, a study by Hillis and Bull NsWM sample was relatively in agreement with the (1993) showed that BCL greater than 70% generated GC content of the spiny sago sample having values a correct tree topology, thus, NsDM sample (93% of 47.3% (SWJ), 49.6% (SDM), 49.9% (SWM), and bootstrap value) would be considered as a 53.8% (SDJ). The decrease in GC% would suggest supported node. Nodes of other sago morphotype the possible influence of epigenetic mechanism on samples that fell below 70% BCL were considered the clustering of the NsWM sample to the spiny unsupported. sago samples. This observation is supported by the Despite having bootstrap values lower than the high degree of methylation percentage of spiny sago bootstrap confidence limit (BCL) of 95%, Neighbor palm with a value of 21.5%, compared to the 11.5% Joining analysis has been nevertheless observed to of the non-spiny sago palm (Novero et al., 2012). generate the correct branching pattern (Klein and Epigenetic silencing had been observed in Klein, 1991). According to Pichon (1993), low Arabidopsis and Phaeodactylum tricornutum bootstrap percentage could also indicate an (diatom) species; in relation to low GC content occurrence of rapid rates of ITS diversification, such (Leon et al., 2007). Furthermore, repetitive DNA that it could reflect unequal rates of concerted content, which is a characteristic of the ITS region, evolution due to low shared nucleotide substitution. has high correlation to relative frequency of DNA From this statement, the divergence of spiny sago methylation (Matzke and Matzke, 1998). This has samples (SWJ, SDM, and SDJ) and the NsWM been observed by Saini et al. (2008) on wild and sample into a separate monophyletic group from cultivated Vigna radiata (mungbean) variants, that of the outgroup could be due to low shared suggesting that epigenetic responses could be the nucleotide substitution causing the observed ITS initial mechanism towards effective ITS sequence polymorphism. This was supported by the high homogenization. percentage of substitutions in the Kimura 2- The Maximum Likelihood (ML) tree (Figure 4) parameter substitution analysis of the sago supported the observed divergence in the Neighbor- morphotype samples when paired with the outgroup. Joining tree (Figure 3), as the same branching The pattern of ITS sequence divergence was pattern was observed. The NsDM sample still observed to be influenced by geographical obtained the highest homology with the outgroup distribution. Based on Figure 3, samples collected with 95% bootstrap percentage, then SWM (36%), from the same sampling site, except for NsWM and NsWJ (23%), NsDJ and NsWM (8%). The only SWM samples, have homologous ITS sequences, difference between the NJ tree and the ML tree was indicating the influence of geographical distribution the clustering of samples, as the NsDJ and NsWM on ITS divergence (Fama et al., 2000). In the NJ (31%) and SDM and SDJ (35%) clustered into a tree, the clustering of the non-spiny sago palm single group. The difference in clustering can be collected from Prosperidad, Agusan del Sur, and the attributed to the algorithm that each analysis Metroxylon salomonense outgroup collected in employed, as NJ analysis utilizes the pairwise Papua New Guinea (Baker et al., 2000) supported distance substitution upon tree construction (Saitou the study of Flach (1997) indicating that the & Nei, 1987), while ML analysis estimates the distribution of high-density sago stands in Agusan probability of a tree topology using heuristic del Sur originated in Papua New Guinea. Further- searches (Felsenstein, 1985). Despite the difference more, spiny sago palm collected from Tagum and in algorithm analysis, both the NJ and ML tree the NsWM sample collected from Prosperidad showed particular agreement in the derived clustered into a single group. The results showed branching pattern of the sago samples, which further that geographical isolation of the spiny sago samples supported the inference of the possible influence of could have contributed to the observed ITS geographic distribution on the observed ITS polymorphism suggesting that geographical polymorphism. isolation could be the driving force that contributed to ITS divergence Chen et al., 2016). However, the inclusion of the NsWM sample in CONCLUSION the spiny group, and SWM in the non-spiny group (Figure 3) could be due to incomplete homo- Internal Sequence Repeat markers enabled the genization of ITS sequences attributed to slow observation of polymorphism among Metroxylon molecular drive caused by intra-species hybri- sagu morphotypes. Based on the obtained Neighbor dization (Saini et al., 2008). 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