Sequence Variation and Phylogenetic Analysis of Sansevieria Trifasciata (Asparagaceae)

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Research Article Bioscience Research, 13(1): 01-07, 2016 Available online at www.isisn.org © Communication Ilennovative Scientific Information & Services Network Print ISSN: 1811-9506 Online ISSN: 2218-3973

Sequence Variation and Phylogenetic Analysis of Sansevieria trifasciata (Asparagaceae)

Trina Ekawati Tallei*, Riano Erlangga Rembet, Johanis Julian Pelealu and Beivy Jonathan Kolondam

Department of Biology, Faculty of Mathematics and Natural Sciences, Sam Ratulangi University, Kampus Unsrat Bahu Manado 95115 Indonesia. *Corresponding author

Sansevieria is an ornamental plant with fairly high economic value. It has many hybrids and varieties which make them difficult to identify. Among Sansevieria, the most commercialized species is S. trifasciata. There are at least 20 cultivars of this species which has been sold around the world. Morphological characters among those cultivars are somehow highly distinct, therefore they maybe considered as different species. This research aimed at analysing the sequence variation among S. trifasciata and their allied taxa in family Asparagaceae. Sequences of their matK gene are analyzed. The evolutionary history was inferred by using the Maximum Likelihood method. Estimation of evolutionary divergence between sequence was analysed using Kimura two-parameter. Evolutionary analysis were conducted in MEGA6. The evolutionary divergence shows that there is no distance within S. trifasciata. The phylogeny indicates that Pleomele is embedded within Dracaena. Overall evolutionary divergence of Asparagaceae shows very low interspecific divergence within Sansevieria and this genus is monophyletic.

Key words: Sansevieria, Sansevieria trifasciata, matK, Asparagaceae, evolutionary divergence. Sansevieria is a genus consisting of many classification of this plant is often hard to do species. Sansevieria is often included in the (Acevedo-Rodríguez and Strong, 2005). A hybrid genus Dracaena in the classification Angiosperm plant is a cross between two or more different Phylogeny Group II system (APG 2003). Both populations, unrelated inbred plants, both Sanserieria and Dracaena are included in family phenotype and genotype. Variety is a population Asparagaceae, subfamily Nolinoideae (formerly of plants within a species that show different the family Ruscaceae), and has also been placed obvious characteristics. A cultivar is race or before in the family Dracaenaceae (Lu and variety of a plant that has been created or Morden, 2014). Sansivieria is an aggressive selected intentionally and maintained through invasive plant that can grow in a wide variety of cultivation and retain a characteristic when habitats (Gilman, 1999). It has a fairly high reproduced, either sexually or asexually. In economic value. In addition to having high fiber Sansevieria, S. trifasciata is the most that can be used as raw material for textiles, this commercialized species. At least there are 20 plant has become one of the export commodities. famous cultivars of this species have been sold According to the Directorate General of around the world (Henley et al 1993). Processing and Marketing of Agricultural Products The unstable genetic characteristic of S. (2010), this plant is exported to foreign countries, trifasciata results in a change of color strokes, especially to Korea. shape and patterns of the leaves, which may be Sansevieria has many hybrids and caused by gene or chromosomal mutations horticultural varieties and cultivars so that the (Purwanto, 2006). In Sansevieria there are many

variegated plants caused by natural variegation. were MatK-1RKIM-f 5’- This natural variegation is very stable so that the ACCCAGTCCATCTGGAAATCTTGGTTC-3’ and color-based identification of variegated plants is MatK-3FKIM-r 5’-CGTACAGTACTTTTGT difficult to perform. In such plants, the genetic GTTTACGAG-3’ (Kuzmina et al 2012). material in the seed will produce offspring with the Amplification was performed as follows: same or similar variegation pattern with its parent Predenaturaion at 95°C for 2 min, followed by 35 (Stein, 2011). Sansevieria hargesiana is one cycles consisted of denaturaion at 95°C for 30 example of the result of natural variegation. sec, annealing at 50°C for 30 sec, and Many variations of these mutations consistent polymerization at 72°C for 50 sec. Amplicons from generation to generation, but others appear were separated using 1% agarose gel. Clear cut randomly. This non-natural mutation is known as bands indicated the success of the amplification. a chimera, or a blend of two different pairs of The PCR products together with their primer pairs chromosomes in an organism. Chimera occurs were sent to 1Base Malaysia for sequencing. when an organism or tissue have at least two different sets of chromosomes, and mostly occurs Sequence and phylogenetic analysis as a result of the fusion of many zygotes. Many Using Genious v5.6, the chromatograms were variegated plants are chimeras, in which that not processed according to Tallei and kolondam all cells in the plant contain identical genetic (2015). The sequences were pairwise aligned information (Stein, 2011). using global alignment with free end gaps to One method for studying the genetic variation identify regions of 95% similiarity. Approximately in plants is by using DNA barcode. The DNA 50 nucleotides at the beginning of DNA barcode can also be used to identify species sequences were trimmed to remove primer based on sequence variation of genes used as sequences. The nucleotide errorness readings DNA barcodes (Kress et al 2005). DNA barcode were corrected. Consensus sequences were provides fast and accurate identification of an generated by pairwaise alignment of forward and organism which its barcode has been deposited in reverse sequences using MUSCLE integrated in sequence library such as GenBank and BOLD. Genious v5.6. The matK sequences from other Ideally, a DNA barcode used for species Asparagaceae (S. trifasciata, Dracaena, Pleomele identification has high enough sequence variation and Polygonatum) were retrieved from GenBank. to differentiate species and has a low intraspecific All sequences were aligned using multiple variation (Kress and Erickson, 2007). One gene sequence alignment with hierarchical clustering used for DNA barcode is matK. Polimorfism in (Corpet, 1988) matK gene has been used for phylogenetic study (http://multalin.toulouse.inra.fr/multalin/) and in various type of plants (Shaw et al 2005). trimmed accordingly to get the core area of matK. Two of the many varieties of S. trifasciata Sequences were subsequently aligned using collected by Sansevieria collectors are S. Clustal O(1.2.1) multiple sequence alignment tool trifasciata var. laurentii and var. hahnii. (www.ebi.ac.uk/Tools/msa/clustalo) to obtain the Morphological characters of both plants are very percent identity matrix. The evolutionary history different so they maybe considered as different was inferred by using the Maximum Likelihood species. Lack of information about the genetic method based on the Kimura two-parameter variation in S. trifasciata encourages research in model (Kimura, 1990). Evolutionary analysis was order to know whether there is a genetic variation conducted in MEGA 6.0 (Tamura et al 2013). in S. trifasciata varieties. The aim of this study is to analyse matK sequence variation and RESULTS AND DISCUSSION phylogenetic of S. trifasciata. The edited sequences of S. trifasciata var. hahnii and laurentii produced 856 bp, respectively. MATERIALS AND METHODS The GC content of the sequences are GC DNA extraction, amplification, and sequencing 31.89%. This is in agreement with the finding of Leaf tissues from S. trifasciata var. hahnii and Guisinger et al (2010) who reported that whole laurentii were collected from living material. The plastid genome of T. latifolia has GC 33.8%. The DNA was extracted from approximately 120 mg of length of matK sequences among 12 taxa being fresh flesh of the leaves using innuPREP Plant analyzed varied based on sequences deposited in DNA Kit (Analytik Jena) with a modification (Tallei GenBank. All sequences were trimmed to obtain and Kolondam, 2015). Gen matK was amplified the core sequence of 648 bp, approximately lies in using 5X Firepol PCR Master Mix Ready-to-load nucleotide 494 - 1377 from 1557 matK gene of S. (Solis Biodyne). Primer pairs used in this study trifasciata obtained from GenBank. Polygonatum

kingianum was used as an out group. Based on exceeds 0.4 for sequences with a very high BLAST search, sequences with high identity with (saturated) substitution rate (Bora, 2010). S. trifasciata var. hahnii and laurentii are listed in Nucleotide variation in matK was 1.1% Table 1. between Pinus contorta and P. thunbergii, and Based on percent identity created by ClustalO can reach 8.4% between two grass genera from (1.2.1), all S. trifasciata has 100% identity. The two different subfamilies (Liang, 1997). In general, percent identity of S. robusta and S. hyacinthoides nucleotide substitution in matK gene is evenly is also 100%. It means that there is no nucleotide distributed in any codon position (Bora, 2010; Hilu variation among them. Lawodi et al (2013) found et al. 2003). Nucleotide variation in matK is higher that there was no matK gene sequence variation than any other chloroplast genes (Bora, 2010). between curly tomato and apple tomato, and also Figure 1 shows evolutionary history inferred between cherry tomatoes. All of them are by using Maximum Likelihood method based on Solanum lycopersicum. Kimura two-parameter model (Kimura, 1990). The According to Tallei and Kolondam (2015), percentage of trees in which the associated taxa there are no matK gene sequence variation clustered together is shown next to branches. among Myristica fragrans, M. fatua, M. maingayi, Initial tree(s) for the heuristic search were and M. globosa. This shows that matK gene alone obtained automatically by applying Neighbor-Join cannot differentiate species in Myristica and BioNJ algorithms to a matrix of pairwise eventhough level of differentiation of matK is distances estimated using the Maximum higher than rbcL. In this research, matK gene Composite Likelihood (MCL) approach, and then alone cannot be used to differentiate intraspecific selecting the topology with superior log likelihood sequence among S. trifasciata and between S. value. The tree is drawn to scale, with branch robusta and S. hyacinthoides. lengths measured in the number of substitutions The identity between S. trifasciata and S. per site. Evolutionary analyses were conducted in robusta or S. hyacinthoides is 99.85% since there MEGA6 (Tamura et al 2013). is only one nucleotide variation between them at Analysis of distance estimation is presented in nucleotide 513. The variation found in the Table 3. Bootstrap method is used for variance sequences is a transition from A↔G. With only estimation with 1000 replications. Kimura two- one nucleotide difference among Sansevieria parameter model was used for nucleotide genera reveals that their interspecific variation is substitution model. Transition and transversion very low. are included. The number of base substitutions Variation of matK gene among Sansevieria per site from between sequences are shown. dengan Dracaena, Pleomele, and Polygonatum Standard error estimate(s) are shown above the shows that transition from A↔G is dominating. diagonal. Analyses were conducted using the Transversion is rarely found. Due to molecular Kimura two-parameter model (Kimura, 1980). mechanism, transition frequency is higher than Intraspecific distance within S. trifasciata is transversion. 0.000, interspecific distance within Sansevieria Transition tends to produce less amino acid ranges from 0-0.002, and intergeneric distance substitution, hence will tend to be stable as silent ranges from 0,003 – 0,020. By relying on substitution in one population as single nucleotide morphological characters, S. trifasciata var. hahnii polymorphisms. The maximum likelihood and var. laurentii are seen as two distinct species. estimation of subsitution matrix is shown in Table Molecular evidence shows that both are the same 2. Each entry shows the probability of substitutin species (Rembet et al 2016). This shows that the (r) from one base (row) to another base (column). morphological characters often cannot be relied Substitution pattern and rates were estimated upon to determine the proximity of a species or under Kimura two-parameter model (Kimura, between species. 1980). Rates of different transititonal substitutions are shown in bold and those of transversional The genetic distance between Sansevieria substitutions are shown in italics. The sum of r and Dracaena ranges from 0,003 – 0,006, value is made equal to 100. Sansevieria and Pleomele sp. is 0,012, and The sum of transitional substitutions is 77.4 Sansevieria and Polygonatum kingianum is 0.020. while the sum of transversional substitutions is The smaller the genetic distance, the closer the 22.64. Therefore the transition/transversion (ti/tv) kinship between the organisms being compared. If ratio is 3.4. In matK gene, transition frequency is there is no genetic distance between organisms higher than transversion. The ti/tv ratio is about 2.0 for recently divergence sequence, and

Table 1. Species used in this study with their accession number Species Family Accession number Identity Sansevieria trifasciata Asparagaceae JQ276422.1 100% Sansevieria trifasciata Asparagaceae HM640584.1 100% Sansevieria robusta Asparagaceae KR734544.1 99% Sansevieria hyacinthoides Asparagaceae JQ024992.1 99% Dracaena aletriformis Asparagaceae JX903540.1 99% Dracaena deremensis Asparagaceae JX903539.1 99% Dracaena aubryana Asparagaceae HM640583.1 99% Dracaena schizantha Asparagaceae HM640582.1 99% Pleomele sp. Asparagaceae JX903541.1 99% Polygonatum kingianum Asparagaceae KJ745691.1 98%

Table 2. Substitution matrix estimated by maximum likelihood A T/U C G A - 2.83 2.83 19.35 T/U 2.83 - 19.35 2.83 C 2.83 19.35 - 2.83 G 19.35 2.83 2.83 -

Sansevieria trifasciata Hanii

61 Sansevieria trifasciata Laurentii JQ276422.1 Sansevieria trifasciata 65 HM640584.1 Sansevieria trifasciata KR734544.1 Sansevieria robusta 67 JQ024992.1 Sansevieria hyacinthoides JX903540.1 Dracaena aletriformis 89 JX903539.1 Dracaena deremensis HM640583.1 Dracaena aubryana JX903541.1 Pleomele sp. HM640582.1 Dracaena schizantha KJ745691.1 Polygonatum kingianum

0.002 Figure 1. Molecular phylogenetic analysis by Maximum Likelihood method. that are compared, then these organisms are the distance between Sansevieria and Dracaena same species. In Figure 1, Sansevieria is closely ranges from 0.002 to 0.009. In Figure 1, Pleomele related with Dracaena and distantly related with sp. is nested together within Dracaena. This is in Polygonatum kingianum. Sansevieria robusta and accordance with the finding of Lu and Morden S. hyacinthoides are closely related to D. (2010) that using psbA-trnH, the maximum aletriformis with genetic distance 0.002. Genetic parsimony tree showed the polytomy of Dracaena distance between S. trifasciata and D. aletriformis and Pleomele. Genetic variance of medicinal is 0.003. As a comparison, the mean interspecific plants in South Africa showed that the intermedian distance of matK in Astragalus was 0.0071 ± interspecific distance (0.232) is significantly higher 0.0064, while the intraspecific distance was than intermedian intraspecific distance (0.000) 0.0014 ± 0.0022 (Zheng et al 2014). Interspecific (Mankga et al 2013). Based on Table 3, there is a distance between Panax ginseng and P. barcode gap in Asparagaceae. Analysis of matK quinquefolius was very close, ranged from 0 to barcode for ornamental trees and shrubs in Egypt 0.002 (Chen et al 2013). This is in agreement with the distance within Sansevieria (0 – 0.0002). The

Table 3. Estimation of evolutionary divergence between sequences Hahnii Laurentii JQ276422 HM640584 KR734544 JQ024992 JX903540 X903539 HM640583 HM640582 JX903541 KJ745691 Hahnii [0.000] [0.000] [0.000] [0.001] [0.001] [0.002] [0.003] [0.003] [0.004] [0.004] [0.005] Laurentii 0.000 [0.000] [0.000] [0.001] [0.001] [0.002] [0.003] [0.003] [0.004] [0.004] [0.005] JQ276422 0.000 0.000 [0.000] [0.001] [0.001] [0.002] [0.003] [0.003] [0.004] [0.004] [0.005] HM640584 0.000 0.000 0.000 [0.001] [0.001] [0.002] [0.003] [0.003] [0.004] [0.004] [0.005] KR734544 0.002 0.002 0.002 0.002 [0.000] [0.001] [0.003] [0.002] [0.003] [0.004] [0.005] JQ024992 0.002 0.002 0.002 0.002 0.000 [0.001] [0.003] [0.002] [0.003] [0.004] [0.005] JX903540 0.003 0.003 0.003 0.003 0.002 0.002 [0.002] [0.002] [0.003] [0.004] [0.005] X903539 0.006 0.006 0.006 0.006 0.005 0.005 0.003 [0.003] [0.004] [0.004] [0.005] HM640583 0.006 0.006 0.006 0.006 0.005 0.005 0.003 0.006 [0.003] [0.004] [0.005] HM640582 0.009 0.009 0.009 0.009 0.008 0.008 0.006 0.009 0.006 [0.004] [0.004] JX903541 0.013 0.013 0.013 0.013 0.011 0.011 0.009 0.012 0.009 0.012 [0.006] KJ745691 0.020 0.020 0.020 0.020 0.019 0.019 0.017 0.020 0.017 0.014 0.024

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