Deciphering Genetic Diversity in Spathodea campanulata Beauv. from South India Based On Randomly Amplified Polymorphic DNA Markers

The African tulip (Spathodea campanulata) is a monophyletic species native to tropical forests of sub-Saharan Africa belonging to family Bignoniaceae. Although its endemism in sub-Saharan Africa, it is listed as potential alien invasive species in Pacific, Indian and Caribbean, but also Singapore, Papua New Guinea and Australia. The expansion of its range from ornamental to invasive is being observed in South India. In this study, RAPD based DNA markers have been used to determine genetic diversity of the monophyletic species. About 14 populations were studied from 6 states of South India using 41 random decamer primers. The total number of 517 bands were scored which are generated by 41 primers producing 12.6 bands on an average per primer, of these 517 bands produced 379 were polymorphic showing 73% polymorphism revealing higher level of genetic polymorphism in the study population. The similarity coefficient ranged from 0.312 to 0.837 and the dendrogram constructed by the UPGMA based on Jaccard's similarity matrix farmed two clusters. In the present study, results showed the high degree of genetic diversity within the populations of S. campanulata supporting high degree of adaptation and its range expansion from ornamental to possible invasive species in South India.


I. INTRODUCTION
The African tulip tree (Spathodea campanulata) is a monophyletic species native to tropical forests in a broad area of sub-Saharan Africa belongs to family Bignoniaceae. It's also known as fountain tree, scarlet bell flower tree, squirt tree, syringe tree in English and neerukayi mara or ucche kayi mara in the local Kannada language of Karnataka, India. It is planted as an ornamental tree for its attractive glossy deep green pinnate leaves and glorious orange scarlet flowers. Spathodea is endemic in Angola, Ethiopia, Ghana, Kenya, Sudan, Tanzania, Uganda, Zambia and exotic to Colombia, Costa Rica, Cuba, India, Jamaica, Puerto Rico, Sri Lanka, Zanzibar [1]. S. campanulata has been introduced pan-tropically for its ornamental value, it was introduced to India in 1800's [2]. The extensive fruiting and the heavy masses of winddispersed seeds credited the capacity of being invasive to the species. It is invasive in many countries like Pacific, Indian and Caribbean but also Singapore, Papua New Guinea and Australia. Removal of trees is highly recommended, particularly when they are in close proximity to natural forests and aquatic ecosystems [3]. S. campanulata grows naturally in secondary forests and prefers wet areas especially the borders of dynamic water resources. It may also appear as a pioneer species in the native range, such as it being one of the species that naturally colonizes grasslands in Uganda. But, it invades both abandoned agricultural land and closed forest being dominant acquiring the weed status [4,5] and is a weed in coffee plantations in Cuba [6]. It is reported as highly invasive in Tahiti, French Polynesia in cloud forests up to 1300 m [7]. In southern part of India, we have observed the preliminary invasion of S. campanulata in barren lands and borders of the forest lines. Introduced species are generally expected to have low genetic diversity due to population bottlenecking of introduction process and founder effect. Low genetic diversity is expected to limit adaptability to the new environment there by limiting the evolutionary potential of introduced population. This relationship between genetic diversity and population viability, in contrast to the success of invaders over native species, constitutes an apparent paradox in invasion biology [8,9].
Understanding Genetic diversity and population structure within introduced populations provide valuable information for assessing the adaptability and evolutionary potential of invasive species and explain successful invasions. The analysis of the population genetic structure through Randomly Amplified polymorphic DNA (RAPD) based markers proved potential in various species. The RAPD technique is quick, require no prior genetic information and relatively inexpensive method which is extensively used to analyze the genetic variability in plants. Despite of lower reproducibility, the RAPD method has become popular and highly practical due to its cost-effectiveness with studies involving a smaller number of populations [10,11]. The present effort is a first attempt to decipher the genetic diversity of S. campanulata populations from different states of south India.

II. MATERIALS AND METHOD 2.1. Sample collection:
Young leaves from the study populations were collected in zip lock bags and brought the laboratory in ice box. On arrival to the laboratory leaf samples were washed in 70% ethyl alcohol and blotted between sterile blotting papers. Leaf samples were wrapped in aluminium foil, labeled and stored at -80ºC until DNA isolation.

Isolation of DNA from leaf samples
The CTAB lysis buffer [12] with certain modification was used for the total DNA isolation from leaf samples. Briefly, the CTAB buffer contained 2% w/v CTAB (HiMedia), 100 mM Tris-HCl (pH = 8.0; HiMedia), 20 mM EDTA (pH = 8.0; HiMedia, 1.4 M NaCl (HiMedia) and PVP 2%. The pH of the lysis buffer was adjusted to 5.0 prior to sterilization by autoclaving. The β-Mercaptoethanol (2%) was added freshly and lysis buffer was prewarmed at 65ºC in a water bath (Grant, India) prior to use. The leaf samples were ground to a fine powder with the help of liquid nitrogen using mortar and pestle. About 2g of powder was ground into a paste in pre warmed CTAB lysis buffer and incubated at 65ºC for 60 minutes. The lysate was cooled to room temperature and extracted with phenol:chloroform:isoamylalcohol (25:24:1) and DNA was precipitated with isopropanol [13]. The DNA pellet obtained was washed in 70% alcohol, air dried and dissolved in 200µl of T10E1 buffer. The genomic DNA samples isolated were separated on 0.8% agarose (SeaKem) gel prepared in 1X TAE in a horizontal electrophoresis Unit. Quality and quantity of DNA were assessed by Biophotometer (Eppendorf) measuring the absorbance at 260nm/280nm.

PCR Amplification
A total number of 60 decamer primers (Eurofin genomics) belonging to OPA, OPB and OPC series were used for the amplification. The PCR reaction was performed in a thermal cycler (Eppendorf, Germany) with the reation volume of 20µl containing 100ng of genomic DNA, 5pm primer, 100µm of dNTP mix (Fermentas), 1.5mMMgCl2, 1X Taq Buffer, 1U Taq DNA polymerase (New England Biolabs) and the total volume was adjusted to 20µl using molecular biology grade water. The cycling conditions were set as follows: initial denaturation temperature set at 95°C for 45 sec followed by 40 cycles of denaturation temperature set at 95°C for 20 sec. The annealing temperature was set depending on the standardized annealing temperature 0f 37°C for 15 sec for all primers. The extension temperature was set at 68°C for 1 min and the final extension for 10 min temperature was set at 68°C.

Agarose gel electrophoresis
The bands amplified using random decamer primers were separated on agorae gel. The gels were prepared using 1.5% agarose dissolved in 1X TAE buffer. 0.5µl of ethidium bromide (10mg/ml) was added to stain bands for better visualization. The PCR products were loaded along with the gel loading dye (Bangalore Genei). The PCR amplicons (bands) were visualized using G;Box Chemi gel documentation unit (Syngene).

Scoring and Statistical analysis
Only brisk and sharp bands were considered as scorable bands and used for scoring. Each amplified product was considered as a unit character and the populations were scored for the presence (1) or absence (0) of a band on the gel. The cluster analysis was performed and dendrogram was drawn using NTSys PC (version 2.0) by Unweighted Pair Group Method with Arithmetic mean (UPGMA) and similarity matrix was deduced using Jacard coefficient [14].

III.
RESULTS AND DISCUSSION The present study is a first attempt to decipher the genetic diversity of S. campanulata from Karnataka, Maharashtra, Telangana, Tamilnadu, Goa and Kerala states belonging to South India. The DNA samples with A260/A280 ratio ranging between 1.6-1.8 were used for the PCR amplification. A total number of 60 decamer primers were tested for 14 plants populations (Table 1), out of which 41primers gave good amplification and the same were used for the analysis of genetic diversity ( Table 2). The OPB-14 primer amplified highest number of bands (18) with 55% polymorphism and OPC-15 showed the least number of bands (7) with 100% polymorphism. The total number of 517 bands was scored which were generated by 41 primers producing 12.6 bands on an average per primer. Out of these 517 bands produced 379 were polymorphic showing 73% polymorphism revealing higher level of genetic diversity in the study population. The primers used in the present study proved very informative suggesting the future possibility of using same for the study of genetic diversity of S. campanulata populations. OPC-20 ACTTCGCCAC 14 5 36%

Fig.1: UPGMA Dendrogram showing genetic diversity in 14 population of S. Campanulata from South India
Based on the sample matching co-efficient (Jaccard index), a genetic similarity matrix was constructed using the binary data resulted from scoring presence or absence of bands to assess the genetic similarity among 14 populations of S. campanulata. The similarity coefficient ranged from 0.312 to 0.837 (Table 3). Interestingly lowest genetic similarity (0.312) was found in population belonging to Dharwad and Belagavi which are comparatively near in its geographical locations with respect to Sirsi and Kolhapur populations (0.837) which are geographically distinct. The dendrogram constructed by the UPGMA based on Jaccard Similarity Matrix farmed two clusters A & B (Fig. 1) [15,16] and cross pollination by birds and bats [1] may be attributed to the higher genetic diversity in the monophyletic species.  [17]. The total genetic diversity of a species has key implications for its long-term survival and continued evolution revealing the insight of the levels and distribution of genetic diversity which is important for designing management strategies for the species [16]. In the present study, results showed the high degree of genetic diversity within the populations of S. campanulata supporting its range expansion from ornamental to possible invasive species in South India. A species being a single taxon by definition, populations of a particular species may exhibit both phenotypic and genotypic diversity. Therefore the successful management of this diversified taxon may be greatly improved by understanding specific genetic variations of the species resulting in phenotypic variation attributable to genetic variation and/or plasticity [18]. Despite the use of morphological markers, there are no reports on molecular phylogeny and genetic diversity of monophyletics species S. campanulata. Hence, RAPD based molecular markers can be efficiently used to decipher the genetic polymorphism and to establish the management strategy for invasive species like S. campanulata.