Ixora chinensis Lam sampled from Karwar, Catunaregam spinosa (Thunb.) Triveng, sampled from Goa and Meyna spinosa Roxb. ex Link sampled from Nasik and their urban (Mumbai) counterparts yielded good quality genomic DNA which gave reproducible results with RAPD analysis. PCR with all five primers (RP01-RP05) yielded amplification products that ranged from approximately 1,500bp- 300bp. Only distinguishable and genuine bands were scored for binary matrix.
From the results of genetic analysis, it can be observed that there is high level of genetic variation between certain sets of plants. The percentage of polymorphism shows 98.67% which signifies highest level of polymorphism among the alleles in these plants. The genetic distance is highest between Catunaregam spinosa (Thunb.) Triveng from Mumbai and Ixora chinensis Lam from Karwar. And as per the Dendrogram, three clades were expected to arise from the ancestor, instead only two clades resulted. Although from the dendrogram it may seem that Catunaregam spinosa (Thunb.) Triveng, from Mumbai and Goa have joined as a clade with Ixora chinensis Lam and Meyna spinosa Roxb. ex. Link respectively, they actually are subdivided from the ancestor directly, as opposed to them together forming a different clade.
To support the above analysis, the binary matrix of the bands generated from all these plants were analyzed under “Groups”. The Percentage of polymorphism between Catunaregam spinosa (Thunb.) Triveng from Goa and Mumbai was high (56%) as compared to Ixora chinensis Lam from Karwar and Mumbai (28%) and Meyna spinosa Roxb. ex Link from Nasik and Mumbai (29.33%). The Nei’s Genetic distance by UPGMA method showed between Catunaregam spinosa (Thunb.) Triveng, from Mumbai and Goa was 41.049.
All these results signify that although morphologically they are the same plants, but Catunaregam spinosa (Thunb.) Triveng from Mumbai and Goa have genetically evolved differently from each other. To probably adapt to the environmental conditions of the respective geographical location, the genome may have acquired or lost some genes. The loss or gain of certain portion of the genome depends on their use or function to the plant’s survival. The function of a gene sometimes depends on the climatic/ soil/ temperature/ stress conditions of the respective geographical location. Therefore, from this data we may conclude that geographical location did affect the gene diversity in Catunaregam spinosa (Thunb.) Triveng.
We can also infer that, Catunaregam spinosa (Thunb.) Triveng, from Mumbai and Goa, both might harbour functionally or structurally different genes of importance, especially those that take part in metabolic pathways of secondary metabolite production. Micronutrients such as Fe, Mo, Cu etc., are known to be co- factors for most of the enzyme’s functioning. As soil nutrients are responsible for the micro and macro nutrients supply to the plants, they in turn may cause certain significant enzymes to be produced. As a part of this study, soil collected from the respective sampling sites was subjected to micro and macro nutrient estimation. Nasik showed least quantities of micronutrients (Fe, Zn, Cu, Mb, Mn & Cl) as compared to other study sites. The Goa sampling site soil revealed less Nitrogen and Phosphorous content as compared to other samples. As opposed to the findings of micronutrients, Nasik sampling site showed highest Magnesium, Nitrogen, Phosphorous, Potassium and Calcium content. Although the concept requires further investigation, the soil composition may be proposed to have an effect on genetic makeup of the plants [Supplementary soil analysis tables 1–5]. In one study involving impact of water and soil nutrients on genetic variation and clonal diversity of Carex nigra in a central alpine fen, it was found that genetic diversity increased with higher phosphorous contents. In contrast, high genetic variation was also observed in low potassium availability, which maybe due to plant’s susceptibility to abiotic stress of this deficiency (Reisch et al. 2020).
From DNA barcoding analysis, the phylogenetic trees generated using matK and rbcL sequences differed from that generated by RAPD. The reason could have been due to targeting a single locus in barcoding as opposed to multiple loci in RAPD. The phylogenetic tree with Vinca major as the outgroup showed Meyna spinosa Roxb. ex Link Nasik and Mumbai as divergent from the ancestor instead of one clade. A closer observation to the tree reveals that Meyna spinosa Roxb. ex Link from Mumbai’s landscape and weather is much similar to that of Goa and Karwar; The reason maybe the effect of topography- Mumbai, Goa and Karwar all being hilly and share the similar climatic conditions and soil profile, would most certainly group together. Studies such as Reisch and Rosbakh, 2021, assessed the pattern of genetic variation in European plant species growing on different altitudinal zones; they also observed genetic variation among spatially and environmentally isolated populations, which could have resulted from restricted gene flow. While studying the effect of geographic environment on genetic variation in 46 species of Erianthus arundinaceus from natural habitats in China, it was observed that the geographic landforms such as oceans and big mountain and river systems played an important role in determining genetic diversity (Zhang et al. 2017). While comparing rare and endemic species of Petunia secreta, the studies showed high genetic diversity. The two species corresponded to two separate sites of species occurrence in different landscapes (Caroline et al. 2016). While assessing microsatellite diversity in 94 individuals of 10 wild barley populations, Hordeum spontaneum (C. Koch) Thell, across Israel, it was observed that gene diversity was highest in stressful arid hot environments (Timo et al. 2001).
It can also be observed that Ixora chinensis Lam from Karwar is most recently evolved from the matK phylogenetic tree and Ixora chinensis Lam from Mumbai being most recently evolved from the rbcL phylogenetic tree. The transitions and transversion substitutions were calculated for barcoding with both the genes; the transition substitutions from C to T and G to A being highest in both the cases. Polymorphism in the gene sequences between a pair of plants from different geographical location is evident, as can be observed in the phylogenetic tree.
Our study reveals the effect of geographical location on genetic make- up of the plants, at least genomic level. Investigation of the similar effect should be checked at expression level by analyzing the RNA, protein, phytochemical fingerprint and bioactivity to obtain a holistic picture if the differences are significantly large or not. The study also provides a preliminary insight into the molecular evolution of select plants representing Rubiaceae family along western ghats of India.