Exploration, Characterization and Phylogenetic Studies in Wild Mangifera indica Relatives

Exploratory surveys were carried out in the Andamans and Nicobar group of islands during 2006 and 2014 to locate wild species viz. Mangifera andamanica King, Mangifera camptosperma Pierre and Mangifera griffithii Hook. Not much variation was observed for fruit shape and size for the species Mangifera andamanica, which was endemic to this region. The species M. griffithii has been reported to be only in Mt. Harriet. However, another plant of M. griffithii in the Shoalbay region was found during the second survey. The foliage & fruit characteristics of the two specimens were similar, with a slight difference in the morphological features, which could be attributed to their origin from seeds. The DNA finger printing carried out showed minor changes in the species. The phylogenetic relationships amongst five Mangifera species viz. M. indica, M. griffithii, M. camptosperma, M. odorata and M. andamanica were analyzed by employing chloroplast markers viz., petB-petD intergenic spacer, rps16 gene, trnL-trnF intergenic spacer and nuclear marker—External Transcribed Spacer (ETS). The nuclear markers and chloroplast markers based on phylogenetic analysis showed that the common mango M. indica L. was closely related to M. griffithii and M. camptosperma, which belonged to subgenus Mangifera. However, M. odorata that belonged to subgenus Limus was grouped separately along with M. andamanica. The above results are in congruent with the accepted classification of genus Mangifera reported by Kostermans and Bompard with the exception of M. andamanica, which has been earlier classified under subgenus Mangifera. Corresponding author.


Introduction
The genus Mangifera is one of the 68 genera in the family Anacardiaceae [1].Mangifera indica L. is the most important species in this genus for commercial fruit production in tropical and sub-tropical regions of the world.The genus Mangifera is believed to have originated somewhere in Myanmar, Thailand, Indo-China and Malaya during the Eocene or an earlier period in the Cretaceous, and then the species have spread to India and Sri Lanka in the west, and to Eastern Malaysia and the Philippines in the east [2].The center of diversity of this genus is thought to be South East Asia, with increased diversity in Peninsular Malaysia.The highest number of Mangifera species is now found in Borneo, Sumatra, Java and Malay Peninsula [3].The most acceptable classification of the genus Mangifera is reported by Kostermans and Bompard [4] who described 69 species in the genus based on the morphological characteristics and classified 58 species into two subgenera Mangifera and Limus with several sections.The 11 remaining species were placed in an uncertain position in the classification.India is reported to be the home of four other species viz., Mangifera andamanica, Mangifera khasiana, Mangifera sylvatica and Mangifera camptosperma [5].Two exploration surveys were carried out at Andamans, one in 2006 and another in 2014 to collect the species viz., Mangifera andamanica, Mangifera camptosperma and Mangifera griffithii.Hence, survey was undertaken to locate diversity and distribution of wild/related Mangifera species within the Andamans.
The phylogenetic relationships among Mangifera species have been described earlier by using genomic restriction fragment length polymorphisms (RFLPs) and amplification of chloroplast DNA (cpDNA) [6].Additionally the sequence analysis of External Transcribed Spacer (ETS) has also been reported to be involved in the construction of phylogeny in plants [7]- [11].For phylogenetic studies at lower taxonomic levels noncoding chloroplast regions have been used frequently and successfully [11]- [13].The rationale behind using noncoding regions is the assumption that they are phylogenetically more informative because they are under less functional constraints [14].
In the present study, morphological (foliage & fruits) and molecular characterization (SSR, ETs and Chloroplast markers) was carried out to infer the evolutionary relationships among the Mangifera Species including Mangifera indica, Mangifera griffithii, Mangifera camptosperma, Mangifera odorata and Mangifera andamanica.

Plant Materials and Methods
An exploration was undertaken in greater Andaman Islands, India to study the genetic diversity of Mangifera species during 2006.Observations were made on the morphological characters of the tree, leaves and fruits wherever it was available.The fruits of the three species of Mangifera viz., Mangifera andamanica, Mangifera camptosperma and Mangifera griffithii were collected and the observations were recorded on fruit weight, fruit length, fruit breadth and total soluble solids.An exploratory mission was again undertaken during 2014 to survey Shoalbay, Chauldhari, Naya Shahar and Chidiyatapu regions besides Mount Harriet, of the Andaman Islands.Wild species like Mangifera andamanica, Mangifera camptosperma and Mangifera griffithii were found distributed in specific isolated pockets, which were identified based on taxonomic keys.Bud sticks and leaf samples were collected from voucher accessions and foliage characteristics were recorded.In situ evaluation of leaf characters for samples collected from these regions and molecular characterization was carried out using total genomic Deoxyribonucleic acid-DNA isolated by the Cetytrimethylammonium Bromide-CTAB method [15], including Mangifera indica cultivars namely Kurukkan, Muvandan, Olour, Alphonso, Raspuri and Langra for phylogenesis; as DNA yields more phylogenetic information among the biomolecules.

PCR Amplification, Sequencing and Analysis
Fluorescence based PCR method [16] was used to amplify the microsatellites in a quick, accurate and efficient manner.Eight microsatellite markers of mango showing high PIC [17] were employed for amplification.SSR markers were amplified in 10 µl volume containing 25 -50 ng mango DNA-3 µl, Taq Buffer 10× (pH 9.0, 10 mM Tris with 15 mM MgCl 2 , 50 mM KCl, 0.01% Gelatin)-1.0 µl, 1mM dNTPs-1.0µl, 25 mM MgCl 2 -0.3 µl, forward labelled specific primer (5 µM)-0.1 µl each thus mixed four PCR products labelled with different fluorophores (FAM, VIC, NED and PET), reverse primer (5 µM)-0.1 µl each, nuclease free water 3.6 µl and 0.3 µl of Taq DNA polymerase (Bangalore Genei, India).PCR was performed on Life Pro Thermocycler (Bioer, Hangzhou, China) with the following temperature profile: 94˚C for 2 min followed by 35 cycles at 94˚C for 30 s, 30 s annealing temperature of 55˚C and 72˚C for 1 min.A final extension reaction was allowed to proceed at 72˚C for 5 min.Amplified products were initially separated on 3% agarose gel for confirmation of the amplification.These samples were separated on the automatic 96 capillary automated DNA Sequencer (ABI 3730) at M/s Eurofin Ltd. facility at Bengaluru.The molecular data was used to generate dendrogram with DARwin 5.0 (Figure 1) [18].
The phylogeny study comprised of five different Mangifera species, namely M.indica, M. griffithii, M. camptosperma, M. odorata and M. andamanica.Within M. indica a total of six cultivars; three polyembryonic cultivars viz Kurukkan, Muvandan and Olour and 3 monoembryonic cultivars viz Alphonso, Raspuri and Langra, were taken for the study.
The PCR reaction mixture compositions and amplification conditions varied among the markers (petB-petD; trnC-trnF; rps16 and ETS) taken for the study.
The rps16 intron was amplified with the rps16F and rps16R2 primers as described [19].For the trnL-trnF intergenic spacer, we used the primers from [20].The petD region was amplified with the forward primer PI-petB1365F and the reverse primer PIpetD738R [21].The PCR reaction mixture and conditions were used according to [22] for rps16 and trnL-trnF, and [21] for petB-petD intergenic spacer.Amplification of ETS region was done according to [23].All the PCR products were separated on 3% agarose gel electrophoresis to check for efficiency of amplification and to ensure that only a single product of the expected size was present, PCR products were purified by MinElute PCR Purification Kit (Qiagen).PCR cleanup products were then sequenced using the same primers as used in initial PCR amplification using in ABI 3730 XL automated sequencer through Big Dye terminator sequencing technology.
Sequence data of all the products were first confirmed through the homology search BLAST of NCBI database and aligned using ClustalW program [24] of BioEdit.Then these aligned files were further converted to.meg file, exported to the MEGA 4.0 software environment for further analysis to construct the phylograms.
The aligned sequence data matrix was analyzed using the Molecular Evolutionary Genetic Analysis (MEGA) 4.0 software program [25].The phylogeny was analyzed using Maximum Parsimony (MP) method, which is a character based on computational approach.The bootstrap test of Phylogeny was computed for all the nuclear and chloroplast markers with 2000 iterations.

Results and Discussion
Identification of Mangifera species involves the observation of vegetative and fruit characteristics.The initial identification is carried out based on the growth habit of the tree, other morphological features and the vernacular names.One of the main problems in the identification is the variation in the morphological characters because of the seedling origin of the progenies and the in-built heterozygosity.The trees belonging to the Mangifera genus are generally tall growing.The phylogenetic taxonomy carried out has shown that the species are included under two sections, depending on the presence or absence of a prominent disc in between the stamen and the carpel [5].

Morphological Analysis
The first survey was carried out during 2006 in the South Andamans and Middle Andaman.Several Mangifera indica varieties were observed in these regions.The survey of Chauldhari and Jirkhatang regions resulted in locating one and two specimens of M. andamanica respectively.One tree of Mangifera camptosperma was located near the coastal region almost by the side of sea at Jirkhatang.In Chidiya Tapu region, one tree of Mangifera griffithii was located.The specimen was evaluated for leaf characteristics and passport data was recorded (Table 1; Table 2), these were further characterised by using microsatellitre markers (Table 3).And compared with other wild species M. odorata maintained in the field genebank.Mangifera andamanica (Figure 3(a)): It is a huge tree with oval shaped fruits borne in clusters.The peel of the fruits was observed to be thin, orange coloured.The pulp is fibrous and juicy with sweet taste (TSS: 22˚ Brix) and yellow in colour.The average weight of fruits was 11.57g.The leaf tip was found to be acute and the leaf base was cuneate with flat margin.The leaf length and breadth were found to be 17.7 and 4.8 cm respectively.The fruit length and breadth were observed to be 3.15 cm and 2.07 cm respectively.

Mangifera camptosperma (Figure 3(b)):
It is a tall tree with sparse foliage and fruits were found to be totally flat and round in shape.The fruit pulp was found to be very fibrous and non-edible.The fruit weight was around 51.8 g.The fruit length and fruit breadth were around 9.86 g and 29.5 g respectively.The pulp recovery was 23% with hardly any edible pulp.

Mangifera griffithii (Figure 3(c)):
The species Mangifera griffithii was located on the hill top of Mt.Hariett.The tree was found to be moderately vigorous.The leaves resembled the leaves of Anacardium occidentale leaves with obtuse base and round tip.The leaves were also leathery, with a length of 13.5 cm and a breadth of 4.3 cm.The fruits were small oval shaped with attractive purple peel color.The fruit weight was observed to be 11.2 g and the pulp recovery was 12%.Although the fruits were observed to have very little pulp, they were juicy with sweet taste (22.6˚Brix).

Mangifera odorata (Figure 3(d)):
The species Mangifera odorata is characterized by the distinct odour of the fruit.It possibly represents hybrid forms between M. indica and M. foetida (Ding Hou, 1978).The fruits on an average weigh about 200 g and the TSS is about 21.4˚ Brix.The pulp recovery is less than 55 per cent.The fruits on ripening have greenish purple peel colour and yellow pulp colour.The panicle is characterized by sparse flowers contrary to the dense flowers of Mangifera indica.Mangifera odorata is also polyembryonic in nature; compared to other wild species the edible quality in M. odorata is far superior, although, much inferior to Mangifera indica.
Mangifera indica (Figure 3(e)): It is a native of the Indian Peninsula with a spreading tree of 20 -45 m; all parts glabrous except inflorescence.Leaves were thinly coriaceous or membranaceous varied in size and shape.Fruit is Drupe large, oblong or subreniform; flesh thick with sweet juice; highly variable in size, shape and coloration of the epicarp in the different cultivated varieties; stone fibrous, very hard, the fibrous are very long in the wild types and inferior cultivated types; cotyledons two, rarely many, unequal.M. indica is economically the most important species of the genus, as it bears one of the most delicious tropical fruits, the mangoes.
In the second exploratory survey carried out in the Andaman Islands, resulted in locating the variants of M. griffithii, in Shoalbay region apart from Mt Harriet.The leaf and fruit characteristics of the two specimens were observed to be similar.The original specimen of M. griffithii from Mt. Harriet (Figure 2 and Figure 3) had slightly longer, wider leaf than the ones observed near the Shoalbay School.The apex of the leaves (Figure 3) observed near the Shoalbay School was slightly more pointed compared to the Mt Harriet leaf sample.The fruits were observed to be similar in both the types but the tree near Shoalbay School was taller compared to the one at  Mt. Harriet.Survey carried out previously by several workers [26]- [28] in the diversity rich regions of Mangifera indica has resulted in several seedling selections in mango, which shows that there is every chance that seedling variants with desirable traits can be identified.The exploratory surveys help in the location of useful types be it wild species or varieties having desirable traits in the places of their diversity.They also help in conserving these types in situ as well as ex situ.

Chloroplast Markers
The Phylograms that are generated for all the 3 chloroplast makers, used in the present study are in congruent to each other by resulting in similar clustering of Mangifera species.

petB-petD
The phylogram of MP (Figure 4) method resulted into two clusters of which M. odorata and M.andamanica have been clustered into one group leaving the rest 3 species into another that have been further subgrouped into 2 subclusters.Here M. camptosperma has been grouped into one subcluster leaving, M. griffithii and M. indica into the other.

trnL-trnF
The phylogram of MP (Figure 5) method resulted into two clusters of which M. andamanica clustered into one leaving the rest 4 species into another that have been further subgrouped into 3 subclusters leaving M. odorata and M. camptosperma into two individual subclusters with M. griffithii and M. indica into the other.

rps16
The clustering pattern of rps16 is similar to that of trnL-trnF.The phylogram by MP (Figure 6) method resulted into two clusters of which M. andamanica clustered into separate group leaving the rest 4 species into another that have been further subgrouped into 3 subclusters leaving M. odorata and M. camptosperma into two individual subclusters with M. griffithii and M. indica into the other.

ETS
The clustering pattern of ETS using MP (Figure 7) method resulted in the phylograms showing two main clusters containing M. andamanica in one cluster leaving the rest 4 species into another that have been further subgrouped into 2 subclusters leaving M. odorata into one subcluster and M. camptosperma, M. griffithii and M. indica into the other.

Phylogenetic Relationships among Mangifera Species
The phylogenetic relationships among Mangifera spp was earlier reported by [2] based on the morphology where he mentioned the existence of 41 valid species of Mangifera that had been classified into two different  sections viz section I and section II.Again in 1993 the most acceptable classification of Mangifera species has been described by [4], which includes 69 species of which 58 species have been classified into subgenera Mangifera and Limus with several sections Marchandra, Euantherae, Rawa and Mangifera under Mangifera subgenus and sections Deciduae and Perennis for the subgenus Limus.According to this classification M.odorata was classified as the section Perennis of the subgenus Limus leaving the other 3 species of the present study, which were classified into the two sections of the subgenus Mangifera; the section Mangifera for M.indica and the section Rawa for M. griffithii and M. andamanica.The remaining final Mangifera species taken in the study is M.camptosperma whose position was not mentioned in this classification.
The results of the present study are in congruence with Kosterman's classification by clustering M. indica and M. griffithii, belonging to the subgenus Mangifera, when compared to M. odorata which belongs to subgenus Limus.The clustering pattern of M.camptosperma with M. indica and M. griffithii shows that they share common ancestry and are evolutionary related hence the position of M. camptosperma has to be considered under the subgenus Mangifera.All the marker analysis in this study showed that M. andamanica grouped separately from the other Mangifera species of subgenus Mangifera showing that it doesn't belong to the genus Mangifera as earlier stated [29].The fruits of M. andamanica are very inferior in quality without any pulp.The shape of the fruits does not resemble any of the indica varieties and fruits have no edible pulp, being juicy and fibrous.The present study results indicate that the taxonomic position of M. andamanica should be reconsidered.

Conclusion
Chloroplast markers (petB-petD; trnL-trnF; rps16) and nuclear marker (ETS) clearly show that M. andamanica is not closely related to M. indica and M. griffithii which belong to subgenus Mangifera.This re-confirms the earlier objections which were raised by Mukherjee [29] about M. andamanica taxonomical position in this subgenus.This study also classifies M. camptosperma under subgenus Mangifera based on our analysis, whose position was not assigned earlier.Thus, finally we conclude that classification of M. andamanica under genus Mangifera needs reconsideration and M. camptosperma has to be included in the subgenus Mangifera.The climatic requirement for these species is very specific.The surveys carried out have shown that the tree observed in Shoalbay region is also M. griffithii.Due to the propagation by seeds, there is difference in certain morphological features between the two specimens.The in situ evaluation, collection and ex situ conservation has to be taken up on priority; otherwise we may lose these species.

Figure 4 .
Figure 4. Phylogram of Mangifera species generated from petB-petD marker using Maximum Parsimony method, with a Bootstrap value of 2000 iterations, of Molecular Evolutionary Genetic Analysis (MEGA) 4.0 program.

Figure 5 .
Figure 5. Phylogram of Mangifera species generated from trnL-trnF marker using Maximum Parsimony method, with a Bootstrap value of 2000 iterations, of Molecular Evolutionary Genetic Analysis (MEGA) 4.0 program.

Figure 6 .
Figure 6.Phylogram of Mangifera species generated from rps16 marker using Maximum Parsimony method, with a Bootstrap value of 2000 iterations, of Molecular Evolutionary Genetic Analysis (MEGA) 4.0 program.

Figure 7 .
Figure 7. Phylogram of Mangifera species generated from ETS marker using Maximum Parsimony method, with a Bootstrap value of 2000 iterations, of Molecular Evolutionary Genetic Analysis (MEGA) 4.0 program.

Table 2 .
Passport data of the Mangifera griffithii located at Shoalbay and Mount Harriet.