4.1. Sites and morphological characterization
A recent survey to Dailong Forest Tamenglong district reported the natural wild population of C. indica, locally known as ‘Garuanthai’ among the Rongmei tribes. In Fig. 2, the collection site of the C. indica in the present study is shown in the red triangle while the sites of earlier explorations are depicted in red circle. Among the earlier exploration, the presence of the species were recorded only in the Garo hills of Meghalaya (Singh and Singh 2003). Our present study reports the re-occurrence of this species in Manipur in Tamenglong District where it was said to be found in earlier studies. Such findings show that detailed research and studies should be carried out in various parts of NE India which remain largely unexplored because of its inaccessibility and difficult topographic nature. The meteorological data of Dailong forest area also support the microclimate of C. indica with low temperature, high rainfall, and humidity which is similar to that of the specific microclimate of Garo hills, which may be the probable reason for occurrence of the plants in this forest.
Morphological characterization of citrus often creates confusion among the taxonomists as there are innumerable hybrids and mutants of citrus due to frequent hybridization, mutation, and polyploidy among the citrus species throughout the world (Susheel Kumar et al. 2013). In the present study, the morphological characters are characterized based on the Descriptors for citrus (IPGRI 1999) protocol which is a standardized tool for the characterization system. In earlier works, morphological classification has relied on a combination of few characters of leaf, seed, flower, and fruit (Swingle 1943; Tanaka 1954; Tanaka 1977; Bhattacharya and Dutta 1956; Hodgson 1965; Swingle and Reece 1967; Singh 1967; Singh and Nath 1969). In the present survey, all the C. indica species observed have identical morphology except for the height and nature of the plants viz., bushy or climber, tall or stunted. The tallest plants were found in Jhoulangpang area of the forest, where the density of the plant is more compared to other locations where the plants are stunted. Potential causes for the differences may be variations in the age of the plants as the forest area was used for jhum cultivation till 1978 except the Joulangpang area. Maybe the plants were newly regenerated from seeds or stem and started acclimatizing in the area. Most of the plants were found in shady and moist slopes under the canopy of the forest. Kumar et al. 2010 observed that the plants preferred cool and shady places under the canopy of other trees. The quantitative and qualitative characterization of leaf, seed, and fruit samples showed that the characters of C. indica described in the present study are more or less similar with the characters of the species found in Garo hills (Malik et al. 2006; Kumar et al. 2010) except for some minor variations. Such differences may be due to variations in meteorological data like temperature, precipitation, location, soil thickness and forest age (Yang et al. 2006).
4.2. Molecular characterization
In the four DNA regions analysed (trnL-trnF, psbI-psbK, matK-5′trnK spacer, and ITS), we detected the sequences obtained from the present study clustered with the C. indica that is found in Garo hills of Meghalaya. The emergence of the DNA barcoding technique has helped in calibrating current taxonomic resolution and provided a straightforward identification system when there exist troubleshooting in morphology-based taxonomy (April et al. 2011). The CBOL plant working group proposed to use rbcL and matK as the standard DNA barcode for plants (CBOL plant working group). However, this DNA barcode-based identification of biologically complex plant groups like Citrus remains challenging due to frequent natural hybridization. So the use of intergenic spacer of chloroplast regions had been preferred for the rapid identification of other secondary citrus species (Mahadani and Ghosh 2014). The ITS region and trnL-trnF regions were successfully used to study the detailed phylogenetic and biogeography of the Zanthoxylum L. and Ivodea which belong to the Rutaceae family (Appelhans and Wen 2020; Appelhans et al. 2018). The ITS region, trnL-trnF intergenic spacer and matK-5′trnK spacer regions were successfully used to resolve the taxonomic uncertainty of Aurantoid Rutaceae (Nguyen et al. 2019; Bayer et al. 2009). The chloroplast psbK-psbI intergenic spacer was proven not only to be the most reliable barcode (Suzuki et al., 2014) but it was also useful for phylogenetic relationships analysis at the taxonomic level (Enan and Ahmed 2016). Moreover, chloroplast intergenic spacer psbK-psbI was included as a potential candidate locus for DNA barcode because of its evolutionary rates and high capability to distinguish independently evolving beings corresponding to taxonomic species (Lahaye et al. 2008).
4.3. Conservation
Citrus indica is unique citrus within the true citrus species. Known for its poor regeneration and endemic nature, this citrus genotype preferred a distinct microclimate for its survival and existence. Though the presence of C. indica has earlier been reported from different restricted pockets in NE India, its existence and conservation cannot be ascertained in many of these locations now. After a long period, the prevalence of C. indica was only recently reported from Behali Reserve Forest of Assam (Dipankar et al. 2018) and thus it is scantily reported even from NE India though the region is regarded as a natural home for Citrus. An assessment of ecological conditions, habitat, and interaction with people inhabiting the locality will enable us to give a clear view for the survival of such rare and endemic citrus genotype. Apart from this, morphological data and clear molecular analysis remain essential for correct identification and further conservation efforts.
Various wild and semi-wild citrus grow in the NE India forest regions which still need to be explored for their proper identification, collection, and conservation (Sharma et al. 2004). Dailong is one such area, which is endowed with a rich biodiversity of flora and fauna. The forest consists of montane wet temperate, tropical evergreen, semi-evergreen and riparian forest. C. indica is one of the seven species of Indian citrus enlisted in the endangered list, which requires urgent and special consideration for conservation because of its endemic nature and also the degree of threat perception is high (Malik et al. 2006). In situ conservation seems to be the most suitable option for C. indica as the previous study by Malik et al. 2006 reported that the plants growing outside its habitat did not show flowering fruiting. This can be related to the complex phenomena of the lack of adaptation to the man-made environment (Zeven and de Wet 1982). Kumar et al. 2010 also observed that after propagation of C. indica from seeds, its growth and survival outside its natural environment became difficult. A study in Canary Island, Spain recommended reintroduction, habitat management, and protection as the most important tools for conservation and management of endangered plants (Marrero-Gómez et al. 2003). However ex situ methods of conservation are also given importance to protect the endangered species (Laskar et al. 2009). National Bureau of Plant Genetic Resources (NBPGR), Delhi has preserved the pollens and seeds of C. indica in Cryogene Bank for long term preservation (Malik et al. 2006). Such an initiative for ex-situ germplasm conservation of citrus can help in the rehabilitation of endangered species (Kumar et al. 2010). Cryopreservation technique and other ex situ conservation actions have been developed for long-term conservation of critically endangered species Rubus humulifolius in Finland (Edesi et al. 2020). Sharma et al. 2004 also suggested the need for more nature reserves, gene sanctuaries, and parks with the inclusion of citrus species. In Burkina Faso, the conservation strategy and survival rate of Zanthoxyllum zanthoxyloides seedlings were suggested to be managed by establishing nurseries, botanical gardens, and target places for safeguarding (Ouédraogo et al. 2019). Those sites were entrusted to the local communities thereby facilitating financial support for the local communities to encourage them to actively participate in the conservation of the species. Likewise, the conservation strategy for C. indica can also be implemented in the present study area. Moreover, any conservation strategies related to natural resources needs public education and proper awareness to the people residing in the area for maintaining a healthy environment (Kaya and Raynal 2001). Thus a holistic approach including both the ex-situ and in situ conservation methods should be developed to contribute to Citrus improvement programs (Malik et al. 2006).
Singh and Singh (2003) also stated that the anthropogenic activities are causing a serious threat to the citrus biodiversity in the NE India region for which, it has been classified as a hotspot. C. indica is seldom cultivated and commercialized as the fruits are small, rarely edible (Bhattaccharcharya and Dutta 1956; Tanaka 1937). The present exploration in Dailong forest area also indicated no domestication and no utility of the species known to the local tribes. It remained as ordinary wild citrus to them with no economic importance. So there was no specific conservation policy taken up in that area particularly for the citrus species. However, in the Nokrek Biosphere reserve buffer zone area, preliminary domestication and conservation measures have been taken up. C. indica locally known as Memang Narang in Garo language is well known to the local tribes of this region and is used for various medicinal and ritual purposes. So the local tribes had cultivated in the area (Singh 1981; Malik et al. 2006). Maybe we can assume that the species had been conserved in Garo hills but not in other parts of the NE India because of its conservation by domestication by Garo tribes. In the other parts of NE India, there are no specific uses and medicinal values known to them. Also, the Rongmei tribes of Dailong Forest have not used the part so far. They know its existence in the forest as wild form; however, there is no domestication history of the species in the place so the conservation motive is very low to the local people as domestication is one of the important ways to conserve the germplasm of species. During the survey, C. indica was observed to be healthy and free from any kind of disease. Such property of C. indica can provide rootstock of high quality for other commercial citrus varieties in the future (Malik et al. 2006). Conservation of C. indica was already initiated around ten decades ago in the Garo hills of Meghalaya by establishing the Citrus Gene Sanctuary which is a part of Nokrek Biosphere Reserve (Singh 1981). Likewise, the protection and rehabilitation of germplasm of the present study area should be proposed and Dailong forest should be recognized as an in situ ‘Citrus Sanctuary’.
Detailed interviews with the local tribes and elders of Dailong area revealed that they have been practicing conservation activities for many generations in the form of protected sacred groves called 'Raengan'. C. indica was reported to be found in abundant according to the elders of Dailong village but now the plant has shrunk its population due to earlier destruction of forest for jhum cultivation. However, an initiative was taken way back in 1978 by Rongmei tribes of Dailong Village by preventing jhum cultivation and other exploitation from the forest. Further discussion was also continued with the members of Dailong Ecology & Environment Preservation Society (DEEPS; local NGO since 1978), we came to know that they played a critical role in spreading awareness among the Rongmei tribes for forest conservation. Despite all the efforts of the local tribes to protect the forest, recent development has hampered the forest ecosystem. There are road and railway construction, and extensive illegal timber logging which led to the loss of large forest area and ultimately leading to the loss of rare species. Even though the local tribes are protecting the forest area, adequate conservation measures have not been implemented so far from the government or other related organizations. There is an urgent need for identifying the priority areas for protection. In southern China, priority protected areas (PPAs) planning methods have been proposed to conserve endangered and threatened plant species in the identified locations (Wang et al. 2015).
Efforts should be given in collection, characterization, and conservation of the wild and semi-wild species of citrus in NE India. The results of our present study try to give insight to develop a conservation management policy for such wild and endangered species. Future studies including these species will give deeper insights into their evolution and geographic origin. It is reported that C. indica is inferred to have originated from NE India region, further studies can be explored whether the ancestor species colonized which location first (likely from Nokrek, Meghalaya) followed by dispersal to other parts of other NE India. In the present study, results derived from morphology and sequence data indicated that ‘Garuanthai’ is identified as C. indica which is grown in wild habitat in Dailong forest, Manipur. The present work also supports the evidence of the origin of citrus in NE India thereby locating a new habitat of the ancestral citrus species. Both the in situ and ex situ conservation strategies should be approached to protect and conserve this species. These measures should also involve the local population by giving them proper information and awareness campaigns about the importance of habitat conservation and management to protect this precious habitat of C. indica.