Introduction
The genus Citrus L. (Family: Rutaceae) has its origins in the northeastern regions of India, northern Burma, Yunnan, and neighbouring areas of China, making these areas as a vital center of origin for diverse Citrus species (Gmitter and Hu, Reference Gmitter and Hu1990; Wu et al., Reference Wu, Terol, Ibanez, López-garcía, Pérez-román, Borredá, Domingo, Tadeo, Carbonell-Caballero, Alonso, Curk, Du, Ollitrault, Roose, Dopazo, Gmitter, Rokhsar and Talon2018). Citrus fruits are renowned for their dietary, nutritional, therapeutic and cosmetic properties and are also good sources for important bio-molecules like citric acid, flavonoids, phenolics, pectins, limonoids, ascorbic acids, etc. (Dugo and Di Giacomo, Reference Dugo and Di Giacomo2002; Kumar et al., Reference Kumar, Jena and Nair2010). Citrus is the third most important fruit crops that occupy larger area and production after Mango and Banana in India. In India, north-eastern Himalayan region and foothills of the central and western Himalayan tracts are rich sources of Citrus genetic diversity which include both cultivated and wild species (Malik et al., Reference Malik, Chaudhury, Dhariwal and Kalia2006). The North-east region of India (comprising eight states such as Assam, Arunachal Pradesh, Meghalaya, Manipur, Mizoram, Nagaland, Sikkim and Tripura) is considered as biodiversity hotspot of Citrus and ancestor for most of the cultivated Citrus (Govind and Yadav, Reference Govind, Yadav, Singh and Ghosh1999; Ray and Deka, Reference Ray and Deka2000; Ghosh, Reference Ghosh2007), from where spread has been taken place to other regions of the world. Multiple closely related but distinct cultivars of Citrus are grown in more than 140 countries around the world (Ma et al., Reference Ma, Zhang, Sugiura, Kato, Talon, Caruso and Gmitter2020).
Northeast India is recognized as a Centre of origin for many Citrus species and their natural hybrids and was documented by numerous earlier studies on their taxonomic classification (Bhattacharya and Dutta, Reference Bhattacharya and Dutta1956; Singh, Reference Singh and Tripathi1984, Reference Singh, Singh and Singh2002; Nair and Nayar, Reference Nair, Nayar, Hajra, Nair and Daniel1997) and genetic diversity (Shampru and Chauhan, Reference Shampru, Chauhan, Singh, Chauhan and Mundar2000; Sharma et al., Reference Sharma, Hore and Gupta2004; Malik et al., Reference Malik, Chaudhury, Dhariwal and Kalia2006, Reference Malik, Chaudhury, Kumar, Dhariwal and Bhandari2012). Based on different Citrus species listed in earlier literatures (Index Kewensis 1985–1988 and Kew Index 1986, 1987 and Botanical Survey of India, Shillong), this north-eastern India harbour about 23 out of 27 species reported in India besides one subspecies and 68 varieties (Sharma et al., Reference Sharma, Hore and Gupta2004). It includes both several indigenous varieties or land races of cultivated species [jambhiri (C. taitensis Risso; C. jambhiri Lush.), karna or khatta orange (C. karna Raf.), sour pummelo (C. megaloxycarpa Lush.), hill lemon (C. pseudolimon Tanaka), ada-jamir or ginger Citrus of Assam (C. assamensis Dutta and Bhattacharya) etc.] found mostly in their home gardens (Kumar et al., Reference Kumar, Jena and Nair2010) and wild or indigenous species (C. ichangensis, C. indica, C. latipes and C. macroptera var. annamensis) occur on the Khasi hills and its adjoining areas (Nair and Nayar, Reference Nair, Nayar, Hajra, Nair and Daniel1997; Malik et al., Reference Malik, Chaudhury, Dhariwal and Kalia2006; Kumar et al., Reference Kumar, Jena and Nair2010; Hazarika, Reference Hazarika2012). A world-famous C. medica is found in wild and semi-wild state at the foothills of the East Himalayas and North-east India (Bhattacharya and Dutta, Reference Bhattacharya and Dutta1956; Tanaka, Reference Tanaka1977; Nair and Nayar, Reference Nair, Nayar, Hajra, Nair and Daniel1997).
Citrus fruits thrive well in tropical and sub-tropical climates with warm to hot summers and mild winters. North-eastern Indian State of Meghalaya with its hilly areas of Garo, Khasi and Jaintia hills are known for its Citrus fruits, including the Khasi Mandarin, C. indica, and C. medica. It is also home to the Nokrek Biosphere Reserve, which is recognized as a “National Citrus Gene Sanctuary” by UNESCO in May, 2009 further underscores the region’s importance in Citrus genetic diversity. In 1981, an effort by ICAR-National Bureau of Plant Genetic Resources (NBPGR), New Delhi through systematic survey had identified the presence of wild populations of Indian wild orange Citrus indica in Tura hills of Meghalaya State, leading to the establishment of the site as National Citrus gene sanctuary to protect them under natural habitat (Singh, Reference Singh1981). This Nokrek Biosphere Reserve (820 km2 which consists of Core Zone [47.48 km2 of Nokrek National Park]; Buffer Zone [227.92 km2]; Transition Zone [544.60 km2]) area is located on Tura range of Meghalaya plateau with average altitude of 600 m (Nokrek peak in Garo hills with altitude of 1412 m), which includes two National Parks located in Garo Hills namely, the Nokrek in West Garo Hills and the Balpakram in South Garo Hills. It protects the important flora and fauna found in evergreen and semi-evergreen forest areas including C. indica and other citrus species (https://indianculture.gov.in/node/2537419).
In case of Citrus, Meghalaya occupies the largest area and production among North-east Indian States (Singh, Reference Singh, Shyam and Naqvi2001) and is also home to many cultivated, semi wild and wild form of Citrus species (C. jambhiri, C. medica, C. medica f. limon [C. limon], C. karna, C. nobilis subf. reticulata [C. aurentifolia], C. indica, C. aurantium, C. sinensis [C. aurantium subf. sinensis], C. maxima [C. grandis], C. assamensis, C. macroptera, C. latipes, C. juko, C. kinesu, C. sechen, C. serotina and C. tanaka) (Sharma et al., Reference Sharma, Hore and Gupta2004). Among them, the Citrus wild species (C. indica, C. latipes, C. macroptera, C. assamensis, C. megaloxycarpa and C. medica) with good agronomic traits, besides having high socio-economic status and health benefit found in this state could be useful for future Citrus breeding programme (Malik et al., Reference Malik, Kumar, Singh, Dhariwal and Chaudhury2013).
Wild species of crops and its wild relatives (CWRs) played a crucial role in directly or indirectly to several crop improvement programmes through breeding methods and also enhanced food security by means of supplying beneficial traits lacking in modern cultivars (Moyers et al., Reference Moyers, Morrell and McKay2018). Hence, the conservation of these valuable CWRs is critical at present climate change scenario. However, the lack of clarity on the many genetic factors involved (population structure, demography, inbreeding, introgression and genetic load) lead to endangered status or extinction of that particular species. These factors making complications while developing tangible recommendations for conserving a species under vulnerable including Citrus (Wang et al., Reference Wang, Cao, Liu, Xiao, Hu, Xu, Chen, Ma, Ye, Chai, Guo, Larkin, Xu, Morrell, Zhou and Deng2023). Wild Citrus species (C. icahangensis Swingle [Disease and cold resistance; rootstock/graft], C. indica [Progenitor, rootstock/graft], C. jambhiri [Engl.] Lush. [rootstock/graft, disease resistance], C. latipes [Cold tolerance]) with economically important traits could be useful in development of improved varieties related to yield, quality and resistant to biotic and abiotic stresses in future (Pandey et al., Reference Pandey, Tomer, Bhandari and Pareek2008; Hazarika, Reference Hazarika2012). Keeping these in view, the present study is undertaken with the following objectives:
1) To explore and collect the Citrus species diversity of North-eastern Indian State of Meghalaya
2) To document the characteristic features of Citrus species diversity collected from North-eastern Indian State of Meghalaya using IPGRI descriptors for their Conservation and Posterity use
3) To compare different Citrus species collected from North-eastern Indian State of Meghalaya based on morphological key traits for effective PGR management
Materials and methods
Field survey, exploration and collection
Field surveys and exploration trips were conducted for collection and documentation of diverse Citrus germplasm accessions comprising wild, semi-wild and cultivated species across various natural habitats of North-eastern Indian State of Meghalaya, the home of National Citrus gene sanctuary over a 2-year period during 2021–22 and 2022–23. The collection sites comprise of various villages encompassing Nine (9) districts of Meghalaya State namely, East Garo hills, West Garo Hills, North Garo hills, South Garo Hills, West Khasi Hills, East Khasi Hills, East Jaintia Hills, West Jaintia Hills and Ri Bhoi. Fruits of different Citrus germplasm were harvested at their peak maturity stage. List of Citrus germplasm accessions collected from Meghalaya is presented in Table 1. Detailed field passport data including GPS coordinates were recorded for each accession and are presented in Table S1. A selective sampling method was employed for collecting of germplasm. Where individual plants treated as distinct accessions, because samples were collected from different natural habitats with varying GPS coordinates, especially altitude and each sample collected representing from more than or equivalent to 10 km2 of area under survey.
The list of 57 accessions of 13 Citrus species collected from Meghalaya, North-eastern India over 2-year period during the 2021–22 to 2022–23
* NGH – North Garo Hills; WGH – West Garo Hills; SGH – South Garo Hills; EGH – East Garo Hills; WKH – West Khasi Hills; EKH – East Khasi Hills; EJH – East Jaintia Hills; RB – Ri Bhoi District; WJH – West Jaintia Hills.
IPGRI descriptors observed
A total of 48 traits (16 quantitative and 32 qualitative) were recorded for the 13 Citrus species collected under study. These traits were categorized into four groups: tree (6 quantitative, 0 qualitative), leaf (4 quantitative, 7 qualitative), fruit (8 quantitative, 16 qualitative) and seed (4 quantitative, 3 qualitative). Data collection was conducted using the IPGRI descriptors (IPGRI, 1999). For each species, one tree, three leaves and three fruits were sampled. Tree and leaf traits were recorded directly at the collecting sites of the mother plants (in situ), while seed and fruit traits were recorded at ICAR-NBPGR Regional Station, Shillong, after being transported and processed.
Data analysis
Data observed on 16 quantitative traits were subjected to basic descriptive statistics and multivariate analysis (cluster and principal component analysis) performed using PAST3 software (Hammer et al., Reference Hammer, Harper and Ryan2001).
Results
Field survey, exploration and collection
Field surveys and exploration trips were made during 2021–22 and 2022–23 across 29 villages at 9 districts in North-eastern Indian State of Meghalaya representing hill areas namely, Garo, Khasi and Jaintia hills. A total of 57 accessions of Citrus belonging to 13 different species were collected from altitudes ranging from 55 to 1698 m from MSL (Fig. 1 and Table S1). Distribution pattern of collected Citrus accession on basis of species, fruit group and district are presented in Fig S1. The collected species under study comprised of four major groups of Citrus: (1) lime and lemon (44%) – C. aurantifolia, C. jambhiri, C. limon, C. pseudolimon, (2) mandarin & orange (32%) – C. reticulata C. sinensis, C. aurantium, (3) papeda (12%) – C. latipes, C. macroptera) and (4) pummelo and others (12%) – C. grandis, C. megaloxycarpa and C. indica (Fig. S1). In terms of geographical distribution of collected Citrus accessions, the Garo hills yielded the maximum number of accessions – 26 (46% of total) followed by Khasi hills – 22 (38%), Ri Bhoi – 5 (9%) and Jaintia hills – 4 (7%). In terms of species-wise, C. reticulata was represented by the highest number of accessions (12) followed by C. pseudolimon and C. macroptera (6 each), whereas, C. medica (cultivated) and three wild Citrus species (C. indica, C. latipes and C. megaloxycarpa) were the least represented, with one accession each. The biological status and nature of different Citrus species collected from North-eastern Meghalaya is presented in Fig. S2.
Map showing distribution of Citrus accessions collected from North-Eastern Indian State of Meghalaya (Red circle denotes the districts where Citrus samples collected).
Documentation of Citrus species diversity using IPGRI descriptors
A total of 48 traits (16 quantitative and 32 qualitative) recorded by using the IPGRI descriptors (1999) for the 13 Citrus species collected under present study are presented in Table 2, S2 and S3. Analysis of 16 quantitative traits showed many diagnostic features among the 13 Citrus species (Table 2). Traits with more variability on basis of coefficient of variation (%) observed among 13 Citrus species under present study were followed as: rind thickness (93.0), number of seeds per fruit (82.5), fruit weight (65.4), mid-diameter of the axis (57.9), pulp vesicle length (46.3), seed weight (44.5), leaf blade length (33.7), leaf blade width (31.2) and fruit height (21.7). Most of the Citrus species under study had exhibited large leaf length (> 75 mm), whereas, the wild species C. latipes had showed smaller leaf length (29.7 mm). Petioles were present only in five species, with lengths ranging from 3.6 mm in C. grandis to 74.9 mm in C. macroptera. Comparatively, Citrus species under acid group showed bigger fruit with thicker rinds than all other groups except a species under papeda group – C. macroptera. Thirty-two qualitative traits of Citrus comprising of tree, leaf, fruit and seed traits observed using the IPGRI descriptors (1999) under study showed many diagnostic features among the collected 13 Citrus species (Table S2 and S3; Fig. 2).
Variability in tree form, leaf, fruit and seed of different Citrus species collected from North-Eastern Indian State of Meghalaya.
The mean value of 16 quantitative traits of 13 Citrus species collected from Meghalaya, North-eastern India
* LL- Leaf blade length (mm); LW- Leaf blade width (mm); PL- Petiole length (mm); PW- Petiole width (mm); FH- Fruit height (mm); FD- Fruit diameter (mm); FWt- Fruit weight (g); RT- Rind thickness (mm); NoS- Number of segment; MDA- Mid diameter of the axis (mm); PVL- Pulp vesicle length (mm); PVW- Pulp vesicle width (mm); NSF- Number of seeds/fruit; SL- Seed length (mm); SW- Seed width (mm); SWt- Seed weight (g).
** Indigenous and Wild.
Comparison of Citrus diversity at species level
The distinguishing diagnostic features identified among collected Citrus species are presented in Fig. 2. In case of tree habit, five species (C. jambhiri, C. reticulata, C. aurantium, C. grandis, C. indica) showed erect nature, whereas, the remaining eight (C. aurantifolia, C. limon, C. pseudolimon, C. medica, C. sinensis, C. macroptera, C. latipes and C. megaloxycarpa) showed spreading type. Among the 13 Citrus species, only two cultivated species (C. sinensis and C. grandis) showed spineless trait. Nine species showed straight spine shape, whereas, two (C. jambhiri and C. megaloxycarpa) showed as curved/angled shape.
Regarding leaf characteristics, most of the Citrus species under study showed sessile type of leaf lamina attachment where petioles are absent. However, brevi-petiolate (short petiole) and longi-petiolate (long petiole) form observed in three (C. aurantifola, C. aurantium and C. grandis) and two (C. latipes and C. macroptera) species, respectively. Three forms of leaf apex were observed among the 13 Citrus species: Obtuse type in nine species, retuse/ rounded in two species (C. jambhiri, C. latipes) and acute in two species (C. reticulata and C. sinensis). In case of leaf margin, sinuate type was observed in 5 Citrus species (C. aurantofolia, C. limon, C. pseudolimon, C. reticulata, C. grandis), entire in three (C. sinensis, C. aurantium, C. macroptera), dentate in two (C. medica, C. latipes) and crenate in one (C. jambhiri) species. Three types of lamina shape were observed: ovate in seven species (C. aurantifolia, C. limon, C. sinensis, C. aurantium, C. latipes, C. macroptera, C. indica), obovate in four species (C. pseudolimon, C. medica, C. grandis, C. megaloxycarpa) and elliptic in two species (C. jambhiri, C. reticulata).
Regarding fruit characteristics, five Citrus species each had showed ellipsoid (C. aurantifolia, C. jambhiri, C. limon, C. pseudolimon and C. medica) and obloid (C. reticulata, C. aurentium, C. latipes, C. macroptera and C. indica) fruit shape, whereas remaining three species (C. sinensis, C. grandis and C. megaloxycarpa) showed spheroid shape. Four types of fruit colour were observed among the 13 Citrus species under study as green-yellow (4), yellow (6), orange (2) and dark orange/ scarlet (1). Seven Citrus species (C. aurantifolia, C. jambhiri, C. limon, C. pseudolimon, C. medica, C. reticulata and C. sinensis) showed mammi-form fruit apex, whereas, truncate in four species (C. aurantium, C. grandis, C. megaloxycarpa and C. indica) and rounded in two species (C. latipes and C. macroptera). Three types of adherence of mesocarp to pulp in fruit were observed among 13 Citrus species under study as weak (3), medium (2), and strong (8). All species showed green with light yellow coloured pulp in fruit except three species (C. reticulata – Orange; C. grandis – Orange-red; C. indica – Yellow). Most of the Citrus species showed smooth seed surface except two (C. macroptera, C. megaloxycarpa) species where rough wrinkled type was found. All species had showed cream coloured seed except three species (C. limon, C. megaloxycarpa and C. indica) with green colored seed.
Comparison of Citrus diversity at major fruit group level
The species collected under study could be categorized into four major groups of Citrus based on their characteristics: (1) lime & lemon (44%) – C. aurantifolia, C. jambhiri, C. limon, C. pseudolimon, (2) mandarin & orange (32%) – C. reticlulata, C. sinensis, C. aurantium, (3) papeda (12%) – C. latipes, C. macroptera) and (4) pummelo and others (12%) – C. grandis, C. megaloxycarpa and C. indica (Fig. S1). The distinguishing diagnostic features identified for major fruit groups are as follows: acid lime (ellipsoid fruit shape with mammiform apex, smooth seed surface), oranges/ mandarins (spheroid/obloid fruit shape, orange to green yellow coloured fruit, smooth surface texture of fruit epicarp and seed, conspicuous oil glands with medium aroma), papeda (spreading tree habit with densely branched, ovate shaped leaf with obtuse apex, long-petiolate leaf lamina attachment, obloid fruit shape with rounded apex, solid fruit axis with firm pulp) and pummelo (obtuse leaf apex, spheroid/obloid fruit shape with truncate apex, ovoid seed shape) (Table S2 and S3).
Cluster analysis
Based on 16 quantitative traits of 13 Citrus species observed under present study, the dendrogram was constructed by using jaccard’s similarity index based UPGMA method (Fig. 3). It grouped 13 Citrus species into 6 clusters as follows: (I) C. jambhiri; (II) C. limon, C. pseudolimon, C. grandis, C. megaloxycarpa, C. medica, C. sinensis; (III) C. reticulata; (IV) C. aurantium, C. latipes and C. macroptera; (V) C. indica, (VI) C. aurantifolia at jaccard’s similarity coefficient value of 0.24. At intra-cluster level, Citrus species belonging to pummelo were grouped together with higher similarity index value of 0.75. Comparatively, the other three groups such as acid lime, mandarins & orange and papeda showed high level of dissimilarity by grouping at lower similarity index value as 0.12 to 0.72, 0.20 to 0.36 and 0.30, respectively.
Dendrogram generated for 16 quantitative traits of 13 Citrus species collected from North-Eastern Indian State of Meghalaya using UPGMA method based on Jaccard’s similarity coefficient.
Principal component analysis
Based on 16 quantitative traits of 13 Citrus species observed under present study, the two-dimensional scatter plot was generated based on jaccard’s similarity coefficient using PCA analysis (Fig. 4). First, second and third principal components explained about 37.7, 20.5 and 15.1% genetic variation, respectively, present in 13 Citrus species as with cumulative variation of 73.3% (Table S4). Traits showed more weightage in three PCAs for grouping were as follows: PCA-1 [fruit height (mm) (0.37), fruit weight (g) (0.36), pulp vesicle length (mm) (0.32) and fruit diameter (mm) (0.32)], PCA-2 [petiole length (mm) (0.48), petiole width (mm) (0.48) and seed length (mm) (0.41)], PCA-3 [pulp vesicle width (mm) (0.52) and seed weight (g) (0.41)] (Table S5).
Two-dimensional scatter-plot of PCA ordination for 16 quantitative traits of 13 Citrus species collected from North-Eastern Indian State of Meghalaya using first two principal components.
Discussion
The passport data presented in Table S1 provides valuable insights into the distribution patterns of collected Citrus species within Meghalaya. Our Citrus sample collection sites covered almost whole state of Meghalaya and the collections also represented 13 different Citrus species out of 23 reported in North eastern India (Sharma et al., Reference Sharma, Hore and Gupta2004). Among them, two Citrus species (C. aurantifolia and C. megaloxycarpa) were not reported in earlier studies (Sharma et al., Reference Sharma, Hore and Gupta2004). Number of collections made per species under present study is directly related to conservation requirements of that particular species as it reflects their wilderness, endemism, vulnerability to genetic erosion to the extent of ease of access by people. The high or low representation of species among collections is directly related to the extent of variability and their frequency in occurrence at that particular area under field survey. Garo and Khasi hills represented by 10 Citrus species each, accounting for 46 and 38% of the total accessions collected, respectively. Whereas, Ri Bhoi and Jaintia hills have lower representation with only 3 species each contributing 9 and 7% of the total accessions, respectively. Among 13 species collected, two cultivated species namely, C. reticulata and C. jambhiri were represented from 6 districts, out of 9 districts in Meghalaya. It indicated the richness of these two species in the different hills of State such as Garo hill, Khasi hill and Jaintia hill. South Garo Hills and West Khasi Hills are identified as key areas for wild Citrus species. Out of five wild species collected, these two districts represented four of them with two each as South Garo hills [C. indica (IC0642129); C. megaloxycarpa (IC0642127); both collected at Tongki Songmong village] and West Khasi Hills [C. medica (IC0642137) collected at Porla A village; C. latipes (IC0648305) collected at Pyndengnongbri village]. Given the concentration of wild species in these two districts, detailed surveys and documentation are crucial for effective conservation strategies for these valuable Citrus genetic resources.
Only one accession of C. indica Tanaka (from Tengki Songmong village, South Garo Hills district of Meghalaya) collected under present study reconfirming the decades-old survey on its spread as it is restricted to the Tura ranges of Garo Hills in this state (Singh and Singh, Reference Singh and Singh2003; Malik et al., Reference Malik, Chaudhury, Kumar, Dhariwal and Bhandari2012) compare to its distribution on five North-eastern Indian states namely, Meghalaya, Assam, Mizoram, Manipur and Nagaland (Bhattacharya and Dutta, Reference Bhattacharya and Dutta1956; Singh, Reference Singh and Tripathi1984, Reference Singh, Singh and Singh2002; Shampru and Chauhan, Reference Shampru, Chauhan, Singh, Chauhan and Mundar2000). However, a few plants only available in Behali Reserve Forest of Assam (Borah et al., Reference Borah, Kafley, Tangjang and Das2018) and also in Dailong Forest of Tamenglong district, Manipur (Devi et al., Reference Devi, Labala, Sanabam, Singh, Modak and Devi2022) revealed its conservation importance as a serious threats posed to its natural population by means of habitat destruction (jhum cultivation and introduction of exotic species) as well as extremely low genetic diversity, high habitat specificity, and low regeneration (Kumar et al., Reference Kumar, Jena and Nair2010). Hence, the diagnostic fruit traits identified under present study for this species (smooth surface texture, obloid shape, dark orange color, thin rind thickness with firm yellow pulp) could aid in identification of species at field level and also help to develop conservation strategies for those identified area for posterity-use. Furthermore, DNA barcodes (three chloroplast regions [trnL-F, psbK-I, matK-50trnK spacer] and one nuclear [ITS] region) developed for taxonomic identification and species conformity (Devi et al., Reference Devi, Labala, Sanabam, Singh, Modak and Devi2022) will enhance its conservation at in situ level.
High CV (>65%) observed on fruit traits (rind thickness, number of seeds per fruit, fruit weight) revealed the presence of considerable variation among collected Citrus species. Hence, traits related to fruit (size, shape, colour, apex shape, surface texture, rind thickness, axis, pulp colour and firmness) were found to be key diagnostic character for identifying explicitly one or more Citrus species or major fruit groups under present study. Fruit size could play a major role in distinguishing the Citrus at species level as large fruit (C. megaloxycarpa with fruit weight >500 g; C. pseudolimon, C. grandis and C. limon with fruit weight > 200 g) to small one (C. indica with fruit weight < 20 g). Whereas, fruit size and rind thickness could be more useful at fruit group level as acid group with bigger fruit (59.7 to 311.0 g) and thicker rinds (2.3 to 6.6 mm) than all other groups (14.3 to 265.3 g and 0.2 to 1.5 mm) except a species under pummelo group- C. megaloxycarpa for fruit weight (525.0 g) and papeda group- C. macroptera for rind thickness (9.0 mm). Besides fruit size and rind thickness, fruit (green-yellow [C. aurantifolia, C. aurantium, C. latipes, C. grandis], orange [C. reticulata, C. sinensis], dark orange/ scarlet [C. indica] and yellow for rest of the species) and pulp (orange [C. reticulata], orange-red [C. grandis], yellow [C. indica] and green with light yellow for rest of the species) colour could also be a better morphological marker for differentiating the Citrus species as obtained in our present study. This pigmentation plays a major role in visual identification of Citrus species at major fruit group level as limes are green/green-yellow, lemons are yellow, mandarins and oranges are orange/ red and pummelos are pale yellow or dark orange.
Citrus leaves have a rare trait known as phyllodes (a petiolar wings and/ or are modified petioles or leaf stems, which are leaf-like in appearance and function (Kaplan, Reference Kaplan1973; Ballve et al., Reference Ballve, Medina-Filho and Bordignon1997)) present only in five among 13 Citrus species reported under present study with a range of 3.6 × 2.1 mm (C. grandis) to 74.9 × 40.2 mm (C. macroptera). Hence, this leaf lamina attachment and petiole wing shape could also provide some valuable clues for their identification (brevi-petiolate [short petiole than lamina] form in three species [C. aurantifolia, C. aurantium, C. grandis] and longi-petiolate [long petiole than lamina] form in two species [C. latipes and C. macroptera]) at species level. This variation in petiole was also reflected in our cluster analysis by forming Cluster IV with two longi-petiolate species (C. latipes and C. macroptera) together and one brevi-petiolate species (C. aurantifolia).
Cluster analysis of 13 Citrus species collected from Meghalaya formed six clusters which had clearly distinguished them at fruit group level. However, it was not happened at major cluster levels, at least two groups (papeda [C. latipes and C. macroptera] at cluster IV and pummelo [C. grandis and C. megaloxycarpa] at cluster II formed groups at low [0.30] and high [0.75] jaccard’s similarity index levels, respectively) had formed at intra-cluster levels. This grouping at intra-cluster level might be due to more similarity in fruit traits (diameter, height, weight, pulp vesicle length) of these species at major fruit group level. Higher levels of complexity existing between Citrus species at inter and intra-cluster levels in forming groups might be owing to hybrid and/or admixture (pummelo introgressions into mandarins) nature of cultivated Citrus species. Earlier studies had also highlighted the existence of complexity in Citrus taxonomy and phylogeny as apomixis, wide cross-compatibility, high frequency of bud mutation, and long history of cultivation (Moore, Reference Moore2001; Pang et al., Reference Pang, Hu and Deng2003). A separate cluster formed by C. indica (Cluster V) under present study at low similarity index value (0.10) indicated its high level of dissimilarity or distance from other Citrus species except C. aurantifolia. It might be due to its tri-hybrid genome origin (C. medica [45%], papeda [30%] and C. reticulata [25%]) as concluded by Garcia-Lor et al (Reference Garcia-Lor, Luro, Ollitrault and Navarro2015) through structure analysis based on SSR, indel and SNP markers data.
A placement of individual Citrus species at specific location in dendrogram generated under present study is also endorsing the well-known involvement of three cultivated Citrus species (C. medica, C. grandis and C. reticulata) in forming majority of cultivated Citrus hybrid species belonging to mandarin (C. reticulata, C. sinensis and C. aurantium), acid lime (C. jambhiri, C. limon, C. pseudolimon) and pummelo (C. megaloxycarpa) groups (Scora, Reference Scora1975; Barrett and Rhodes, Reference Barrett and Rhodes1976; Nicolosi et al., Reference Nicolosi, Deng, Gentile, La Malfa, Continella and Tribulato2000; Hynniewta et al., Reference Hynniewta, Malik and Rao2014; Wu et al., Reference Wu, Terol, Ibanez, López-garcía, Pérez-román, Borredá, Domingo, Tadeo, Carbonell-Caballero, Alonso, Curk, Du, Ollitrault, Roose, Dopazo, Gmitter, Rokhsar and Talon2018). A recent study on Citrus genealogy had also reconfirmed the existence of three types of mandarins (Type-1 as pure mandarin; Type-2 as early admixture from pummelo; Type-3 as late admixture from pummelo) due to pummelo introgressions by studying 46 accessions through ancestry-informative single-nucleotide polymorphism (SNPs) derived from three species, C. medica, C. grandis and C. reticulata (Wu et al., Reference Wu, Terol, Ibanez, López-garcía, Pérez-román, Borredá, Domingo, Tadeo, Carbonell-Caballero, Alonso, Curk, Du, Ollitrault, Roose, Dopazo, Gmitter, Rokhsar and Talon2018). Traits related to fruit (weight, height, diameter, pulp vesicle length and rind thickness) were played a major role in grouping of 13 Citrus species under present study and it was evidenced from more weightage obtained by these traits in first two PCAs. The grouping formed in cluster analysis was confirmed by PCA analysis and it had also revealed the cumulative genetic variation of 73.3% from first three PCs. Hence, the diagnostic morphological key traits (fruit size, shape, colour, rind thickness, petiole presence and leaf morphology) identified for Citrus species at fruit group level under present study could provide clues to identify and compare species at field level and also be useful for curators at gene bank level for PGR management.
Future prospects
A recent study on ecological niche of cultivated kumquat (Fortunella spp. Swingle – are the smallest trees among Citrus species and is native to Southeast Asia) revealed that ‘the extensive gene flow from the cultivated population into its wild population might be owing to the spread of cultivated varieties by anthropogenic activities’ (Wang et al., Reference Wang, Cao, Liu, Xiao, Hu, Xu, Chen, Ma, Ye, Chai, Guo, Larkin, Xu, Morrell, Zhou and Deng2023). Hence, it emphasizes the role of land use and land cover (LULC) map of any area for prioritizing the conservation strategies on basis of several factors including anthropogenic activities (urbanization, environmental degradation, deforestation, high selection pressure and crop monoculture) and it may also lead to significant decline in Citrus diversity in the Meghalaya state in particular (Fig. S3) and NE region as a whole (Malik et al., Reference Malik, Chaudhury, Kumar, Dhariwal and Bhandari2012). Consequences of habitat threat led to declaration of seven wild Citrus species (Citrus indica Tanaka, C. latipes Tanaka, C. assamensis Dutta and Bhattacharya, C. ichangensis Swingle, C. megaloxycarpa Lushington, C. rugulosa Tanaka and C. macroptera) as threatened, vulnerable by the IUCN and also urged to conserve them for posterity use (Singh and Singh, Reference Singh and Singh2003; Malik et al., Reference Malik, Chaudhury, Dhariwal and Kalia2006; Hynniewta et al., Reference Hynniewta, Malik and Rao2011). Systematic characterization of any rare and wild species should include both morphological and molecular identification to confirm their taxonomic identity. Hence, the following suggestions are made for future research programmes on Citrus species:
• DNA barcoding: Taxonomic identity/species confirmation to be done by genomic validation to address the existence of high level of complexity in Citrus genetic resources.
• Upscaling of PGR activities: All PGR activities starting from field survey and exploration to end of its utilization including conservation aspects to be upscaled to pin-point the exact precise locations of micro-hot spots for in-situ conservation of those identified areas.
• Thematic map-based threat assessment: Thematic maps to be prepared by displaying the different types of land use and land cover (LULC) found in a specific area to show the current state of its cover and use at a specific point in time or period using remote sensing and Geographic Information System (GIS) software.
• Conservation strategies to be developed: On basis of the above thematic maps for threat assessment using LULC map of concerned areas where more anthropogenic activates taking place, effective conservation strategies to be developed for those identified endangered species include habitat protection and restoration, species reintroduction programs, control of invasive species, and public education and awareness. Moreover, suggesting sustainable land-use practices, research and monitoring, and international collaboration are crucial for its long-term success at grass root level.
• Community involvement: A holistic approach (both in situ and ex situ) to be prepared by involving all stakeholders including tribal community for effective implementation to achieve long-term success in this aspect, as they are often the most directly impacted by these conservation decisions.
• Reducing carbon footprint: Mitigating climate change, a significant threat to many species including Citrus, requires reducing greenhouse gas emissions and promoting sustainable practices.
Conclusion
This study delved into the rich Citrus diversity of Meghalaya, a North-eastern Indian state renowned as the home of the National Citrus Gene Sanctuary. Through comprehensive field surveys and morphological characterization, we collected and documented 57 accessions belonging to 13 Citrus species. Our findings revealed that Garo and Khasi hills are hotspots for Citrus diversity, harboring a significant number of species. Notably, South Garo Hills and West Khasi Hills emerged as critical regions for the conservation of wild Citrus species, particularly C. indica in South Garo Hills. Morphological traits such as fruit size, shape, colour, rind thickness, petiole presence and leaf morphology proved to be valuable diagnostic features for differentiating Citrus species. These traits can be instrumental in field identification, germplasm management, and breeding programmes. The study underscores the importance of continued exploration, conservation, and utilization of Citrus genetic resources in Meghalaya. The detailed morphological characterization and distribution data generated in this study could provide a strong foundation for future research and conservation efforts.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S147926212510018X.
Acknowledgements
The authors are thankful to The Director, ICAR-NBPGR, New Delhi and Department of Biotechnology, Ministry of Science and Technology, Government of India (DBT Project: Collection, conservation, and morpho-phenological characterization of citrus germplasm of NE region; Cost: Rs. 40.1 lakhs; Duration - 4 Years (16 February 2021 to 15 February 2025]) for providing facilities for undertaking the work.
Author contributions
J.U.: Conceptualization, exploration and collection, Writing – Original draft; K.V.: Data analysis, Writing – review & editing; D.T.: Co-supervision at Meghalaya; P.K.S.: Overall supervision and Writing – review & editing; B.L.: Co-supervision at Meghalaya; S: Literature review; P.H.N.: Co-supervision at Meghalaya; H.G.D.: Logistic arrangements during exploration and morphological characterization; SH: Communication during exploration with local tribal communities; A.D.: Exploration and collection, morphological characterization; A.K.: Exploration and collection, morphological characterization; K.K.M.: Exploration and collection, morphological characterization in Garo hills; O.M.: Exploration and collection, morphological characterization in East Khasi hills; G.L.M.: Exploration and collection, morphological characterization in West Khasi & Jaintia hills; A.D.: Passport data collection. All authors have read and approved the final version of the manuscript.
Competing interests
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors also declare that no ethical issues are raised during research.
